BR122024025431B1 - Use of an anti-CCR8 antibody in simultaneous, separate, or sequential combination with one or more additional therapeutically active compounds to induce ADCP in a human patient with a tumor. - Google Patents

Abstract

CCR8 ANTIBODIES FOR THERAPEUTIC APPLICATIONS, THEIR USE AND PRODUCTION METHOD, CONJUGATE, PHARMACEUTICAL COMPOSITION, POLYNUCLEOTIDE, VECTOR, ISOLATED CELL AND KIT. The present invention relates to tools and methods for generating antibodies that bind specifically to chemokine receptors, such as CC or CXC chemokine receptors. Isolated sulfated polypeptides and their conjugates are provided, which can be used, for example, as antigens or for off-target panning to facilitate the generation of anti-human, anti-cynomolgus and/or anti-mouse chemokine receptor antibodies, for example for the generation of antibodies with fully human CDRs and/or other properties favorable for therapeutic use. Furthermore, the invention relates to antibodies and their conjugates that can be obtained by applying such tools and methods. Antibodies that bind specifically to human, cynomolgus, and/or murine CCR8 with properties favorable for therapeutic use are provided, such as cross-reacting antibodies, fully human antibodies, low internalization antibodies (including non-internalizing antibodies), and antibodies that effectively induce ADCC and/or ADCP in Treg cells. Medical uses of the inventive antibodies or conjugates and/or treatment methods comprising administering the antibodies to a patient or (...) are also provided.

Description

DESCRIPTION Technical Field

[001] The present invention relates to tools and methods for generating antibodies that bind specifically to chemokine receptors, such as CC or CXC chemokine receptors. Isolated and conjugated sulfated polypeptides are provided, which can be used, for example, as antigens or for off-target panning to facilitate the generation of anti-human, anti-cynomolgus and/or anti-mouse chemokine receptor antibodies, for example for the generation of antibodies with fully human CDRs and/or other properties favorable for therapeutic use.

[002] In addition, the present invention relates to antibody conjugates thereof that can be obtained by applying the tools and methods mentioned above. Antibodies are provided that bind specifically to human, cynomolgus and/or murine CCR8 with properties favorable for therapeutic use, such as cross-reacting antibodies, fully human antibodies, low internalization antibodies (including non-internalizing) and antibodies that effectively induce ADCC and/or ADCP in Treg cells.

[003] Medical uses of the inventive antibodies or conjugates and/or treatment methods comprising administering these antibodies to a patient or individual, alone or in combination, are also provided. Biomarkers, stratification methods and diagnostic methods are finally provided to predict or assess responsiveness to monotherapy or combination therapy of anti-CCR8 antibodies.

[004] In addition, the invention provides tools and methods for producing the aforementioned antibodies, pharmaceutical compositions, diagnostic uses of the antibodies, and kits with instructions for use.

Technical Problem Generation of antibodies for CC and CXC chemokine receptors.

[005] CXC and CC chemokine receptors are seven-transmembrane G protein-coupled specific receptors that mediate cell migration in chemotactic gradients. Chemokine receptors are a challenging target class for antibody generation due to their inherent structural, biophysical, and biological properties. There are several reasons for the difficulties in obtaining ideal antibodies against chemokine receptors, which will be discussed in an exemplary manner, with emphasis on human CCR8.

[006] Chemokine receptors are characterized by seven domains embedded in the cell membrane and therefore cannot be easily purified into their native conformation. A purified native chemokine receptor will have been removed from the membrane environment and is therefore likely to be conformationally compromised. These corrupted structures are generally unsuitable for antibody generation because antibodies are required to recognize the intact antigen as it is presented on the cell surface. Although some chemokine receptors, such as CXCR4 and CCR5, possess some inherent stability that allows purification in mild detergents, this does not apply to most chemokine receptors (Hutchings, Catherine J., et al. "Opportunities for therapeutic antibodies directed at G-protein-coupled receptors." Nature reviews Drug discovery 16.11 (2017): 787.). This is emphasized by the fact that to date no X-ray crystal structure is available for CCR8 in the Protein Data Bank PDB (rcsb.org).

[007] Consequently, solubilization to obtain the necessary amounts of protein in the native conformation, as well as the correct orientation and folding for use as an immunogen, is difficult for chemokine receptors (as per, Klarenbeek, Alex et al. "Targeting chemokines and chemokine receptors with antibodies." Drug Discovery Today: Technologies 9.4 (2012): e237-e244.).

[008] A schematic representation of the general structure of human CCR8 is shown in Fig. 2b. Of the 355 amino acids, residues 1 to 35 (N term), 94 to 107 (ECL1), 172 to 202 (ECL2), and 264 to 280 (ECL3) have been predicted to be extracellular domains (uniprot.org). These extracellular domains are assumed to adopt a tertiary structure, stabilized by disulfide bonds. On average, less than 30% of a chemokine receptor is thus exposed on the cell surface. Consequently, chemokine receptors, and particularly CCR8, are not readily accessible for antibody binding, as also described in WO200744756.

[009] Furthermore, antibodies generated against peptides corresponding to the extracellular domains of chemokine receptors often fail to recognize the intact receptor on the cell, possibly due to differences in secondary structure. Consequently, researchers in this field have had a low success rate in developing antibodies (Tschammer, Nuska, ed. Chemokines: chemokines and their receivers in drug discovery. Vol. 14. Springer, 2015, Chapter by JE Pease & R. Horuk, section 6, page 12, 2nd paragraph).

[0010] Generating antibodies to obtain antimurine CCR8 antibodies seems somewhat less difficult, and conventional approaches such as that described by Kremer and Márquez can be reproduced by the inventors (D'Ambrosio, Daniele and Francesco Sinigaglia. Cell Migration in Inflammation and Immunity. Springer, 2004., Chapter by Kremer and Márquez pp. 243-260). Murine and human CCR8 have a sequence identity of ~70%, which is even lower for the extracellular domains, as described in more detail in Example 2. However, Kremer and Márquez discuss that producing monoclonal antimouse chemokine receptor antibodies is a demanding challenge and that antibodies against murine chemokine receptors are comparatively scarce, despite the time that has passed since their amino acid sequences were first reported.

Therapeutic antibodies for CCR8 and their medical uses

[0011] Despite the difficulties associated with their generation, antibodies targeting CC chemokine receptors have been suggested as promising therapeutic tools in several diseases, for example, based on mechanistic insights in diseases involving immune cells or in various cancer indications characterized by aberrant expression of chemokine receptors.

[0012] In 2004, Curiel et al. were able to show for CD4+CD25+FOXP3+ regulatory T cells (Tregs or Tregs) in 104 individuals affected with ovarian carcinoma, that human tumor Treg cells suppress tumor-specific T cell immunity and contribute to the growth of human tumors in vivo (Curiel, Tyler J., et al., "Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival." Nature medicine 10.9 (2004): 942-949.)

[0013] Tumor cells and microenvironmental macrophages produce chemokines, such as CCL22, which mediates the trafficking of Treg cells to the tumor. High levels of Tregs in the tumor microenvironment are not only associated with a poor prognosis in many cancers, such as ovarian, breast, kidney, and pancreatic cancer, but also cause suppression of the immune response against these cancers, for example, by suppressing the action of effector cells of the immune system. This specific recruitment of Treg cells therefore represents a mechanism by which tumors can promote immunological privilege. Therefore, it has been suggested that blocking the migration or function of Treg cells can defeat human cancer.

[0014] Aware of Curiel's discovery, two independent teams around Plitas/Rudensky and De Simone/Abrignani/Pagani discovered that tumor-infiltrating Treg cells are characterized by the selective expression of CCR8. In fact, the selectivity of Treg cell depletion for tumor-infiltrating Tregs is important because systemic depletion of Treg cells can cause severe autoimmunity (Nishikawa, Hiroyoshi, and Shimon Sakaguchi. "Regulatory T cells in tumor immunity." International journal of cancer 127.4 (2010): 759-767.). The search for a specific Treg marker is complicated by the fact that Treg cells exhibit a molecular pattern that resembles effector lymphocytes, as shown in Example 11.2. Neither peripheral Tregs nor tumor-specific effector cells should suffer friendly fire during the depletion of intratumoral Tregs, because peripheral Tregs are important for preventing autoimmunity, while tumor-specific effector cells help keep the tumor under control.

[0015] Based on these findings, several teams have suggested the use of CCR8 antibodies for the selective depletion of tumor-infiltrating regulatory T cells. Although some have presented data confirming tumor reduction in tumor models using anti-murine CCR4 or anti-murine CCR8 antibodies, thus confirming the mechanistic concept of Treg depletion, there is a need for therapeutic anti-human CCR8 antibodies, for example, with superior properties in formulations.

PREVIOUS TECHNIQUE 1.1 Antigens, methods and antibodies

[0016] The selection of an antigen in antibody generation is crucial for the properties of the resulting antibodies and can present serious problems, as described elsewhere here, for chemokine receptors such as CCR8. In some cases, antibodies have been obtained using whole cells as antigens, which were engineered for overexpression of chemokine receptors. At least for some chemokine receptors, these approaches appear to suffer from low ligand counts. Furthermore, antibodies obtained with these antigens are often characterized by off-target binding and low specificity for the chemokine receptor. If whole cells are used as antigens, immunodominant epitopes can mask other less antigenic ones, where the less antigenic ones would have the potential to generate the desired selective and specific antibodies.

[0017] For CCR8, a successful "whole cell" approach was described in WO2007044756 (ICOS). In summary, anti-CCR8 monoclonal antibodies were developed by immunizing Balb/c mice with irradiated cells transfected with CCR8 that had high levels of CCR8 expressed on the cell surface. Spleen cells from these mice were fused by standard methods to create hybridomas that produce the antibodies. Positive pools were identified by FACS and cloned by limiting dilution. Two of the antibodies, 433H and 459M, showed specific reactivity to human CCR8 in immunohistochemistry. The 433H antibody is still available and can be purchased from BD.

[0018] Biolegend distributes the L263G8 clone, a purified mouse IgG2a anti-human CCR8 antibody that was generated using human CCR8 transfectants as an immunogen.

[0019] Kremer and Márquez described the generation of monoclonal antibodies against murine CCR8 (D'Ambrosio, Daniele and Francesco Sinigaglia. Cell Migration in Inflammation and Immunity. Springer, 2004., Chapter by Kremer and Márquez p. 243 - 260). In summary, Kremer and Márquez describe the use of peptides derived from the extracellular domains of murine CCR8 as immunogens, but do not suggest sulfation of tyrosines. In fact, the inventors found that the approach described by Kremer and Márquez can be successfully applied to antibodies that recognize murine CCR8, but has low success rates for antibodies that recognize human CCR8.

[0020] Schaerli et al. generated anti-human CCR8 antibodies by immunizing rabbits with a human CCR8 peptide conjugate, corresponding to positions 1 to 34 of the N-terminal region of CCR8 coupled to KLH or BSA, but, again, they do not suggest sulfation of the tyrosines (Schaerli, Patrick, et al., "A skin-selective homing mechanism for human immune surveillance T cells."

[0021] A murine monoclonal antibody that binds to a 26-amino acid-containing peptide from the extracellular N-terminal portion of CCR8 was described by Haque et al. (Haque, Nasreen S., et al. "The chemokine receptor CCR8 mediates human endothelial cell chemotaxis induced by I-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and by lipoprotein (a)-stimulated endothelial cell conditioned medium." Blood, The Journal of the American Society of Hematology 97.1 (2001): 39-45.).

[0022] None of these methods for generating antibodies uses an isolated sulfated polypeptide comprising the tyrosine-rich domain (TRD) of a chemokine receptor or transmembrane protein as an antigen. The use of an isolated sulfated polypeptide comprising the TRD of a chemokine receptor or transmembrane protein as an antigen influences the structural and functional characteristics of the antibody set obtained, as described elsewhere herein.

[0023] Consequently, it is assumed that the antibodies according to the present invention deviate in structure and function from the antibodies of the prior art mentioned above, for example, at least in their affinity for sulfated CCR8, in their affinity for non-sulfated CCR8, in their shape and degree of modulation of the signaling receptor, in their internalization behavior, in cross-reactivity, in clearance and pharmacokinetic behavior and finally in Treg depletion and/or efficacy for therapeutic applications.

[0024] Furthermore, the use of the antigens described herein allowed the generation of fully human antibodies. In contrast, the antibodies described in the prior art for CCR8 are of non-human origin and deviate from some of the inventive antibodies at least because they do not comprise human CDRs.

1.2 Medical uses and mode of action

[0025] Cancer immunotherapy involves using an individual's immune system to treat or prevent cancer. Immunotherapies typically exploit the fact that cancer cells often have subtly different molecules on their surface that can be detected by the immune system—cancer antigens. Immunotherapy, therefore, involves provoking the immune system to attack tumor cells using these cancer antigens. However, some cancers, such as solid tumors or hematological cancers, may escape immune surveillance. For example, tumor infiltration by regulatory T cells (Treg cells or Tregs) and, more specifically, the low ratio of effector T cells (Teff) to Tregs have been proposed as critical factors in hiding the tumor from the immune system (Smyth, Mark J., Shin Foong Ngiow, and Michele WL Teng. "Targeting regulatory T cells in tumor immunotherapy." Immunology and cell biology 92.6 (2014): 473-474.).

[0026] Regulatory T cells expressing Foxp3, which are essential for preventing autoimmunity, are known to effectively suppress tumor immunity. Treg cells infiltrate tumor tissues abundantly, which is often associated with a poor prognosis in cancer patients. Removing Treg cells increases antitumor immune responses but can also trigger autoimmunity. A key issue in adapting Treg-targeted cancer immunotherapy lies in the specific depletion of Treg cells that infiltrate tumor tissues without affecting tumor-reactive effector T cells, while suppressing autoimmunity.

[0027] In 2010, several groups had already investigated that the removal of Tregs, for example, by depletion with anti-CD25 antibodies, could improve antitumor immunity in mice. It was demonstrated that depletion of Tregs before inoculation of tumor cells led to their efficient rejection, while Treg depletion occurring simultaneously with or after tumor inoculation did not result in tumor regression. Furthermore, it was suggested that this is due to the fact that the administered depleting antibodies also removed effector T cells expressing CD25, while CD25-Foxp3+ Tregs persisted (Klages, Katjana, et al. "Selective depletion of Foxp3+ regulatory T cells improves effective therapeutic vaccination against established melanoma." Cancer research 70.20 (2010): 77887799.).

[0028] In 2015, the non-fucosylated humanized anti-murine CCR4 monoclonal antibody, mogamulizumab (KW-0761), was evaluated in a clinical study for patients with CCR4-positive cancers (Kurose, Koji, et al. "Phase Ia study of FoxP3+ CD4 Treg depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer patients." Clinical Cancer Research 21.19 (2015): 4327-4336.). CCR4 is expressed on regulatory T cells. In fact, mogamulizumab effectively depleted Treg cells and an increase or induction of specific immune responses to cancer antigens was observed. However, mogamulizumab targets both peripheral and intratumoral Tregs and other effector cell populations, leading to immunological side effects such as skin rashes or Stevens-Johnson syndrome.

[0029] Thus, although the great potential for Treg depletion in cancer therapies was obvious, a concept for selectively targeting Tregs and avoiding overt autoimmunity was lacking. This was changed with the work of two teams around Plitas/Rudensky and De Simone/Abrignani/Pagani in 2015 and 2016.

[0030] Plitas et al. demonstrated that CCR8 is selectively expressed by Treg cells infiltrating human breast cancer and immediately concluded that targeting CCR8 represents a promising immunotherapeutic approach for the treatment of patients with breast cancer and other tumors (Plitas, G., et al. "Abstract P404-11: Preferred expression of chemokine receptor 8 (CCR8) on regulatory T cells (Treg) infiltrating human breast cancer represents a novel immunotherapeutic target."(2016): P4-04.; Plitas, George, et al. "Regulatory T cells exhibit distinct features in human breast cancer." Immunity 45.5 (2016): 1122-1134.; US10087259).

[0031] Just after the publication of Plitase et al.'s initial work, De Simone et al. published an analysis of the transcriptional landscape of tumor-infiltrating regulatory T cells and found that tumor-infiltrating Treg cells were highly immunosuppressive and expressed CCR8 on their cell surface as a specific signature molecule (De Simone, Marco, et al. "Transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating T regulatory cells." Immunity 45.5 (2016): 1135-1147., WO2017198631). The authors described that CCR8 correlated with poor prognosis and concluded that CCR8 could be an interesting therapeutic target for inhibiting Treg cell trafficking to tumor sites without disrupting the recruitment of other effector T cells that do not express CCR8.

[0032] WO2018112032 and WO2018112033 describe methods for decreasing the number or activity of tumor infiltrating regulatory T cells (TITR) in a tumor comprising administering an agent that induces cytotoxicity in cells expressing a gene product, for example, CCR8.

[0033] WO2018112033 and EP3431105 describe a molecule that can modulate the expression and/or function of at least one marker that is selectively downregulated in tumor-infiltrating regulatory T cells for use in the prevention and/or treatment of this tumor.

[0034] WO2018181425 refers to an antibody against CCR8 with ADCC activity, for use in a cancer treatment method, wherein the antibody against CCR8 is a CCR8-neutralizing antibody. WO2018181425 does not disclose specific antibody sequences, but refers to the mouse anti-mouse CCR8 clone SA214G2 distributed by BioLegend under Cat. No. 150302. This antibody was used as a tool antibody to recapitulate the previously described antitumorigenic effects of Treg depletion in mice.

[0035] According to the present invention, a method is provided that facilitates the generation of anti-CCR8 antibodies and produces antibodies with superior properties for therapeutic uses. The antibodies according to the present invention were compared with antibodies of the prior art and deviations in structure, function and therapeutic efficacy were found, as described elsewhere herein.

Solution to the problem 1.1 Generation of antibodies for CC and CXC chemokine receptors

[0036] Since prior art approaches to generating antibodies to CC chemokine receptors suffer from low success rates and the resulting antibodies generally show poor performance (as, for example, in Example 3), the inventors developed a new method to generate antibodies to chemokine receptors in general and, in particular, to generate anti-CCR8 antibodies.

[0037] The use of a specifically modified isolated polypeptide as an antigen for the selection of human anti-human CCR8 antibodies solved the problem of providing an improved method for generating antibodies against CC and CXC chemokine receptors, so that, for example, fully human anti-CCR8 antibodies could be obtained for the first time, as per Example 6. In greater detail, a polypeptide comprising the tyrosine-rich domain of human CCR8, optionally including the LID domain, was modified by introducing sulfate modifications at specific positions to form the antigen, as per Example 4, Table 4.1. A phage display approach was optionally used in conjunction with this specifically modified antigen to select fully human anti-human CCR8 antibodies. Alternatively, sulfated antigens can be used in several other methods, for example in conventional immunization approaches.

[0038] The antibodies obtained showed an excellent binding profile for the sulfated antigen and the biological target expressed under physiological conditions, as per Examples 6, 10.1.1, but a comparatively low or no affinity for the unmodified antigen, as per Examples 10.1.2, 10.1.3, demonstrating that the sulfated residues were indeed crucial for antibody-antigen binding. Furthermore, the inventive antibodies showed excellent and specific binding to cell lines engineered to express human, cynomolgus, or murine CCR8, and also to activated human Tregs, as per Example 10.1.1.

[0039] Of the six CDR loops of an antibody, the H3 loop shows the greatest structural diversity and is located in the center of the binding site. It also acquires more mutations through affinity maturation and has, on average, the highest number of contacts with the antigen. Therefore, it plays a crucial role in antigen binding. After analysis of the specific structure of antibodies obtained using the method according to the present invention, it was surprisingly found that the composition of HCDR3 was structurally different from the usual human HCDR3 domains, as shown in Example 9. In particular, the HCDR3 domains of antibodies with the most therapeutically beneficial properties and specific binding to CCR8 were characterized by an average frequency of ~21% tyrosine residues and an average histidine content of ~10%, emphasizing a beneficial impact of these residues on the recognition of the specific sulfated antigen (as shown in Table 9.2).

[0040] Without being limited by theory, the inventors believe that these structural characteristics translate into certain functional characteristics that make the obtained antibodies more suitable for therapeutic applications. For example, and unlike the prior art antibodies tested, several antibodies according to the invention block G protein-dependent signaling, as per Example 10.4.3, but do not affect G protein-independent pathways, as per Examples 10.4, 10.4.1, 10.4.2, and do not substantially internalize into cells with endogenous expression of the target chemokine receptor, as per Example 10.5. Furthermore, the particularly preferred antibodies according to the present invention were specific to the target receptor and target cells, as per Examples 10.2, 11, and are especially suitable/have demonstrated superior properties in cancer treatment methods, as per Examples 12 et seq. Furthermore, the use of these synthetic polypeptides, either as antigens or for off-target panning, facilitated or enabled the generation of cross-reacting antibodies, such as antibodies that bind specifically to human CCR8 and/or cynomolgus CCR8, as per Example 10.1.1.

1.2 Delivery of therapeutic antibodies that bind specifically to chemokine receptors 1.2.1 Obtaining chemokine receptor antibodies with human CDRs

[0041] Although humanization of antibodies with murine CDRs may improve the immunogenicity of an antibody, residual immunogenicity resides in the CDR regions (Harding, Fiona A., et al. "The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions." MAbs. Vol. 2. No. 3. Taylor & Francis, 2010.). Antibodies comprising human CDRs are therefore assumed to have superior suitability as therapeutic agents for humans compared to antibodies comprising CDRs from other species.

[0042] For example, antibodies with human CDRs can be produced through phage display technologies or using transgenic animals capable of generating fully human antibodies. However, obtaining fully human antibodies is not always trivial. While, in theory, the diversity of HCDR3 regions is almost unlimited, in practice, the generation of antibody repertoire diversity appears to be carefully regulated by multiple mechanisms, which produce an in vivo repertoire that is restricted in its diversity and restricted in its range of antigen-binding sites. This is also the case for human phage display libraries, which are often designed to reflect the in vivo repertoire. Thus, in some cases, the structural requirements of a "complicated" antigen may be met by rodent CDRs, while the same structure may not be readily recognized by human CDRs. Based on the observation that tyrosine representation is, on average, approximately 50% lower in human CCR3 than in murine CCR3, it can be assumed that it is clearly more challenging to find a human anti-human antibody in cases where the unique binding abilities of tyrosine and histidine are required to enable binding to the target. In fact, the inventors are not aware of fully human anti-human CCR8 antibodies of the prior art. According to the present invention, CCR8 antibodies comprising human-derived CDRs are provided, as well as fully human antibodies, as per Examples 6, 7, 8.

1.2.2 Obtaining antibodies from the cross-reacting chemokine receptor

[0043] Cross-reacting anti-chemokine receptor antibodies, such as cross-reacting anti-CCR8 antibodies, are advantageous for the development of a therapeutic antibody because they can be used in non-human animal models to characterize therapeutic agents with respect to pharmacological and safety data before the antibodies are administered to humans. However, cross-reacting antibodies, for example, with similar binding behavior in two species, are difficult to generate and cannot be easily matured by affinity, for example, because the extracellular parts of chemokine receptors, such as CCR8, have low homology between species (see example 2). According to the present invention, cross-reacting antibodies to CCR8 can be generated, in particular, using small motifs containing sulfated tyrosine that have greater conservation between species, so that cross-reacting antibodies that bind to a chemokine receptor, such as CCR8, of two or more species, for example with affinities of the same order of magnitude, can be obtained easily and conveniently, as per Examples 6, 7, 10.1.1.

1.2.3 Obtaining chemokine receptor antibodies for therapy

[0044] To induce the death of cells expressing a target chemokine receptor or CCR8, several modes of action can be predicted. One mode of action is the conjugation of an antibody targeting the chemokine receptor or CCR8 to a drug in the form of an antibody-drug conjugate (ADC). Other possible modes of action are the induction of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). For ADCC, CDC, and ADCP, a two-step mechanism is involved: on the one hand, the antibody or fragment is required to bind effectively to the target cell, for example, Treg via CCR8; on the other hand, the FC portion of the antibody (or an alternative binding portion that may be conjugated to the antibody or fragment as described elsewhere here) has to bind to an effector cell, which will then mediate the death of the target cell. For ADCP, binding to macrophages as effector cells typically occurs through the interaction of the Fc portion of antibodies with FcYRIIIa (CD32a) expressed by macrophages. In contrast, ADCC is mediated through the interaction of the antibody or fragment with FcYRIIIa. In humans, FcYRIII exists in two different forms: FcYRIIIa (CD16a) and FcYRIIIb (CD16b). While FcYRIIIa is expressed on mast cells, macrophages, and natural killer cells as a transmembrane receptor, FcYRIIIb is expressed only on neutrophils. These receptors bind to the Fc portion of IgG antibodies, which then activates antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

1.2.4 Obtaining low-internalization (including non-internalization) chemokine receptor antibodies

[0045] When the inventors analyzed anti-human CCR8 antibodies from the prior art, they found that these antibodies readily internalized into a cell with endogenous target expression, but did not reside on the cell surface for extended periods of time. The internalization behavior of an antibody influences not only its clearance and pharmacological behavior, but also its suitability for a specific mode of action for therapeutic uses.

[0046] Although high internalization is desirable for the generation of certain antibody-drug conjugates, it is undesirable for ADCC-induced depletion of tumor cells or Treg cells. More specifically, an antibody-drug conjugate is necessary to transport the drug into the cell to achieve efficient and specific depletion of the target cell. In contrast, the ADCC mode of action requires exposure of the antibody and its Fc domain on the outer part of the target cell, where immune effector cells can bind to the Fc domain for lysis of the target cell. A low or even absent internalization rate thus increases the time of effect for an ADCC/ADCP-inducing antibody.

[0047] After characterizing the antibodies obtained with the new method, the inventors surprisingly discovered that several of the antibodies according to the present invention showed a particularly low or even absent internalization profile in cells with endogenous CCR8 expression levels, as per Example 10.5, while prior art antibodies recognizing the same target had a higher internalization rate. For example, the 433H and L263G8 antibodies of the prior art are readily internalized into the target cell and do not reside on the cell surface for long periods of time. Due to their low internalization properties, some of the antibodies according to the present invention are therefore particularly useful in an ADCC, ADCP, CDC, or mixed approach, such as a combined ADCC/ADCP approach.

[0048] Typically, target selection is an important factor that strongly influences antibody internalization into a cell. According to Islam SA et al., CCL1 ligand binding induces Ca2+ influx and rapid internalization of the CCR8 receptor (Islam, Sabina A., et al. "Identification of human CCR8 as a CCL18 receptor. CCR8 is a CCL18 receptor." The Journal of experimental medicine 210.10 (2013): 1889-1898). The inventors found it surprising that the antibody itself was able to influence internalization properties to such a substantial degree. However, when they characterized the ability of the inventive antibodies to modulate the G protein-independent signaling pathways of their target chemokine receptor, they found that all antibodies tested from the prior technique not only blocked G protein-dependent signaling but also modulated G protein-independent signaling, as per Examples 10.4 et seq. G protein-independent signaling has previously been linked to internalization behavior, as per Fox, James M., et al. "Structure/function relationships of CCR8 agonists and antagonists: Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist." Journal of Biological Chemistry 281.48 (2006): 36652-36661.

1.2.5 Obtaining chemokine receptor antibodies by modulating target receptor signaling

[0049] There are several different ways in which an antibody can modulate a chemokine receptor. For example, an antibody can a) block G protein-independent signaling, b) block G protein-dependent signaling, c) block both G protein-dependent and G protein-independent signaling, d) increase G protein-independent signaling, e) increase G protein-dependent signaling, f) increase both G protein-dependent and G protein-independent signaling.

[0050] Without being constrained by theory, the inventors hypothesize that the sulfated TRD of the respective chemokine receptor may be relevant to ligand-induced signaling of the chemokine receptor. Interestingly, most of the inventive antibodies effectively blocked ligand-induced G protein-dependent signaling but did not influence G protein-independent signaling, as shown in Examples 10.4 et seq. In contrast, all prior art antibodies also blocked ligand-induced G protein-dependent signaling but agonized G protein-independent signaling. Without being constrained by theory, these differences may contribute to the differences in internalization behavior.

[0051] Avoiding the induction of G-protein-independent signaling by a therapeutic antibody may also be advantageous, because nonspecific signaling can result in uncontrollable downstream effects, such as increased cell proliferation, as per Hutchings, Catherine J., et al. (2017); Webb, David R., et al. "Opportunities for functional selectivity in GPCR antibodies." Biochemical pharmacology 85.2 (2013): 147-152.; Fox, James M., et al. (2006).

1.2.6 Obtaining antibodies for ADCC/ADCP-inducing chemokine receptors

[0052] The antibodies according to the present invention are characterized by a superior induction of ADCC and ADCP, as shown, for example, in Examples 10.3.3, 10.3.4. In some preferred embodiments, the antibodies were manipulated by fucose elimination in N297 (afucosylation) to obtain the high ADCC rates, as per Example 10.3.1. Interestingly, many of the inventive anti-CCR8 antibodies appeared to induce target cell depletion through a combined mode of action using both mechanisms, i.e., ADCC and ADCP.

1.3 Treatment Methods

[0053] Although the use of CCR8-binding antibodies in treatment methods based on mechanistic insights has been suggested by several research groups, the path to providing antibodies with ideal therapeutic properties has been tedious. By providing a method for generating antibodies according to the present invention, antibodies to chemokine receptors with superior therapeutic properties can now be easily obtained. According to the present invention, several sequence-defined antibodies with favorable properties are described, which can be used in a treatment method, for example, as part of a conjugate, in an ADCC-based approach, in an ADCP-based approach, in a CDC-based approach, or in a mixed ADCC/ADCP-based approach. Examples 12 et seq. show remarkable efficacies in Treg depletion, overall response rate, and tumor-control ratio for surrogate antibodies generated with a method according to the present invention.

1.4 Combined Treatment

[0054] Although remarkable response rates have been obtained in monotherapy with the inventive anti-CCR8 antibodies, it has been surprisingly discovered that the therapeutic benefit could be further enhanced by combining the antibodies with immune checkpoint inhibitors, with other targeted therapies, and even with nonspecific chemotherapy or radiotherapy. These combined treatments have been particularly beneficial in challenging tumor models. In particular, one specific combination treatment regimen has been found effective where the second therapeutic agent or therapy was administered only after the anti-CCR8 antibody has caused substantial Treg depletion (see Examples 12.6 and following). Furthermore, even a single treatment with anti-CCR8 antibody has been observed to show a substantial therapeutic effect (see Example 12.4.2).

1.5 Stratification Schemes and Diagnostic Methods

[0055] By evaluating the responsiveness of different syngeneic mouse models to anti-CCR8 antibody treatment, the inventors were able to identify several mechanisms, biomarkers, and biomarker combinations that were predictive of treatment efficacy and overall response. For example, it was surprisingly found that the degree of responsiveness to immune checkpoint inhibitors, such as PD-1, PD-L1, or CTLA4 antibodies, was also predictive of response to anti-CCR8 antibody treatment. Since PD-L1 expression is sometimes used as a surrogate marker to predict responsiveness to immune checkpoint inhibitors, the inventors evaluated whether PD-L1 could also be used to predict responsiveness to anti-CCR8 antibody treatment. This hypothesis can ultimately be confirmed by correlation data, see Example 12.7.1. In another attempt to identify individuals who would likely benefit from anti-CCR8 treatment, other marker genes, such as immune cell and Treg markers, were evaluated as biomarkers (see Examples 12.1.2, 12.7.2, 12.8).

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Fig. 1: Alignment of human CC chemokine receptors and CXC chemokine receptors.

[0057] Figure 2: a. Human CCR8, cynomolgus CCR8, and murine CCR8 sequences were retrieved from Uniprot and aligned with Clustal Omega. b. Schematic representation of the CCR8 receptor structure containing 7 transmembrane domains (black boxes), three extracellular loops (ECL), several disulfide bridges (SS), and an intracellular C-terminal portion, as well as the extracellular N-terminal domain composed of the "LID" and the tyrosine-rich domain (TRD).

[0058] Fig. 3: Evaluation of three antibodies from the prior technique by FACS staining in CCR8-positive target cells. None of the prior technique antibodies shown exhibited a deviation in FACS staining compared to the isotype control. hAP-0068 and MAB1429-100 were tested in 293T cells stably transfected with human CCR8, while MAB8324 was tested in BW5147.3 cells expressing murine CCR8.

[0059] Fig. 4: Panning strategy for lead discovery: Two main strategies for selection in sulfated peptides are represented. In all strategies, before each round of selection, a depletion step of a relevant or irrelevant biotinylated protein was included.

[0060] Fig. 5: Panning strategy to obtain CCR8 antimurine antibodies: The main selection strategy based on a mixture of sulfated/non-sulfated peptides is described. Before each selection round, a depletion step of a relevant biotinylated protein was performed.

[0061] Fig. 6: FACS data of inventive antibodies that bind to CHO cells expressing human CCR8.

[0062] Fig. 7: FACS data of inventive antibodies that bind to CHO cells expressing cynomolgus CCR8.

[0063] Fig. 8: FACS data from candidates that bind to activated human Tregs (donor 1). Right side: Percentage of CCR8 expression determined using BioLegend Clone L263G8.

[0064] Fig. 9: FACS data of inventive antibodies TPP-21181 and TPP-23411 that bind to CHO cells expressing human CCR8.

[0065] Fig. 10: FACS data of inventive antibodies TPP-21181 and TPP-23411 that bind to CHO cells expressing cynomolgus CCR8.

[0066] Fig. 11: Binding of TPP-21360, L263G8 and 433H to CHO cells expressing human CCR8.

[0067] Fig. 12: Binding of TPP-21360, L263G8 and 433H to CYNOMLOCs CCR8-expressing CHO cells. The antibodies from the previous technique showed only very low overall binding to CYNOMLOCs CCR8 (low saturation). However, EC50 values could be determined for each antibody.

[0068] Fig. 13: Staining of human Tregs activated with the inventive antibody TPP-23411, gating strategy. Classified CD4+ and CD25+ peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28 beads (Treg expansion kit, Miltenyi). Center panel: cells that are CD4+ CD25+ CD127low and Foxp3+. Bottom panel: CCR8 expression in Tregs after staining with TPP-23411 or L263G8.

[0069] Fig. 14: Binding of TPP-21360 to CHO cells with simulated transfectants. No nonspecific binding was observed.

[0070] Fig. 15: Off-target binding in HEK cells transiently transfected with human CCR1. Most inventive candidates showed only low target binding for CCR1.

[0071] Fig. 16: Off-target binding in HEK cells transiently transfected with human CCR4. Most inventive candidates showed only low target binding for CCR4.

[0072] Figure 17: Staining for CCR8 expression in different immune cell populations of PBMCs from healthy donors (CD4+ T cells, CD8+ T cells, B cells labeled for CD19+ expression, myeloid cells labeled for CD11b+ expression, CD4+ T cells activated with anti-CD3 and anti-CD28 beads for 4 days, CD8+ T cells activated with anti-CD3 and anti-CD28 beads for 4 days).

[0073] Figure 18: CCR8 is upregulated in human Tregs after activation. After activation, the percentage of CCR8+ cells increased significantly in purified CD4+CD25+ cells with increased MFI, while the percentage of CCR8+ cells increased slightly in the PBMC population. BioLegend anti-CR8 antibody was used for these experiments. N=4 donors.

[0074] Fig. 19: ADCC induced by inventive wild-type or non-fucosylated antibodies TPP-19546, TPP-21360 or TPP-23411 in HEK cells expressing human CCR8 as target cells. Isotype control: TPP-9808.

[0075] Fig. 20: Left: Percentage of CCR8+ expression on day 3 for activated human Tregs as target cells. Right: ADCC induced by the inventive antibody TPP-21360, wild-type or non-fucosylated TPP-23411 in activated human Tregs as target cells. Isotype control: TPP-9808. Results were reproducible in different donors (data not shown).

[0076] Fig. 21: Protocol for ADCP test.

[0077] Fig. 22: The ADCP assay for wild-type and non-fucosylated versions of inventive antibodies shows the induction of phagocytosis in HEK cells expressing human CCR8 as target cells with in vitro differentiated M2c macrophages as effector cells. Wild-type and non-fucosylated versions of inventive antibodies TPP-19546, TPP-21360, and TPP-23411 induced ADCP. TPP-9808 is the isotype control. Mogamulizumab is a commercially available anti-CCR4 antibody.

[0078] Fig. 23: The ADCP assay for wild-type and non-fucosylated versions of inventive antibodies TPP-21360 and TPP-23411 shows induction of phagocytosis in activated human Tregs from two different donors as target cells (upper or lower panel) and M2c macrophages as effector cells. TPP-9808 is the isotype control.

[0079] Fig. 24: The ADCP assay for wild-type and non-fucosylated versions of inventive antibodies TPP-21360 and TPP-23411 shows the induction of phagocytosis in activated human Tregs as target cells and M1 macrophages as effector cells.

[0080] Fig. 25: Activation of β-arrestin signaling measured as a DiscoverX assay for CCL1, TPP-23411, L263G8, or 433H in CHO cells co-expressing ProLink-labeled human CCR8 and EA. As expected, the CCR8 CCL1 (human) ligand induces β-arrestin signaling. An extra time point was analyzed for TPP-23411 to ensure that no β-arrestin activation occurred at earlier time points. None of the antibodies analyzed induced β-arrestin signaling activation.

[0081] Fig. 26: Evaluation of the blockade of CCL1-induced β-arrestin signaling by TP-23411, L263G8, or 433H, measured with the DiscoverX assay. CCL1 was used at its EC80 (20 ng/ml). The prior art antibodies L263G8 and 433H blocked CCL1-induced β-arrestin signaling at low antibody concentrations, while the inventive antibody TPP-23411 showed no effect at these concentrations. It has been suggested that β-arrestin signaling plays a role in internalization.

[0082] Fig. 27: Phospho Erk1/2 ELISA assay. CHO cells expressing human CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8 or BD 433H antibody and cell lysates were collected at the respective time point.

[0083] Fig. 28: Phospho Erk 1/2 ELISA assay. Activated human Treg cells expressing CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8 or BD 433H antibody and cell lysates were collected at the respective time point. The antibodies from the prior technique, Biolegend L263G8 and BD 433H antibody, induced a significant increase in phosphorylated Erk1/2 levels, for example, after 15 minutes in activated human Tregs, while this was not the case for the inventive antibody.

[0084] Fig. 29: Phospho AKT ELISA assay. CHO cells expressing human CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8 or BD 433H antibody and cell lysates were collected at the respective time point.

[0085] Fig. 30: Phospho AKT ELISA assay. Activated human Treg cells expressing CCR8 were treated with CCL1, TPP-23411, Biolegend L263G8 or BD 433H antibody and cell lysates were collected at the respective time point. The prior technique antibodies Biolegend L263G8 and BD 433H induced a significant increase in phospho AKT levels in activated human Tregs, for example, after 15 minutes, while this was not the case for the inventive antibodies.

[0086] Fig. 31: Internalization studies. a. FACS analysis of commercial anti-human CCR8 antibodies Biolegend L263G8 or BD 433H antibody with the HuT78 cell line that endogenously expresses CCR8. b. Internalization studies of commercial anti-human CCR8 antibodies with the HuT78 cell line that endogenously expresses CCR8 based on cytoplasmic intensity staining. TPP-5657: Isotype control.

[0087] Fig. 32: Internalization studies. a. FACS analysis of the commercial anti-murine CCR8 antibody SA214G2 with the BW5147.3 cell line that endogenously expresses murine CCR8. b. Internalization study of the commercial anti-murine CCR8 antibody SA214G2 with the BW5147.3 cell line that endogenously expresses murine CCR8. The anti-murine CCR8 antibody from the previous technique SA214G2 induces internalization.

[0088] Fig. 33: Internalization study of inventive anti-human CCR8 antibodies TPP-21360 and TPP-23411 with cell lines that endogenously express CCR8 TALL-1 (a) and HuT78 (b). Both antibodies reveal identical internalization behavior, comparable to the TPP-5657 isotype control.

[0089] Fig. 34: FACS analysis of inventive anti-human CCR8 antibodies TPP-21360 and TPP-23411 with cell lines that endogenously express CCR8 TALL-1 (a) and HuT78 (b). Both antibodies show the same binding potency to the cell lines.

[0090] Fig. 35: CCR8 mRNA expression in 11642 samples representing a comprehensive set of human tissues and cell types measured by the affymetrix 208059_at probe. All samples were conormalized using the refRMA method (Katz, Simon, et al. "A summarization approach for Affymetrix Genechip data using a reference training set from a large, biologically diverse database." BMC bioinformatics 7.1 (2006): 1-11.). The dark gray box coloring indicates that the median sample in the corresponding group has a probe signal intensity significantly above the background noise (as estimated by the affymetrix MAS5 algorithm, as per Pepper, Stuart D., et al. "The utility of MAS5 expression summary and detection call algorithms." BMC bioinformatics 8.1 (2007): 1-12.). Median CCR8 expression significantly above background noise is observed only in activated regulatory T cells, as well as in tumor-infiltrating lymphocytes. Groups are classified based on decreasing mean expression.

[0091] Fig. 36: CCR8 mRNA expression, measured by RNA-seq, in 50 different TCGA tumor indications (https://www.cancer.gov/tcga) (dark gray boxes), corresponding normal tissues (white boxes), as well as across the entire CCLE tumor cell line panel (Barretina, Jordi, et al. "The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity." Nature 483.7391 (2012): 603-607.) also grouped by tumor indications (light gray boxes). Groups are ranked based on mean expression. Indications with the highest CCR8 expression are breast cancer, lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), head and neck malignancies, as well as esophageal tumors. In all indications except pancreatic adenocarcinoma and melanoma, expression appears higher in the tumor compared to the corresponding normal tissue. Little or no expression is observed in corresponding tumor cell lines of epithelial origin, indicating that CCR8 is not expressed by tumor cells, but exclusively by tumor-infiltrating T cells. In principle, every indication of tumor with CCR8+ Treg cell infiltration should be eligible for treatment with anti-CCR8 antibodies.

[0092] Fig. 37: Immunohistochemistry with Treg FOXP3 and CCR8 (clone 433H) marker staining in human non-small cell lung cancer (NSCLC) tissue or human melanoma tissue.

[0093] Fig. 38: Staining of T cell populations classified by FACS based on CCR4, CCR8, OX40, GITR, and CD25. Only CCR8 is specific for activated Tregs. OX40, GITR, and CD25 are significantly expressed in stimulated CD8+ Teff cells and CD4+ T cells (CD4+CD25+Foxp3-). Dark gray: isotype control, light gray: target staining.

[0094] Fig. 39: CCR8 expression in different subsets of T cells extracted from different tumor entities, as measured by single-cell RNA-seq. For these tumors, CCR8 mRNA expression is largely restricted to regulatory T cells (light gray boxes) residing in tumor tissue, while it is largely absent in regulatory T cells from normal tissues, as well as from CD4 helper and CD8 cytotoxic T cells (medium and dark gray boxes, respectively). Top panel: Colorectal tumor tissue (Tumor) or adjacent normal tissue (Normal). Middle panel: Liver cancer tissue (Tumor) or adjacent normal tissue (Normal). Bottom panel: Lung cancer tissue (tumor) or adjacent normal lung tissue and peripheral blood (normal). Sample sizes, N, indicate the number of cells in each category. The cells were designated as regulatory T cells, helper T cells, or cytotoxic T cells based on the expression of the marker genes FOXP3, CD4 (but not FOXP3), and CD8A/B, respectively.

[0095] Fig. 40: Tregs, macrophages, T cells, or tumor cell populations from NSCLC, CRC (colorectal cancer), or RCC (renal carcinoma) were stained with anti-CCR8 antibody or isotype control and analyzed by flow cytometry. X-axis: Log shift up to 105; Y-axis: Intensity normalized to the mode. Lighter gray: Isotype control. Darker gray: CCR8. The specific expression of CCR8 in intratumoral human Tregs is indicated by arrows.

[0096] Fig. 41: Growth of CT26 tumor after treatment with different doses of anti-CCR8 antibodies or anti-PDL1 antibody.

[0097] Fig. 42: Spider graphs of mice carrying CT26 tumor after treatment with different doses of anti-CCR8 antibodies, anti-PDL1 antibody or isotype controls.

[0098] Fig. 43: FACS analysis of intratumoral immune cells 24 hours after treatment with the second antibody. Analysis of intratumoral Tregs. The anti-CCR8 antibody TPP15285 leads to an average decrease in intratumoral Tregs to 13%, 33%, or 47% of Tregs in the isotype control, for 10 mg/kg, 1 mg/kg, or 0.1 mg/kg, respectively. The anti-CCR8 antibody TPP15286 leads to an average decrease in intratumoral Tregs to 10%, 9%, or 14% of Tregs in the isotype control, for 10 mg/kg, 1 mg/kg, or 0.1 mg/kg, respectively, as shown in Table 12.2.1. Analysis of intratumoral CD8+ T cells. The average % increase in isotype control is shown in Table 12.2.1. Analysis of the ratio of CD8+ T cells to Treg cells. The anti-CCR8 antibody TPP15286 leads to an average increase in the ratio of CD8+ cells to Treg cells of 44 (10 mg/kg), 87 (1 mg/kg), or 68 (0.1 mg/kg). The anti-CCR8 antibody TPP15285 leads to an average increase in the ratio of CD8+ cells to Treg cells of 64 (10 mg/kg), 16 (1 mg/kg), or 10 (0.1 mg/kg). Analysis of the ratio of converting CD4+ T cells to Treg cells.

[0099] Fig. 44: CT26 tumor growth after treatment with glycosylated (TPP-14095, TPP-14099, TPP-15285, TPP-15286) or aglycosylated (TPP-18208, TPP-18209) anti-CCR8 antibodies. Aglycosylation largely abolished the antitumor effect.

[00100] Fig. 45: Spider plots of mice bearing CT26 tumor after treatment with glycosylated (TPP-14095, TPP-14099, TPP-15285, TPP-15286) or aglycosylated (TPP-18208, TPP-18209) anti-CCR8 antibodies. Aglycosylation largely abolished the antitumor effect, suggesting an ADCC/ADCP-dependent mechanism of antitumor efficacy.

[00101] Fig. 46: FACS analysis of immune cells 24 hours after treatment with the second antibody. Absolute numbers of intratumoral CD45+ cells. Absolute numbers of intratumoral CD45+CD8+ T cells. Absolute numbers of intratumoral CD8+ T cells. Absolute numbers of conventional intratumoral CD4+ cells. Analysis of intratumoral Treg depletion by flow cytometry showing absolute cell numbers. The mean percentage of residual Tregs relative to the respective isotype control after antibody administration was 15.6% for TPP-14095, 28.2% for TPP-14099, 8.7% for TPP-15285, and 36.5% for TPP-15286. No reduction in Tregs was observed for the non-glycosylated antibodies. % of intratumoral CD8+ T cells of CD45+ cells. CD8+:Treg T cell ratio. CD4+:Treg conversion rate. Percentage of Treg cells to CD4+ T cells. Absolute number of intratumoral 4-1BB+ Treg cells. Absolute number of intratumoral 4-1BB+ CD4+ cells converted.

[00102] Fig. 47: EMT6 tumor growth after treatment with different doses of anti-CCR8 antibody TPP-15285. Significance was determined by 1-Way ANOVA plus Sidak's post-hoc test after logarithmic transformation.

[00103] Fig. 48: EMT6 tumor growth on day 19 after treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody.

[00104] Fig. 49: Spider graphs of mice bearing EMT6 tumor after treatment with different doses of anti-CCR8 TPP-15285 antibody or anti-CTLA4 antibody.

[00105] Fig. 50: FACS analysis of immune cells from mice bearing EMT6 tumor after treatment with different doses of CCR8 TPP-15285 antibody or anti-CTLA4 antibody, 24 hours after the second treatment or at the end of the study. Absolute numbers of intratumoral Treg cells. Significant differences were observed, for example, for 10 mg/kg of anti-CCR8 antibody at both time points. Ratio of CD8-positive cells to Treg cells. CD4+ cells converting to Treg cells. Percentage of Treg cells to CD4+ T cells.

[00106] Fig. 51: FACS analysis of immune cells from mice bearing EMT6 tumors after treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-CTLA4 antibody, 24 hours after the second treatment or at the end of the study. Absolute numbers of intratumoral CD45+ cells. Absolute numbers of intratumoral CD4+ cells. Absolute numbers of intratumoral CD4+ T cells. Absolute numbers of intratumoral activated CD8+ T cells. Absolute numbers of intratumoral NK cells. Absolute numbers of intratumoral CD8+ T cells. Differences were observed, for example, for 10 mg/kg of anti-CCR8 antibody at the end of the study.

[00107] Fig. 52: Growth of F9 tumor after treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-PDL1 antibody.

[00108] Fig. 53: Tumor volume F9 on day 16 after the start of treatment with different doses of anti-CCR8 antibody TPP-15285 or anti-PDL1 antibody. Significant improvement was observed at least for an antibody dose of 10 mg/kg.

[00109] Fig. 54: Spider graphs of mice bearing F9 tumor after treatment with different doses of anti-CCR8 antibodies or anti-PDL1 antibody.

[00110] Fig. 55: Effects of anti-CCR8 or anti-PDL1 antibodies on immune cell populations and their relationships in F9 tumors analyzed by FACS 24 hours after treatment with the second antibody. The anti-CCR8 antibody increased the absolute number of intratumoral CD45+ T cells, intratumoral CD4+ T cells, intratumoral CD8+ T cells, intratumoral CD4+conv T cells, and intratumoral NK cells compared to the matched isotype control. The anti-CCR8 antibody at 10 mg/kg decreased the absolute number of intratumoral Treg cells (CD4+, CD25+, FoxP3+).

[00111] Fig. 56: Effects of anti-CCR8 or anti-PDL1 antibodies on immune cell populations and their ratios in F9 tumors analyzed by FACS 24 hours after the second treatment plotted as CD8+ T cell to Treg ratio, Treg to CD4+ T cell frequency, 4-1BB+ CD8+ T cell frequency, 4-1BB+ Treg cell frequency, and 4-1BB+ conventional CD4+ T cell frequency. 10 mg/kg of anti-CCR8 antibody increased the CD8+ to Treg cell ratio to approximately 54 or higher, as shown in Table 12.5.1.

[00112] Fig. 57: Efficacy of combination treatments in C38 tumor-bearing mice. a. C38 tumor growth after treatment with anti-CCR8 surrogate antibodies TPP-14099 or TPP-15285 or anti-PD-L1 antibody or after combination treatment with 3 mg/kg of anti-PDL1 antibody, 10 mg/kg of TPP-15285. Significance determined by 1-Way ANOVA plus Sidak's post-hoc test after log transformation. b. Survival graph of C38 tumor-bearing mice after treatment with antimurine CCR8 antibody TPP-15285 (10 mg/kg), antimurine PD-L1 antibody (PDL1, 3 mg/kg) or a combination of TPP-15285 (10 mg/kg) and antimurine PD-L1 antibody (3 mg/kg).

[00113] Fig. 58: Analysis of tumor Treg depletion in C38 tumors by flow cytometry (sampling 24 hours after treatment with the second antibody). Mice bearing C38 tumors were treated with anti-CCR8 surrogate antibodies TPP-14099 or TPP-15285 or anti-PD-L1 antibody in monotherapy, or with TPP-15285 in combination with anti-PD-L1 antibody. Absolute Treg depletion. CD8+ T cell/Treg cell ratios.

[00114] Fig. 59: CD11+ F4/80+ macrophages in C38 tumors as analyzed by flow cytometry (sampling at the end of the study).

[00115] Fig. 60: Growth of B16F10-OVA tumor after treatment with TPP-15285, anti-CTLA4 antibody or both.

[00116] Fig. 61: EMT-6 tumor growth after treatment with CCR8 antimurine antibody TPP-15285 (1 mg/kg), PD-1 antimurine antibody (CDRs: atezolizumab, 10 mg/kg) or a combination of TPP-15285 (1 mg/kg) and PD-1 antimurine antibody (10 mg/kg), (twice weekly i.p.). Mean with standard deviation.

[00117] Fig. 62: FACS analysis of immune cell populations in EMT-6 tumors analyzed by flow cytometry, sampling 24 hours after the second antibody treatment. CD4+ T cells, Tregs, CD8+ T cells, NK cells, CD8+ T cell to Treg ratios. Box and whiskers, min to max with median (GraphPad).

[00118] Fig. 63: a. Analysis of C38 tumor-bearing mice after treatment with anti-murine CCR8 antibody TPP-15285 (5 mg/kg), anti-murine PD-1 antibody (CDRs: atezolizumab, 5 mg/kg) or a combination of TPP-15285 (5 mg/kg) and anti-murine PD-1 antibody (5 mg/kg), (twice weekly i.p.). a. Tumor volume. Mean with standard deviation. b. Survival graph.

[00119] Fig. 64: Analysis of tumor CD4+ T cells, Tregs, CD8+ T cells, NK cells or CD8+ T cell to Treg ratios in C38 tumors by flow cytometry (sampled 24 hours after the second antibody treatment).

[00120] Fig. 65: MB49 tumor growth after treatment with anti-CCR8 antibody TPP-15285 (10 mg/kg) alone, anti-PD-1 antibody (aPD-1, CDRs: atezolizumab, 10 mg/kg) alone or sequential combination treatment of TPP-15285 (10 mg/kg) and anti-PD-1 antibody (10 mg/kg) (both twice weekly i.p.).

[00121] Fig. 66: Analysis of CD45+ tumor cells, Tregs, CD8+ T cells, NK cells and CD8 to Treg ratio in MB49 tumors by flow cytometry (sampling at the end of the study).

[00122] Fig. 67: EMT-6 tumor growth after treatment with anti-CCR8 antibody TPP-15285 (5 mg/kg, q3/4d ip), Oxaliplatin (5 mg/kg, q4d ip), Doxorubicin (6 mg/kg, iv SD), Docetaxel (10 mg/kg, q2dx5, iv) or a combination of TPP-15285 with Oxaliplatin, Doxorubicin or Docetaxel.

[00123] Fig. 68: Growth of MB49 tumor after treatment with anti-CCR8 antibody TPP-15285, gemcitabine or a combination of TPP-15285 with gemcitabine.

[00124] Fig. 69: Growth of Lewis lung tumor after treatment with anti-CCR8 antibody TPP-15285 (10 mg/kg) alone, anti-PD-1 antibody (aPD-1, CDRs: atezolizumab, 10 mg/kg) alone, anti-PD-L1 antibody (10 mg/kg) alone, anti-CTLA4 antibody alone, TPP-15285 (10 mg/kg) and anti-murine PD-1 antibody (10 mg/kg) in combination, or TPP-15285 (10 mg/kg) and anti-PD-L1 antibody (10 mg/kg) in combination.

[00125] Fig. 70: Growth of EMT-6 tumor after treatment with anti-CCR8 antibody TPP-15285 (3 mg/kg) or radiotherapy (RT, 3 x 2 Gy) alone or in combination. Significance was determined by 1-Way ANOVA plus Sidak's post-hoc test after logarithmic transformation.

[00126] Fig. 71: mRNA expression levels of different immune cell markers in different syngeneic tumor models treated with isotype control (TPP-9809) or anti-CCR8 antibody (TPP-14099). Median expression in isotype-treated control groups is defined as zero. Gene expression is measured in transcripts per million (TPM) as estimated by the RSEM algorithm. Treg Foxp3 marker. Significantly higher Foxp3 levels are observed in CT26, H22, Hepa1-6, and RM1 models treated with TPP-14099, indicating increased Treg infiltration after administration of at least three doses of TPP-14099.

[00127] Fig. 72: Inflammation marker lfng. Significantly higher lfng levels are observed in the H22, CT26, and RM1 models treated with TPP-14099, pointing to strong pro-inflammatory activity induced by TPP-14099.

[00128] Fig. 73: Macrophage marker Ms4a7. Significantly higher levels of Ms4a7 are observed in CT26, H22, and Hepa1-6 models treated with TPP-14099, indicating increased macrophage infiltration caused by TPP-14099.

[00129] Fig. 74: Cytotoxic T cell markers Cd8a and Cd8b1. Significantly higher levels of cytotoxic T cells are observed in CT26, H22, RM1 and Hepa1-6 models treated with TPP-14099, indicating the induction of cytotoxic T cell infiltration and/or proliferation by TPP-14099.

[00130] Fig. 75: Natural killer (NK) cell marker Ncr1. Significantly higher NK cell levels are observed in CT26, H22, and Hepa1-6 models treated with TPP-14099.

[00131] Fig. 76: Pan T cell markers Cd3e/d/g. Significantly higher T cell levels are observed in CT26, H22, and Hepa1-6 models treated with TPP-14099.

[00132] Fig. 77: Activated Treg marker and target antibody Ccr8. Significantly higher levels of Ccr8 are observed in CT26, H22, and Hepa1-6 models treated with TPP-14099.

[00133] Fig. 78: Highly specific pro-inflammatory M1 macrophage marker Acod1 (a) and highly specific anti-inflammatory M2 macrophage marker Mrc1 (b). Markedly higher M1 macrophage levels are observed in the CT26, 4T1, H22, Hepa1-6, and RM1 models treated with TPP-14099, while markedly higher M2 macrophage levels are not observed in any of the TPP-14099-treated models.

[00134] Fig. 79: Ratio of highly specific pro-inflammatory M1 macrophage marker Acod1 and highly specific anti-inflammatory M2 macrophage marker Mrc1. Taken together, multiple doses of anti-CCR8 antibody TPP-14099 increased the M1/M2 macrophage ratio in these tumor models.

[00135] Fig. 80: B cell markers Cd19 and Cd22. Markedly higher B cell levels are observed in CT26, H22, RM1, and Hepa1-6 models treated with TPP-14099. The anti-CCR8 antibody appears to recruit B cells to the tumor. Without being limited by theory, B cell recruitment may affect the antitumor response induced by TPP-14099.

[00136] Fig. 81: Expression levels for LTta/b as well as Cxcr5 and its ligand Cxcl13. These markers are crucial for the development of lymph nodes, as well as tertiary lymphoid structures (as per Cyster, Jason G. "Blown away: the unexpected role of lym-photoxin in lymphoid organ development." The Journal of Immunology 192.5 (2014): 2007-2009., and Cupedo, Tom, et al. "Induction of secondary and tertiary lymphoid structures in the skin." Immunity 21.5 (2004): 655-667.) which are the main drivers of an antitumor effect in humans (as per Dieu-Nosjean, Marie-Caroline, et al. "Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers." Immunological reviews 271.1 (2016). All three publications are incorporated here by reference in their entirety.

[00137] Fig. 82: Spider plots of tumor growth (measured in mm3) over time in the TPP-14099 treatment group (shown as triangles) and isotype-treated control group (shown as circles) for different tumor models. T-cell infiltration levels at the end of the study, as judged by Cd8a mRNA levels in corresponding mass tumor samples, are indicated by shades of gray (black and light gray correspond to the highest and lowest Cd8a levels, respectively). In H22 and CT26, tumor size showed an excellent anticorrelation with Cd8a levels.

[00138] Fig. 83: Correlation between tumor infiltration by different immune cell populations as judged by mRNA levels of different immune cell markers and tumor size. Tumors treated with TPP-14099 are shown as triangles and tumors treated with the isotype are shown as circles. Tumor size is measured in mm3. Strong negative correlation between cytotoxic T cell infiltration (as assessed by Cd8a+Cd8b1 mRNA levels) and CT26 tumor sizes. Tumors treated with TPP-14099 are smaller in size and show higher levels of Cd8a+Cd8b1 T cell infiltration than controls, indicating that increased T cell infiltration after TPP-14099 treatment causes reduced tumor growth. Strong negative correlation between T cell infiltration (as measured by Cd3 mRNA levels) and CT26 tumor sizes in tumors treated with TPP-14099. Tumors treated with TPP-14099 are smaller in size and exhibit higher levels of T cell infiltration; that is, the higher the Cd3 mRNA level, the smaller the tumors. Negative correlation between NK cell infiltration (as assessed by the expression of the NK marker Ncr1) and CT26 tumor sizes. Tumors treated with TPP-14099 are smaller in size and exhibit greater NK cell infiltration. Tumor size is strongly negatively correlated with NK infiltration; the higher the NK level, the smaller the tumors. Correlation between the postulated induction of tertiary lymphoid structures as judged by Lta/Ltb/Cxcr5 and Cxcl13 mRNA levels and CT26 tumor sizes in tumors treated with TPP-14099. For each of the four markers, the level is strongly increased after treatment with TPP-14099, and tumor size is strongly negatively correlated with the expression level, indicating that the increased formation of tertiary lymphoid structures after treatment with TPP-14099 causes reduced tumor growth.

[00139] Fig. 84: Correlation between Cd8a infiltration levels, as judged by Cd8a mRNA expression, and tumor sizes in tumors treated with TPP-14099 (shown as triangles) and in the isotype-treated control group (shown as circles) for different tumor models. Tumors treated with TPP-14099 are smaller in size and show higher levels of Cd8a infiltration. Tumor size is strongly negatively correlated with Cd8a infiltration; the higher the Cd8a level, the smaller the tumors.

[00140] Fig. 85: Efficacy of the CCR8 antibody TPP-15285 in EMT6 tumor-bearing mice treated with four different treatment regimens: single treatment, twice-BIW treatment, three times-BIW treatment, or four times-BIW treatment. Each group was treated with vehicle control or 0.1 mg/kg, 1 mg/kg, or 5 mg/kg of TPP-15285.

[00141] Fig. 86: Therapeutic efficacies of the inventive anti-CCR8 antibody, anti-PD-1 antibody, anti-PD-L1 antibody or Paclitaxel tested alone or in combination in MBT2 syngeneic tumor-bearing mice as shown in Table 12.6.9.1.

[00142] Fig. 87: Therapeutic efficacy of the inventive anti-CCR8 antibody after quantitative depletion of CD8a+ expression in DTR mice treated with diphtheria toxin. Depletion of CD8+ T cells abolishes the antitumor growth effect of TPP15825 (as per group 02b vs group 01b).

[00143] Fig. 88: Therapeutic efficacy of the inventive anti-CCR8 antibody after quantitative depletion of CD19+ expression in DTR mice treated with diphtheria toxin. Depletion of CD19+ B cells increases the antitumor growth effect of TPP15825 (as per group 02b vs group 01b).

[00144] Fig. 89: Internalization curves for several inventive anti-CCR8 antibodies in HUVEC cells. An anti-CD71 antibody with a known internalization profile was used as a positive control. The corresponding data are shown in Table 10.5.2. None of the antibodies obtained with a method according to the present invention showed substantial internalization, making them particularly useful for ADCC/ADCP-based approaches.

BRIEF DESCRIPTION OF SEQUENCE IDs

[00145] The sequence listing provided with the application via electronic filing is included herein in its entirety. SEQ ID NO:1 to SEQ ID NO:108 and SEQ ID NO:157 to SEQ ID NO:168 refer to isolated polypeptides that can be used as antigens or for off-target panning. SEQ ID NO:109 to SEQ ID NO:156 refer to chemokine receptor proteins of different species. SEQ ID NO:201 to SEQ ID NO:965 refer to inventive antibodies. The sulfation column is provided for convenience only and is not intended to restrict the respective sequence in any way.

DEFINITIONS

[00146] Unless otherwise defined, all scientific and technical terms used in the description, figures, and claims have their common meaning as commonly understood by one skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, shall prevail. If two or more documents incorporated by reference contain conflicting and/or inconsistent information, then the document with the later effective date shall control. When a database is referenced, the effective date is the applicable version number 26.05.2021, unless otherwise indicated. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. Unless otherwise indicated, the following terms used herein, including the description and claims, have the definitions provided below.

[00147] The expression "about" or "~", as used herein, refers to a value that is within an acceptable error range for the specific value determined by someone skilled in the art, which will depend in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, according to practice in the art. The term "about" is also used to indicate that the quantity or value in question may be the designated value or some other approximately equal value. The phrase is intended to convey that similar values promote equivalent results or effects, as described herein. In this context, "about" may refer to a range above and/or below up to 10%. Whenever the term "about" is specified for a particular assay or embodiment, that definition shall prevail for the specific context.

[00148] The terms "comprising", "including", "containing", "possessing", etc. should be read in an expansive or open-ended manner and without limitation. The term comprising, when used in the descriptive report, includes "consisting of".

[00149] Singular forms such as "a", "an", or "the" include plural references unless the context clearly indicates otherwise. Thus, for example, the reference to "a monoclonal antibody" includes a single monoclonal antibody as well as a plurality of monoclonal antibodies, whether the same or different.

[00150] Similarly, the reference to "cell" includes a single cell as well as a plurality of cells.

[00151] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood as referring to all elements of the series. The terms "at least one" and "at least one of" include, for example, one, two, three, four, five or more elements.

[00152] Furthermore, it is understood that small variations above and below a given range may be used to achieve substantially the same results as a value within the range. Additionally, unless otherwise indicated, the description of ranges is intended to be a continuous range including all values between the minimum and maximum values.

[00153] The term "amino acid" or "amino acid residue," as used herein, typically refers to a naturally occurring amino acid. The one-letter code is used herein to refer to the respective amino acid. As used herein, a "charged amino acid" is an amino acid that is negatively charged or positively charged. The "negatively charged amino acids" are aspartic acid (D) and glutamic acid (E). "Positively charged amino acids" are arginine (R), lysine (K), and histidine (H). "Polar amino acids" are all amino acids that form hydrogen bonds as donors or acceptors. These are all the charged amino acids and asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine (C). "Uncharged polar amino acids" are asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine (C). "Amphiphatic amino acids" are tryptophan (W), tyrosine (Y), and methionine (M). "Aromatic amino acids" are phenylalanine (F), tyrosine (Y), and tryptophan (W). "Hydrophobic amino acids" are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and cysteine. "Small amino acids" are glycine (G), alanine (A), serine (S), proline (P), threonine (T), aspartic acid (D), and asparagine (N).

[00154] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids. Polypeptides include any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, the term refers both to short chains, which are also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which are generally referred to in the art as proteins, of which there are many types.

[00155] "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[00156] When generic reference is made to a gene or protein of a certain species, such as a mouse, the human analogue should be understood similarly, unless otherwise indicated or obviously incompatible. This is particularly true in the context of biomarkers.

[00157] The term "isolate" when applied to a nucleic acid, polypeptide, protein, or antibody, denotes that the nucleic acid, polypeptide, protein, or antibody is essentially free from other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It may be in dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A protein, polypeptide, or antibody that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein different from the gene of interest. However, an isolated polypeptide can be immobilized, for example, on beads or particles, for example, by means of a suitable ligand.

[00158] The term "purified" denotes that a nucleic acid or protein essentially gives rise to a band on an electrophoretic gel. In particular, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[00159] As used herein, the term "synthetic," with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by synthetic methods. As used herein, production by recombinant means using recombinant DNA methods means the use of well-known molecular biology methods to express proteins encoded by cloned DNA.

[00160] "Post-translational modification(s)" (PTM) refers to the covalent modification(s) of peptides or proteins that are introduced after protein biosynthesis under natural conditions. The term includes, without limitation, glycosylation, phosphorylation, acylation, adenylation, farnesylation, ubiquitination, and sulfation. Post-translational modifications can influence the activity of peptides or proteins.

[00161] In 2004, Gutierrez et al. described the sulfation and glycosylation state of the murine CCR8 chemokine receptor and the way in which these post-translational modifications affect CCR8 activity. They suggest that the tyrosines at positions 14 and 15 in mouse CCR8 are sulfated amino acid residues, while asparagine 8 and threonines 10 and 12 are glycosylated. Furthermore, they show that sulfations are important for CCR8 activity (Gutiérrez, Julio, et al. "Analysis of post-translational CCR8 modifications and their influence on receptor activity." Journal of Biological Chemistry 279.15 (2004): 14726-14733.).

[00162] A "sulfation" is a post-translational modification in which a sulfate group is added to an amino acid, such as a tyrosine residue of a polypeptide or protein. Tyrosine sulfation occurs in all multicellular organisms. Under physiological conditions, it is catalyzed by tyrosylprotein sulfotransferases (TPSTs) 1 and 2, Golgi-resident enzymes that transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependent tyrosine on a protein substrate. Synthetic tyrosine sulfation can be performed using a technique known in the art, for example, as described in Bunschoten, Anton, et al. "A general sequence-independent solid phase method for the site-specific synthesis of multiple sulfated-tyrosine-containing peptides." Chemical Communications 21 (2009): 2999-3001. A sulfated polypeptide is a polypeptide comprising at least one sulfation. A non-sulfated polypeptide is a polypeptide that does not comprise sulfation.

[00163] A "tyrosine-rich domain" (TRD) is a conserved domain that characterizes seven transmembrane proteins, such as the CXC and CC chemokine receptors. The TRD is typically located at the N-terminus of the chemokine receptor and is typically linked to a LID domain via a cysteine, as in Fig. 2b. Thus, as used herein, the term TRD refers to the amino acid sequence or protein of a CXC or CC chemokine receptor that is located at the N-terminus of the first cysteine counted from the N-terminus. The TRD may or may not comprise a signal peptide. The TRD may or may not be modified. In addition to tyrosine, a TRD generally comprises negatively charged amino acid residues, such as aspartic acid. TRDs have been proposed as important structures for the interaction of chemokine receptors with their endogenous ligands. Under physiological conditions, tyrosine residues in a TRD can be sulfated, non-sulfated, or partially sulfated. The specific sequences for the TRDs of the respective chemokine receptors are provided in Table 4.1.

[00164] However, it is obvious that mutations can be introduced into the TRD sequence without altering the overall loading and interaction pattern. Preferably, a TRD has at least 90%, 95%, or 99% sequence identity or sequence similarity with at least one TRD sequence according to Table 4.1.

[00165] The term "N-terminal" or "N-term" of a chemokine receptor, as used herein, refers to the N-terminal amino acids of the chemokine receptor comprising at least the TRD. When a polypeptide or protein comprises a signal peptide, the N-terminal may also refer to the N-terminal sequence behind the natural cleavage site of the polypeptide or protein. According to some preferred embodiments, the N-terminal comprises the LID domain and the TRD domain of a chemokine receptor, but does not comprise the natural cysteine between these two domains. Instead, the cysteine may be removed or replaced with a different amino acid.

[00166] The "LID" domain of a chemokine receptor, as used herein, refers to an amino acid sequence located at the C-terminus of the chemokine receptor's TRD. The TRD and LID domains are normally separated by a single cysteine.

[00167] "Sequence identity" or "percent identity" is a number that describes how similar a query sequence is to a target sequence, more precisely how many characters in each sequence are identical after alignment. The most popular tool for calculating sequence identity is BLAST (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/), which performs pairwise comparisons of sequences, searching for regions of local similarity. Suitable alignment methods are known in the art, for example, the Needleman-Wunsch algorithm for global-global alignment, using the BLOSUM62 matrix, with a gap opening penalty of 11 and a gap extension penalty of 1. Then, the aligned identical residue pairs can be counted and then divided by the total length of the alignment (including gaps, internal and external) to arrive at the percent identity value.

[00168] For "% similarity" or "sequence similarity" values, the same approach as for percent identity values can be used, except that what is counted, instead of identical residual pairs, are the residual pairs aligned with BLOSUM62 values that are not negative (i.e., >0).

[00169] "Seven-transmembrane receptors" (7-TM receptors) are integral membrane proteins that contain seven transmembrane helices. As used herein, 7-TM receptors are G protein-coupled receptors.

[00170] "Chemokine receptors" are seven-transmembrane receptors. The chemokine receptor family contains 24 members in humans and can be subdivided, based on the class of chemokines they bind to, into four subfamilies: CX3CR, CXCR, CCR, and XCR, all of which are G protein activators, and ACKR, containing 6 atypical receptors, incapable of activating G proteins after ligand binding.

[00171] "CXC chemokine receptors" (CXCRs) are integral membrane proteins that bind to and respond specifically to cytokines of the CXC chemokine family. They represent a subfamily of chemokine receptors, a large family of G protein-coupled receptors known as seven-transmembrane proteins (7-TMs) because they span the cell membrane seven times. There are currently seven known CXC chemokine receptors in mammals, designated CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, and CXCR6. CXCR6 is more closely related structurally to CC chemokine receptors than to other CXC chemokine receptors.

[00172] "CC chemokine receptors" (CCRs, also beta chemokine receptors) are integral membrane proteins that bind to and respond specifically to cytokines of the CC chemokine family. They represent a subfamily of chemokine receptors, a large family of G protein-coupled receptors known as seven transmembrane proteins (7-TMs) because they cross the cell membrane seven times. The CC chemokine receptor subfamily comprises CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10.

[00173] The term "CCR8" refers to the CC chemokine receptor type 8. The CCR8 protein is encoded by the CCR8 gene (NCBI gene ID 1237). Synonyms for CCR8 are, inter alia, CC-CKR-8, CCR-8, CDw198, CKRL1, CMKBR8, CMKBRL2, GPRCY6, CY6, TER1. The CCR8 protein comprises humans, mice, rats, rhesus monkeys, and other mammalian and non-mammalian homologs. The sequence(s) for human CCR8 are accessible through the UniProt identifier P51685 (CCR8_HUMAN), for example, human isoform P51685-1 or P51685-2 (UniProt, November 29, 2019).

[00174] The sequence(s) for murine CCR8 are accessible via UniProt identifier P56484 (CCR8_MOUSE). The sequence(s) for rhesus monkey CCR8 are accessible via UniProt identifier O97665 (CCR8_MACMU). Different isoforms and variants may exist for different species and are all encompassed by the term CCR8. Also included are CCR8 molecules before and after maturation, i.e., independent of the cleavage of one or more prodomains. In addition, synthetic variants of the CCR8 protein can be generated and are encompassed by the term CCR8. The CCR8 protein may also be subject to various modifications, for example, synthetic or natural modifications, such as post-translational modifications. Recombinant human CCR8 is commercially available or can be manufactured as known in the art. CCR8 is a receptor for the chemokine CCL1/SCYA1/I-309. Barington et al. reported the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation at the CCR8 chemokine receptor (Barington, Line, et al. "Role of conserved disulfide bridges and aromatic residues in extracellular loop 2 of chemokine receptor CCR8 for chemokine and small molecule binding." Journal of Biological Chemistry 291.31 (2016): 16208-16220.). Furthermore, they discovered that two distinct aromatic residues in ECL-2, Tyr184 (Cys + 1) and Tyr187 (Cys + 4), were crucial for the binding of the chemokines CC CCL1 (agonist) and MC148 (antagonist), respectively, but not for the binding of small molecules.

[00175] "Programmed Death 1 (PD-1)" refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is predominantly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1," as used herein, includes, without limitation, human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs with at least one epitope in common with hPD-1. The complete sequence of hPD-1 can be found under GenBank accession number U64863 (November 29, 2019).

[00176] "Programmed Death Ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that negatively regulate T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1," as used herein, includes, without limitation, human PD-L1 (hPDL1), variants, isoforms, and species homologs of hPD-L1, and analogs with at least one epitope in common with hPD-L1. The complete sequence of hPD-L1 can be found in GenBank Accession No. Q9NZQ7 (November 29, 2019).

[00177] "Tumor Proportion Score" (TPS) is the percentage of viable tumor cells that exhibit partial or complete membrane staining at any intensity. For example, a sample should be considered to have PD-L1 expression if TPS > 1% and high PD-L1 expression if TPS > 50%. For example, PD-L1 protein expression in NSCLC is commonly determined using Tumor Proportion Score (TPS).

[00178] "Combined Positive Score" (CPS) is the number of staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. For example, a sample should be considered to have PD-L1 expression if CPS > 1 and to have high PD-L1 expression if CPS > 10. The FDA has approved the use of the PD-L1 IHC 22C3 pharmDx assay to determine a patient's eligibility for the therapeutic antibody pembrolizumab.

[00179] "FOXP3" is a 50-55 kD transcription factor, also known as Forkhead Box Protein P3, Scurfin, JM2, or IPEX. It is proposed to be a master regulatory gene and a more specific marker of regulatory T cells than most cell surface markers. Transduced expression of FOXP3 in CD4+/CD25 cells has been shown to induce GITR, CD103, and CTLA4 and confer a regulatory T cell phenotype. The antibody clones Biolegend 206D and 259D recognize a human epitope of FOXP3 in the amino acid region 105 to 235. Poly6238 recognizes human and mouse FOXP3 and was engineered against the N-terminal portion of FOXP3.

[00180] The term "modulation" refers to any alteration of an existing process or behavior, such as blocking (antagonism) and induction (agonism). For example, modulation of G protein-independent signaling refers to any significant alteration of G protein-independent signaling.

[00181] The term "internalization" of an antibody, fragment, or conjugate refers to the uptake of the antibody, fragment, or conjugate into a cell. Preferably, internalization is determined for a cell line with endogenous target expression, for example, as described elsewhere herein for human or murine CCR8. Preferably, internalization is determined by measuring the total internalized fluorescence intensity per cell and is quantified relative to an isotype control, for example, as described in Example 10.5. In summary, the antibody, fragment, or conjugate and a corresponding isotype control are labeled with a dye, and the internalized fluorescence is determined and quantified for the antibody, fragment, or conjugate relative to the isotype control.

[00182] A "non-internalizing antibody" is defined as an antibody that shows substantially the same internalization as a matching isotype control. A "low internalizing antibody" is defined as an antibody that shows internalization that is equal to or less than 10 times the internalization of the isotype control, preferably less than 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1.4, 1.3, 1.2, or 1.1 times the internalization of the isotype control. A "medium internalizing antibody" is defined as an antibody that shows internalization that is equal to or less than 21 times the internalization of the isotype control and greater than 10 times the internalization of the isotype control. A "high internalization antibody" is defined as an antibody that shows internalization that is more than 21 times the internalization of the isotype control.

[00183] Alternatively, internalization can also be quantified based on t(1/2), that is, as the time until half of the amount of antibody, fragment or conjugate has been internalized. Preferably, the antibodies according to the present invention are characterized by a time until half of the amount of antibody, fragment or conjugate has been internalized that is > 2 hours, preferably > 4, > 5, > 6, > 7, > 8, > 9, > 10, > 11, > 12, > 13, > 14, > 15, > 16, > 17, > 18, > 19, > 20, > 21, > 22, > 23, > 24, > 26, > 28, > 30 or > 48 hours. More preferably, the antibodies according to the present invention are not internalized, that is, no time can be defined until half of the quantity of antibody, fragment or conjugate has been internalized.

[00184] An "isotype control" is an antibody or fragment that does not bind to a target but has the same class and type as the reference antibody or fragment that recognizes the target.

[00185] An antibody or fragment is called "cross-reactive" or "cross-reactive" if the antibody or fragment binds to an antigen of two or more different species, for example, with a KD value of 10 to 7 M or less, more preferably less than 10 to 8 M, even more preferably in the range of 10 to 9 M to 10 to 11 M.

[00186] By the term "specifically binds" as used herein in relation to an antibody, is meant an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample: An antibody characterized by substantial nonspecific binding would not have therapeutic applicability, so these modalities are excluded. However, as is known in the art, specific binding of an antibody or ligand does not necessarily exclude binding of an antibody or ligand to other antigens/target molecules. An antibody that specifically binds to an antigen of one species may also bind to that antigen of one or more additional species. This cross-species reactivity does not in itself alter the classification of an antibody as specific.

[00187] In some cases, the terms "specific binding" or "specifically ligand" may be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[00188] In case of doubt, the specific binding of an antibody or ligand preferably describes the binding of an antibody, antibody fragment or ligand to its antigen/target with an affinity of at least 10 to 7 M (as KD value; that is, preferably those with KD values less than 10 to 7 M), with the antibody or ligand having at least half the affinity for a non-specific antigen that is not the predetermined target antigen/molecule or a closely related target antigen/molecule.

[00189] "Polyspecificity," also "polyreactivity" or "nonspecific binding," refers to the ability of ligands or antibodies to bind to a defined set of unrelated antigens. Nonspecific binding is substantial if the (therapeutic) applicability of the antibody is compromised. Polyspecificity to nonprotein structures, including but not limited to negative target cell lines or tissues, baculovirus particle (BVP), insulin, or DNA, can be assessed as known in the art and as described herein. For example, nonspecific binding to negative target human cell lines can be determined, for example, by FACS analysis using sham-transfected CHO or HEK cells. In a second example, nonspecific binding to different tissues can be analyzed by FACS analysis of a cell line or panel of cell lines derived from the respective tissue. In a third example, nonspecific binding to immune cell populations can be analyzed by FACS after classifying immune cell populations as known in the art. In a fourth example, nonspecific binding to BVP, insulin, or DNA can be analyzed using ELISA, for example, as described in Hotzel, Isidro, et al. "A strategy for risk mitigation of antibodies with fast clearance." MAbs. Vol. 4. No. 6. Taylor & Francis, 2012; Avery, Lindsay B., et al. "Establishing in vitro, in vivo correlations to screen monoclonal antibodies for physico-chemical properties related to favorable human pharmacokinetics." MAbs. Vol. 10. No. 2. Taylor & Francis, 2018; and Jain, Tushar, et al. "Biophysical properties of the clinical-stage antibody landscape." Proceedings of the National Academy of Sciences 114.5 (2017): 944-949, incorporated herein in their entirety and in particular with respect to the technical details necessary to analyze and quantify nonspecific binding. An antibody without substantial nonspecific binding is preferably characterized by nonspecific binding that is less than the nonspecific binding of the reference antibody Gantenerumab (Roche) and more preferably less than the nonspecific binding of the reference antibody Remicade (Janssen Biotech).

[00190] The term "off-target binding" refers to the ability of an antibody to bind to individual proteins other than the intended target, for example, proteins from the target protein family. Off-target binding can be assessed using commercially known assays in the art, such as the Retrogenix off-target profiling assay. In summary, antibodies are tested on microarrays containing HEK293 cells that individually express several thousand human membrane proteins and secreted proteins. Antibody binding to an off-target potential must be confirmed by FACS using cells that overexpress the off-target potential.

[00191] The term "affinity" is a term of the art and describes the binding strength between a ligand, antibody or antibody fragment and a target. The "affinity" of antibodies and antibody fragments for a target can be determined using techniques well known in the art or described herein, for example, by ELISA assays, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), flow cytometry or fluorescence polarization. Preferably, the affinity is given as the dissociation constant KD.

[00192] The "dissociation constant" (KD) has molar units (M) and corresponds to the concentration of the ligand/antibody at which half of the target proteins are occupied in equilibrium. The smaller the dissociation constant, the greater the affinity between the ligand or antibody and its target.

[00193] According to the present invention, the antibodies preferably have a target affinity of at least 10 to 7 M (as the KD value), more preferably at least 10 to 8 M, even more preferably in the range of 10 to 9 M to 10 to 11 M. The KD values can preferably be determined by surface plasmon resonance spectroscopy, for example, as described elsewhere herein. When assay conditions influence the determined KD, the assay setting with the lowest standard deviation should be used.

[00194] "Half-maximum effective concentration" (EC50) refers to the concentration of a drug, antibody, fragment, conjugate, or molecule that induces a response halfway between baseline and maximal after a specified incubation time. In the context of antibody binding, EC50 thus reflects the antibody concentration required for semi-maximal binding. An EC50 can be determined if an inflection point can be determined by mathematical modeling (e.g., nonlinear regression) of the dose-response curve describing the relationship between the applied drug, antibody, fragment, conjugate, or molecule concentration and signal. For example, if the dose-response curve follows a sigmoidal curve, an EC50 can be determined. When the response is an inhibition, the EC50 is termed half-maximum inhibitory concentration (IC50). EC80 can be determined mutatis mutandis.

[00195] The term "antibody" (Ab) refers to an immunoglobulin molecule (e.g., without limitation, human IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgE, IgA1, IgA2, mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, IgD, IgE or IgM, rat IgG1, IgG2a, IgG2b, IgG2c, IgA, IgD, IgE or IgM, rabbit IgA1, IgA2, IgA3, IgE, IgG, IgM, goat IgA, IgE, IgG1, IgG2, IgE, IgM or chicken IgY) that specifically binds to, or is immunologically reactive with, a particular antigen. Antibodies or antibody fragments comprise complementarity-determining regions (CDRs), also known as hypervariable regions, in the variable domains of the light and heavy chains. The most highly conserved portions of the variable domains are called frameworks (FRs). As is known in the art, the amino acid position/boundary that delineates a hypervariable region of an antibody can vary depending on the context and the various definitions known in the art. As used herein, the numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al. The native heavy and light chain variable domains each comprise four FR regions. The three CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the antibody antigen-binding site, see Kabat, E. A., et al. "Sequences of Proteins of Immunological Interest (Natl. Inst. Health, Bethesda, MD), GPO Publ." No. 165-462 (1987). The term antibody, as used herein, also refers to antibody fragments, except where explicitly indicated otherwise. Depending on the context, the term antibody may also refer to any protein-binding molecule with immunoglobulin-like function.

[00196] The term "CDR" refers to the complementary determinant region of the antibody. As is known in the art, complementarity-determining regions (CDRs) are part of the variable chains in antibodies and T-cell receptors. A set of CDRs constitutes a paratope. CDRs are crucial for the diversity of antigen specificities. There are three CDRs (CDR1, CDR2, and CDR3), arranged non-consecutively in the amino acid sequence of a variable domain of an antigen receptor. Since antigen receptors are typically composed of two variable domains (in two different polypeptide chains, heavy and light chain), there are generally six CDRs for each antigen receptor that can collectively come into contact with the antigen. The light chain CDRs are LCDR1, LCDR2, and LCDR3. The heavy chain CDRs are designated HCDR1, HCDR2, and HCDR3. HCDR3 is the most variable complementary determining region (see, for example, Chothia, Chothia, Cyrus, and Arthur M. Lesk. "Canonical structures for the hypervariable regions of immunoglobulins." Journal of molecular biology 196.4 (1987): 901-917.; Kabat, E. A., et al. "Sequences of proteins of immunological interest. Bethesda, MD: US Department of Health and Human Services." Public Health Service, National Institutes of Health (1991): 103-511.).

[00197] The "constant region" refers to the portion of the antibody molecule that confers effector functions. The constant region of the heavy chain can be selected from any of the five isotypes: alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ).

[00198] The term "Fc domain," "Fc region," or "Fc portion," as used herein, refers to a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. For example, a human IgG heavy chain Fc region may extend from Cys226, or from Pro230, to the carboxyl terminal of the heavy chain.

[00199] Antibodies or binding fragments according to the present invention may have been modified to alter at least one constant region-mediated biological effector function. For example, in some embodiments, an antibody may be modified to reduce or increase at least one constant region-mediated biological effector function relative to the unmodified antibody, for example, reduced or enhanced binding to the Fc receptor (FCYR). FcyR binding can be reduced, for example, by mutation of the immunoglobulin constant region segment of the antibody in specific regions required for FcyR interactions (see, for example, Canfield, Stephen M. and Sherie L. Morrison. "The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region." The Journal of Experimental Medicine 173.6 (1991): 1483-1491; and Lund, John, et al. "Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG." The Journal of Immunology 147.8 (1991): 2657-2662). FcyR binding can be increased, for example, by afucosylation. Reduced FcyR binding can also reduce other effector functions that depend on FcyR interactions, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity ("ADCC").

[00200] Furthermore, addressing the Fc interaction with FcRn allows modulating the half-life of antibodies in vivo. Revoking the interaction, for example, by introducing the H435A mutation, leads to an extremely short half-life, since the antibody is no longer protected from lysosomal degradation by FcRn recycling. In some respectfully preferred embodiments, the antibody according to the present invention comprises the H435A mutation or has been modified for a reduced half-life.

[00201] Conversely, antibodies comprising "YTE" mutations (M252Y/S254T/T256E) and/or equivalent mutations, such as "LS" mutations (M428L/N434S), have been shown to significantly extend half-life through more efficient endosome recycling in both preclinical species and humans (Dall’Acqua, William F., et al. "Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences." The Journal of Immunology 169.9 (2002): 5171-5180.; Zalevsky, Jonathan, et al. "Enhanced antibody half-life improves in vivo activity." Nature biotechnology 28.2 (2010): 157-159.). In some preferred embodiments in all respects, the antibody according to the present invention comprises YTE mutations (M252Y/S254T/T256E) and/or equivalent mutations, such as LS mutations (M428L/N434S), or has been engineered for an improved half-life. Suitable Fc engineering approaches for half-life extension can be found in Haraya, Kenta, Tatsuhiko Tachibana, and Tomoyuki Igawa, "Improvement of pharmacokinetic properties of therapeutic antibodies by antibody engineering," Drug Metabolism & Pharmacokinetics 34.1 (2019): 25-41, and/or Lee, Chang-Han, et al., "An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence," Nature Communications 10.1 (2019): 1-11, both incorporated herein by reference.

[00202] "Non-fucosylated" antibodies are designed so that the oligosaccharides in the Fc region of the antibody do not have fucose sugar units. Glycosylation of an antibody can alter its function. For example, if glycosylation at N297 in the CH2 domain of an IgG is completely eliminated, binding to FCYRS is lost. However, modulation of the specific carbohydrate composition at N297 can have the opposite effect and increase the ADCC activity of the antibody. In summary, the affinity of an antibody for activating FCYRS depends on the composition of the N297-linked oligosaccharide. There are 32 different possible combinations of oligosaccharides that can occur at this site. Naturally occurring human IgGs and those produced by hybridomas or other common expression systems are generally composed of N-acetylglucosamine (GlcNAc) and three mannose residues that form a core carbohydrate. This core is linked to two additional GlcNAc groups to form biennial branches. The addition of galactose to each branch can occur, as well as the terminal addition of sialic acid to these galactose molecules. Fucose is often part of the GlcNAc core. This fucose, through steric hindrance, obstructs the interaction of the antibody with FcyRIIIA. Thus, the elimination of this fucose molecule, while maintaining other forms of glycosylation at this site, increases the binding of the antibody to activating FcyRs, increasing its ability to induce ADCC and/or ADCP (Almagro, Juan C., et al. "Progress and challenges in the design and clinical development of antibodies for cancer therapy." Frontiers in Immunology 8 (2018): 1751). Methods for preparing fucose-free antibodies include growth in YB2/0 mouse myeloma cells (ATCC CRL 1662). YB2/0 cells express low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme required for the fucosylation of polypeptides. Non-fucosylated antibodies are preferred for the present invention.

[00203] "Antibody-dependent cellular cytotoxicity" ("ADCC"), also known as "antibody-dependent cell-mediated cytotoxicity," is a cell-mediated immune defense mechanism whereby an immune cell actively lyses a target cell whose membrane surface antigens have been bound by specific antibodies. ADCC is mediated through the interaction of the antibody or fragment with FcYRIIIa. In humans, FcYRIII exists in two different forms: FcYRIIIa (CD16a) and FcYRIIIb (CD16b). While FcYRIIIa is expressed on monocytes, neutrophils, mast cells, macrophages, and natural killer cells as a transmembrane receptor, FcYRIIIb is expressed only on neutrophils. These receptors bind to the Fc portion of IgG antibodies, which then activates antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

[00204] Different assay systems for determining ADCC induction in humans have been described in the literature and are suitable for characterizing the subject described herein. For example, Yao-Te Hsieh et al. studied different ADCC assay systems, namely assays based on (i) natural killer cells from human donors (FcYRIIIA + primary NK), (ii) FcYRIIIA-manipulated NK-92 cells, and (iii) FcYRIIIA/NFAT-RE/luc2-manipulated Jurkat T cells (Hsieh, Yao-Te, et al. "Characterization of FcYRIIIA effector cells used in in vitro ADCC bioassay: comparison of primary NK cells with engineered NK-92 and Jurkat T cells." Journal of Immunological Methods 441 (2017): 56-66, incorporated herein in full; in particular, reference is made to the method description for these assays). In summary, all three effector cell systems differentially express FcYRIIIA and provide dose-dependent ADCC pathway activity, but only primary NK cells and engineered NK-92 cells are capable of inducing ADCC-mediated cell lysis. For functional assessment of ADCC activity, primary NK or NK-92 (V-158) cells best reflect the physiologically relevant ADCC mechanism of action. As a modified cell line, NK-92 cells can behave more reproducibly than primary NK cells and are therefore the preferred assay system for determining the ADCC response in humans, for example, in cases of doubt.

[00205] An antibody or antigen-binding fragment that induces ADCC is an antibody that can induce a substantial amount of lysis of target cells in the presence of effector NK cells. Preferably, ADCC induction results in the lysis of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of target cells.

[00206] "Antibody-dependent cellular phagocytosis" ("ADCP") is the mechanism by which antibody-opsonized target cells activate FcYRs on the surface of macrophages to induce phagocytosis, resulting in the internalization and degradation of the target cell. For ADCP, binding to macrophages as effector cells typically occurs through the interaction of the Fc portion of antibodies with FcYR1Ia (CD32a) expressed by macrophages.

[00207] An antibody or antigen-binding fragment that induces ADCP is an antibody that can induce a substantial amount of phagocytosis of target cells in the presence of macrophages. Preferably, ADCP induction results in the phagocytosis of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of target cells.

[00208] "Complement-dependent cytotoxicity" ("CDC") is an effector function of IgG and IgM antibodies. When they are bound to a surface antigen on a target cell (e.g., a cell infected by bacteria or viruses), the classical complement pathway is triggered by the binding of the C1q protein to these antibodies, resulting in the formation of a membrane attack complex (MAC) and lysis of the target cell. The complement system is effectively activated by human IgG1, IgG3, and IgM antibodies, weakly by IgG2 antibodies, and not activated by IgG4 antibodies. It is a mechanism of action by which therapeutic antibodies—in addition to specific antibody modalities according to the present invention—can achieve an antitumor effect. Several laboratory methods exist to determine the effectiveness of CDC and are known in the art.

[00209] An antibody or antigen-binding fragment that induces CDC is an antibody that can induce a substantial amount of membrane attack complex formation and lysis of target cells. Preferably, CDC induction results in the lysis of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of target cells.

[00210] Antibodies comprising an Fc region may or may not comprise a modification that promotes the association of the first and second subunits of the Fc domain.

[00211] A "modification that promotes the association of the first and second Fc domain subunits" is a manipulation of the peptide backbone or post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. Antibodies comprising an Fc region may or may not comprise a modification that promotes the association of the first and second Fc domain subunits. A modification-promoting association, as used herein, particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e., the first and second Fc domain subunits), wherein the modifications are complementary to each other so as to promote the association of the two Fc domain subunits. For example, a modification-promoting association may alter the structure or charge of one or both Fc domain subunits so as to make their association stereostatic or electrostatically favorable. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be non-identical, for example, in the sense that other components fused to each of the subunits (e.g., antigen-binding halves) are not the same. In some modalities, the modification-promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification-promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two Fc domain subunits.

[00212] An antibody "fragment" as used herein is necessary to retain substantially the desired affinity of the full-length antibody. As such, suitable fragments of a human anti-CCR8 antibody will retain the ability to bind to the target chemokine receptor, for example, to bind to the human CCR8 receptor. Antibody fragments comprise a portion of a full-length antibody, usually the antigen-binding site or its variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain antibody molecules, diabodies, and domain antibodies; see Holt, Lucy J., et al. "Domain antibodies: proteins for therapy." Trends in biotechnology 21.11 (2003): 484-490.

[00213] A "Fab fragment" contains the constant domain of the light chain and the first constant domain (CH2) of the heavy chain.

[00214] “Fab fragments” differ from Fab fragments by the addition of some residues at the carboxyl terminal of the CH2 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody.

[00215] "F(ab') fragments" are produced by cleavage of the disulfide bond in the hinge cysteines of the pepsin digestion product F(ab')2. Additional chemical couplings of antibody fragments are known to those skilled in the art. Fab and F(ab')2 fragments lack the intact Fc fragment of the antibody, are cleared more rapidly from animal circulation, and may have less nonspecific tissue binding than an intact antibody; see, for example, Wahl, Richard L., Charles W. Parker, and Gordon W. Philpott. "Improved radioimaging and tumor localization with monoclonal F(ab')2." Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine 24.4 (1983): 316-325.

[00216] An "Fv fragment" is the smallest fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in a narrow, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Often, all six CDRs confer antigen-binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv comprising only three target-specific CDRs) may have the ability to recognize and bind to the antigen, albeit with a lower affinity than the entire binding site.

[00217] "Single-chain Fv" or "scFv" comprises the VH and VL domains of an antibody in a single polypeptide chain. Generally, the Fv polypeptide also comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.

[00218] "Single domain antibodies" are composed of single VH or VL domains that exhibit sufficient affinity for the target. In one specific embodiment, the single domain antibody is a camelized antibody, see, for example, Riechmann, Lutz and Serge Muyldermans. "Single domain antibodies: comparison of camel VH and camelized human VH domains." Journal of immunological methods 231.1-2 (1999): 25-38.

[00219] "Bispecific antibodies" are monoclonal antibodies that possess binding specificities for at least two different epitopes on the same or different antigens. In the present invention, one of the binding specificities may be directed to the target chemokine receptor, such as CCR8, the other may be to any other antigen, for example, without limitation to a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virus-derived protein, virus-encoded envelope protein, bacterial-derived protein, or bacterial surface protein. Bispecific antibody constructs according to the invention also encompass multispecific antibody constructs comprising multiple binding domains/binding sites, such as trispecific antibody constructs, where the construct comprises three binding domains.

[00220] "Derived antibodies" are typically modified by glycosylation, acetylation, pegylation, phosphorylation, sulfation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein. Any of the numerous chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative may contain one or more unnatural amino acids, for example, using ambrx technology, see, for example, Wolfson, Wendy. "Amber codon flashing ambrx augments proteins with unnatural amino acids." Chemistry & biology 13.10 (2006): 1011-1012. The antibodies according to the present invention may be derived, for example, glycosylated or sulfated.

[00221] "Monoclonal antibodies" are substantially homogeneous populations of antibodies that bind to a specific antigen. Monoclonal immunoglobulins can be obtained by methods well known to those skilled in the art (see, for example, Kohler, Georges, and Cesar Milstein. "Continuous cultures of fused cells secreting antibody of predefined specificity." nature 256,5517 (1975): 495-497, and U.S. Patent No. 4,376,110). An immunoglobulin or immunoglobulin fragment with specific binding affinity can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art allow the production of immunoglobulins or immunoglobulin fragments and protein-binding molecules with immunoglobulin-like functions in prokaryotic and eukaryotic organisms. The antibodies according to the present invention are preferably monoclonal.

[00222] "Humanized antibodies" contain CDR regions derived from a non-human species, such as a mouse, which have been, for example, grafted, along with any necessary structural mutations, onto human sequence-derived V regions. Thus, most humanized antibodies are human immunoglobulins (receptor antibody) in which residues from a hypervariable region of the receptor are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, with the desired specificity, affinity, and capability. See, for example, U.S. Patents 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205, each incorporated herein by reference. In some cases, structural residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain the desired affinity). In general, the humanized antibody will comprise substantially all of at least one and typically two variable domains, where all or substantially all hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all structural regions are those of a human immunoglobulin sequence. The humanized antibody optionally comprises at least a portion of an immunoglobulin constant (Fc) region, typically that of a human immunoglobulin. For more details, see Jones, Peter T., et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse." Nature 321.6069 (1986): 522-525.; Riechmann, Lutz, et al. "Reshaping human antibodies for therapy." Nature 332.6162 (1988): 323-327; and Presta, Leonard G. "Antibody engineering." Current Opinion in Structural Biology 2.4 (1992): 593-596, each incorporated herein by reference.

[00223] Fully human antibodies (human antibodies) comprise human-derived CDRs, i.e., CDRs of human origin. Preferably, a fully human antibody according to the present invention is an antibody with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with the closest human germline VH gene (e.g., sequence extracted from the recommended list and analyzed in IMGT/Domain-gap-align).

[00224] As accepted by usual nomenclature systems, such as the INN species subsystem in effect until 2017, fully human antibodies may comprise a low number of germline deviations compared to the nearest human germline reference determined based on the IMGT database (http://www.imgt.org, November 29, 2019). For example, a fully human antibody according to the present invention may comprise up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15 germline deviations in the CDRs compared to the nearest human germline reference. Fully human antibodies may be developed from human-derived B cells by cloning techniques in combination with a cell enrichment or immortalization step. Most fully human antibodies in clinical use, however, have been isolated from transgenic immunized mice for the human IgG locus or from sophisticated combinatorial libraries by phage display (Brüggemann, Marianne, et al. "Human antibody production in transgenic animals." Archivum immunologiae et therapiae experimentalis 63.2 (2015): 101-108.; Carter, Paul J. "Potent antibody therapeutics by design." Nature reviews immunology 6.5 (2006): 343-357.; Frenzel, André, Thomas Schirrmann, and Michael Hust. "Phage display-derived human antibodies in clinical development and therapy." MAbs. Vol. 8. No. 7. Taylor & Francis, 2016.; Nelson, Aaron L., Eugen Dhimolea, and Janice M. Reichert. "Development trends for human monoclonal antibody therapeutics." Nature reviews drug discovery 9.10 (2010): 767-774.).

[00225] Several techniques are available for generating fully human antibodies or for generating antibodies comprising human-derived CDRs (as per WO2008112640). Cambridge Antibody Technologies (CAT) and Dyax have obtained antibody cDNA sequences from peripheral B cells isolated from immunized humans and created phage display libraries for the identification of human variable region sequences of a particular specificity. In short, the antibody variable region sequences are fused with the structure of Gene III or Gene VIII of bacteriophage M13. These antibody variable region sequences are expressed as single-stranded Fab or Fv (scFv) structures at the tip of the phage carrying the respective sequences. Through cycles of a panning process using different levels of antigen-binding conditions (constraints), phages expressing Fab or scFv structures that are specific for the antigen of interest can be selected and isolated. The cDNA sequences of the variable region of the selected phage antibody can then be elucidated using standard sequencing procedures. These sequences can then be used to reconstruct a complete antibody with the desired isotype using established antibody engineering techniques. Antibodies constructed according to this method are considered fully human antibodies (including CDRs). In order to improve the immunoreactivity (affinity and specificity of antigen binding) of the selected antibody, an in vitro maturation process can be introduced, including a combinatorial association of different heavy and light chains, deletion/addition/mutation in CDR3 of the heavy and light chains (to mimic VJ and VDJ recombination) and random mutations (to mimic somatic hypermutation). An example of a "fully human" antibody generated by this method is the antitumor necrosis factor α antibody, Humira (adalimumab).

[00226] The term "polynucleotide" refers to a recombinant or synthetically produced polymeric deoxyribonucleotide or an analog thereof, or a modified polynucleotide. The term includes double-stranded and single-stranded DNA or RNA. The polynucleotide may be integrated, for example, into minicircles, plasmids, cosmids, minichromosomes, or artificial chromosomes. The polynucleotide may be isolated or integrated into another nucleic acid molecule, for example, into an expression vector or chromosome of a eukaryotic host cell.

[00227] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating a nucleic acid molecule to which it is attached. The term also includes (non-viral) plasmids and viral vectors. Certain vectors are capable of directing the expression of nucleic acids or polynucleotides to which they are operatively attached. Such vectors are referred to herein as "expression vectors." Expression vectors for eukaryotic use can be constructed by inserting a polynucleotide sequence encoding at least one protein of interest (POI) into a suitable vector structure. The vector structure may comprise the elements necessary to ensure vector maintenance and, if desired, to provide amplification within the host. For viral vectors, for example, lentiviral or retroviral vectors, other virus-specific elements, such as structural elements or other elements, may be required and are well known in the art. These elements may be provided, for example, in cis (in the same plasmid) or in trans (in a separate plasmid). Viral vectors may require helper viruses or packaging strains for large-scale transfection. Vectors may contain other elements, such as, for example, enhancer elements (e.g., viral, eukaryotic), introns, and viral plasmid replication origins for replication in mammalian cells. According to the present invention, expression vectors typically have a promoter sequence that drives POI expression. POI and/or selective marker protein expression may be constitutive or regulated (e.g., inducible by the addition or removal of small molecule inducers). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of POI expression in mammalian cells, such as regulatory elements, promoters, and/or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus late major Ad LP promoter), or polyoma. For further description of viral regulatory elements and their sequences, see, for example, US 5,168,062, US 4,510,245, and US 4,968,615.

[00228] The term "ligand" or "spacer," as used herein, refers to any molecule that allows a direct topological connection between two portions. A portion may be, inter alia, a polypeptide, a protein, an antibody, an antibody fragment, a cytotoxic portion, a binding portion, a detection portion, such as a fluorophore, an immobilization or retrieval portion, such as beads or magnetic beads, a reactive portion, or any other molecule. The two portions may be of the same type or different. Ligands may form part of conjugates and may even contribute to their function. For example, for a conjugate comprising a polypeptide and biotin, the presence of a spacer of approximately 4 Å (~5 atoms) between the carboxy group of biotin and the bulky first amino acid of the peptide allows biotin to reach the (strept)avidin binding pocket. Several ligands are known in the art and can be selected based on the portions that need to be connected. The ligand length typically ranges from 4 atoms to over 200 atoms. Ligands exceeding 60 atoms in length generally comprise a population of compounds with an average length.

[00229] "Polypeptide ligands" can be linked via an amide linkage or any other functional residue. Polypeptide ligands can be linked to the N-terminus or C-terminus of the polypeptide or can be linked via a reactive functional group or amino acid side chain. Polypeptides can be coupled, for example, to biotin, proteins such as human serum albumin (HSA), carrier proteins such as limpet hemocyanin (KLH), ovalbumin (OVA) or bovine serum albumin (BSA), fluorescent dyes, short amino acid sequences such as Flag marker, HA marker, Myc marker or His marker, reactive markers such as maleimides, iodoacetamides, alkyl halides, 3-mercaptopropyl or 4-azidobutyric acid, or various other suitable moieties. Non-limiting examples of suitable ligands, for example, for polypeptide conjugation, include beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), PEG2 spacer (8-amino-3,6-dioxaoctanoic acid), PEG3 spacer (12-amino-4,7,10-trioxadodecanoic acid), PEG4 spacer (15-amino-4,7,10,13-tetraoxapentadecanoic acid), and Ttds (trioxapentasuccinamic acid). In some cases, the ligand may be derived from a reactive moiety, for example, maleimides, iodoacetamides, alkyl halides, 3-mercaptopropyl, or 4-azidobutyric acid. In some cases, the binder may comprise polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol or polypropylene glycol.

[00230] "Antibody ligands" are ligands that establish a covalent connection between different portions of antibodies and include peptide ligands and non-proteinaceous polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol, polypropylene glycol.

[00231] "Treating" a disease in an individual or "treating" an individual with a disease refers to subjecting the individual to pharmaceutical treatment, for example, the administration of a medication, so that at least one symptom of the disease is reduced or prevented from worsening.

[00232] The terms "prevent", "preventing", "prevention" and the like refer to reducing the likelihood of developing a disease, disorder or condition in an individual who does not have, but is at risk of or susceptible to developing a disease, disorder or condition.

[00233] The term "effective amount" or "therapeutically effective amount" is used interchangeably herein and refers to an amount sufficient to achieve a specific biological outcome or to modulate or improve a symptom in an individual, or the time to onset of a symptom, typically by at least about 10%; generally by at least about 20%, preferably at least about 30%, or more preferably at least about 50%. The effectiveness of using an antibody in cancer therapy can be assessed based on the change in tumor burden. Both tumor shrinkage (objective response) and time to disease progression are important outcomes in cancer clinical trials. Standardized response criteria, known as RECIST (Response Assessment Criteria in Solid Tumors), were published in 2000. An update (RECIST 1.1) was released in 2009. RECIST criteria are typically used in clinical trials where objective response is the primary outcome of the study, as well as in trials where assessment of stable disease, tumor progression, or time-to-progression analyses are performed because these outcome measures are based on an assessment of anatomical tumor burden and its change over the course of the trial. An effective amount for a given individual may vary depending on factors such as the condition being treated, the individual's overall health, the method, route, and dose of administration, and the severity of side effects. When combined, an effective amount is proportional to a combination of components, and the effect is not limited to the individual components alone.

[00234] Unless otherwise defined, "Complete Response" (CR) is defined as the disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have a short-axis reduction to <10 mm. For "Partial Response" (PR), a decrease of at least 30% in the sum of the diameters of the target lesions must be achieved, taking as a reference the diameters of the baseline sum. For "Progressive Disease" (PD), at least a 20% increase in the sum of the diameters of the target lesions, taking as a reference the smallest sum in the study (this includes the baseline sum if it is the smallest in the study). In addition to the 20% relative increase, the sum must also demonstrate an absolute increase of at least 5 mm. In "Stable Disease" (SD), there is not sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as a reference the diameters of the smallest sum during the study.

[00235] Secondary outcome measures that can be used to determine the therapeutic benefit of the inventive antibodies described herein include the following: "Objective response rate" (ORR) is defined as the proportion of individuals who achieve a complete response (CR) or partial response (PR). "Progression-free survival" (PFS) is defined as the time from the date of the first dose of an antibody until disease progression or death, whichever occurs first. "Overall survival" (OS) is defined as the period of time from the date of diagnosis or initiation of treatment of a disease that patients diagnosed with the disease are still alive. "Duration of overall response" (DOR) is defined as the time from the participant's initial CR or PR until the time of disease progression. "Depth of response" (DpR) is defined as the percentage of tumor reduction observed at the point of maximum response compared to the initial tumor burden. Clinical outcomes for ORR and PFS can be determined based on the RECIST 1.1 criteria described above.

[00236] When non-human subjects are analyzed, the above-mentioned parameters for determining therapeutic efficacy and benefit should be adapted as described elsewhere herein, as per Example 12ff.

[00237] Typical "individuals" according to the present invention include human and non-human individuals. Individuals may be mammals, such as mice, rats, cats, dogs, primates and/or humans.

[00238] "Pharmaceutical compositions" (also "therapeutic formulations") of the antibody, fragment or conjugate may be prepared by mixing the antibody to the desired degree of purity with optional physiologically acceptable vehicles, excipients or stabilizers, for example, according to Remington's Pharmaceutical Sciences (18th ed.; Mack Pub. Co.: Eaton, Pa., 1990), for example in the form of lyophilized formulations or aqueous solutions. Acceptable vehicles, excipients or stabilizers are not toxic to receptors at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; Preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as Tween®, Pluronic®, or polyethylene glycol (PEG).

[00239] A "host cell" is a cell that is used to receive, maintain, reproduce, and amplify a vector. A host cell may also be used to express the polypeptide, for example, an antibody or fragment thereof encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thus amplifying the nucleic acids. Preferred host cells are mammalian cells, such as CHO cells or HEK cells. Other preferred host cells are YB2/0 mouse myeloma cells.

[00240] An "endogenously targeted expression cell" is a cell that expresses a target protein at a level comparable to the physiological or disease state. Typically, cells that have been manipulated for overexpression express a target protein at much higher levels.

[00241] The term "intratumoral", "intratumoral", "tumor infiltration" or "tumor" in the context of cells, structures, proteins, antibodies or markers refers to their location within the tumor tissue.

[00242] Cells that are "positive" or "+" for a given marker or protein are cells characterized by substantial expression of that marker or protein. The expression of the marker or protein can be determined and quantified as is known in the art, for example, to define different cell populations. For the characterization of (immune) cell populations, marker expression can be determined by FACS or using any other technique described herein.

[00243] "Leukocytes" are immune cells that express CD45. "CD45+ cells," as used herein, refers to all leukocytes. CD45 can be used as a marker to distinguish immune cells from non-immune cells.

[00244] The term "lymphocyte" refers to all populations of immature, mature, undifferentiated, and differentiated white lymphocytes, including tissue-specific and specialized varieties. It includes, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages, including pre-B cells, B progenitor cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B1 cells, B-2 cells, and anergic AN1/T3 cell populations.

[00245] "T cells" are immune cells that express TCRαβ, CD3, and CD8 or CD4. As used herein, the term includes naive T cells, CD4+ T cells, CD8+ T cells, regulatory T cells, memory T cells, activated T cells, anergic T cells, tolerant T cells, chimeric B cells, and antigen-specific T cells, and other T cell populations known in the art. In some modalities, the presence of a T cell receptor (TCR) on the cell surface distinguishes T cells from other lymphocytes.

[00246] "CD8+ T cells" (also "cytotoxic T cell", "TC", "cytotoxic T lymphocyte", "CTL", "killer T cell", "cytolytic T cell", "CD8+ T cell" or "exterminator T cell") are T cells that express CD3, CD45 and CD8. CD8+ T cells can kill cancerous cells, infected cells (particularly with viruses) or damaged cells.

[00247] "CD4+ T cells" (also "helper T cells", "Th cells") are immune cells that express CD3, CD4, and CD45. There are several subsets of helper T cells, such as, but not limited to, Th1, Th2, and Th17. CD4+ T cells help suppress or regulate immune responses. They are essential in antibody class switching of B cells, in the activation and growth of cytotoxic T cells, and in maximizing the bactericidal activity of phagocytes, such as macrophages.

[00248] As used herein, the term "Treg cells" (also "Tregs", "regulatory T cells", "suppressor T cells") refers to immune cells that express CD3, CD4, CD45, and FoxP3, and in addition express high levels of CD25 and low levels of CD127. Identification of Treg cells can be performed as described elsewhere herein. Treg cells also typically express high levels of CTLA-4, GITR, and LAG-3. In the literature, Tregs have also been classified based on the memory marker CD45RO.

[00249] Under physiological conditions, Treg cells maintain immunological tolerance. During an immune response, Treg cells disrupt T cell-mediated immunity and suppress autoreactive T cells that have escaped negative selection within the thymus. Treg cells can also suppress other types of immune cells, such as NK cells and B cells. Adaptive Treg cells (called Th3 or Tr1 cells) are believed to be generated during an immune response.

[00250] Furthermore, Treg cells play an important role in immune escape, suppressing antitumor immunity, thus providing an environment of immune tolerance. T cells that recognize cancer cells are often present in large numbers in tumors, but their cytotoxic function is suppressed by nearby immunosuppressive cells. Tregs are abundant in various types of cancer, are highly enriched in the tumor microenvironment, and are well known for their role in tumor progression.

[00251] "Activated Treg cells" express CD4, CD45, FoxP3, CD69, and CCR8, and in addition, they show high expression of CD25 and low expression of CD127. CD69 is a marker of T cell activation.

[00252] "CCR8-positive regulatory T cells" or "CCR8+ regulatory T cells" are Tregs that express CCR8. "CD4conv cells" are conventional CD4+ and CD25 T cells.

[00253] "Gamma delta T cells" are T cells that express a distinct T cell receptor, TCRYδ, on their surface. Gamma delta T cells also express CD3.

[00254] "B cells" are immune cells that express CD19, and mature B cells express CD20 and CD22. After activation via CD40, B cells undergo differentiation where somatic hypermutation and enhanced immunoglobulin class switching occur, resulting in mature B cells or plasma cells (capable of secreting antibodies). B cells are involved in the humoral immunity of the adaptive immune system and are antigen-presenting cells.

[00255] "Macrophages" are immune cells that express low CD14, high CD16, CD11b, CD68, CD163, and CD206. Macrophages engulf and digest cellular debris, foreign substances, microbes, or cancer cells by phagocytosis. In addition to phagocytosis, macrophages play a critical role in innate immunity and also help initiate adaptive immunity by recruiting other immune cells. For example, macrophages are important as antigen presenters to T cells. Macrophages that stimulate inflammation are called M1 macrophages, while those that decrease inflammation and stimulate tissue repair are called M2 macrophages.

[00256] As used herein, "M1 macrophages" are a subset of macrophages that express ACOD1. M1 macrophages have pro-inflammatory, bactericidal, and phagocytic functions.

[00257] As used herein, "M2 macrophages" are a subset of macrophages that express MRC1 (CD206). M2 macrophages secrete anti-inflammatory interleukins, play a role in wound healing, and are required for revascularization and re-epithelialization. Tumor-associated macrophages are primarily of the M2 phenotype and appear to actively promote tumor growth.

[00258] Dendritic cells (DCs) are bone marrow-derived leukocytes and are the most potent type of antigen-presenting cells. DCs are specialized in capturing and processing antigens, converting proteins into peptides that are presented to major histocompatibility complex (MHC) molecules recognized by T cells. As defined herein, DCs are characterized by the expression of CD1c, CD14, CD16, CD141, CD11c, and CD123. There are different subpopulations of dendritic cells. In humans, DC1 cells are immunogenic while DC2 cells are tolerogenic. Mature DCs express CD83, while plasmacytoid DCs express CD123.

[00259] "NK cells" (also natural killer cells) are immune cells that express CD45, CD16, CD56, NKG2D, but are negative for CD3. NK cells do not require activation to kill cells that do not possess "self" MHC class 1 markers. NCR1 (also known as CD335 or NKp46) is expressed on NK cells and a subset of NKT cells.

[00260] "Natural killer (NKT) T cells" are a heterogeneous group of T cells that share properties of both T cells and natural killer cells.

[00261] "iNKT cells" (also "invariant natural killer T cells") express invariant αβ TCR (Vα24-Jα18, CD24lo), CD44hi, NK1.1 (mouse) and NKG2D. The invariant TCR recognizes the glycolipid antigen presented by the non-polymorphic MHC class I molecule, CD1d. These cells can influence an immune response by rapidly producing large amounts of cytokines, i.e., IFNγ.

[00262] As known in the art, "effector cells" are immune cells that actively support the immune response after stimulation. As used herein, effector cells refer to immune cells that express FCY receptors and are therefore capable of mediating ADCC or ADCP. Non-limiting examples of effector cells are monocytes, neutrophils, mast cells, and preferably macrophages and natural killer cells.

[00263] "Tertiary lymphoid structures" are intratumoral structures characterized by increased expression of LTta, LTtb, Cxcr5 and Cxcl13.

[00264] The term "chimeric antigen receptor" or "CAR," as used herein, refers to an artificial T cell surface receptor that is designed to be expressed on an immune effector cell and specifically binds to an antigen. CARs can be used as adoptive cell transfer therapy. Monocytes are removed from a patient (blood, tumor, or ascitic fluid) and modified to express receptors specific to a particular form of antigen. In some embodiments, CARs have been expressed with specificity for a tumor-associated antigen. CARs may also comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen-binding region. In some aspects, CARs comprise fusions of monoclonal antibodies derived from single-chain variable fragments (scFv), fused to the CD3-zeta transmembrane and intracellular domain. The specificity of CAR designs may be derived from receptor ligands (e.g., peptides). In some modalities, a CAR can target cancers by redirecting a monocyte/macrophage expressing the specific CAR to tumor-associated antigens.

[00265] Dosage schedules are abbreviated as known in the art, for example, every day (QD), every 2 days (Q2D), or every 3 days (Q3D).

MODALITIES ANTIGEN- AND ANTIBODIAL-BINDING CHEMOKINE RECEPTORS ASPECT 1 - ANTIGEN

[00266] According to a first aspect, an isolated sulfated polypeptide comprising the tyrosine-rich domain (TRD) of a seven-membrane receptor is provided. A tyrosine-rich domain is a conserved N-terminal domain that characterizes seven-transmembrane receptors, such as CXC and CC chemokine receptors, as in Example 1. As used herein, the term TRD refers to the amino acid sequence or protein of a CXC or CC chemokine receptor that is located at the N-terminal of the first cysteine counted from the N-terminal. In addition to tyrosine, a TRD typically comprises negatively charged amino acid residues, such as aspartic acid. The TRDs for all CC and CXC chemokine receptors are listed in Example 4, Table 4.1 for mouse, monkey, and human.

[00267] Tyrosine sulfation is a ubiquitous post-translational protein modification that occurs in all multicellular organisms. It is catalyzed by tyrosylprotein sulfotransferases (TPSTs) 1 and 2, Golgi-resident enzymes that transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependent tyrosine on a protein substrate. Currently, only a small portion of sulfated proteins are known, and understanding the biological mechanisms of sulfation and specific modification sites is still ongoing, as shown in Example 4. Although post-translational modifications such as glycosylation, phosphorylation, acylation, adenylation, farnesylation, ubiquitination, and sulfation are frequent in proteins from living systems, antibody generation is typically performed based on the unmodified target sequence.

[00268] The inventors developed an unusual approach to antibody generation, and surprisingly found that synthetically sulfated polypeptides according to the first aspect could be used to increase both the success rates for generating antibodies to chemokine receptors per se, and also the success rates for chemokine receptor antibodies with superior functional properties for therapeutic uses, as described elsewhere herein. The increased success rate for highly specific chemokine receptor antibodies was particularly surprising because research antibodies that are designed for independent detection of sulfated tyrosine sequence as a post-translational modification are rare.

[00269] Seven transmembrane receptors can be from any species that expresses chemokine receptors characterized by a TRD, for example, human, monkey, macaca fascicularis (cynomolgus monkey), macaca mulatta (rhesus monkey), rodent, mouse, rat, horse, bovine, pig, dog, cat and camel.

[00270] According to some early embodiments of the first aspect, an isolated polypeptide is provided, wherein the isolated polypeptide comprises the tyrosine-rich domain (TRD) of a seven-transmembrane receptor, further characterized in that at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated.

[00271] For example, in a highly successful antibody campaign, the TRD of human or cynomolgus CCR8 was sulfated at positions Y3, Y15, and Y17, while Y16 was omitted, i.e., 75% of the tyrosines in the TRD were sulfated (Table 6.1). In another example, the TRD of human CCR4 was sulfated at positions 19 and 22 and was used for off-target binding, i.e., 50% of the tyrosines were sulfated (Table 8.1). In yet another approach, the TRD of murine CCR4 was sulfated at position 22 and used for off-target binding, i.e., 25% of the tyrosines in the TRD were sulfated (Table 6.1).

[00272] Table 4.1 shows a list of sulfated peptides including preferred positions for tyrosine sulfations. Without being limited by theory, the inventors believe that the introduction of additional charges in the form of tyrosine sulfations adapts the antibody to recognize a specific pattern of negative charges. This recognition appears to require a higher percentage of tyrosines and positively charged amino acids, at least in the antibody's HCDR3, i.e., the use of the isolated sulfated polypeptides according to the present invention also influenced the structural composition of the antibodies and in particular the amino acid composition of HCDR3, as per Example 9.

[00273] According to some second embodiments of the first aspect, which may or may not be the same as the first embodiments according to the first aspect, an isolated polypeptide is provided, in which the seven-transmembrane receptor is human, cynomolgus or mouse.

[00274] In some of these second embodiments, the seven-transmembrane receptor is murine. In some of these preferred second embodiments, the seven-transmembrane receptor is human and/or cynomolgus. In some of these preferred second embodiments, the seven-transmembrane receptor is human. In some of these second embodiments, the seven-transmembrane receptor is cynomolgus.

[00275] According to some third embodiments of the first aspect, which may or may not be the same as the first and/or second embodiments according to the first aspect, an isolated polypeptide is provided, wherein the seven-transmembrane receptor is a chemokine receptor, preferably a) a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10, b) a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 or CXCR6, or c) CX3CR1 or CXCR1.

[00276] In some of these third embodiments, the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor. In some of these third embodiments, the seven-transmembrane receptor is a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10. In some of these preferred third embodiments, the seven-transmembrane receptor is CCR8 or CCR4. In some of these highly preferred third embodiments, the seven-transmembrane receptor is CCR8. In some of these third embodiments, the seven-transmembrane receptor is a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6. In some of these third embodiments, the seven-transmembrane receptor is CX3CR1 or CXCR1.

[00277] In some preferred embodiments of the first aspect, an isolated polypeptide is provided comprising the TRD of a human or cynomolgus seven-transmembrane receptor, further characterized in that at least 25%, at least 50% or at least 75% of the tyrosine residues of the TRD are sulfated, wherein the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor and, preferably, wherein the seven-transmembrane receptor is CCR8 or CCR4.

[00278] According to some embodiments of the third embodiment of the first aspect, the isolated sulfated polypeptide comprises a sequence conforming to or having at least 90%, 95% or 98% sequence identity with: a) SEQ ID NO:1 (CCR1_human_TRD), preferably in which at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:7 (CCR2_human_TRD), preferably in which at least Y26 has been sulfated, or c) SEQ ID NO:13 (CCR3_human_TRD), preferably in which Y16 and/or Y17 have been sulfated, or d) SEQ ID NO:19 (CCR4_human_TRD), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:25 (CCR5_human_TRD), preferably in which two, three, or all of Y3, Y10, Y14, and Y15 were sulfated, or f) SEQ ID NO:31 (CCR6_human_TRD), preferably in which at least two or three of Y18, Y26, and Y27 were sulfated, or g) SEQ ID NO:37 (CCR7_human_TRD), preferably in which one or both Y8 and Y17 were sulfated, or h) SEQ ID NO:43 (CCR8_human_TRD), preferably in which at least two or all of Y3, Y15, and Y17 were sulfated, or i) SEQ ID NO:61 (CCR9_human_TRD), in which at least Y28 and preferably also Y17 and/or Y37 were sulfated, or j) SEQ ID NO:67 (CCR10_human_TRD), preferably in which at least one or both of Y14 and Y22 were sulfated, ouk) SEQ ID NO:73 (CXCR1_humano_TRD), preferably where Y27 was sulfated, oul) SEQ ID NO:79 (CXCR2_humano_TRD), preferably where Y23 and/or Y25 were sulfated, oum) SEQ ID NO:85 (CXCR3_humano_TRD), preferably where at least one or both of Y27 and Y29 were sulfated, oun) SEQ ID NO:91 (CXCR4_humano_TRD), preferably where at least Y12 and/or Y21 were sulfated, ouo) SEQ ID NO:97 (CXCR5_humano_TRD), preferably where at least one of Y3 and Y27 were sulfated, ouup) SEQ ID NO:103 (CXCR6_humano_TRD), preferably where at least One or both of Y6 and Y10 were sulfated, or) SEQ ID NO:157 (CX3CR1_humano_TRD), preferably wherein at least Y14 was sulfated, or) SEQ ID NO:163 (CXCR1_humano_TRD), preferably wherein at least Y27 was sulfated.

[00279] According to some embodiments B of the third embodiments of the first aspect, the isolated sulfated polypeptide comprises a sequence according to or having at least 90%, 95% or 98% sequence identity with a) SEQ ID NO:3 (CCR1_mouse_TRD), preferably in which at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:9 (CCR2_mouse_TRD), preferably in which at least Y37 and/or Y39 have been sulfated, or c) SEQ ID NO:15 (CCR3_mouse_TRD), preferably in which Y20 and/or Y22 have been sulfated, or d) SEQ ID NO:21 (CCR4_mouse_TRD), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:27 (CCR5_camundongo_TRD), preferably in which two or three of Y10, Y12 and Y16 were sulfated, or f) SEQ ID NO:33 (CCR6_camundongo_TRD), preferably in which at least two or three of Y13, Y18 and Y19 were sulfated, g) SEQ ID NO:39 (CCR7_camundongo_TRD), preferably in which one or both Y8 and Y17 and optionally Y20 were sulfated, or h) SEQ ID NO:45 (CCR8_camundongo_TRD), preferably in which at least two or all of Y3, Y14 and Y15 were sulfated, or i) SEQ ID NO:63 (CCR9_camundongo_TRD), preferably in which at least Y28 was sulfated, and preferably also Y19 was sulfated, or j) SEQ ID NO:69 (CCR10_mouse_TRD), preferably in which at least one, two, or all of Y14, Y17, and Y22 were sulfated, or k) SEQ ID NO:75 (CXCR1_mouse_TRD), preferably in which at least Y6 was sulfated, or l) SEQ ID NO:81 (CXCR2_mouse_TRD), preferably in which Y24 was sulfated, or m) SEQ ID NO:87 (CXCR3_mouse_TRD), preferably in which at least one or both of Y27 and Y29 were sulfated, or n) SEQ ID NO:93 (CXCR4_mouse_TRD), preferably in which at least Y23, Y13, and/or Y14 were sulfated, or o) SEQ ID NO:99 (CXCR5_mouse_TRD), preferably in which at least Y3 and/or Y14 and/or Y20 and/or Y26 were sulfated, or) SEQ ID NO:105 (CXCR6_camundongo_TRD), preferably where at least one or both of Y11 and Y15 were sulfated, or) SEQ ID NO:159 (CX3CR1_camundongo_TRD), preferably where at least Y15 was sulfated, or) SEQ ID NO:165 (CXCR1_camundongo_TRD), preferably where at least Y6 was sulfated.

[00280] According to some embodiments of the third embodiments of the first aspect, the isolated sulfated polypeptide comprises a sequence conforming to or having at least 90%, 95% or 98% sequence identity with e) SEQ ID NO:26 (CCR5_MACMU_TRD), preferably in which two, three or all of Y3, Y10, Y14 and Y15 have been sulfated, or f) SEQ ID NO:32 (CCR6_MACFA_TRD), preferably in which at least two or three of Y23, Y31 and Y32 have been sulfated, or g) SEQ ID NO:38 (CCR7_MACFA_TRD), preferably in which one or both Y8 and Y17 have been sulfated, or h) SEQ ID NO:44 (CCR8_MACFA_TRD), preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated. sulfated, or i) SEQ ID NO:62 (CCR9_MACFA_TRD), preferably in which at least Y28, and preferably also Y17 and/or Y37 were sulfated, or j) SEQ ID NO:68 (CCR10_MACFA_TRD), preferably in which at least one or both of Y14 and Y22 were sulfated, or k) SEQ ID NO:74 (CXCR1_MACFA_TRD), preferably in which at least one of Y14 and Y28 was sulfated, or l) SEQ ID NO:80 (CXCR2_MACFA_TRD), preferably in which Y20 and/or Y22 were sulfated, or m) SEQ ID NO:86 (CXCR3_MACFA_TRD), preferably in which at least one or both of Y27 and Y29 were sulfated, or n) SEQ ID NO:92 (CXCR4_MACFA_TRD), preferably in which at least Y12 and/or Y21 have been sulfated, or) SEQ ID NO:98 (CXCR5_MACFA_TRD), preferably in which at least one of Y3 and Y27 has been sulfated, or) SEQ ID NO:104 (CXCR6_MACFA_TRD), preferably in which at least two or all of Y4, Y7 and Y39 have been sulfated, or) SEQ ID NO:158 (CX3CR1_MACFA_TRD), preferably in which at least Y20 has been sulfated, or) SEQ ID NO:164 (CXCR1_MACMU_TRD), preferably in which at least Y14 has been sulfated.

[00281] In some fourth embodiments of the first aspect, which may or may not be the same as the first, second and/or third embodiments according to the first aspect, the isolated polypeptide comprises the N-terminal of seven transmembrane receptors including TRD and LID domains, preferably wherein at least the cysteine between the TRD and LID domains has been removed or has been altered to a different amino acid.

[00282] The extracellular domains of chemokine receptors can be structured into four regions:(1) an N-terminal domain that can be subdivided into(a) the membrane-distal tyrosine-rich domain (TRD),(b) a cysteine, and(c) a LID domain,(iii) an extracellular domain 1 (ECL1),(iii) an extracellular domain 2 (ECL2), and(iv) an extracellular domain 3 (ECL3).

[00283] The polypeptides according to the present invention are difficult to handle, for example, due to a high tendency for aggregation. Without being limited by theory, the inventors believe that the high "adhesion" results from the increased number of charged amino acids and charged sulfate residues. For polypeptides comprising the N-terminal of a chemokine receptor, the aggregation properties can be improved by removing or exchanging cysteine amino acids between the TRD and LID domains. Ideal results were obtained by changing the cysteine to a serine (Example 5, Table 4.1).

[00284] In some of the fourth embodiments according to the first aspect, cysteine may be omitted and TRD and LID are directly connected. In some of the different fourth embodiments according to the first aspect, cysteine may be replaced by a different uncharged polar amino acid. In some of the preferred fourth embodiments according to the first aspect, cysteine is replaced by a serine (as per Table 4.1). In some of the fourth embodiments according to the first aspect, cysteine may be replaced by at least one different amino acid.

[00285] According to some embodiments of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises a sequence with or with at least 90%, 95% or 98% sequence identity with a) SEQ ID NO:4 (CCR1_human_N term), wherein at least Y10 and/or Y18 have been sulfated, b) SEQ ID NO:10 (CCR2_human_N term), wherein at least Y26 has been sulfated, c) SEQ ID NO:16 (CCR3_human_N term), wherein Y16 and/or Y17 have been sulfated, d) SEQ ID NO:22 (CCR4_human_N term), wherein at least Y22 has been sulfated and, preferably, in addition Y16, Y19 and/or Y20 have been sulfated, e) SEQ ID NO:28 (CCR5_human_N term), wherein two, three or all of Y3, Y10, Y14 and Y15 were sulfated,f) SEQ ID NO:34 (CCR6_humano_N term), in which at least two or three of Y18, Y26 and Y27 were sulfated,g) SEQ ID NO:40 (CCR7_humano_N term), in which one or both Y8 and Y17 were sulfated,h) SEQ ID NO:46 (CCR8_humano_N term), in which at least two or all of Y3, Y15 and Y17 were sulfated,i) SEQ ID NO:64 (CCR9_humano_N term), in which at least Y28, and preferably also Y17 and/or Y37 were sulfated,j) SEQ ID NO:70 (CCR10_humano_N term), in which at least one or both of Y14 and Y22 were sulfated,k) SEQ ID NO:76 (CXCR1_human_N term), in which Y27 was sulfated, l) SEQ ID NO:82 (CXCR2_human_N term), in which Y23 and/or Y25 were sulfated, m) SEQ ID NO:88 (CXCR3_human_N term), in which at least one or both of Y27 and Y29 were sulfated, n) SEQ ID NO:94 (CXCR4_human_N term), in which at least Y12 and/or Y21 were sulfated, o) SEQ ID NO:100 (CXCR5_human_N term), in which at least one of Y3 and Y27 was sulfated, oup) SEQ ID NO:106 (CXCR6_human_N term), in which at least one or both of Y6 and Y10 were sulfated, ouq) SEQ ID NO:160 (CX3CR1_human_N), preferably in that at least Y14 was sulfated, our) SEQ ID NO:166 (CXCR1_humano_N term), preferably in which at least Y27 was sulfated.

[00286] According to some embodiments B of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises a sequence according to or having at least 90%, 95% or 98% sequence identity with a) SEQ ID NO:6 (CCR1_mouse_N term), preferably in which at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:12 (CCR2_mouse_N term), preferably in which at least Y37 and/or Y39 have been sulfated, or c) SEQ ID NO:18 (CCR3_MOUSE_N term), preferably in which Y20 and/or Y22 have been sulfated, or d) SEQ ID NO:24 (CCR4_mouse_N term), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:30 (CCR5_mouse_N term), preferably in which two or three of Y10, Y12, and Y16 were sulfated, or f) SEQ ID NO:36 (CCR6_mouse_N term), preferably in which at least two or three of Y13, Y18, and Y19 were sulfated, g) SEQ ID NO:42 (CCR7_mouse_N term), preferably in which one or both Y8 and Y17 and optionally Y20 were sulfated, or h) SEQ ID NO:48 (CCR8_mouse_N term with C = X or S), preferably in which at least two or all of Y3, Y14, and Y15 were sulfated, or i) SEQ ID NO:66 (CCR9_mouse_N term), preferably in which at least Y28 was sulfated, and preferably also Y19 was sulfated, or j) SEQ ID NO:72 (CCR10_mouse_N term), preferably in which at least one, two, or all of Y14, Y17, and Y22 were sulfated, ouk) SEQ ID NO:78 (CXCR1_mouse_N term), preferably in which at least Y6 was sulfated, oul) SEQ ID NO:84 (CXCR2_mouse_N term), preferably in which Y24 was sulfated, oum) SEQ ID NO:90 (CXCR3_mouse_N term), preferably in which at least one or both of Y27 and Y29 were sulfated, oun) SEQ ID NO:96 (CXCR4_mouse_N term), preferably in which at least Y23 and/or Y14 were sulfated, ouo) SEQ ID NO:102 (CXCR5_mouse_N term), preferably in which at least Y3 and/or Y14 and/or Y20 and/or Y26 were sulfated, or) SEQ ID NO:108 (CXCR6_mouse_N term), preferably wherein at least one or both of Y11 and Y15 were sulfated, or) SEQ ID NO:162 (CX3CR1_mouse_N term), preferably wherein at least Y15 was sulfated, or) SEQ ID NO:168 (CXCR1_mouse_N term), preferably wherein at least Y6 was sulfated.

[00287] According to some embodiments of the fourth embodiment of the first aspect, the isolated sulfated polypeptide comprises a sequence according to or having at least 90%, 95% or 98% sequence identity with a) SEQ ID NO:5 (CCR1_MACFA_N term), preferably in which at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:11 (CCR2_MACMU_N term), preferably in which at least Y26 has been sulfated, or c) SEQ ID NO:17 (CCR3_MACFA_N term), preferably in which Y16 has been sulfated, or d) SEQ ID NO:23 (CCR4_MACFA_N term), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:29 (CCR5_MACMU_N term), preferably in which two, three, or all of Y3, Y10, Y14, and Y15 were sulfated, or f) SEQ ID NO:35 (CCR6_MACFA_N term), preferably in which at least two or three of Y23, Y31, and Y32 were sulfated, or g) SEQ ID NO:41 (CCR7_MACFA_N term), preferably in which one or both of Y8 and Y17 were sulfated, or h) SEQ ID NO:47 (CCR8_MACFA_N term), preferably in which at least two or all of Y3, Y15, and Y17 were sulfated, or i) SEQ ID NO:65 (CCR9_MACFA_N term), preferably in which at least Y28, and preferably also Y17 and/or Y37 were sulfated, or j) SEQ ID NO:71 (CCR10_MACFA_N term), preferably in which at least one or both of Y14 and Y22 were sulfated, ouk) SEQ ID NO:77 (CXCR1_MACFA_N term), preferably in which at least one of Y14 and Y28 was sulfated, oul) SEQ ID NO:83 (CXCR2_MACFA_N term), preferably in which Y20 and/or Y22 were sulfated, oum) SEQ ID NO:89 (CXCR3_MACFA_N term), preferably in which at least one or both of Y27 and Y29 were sulfated, oun) SEQ ID NO:95 (CXCR4_MACFA_N term), preferably in which at least Y12 and/or Y21 were sulfated, ouo) SEQ ID NO:101 (CXCR5_MACFA_N term), preferably in which at least one of Y3 and Y27 was sulfated, oup) SEQ ID NO:107 (CXCR6_MACFA_N term), preferably in which at least two or all of Y4, Y7 and Y39 were sulfated, ouq) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferably in which at least Y20 or Y22 was sulfated, our) SEQ ID NO:167 (CXCR1_MACMU_N term), preferably in which at least Y14 or Y28 was sulfated.

[00288] In some of the fourth embodiments of the first aspect, the isolated sulfated polypeptide comprises a sequence according to a) SEQ ID NO:4 (CCR1_human_N term), SEQ ID NO:5 (CCR1_MACFA_N) or SEQ ID NO:6 (CCR1_mouse_N), preferably wherein at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:10 (CCR2_human_N term), or SEQ ID NO:11 (CCR2_MACMU_N), preferably wherein at least Y26 has been sulfated, or c) SEQ ID NO:12 (CCR2_mouse_N term), preferably wherein at least Y37 and/or Y39 have been sulfated, or d) SEQ ID NO:16 (CCR3_human_N term), preferably wherein Y16 and/or Y17 have been sulfated, or e) SEQ ID NO:17 (CCR3_MACFA_N term), preferably where Y16 has been sulfated, or f) SEQ ID NO:18 (CCR3_MOUSE_N term), preferably where Y20 and/or Y22 have been sulfated, or g) SEQ ID NO:22 (CCR4_human_N term), SEQ ID NO:23 (CCR4_MACFA_N) or SEQ ID NO:24 (CCR4_mouse_N), preferably where at least Y22 has been sulfated and, preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or h) SEQ ID NO:28 (CCR5_human_N term), or SEQ ID NO:29 (CCR5_MACMU_N), preferably where two, three or all of Y3, Y10, Y14 and Y15 have been sulfated, or i) SEQ ID NO:30 (CCR5_mouse_N term), preferably in which two or three of Y10, Y12 and Y16 were sulfated, or j) SEQ ID NO:34 (CCR6_human_N term), preferably in which at least two or three of Y18, Y26 and Y27 were sulfated, or k) SEQ ID NO:35 (CCR6_MACFA_N term), preferably in which at least two or three of Y23, Y31 and Y32 were sulfated, or l) SEQ ID NO:36 (CCR6_mouse_N term), preferably in which at least two or three of Y13, Y18 and Y19 were sulfated, or m) SEQ ID NO:40 (CCR7_human_N term), or SEQ ID NO:41 (CCR7_MACFA_N), preferably in which one or both Y8 and Y17 were sulfated, or n) SEQ ID NO:42 (CCR7_mouse_N term), preferably in which one or both Y8 and Y17 and optionally Y20 were sulfated, or) SEQ ID NO:46 (CCR8_human_N term with C = X or S), or SEQ ID NO:47 (CCR8_MACFA_N term with C = X or S), preferably in which at least two or all of Y3, Y15 and Y17 were sulfated, or) SEQ ID NO:48 (CCR8_mouse_N term with C = X or S), preferably in which at least two or all of Y3, Y14 and Y15 were sulfated, or) SEQ ID NO:64 (CCR9_human_N term), or SEQ ID NO:65 (CCR9_MACFA_N), preferably in which at least Y28, and preferably also Y17 and/or Y37 were sulfated, or) SEQ ID NO:66 (CCR9_mouse_N term), preferably in which at least Y28 has been sulfated, and preferably also Y19 has been sulfated, ous) SEQ ID NO:70 (CCR10_human_N term), or SEQ ID NO:71 (CCR10_MACFA_N), preferably in which at least one or both of Y14 and Y22 have been sulfated, ou) SEQ ID NO:72 (CCR10_mouse_N term), preferably in which at least one, two or all of Y14, Y17 and Y22 have been sulfated, ouu) SEQ ID NO:76 (CXCR1_human_N term), preferably in which Y27 has been sulfated, ouv) SEQ ID NO:77 (CXCR1_MACFA_N term), preferably in which at least one of Y14 and Y28 has been sulfated, ou w) SEQ ID NO:78 (CXCR1_mouse_N term), preferably in which at least Y6 has been sulfated, or x) SEQ ID NO:82 (CXCR2_human_N term), preferably in which Y23 and/or Y25 have been sulfated, or y) SEQ ID NO:83 (CXCR2_MACFA_N), preferably in which Y20 and/or Y22 have been sulfated, or z) SEQ ID NO:84 (CXCR2_mouse_N term), preferably in which Y24 has been sulfated, or aa) SEQ ID NO:88 (CXCR3_human_N term), SEQ ID NO:89 (CXCR3_MACFA_N term) or SEQ ID NO:90 (CXCR3_mouse_N), preferably in which at least one or both of Y27 and Y29 have been sulfated, or bb) SEQ ID NO:94 (CXCR4_human_N term), or SEQ ID NO:95 (CXCR4_MACFA_N term), preferably in which at least Y12 and/or Y21 have been sulfated, oucc) SEQ ID NO:96 (CXCR4_mouse_N term), preferably in which at least Y23 and/or Y14 have been sulfated, oudd) SEQ ID NO:100 (CXCR5_human_N term), or SEQ ID NO:101 (CXCR5_MACFA_N term), preferably in which at least one of Y3 and Y27 have been sulfated, ouee) SEQ ID NO:102 (CXCR5_mouse_N term), preferably in which at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, ouuff) SEQ ID NO:106 (CXCR6_human_N term), preferably in which at least one or both of Y6 and Y10 were sulfated, ougg) SEQ ID NO:107 (CXCR6_MACFA_N term), preferably in which at least two or all of Y4, Y7 and Y39 were sulfated, ouhh) SEQ ID NO:108 (CXCR6_mouse_N term), preferably in which at least one or both of Y11 and Y15 were sulfated, ouii) SEQ ID NO:160 (CX3CR1_human_N term), preferably in which at least Y14 was sulfated, oujj) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferably in which at least Y20 or Y22 was sulfated, oukk) SEQ ID NO:162 (CX3CR1_mouse_N term), preferably in which at least Y15 was sulfated, ou11) SEQ ID NO:166 (CXCR1_humano_N term), preferably in which at least Y27 has been sulfated, oumm) SEQ ID NO:167 (CXCR1_MACMU_N term), preferably in which at least Y14 or Y28 has been sulfated, ounn) SEQ ID NO:168 (CXCR1_camundongo_N term), preferably in which at least Y6 has been sulfated.

[00289] In some of the first, second, third or fourth embodiments of the first aspect, the isolated sulfated polypeptide comprises a sequence conforming to or having at least 90% sequence identity with a) SEQ ID NO:1 (CCR1_human_TRD), SEQ ID NO:4 (CCR1_human_N term), SEQ ID NO:2 (CCR1_MACFA_TRD), SEQ ID NO:5 (CCR1_MACFA_N term), SEQ ID NO:3 (CCR1_mouse_TRD) or SEQ ID NO:6 (CCR1_mouse_N), preferably wherein at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:7 (CCR2_human_TRD), SEQ ID NO:10 (CCR2_human_N term), SEQ ID NO:8 (CCR2_MACMU_TRD) or SEQ ID NO:11 (CCR2_MACMU_N term), preferably in which at least Y26 has been sulfated, or c) SEQ ID NO:9 (CCR2_mouse_TRD) or SEQ ID NO:12 (CCR2_mouse_N term), preferably in which at least Y37 and/or Y39 have been sulfated, or d) SEQ ID NO:13 (CCR3_human_TRD) or SEQ ID NO:16 (CCR3_human_N term), preferably in which Y16 and/or Y17 have been sulfated, or e) SEQ ID NO:14 (CCR3_MACFA_TRD) or SEQ ID NO:17 (CCR3_MACFA_N term), preferably in which Y16 has been sulfated, or f) SEQ ID NO:15 (CCR3_mouse_TRD) or SEQ ID NO:18 (CCR3_mouse_N term), preferably in which Y20 and/or Y22 were sulfated, or g) SEQ ID NO:19 (CCR4_human_TRD), SEQ ID NO:22 (CCR4_human_N term), SEQ ID NO:20 (CCR4_MACFA_TRD), SEQ ID NO:23 (CCR4_MACFA_N term), SEQ ID NO:21 (CCR4_mouse_TRD) or SEQ ID NO:24 (CCR4_mouse_N term), preferably wherein at least Y22 was sulfated and preferably, in addition, Y16, Y19 and/or Y20 were sulfated, or h) SEQ ID NO:25 (CCR5_human_TRD), SEQ ID NO:28 (CCR5_human_N term), SEQ ID NO:26 (CCR5_MACMU_TRD) or SEQ ID NO:29 (CCR5_MACMU_N term), preferably in which two, three, or all of Y3, Y10, Y14, and Y15 were sulfated, or i) SEQ ID NO:27 (CCR5_mouse_TRD) or SEQ ID NO:30 (CCR5_mouse_N term), preferably in which two or three of Y10, Y12, and Y16 were sulfated, or j) SEQ ID NO:31 (CCR6_human_TRD) or SEQ ID NO:34 (CCR6_human_N term), preferably in which at least two or three of Y18, Y26, and Y27 were sulfated, or k) SEQ ID NO:32 (CCR6_MACFA_TRD) or SEQ ID NO:35 (CCR6_MACFA_N term), preferably in which at least two or three of Y23, Y31, and Y32 were sulfated, or l) SEQ ID NO:33 (CCR6_mouse_TRD) or SEQ ID NO:36 (CCR6_mouse_N term), preferably in which at least two or three of Y13, Y18 and Y19 were sulfated, or) SEQ ID NO:37 (CCR7_human_TRD), SEQ ID NO:40 (CCR7_human_N term), SEQ ID NO:38 (CCR7_MACFA_TRD) or SEQ ID NO:41 (CCR7_MACFA_N term), preferably in which one or both Y8 and Y17 were sulfated, or) SEQ ID NO:39 (CCR7_mouse_TRD) or SEQ ID NO:42 (CCR7_mouse_N term), preferably in which one or both Y8 and Y17 and optionally Y20 were sulfated, or) SEQ ID NO:43 (CCR8_human_TRD), SEQ ID NO:44 (CCR8_MACFA_TRD), SEQ ID NO:46 (CCR8_humano_N term with C = X or S), or SEQ ID NO:47 (CCR8_MACFA_N term with C = X or S), preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated, or SEQ ID NO:45 (CCR8_camundongo_TRD) or SEQ ID NO:48 (CCR8_camundongo_N term with C = X or S), preferably in which at least two or all of Y3, Y14 and Y15 have been sulfated, or SEQ ID NO:61 (CCR9_humano_TRD), SEQ ID NO:64 (CCR9_humano_N term), SEQ ID NO:62 (CCR9_MACFA_TRD) or SEQ ID NO:65 (CCR9_MACFA_N term), preferably in which at least Y28, and preferably also Y17 and/or Y37 has been sulfated, our) SEQ ID NO:63 (CCR9_mouse_TRD) or SEQ ID NO:66 (CCR9_mouse_N term), preferably in which at least Y28 has been sulfated, and preferably also Y19 has been sulfated, ous) SEQ ID NO:67 (CCR10_human_TRD), SEQ ID NO:70 (CCR10_human_N term), SEQ ID NO:68 (CCR10_MACFA_TRD) or SEQ ID NO:71 (CCR10_MACFA_N term), preferably in which at least one or both of Y14 and Y22 have been sulfated, out) SEQ ID NO:69 (CCR10_mouse_TRD) or SEQ ID NO:72 (CCR10_mouse_N term), preferably in which at least one, two, or all of Y14, Y17, and Y22 have been sulfated, oru) SEQ ID NO:73 (CXCR1_human_TRD) or SEQ ID NO:76 (CXCR1_human_N), preferably in which Y27 has been sulfated, orv) SEQ ID NO:74 (CXCR1_MACFA_TRD) or SEQ ID NO:77 (CXCR1_MACFA_N term), preferably in which at least one of Y14 and Y28 has been sulfated, orw) SEQ ID NO:75 (CXCR1_mouse_TRD) or SEQ ID NO:78 (CXCR1_mouse_N term), preferably in which at least Y6 has been sulfated, orx) SEQ ID NO:79 (CXCR2_human_TRD) or SEQ ID NO:82 (CXCR2_human_N term), preferably where Y23 and/or Y25 have been sulfated, or y) SEQ ID NO:80 (CXCR2_MACFA_TRD) or SEQ ID NO:83 (CXCR2_MACFA_N term), preferably where Y20 and/or Y22 have been sulfated, or z) SEQ ID NO:81 (CXCR2_mouse_TRD) or SEQ ID NO:84 (CXCR2_mouse_N term), preferably where Y24 has been sulfated, or aa) SEQ ID NO:85 (CXCR3_human_TRD), SEQ ID NO:88 (CXCR3_human_N term), SEQ ID NO:86 (CXCR3_MACFA_TRD), SEQ ID NO:89 (CXCR3_MACFA_N term), SEQ ID NO:87 (CXCR3_mouse_TRD) or SEQ ID NO :90 (CXCR3_mouse_N), preferably in which at least one or both of Y27 and Y29 have been sulfated, or bb) SEQ ID NO:91 (CXCR4_human_TRD), SEQ ID NO:94 (CXCR4_human_N term), SEQ ID NO:92(CXCR4_MACFA_TRD) or SEQ ID NO:95 (CXCR4_MACFA_N term), preferably in which at least Y12 and/or Y21 have been sulfated, oucc) SEQ ID NO:93 (CXCR4_mouse_TRD) or SEQ ID NO:96 (CXCR4_mouse_N term), preferably in which at least Y23 and/or Y14 have been sulfated, oudd) SEQ ID NO:97 (CXCR5_human_TRD), SEQ ID NO:100 (CXCR5_human_N term), SEQ ID NO:98(CXCR5_MACFA_TRD) or SEQ ID NO:101 (CXCR5_MACFA_N term), preferably in which at least one of Y3 and Y27 were sulfated, ouee) SEQ ID NO:99 (CXCR5_mouse_TRD) or SEQ ID NO:102 (CXCR5_mouse_N term), preferably in which at least Y3 and/or Y14 and/or Y20 and/or Y26 were sulfated, ouff) SEQ ID NO:103 (CXCR6_human_TRD) or SEQ ID NO:106 (CXCR6_human_N term), preferably in which at least one or both of Y6 and Y10 were sulfated, ougg) SEQ ID NO:104 (CXCR6_MACFA_TRD) or SEQ ID NO:107 (CXCR6_MACFA_N term), preferably in which at least two or all of Y4, Y7, and Y39 have been sulfated, or) SEQ ID NO:105 (CXCR6_mouse_TRD) or SEQ ID NO:108 (CXCR6_mouse_N term), preferably in which at least one or both of Y11 and Y15 have been sulfated, or) SEQ ID NO:157 (CX3CR1_human_TRD) or SEQ ID NO:160 (CX3CR1_human_N term), preferably in which at least Y14 has been sulfated, or) SEQ ID NO:158 (CX3CR1_MACFA_TRD), preferably in which at least Y20 has been sulfated, or) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferably in which at least Y20 or Y22 was sulfated, oull) SEQ ID NO:159 (CX3CR1_mouse_TRD) or SEQ ID NO:162 (CX3CR1_mouse_N term), preferably wherein at least Y15 was sulfated, oumm) SEQ ID NO:163 (CXCR1_human_TRD) or SEQ ID NO:166 (CXCR1_human_N term), preferably wherein at least Y27 was sulfated, ounn) SEQ ID NO:164 (CXCR1_MACMU_TRD), preferably wherein at least Y14 was sulfated, ouoo) SEQ ID NO:167 (CXCR1_MACMU_N term), preferably wherein at least Y14 or Y28 was sulfated, oupp) SEQ ID NO:165 (CXCR1_mouse_TRD) or SEQ ID NO:168 (CXCR1_mouse_N term), preferably in which at least Y6 has been sulfated.

[00290] Preferably, the isolated polypeptide according to the current aspect is immobilized, for example, by means of a ligand. Immobilization can occur without limitation on granules, particles, proteins or suitable solid supports.

ASPECT 2 - CONJUGATE COMPRISING ISOLATED POLYPEPTIDE

[00291] According to a second aspect of the present invention, a conjugate comprising an isolated sulfated polypeptide according to the first aspect is provided.

[00292] For example, the conjugate may comprise a polypeptide according to the first embodiments of the first aspect. For example, the conjugate may comprise a polypeptide according to the second embodiments of the first aspect. For example, the conjugate may comprise a polypeptide according to the third embodiments of the first aspect. For example, the conjugate may comprise a polypeptide according to some embodiments A, B, C of the third embodiments of the first aspect. For example, the conjugate may comprise a polypeptide according to the fourth embodiment of the first aspect. For example, the conjugate may comprise a polypeptide according to some embodiments A, B, C of the fourth embodiments of the first aspect.

[00293] For example, the isolated sulfated polypeptide can be linked to a label or ligand for immobilization or retrieval. Suitable labels are selected from labels known in the art, for example, small organic molecules such as biotin (which binds strongly and non-covalently to streptavidin), derivatives thereof, or short peptide sequences such as Flag label, HA label, Myc label, or His label (as per Table 4.1 or Example 10.1.2). For some applications, the label may be a protein, such as human serum albumin (as per Table 4.1 or Example 10.1.2), or a carrier protein, such as KLH, OVA, or BSA, or a larger structure, such as a bead or magnetic bead. Preferably, the label can be linked through the C-term or the N-term of the TRD or the N-terminal of the chemokine receptor, but it can also be linked through a reactive residue or amino acid side chain, such as a lysine within the TRD. In some embodiments, the label is linked via a ligand, which can be any ligand known in the art. Suitable ligands include trioxatridecanesuccinamic acid (Ttds) ligand (as per Table 4.1), beta-alanine, GABA, AEA, Ava, Ahx, PEG2 spacer, PEG3 spacer, PEG4 spacer, O1Pen, O2Oc, or O1Pen-O1.

ASPECT 3 - ANTIGEN PRODUCTION METHOD

[00294] Sulfated peptides can be difficult to synthesize, for example, because sulfate is unstable under acidic conditions (Houben-Weyl, Methods of Organic Chemistry Vol. E 22b, Synthesis of Peptides and Peptidomimetics, 4th Edition, section 6.6.1.2 Synthesis of Sulfated Tyrosine Peptides with Tyrosine O-Sulfate Synthons, p.440 et seq. in: Felix, Arthur et al.:2004).

[00295] According to a third aspect, a method is provided for producing the isolated sulfated polypeptide according to the first aspect or the conjugate according to the second aspect, wherein the method comprises synthesizing the isolated polypeptide and sulfating the respective tyrosine residues.

[00296] The synthesis of the isolated polypeptide according to the first aspect and the sulfation of the respective tyrosine residues can occur as described in Example 5, or can occur according to any other method known in the art. In chapter 6.6.1 "Methods of Organic Chemistry Vol. E 22b, Synthesis of Peptides and Peptidomimetics", Houben-Weyl describes several chemical approaches for the synthesis of sulfated tyrosine peptides. The chapter, and in particular these methods, are incorporated herein by reference in their entirety.

[00297] For example, the synthesis can be carried out using the sequence-independent solid-phase method previously described by Bunschoten et al. (Bunschoten, Anton, et al. "A general sequence independent solid phase method for the site-specific synthesis of multiple sulfated-tyrosine-containing peptides." Chemical communications 21 (2009): 2999-3001.), incorporated herein in its entirety. In summary, a peptide is synthesized according to the Fmoc-tBu strategy, followed by selective deprotection of the tyrosine residues to be sulfated and introduction of a protected sulfate group. After completion of the synthesis of the sulfated peptide, it is cleaved from the resin by acidolysis and the protecting groups are removed except for the sulfate protecting group, thus avoiding unwanted acid-induced removal of the sulfate group(s) during this step. Finally, the sulfate protecting groups can be removed in a mildly acidic reducing step, leaving the sulfate groups untouched.

[00298] Chen et al. reported a short and efficient one-step route for sY-containing peptides, in which Fmoc-protected fluorosulfated tyrosine (Y(OSO2F)) is incorporated into the peptide of interest via an Fmoc-based solid-phase synthetic strategy (as described in Chen, Wentao, et al. "Synthesis of Sulfotyrosine-Containing Peptides by Incorporating Fluorosulfated Tyrosine Using an Fmoc-Based Solid-Phase Strategy." Angewandte Chemie 128.5 (2016): 1867-1870.), incorporated here in its entirety. Standard simultaneous peptide-resin cleavage and removal of acid-labile side-chain protecting groups yield crude fluorosulfated tyrosine-containing peptides. Basic ethylene glycol, serving as both solvent and reagent, transforms fluorosulfated tyrosine peptides into sulfotyrosine peptides in high yield.

[00299] Global sulfation of polypeptides is also possible, for example, using sulfur trioxide-pyridine. According to the present invention, this route can be used if all tyrosines have to be sulfated or if "crude" mixtures of partially sulfated polypeptides are to be used for further steps. Furthermore, sulfation can occur enzymatically, for example, in a biotransformation reaction using natural sulfation enzymes or their modified versions. Preferably, the sulfation of the respective tyrosine can occur chemically or enzymatically. Preferably, the synthesis of the isolated polypeptide occurs using the Fmoc-tBu strategy.

[00300] According to some embodiments of the 3rd aspect, the method comprises the purification of the obtained polypeptides. Purification can occur, for example, by HPLC, for example, using a C18 column. According to some embodiments of the 3rd aspect, the method comprises the analytical characterization of the polypeptide. Without limitation, the analytical characterization can be performed using spectroscopic methods or mass spectrometry.

ASPECT 4 - USES OF THE ANTIGEN/METHODS OF USE OF THE ANTIGEN

[00301] According to a fourth aspect, the use of the isolated sulfated polypeptide according to the first aspect or conjugated according to the second aspect is provided for the generation of antibodies, as an antigen or for off-target panning and/or for the characterization of an antibody. For example, the isolated sulfated polypeptide according to the first aspect or conjugated according to the second aspect can be used for the generation of a fully human antibody or a fragment thereof. For example, the isolated sulfated polypeptide according to the first aspect or conjugated according to the second aspect can be used for the generation of a cross-reactive antibody.

[00302] According to the fourth aspect, the use of the isolated sulfated polypeptide according to the first aspect or the conjugate according to the second aspect is provided for the generation of antibodies. In particular, the isolated sulfated polypeptide according to the first aspect can be used to facilitate the generation of antibodies that specifically recognize chemokine receptors, as described elsewhere herein.

[00303] According to the 4th aspect, which may be the same as or different from the first embodiments according to the 4th aspect, the isolated sulfated polypeptide according to the first aspect or conjugated according to the second aspect is used as an antigen, for example, to select antibodies, antibody fragments or molecules that bind specifically to chemokine receptors, as per Examples 6 and 8.

[00304] According to the 4th aspect, which may be the same as or different from the first and/or second embodiments according to the 4th aspect, the sulfated polypeptide isolated according to the first aspect or conjugated according to the second aspect is used for off-target panning to select antibodies that do not bind to a certain seven-transmembrane receptor, for example, a chemokine receptor (off-target receptor), as per Examples 6 and 8.

[00305] According to the 4th aspect, it comprises the use of the isolated sulfated polypeptide according to the first aspect or conjugated according to the second aspect for characterization of an antibody. Preferably, the antibody is an antibody according to the present invention. For example, the characterization of the antibody may comprise the use of ELISA, surface plasmon resonance, mass spectrometry, competition assays, staining, IHC, FACS or various other assays known in the art.

ASPECT 5 - ANTIBODY PRODUCTION METHOD

[00306] According to a fifth aspect, a method is provided for obtaining an antibody or ligand, the method comprising the use of the isolated sulfated polypeptide according to the first aspect or conjugated according to the second aspect.

[00307] According to some early embodiments, the method according to the 5th aspect comprises the use of the sulfated polypeptide isolated according to the first aspect or conjugated according to the second aspect as an antigen.

[00308] According to some of the first preferred embodiments of the 5th aspect, the method comprises the use of at least one more isolated or conjugated polypeptide, wherein the at least one more isolated polypeptide comprises a TRDa) of a seven-transmembrane receptor different from the first seven-transmembrane receptor, or b) of the first seven-transmembrane receptor derived from a different species, preferably wherein the at least one additional isolated polypeptide is an isolated polypeptide according to the first aspect.

[00309] According to some embodiments of the first embodiments of the 5th aspect, the method comprises the use of at least one additional isolated polypeptide or conjugate, preferably according to the first or second aspect, preferably as an antigen or for off-target panning. For these embodiments, the first isolated sulfated polypeptide comprises a TRD of a first seven-transmembrane receptor (e.g., a chemokine receptor) and the additional isolated polypeptide or conjugate comprises a TRD of a seven-transmembrane receptor other than the first seven-transmembrane receptor. When the additional isolated polypeptide or conjugate is used as an antigen, the method is a method for producing antibodies or ligands that recognize at least two different seven-transmembrane receptors, for example, two different members of the chemokine receptor family.

[00310] When additional isolated polypeptide or conjugate is used for off-target panning, the method is a method for producing antibodies or ligands that recognize only one specific seven-transmembrane receptor, for example, to avoid off-target binding to additional chemokine receptor family members.

[00311] According to some embodiments B of the first embodiments of the 5th aspect, the method comprises the use of at least one additional isolated polypeptide or conjugate, preferably according to the first or second aspect, as an antigen or for off-target panning. For these embodiments, the first isolated polypeptide comprises a TRD of a first seven-transmembrane receptor of a first species (e.g., a human chemokine receptor) and the additional isolated polypeptide comprises a TRD of the same seven-transmembrane receptor derived from a different species (e.g., a cynomolgus chemokine receptor). For example, the first polypeptide may comprise the TRD of a human chemokine receptor and the second polypeptide may comprise the TRD of a cynomolgus chemokine receptor.

[00312] These B embodiments are particularly advantageous because generating antibodies or chemokine receptor ligands that are cross-reactive for both human species and a suitable model species is difficult for chemokine receptors, particularly for CCR8. Although general consensus among transmembrane domains is comparatively high, extracellular domains of chemokine receptors have low consensus among different members of the chemokine receptor family and also among species for a given chemokine receptor (Fig. 1, Fig. 2a). However, according to the present invention, it has recently been discovered that small motifs comprising sulfated tyrosine within the TRD are sufficiently conserved for a given chemokine receptor to be used to obtain high numbers of cross-reacting antibodies. Example 6 describes the generation of cross-reacting antibodies for humans and cynomolgus, and Example 10.1.1 shows excellent affinities in both species for several antibodies according to the present invention. Compared to rodents and mice, cynomolgus is a preferred model system because mouse models failed to predict immunological side effects.

[00313] The method according to the 5th aspect may be any method for generating antibodies known in the art. For example, the method may be a conventional immunization method, for instance, where the polypeptide according to the first aspect is conjugated with KLH and administered to an animal suitable for immunization.

[00314] For example, after immunization, splenocytes from immunized animals can be used to generate hybridoma cells, which produce the animals' antibodies, and in a second step, antibodies that bind specifically to the antigen can be selected by methods known in the art such as, for example, ELISA, as well as off-target binding, if applicable. Alternatively, splenocytes can be directly analyzed for the production of antibodies that bind to the antigen in droplet-based microfluidic systems or in cell culture assays. Then, only selected splenocytes are directly applied to sequencing to obtain the antibody sequence or hybridoma generation. Another method may comprise the use of the antigen for selection with an antibody library which, for example, could be a phage display library, for example, without limitation, as described elsewhere herein, or alternatively a mammalian library. After selecting the library for the antigen, the enriched antibodies can be analyzed for specific antigen binding using methods known in the art, such as ELISA or SPR, as described in more detail here.

[00315] According to some second embodiments of the 5th aspect, which may be the same as or different from the first embodiments of the 5th aspect, the method for obtaining an antibody according to the 5th aspect is a method comprising the use of a phage display library, a transgenic animal, or any other technique available in the art for the generation of antibodies comprising human CDRs.

[00316] In some embodiments of the second embodiments of the 5th aspect, the method comprises the use of a human phage display library, as per Example 6 and Example 8. For example, the phage display library may be a fully human antibody phage display library, such as the BioInvent n-CoDeR Fab lambda library. In some preferred embodiments, the phage display library is enriched with respect to tyrosine and/or histidine content. In a first step, the phage display library comprising bacteriophages displaying antibodies or antibody fragments on their exterior may be combined with the isolated polypeptide immobilized according to the first aspect or conjugated according to the second aspect to enable binding to the isolated or conjugated polypeptide.

[00317] In a second optional depletion step, which may occur before or after the first step, the phage display library comprising bacteriophages displaying antibodies or antibody fragments on their exterior may be combined with an immobilized or conjugated isolated polypeptide, depending on the first aspect, being different from the isolated polypeptide of the first step, to deplete the ligands off-target.

[00318] In an optional third step, which may occur before or after the first step, or may occur before or after the second step, the phage display library comprising bacteriophages displaying antibodies or antibody fragments on their exterior may be combined with an immobilized isolated polypeptide according to the first aspect or conjugated according to the second aspect different from the isolated polypeptide of the first step and the isolated polypeptide of the optional second step, to obtain cross-reacting ligands.

[00319] Each of the steps can be repeated several times, for example, one, two, three, four, five, six, seven, eight, nine, ten or more times. Binding antibodies or fragments can be recovered for expansion by infection of suitable bacterial hosts and the DNA can be sequenced as known in the art to obtain the sequence of the antibody or seven-transmembrane receptor fragment.

[00320] In some embodiments of the second embodiments of the 5th aspect, the method for obtaining an antibody according to the 5th aspect is a method comprising the use of transgenic animals as described by Lonberg (Lonberg, Nils. "Human antibodies from transgenic animals." Nature biotechnology 23.9 (2005): 1117-1125.), incorporated herein in its entirety. For example, the transgenic animal may be a XenoMouse (Abgenix Inc., Fremont, CA, for example, U.S. Patent No. 5,939,598), the HuMAb Mouse (Gen-Pharm-Medarex, San Jose, CA), the RenMab Mouse (Biocytogen), or any other animal known in the art for the generation of fully human antibodies.

[00321] In some embodiments of the second embodiments of the 5th aspect, the method for obtaining an antibody according to the 5th aspect is a method comprising the use of activated B cells in vitro (see U.S. Patents 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety).

[00322] According to some third embodiments of the 5th aspect, a method is provided for obtaining an antibody or antibody fragment or a ligand that binds specifically to a human and/or cynomolgus and/or murine CC or CXC chemokine receptor, the method comprising: a) (synthetically) sulfated a polypeptide comprising a tyrosine-rich domain (TRD) and b) selecting an antibody, antibody fragment or ligand recognizing the sulfated polypeptide, and c) optionally producing the antibody, antibody fragment or ligand.

[00323] In some embodiments, the use according to aspect 4 or the method according to aspect 5 is a use/method for obtaining an antibody with favorable properties as described elsewhere herein, preferably wherein the antibody: a) comprises human-derived CDRs and/or b) is a human, mouse or murine IgG antibody, preferably a human IgG1 antibody or a murine IgG2a antibody, and/or c) is cross-reactive for two different seven-transmembrane receptors, and/or d) is cross-reactive for a human and a cynomolgus seven-transmembrane receptor, and/or e) is characterized by an HCDR3 region comprising between 10 and 34% tyrosine and/or between 2 and 20% histidine, preferably between 7 and 20% histidine and/or f) does not modulate chemokine receptor G protein-independent signaling and/or g) is a non-internalizing antibody or is characterized by a internalization into a cell with endogenous target expression that is less than 1.5, 2, 3, 4, 5, 6, 7, or 10 times the internalization of isotype control.

[00324] The method according to the present aspect can be used to obtain high numbers of antibodies that bind to chemokine receptors. Interestingly, a comparatively high number of these antibodies were characterized by properties that differentiate them from known antibodies obtained by conventional methods, as described elsewhere here.

ASPECT 6 - ANTIBODY DEFINED BY THE ANTIGEN

[00325] According to the present invention, an isolated antibody or antigen-binding fragment thereof or a ligand obtained by a method or use according to a prior aspect is provided.

[00326] As will be appreciated by those skilled in the art, antibodies and/or binding fragments are of a "modular" nature. Throughout the invention, various specific aspects and embodiments of the various "modules" that comprise the antibodies and/or binding fragments are described. As specific, non-limiting examples, various specific embodiments of VH CDRs, VH chains, VL CDRs, and VL chains or functional features are described. It is intended that all specific embodiments can be combined with each other as if each specific combination were explicitly described individually. As specific, non-limiting examples, various specific functional embodiments are described. It is intended that all specific embodiments can be combined with each other as if each specific combination were explicitly described individually.

[00327] According to a sixth aspect, an isolated antibody or antigen-binding fragment thereof is provided, (specifically) binding to a first isolated sulfated polypeptide comprising the tyrosine-rich domain (TRD) of a seven-transmembrane receptor, wherein at least 25%, at least 50% or at least 75% of the tyrosine residues of the TRD are sulfated. Preferably, the first isolated sulfated polypeptide also comprises the LID domain of the seven-transmembrane receptor. Preferably, the cysteine between the TRD and the LID domain has been removed or replaced with a different amino acid.

[00328] Preferably, the first isolated sulfated polypeptide comprises the N-terminal of a seven-transmembrane receptor including its tyrosine-rich domain (TRD) and optionally including its LID domain, and even more preferably, at least 25%, at least 50% or at least 75% of the tyrosine residues of the TRD are sulfated.

[00329] In preferred embodiments, the isolated antibody or antigen-binding fragment thereof binds to its target(s) with a KD value of < 5E-8 M, < 4E-8 M, < 3E-8 M, < 2E-8 M, < 1E-8 M, < 9E-9 M, < 8E-9 M, < 7E-9 M, < 6E-9 M, < 5E-9 M, < 4E-9 M, < 3E-9 M, < 2.5E-9 M, < 2E-9 M, < 1.5E-9 M, < 1E-9 M, < 9E-10 M, < 8E-10 M, < 7E-10 M, < 6E-10 M, < 5E-10 M, < 4E-10 M, < 3E-10 M, < 2.5E-10 M, < 2E-10 M, < 1.5E-10 M, < 1E-10 M, or < 9E-11 M. For example, the inventive antibody may bind to its target(s) with a KD value between < 8E-9 M and > 4E-10 M. When the isolated antibody or antigen-binding fragment thereof binds to more than one target, it most preferably binds to its targets with affinity of the same order of magnitude.

[00330] According to some preferred embodiments, the isolated antibody or antigen-binding fragment thereof a) comprises human-derived CDRs and/or b) is cross-reactive for humans and cynomolgus, and/or c) is characterized by an HCDR3 region comprising between 10 and 34% tyrosine and/or between 7 and 20% histidine, and/or d) does not modulate G protein-independent signaling of seven transmembrane receptors and/or e) is a non-internalizing antibody or is characterized by internalization into a cell with endogenous target expression that is less than 1.5, 2, 3, 4, 5, 6, 7 or 10 times the internalization of the isotype control, and/or f) induces ADCC and/or ADCP and/or g) is a human, mouse or murine IgG antibody, preferably a human IgG1 antibody or a murine IgG2a antibody, and/or h) is a scFv, Fab, Fab' or F(ab')2 fragment.

[00331] According to some early embodiments of the 6th aspect, the seven-transmembrane receptor is a chemokine receptor. In some of these early embodiments, the seven-transmembrane receptor is a CC chemokine receptor or a CXC chemokine receptor. In some of these early embodiments, the seven-transmembrane receptor is a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10. In some of these preferred early embodiments, the seven-transmembrane receptor is CCR8 or CCR4. In some of these most preferred early embodiments, the seven-transmembrane receptor is CCR8. In some of the early embodiments, the seven-transmembrane receptor is a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6. In some of the earliest forms, the seven-transmembrane receptor is CX3CR1 or CXCR1.

[00332] According to some second embodiments of the 6th aspect, which may or may not be the same as the first embodiments of the 6th aspect, seven-transmembrane receptors can be from any species that expresses chemokine receptors characterized by a TRD, for example, human, monkey, macaca fascicularis (cynomolgus monkey), macaca mulatta (rhesus monkey), rodent, mouse, rat, horse, bovine, pig, dog, cat, and camel. In some of these second embodiments of the 6th aspect, the seven-transmembrane receptor is murine. In some of these more preferred second embodiments of the sixth aspect, the seven-transmembrane receptor is human. In some of these second embodiments of the sixth aspect, the seven-transmembrane receptor is cynomolgus. In some of these preferred embodiments, the seven-transmembrane receptor is human, cynomolgus, or mouse. In some of these preferred second-part modalities of the sixth aspect, the seven-transmembrane receptor is human or cynomolgus.

[00333] In some preferred embodiments, the seven-transmembrane receptor is a human, cynomolgus or mouse seven-transmembrane receptor and the seven-transmembrane receptor is a) a CC chemokine receptor, preferably CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10, b) a CXC chemokine receptor, preferably CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 or CXCR6, or c) CX3CR1 or CXCR1.

[00334] According to some third embodiments of the 6th aspect, which may or may not be the same as the first and/or second embodiments according to the 6th aspect, the (first) isolated sulfated polypeptide comprises or consists of a sequence according to: a) SEQ ID NO:1 (CCR1_human_TRD), SEQ ID NO:4 (CCR1_human_N term), SEQ ID NO:2 (CCR1_MACFA_TRD), SEQ ID NO:5 (CCR1_MACFA_N term), SEQ ID NO:3 (CCR1_mouse_TRD) or SEQ ID NO:6 (CCR1_mouse_N term), preferably wherein at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:7 (CCR2_human_TRD), SEQ ID NO:10 (CCR2_human_N term), SEQ ID NO:8 (CCR2_MACMU_TRD) or SEQ ID NO:11 (CCR2_MACMU_N term), preferably in which at least Y26 has been sulfated, or c) SEQ ID NO:9 (CCR2_mouse_TRD) or SEQ ID NO:12 (CCR2_mouse_N term), preferably in which at least Y37 and/or Y39 has been sulfated, or d) SEQ ID NO:13 (CCR3_human_TRD) or SEQ ID NO:16 (CCR3_human_N term), preferably in which Y16 and/or Y17 have been sulfated, or e) SEQ ID NO:14 (CCR3_MACFA_TRD) or SEQ ID NO:17 (CCR3_MACFA_N term), preferably in which Y16 has been sulfated, or f) SEQ ID NO:15 (CCR3_mouse_TRD) or SEQ ID NO:18 (CCR3_CAMUNDONGO _N term), preferably in which Y20 and/or Y22 have been sulfated, or g) SEQ ID NO:19 (CCR4_humano_TRD), SEQ ID NO:22 (CCR4_humano_N term), SEQ ID NO:20 (CCR4_MACFA_TRD), SEQ ID NO:23 (CCR4_MACFA_N term), SEQ ID NO:21 (CCR4_camundongo_TRD) or SEQ ID NO:24 (CCR4_camundongo_N term), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or h) SEQ ID NO:25 (CCR5_humano_TRD), SEQ ID NO:28 (CCR5_humano_N term), SEQ ID NO:26 (CCR5_MACMU_TRD) or SEQ ID NO:29 (CCR5_MACMU_N term), preferably in which two, three, or all of Y3, Y10, Y14, and Y15 were sulfated, or i) SEQ ID NO:27 (CCR5_mouse_TRD) or SEQ ID NO:30 (CCR5_mouse_N term), preferably in which two or three of Y10, Y12, and Y16 were sulfated, or j) SEQ ID NO:31 (CCR6_human_TRD) or SEQ ID NO:34 (CCR6_human_N term), preferably in which at least two or three of Y18, Y26, and Y27 were sulfated, or k) SEQ ID NO:32 (CCR6_MACFA_TRD) or SEQ ID NO:35 (CCR6_MACFA_N term), preferably in which at least two or three of Y23, Y31, and Y32 were sulfated, or l) SEQ ID NO:33 (CCR6_mouse_TRD) or SEQ ID NO:36 (CCR6_mouse_N term), preferably wherein at least two or three of Y13, Y18, and Y19 were sulfated, or m) SEQ ID NO:37 (CCR7_human_TRD), SEQ ID NO:40 (CCR7_human_N term), SEQ ID NO:38 (CCR7_MACFA_TRD), or SEQ ID NO:41 (CCR7_MACFA_N term), preferably wherein one or both Y8 and Y17 were sulfated, or n) SEQ ID NO:39 (CCR7_mouse_TRD) or SEQ ID NO:42 (CCR7_mouse_N term), preferably wherein one or both Y8 and Y17 and optionally Y20 were sulfated. sulfated were sulfated, or) SEQ ID NO:43 (CCR8_human_TRD), SEQ ID NO:44 (CCR8_MACFA_TRD), SEQ ID NO:46 (CCR8_human_N term with C = X or S), or SEQ ID NO:47 (CCR8_MACFA_N term with C = X or S), preferably wherein at least two or all of Y3, Y15 and Y17 were sulfated, or) SEQ ID NO:45 (CCR8_mouse_TRD) or SEQ ID NO:48 (CCR8_mouse_N term with C = X or S), preferably wherein at least two or all of Y3, Y14 and Y15 were sulfated, or) SEQ ID NO:61 (CCR9_human_TRD), SEQ ID NO:64 (CCR9_human_N term), SEQ ID NO:62 (CCR9_MACFA_TRD) or SEQ ID NO:65 (CCR9_MACFA_N term), preferably in which at least Y28, and preferably also Y17 and/or Y37 has been sulfated, our) SEQ ID NO:63 (CCR9_mouse_TRD) or SEQ ID NO:66 (CCR9_mouse_N term), preferably in which at least Y28 has been sulfated, and preferably also Y19 has been sulfated, ous) SEQ ID NO:67 (CCR10_human_TRD), SEQ ID NO:70 (CCR10_human_N term), SEQ ID NO:68 (CCR10_MACFA_TRD) or SEQ ID NO:71 (CCR10_MACFA_N term), preferably in which at least one or both of Y14 and Y22 has been sulfated, out) SEQ ID NO:69 (CCR10_mouse_TRD) or SEQ ID NO:72 (CCR10_mouse_N term), preferably in which at least one, two, or all of Y14, Y17, and Y22 have been sulfated, oru) SEQ ID NO:73 (CXCR1_human_TRD) or SEQ ID NO:76 (CXCR1_human_N term), preferably in which Y27 have been sulfated, orv) SEQ ID NO:74 (CXCR1_MACFA_TRD) or SEQ ID NO:77 (CXCR1_MACFA_N term), preferably in which at least one of Y14 and Y28 have been sulfated, orw) SEQ ID NO:75 (CXCR1_mouse_TRD) or SEQ ID NO:78 (CXCR1_mouse_N term), preferably in which at least Y6 have been sulfated, orx) SEQ ID NO:79 (CXCR2_human_TRD) or SEQ ID NO:82 (CXCR2_human_N term), preferably where Y23 and/or Y25 have been sulfated, or y) SEQ ID NO:80 (CXCR2_MACFA_TRD) or SEQ ID NO:83 (CXCR2_MACFA_N term), preferably where Y20 and/or Y22 have been sulfated, or z) SEQ ID NO:81 (CXCR2_mouse_TRD) or SEQ ID NO:84 (CXCR2_mouse_N term), preferably where Y24 has been sulfated, or aa) SEQ ID NO:85 (CXCR3_human_TRD), SEQ ID NO:88 (CXCR3_human_N term), SEQ ID NO:86 (CXCR3_MACFA_TRD), SEQ ID NO:89 (CXCR3_MACFA_N term), SEQ ID NO:87 (CXCR3_mouse_TRD) or SEQ ID NO:90 (CXCR3_mouse_N term), preferably in which at least one or both of Y27 and Y29 have been sulfated, or bb) SEQ ID NO:91 (CXCR4_human_TRD), SEQ ID NO:94 (CXCR4_human_N term), SEQ ID NO:92 (CXCR4_MACFA_TRD) or SEQ ID NO:95 (CXCR4_MACFA_N term), preferably in which at least Y12 and/or Y21 have been sulfated, or cc) SEQ ID NO:93 (CXCR4_mouse_TRD) or SEQ ID NO:96 (CXCR4_mouse_N term), preferably in which at least Y23 and/or Y14 have been sulfated, oudd) SEQ ID NO:97 (CXCR5_humano_TRD), SEQ ID NO:100 (CXCR5_humano_N term), SEQ ID NO:98(CXCR5_MACFA_TRD) or SEQ ID NO:101 (CXCR5_MACFA_N term), preferably in which at least one of Y3 and Y27 has been sulfated, ouee) SEQ ID NO:99 (CXCR5_camundongo_TRD) or SEQ ID NO:102 (CXCR5_camundongo_N term), preferably in which at least Y3 and/or Y14 and/or Y20 and/or Y26 has been sulfated, ouff) SEQ ID NO:103 (CXCR6_humano_TRD) or SEQ ID NO:106 (CXCR6_humano_N term), preferably in which at least one or both of Y6 and Y10 has been sulfated, ougg) SEQ ID NO:104 (CXCR6_MACFA_TRD) or SEQ ID NO:107 (CXCR6_MACFA_N term), preferably in which at least two or all of Y4, Y7 and Y39 have been sulfated, ouhh) SEQ ID NO:105 (CXCR6_mouse_TRD) or SEQ ID NO:108 (CXCR6_mouse_N term), preferably in which at least one or both of Y11 and Y15 have been sulfated, ou11) SEQ ID NO:157 (CX3CR1_human_TRD) or SEQ ID NO:160 (CX3CR1_human_N term), preferably in which at least Y14 has been sulfated, oujj) SEQ ID NO:158 (CX3CR1_MACFA_TRD), preferably in which at least Y20 has been sulfated, or kk) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferably in which at least Y20 or Y22 has been sulfated, or 11) SEQ ID NO:159 (CX3CR1_mouse_TRD) or SEQ ID NO:162 (CX3CR1_mouse_N term), preferably in which at least Y15 has been sulfated, or mm) SEQ ID NO:163 (CXCR1_human_TRD) or SEQ ID NO:166 (CXCR1_human_N term), preferably in which at least Y27 has been sulfated, or nn) SEQ ID NO:164 (CXCR1_MACMU_TRD), preferably in which at least Y14 has been sulfated, or ooo) SEQ ID NO:167 (CXCR1_MACMU_N term), preferably in which at least Y14 or Y28 has been sulfated, oupp) SEQ ID NO:165 (CXCR1_camundongo_TRD) or SEQ ID NO:168 (CXCR1_camundongo_N term), preferably in which at least Y6 has been sulfated.

[00335] According to some embodiments of the third embodiments of the 6th aspect, the (first) isolated sulfated polypeptide comprises or consists of a sequence according to a. SEQ ID NO:1 (CCR1_human_TRD), preferably in which at least Y10 and/or Y18 have been sulfated, or b. SEQ ID NO:7 (CCR2_human_TRD), preferably in which at least Y26 has been sulfated, or c. SEQ ID NO:13 (CCR3_human_TRD), preferably in which Y16 and/or Y17 have been sulfated, or d. SEQ ID NO:19 (CCR4_human_TRD), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e. SEQ ID NO:25 (CCR5_humano_TRD), preferably in which two, three, or all of Y3, Y10, Y14, and Y15 were sulfated, ouf. SEQ ID NO:31 (CCR6_humano_TRD), preferably in which at least two or three of Y18, Y26, and Y27 were sulfated, oug. SEQ ID NO:37 (CCR7_humano_TRD), preferably in which one or both Y8 and Y17 were sulfated, ouh. SEQ ID NO:43 (CCR8_humano_TRD), preferably in which at least two or all of Y3, Y15, and Y17 were sulfated, oui. SEQ ID NO:61 (CCR9_humano_TRD), preferably also Y17 and/or Y37 were sulfated, ouj. SEQ ID NO:67 (CCR10_humano_TRD), preferably in which at least one or both of Y14 and Y22 have been sulfated, ouk. SEQ ID NO:73 (CXCR1_humano_TRD), preferably in which Y27 has been sulfated, oul. SEQ ID NO:79 (CXCR2_humano_TRD), preferably in which Y23 and/or Y25 have been sulfated, oum. SEQ ID NO:85 (CXCR3_humano_TRD), preferably in which at least one or both of Y27 and Y29 have been sulfated, oun. SEQ ID NO:91 (CXCR4_humano_TRD), preferably in which at least Y12 and/or Y21 have been sulfated, ouo. SEQ ID NO:97 (CXCR5_humano_TRD), preferably in which at least one of Y3 and Y27 have been sulfated, ouup. SEQ ID NO:103 (CXCR6_humano_TRD), preferably in which at least one or both of Y6 and Y10 have been sulfated, or SEQ ID NO:157 (CX3CR1_humano_TRD), preferably in which at least Y14 has been sulfated, or SEQ ID NO:163 (CXCR1_humano_TRD), preferably in which at least Y27 has been sulfated.

[00336] According to some embodiments B of the third embodiments of the 6th aspect, the (first) isolated sulfated polypeptide comprises or consists of a sequence according to a) SEQ ID NO:3 (CCR1_mouse_TRD), preferably in which at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:9 (CCR2_mouse_TRD), preferably in which at least Y37 and/or Y39 have been sulfated, or c) SEQ ID NO:15 (CCR3_mouse_TRD), preferably in which Y20 and/or Y22 have been sulfated, or d) SEQ ID NO:21 (CCR4_mouse_TRD), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:27 (CCR5_mouse_TRD), preferably in which two or three of Y10, Y12 and Y16 were sulfated, or f) SEQ ID NO:33 (CCR6_mouse_TRD), preferably in which at least two or three of Y13, Y18 and Y19 were sulfated, g) SEQ ID NO:39 (CCR7_mouse_TRD), preferably in which one or both Y8 and Y17 and optionally Y20 were sulfated, or h) SEQ ID NO:45 (CCR8_mouse_TRD), preferably in which at least two or all of Y3, Y14 and Y15 were sulfated, or i) SEQ ID NO:63 (CCR9_mouse_TRD), preferably in which at least Y28 was sulfated, and preferably also Y19 was sulfated, or j) SEQ ID NO:69 (CCR10_mouse_TRD), preferably in which at least one, two, or all of Y14, Y17, and Y22 have been sulfated, or k) SEQ ID NO:75 (CXCR1_mouse_TRD), preferably in which at least Y6 has been sulfated, or l) SEQ ID NO:81 (CXCR2_mouse_TRD), preferably in which Y24 has been sulfated, or m) SEQ ID NO:87 (CXCR3_mouse_TRD), preferably in which at least one or both of Y27 and Y29 have been sulfated, or n) SEQ ID NO:93 (CXCR4_mouse_TRD), preferably in which at least Y23 and/or Y14 have been sulfated, or o) SEQ ID NO:99 (CXCR5_mouse_TRD), preferably in which at least Y3 and/or Y14 and/or Y20 and/or Y26 were sulfated, or) SEQ ID NO:105 (CXCR6_camundongo_TRD), preferably where at least one or both of Y11 and Y15 were sulfated, or) SEQ ID NO:159 (CX3CR1_camundongo_TRD), preferably where at least Y15 was sulfated, or) SEQ ID NO:165 (CXCR1_camundongo_TRD), preferably where at least Y6 was sulfated.

[00337] According to some embodiments C of the third embodiments of the 6th aspect, the (first) isolated sulfated polypeptide comprises or consists of a sequence according to a) SEQ ID NO:2 (CCR1_MACFA_TRD), preferably in which at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:8 (CCR2_MACMU_TRD), preferably in which at least Y26 has been sulfated, or c) SEQ ID NO:14 (CCR3_MACFA_TRD), preferably in which Y16 has been sulfated, or d) SEQ ID NO:20 (CCR4_MACFA_TRD), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:26 (CCR5_MACMU_TRD), preferably in which two, three or all of Y3, Y10, Y14, and Y15 were sulfated, or f) SEQ ID NO:32 (CCR6_MACFA_TRD), preferably in which at least two or three of Y23, Y31, and Y32 were sulfated, or g) SEQ ID NO:38 (CCR7_MACFA_TRD), preferably in which one or both Y8 and Y17 were sulfated, or h) SEQ ID NO:44 (CCR8_MACFA_TRD), preferably in which at least two or all of Y3, Y15, and Y17 were sulfated, or i) SEQ ID NO:62 (CCR9_MACFA_TRD), preferably in which at least Y28, and preferably also Y17 and/or Y37 were sulfated, or j) SEQ ID NO:68 (CCR10_MACFA_TRD), preferably in which at least one or both of Y14 and Y22 were sulfated, ouk) SEQ ID NO:74 (CXCR1_MACFA_TRD), preferably in which at least one of Y14 and Y28 has been sulfated, oul) SEQ ID NO:80 (CXCR2_MACFA_TRD), preferably in which Y20 and/or Y22 has been sulfated, oum) SEQ ID NO:86 (CXCR3_MACFA_TRD), preferably in which at least one or both of Y27 and Y29 has been sulfated, oun) SEQ ID NO:92 (CXCR4_MACFA_TRD), preferably in which at least Y12 and/or Y21 has been sulfated, ouo) SEQ ID NO:98 (CXCR5_MACFA_TRD), preferably in which at least one of Y3 and Y27 has been sulfated, ouup) SEQ ID NO:104 (CXCR6_MACFA_TRD), preferably in which at least two or all of Y4, Y7 and Y39 were sulfated, or q) SEQ ID NO:158 (CX3CR1_MACFA_TRD), preferably in which at least Y20 was sulfated, or r) SEQ ID NO:164 (CXCR1_MACMU_TRD), preferably in which at least Y14 was sulfated.

[00338] According to some fourth embodiments of the 6th aspect, which may or may not be the same as the first, second and/or third embodiments of the 6th aspect, the first isolated sulfated polypeptide comprises the N-terminal of a seven-transmembrane receptor including a tyrosine-rich domain (TRD) and preferably a LID domain, and at least 25%, at least 50% or at least 75% of the tyrosine residues of the TRD are sulfated, preferably in which at least one cysteine between the TRD and the LID domain has been removed or has been replaced by a different amino acid.

[00339] According to some embodiments A of the fourth embodiment of the 6th aspect, the (first) isolated sulfated polypeptide comprises or consists of a sequence according to a) SEQ ID NO:4 (CCR1_human_N term), preferably in which at least Y10 and/or Y18 have been sulfated, b) SEQ ID NO:10 (CCR2_human_N term), preferably in which at least Y26 has been sulfated, c) SEQ ID NO:16 (CCR3_human_N term), preferably in which Y16 and/or Y17 have been sulfated, d) SEQ ID NO:22 (CCR4_human_N term), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, e) SEQ ID NO:28 (CCR5_human_N term), preferably in which two, three or all of Y3, Y10, Y14, and Y15 were sulfated, f) SEQ ID NO:34 (CCR6_human_N term), preferably in which at least two or three of Y18, Y26, and Y27 were sulfated, g) SEQ ID NO:40 (CCR7_human_N term), preferably in which one or both Y8 and Y17 were sulfated, h) SEQ ID NO:46 (CCR8_human_N term with C = X or S), preferably in which at least two or all of Y3, Y15, and Y17 were sulfated, i) SEQ ID NO:64 (CCR9_human_N term), preferably in which at least Y28, and preferably also Y17 and/or Y37 were sulfated, j) SEQ ID NO:70 (CCR10_human_N term), preferably in which at least one or both of Y14 and Y22 were sulfated, k) SEQ ID NO:76 (CXCR1_human_N term), preferably in which Y27 has been sulfated, l) SEQ ID NO:82 (CXCR2_human_N term), preferably in which Y23 and/or Y25 have been sulfated, m) SEQ ID NO:88 (CXCR3_human_N term), preferably in which at least one or both of Y27 and Y29 have been sulfated, n) SEQ ID NO:94 (CXCR4_human_N term), preferably in which at least Y12 and/or Y21 have been sulfated, o) SEQ ID NO:100 (CXCR5_human_N term), preferably in which at least one of Y3 and Y27 has been sulfated, oup) SEQ ID NO:106 (CXCR6_human_N term), preferably in which at least one or both of Y6 and Y10 have been sulfated, q) SEQ ID NO:160 (CX3CR1_humano_N term), preferably in which at least Y14 has been sulfated, our) SEQ ID NO:166 (CXCR1_humano_N term), preferably in which at least Y27 has been sulfated.

[00340] According to some embodiments B of the fourth embodiment of the 6th aspect, the (first) isolated sulfated polypeptide comprises or consists of a sequence according to a) SEQ ID NO:6 (term CCR1_mouse_N), preferably in which at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:12 (term CCR2_mouse_N), preferably in which at least Y37 and/or Y39 have been sulfated, or c) SEQ ID NO:18 (term CCR3_MOUSE_N), preferably in which Y20 and/or Y22 have been sulfated, or d) SEQ ID NO:24 (term CCR4_mouse_N), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:30 (term f) SEQ ID NO:36 (term CCR6_camundongo_N), preferably in which at least two or three of Y13, Y18 and Y19 were sulfated, g) SEQ ID NO:42 (term CCR7_camundongo_N), preferably in which one or both Y8 and Y17 and optionally Y20 were sulfated, h) SEQ ID NO:48 (term CCR8_camundongo_N with C = X or S), preferably in which at least two or all of Y3, Y14 and Y15 were sulfated, i) SEQ ID NO:66 (term CCR9_camundongo_N), preferably in which at least Y28 was sulfated, and preferably also Y19 was sulfated, j) SEQ ID NO:72 (term CCR10_mouse_N), preferably in which at least one, two, or all of Y14, Y17, and Y22 were sulfated, ouk) SEQ ID NO:78 (term CXCR1_mouse_N), preferably in which at least Y6 was sulfated, oul) SEQ ID NO:84 (term CXCR2_mouse_N), preferably in which Y24 was sulfated, oum) SEQ ID NO:90 (term CXCR3_mouse_N), preferably in which at least one or both of Y27 and Y29 were sulfated, oun) SEQ ID NO:96 (term CXCR4_mouse_N), preferably in which at least Y13 and/or Y14 were sulfated, ouo) SEQ ID NO:102 (term CXCR5_mouse_N), preferably in which at least Y3 and/or Y14 and/or Y20 and/or Y26 were sulfated, or) SEQ ID NO:108 (term CXCR6_mouse_N), preferably where at least one or both of Y11 and Y15 were sulfated, or) SEQ ID NO:162 (term CX3CR1_mouse_N), preferably where at least Y15 was sulfated, or) SEQ ID NO:168 (term CXCR1_mouse_N), preferably where at least Y6 was sulfated.

[00341] According to some embodiments C of the fourth embodiment of the 6th aspect, the (first) isolated sulfated polypeptide comprises or consists of a sequence according to a) SEQ ID NO:5 (CCR1_MACFA_N term), preferably in which at least Y10 and/or Y18 have been sulfated, or b) SEQ ID NO:11 (CCR2_MACMU_N term), preferably in which at least Y26 has been sulfated, or c) SEQ ID NO:17 (CCR3_MACFA_N term), preferably in which Y16 has been sulfated, or d) SEQ ID NO:23 (CCR4_MACFA_N term), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) SEQ ID NO:29 (CCR5_MACMU_N term), preferably in which two, three or all of Y3, Y10, Y14 and Y15 were sulfated, or f) SEQ ID NO:35 (CCR6_MACFA_N term), preferably wherein at least two or three of Y23, Y31 and Y32 were sulfated, or g) SEQ ID NO:41 (CCR7_MACFA_N term), preferably wherein one or both Y8 and Y17 were sulfated, or h) SEQ ID NO:47 (CCR8_MACFA_N with term C = X or S), preferably wherein at least two or all of Y3, Y15 and Y17 were sulfated, or i) SEQ ID NO:65 (CCR9_MACFA_N term), preferably wherein at least Y28, and preferably also Y17 and/or Y37 were sulfated, or j) SEQ ID NO:71 (CCR10_MACFA_N term), preferably wherein at least one or both of Y14 and Y22 were sulfated, ouk) SEQ ID NO:77 (CXCR1_MACFA_N term), preferably where at least one of Y14 and Y28 was sulfated, oul) SEQ ID NO:83 (CXCR2_MACFA_N term), preferably where Y20 and/or Y22 were sulfated, oum) SEQ ID NO:89 (CXCR3_MACFA_N term), preferably where at least one or both of Y27 and Y29 were sulfated, oun) SEQ ID NO:95 (CXCR4_MACFA_N term), preferably where at least Y12 and/or Y21 were sulfated, ouo) SEQ ID NO:101 (CXCR5_MACFA_N term), preferably where at least one of Y3 and Y27 was sulfated, ouup) SEQ ID NO:107 (CXCR6_MACFA_N term), preferably in which at least two or all of Y4, Y7 and Y39 were sulfated, ouq) SEQ ID NO:161 (CX3CR1_MACFA_N term), preferably in which at least Y20 or Y22 was sulfated, ouq) SEQ ID NO:167 (CXCR1_MACMU_N term), preferably in which at least Y14 or Y28 was sulfated.

[00342] According to some fifth embodiments of the 6th aspect, which may or may not be the same as the first, second, third and/or fourth embodiments according to the 6th aspect, the isolated antibody or antigen-binding fragment thereof (specifically) binds to a second isolated sulfated polypeptide comprising the tyrosine-rich domain (TRD) of a seven-transmembrane receptor.

[00343] Preferably, the seven-transmembrane receptor of the TRD comprised by the second isolated sulfated polypeptide a) is different from the seven-transmembrane receptor of the TRD comprised by the first isolated sulfated polypeptide, or b) the corresponding seven-transmembrane receptors of the TRD comprised by the first isolated sulfated polypeptide, but of a different species.

[00344] According to some preferred embodiments, the second isolated sulfated polypeptide comprising the tyrosine-rich domain (TRD) of a seven-transmembrane receptor is different from the first isolated sulfated polypeptide comprising the tyrosine-rich domain (TRD) of a seven-transmembrane receptor and is an isolated polypeptide with a sequence according to any of the embodiments A, B, or C according to the third and/or fourth embodiments of the 6th aspect. For example, the first isolated sulfated polypeptide may comprise the TRD of a seven-transmembrane receptor of a first species and the second isolated sulfated polypeptide may comprise the TRD of a seven-transmembrane receptor of a second species, preferably wherein the species are humans and cynomolgus, or humans and mice, or humans and rats.

[00345] According to some embodiments of the fifth embodiment of the sixth aspect, the antibody or fragment binds specifically to a) a first isolated sulfated polypeptide comprising SEQ ID NO:1 (CCR1_human_TRD), preferably in which at least Y10 and/or Y18 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:2 (CCR1_MACFA_TRD), preferably in which at least Y10 and/or Y18 have been sulfated, or b) a first isolated sulfated polypeptide comprising SEQ ID NO:7 (CCR2_human_TRD), preferably in which at least Y26 has been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:8 (CCR2_MACMU_TRD), preferably in which at least Y26 has been sulfated, or c) a first isolated sulfated polypeptide comprising SEQ ID NO:13 (CCR3_human_TRD), preferably in which Y16 and/or Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:14 (CCR3_MACFA_TRD), preferably in which Y16 has been sulfated, or d) a first isolated sulfated polypeptide comprising SEQ ID NO:19 (CCR4_human_TRD), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:20 (CCR4_MACFA_TRD), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or e) a first isolated sulfated polypeptide comprising SEQ ID NO:25 (CCR5_human_TRD), preferably in wherein two, three, or all of Y3, Y10, Y14, and Y15 were sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:26 (CCR5_MACMU_TRD), preferably wherein two, three, or all of Y3, Y10, Y14, and Y15 were sulfated, or f) a first isolated sulfated polypeptide comprising SEQ ID NO:31 (CCR6_humano_TRD), preferably wherein at least two or three of Y18, Y26, and Y27 were sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:32 (CCR6_MACFA_TRD), preferably wherein at least two or three of Y23, Y31, and Y32 were sulfated, or g) a first isolated sulfated polypeptide comprising SEQ ID NO:37 (CCR7_humano_TRD), preferably wherein one or both Y8 and Y17 were sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:38 (CCR7_MACFA_TRD), preferably in which one or both Y8 and Y17 were sulfated, or h) a first isolated sulfated polypeptide comprising SEQ ID NO:43 (CCR8_humano_TRD), preferably in which at least two or all of Y3, Y15 and Y17 were sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:44 (CCR8_MACFA_TRD), preferably in which at least two or all of Y3, Y15 and Y17 were sulfated, or i) a first isolated sulfated polypeptide comprising SEQ ID NO:61 (CCR9_humano_TRD), preferably also Y17 and/or Y37 were sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:62 (CCR9_MACFA_TRD), preferably in which at least Y28, and preferably also Y17 and/or Y37 have been sulfated, or j) a first isolated sulfated polypeptide comprising SEQ ID NO:67 (CCR10_human_TRD), preferably in which at least one or both of Y14 and Y22 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:68 (CCR10_MACFA_TRD), preferably in which at least one or both of Y14 and Y22 have been sulfated, or k) a first isolated sulfated polypeptide comprising SEQ ID NO:73 (CXCR1_human_TRD), preferably in which Y27 has been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:74 (CXCR1_MACFA_TRD), preferably in which at least one of Y14 and Y28 has been sulfated, oul) a first isolated sulfated polypeptide comprising SEQ ID NO:79 (CXCR2_human_TRD), preferably wherein Y23 and/or Y25 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:80 (CXCR2_MACFA_TRD), preferably wherein Y20 and/or Y22 have been sulfated, oum) a first isolated sulfated polypeptide comprising SEQ ID NO:85 (CXCR3_human_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:86 (CXCR3_MACFA_TRD), preferably wherein at least one or both of Y27 and Y29 have been sulfated, oun) a first isolated sulfated polypeptide comprising SEQ ID NO:91 (CXCR4_human_TRD), preferably wherein at least Y12 and/or Y21 were sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:92 (CXCR4_MACFA_TRD), preferably in which at least Y12 and/or Y21 were sulfated, or a first isolated sulfated polypeptide comprising SEQ ID NO:97 (CXCR5_humano_TRD), preferably in which at least one of Y3 and Y27 were sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:98 (CXCR5_MACFA_TRD), preferably in which at least one of Y3 and Y27 were sulfated, or a first isolated sulfated polypeptide comprising SEQ ID NO:103 (CXCR6_humano_TRD), preferably in which at least one or both of Y6 and Y10 were sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:104 (CXCR6_MACFA_TRD), preferably in which at least two or all of Y4, Y7 and Y39 have been sulfated; or) a first isolated sulfated polypeptide comprising SEQ ID NO:157 (CX3CR1_humano_TRD), preferably in which at least Y14 has been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:158 (CX3CR1_MACFA_TRD), preferably in which at least Y20 has been sulfated; or) a first isolated sulfated polypeptide comprising SEQ ID NO:163 (CXCR1_humano_TRD), preferably in which at least Y27 has been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:164 (CXCR1_MACMU_TRD), preferably in which at least Y14 has been sulfated.

[00346] According to some embodiments B of the fifth embodiment of the 6th aspect, the antibody or fragment binds specifically to a) a first isolated sulfated polypeptide comprising SEQ ID NO:4 (CCR1_human_N term), preferably in which at least Y10 and/or Y18 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:5 (CCR1_MACFA_N term), preferably in which at least Y10 and/or Y18 have been sulfated, b) a first isolated sulfated polypeptide comprising SEQ ID NO:10 (CCR2_human_N term), preferably in which at least Y26 has been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:11 (CCR2_MACMU_N term), preferably in which at least Y26 has been sulfated, c) a first isolated sulfated polypeptide comprising SEQ ID NO:16 (CCR3_human_N term), preferably in which Y16 and/or Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:17 (CCR3_MACFA_N term), preferably in which Y16 has been sulfated, d) a first isolated sulfated polypeptide comprising SEQ ID NO:22 (CCR4_human_N term), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:23 (CCR4_MACFA_N term), preferably in which at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, e) a first isolated sulfated polypeptide comprising SEQ ID NO:28 (CCR5_human_N term), preferably in which two, three or all of Y3, Y10, Y14, and Y15 were sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:29 (CCR5_MACMU_N term), preferably in which two, three, or all of Y3, Y10, Y14, and Y15 were sulfated, f) a first isolated sulfated polypeptide comprising SEQ ID NO:34 (CCR6_human_N), preferably in which at least two or three of Y18, Y26, and Y27 were sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:35 (CCR6_MACFA_N term), preferably in which at least two or three of Y23, Y31, and Y32 were sulfated, g) a first isolated sulfated polypeptide comprising SEQ ID NO:40 (CCR7_human_N term), preferably in which one or both of Y8 and Y17 were sulfated and a second poly- a) an isolated sulfated peptide comprising SEQ ID NO:41 (CCR7_MACFA_N term), preferably wherein one or both Y8 and Y17 have been sulfated; b) a first isolated sulfated polypeptide comprising SEQ ID NO:46 (CCR8_human_N term with C = X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:47 (CCR8_MACFA_N term with C = X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated; c) a first isolated sulfated polypeptide comprising SEQ ID NO:64 (CCR9_human_N term), preferably wherein at least Y28, and preferably also Y17 and/or Y37 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:64 (CCR9_human_N term), preferably wherein at least Y28, and preferably also Y17 and/or Y37 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:64 (CCR8_human_N term). ID NO:65 (CCR9_MACFA_N term), preferably in which at least Y28, and preferably also Y17 and/or Y37 has been sulfated,j) a first isolated sulfated polypeptide comprising SEQ ID NO:70 (CCR10_human_N term), preferably in which at least one or both of Y14 and Y22 have been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:71 (CCR10_MACFA_N term), preferably in which at least one or both of Y14 and Y22 have been sulfated,k) a first isolated sulfated polypeptide comprising SEQ ID NO:76 (CXCR1_human_N term), preferably in which Y27 has been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:77 (CXCR1_MACFA_N term), preferably in which at least one of Y14 and Y28 has been sulfated sulfated, l) a first isolated sulfated polypeptide comprising SEQ ID NO:82 (CXCR2_human_N term), preferably wherein Y23 and/or Y25 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:83 (CXCR2_MACFA_N term), preferably wherein Y20 and/or Y22 have been sulfated, m) a first isolated sulfated polypeptide comprising SEQ ID NO:88 (CXCR3_human_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:89 (CXCR3_MACFA_N term), preferably wherein at least one or both of Y27 and Y29 have been sulfated, h) a first isolated sulfated polypeptide comprising SEQ ID NO:94 (CXCR4_human_N term), preferably wherein at least Y12 and/or Y21 have been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:95 (CXCR4_MACFA_N term), preferably wherein at least Y12 and/or Y21 have been sulfated, o) a first isolated sulfated polypeptide comprising SEQ ID NO:100 (CXCR5_human_N term), preferably wherein at least one of Y3 and Y27 has been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:101 (CXCR5_MACFA_N term), preferably wherein at least one of Y3 and Y27 has been sulfated, oup) a first isolated sulfated polypeptide comprising SEQ ID NO:106 (CXCR6_human_N term), preferably wherein at least one or both of Y6 and Y10 have been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:107 (CXCR6_MACFA_N term), preferably in which at least two or all of Y4, Y7 and Y39 have been sulfated, or a first isolated sulfated polypeptide comprising SEQ ID NO:160 (CX3CR1_humano_N term), preferably in which at least Y14 has been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:161 (CX3CR1_MACFA_N term), preferably in which at least Y20 or Y22 has been sulfated, or a first isolated sulfated polypeptide comprising SEQ ID NO:166 (CXCR1_humano_N term), preferably in which at least Y27 has been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:167 (CXCR1_MACMU_N term), preferably in which at least Y14 or Y28 has been sulfated.

[00347] According to some C1 embodiments of the fifth embodiments of the 6th aspect, the antibody or fragment binds specifically to a first isolated sulfated polypeptide comprising a sequence according to some A embodiments of the third embodiments of the 6th aspect and binds to a second isolated sulfated polypeptide comprising a sequence according to some B or C embodiments of the third embodiments of the 6th aspect, wherein preferably the first and second polypeptide comprise the TRD of the same receptor, but of different species.

[00348] According to some C2 embodiments of the fifth embodiments of the 6th aspect, the antibody or fragment binds specifically to a first isolated sulfated polypeptide comprising a sequence according to some A embodiments of the fourth embodiments of the 6th aspect and binds to a second isolated sulfated polypeptide comprising a sequence according to some B or C embodiments of the fourth embodiment of the 6th aspect, wherein preferably the first and second polypeptide comprise the TRD of the same receptor, but of different species.

[00349] According to some sixth embodiments of the 6th aspect, which may or may not be the same as the first, second, third, fourth and/or fifth embodiments according to the 6th aspect, the dissociation constant or EC50 of the antibody or antigen-binding fragment for binding the first isolated sulfated polypeptide and/or for binding said seven-transmembrane receptor is below 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

[00350] According to some embodiments A of the sixth embodiments of the 6th aspect, the dissociation constant or EC50 of the antibody or antigen-binding fragment for binding the first isolated sulfated polypeptide and/or for binding said seven-transmembrane receptor is below 200 nM, 199 nM, 198 nM, 197 nM, 196 nM, 195 nM, 194 nM, 193 nM, 192 nM, 191 nM, 190 nM, 189 nM, 188 nM, 187 nM, 186 nM, 185 nM, 184 nM, 183 nM, 182 nM, 181 nM, 180 nM, 179 nM, 178 nM, 177 nM, 176 nM, 175 nM, 174nM, 173 nM, 172 nM, 171 nM, 170 nM,169 nM, 168 nM, 167 nM, 166nM, 165 nM, 164 nM, 163 nM, 162 nM, 161 nM, 160 nM, 159 nM, 158nM, 157 nM, 156 nM, 155 nM, 154 nM, 153 nM, 152 nM, 151 nM, 150nM, 149 nM, 148 nM, 147 nM, 146 nM, 145 nM, 144 nM, 143 nM, 142nM, 141 nM, 140 nM, 139 nM, 138 nM, 137 nM, 136 nM, 135 nM, 134nM, 133 nM,132 nM, 131 nM, 130 nM, 129 nM, 128 nM, 127 nM, 126nM, 125 nM, 124 nM, 123 nM, 122 nM, 121 nM, 120 nM, 119 nM, 118 nM, 117 nM, 116 nM, 115 nM, 114 nM, 113 nM, 112 nM, 111 nM, 110 nM, 109 nM, 108 nM, 107 nM, 106 nM, 105 nM, 104 nM, 103 nM, 102nM, 101 nM, 100 nM, 99 nM, 98 nM, 97 nM, 96 nM, 95 nM, 94 nM, 93 nM, 92 nM, 91 nM, 90 nM, 89 nM, 88 nM,87 nM, 86 nM, 85 nM, 84 nM, 83 nM, 82 nM, 81 nM, 80 nM, 79 nM, 78 nM, 77 nM, 76 nM, 75 nM, 74 nM, 73 nM, 72 nM, 71 nM, 70 nM, 69 nM, 68 nM, 67 nM, 66 nM, 65nM, 64nM, 63nM, 62nM, 61nM, 60nM, 59nM, 58nM, 57nM, 56nM, 55nM, 54nM, 53nM, 52nM, 51nM, 50nM, 49nM, 48nM, 47nM, 46 nM, 45 nM, 44 nM, 43 nM, 42 nM, 41 nM, 40 nM, 39 nM, 38 nM, 37 nM, 36 nM, 35 nM, 34 nM, 33 nM, 32 nM, 31 nM, 30 nM, 29 nM, 28 nM, 27 nM, 26 nM, 25 nM, 24 nM, 23 nM, 22 nM, 21 nM, 20nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15 nM, or 0.1 nM.

[00351] Preferably, the dissociation constant or EC50 of the antibody or antigen-binding fragment for binding the first isolated sulfated polypeptide and/or for said seven-transmembrane receptor is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, 0.25 nM, 0.2 nM, 0.15 nM or 0.1 nM.

[00352] Preferably, the EC50 can be determined in CHO cells that overexpress the target. As described, for example, in Example 10.1.1, antibodies obtained with a method described herein have excellent affinities for their respective targets. For example, TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bound human CCR8 with an EC50 of 4.8 nM, 1.7 nM, 0.8 nM, 0.6 nM, ~0.9 nM, or 1.7 nM in CHO cells engineered to overexpress CCR8. Furthermore, TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bound cynomolgus CCR8 cells with an EC50 of 1.8 nM, 1 nM, 0.5 nM, 0.7 nM, ~0.55 nM, or 0.9 nM. Additionally, TPP-17578, TPP-19546, TPP-18206, and TPP-21360 bound human regulatory T cells with an EC50 of 25 nM, 15 nM, 23 nM, or 10 nM. Furthermore, the anti-murine CCR8 antibody TPP-14099 binds murine CCR8-expressing CHO cells with an EC50 of 3 nM and murine iTregs with an EC50 of 13.2 nM, as shown in Table 10.1.1.5.

[00353] According to some B embodiments of the sixth embodiments of the 6th aspect, which may or may not be the same as the A embodiments of the sixth embodiments of the 6th aspect, the dissociation constant or EC50 of the antibody or antigen-binding fragment for binding of the second isolated sulfated polypeptide and/or for binding of the second seven-transmembrane receptor is below 200 nM, 199 nM, 198 nM, 197 nM, 196 nM, 195 nM, 194 nM, 193 nM, 192 nM, 191 nM, 190 nM, 189 nM, 188 nM, 187 nM, 186 nM, 185 nM, 184 nM, 183 nM, 182 nM, 181 nM, 180 nM, 180 nM, 179 nM, 178 nM, 177 nM,176 nM, 175 nM, 173 nM, 172 nM, 171 nM, 170 nM, 169 nM, 168 nM, 167 nM, 166 nM, 165 nM, 164 nM, 163 nM, 162 nM, 161 nM, 160 nM,159 nM, 158 nM, 156 nM, 155 nM, 154 nM, 153 nM, 152 nM, 151 nM, 150 nM, 149 nM, 148 nM, 147 nM, 146 nM, 145 nM, 144 nM, 143nM, 142 nM, 141 nM, 140 nM, 139 nM, 138 nM, 137 nM, 136 nM, 135nM, 134 nM, 133 nM, 132 nM, 131 nM, 130 nM, 129 nM, 128 nM, 127nM, 126 nM, 125 nM, 124 nM, 123nM, 122 nM, 121 nM, 120 nM, 119nM, 118 nM, 117 nM, 116 nM, 115 nM, 114 nM, 113 nM, 112 nM, 111nM, 110 nM, 109nM, 108nM, 107 nM, 106 nM, 105 nM, 104 nM, 103nM, 102 nM, 101 nM, 100 nM, 99 nM, 98 nM, 97 nM, 96 nM, 95 nM, 94 nM, 93 nM, 92 nM, 91 nM, 90 nM, 89 nM, 88 nM, 87 nM, 86 nM, 85 nM, 84 nM, 83 nM, 82 nM, 82 nM, 81 nM, 80 nM, 79 nM, 78 nM, 77 nM, 76 nM, 75 nM, 74 nM, 73 nM, 72 nM, 71 nM, 70 nM, 69 nM, 68nM, 67nM, 66nM, 65nM, 64nM, 64nM, 63nM, 62nM, 61nM, 60nM, 59nM, 58nM, 57nM, 56nM, 55nM, 54nM, 53nM, 52nM, 51nM, 50 nM, 49 nM, 48 nM, 47 nM, 46 nM, 45 nM, 44 nM, 43 nM, 42nM, 41 nM, 40 nM, 39 nM, 38 nM, 37 nM, 36 nM, 35 nM, 34 nM, 33nM, 32 nM, 31 nM, 30 nM, 29 nM, 28 nM, 27 nM, 26 nM, 25 nM, 24nM, 23 nM, 22 nM, 21 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15nM, 14 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2nM, 0.15nM or 0.1 nM.

[00354] Preferably, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment for binding the second isolated sulfated polypeptide and/or for binding the second seven-transmembrane receptor is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM.

[00355] According to some AB embodiments of the sixth embodiment of the 6th aspect, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment for binding the first isolated sulfated polypeptide and/or the first seven-transmembrane receptor is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM and the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment for binding the second isolated sulfated polypeptide and/or the second seven-transmembrane receptor is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

[00356] According to some seventh embodiments of the 6th aspect, which may or may not be the same as the first, second, third, fourth, fifth and/or sixth embodiments according to the 6th aspect, the antibody dissociation constant (KD) for binding the first isolated sulfated polypeptide is below the antibody dissociation constant (KD) for binding a first isolated non-sulfated polypeptide with the same sequence as the first isolated sulfated polypeptide. Preferably, the antibody does not substantially bind to a first isolated non-sulfated polypeptide with the same sequence as the first isolated sulfated polypeptide.

[00357] According to some embodiments A of the seventh embodiments of the 6th aspect, the dissociation constant or EC50 of the antibody for binding the first isolated non-sulfated polypeptide is greater than 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, or 2.5 μM, 2.75 μM 3 μM, or is not detectable. Preferably, the dissociation constant or EC50 of the antibody for binding the first isolated non-sulfated polypeptide is greater than 100 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or it is not detectable.

[00358] According to some embodiments B of the seventh embodiments of the 6th aspect, which may be the same as the embodiments A of the seventh embodiments of the 6th aspect, the dissociation constant or EC50 of the antibody or fragment for binding the first isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM, and the dissociation constant or EC50 of the antibody or fragment for binding the first isolated non-sulfated polypeptide is greater than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM or 500 nM, or is not detectable.

[00359] The antibody according to aspect 6 may comprise CDRs derived from human, monkey, Macaca fascicularis (cynomolgus monkey), Macaca mulatta (rhesus monkey), rodent, mouse, rat, horse, bovine, pig, dog, cat and camel. Preferably, the antibody according to aspect 6 comprises CDRs derived from humans, rats or mice.

[00360] According to some 8a embodiments of the 6th aspect, which can be and are suggested to be combined with the first, second, third, fourth, fifth, sixth and/or seventh embodiments of the sixth aspect, the isolated antibody or antigen-binding fragment comprises human-derived CDRs. According to some highly preferred embodiments, the human-derived CDRs do not comprise deviations or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 deviations from the nearest human germline. The closest human germline can be determined as known in the art, for example, in silico using IgBLAST (Ye, Jian, et al. "IgBLAST: an immunoglobulin variable domain sequence analysis tool." Nucleic acids research 41.W1 (2013): W34-W40.) with data retrieved from the IMGT human germline database. Antibodies according to these 8a embodiments can be obtained as described, for example, in Example 6 or 8, or as described elsewhere herein.

[00361] According to some 9th embodiments of the 6th aspect, which can be and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh or 8th embodiments of the 6th aspect, the isolated antibody or antigen-binding fragment is cross-reactive for humans and cynomolgus, as per Example 10.1.1. Preferably, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment for binding to a human chemokine receptor is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM, such as below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. Preferably, the dissociation constant (KD) or EC50 of the antibody or antigen-binding fragment for binding to a cynomolgus chemokine receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5, or 0.25 nM.

[00362] According to some 10a embodiments of the 6th aspect, which can be and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth and/or ninth embodiments of the 6th aspect, the isolated antibody or antigen-binding fragment is characterized by an HCD3 region that deviates in composition from the average HCD3 regions. Preferably, the isolated antibody or antigen-binding fragment is characterized by an HCD3 region comprising between 10 and 34% tyrosine and/or at least one histidine, preferably between 2 and 20% histidine, more preferably between 7 and 20% histidine.

[00363] In some embodiments of the 10 embodiments of aspect 6, the isolated antibody or antigen-binding fragment comprises an HCDR3 with a) > 0 and < 35%, > 8 and < 34%, > 10 and < 34%, or > 15 and < 31% of tyrosine (Y) residues, and/or b) > 0 and < 16%, > 2 and < 20%, or > 7 and < 20% of histidine (H) residues, and/or c) preferably > 0 and < 18%, > 7 and < 10%, or > 0 and < 7% of arginine (R) residues, and/or d) preferably > 0 and < 25%, > 7 and < 16%, or > 7 and < 13% of aspartic acid (D) residues, and/or e) preferably without lysine residues (K), and/or (E) preferably without glutamic acid residues.

[00364] In some embodiments B of the 10 embodiments of aspect 6, which may be the same as or different from embodiments A, the isolated antibody or antigen-binding fragment comprises an HCR3 with a) > 0 and < 47%, > 22 and < 50%, > 10 and < 34%, or > 15 and < 47% of charged amino acids, and/or b) > 0 and < 32%, > 8 and < 30%, or > 10 and < 37% of positively charged amino acids, and/or c) > 0 and < 26%, > 7 and < 16%, or > 7 and < 14% of negatively charged amino acids.

[00365] In some C embodiments of the 10a embodiments of aspect 6, which may be the same as or different from embodiments A and/or B, the isolated antibody or antigen-binding fragment comprises an HCD3 with > 0 and < 42%, > 10 and < 42%, or > 36 and < 43% of total histidine and tyrosine residues.

[00366] Each of the embodiments and descriptions described for CCR8, according to aspect 10, is described here for chemokine receptor antibodies in general, for example, with the necessary modifications. The inventors believe that the sulfated TRD motif instead of the specific CCR8 sequence promotes increased tyrosine and/or histidine frequencies.

[00367] According to some 11th modalities of the 6th aspect, which can be and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and/or 10th modalities of the 6th aspect, the isolated antibody or antigen-binding fragment at least partially modulates CCR8 signaling.

[00368] For example, an antibody according to the present invention can a) block G protein-independent signaling and/or b) block G protein-dependent signaling and/or c) block both G protein-dependent and G protein-independent signaling, and/or d) increase G protein-independent signaling and/or e) increase G protein-dependent signaling and/or f) increase both G protein-dependent and G protein-independent signaling.

[00369] In this particular context, modulation refers to the blocking of ligand-induced G protein-independent signaling and the induction of G protein-independent signaling.

[00370] According to some preferred embodiments, the antibody or fragment does not modulate G protein-independent signaling of the seven-transmembrane receptor. According to some preferred embodiments, the antibody or fragment does not block ligand-induced G protein-independent signaling of the target protein. According to some preferred embodiments, the antibody or fragment does not induce G protein-independent signaling.

[00371] According to some preferred embodiments, the antibody or fragment blocks G protein-dependent signaling. G protein-dependent signaling can be analyzed by chemotaxis assay or preferably by calcium flux assay, as described elsewhere here.

[00372] According to some preferred embodiments, the antibody or fragment blocks chemokine receptor G protein-dependent signaling, but does not block chemokine receptor G protein-independent signaling, as per Example 10.4. Chemokine receptor G protein-independent signaling is any signaling activity that is not G protein-dependent signaling.

[00373] According to some 12th modalities of the 6th aspect, which can be and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh modalities of the 6th aspect, the isolated antibody or antigen-binding fragment is a low-internalization or non-internalization antibody or antigen-binding fragment.

[00374] Since overexpression can affect internalization behavior and is less suitable for modeling internalization in a therapeutic setting, internalization is preferably determined using a model cell line with endogenous expression of the target chemokine receptor, as in Example 10.5. For example, internalization can be determined over a period of time or for specific time points. Preferably, internalization can be determined after 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, 24 h, or 48 h in a cell endogenously expressing the target.

[00375] According to some of the 12a modalities of the 6th aspect, the antibody or antigen-binding fragment has an internalization rate of the same order of magnitude as the internalization rate of the isotype control.

[00376] According to some embodiments B of the 12a embodiments of aspect 6, the antibody or antigen-binding fragment is characterized by internalization into a cell with endogenous target expression that is less than 1.5, 2, 3, 4, 5, 6, 7, or 10 times the internalization of the isotype control. The isotype control for an antibody may be selected as known in the art to match the antibody isotype as closely as possible, but without binding to the target.

[00377] According to some C modalities of the 12a modalities of the 6th aspect, the antibody or antigen-binding fragment is characterized by an internalization in a cell with endogenous target expression that is less than 150%, 175%, 200%, 300%, 400%, or 500% of the internalization of the isotype control, for example, after 15 min, 30 min, 1h, 2h, 3h, 6h, 12h, 24h, or 48h.

[00378] According to some 13th embodiments of the 6th aspect, which can be and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and/or twelfth embodiments of the 6th aspect, the isolated antibody or antigen-binding fragment induces ADCC and/or ADCP of cells expressing the antibody's target receptor.

[00379] In some preferred embodiments of the 13 embodiments of aspect 6, the antibody or antigen-binding fragment is fucosylated.

[00380] In some A1 embodiments of the 13a embodiments of aspect 6, the antibody or antigen-binding fragment binds to the human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM, or 200 nM. Preferably, the KD can be determined using surface plasmon resonance.

[00381] In some B1 embodiments of the 13a embodiments of aspect 6, which may be the same as or different from A1 embodiments, the antibody or antigen-binding fragment induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing the target receptor via human effector cells, such as human NK cells. ADCC induction can be analyzed with an assay known in the art, for example, as described in Examples 10.3.3 ff. or elsewhere herein. Preferably, the assay is performed with Treg cells in which at least 80% or 85% of the Treg cells express CCR8.

[00382] In some C1 modalities of the 13 modalities of the 6th aspect, which may be the same as or different from the A1 or B1 modalities, the maximum ADCC-induced depletion of cells expressing the target receptor is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%.

[00383] In some D1 modalities of the 13th modality of the 6th aspect, which may be the same as or different from modalities A1, B1 or C1, the EC50 for ADCC-induced depletion of target-expressing cells is less than 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 12.5 pM, 10 pM, 5 pM or 2.5 pM.

[00384] In some A2 embodiments of the 13a embodiments of aspect 6, which may or may not be the same as embodiments A1, B1, C1 and/or D1, the antibody or antigen-binding fragment binds to human Fc gamma RIIA (CD32a) with a dissociation constant (KD) less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

[00385] In some B2 embodiments of the 13th embodiment of aspect 6, which may be the same as A1, B1, C1 and/or D1 embodiments, and which may be the same as A2 embodiments, the antibody or antigen-binding fragment induces antibody-dependent cell-mediated phagocytosis (ADCP) in cells expressing the target receptor via human effector cells, such as human macrophages. For example, human macrophages may be M2 or M1 macrophages.

[00386] In some C2 modalities of the 13 modalities of the 6th aspect, which may be the same as modalities A1, B1, C1 and/or D1, and which may be the same as modalities A2 and/or B2, the ADCP - the maximum induced depletion of cells expressing the target receptor is at least 5, 10, 15, 20, 25, 30, 40 or 50%.

[00387] In some D2 modalities of the 13a modalities of aspect 6, which may be the same as modalities A1, B1, C1 and/or D1, and which may be the same as modalities A2, B2 and/or C2, the EC50 for ADCP-induced depletion of activated human regulatory T cells is below 1500 pM, 1000 pM, 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25 pM or 10 pM.

[00388] In some preferred embodiments of the 13 embodiments of aspect 6, an isolated antibody or antigen-binding fragment thereof is provided, specifically binding to a chemokine receptor, wherein the antibody or antigen-binding fragment a) binds to human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM and/or b) binds to human Fc gamma receptor RIIA (CD32a) with a dissociation constant (KD) less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

[00389] In some preferred embodiments of the 13 embodiments of aspect 6, an isolated antibody or antigen-binding fragment thereof is provided, specifically binding to a chemokine receptor, wherein the antibody or antigen-binding fragment a) induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing the human chemokine receptor via human effector cells, such as human NK cells and/or b) induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing the human chemokine receptor via human effector cells, such as human macrophages.

[00390] In some preferred embodiments of the 13 embodiments of aspect 6, an isolated antibody or antigen-binding fragment thereof is provided, specifically binding to a chemokine receptor, wherein a) the maximum ADCC-induced depletion in target cells expressing the human chemokine receptor is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, and/or b) the maximum ADCP-induced depletion in target cells expressing the human chemokine receptor is at least 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, and/or c) the maximum depletion of target cells expressing the chemokine receptor The human percentage is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99%.

[00391] In some preferred embodiments of the 13th embodiment of aspect 6, an isolated antibody or antigen-binding fragment thereof is provided, specifically binding to a chemokine receptor, wherein a) the EC50 for ADCC-induced depletion of target cells expressing the human chemokine receptor is less than 200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM or 5 pM and/or b) the EC50 for ADCP-induced depletion of target cells expressing the human chemokine receptor is less than 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM or 25 pM.

[00392] According to some 14 embodiments of the 6th aspect, which can be and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, 8th, 9th, 10th, 11th, 12th and/or 13th embodiments of the 6th aspect, the isolated antibody or antigen-binding fragment comprises a human, mouse or murine IgG antibody, preferably a human IgG1 antibody or a murine IgG2a antibody, as per Examples 6 and 8.

[00393] According to some 15th embodiments of the 6th aspect, which can be and are suggested to be combined with the first, second, third, fourth, fifth, sixth, seventh, 8th, 9th, 10th, 11th, 12th, 13th and/or 14th embodiments of the 6th aspect, the isolated antibody or antigen-binding fragment is an scFv, Fab, Fab' or F(ab')2 fragment, as per Example 6.

[00394] Antibodies according to the current aspect can be conjugated, for example, as described elsewhere here. Antibodies according to the current aspect can be used in the treatment of a tumor or a disease characterized by the involvement of cells expressing the seven-transmembrane receptor, for example, as described elsewhere here.

[00395] Antibodies according to the current aspect can be used as a diagnostic agent in vivo or in vitro, for example, as described elsewhere herein. In addition, a kit comprising one antibody according to the current aspect with instructions for use is provided.

Preferred combinations according to the 6th aspect.

[00396] The following embodiments describe preferred combinations of the sixth aspect, thus emphasizing the modular nature of the invention. According to a preferred embodiment I, an isolated antibody or antigen-binding fragment thereof is provided, which specifically binds to an isolated first sulfated polypeptide comprising the tyrosine-rich domain (TRD) of a seven-transmembrane receptor and, optionally, its LID domain, wherein at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated. According to a preferred embodiment II, the isolated antibody or antigen-binding fragment according to the preferred embodiment I is provided, wherein the cysteine between the TRD and the LID domain has been removed or replaced with a different amino acid. According to a preferred embodiment III, the isolated antibody or antigen-binding fragment is provided according to preferred embodiment I or II, wherein the seven-transmembrane receptor is a human, cynomolgus, or mouse seven-transmembrane receptor. According to a preferred embodiment IV, the isolated antibody or antigen-binding fragment is provided according to any of preferred embodiments I, II, or III, wherein the seven-transmembrane receptor is a) a CC chemokine receptor, preferably CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, or CCR10, b) a CXC chemokine receptor, preferably CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, or CXCR6, or c) CX3CR1 or CXCR1. Provided according to a preferred embodiment V is an isolated antibody or antigen-binding fragment according to any of the preferred embodiments I, II, III or IV, said first isolated sulfated polypeptide comprising or consisting of a sequence according to a. SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:3 or SEQ ID NO:6, preferably wherein at least Y10 and/or Y18 have been sulfated, or b. SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:8 or SEQ ID NO:11, preferably wherein at least Y26 has been sulfated, or c. SEQ ID NO:9 or SEQ ID NO:12, preferably wherein at least Y37 and/or Y39 have been sulfated, or d. SEQ ID NO:13 or SEQ ID NO:16, preferably wherein Y16 and/or Y17 have been sulfated, or e. SEQ ID NO:14 or SEQ ID NO:17, preferably wherein Y16 has been sulfated, or f. SEQ ID NO:15 or SEQ ID NO:18 preferably wherein Y20 and/or Y22 have been sulfated, or g. SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:21 or SEQ ID NO:24, preferably wherein at least Y22 has been sulfated and preferably, in addition, Y16, Y19 and/or Y20 have been sulfated, or h. SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:26 or SEQ ID NO:29, preferably in which two, three or all of Y3, Y10, Y14 and Y15 were sulfated, oui. SEQ ID NO:27 or SEQ ID NO:30, preferably in which two or three of Y10, Y12 and Y16 were sulfated, ouj. SEQ ID NO:31 or SEQ ID NO:34, preferably in which at least two or three of Y18, Y26 and Y27 were sulfated, ouk. SEQ ID NO:32 or SEQ ID NO:35, preferably in which at least two or three of Y23, Y31 and Y32 were sulfated, oul. SEQ ID NO:33 or SEQ ID NO:36, preferably in which at least two or three of Y13, Y18 and Y19 were sulfated, oum. SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:38 or SEQ ID NO:41, preferably in which one or both Y8 and Y17 have been sulfated, oun. SEQ ID NO:39 or SEQ ID NO:42, preferably in which one or both Y8 and Y17 and optionally Y20 have been sulfated, ouo. SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46 or SEQ ID NO:47, preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated, ouup. SEQ ID NO:45 or SEQ ID NO:48, preferably in which at least two or all of Y3, Y14 and Y15 have been sulfated, ouq. SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:62 or SEQ ID NO:65, preferably in which at least Y28, and preferably also Y17 and/or Y37 have been sulfated, or r. SEQ ID NO:63 or SEQ ID NO:66, preferably in which at least Y28 has been sulfated, and preferably also Y19 has been sulfated, ous. SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:68 or SEQ ID NO:71, preferably in which at least one or both of Y14 and Y22 have been sulfated, ouu. SEQ ID NO:69 or SEQ ID NO:72, preferably in which at least one, two or all of Y14, Y17 and Y22 have been sulfated, ouu. SEQ ID NO:73 or SEQ ID NO:76, preferably in which Y27 has been sulfated, ouv. SEQ ID NO:74 or SEQ ID NO:77, preferably wherein at least one of Y14 and Y28 has been sulfated, ouw. SEQ ID NO:75 or SEQ ID NO:78, preferably wherein at least Y6 has been sulfated, oux. SEQ ID NO:79 or SEQ ID NO:82, preferably wherein Y23 and/or Y25 have been sulfated, ouy. SEQ ID NO:80 or SEQ ID NO:83, preferably wherein Y20 and/or Y22 have been sulfated, ouz. SEQ ID NO:81 or SEQ ID NO:84, preferably wherein Y24 has been sulfated, ouaa. SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:87 or SEQ ID NO:90, preferably in which at least one or both of Y27 and Y29 have been sulfated, oubb. SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:92 or SEQ ID NO:95, preferably in which at least Y12 and/or Y21 have been sulfated, oucc. SEQ ID NO:93 or SEQ ID NO:96, preferably in which at least Y23 and/or Y14 have been sulfated, oudd. SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:98 or SEQ ID NO:101, preferably in which at least one of Y3 and Y27 have been sulfated, ouee. SEQ ID NO:99 or SEQ ID NO:102, preferably where at least Y3 and/or Y14 and/or Y20 and/or Y26 have been sulfated, ouff. SEQ ID NO:103 or SEQ ID NO:106, preferably where at least one or both of Y6 and Y10 have been sulfated, ougg. SEQ ID NO:104 or SEQ ID NO:107, preferably where at least two or all of Y4, Y7 and Y39 have been sulfated, ouhh. SEQ ID NO:105 or SEQ ID NO:108, preferably where at least one or both of Y11 and Y15 have been sulfated, ouii. SEQ ID NO:157 or SEQ ID NO:160, preferably where at least Y14 has been sulfated, oujj. SEQ ID NO:158, preferably where at least Y20 has been sulfated, oukk. SEQ ID NO:161, preferably in which at least Y20 or Y22 was sulfated, ou11. SEQ ID NO:159 or SEQ ID NO:162, preferably in which at least Y15 was sulfated, oumm. SEQ ID NO:163 or SEQ ID NO:166, preferably in which at least Y27 was sulfated, ounn. SEQ ID NO:164, preferably in which at least Y14 was sulfated, ouoo. SEQ ID NO:167, preferably in which at least Y14 or Y28 was sulfated, oupp. SEQ ID NO:165 or SEQ ID NO:168, preferably in which at least Y6 was sulfated.

[00397] According to a preferred embodiment VI, an isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments I, II, III, IV or V, wherein the dissociation constant or EC50 of the antibody for binding to the first isolated sulfated polypeptide and/or to said seven-transmembrane receptor is less than 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. According to a preferred embodiment VII, an isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments I to VI, wherein the isolated antibody or antigen-binding fragment specifically binds to a second isolated sulfated polypeptide comprising the seven-transmembrane receptor DRT, preferably wherein the seven-transmembrane receptor of the DRT comprises the second isolated sulfated polypeptide. It is different from the seven transmembrane receptors of the TRD comprised by the first isolated sulfated polypeptide, or it is the corresponding seven transmembrane receptor of the TRD constituted by the first isolated sulfated polypeptide but of a different species.

[00398] Provided according to a preferred embodiment VIII is the isolated antibody or antigen-binding fragment according to preferred embodiment VII, wherein the dissociation constant or EC50 of the antibody for binding the second isolated sulfated polypeptide and/or for binding the second seven-transmembrane receptor is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. Provided according to a preferred embodiment IX is an isolated antibody or antigen-binding fragment according to any of preferred embodiments I to VIII, wherein the dissociation constant (KD) of the antibody for binding the first isolated sulfated polypeptide is less than the dissociation constant (KD) of the antibody for binding a first isolated non-sulfated polypeptide with the same sequence as the first isolated sulfated polypeptide. According to a preferred embodiment X, the isolated antibody or antigen-binding fragment is provided, according to preferred embodiment IX, wherein the dissociation constant and/or EC50 of the antibody for binding to the first isolated non-sulfated polypeptide is greater than 150 nM, 250 nM, 500 nM, 1 μM, 2 μM or 3 μM, or is not detectable. According to a preferred embodiment XI, the isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments IX or X, wherein the dissociation constant or EC50 of the antibody or fragment for binding to the first isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM, and wherein the dissociation constant of the antibody or fragment for binding to the first isolated non-sulfated polypeptide is greater than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, or 500 nM, or is not detectable. According to a preferred embodiment XII, the isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments I to XI, wherein the antibody comprises CDRs derived from humans, rats, or mice. According to a preferred embodiment XIII, the isolated antibody or antigen-binding fragment is provided according to any of preferred embodiments I to XII, wherein the antibody a. comprises human-derived CDRs and/or b. is cross-reactive for humans and cynomolgus, and/or c. is characterized by an HCDR3 region comprising between 10 and 34% tyrosine and/or between 2 and 20% histidine, and/or d. does not modulate G protein-independent signaling of seven transmembrane receptors and/or e. is a non-internalizing antibody or is characterized by internalization into a cell with endogenous target expression that is less than 1.5, 2, 3, 4, 5, 6, 7 or 10 times the internalization of the isotype control, and/or f. induces ADCC and/or ADCPγ. is a human, mouse, or murine IgG antibody, preferably a human IgG1 antibody or a murine IgG2a antibody, and/or h. is a scFv, Fab, Fab', or F(ab')2 fragment.

[00399] A conjugate comprising an antibody or antigen-binding fragment according to any of the preferred embodiments I to XIII is provided, according to a preferred embodiment XIV, preferably wherein the conjugate comprises: a. a radioactive element, b. a cytotoxic agent, such as an auristatin, a maytansinoid, a kinesin spindle protein inhibitor, a nicotinamide phosphoribosyltransferase inhibitor or a pyrrolobenzodiazepine derivative, c. an additional antibody or antigen-binding fragment, or d. a chimeric antigen receptor.

[00400] An antibody or antigen-binding fragment according to any of the preferred embodiments I to XIII or a conjugate according to the preferred embodiment XV is provided for use in the treatment of a tumor or disease characterized by the involvement of cells expressing seven transmembrane receptors, optionally in combination with an antibody directed to a checkpoint inhibitor. An antibody or antigen-binding fragment according to any of the preferred embodiments I to XIII or a conjugate according to the preferred embodiment XIV is provided for use as an in vivo or in vitro diagnostic agent. A kit comprising an antibody or antigen-binding fragment according to any of the preferred embodiments I to XIII or a conjugate according to the preferred embodiment XIV with instructions for use is provided according to the preferred embodiment XVII.

CCR8 Antibody Binding

[00401] The following aspects 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 refer to antibodies that bind specifically to CCR8. Although each aspect is described individually, the different structural or functional aspects can be combined and it is suggested that they be combined with each other, except when obviously incompatible. Furthermore, each modality in each aspect can be combined with each modality in the same aspect or in a different aspect, except when obviously incompatible.

[00402] Unless otherwise indicated, CCR8 can be from any species, for example, human, monkey, macaque (cynomolgus macaque), macaque (rhesus macaque), rodent, mouse, rat, horse, bovine, pig, dog, cat, and camel CCR8. Antibodies that bind to CCR8 from at least two species, wherein one species is human, are highly preferred and are suggested to be combined with each embodiment described for these aspects. Antibodies with CDRs derived from humans are highly preferred and are suggested to be combined with each embodiment described for these aspects. According to the current aspects, antibodies with an HCDR3 domain characterized by a frequency of at least 21% tyrosine residues and/or at least 2%, 7%, or 10% histidine are highly preferred and are suggested to be combined with each embodiment described for the current aspect. According to the current aspects, antibodies that induce both ADCC and ADCP, such as non-fucosylated antibodies, are highly preferred and it is suggested that they be combined with each modality described for these aspects. According to the current aspects, low-internalization or non-internalization antibodies or fragments are highly preferred and it is suggested that they be combined with each modality described for these aspects. Some highly preferred characterization features that are applicable to each of the described aspects are described in the section "Preferred combinations according to 'all aspects'".

ASPECT 7 - CCR8 ANTIBODY BINDING SULFATE TRD

[00403] The extracellular domains of human CCR8 can be structured into four regions:(1) an N-terminal domain that can be subdivided into a) the membrane-distal tyrosine-rich domain (TRD), formed by amino acids 1 to 24 (SEQ ID NO:43)b) a cysteine at amino acid position 25, and c) a LID domain, formed by amino acids 26 to 35 (SEQ ID NO:49)(iii) an extracellular domain 1 (ECL1) according to SEQ ID NO:52,(iii) an extracellular domain 2 (ECL2) according to SEQ ID NO:55, and(iv) an extracellular domain 3 (ECL3) according to SEQ ID NO:58.

[00404] According to a 7th aspect, which may or may not be the same as the 6th aspect, an isolated antibody or antigen-binding fragment is provided that specifically binds to the sulfated tyrosine-rich domain of CCR8.

[00405] For example, the antibody or antigen-binding fragment binds to a first isolated sulfated polypeptide comprising: a) the tyrosine-rich domain (TRD) of CCR8 or b) the N-term of CCR8 including the TRD, optionally wherein at least the cysteine between the TRD and the LID domain has been removed or replaced with a different amino acid. A sulfated polypeptide according to the present aspect is a polypeptide in which preferably at least 25%, at least 50%, or at least 75% of the tyrosine residues of the TRD are sulfated. When the polypeptide comprises the N-term of CCR8 including the TRD, preferably the cysteine between the TRD and the LID domain has been replaced with a serine or removed.

[00406] Without being limited by theory, specific recognition of the sulfation pattern provided for CCR8 appears to influence whether the antibody competes with CCL1, the natural ligand of CCR8, and also whether and how the antibody agonizes or antagonizes CCR8 signaling, for example, as described in relation to aspect 11.

[00407] In some preferred embodiments according to the present aspect, the inventive antibody binds sulfated TRDs of human and/or cynomolgus CCR8 with a KD value of < 5E-8 M, < 4E-8 M, < 3E-8 M, < 2E-8 M, < 1E-8 M, < 9E-9 M, < 8E-9 M, < 7E-9 M, < 6E-9 M, < 5E-9 M, < 4E-9 M, < 3E-9 M, < 2.5E-9 M, < 2E-9 M, < 1.5E-9 M, < 1E-9 M, < 9E-10 M, < 8E-10 M, < 7E-10 M, < 6E-10 M, < 5E-10 M, < 4E-10 M, < 3E-10 M, < 2.5E-10 M, < 2E-10 M, < 1.5E-10 M, < 1E-10 M, or < 9E-11 M. For example, the inventive antibody can bind sulfated TRDs from human and/or cynomolgus CCR8 with a KD value between <8E-9 M and > 4E-10 M. For example, the inventive antibody can bind sulfated TRDs from human and/or cynomolgus CCR8 with a KD value between < 8E-9 M and > 5.5E-10 M. In some other preferred embodiments, the inventive antibody binds to sulfated N-terminus (i.e., comprising the sulfated TRD mentioned above) from human and/or cynomolgus CCR8 with substantially the same KD value. In some of these preferred embodiments, the inventive antibody does not bind substantially to non-sulfated TRDs of human and/or canine CCR8.

[00408] In some preferred embodiments according to the present aspect, the inventive antibody binds to N-termined sulfated human and/or cynomolgus CCR8 with a KD value of < 5E-8 M, < 4E-8 M, < 3E-8 M, < 2E-8 M, < 1E-8 M, < 9E-9 M, < 8E-9 M, < 7E-9 M, < 6E-9 M, < 5E-9 M, < 4E-9 M, < 3E-9 M, < 2.5E-9 M, < 2E-9 M, < 1.5E-9 M, < 1E-9 M, < 9E-10 M, < 8E-10 M, < 7E-10 M, < 6E-10 M, < 5E-10 M, < 4E-10 M, < 3E-10 M, < 2.5E-10 M, < 2E-10 M, < 1.5E-10 M, < 1E-10 M, < 9E-11 M, < 8E-11 M, < 7E-11 M, < 6E-11 M, or < 5E-11 M. For example, the inventive antibody can bind to the sulfated N-term of human and/or cynomolgus CCR8 with a KD value between < 8E-9 M and > 8E-11 M. For example, the inventive antibody can bind to the sulfated N-term of human and/or cynomolgus CCR8 with a KD value between < 8E-9 M and > 7.5E-11 M. In some other preferred embodiments, the inventive antibody binds to sulfated TRD (i.e., a subsequence of the aforementioned sulfated N-term) of human and/or cynomolgus CCR8. with substantially the same KD value or a KD value within the same order of magnitude. In some of these more preferred embodiments, the inventive antibody does not bind substantially to non-sulfated human CCR8 and/or cynomolgus TRDs.

[00409] According to some early embodiments of the 7th aspect, the first isolated sulfated polypeptide comprises: a) SEQ ID NO:43 (CCR8_human_TRD), preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated, b) SEQ ID NO:44 (CCR8_MACFA_TRD), preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated and/or c) SEQ ID NO:45 (CCR8_mouse_TRD), preferably in which at least two or all of Y3, Y14 and Y15 have been sulfated.

[00410] According to some second embodiments of the 7th aspect, the first isolated sulfated polypeptide comprises: a) SEQ ID NO:46 (CCR8_human_N term with C = X or S), preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated, b) SEQ ID NO:47 (CCR8_MACFA_N term with C = X or S), preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated and/or c) SEQ ID NO:48 (CCR8_mouse_N term with C = X or S), preferably in which at least two or all of Y3, Y14 and Y15 have been sulfated.

[00411] According to some third embodiments of the 7th aspect, which may be the same as or different from the first and/or second embodiments, the antibody or antigen-binding fragment binds specifically to the first isolated sulfated polypeptide with a dissociation constant or EC50 of < 15 nM, < 10 nM, < 5 nM, < 1 nM or < 0.6 nM.

[00412] According to some of these preferred embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds with a dissociation constant or EC50 of < 15 nM, < 10 nM, < 5 nM, < 1 nM or < 0.6 nM) to human CCR8 or to a polypeptide isolated according to SEQ ID NO:46, in which at least two or all of Y3, Y15 and Y17 have been sulfated, b) to cynomolgus CCR8 or to a polypeptide isolated according to SEQ ID NO:47, in which at least two or all of Y3, Y15 and Y17 have been sulfated and/or c) to murine CCR8 or to a polypeptide isolated according to SEQ ID NO:48, in which at least two or all of Y3, Y14 and Y15 have been sulfated.

[00413] According to some fourth embodiments of the 7th aspect, which can be and are suggested to be combined with the first, second and/or third embodiments of the 7th aspect, the antibody dissociation constant (KD) for binding the first isolated sulfated polypeptide is smaller than the antibody dissociation constant (KD) for binding an isolated non-sulfated polypeptide with the same sequence as the first isolated sulfated polypeptide.

[00414] According to some embodiments A of these fourth embodiments, the dissociation constant or EC50 of the antibody for binding the isolated non-sulfated polypeptide is greater than 1 pM, 10 nM, 25 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.75 μM, or 3 μM, 5 μM or is not detectable. Preferably, the dissociation constant or EC50 of the antibody for binding the isolated non-sulfated polypeptide is greater than 100 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or it is not detectable.

[00415] According to some embodiments B of these fourth embodiments, which may be the same as embodiments A of these second embodiments, the dissociation constant or EC50 of the antibody for binding the first isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM, and the dissociation constant or EC50 of the antibody for binding the isolated non-sulfated polypeptide is greater than 10 nM, 25 nM, 50 nM, 100 nM, 250 nM or 500 nM, or is not detectable.

ASPECT 8 - CCR8 ANTIBODY COMPRISING HUMAN CDRs

[00416] According to an 8th aspect, which may or may not be the same as the 6th or 7th aspect, an isolated antibody or antigen-binding fragment thereof is provided, (specifically) binding to CCR8, wherein the antibody comprises human-derived CDRs. More preferably, the antibody or antigen-binding fragment thereof (specifically) binds to the sulfated TRD of CCR8. According to some preferred embodiments, a single CDR comprises fewer than 1, 2, 3, 4, 5, or 6 deviations from the nearest human germline. According to some highly preferred embodiments, the human-derived CDRs comprise no deviations or fewer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 deviations from the nearest human germline. The closest human germline can be determined as known in the art, for example, in silico using IgBLAST with data retrieved from the IMGT human germline database, as described elsewhere here.

[00417] Although humanization of antibodies with non-human CDRs can improve immunogenicity, residual immunogenicity resides in the CDR regions (Harding, Fiona A., et al. "The immunogenicity of humanized and fully human antibodies: residual immunogenicity re-sides in the CDR regions." MAbs. Vol. 2. No. 3. Taylor & Francis, 2010.). Antibodies according to the current aspect comprising human-derived CDRs and a low number of germline deviations are therefore assumed to have superior suitability as therapeutic agents.

[00418] Antibodies comprising human-derived CDRs can be obtained as described herein, for example, using a human phage display library as described in Examples 4 and 6. Alternatively, antibodies with human CDRs can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. In addition, antibodies comprising human CDRs can also be generated using activated B cells in vitro (see U.S. Patents Nos. 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety).

[00419] The supplied isolated antibody or antigen-binding fragment comprising human-derived CDRs is preferably also an antibody according to any one of aspects 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 or a combination thereof.

ASPECT 9 - CCR8 CROSS-REACTIVATED ANTIBODY

[00420] According to a 9th aspect, which may or may not be the same as the 6th, 7th and/or 8th aspects, an isolated antibody or antigen-binding fragment is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment is cross-reactive for CCR8 of at least two species, preferably selected from humans, monkeys, Macaca fascicularis (cynomolgus monkey), Macaca mulatta (rhesus monkey), rodents, mice, rats, horses, cattle, pigs, dogs, cats and camels, and even more preferably selected from humans, cynomolgus monkeys and mice. According to some more preferred embodiments, the antibody or antigen-binding fragment is cross-reactive for human and cynomolgus CCR8.

[00421] In preferred embodiments, the antibody or antigen-binding fragment binds to CCR8 of a first species with a first dissociation constant KD and binds to CCR8 of a second species with a second dissociation constant KD, wherein the first and second dissociation constants are of the same order of magnitude. As understood by a person skilled in the art, a difference of order of magnitude between two values is a factor of 10.

[00422] Cross-reacting anti-CCR8 antibodies are advantageous for the development of a therapeutic antibody because they can be used in non-human animal models to characterize therapeutic agents with respect to pharmacological and safety data before the antibodies are administered to humans. However, cross-reacting antibodies with similar binding behavior in two species are difficult to generate because the CCR8 parts that can be used for antibody recognition have low homology between species (see Example 2). According to the present invention, cross-reacting antibodies to CCR8 can be generated using small sulfated tyrosine comprising motifs that have greater conservation between species, so that cross-reacting antibodies that bind CCR8 from two or more species with affinities on the same order of magnitude can be obtained easily and conveniently. In more detail, the antibodies bind specifically to the sulfated TRD motifs and cross-reacting antibodies can be obtained because these sulfated TRD motifs are conserved between species.

[00423] In some preferred embodiments according to the present aspect, the inventive antibody binds to the sulfated TRD of human and cynomolgus CCR8 with a KD value of < 5E-8 M, < 4E-8 M, < 3E-8 M, < 2E-8 M, < 1E-8 M, < 9E-9 M, < 8E-9 M, < 7E-9 M, < 6E-9 M, < 5E-9 M, < 4E-9 M, < 3E-9 M, < 2.5E-9 M, < 2E-9 M, < 1.5E-9 M, < 1E-9 M, < 9E-10 M, < 8E-10 M, < 7E-10 M, < 6E-10 M, < 5E-10 M, < 4E-10 M, < 3E-10 M, < 2.5E-10 M, < 2E-10 M, < 1.5E-10 M, < 1E-10 M, or < 9E-11 M. For example, the inventive antibody can bind sulfated TRDs of human and cynomolgus CCR8 with a KD value between < 8E-9 M and > 4E-10 M. For example, the inventive antibody can bind sulfated TRDs of human and cynomolgus CCR8 with a KD value between < 8E-9 M and > 5.5E-10 M. In some other preferred embodiments, the inventive antibody binds to sulfated N-terminus (i.e., comprising the sulfated TRD mentioned above) of human and cynomolgus CCR8 with substantially the same KD value or a KD within an order of magnitude. In some of these preferred embodiments, the inventive antibody does not bind substantially to the non-sulfated TRD of human CCR8 and cynomolgus.

[00424] In some preferred embodiments according to the present aspect, the inventive antibody binds to N-termined sulfated human CCR8 and cynomolgus with a KD value of < 5E-8 M, < 4E-8 M, < 3E-8 M, < 2E-8 M, < 1E-8 M, < 9E-9 M, < 8E-9 M, < 7E-9 M, < 6E-9 M, < 5E-9 M, < 4E-9 M, < 3E-9 M, < 2.5E-9 M, < 2E-9 M, < 1.5E-9 M, < 1E-9 M, < 9E-10 M, < 8E-10 M, < 7E-10 M, < 6E-10 M, < 5E-10 M, < 4E-10 M, < 3E-10 M, < 2.5E-10 M, < 2E-10 M, < 1.5E-10 M, < 1E-10 M, < 9E-11 M, < 8E-11 M, < 7E-11 M, < 6E-11 M, or < 5E-11 M. For example, the inventive antibody can bind to the sulfated N-term of human and cynomolgus CCR8 with a KD value between < 8E-9 M and > 8E-11 M. For example, the inventive antibody can bind to the sulfated N-term of human and cynomolgus CCR8 with a KD value between < 8E-9 M and > 7.5E-11 M. In some other preferred embodiments, the inventive antibody binds to sulfated TRD (i.e., a subsequence of the aforementioned sulfated N-term) of human and cynomolgus CCR8 with substantially the same KD value or a value of the same order of magnitude. In some of these more preferred embodiments, the inventive antibody does not bind substantially to the non-sulfated TRD of human CCR8 and cynomolgus.

[00425] According to some early embodiments of the 9th aspect, the antibody binds specifically to a) a first isolated sulfated polypeptide comprising SEQ ID NO:46 (CCR8_human_N term with C = X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:47 (CCR8_MACFA_N term with C = X or S), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, or b) a first isolated sulfated polypeptide comprising SEQ ID NO:43 (CCR8_human_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:44 (CCR8_MACFA_TRD), preferably wherein at least two or all of Y3, Y15 and Y17 have been sulfated. At least two or all of Y3, Y15, and Y17 were sulfated.

[00426] Preferably, the EC50 of the antibody or antigen-binding fragment for binding to human CCR8 or for binding to the first isolated sulfated polypeptide is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM, such as below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. Preferably, the EC50 of the antibody or antigen-binding fragment for binding to cynomolgus CCR8 or for binding to the second isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. In some preferred embodiments, antibodies bind to the first isolated sulfated polypeptide and the second isolated sulfated polypeptide with substantially the same affinity.

[00427] According to some second embodiments of the 9th aspect, the antibody binds specifically to a) a first isolated sulfated polypeptide comprising SEQ ID NO:46 (CCR8_human_N term with C = X or S), preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated and a second isolated sulfated polypeptide comprising SEQ ID NO:48 (CCR8_mouse_N term with C = X or S), preferably in which at least two or all of Y3, Y14 and Y15 have been sulfated, or b) a first isolated sulfated polypeptide comprising SEQ ID NO:43 (CCR8_human_TRD), preferably in which at least two or all of Y3, Y15 and Y17 have been sulfated, and a second isolated sulfated polypeptide comprising SEQ ID NO:45 (CCR8_mouse_TRD), preferably in which At least two or all of Y3, Y14, and Y15 were sulfated.

[00428] Preferably, the EC50 of the antibody or antigen-binding fragment for binding to human CCR8 or for binding to the first isolated sulfated polypeptide is less than 200 nM, 150 nM, 100 nM, 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM, such as below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. Preferably, the EC50 of the antibody or antigen-binding fragment for binding to murine CCR8 or for binding to the second isolated sulfated polypeptide is less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 or 0.25 nM. In some preferred embodiments, antibodies bind to the first isolated sulfated polypeptide and the second isolated sulfated polypeptide with substantially the same affinity.

[00429] The supplied isolated antibody or antigen-binding fragment is preferably also an antibody according to any one of aspects 7, 8, 10, 11, 12, 13, 14, 15, 16, 17 or 18 or a combination thereof. ASPECT 10 - CCR8 ANTIBODY DEFINED BY HCDR3 STRUCTURE

[00430] According to a 10th aspect, which may or may not be the same as the 6th, 7th, 8th and/or 9th aspect, an isolated antibody or antigen-binding fragment is provided, specifically binding to CCR8, wherein the antibody or antigen-binding fragment is characterized by an HCDR3 region with an increased frequency of histidine and/or tyrosine. As understood by a person skilled in the art, although the frequency for each amino acid is described separately, the respective frequencies are interrelated and it is suggested that they be combined, except when the person skilled immediately recognizes their incompatibility. For the determination of CDR regions, the Kabat immunoglobulin amino acid residue numbering system was used here and should be decisive in case of doubt.

[00431] It was surprisingly found that the CCR8 antibodies according to the present invention were characterized by an HCDR3 with substantially higher frequencies of tyrosine residues than would be expected for a random, fully human HCDR3 of corresponding length (~10%, as per Zemlin, Michael, et al. "Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures." Journal of molecular biology 334.4 (2003): 733-749.). On average, the initial set of inventive antibodies comprised ~20.6% tyrosine residues, while the optimized set of human CCR8-binding specific antibodies comprised on average 21% tyrosine residues in the HCDR3.

[00432] Furthermore, the number of positively charged amino acids, and in particular the number of histidine residues, was higher than expected, particularly in the optimized antibody set with improved therapeutic characteristics, as shown in Example 9. On average, when summed, histidine and tyrosine residues accounted for 31% of all amino acids in the HCDR3 of the optimized CCR8-binding antibody set.

[00433] Interestingly, the antigen used, comprising the sulfated TRD, is characterized by tyrosine residues and a particularly high number of negative charges, which was further increased by sulfation. Without being limited by theory, the inventors believe that the preference for a high tyrosine and histidine content in the CDRH3 of the inventive antibodies is caused by the particular sulfated motif of the antigen and that a high tyrosine/histidine content in the CDRH3 promotes specific antigen recognition. Without being limited by theory, the inventors further believe that this specific recognition influences the characteristics of the antibodies obtained as a CCR8 modulator, for example, as described in aspect 11.

[00434] In some highly preferred embodiments of the 10th aspect, an isolated antibody or antigen-binding fragment is provided that binds specifically to CCR8, wherein the antibody or fragment is characterized by an HCDR3 region comprising between 10 and 34% tyrosine and/or between 2 and 20% histidine.

[00435] The supplied isolated antibody or antigen-binding fragment is preferably also an antibody according to any one of aspects 7, 8, 9, 11, 12, 13, 14, 15, 16, 17 or 18 or a combination thereof.

[00436] In some early embodiments of the 10th aspect, the isolated antibody or antigen-binding fragment comprises an HCR3 having: a) > 0 and < 35%, > 8 and < 34%, > 10 and < 34%, or > 15 and < 31% of tyrosine (Y) residues, and/or b) > 0 and < 16%, > 2 and < 20%, or > 7 and < 20% of histidine (H) residues, and/or c) preferably > 0 and < 18%, > 7 and < 10%, or > 0 and < 7% of arginine (R) residues, and/or d) preferably > 0 and < 25%, > 7 and < 16%, or > 7 and < 13% of aspartic acid (D) residues, and/or e) preferably without lysine (K) residues, and/or) preferably without glutamic acid residues (E).

[00437] In some second embodiments of the 10th aspect, which may be the same as or different from the first embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with a) > 0 and < 47%, > 22 and < 50%, > 10 and < 34%, or > 15 and < 47% of charged amino acids, and/or b) > 0 and < 32%, > 8 and < 30%, or > 10 and < 37% of positively charged amino acids, and/or c) > 0 and < 26%, > 7 and < 16%, or > 7 and < 14% of negatively charged amino acids.

[00438] In some third embodiments of the 10th aspect, which may be the same as or different from the first and/or second embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with > 0 and < 42%, > 10 and < 42%, or > 36 and < 43% of total histidine and tyrosine residues.Tyrosine (Y)

[00439] In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with 33%, 31%, 25%, 23%, 21%, 20%, 18%, 15%, 10%, 9%, 8%, or 0% tyrosine. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with (a) > 0 and < 35%, (b) > 8 and < 34%, (c) > 10 and < 34%, or (d) > 15 and < 31% tyrosine.

[00440] In some embodiments, the isolated antibody or antigen-binding fragment comprises an HCDR3 with > 8, > 9, > 10, > 11, > 12, > 13, > 14, > 15, > 16, > 17, > 18, > 19, > 20, > 21, > 22, > 23, > 24, > 25, > 26, > 27, > 28, > 29, > 30, > 31, > 32, > 33% tyrosine residues. In some embodiments, the isolated antibody or antigen-binding fragment comprises an HCDR3 with < 35, < 34, < 33, < 32, < 31, < 30, < 29, < 28, < 27, < 26, < 25, < 24, < 23, < 22, < 21, < 20, < 19, < 18, < 17, < 16, < 15, < 14, < 13, < 12, < 11, < 10, < 9 or < 8% tyrosine residues.

[00441] In some embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with > 0 and < 34%, > 1 and < 34%, > 2 and < 34%, > 3 and < 34%, > 4 and < 34%, > 5 and < 34%, > 6 and < 34%, > 7 and < 34%, > 8 and < 34%, > 9 and < 34%, > 10 and < 34%, > 11 and < 34%, > 12 and < 34%, > 13 and < 34%, > 14 and < 34%, > 15 and < 34%, > 16 and < 34%, > 17 and < 34%, > 18 and < 34%, > 19 and < 34%, > 20 and < 34%, > 21 and < 34%, > 22 and < 34%, > 23 and < 34%, > 24 and < 34%, > 25 and < 34%, > 26 and < 34%, > 27 and < 34%, > 28 and < 34%, > 29 and < 34% or > 30 and < 34% tyrosine residues.

[00442] In some embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with > 0 and < 36%, > 0 and < 35%, > 0 and < 34%, > 0 and < 33%, > 0 and < 32%, > 0 and < 31%, > 0 and < 30%, > 0 and < 29%, > 0 and < 28%, > 0 and < 27%, > 0 and < 26%, > 0 and < 25%, > 0 and < 24%, > 0 and < 23%, > 0 and < 22%, > 0 and < 21%, > 0 and < 20%, > 0 and < 19%, > 0 and < 18%, > 0 and < 17%, > 0 and < 16%, or > 0 and < 15%, > 0 and < 14%, > 0 and < 13%, > 0 and < 12%, > 0 and < 11%, > 0 and < 10%, > 0 and < 9%, > 0 and < 8%, > 0 and < 7%, > 0 and < 6%, > 0 and < 5% tyrosine residues.

A loaded

[00443] In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with 47%, 39%, 31%, 28%, 27%, 25%, 23%, 20%, 18%, 17%, 16%, 15%, 9%, or 0% of charged amino acids. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with (a) > 0 and < 47%, (b) > 22 and < 50%, (c) > 10 and < 34%, or (d) > 15 and < 47% of charged amino acids.

Positively charged

[00444] In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with 31%, 23%, 18%, 15%, 10%, 8%, 7%, or 0% positively charged amino acids. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with (a) > 0 and < 32%, (b) > 8 and < 30%, or (c) > 10 and < 37% positively charged amino acids.

A negatively charged

[00445] In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with 25%, 17%, 15%, 11%, 10%, 9%, 8%, 7%, or 0% negatively charged amino acids. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with (a) > 0 and < 26%, (b) > 7 and < 16%, or (c) > 7 and < 14% negatively charged amino acids.

Histidine (H)

[00446] In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with 0%, 3%, 7%, 8%, 10%, or 15% histidine residues. Preferably, the isolated antibody or antigen-binding fragment comprises an HCR3 with at least one histidine residue. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with (a) > 0 and < 16%, (b) > 2 and < 20%, or (c) > 7 and < 20% histidine residues.

[00447] In some embodiments, the isolated antibody or antigen-binding fragment comprises an HCDR3 with > 0, > 0, > 1, > 2, > 3, > 4, > 5, > 6, > 7, > 8, > 9, > 10, > 11, > 12, > 13, > 14, > 15, > 16, > 17, > 18, > 19, > 20% histidine residues. In some embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with < 20, < 19, < 18, < 17, < 16, < 15, < 14, < 13, < 12, < 11, < 10, < 9, < 8, < 7, < 6, < 5, < 4, < 3, < 2, or < 1% of histidine residues.

[00448] In some embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with > 0 and < 24%, > 1 and < 24%, > 2 and < 24%, > 3 and < 24%, > 4 and < 24%, > 5 and < 24%, > 6 and < 24%, > 7 and < 24%, > 8 and < 24%, > 9 and < 24%, > 10 and < 24%, > 11 and < 24%, > 12 and < 24%, > 13 and < 24%, > 14 and < 24%, > 15 and < 24%, > 16 and < 24%, > 17 and < 24%, > 18 and < 24%, > 19 and < 24%, > 20% and < 24%, > 21% and < 24%, > 22% and < 24%, or > 23% and < 24% of histidine residues.

[00449] In some embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with > 0 and < 24%, > 0 and < 23%, > 0 and < 22%, > 0 and < 21%, > 0 and < 20%, > 0 and < 19%, > 0 and < 18%, > 0 and < 17%, > 0 and < 16%, or > 0 and < 15%, > 0 and < 14%, > 0 and < 13%, > 0 and < 12%, > 0 and < 11%, > 0 and < 10%, > 0 and < 9%, > 0 and < 8%, > 0 and < 7%, > 0 and < 6%, > 0 and < 5%, > 0 and < 4%, >0 and <3%, >0 and <2%, or >0 and <1% histidine residues.

Histidine + Tyrosine

[00450] In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with 42%, 38%, 33%, 31%, 30%, 24%, 23%, 18%, 15%, 10%, 9%, or 0% of total histidine and tyrosine residues. Preferably, the isolated antibody or antigen-binding fragment comprises an HCR3 with at least one histidine residue. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with (a) > 0 and < 42%, (b) > 10 and < 42%, or (c) > 36 and < 43% total histidine and tyrosine residues.

Arginine (R)

[00451] In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with 0%, 7%, 8%, 10%, 15%, or 18% arginine residues. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with (a) > 0 and < 18%, (b) > 7 and < 10%, or (c) > 0 and < 7% arginine residues.

Lysine (K)

[00452] In some embodiments, the isolated antibody or antigen-binding fragment comprises a HCDR3 with 0% to 8% lysine residues. In some embodiments, the isolated antibody or antigen-binding fragment comprises a HCDR3 without lysine residues.

Aspartic acid (D)

[00453] In some preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with 0%, 7%, 8%, 9%, 10%, 11%, 15%, 16%, 17%, or 25% aspartic acid residues. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises an HCR3 with (a) > 0 and < 25%, (b) > 7 and < 16%, or (c) > 7 and < 13% aspartic acid residues.

Glutamic acid (E)

[00454] In some embodiments, the isolated antibody or antigen-binding fragment comprises a HCDR3 with 0%, 8%, or 9% glutamic acid residues. In some highly preferred embodiments, the isolated antibody or antigen-binding fragment comprises a HCDR3 without glutamic acid residues.

Length

[00455] In some embodiments, the isolated antibody or antigen-binding fragment comprises a HCDR3 with a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Preferably, the isolated antibody or antigen-binding fragment comprises a HCDR3 with a length of 10, 11, 12 or 13 amino acid residues or a length between 8 and 13 amino acid residues.

ASPECT 11 - BLOCKADE/NEUTRAL ANTIBODY CCR8

[00456] There are several different ways in which an antibody can modulate CCR8 signaling. For example, an antibody can: a) block G protein-independent signaling, b) block G protein-dependent signaling, c) block both G protein-dependent and G protein-independent signaling, d) increase G protein-independent signaling, e) increase G protein-dependent signaling, f) increase both G protein-dependent and G protein-independent signaling.

[00457] According to an 11th aspect, which may or may not be the same as the 6th, 7th, 8th, 9th and/or 10th aspects, an isolated antibody or antigen-binding fragment is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment at least partially modulates CCR8 signaling.

[00458] For CCR8, the G protein-independent signaling pathways are β-arrestin signaling (Example 10.4.1), phospho Erk1/2 signaling (phosphorylation of Erk1/2), and phospho Akt signaling (phosphorylation of AKT) (Example 10.4.2). Other antagonist or agonist subtypes can be defined based on their impact on these three G protein-independent signaling pathways.

[00459] According to some early embodiments of the 11th aspect, the antibody or antigen-binding fragment a) does not block CCL1-induced β-arrestin signaling and/or b) does not induce ERK1/2 phosphorylation and/or c) does not induce AKT phosphorylation.

[00460] Example 10.4.1 shows that the prior art antibodies 433H and L268G8 effectively blocked CCL1-induced β-arrestin signaling, for example, with IC50 values below 20 nM (as per Table 10.4.1.1), whereas no IC50 value could be determined for inventive antibodies such as TPP-23411.

[00461] According to some embodiments of the first embodiments of aspect 11, the antibody or antigen-binding fragment does not block CCL1-induced β-arrestin signaling. In case of doubt, an antibody blocks CCL1-induced β-arrestin signaling if the IC50 is below 100 nM. In case of doubt, an antibody does not block CCL1-induced β-arrestin signaling if the IC50 is > 100 nM, or if no IC50 can be determined with the assay system described herein. CCL1-induced β-arrestin signaling has been associated with receptor internalization, as described in aspect 12.

[00462] Example 10.4.2, Fig. 27, 28 shows that the antibodies of the prior art induce Erk1/2 phosphorylation in CHO cells expressing human CCR8 or activated human Tregs, for example, after 15 minutes, by at least a factor of 1.5. Instead, the TPP-23411 antibody of the invention did not induce significant Erk1/2 phosphorylation.

[00463] According to some embodiments B of the first embodiments of the 11th aspect, the antibody or antigen-binding fragment does not induce ERK1/2 phosphorylation. In case of doubt, an antibody induces Erk1/2 phosphorylation if – using an assay as described herein – at least a 1.5-fold increase in Erk1/2 phosphorylation levels over the control can be detected. In case of doubt, an antibody does not induce Erk1/2 phosphorylation if no significant increase in Erk1/2 phosphorylation levels is detected or if the increase is less than 1.5-fold relative to the control.

[00464] Example 10.4.2, Fig. 30, shows that the antibodies of the prior art induce AKT phosphorylation, for example, after 15 minutes by a factor of 1.5. Instead, the TPP-23411 antibody of the invention did not induce significant AKT phosphorylation.

[00465] According to some embodiments C of the first embodiments of the 11th aspect, the antibody or antigen-binding fragment does not induce AKT phosphorylation. In case of doubt, an antibody induces AKT phosphorylation if – using an assay as described herein – at least a 1.5-fold increase in AKT phosphorylation levels over the control can be detected. In case of doubt, an antibody does not induce AKT phosphorylation if no significant increase in AKT phosphorylation levels is detected or if the increase is less than 1.5-fold relative to the control.

[00466] It is assumed that antibodies or fragments that do not show induction of G protein-independent signaling pathways, such as AKT or ERK1/2 phosphorylation, have advantages in therapy because induction of G protein-dependent signaling can lead to undesirable effects and side effects.

[00467] To analyze G protein-dependent signaling, the Ca flux assay as known in the art and as described herein can be used as a readout. Most of the inventive antibodies tested were found to be fully effective antagonists of G protein-dependent Ca signaling; however, differences in IC50 can be determined in comparison with antibodies from the prior art.

[00468] Example 10.4.3 shows that several of the anti-CCR8 antibodies tested blocked CCL1-induced G protein-dependent Ca signaling, for example, with IC50 values in the low nM or even sub-nM range.

[00469] According to some second embodiments of the 11th aspect, which may be the same as or different from the first embodiments, the antibody or antigen-binding fragment blocks CCL1-induced calcium signaling. In some of these preferred embodiments, the antibody or antigen-binding fragment blocks CCL1-induced calcium signaling, for example, with an IC50 < 1 nM, < 0.5 nM, or < 0.01 nM.

[00470] According to some third embodiments of the 11th aspect, which may be the same as or different from the first embodiments, the antibody or antigen-binding fragment does not block CCL1-induced G protein-dependent calcium signaling.

Aspect 12 - CCR8 antibody with no and/or low internalization.

[00471] An anti-CCR8 antibody can be a non-internalizing, low-internalizing, medium-internalizing, or high-internalizing antibody or antigen-binding fragment.

[00472] According to a 12th aspect, which may or may not be the same as the 6th, 7th, 8th, 9th, 10th and/or 11th aspects, an isolated antibody or antigen-binding fragment is provided that binds specifically to CCR8, wherein the antibody or antigen-binding fragment is a non-internalizing or low-internalizing antibody or antigen-binding fragment.

[00473] Depending on the specific mode of action of an antibody, fragment, or conjugate, internalization into a cell may be desired or should be avoided, as described elsewhere here. Antibodies according to the 12th aspect are particularly well-suited for an ADCC/ADCP approach, or any other antibody- or fragment-based antigen-binding mode of action to recruit effector cells.

[00474] Because overexpression can affect internalization behavior and is less suitable for modeling internalization in a therapeutic setting, internalization is preferably determined using a model cell line with endogenous expression of the target. When the target is human CCR8, the cell endogenously expressing the target is preferably HuT78. When the target is murine CCR8, the cell endogenously expressing the target is preferably murine BW5147.3. HuT78 and mBW5147.3 can be obtained from ATCC.

[00475] Although all antibodies of the prior art are readily internalized into cells with endogenous target expression, as shown in Example 10.5, the tested antibodies TPP-21360, TPP-21047 (data not shown) and TPP-23411 and several other antibodies according to the present invention did not internalize significantly.

[00476] For example, internalization can be determined over a period of time or for specific time points. Preferably, internalization can be determined after 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, 24 h, or 48 h in a cell that endogenously expresses the target.

[00477] According to some early embodiments of the 12th aspect, the antibody or antigen-binding fragment is characterized by internalization into a cell with endogenous target expression that is less than 1.5, 2, 3, 4, 5, 6, 7, or 10 times the internalization of the isotype control. The isotype control for an antibody may be selected as known in the art to match the antibody isotype as closely as possible, but without binding to the target.

[00478] According to some of these embodiments, the antibody or antigen-binding fragment is characterized by internalization into a cell with endogenous target expression that is less than 150%, 175%, 200%, 300%, 400%, or 500% of the internalization of the isotype control, for example, after 15 min, 30 min, 1h, 2h, 3h, 6h, 12h, 24h, or 48h, preferably where the cell with endogenous target expression is a HuT78 lymphoma cell.

[00479] According to some of these preferred embodiments, the antibody or antigen-binding fragment has an internalization rate on the same order of magnitude as the internalization rate of the isotype control.

[00480] According to some second embodiments of the 12th aspect, which may be the same as or different from the first embodiments, the antibody or antigen-binding fragment binds specifically to human CCR8 and is characterized by a time until half the amount of antibody, fragment or conjugate is internalized that is > 2 hours, preferably > 4, > 5, > 6, > 7, > 8, > 9, > 10, > 11, > 12, > 13, > 14, > 15, > 16, > 17, > 18, > 19, > 20, > 21, > 22, > 23, > 24, > 26, > 28, > 30 or > 48 hours in a cell endogenously expressing the target, or no time can be determined, preferably in which the cell endogenously expressing the target is a HuT78 lymphoma cell. For example, the anti-CCR8 antibody has a lower internalization rate than the 433H and L263G8 antibodies (BioLegend Cat. No. 360602).

[00481] According to some third embodiments of the 12th aspect, which may be the same as or different from the first or second embodiments, the antibody or antigen-binding fragment binds specifically to murine CCR8 and has a lower internalization rate than SA214G2 antibody in a cell endogenously expressing the target, preferably where the cell endogenously expressing the target is a murine lymphoma cell line BW5147.3.

[00482] The supplied isolated antibody or antigen-binding fragment is preferably also an antibody according to any one of aspects 7, 8, 9, 10, 11, 13, 14, 15, 16, 17 or 18 or a combination thereof.

ASPECT 13 - CCR8 ADCC/ADCP ANTIBODY THAT INDUCES ADCC/ADCP

[00483] To induce the death of cells expressing CCR8, such as activated Tregs, several modes of action can be predicted. One mode of action is the conjugation of an antibody targeting CCR8 to a drug in the form of an antibody-drug conjugate (ADC). Other possible modes of action are ADCC, CDC, and ADCP. For ADCC, CDC, and ADCP, a two-step mechanism is involved: on the one hand, the antibody or fragment is required to bind effectively to the target cell, for example, the Treg via CCR8; on the other hand, the FC portion of the antibody (or an alternative binding portion that may be conjugated to the antibody or fragment as described elsewhere here) has to bind to an effector cell, which will then mediate the death of the target cell. For ADCP, binding to macrophages as effector cells typically occurs through the interaction of the FC portion of the antibodies with FcYRIIa (CD32a) expressed by macrophages. In contrast, ADCC is mediated through the interaction of the antibody or fragment with FcYRIIIa. In humans, FcYRIII exists in two different forms: FcYRIIIa (CD16a) and FcYRIIIb (CD16b). While FcYRIIIa is expressed on mast cells, macrophages, and natural killer cells as a transmembrane receptor, FcYRIIIb is expressed only on neutrophils. These receptors bind to the Fc portion of IgG antibodies, which then activates antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

[00484] In case of doubt, where ADCC and/or ADCP induction is analyzed, at least 80% and preferably at least 85% of the target cells are required to show CCR8 expression.

[00485] According to a 13th aspect, which may or may not be the same as aspects 6, 7, 8, 9, 10, 11 and/or 12, an isolated antibody or antigen-binding fragment is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment induces ADCC and/or ADCP.

[00486] For example, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment is non-fucosylated and a) induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells, such as human NK cells, and b) induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells, such as human macrophages, preferably wherein the maximum ADCC and ADCP induced upon in vitro depletion of target cells expressing human CCR8 is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%. In case of doubt, ADCC and ADCP should be determined using target cells in which at least 85% of the cells express CCR8.

[00487] In some preferred embodiments of the 13th aspect, the antibody or antigen-binding fragment is non-fucosylated. Non-fucosylated antibodies are antibodies designed so that the oligosaccharides in the Fc region of the antibody do not have fucose sugar units. Non-fucosylated antibodies can be obtained as is known in the art, for example, as described in Example 10.3. Example 10.3.2 demonstrates the improved binding of the non-fucosylated versions of the inventive antibodies to FcYRIIIa. Binding to FcYRIIa was also slightly improved. In addition, fucosylation increased binding to cynomolgus Fc gamma RIII, as shown in Table 10.3.2.1. ADCC

[00488] In some early embodiments of the 13th aspect, the antibody or antigen-binding fragment binds to the human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM, or 200 nM.

[00489] In some early embodiments B of aspect 13, which may be the same as or different from early embodiments A, the antibody or antigen-binding fragment induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells, such as human NK cells. The induction of ADCC can be analyzed with an assay known in the art, for example, as described in Examples 10.3.3 ff.

[00490] In some early C embodiments of aspect 13, which may be the same as or different from early A or B embodiments, the maximum ADCC-induced depletion of activated human regulatory T cells is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, preferably, where at least 85% of activated human regulatory T cells have CCR8 expression, as per Table 10.3.3.1.2 and Table 10.3.3.1.3.

[00491] In some early D modalities of aspect 13, which may be the same as or different from early A, B, or C modalities, the EC50 for ADCC-induced depletion of activated human regulatory T cells is less than 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 20 pM, 12.5 pM, 10 pM, 5 pM, or 2.5 pM. Preferably, at least 85% of activated human regulatory T cells have CCR8 expression. ADCP

[00492] In some second embodiments A of the 13th aspect, which may or may not be the same as the first embodiments A, B, C and/or D, the antibody or antigen-binding fragment binds to human Fc gamma RIIA (CD32a) with a dissociation constant (KD) less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

[00493] In some second-part B embodiments of the 13th aspect, which may be the same as the first embodiments A, B, C, and/or D, and which may be the same as the second-part A embodiments, the antibody or antigen-binding fragment induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells, such as human macrophages. For example, human macrophages may be M2c or M1 macrophages.

[00494] In some second C modalities of the 13th aspect, which may be the same as the first modalities A, B, C and/or D, and which may be the same as the second modalities A and/or B, the maximum ADCP-induced depletion of activated human regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%.

[00495] In some second D modalities of the 13th aspect, which may be the same as the first modalities A, B, C and/or D, and which may be the same as the second modalities A, B and/or C, the EC50 for ADCP-induced depletion of activated human regulatory T cells is less than 1500 pM, 1000 pM, 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25 pM or 10 pM.

[00496] In some preferred embodiments, an isolated antibody or antigen-binding fragment thereof is provided that binds specifically to CCR8, wherein the antibody or antigen-binding fragment a) binds to human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM and/or b) binds to human Fc gamma receptor RIIA (CD32a) with a dissociation constant (KD) less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

[00497] In some preferred embodiments, an isolated antibody or antigen-binding fragment thereof is provided that binds specifically to CCR8, wherein the antibody or antigen-binding fragment a) induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells such as human NK cells and/or b) induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells such as human macrophages.

[00498] In some preferred embodiments, an isolated antibody or antigen-binding fragment thereof is provided that binds specifically to CCR8, wherein a) the maximum ADCC-induced depletion of activated human regulatory T cells is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, and/or b) the maximum ADCP-induced depletion of activated human regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, and/or c) the maximum depletion of intratumoral regulatory T cells, in vitro or in an individual, is at least 50%, 60%, 70%, 80%, 90%, 95% or 99%.

[00499] In some preferred embodiments, an isolated antibody or antigen-binding fragment thereof is provided that binds specifically to CCR8, wherein a) the EC50 for ADCC-induced depletion of activated human regulatory T cells is less than 200 pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM or 5 pM and/or b) the EC50 for ADCP-induced depletion of activated human regulatory T cells is less than 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM or 25 pM.

[00500] The supplied isolated antibody or antigen-binding fragment is preferably also an antibody according to any of aspects 14, 15, 16, 17 or 18 or a combination thereof.

ASPECT 14 - TREG MODULATORY CCR8 ANTIBODY

[00501] The in vivo therapeutic effects of antibodies according to the present invention are shown in Examples 12 et seq. The ability of an antibody to modulate absolute and relative numbers of various immune cell populations is structurally realized by a combination of target-binding properties and interaction with the FC receptor, for example, as described according to the 7th, 10th, 12th and/or 13th aspects or a combination thereof.

[00502] According to a 14th aspect, which may or may not be the same as aspects 6, 7, 8, 9, 10, 11, 12, and/or 13, an antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment depletes activated regulatory T cells, preferably intratumoral Tregs. Example 12.1.1 shows high levels of Treg depletion obtained with the inventive antibodies in several mouse models, wherein the surrogate antibodies are characterized by functional features comparable to the provided anti-human CCR8 antibodies. Interestingly, a superior therapeutic response was observed in those cases where Treg depletion was at least 50%. Treg depletion can be measured in vitro or in vivo. As understood by someone skilled in the art, Treg depletion can be a temporal depletion. For example, Treg depletion can be analyzed 24, 48, or 72 hours after treatment.

[00503] According to some early embodiments of the 14th aspect, an effective dose of the antibody or antigen-binding fragment is characterized by a maximal depletion of activated or intratumoral regulatory T cells, in vitro or in an individual, of at least 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%.

[00504] In vitro or in vivo determination may occur as known in the art. In vivo determination may occur as described in Examples 12 et seq. Suitable subjects include, for example, humans and non-humans such as mice (e.g., CT26 model or EMT-6 model), rodents, or cynomolgus.

[00505] According to some second embodiments of the 14th aspect, which may or may not be the same as the first embodiments, an effective dose of the antibody or antigen-binding fragment decreases the number of activated or intratumoral regulatory T cells, in vitro or in an individual, to less than 55%, 50%, 40%, 30%, 25%, 20%, 10%, 5%, or 1%. The decrease may be temporary. For example, and without being limited by the theory, the pool of intratumoral Tregs may be subsequently replenished.

[00506] According to some third embodiments of the 14th aspect, which may or may not be the same as the first and/or second embodiments, an effective dose of the antibody or antigen-binding fragment increases the ratio of intratumoral CD8+ T cells to tumor-Tregs, in vitro or in an individual, to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or higher. The increase may be a temporary increase.

[00507] According to some fourth embodiments of the 14th aspect, which may or may not be the same as the first, second and/or third embodiments, an effective dose of the antibody or antigen-binding fragment decreases the percentage of intratumoral CD4+ T cells, in vitro or in an individual, to < 30%, < 20%, < 10% or < 5%. The decrease may be a temporary decrease.

[00508] In preferred embodiments, an effective dose of the antibody or antigen-binding fragment a) decreases the number of activated or intratumoral regulatory T cells, in vitro or in an individual, to less than 30%, 25%, 20%, 10%, 5% or 1% and/or b) increases the ratio of intratumoral CD8+ T cells to intratumoral Tregs, in vitro or in an individual, to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more and/or c) decreases the percentage of regulatory T cells to intratumoral CD4+ T cells, in vitro or in an individual, to < 30%, < 20%, < 10% or < 5%.

[00509] Antibodies according to the present aspect were superior in terms of therapeutic efficacy, see Examples 12 et seq. The single antibody or antigen-binding fragment provided is preferably also an antibody according to any of aspects 15, 16, 17 or 18 or a combination thereof.

ASPECT 15 - CCR8 ANTIBODY IMMUNE CELL MODULATOR

[00510] According to a 15th aspect, which may or may not be the same as the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th and/or 14th aspect, an isolated antibody or antigen-binding fragment thereof is provided which binds specifically to CCR8, wherein an effective dose of the antibody or antigen-binding fragment increases the absolute number of immune cells in a defined tumor volume, for example, by at least a factor of 1.5, 2, 2.5, 3, 3.5 or 4.

[00511] Example 12.8 shows a massive increase in mRNA expression levels for several global or specific immune cell markers in responsive tumors from different individuals at the end of the study. An increase was observed, for example, for macrophages (in particular M1 macrophages), CD8+ T cells, NK cells, CD3+ T cells, B cells and, interestingly, also activated Tregs, suggesting an increased infiltration of these immune cell populations into the tumor after chronic treatment. The increase in the absolute number of immune cells was also confirmed by FACS, see Example 12.3 (CT26), Example 12.4 (EMT6), Example 12.5 (F9). Suitable individuals include, for example, human and non-human individuals such as mice, rodents or cynomolgus. For example, an increase in immune cells can be determined after one, two, three, or four effective doses of the antibody, as per Examples 12.3, 12.4.2, and also after chronic treatment, for example, after the final treatment. For example, the tumor may be a tumor characterized by tumor-infiltrating lymphocytes, for example, a tumor characterized by tumor-infiltrating T cells, or a tumor characterized by the expression of pro-inflammatory cytokines.

[00512] According to some early embodiments of the 15th aspect, immune cells are at least one, two, three, four, five, six, seven, eight, or nine selected from a) CD45+ cells (intratumoral), b) CD8+ T cells (intratumoral), c) CD4+ T cells (intratumoral), d) macrophages (intratumoral), such as M1 macrophages or M2 macrophages, e) NK cells (intratumoral), f) B cells (intratumoral), g) dendritic cells (intratumoral), h) gamma delta T cells (intratumoral) (non-conventional T cells), and i) iNKT cells.

[00513] The respective cell types and populations are defined as known in the art and, in particular, as defined elsewhere herein.

[00514] According to some of these highly preferred early modalities, three or more effective doses of antibody or antigen-binding fragment in a tumor increase a) the number of intratumoral CD8+ T cells by at least 150%, 200%, 250%, 300%, or 350%, b) the ratio of intratumoral CD8+ T cells to intratumoral regulatory T cells by at least 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or higher, c) the number of intratumoral macrophages by at least a factor of 2, 3, 4, or 5, d) the number of intratumoral ACOD1+ macrophages (M1 macrophages) by at least a factor of 2, 3, 4, 5, 6, 7, 8, 9 or 10, e) the ratio of ACOD1+ macrophages (M1 macrophages) to MRC1+ macrophages (M2 macrophages) by at least 1, 5, 2, 3, 4, 5, 10 or greater, f) the number of intratumoral NK cells by at least 140% or 200%, g) the number of intratumoral CD3+ T cells by at least 150%, 200%, 300% or 400%, h) the number of intratumoral B cells by at least a factor of 2, 5, 10, 20, 30 or 40, i) the number of intratumoral CD45+ T cells by at least 150%, 200% or 300%, and/or j) the number of Intratumoral CD4+ T cells in at least 150%, 200%, 300%, or 400%.

[00515] The tumor is preferably a tumor characterized by tumor-infiltrating lymphocytes. As understood by a person skilled in the art, the tumor is characterized by cells expressing the antibody-target CCR8 or inventive fragment, for example, intratumoral Tregs. Preferably, the tumor is a tumor in a syngeneic tumor model derived from a BALB/c strain. For example, as shown in Example 12.1.1, the tumor may be derived from the CT26, EMT-6, F9, C38, H22, or B16F10-OVA models, where the antibody specifically binds to murine CCR8. For example, when the antibody binds specifically to human CCR8, the tumor can be selected from adrenal cancer (e.g., adrenocortical carcinoma or pheochromocytoma), bladder cancer (e.g., transitional cell carcinoma, transitional cell-papillary carcinoma), brain cancer (e.g., glioma-astrocytoma, glioma-astrocytoma-glioblastoma, glioma-oligoastrocytoma, glioma-oligodendroglioma), breast cancer (e.g., ADC, ADC-ductal, ADC-ductal-TNBC, ADC-ductal-TPBC, ADC-lobular), colorectal cancer (e.g., ADC), esophageal cancer (e.g., ADC), esophageal cancer (e.g., SCC), gastric cancer (e.g., ADC, diffuse ADC, ADC-intestinal, ADC-intestinal-tubular), head and neck cancer (e.g., Laryngeal cancer (SCC, SCC, oral cancer), kidney cancer (e.g., ccRCC, chromophobe, papillary, papillary type I, papillary type II), liver cancer (e.g., HCC), lung cancer (e.g., NSCLC-ADC, NSCLC-ADC-Mixed, NSCLC-SCC, SCLC), mesothelioma (e.g., epithelioid), ovarian cancer (e.g., ADC-Serous papillary cystadenocarcinoma), pancreatic cancer (e.g., ADC-ductal), prostate cancer (e.g., ADC-Acinar type), sarcoma (e.g., leiomyosarcoma, undifferentiated liposarcoma, malignant fibrous histiocytoma), skin cancer (e.g., melanoma), testicular cancer (e.g., germ cell tumor-seminoma), thymoma, thyroid cancer (e.g., follicular carcinoma, carcinoma papillary-classical variant), uterine cancer (e.g., Cervical SCC, Keratinizing Cervical SCC, Non-keratinizing Cervical SCC, Endometrial-ADC-Endometrioid, Endometrial-ADC-Serous Papillary, Endometrial-Carcinosarcoma-Malignant Müllerian Tumor), as per Table 11.1.2.

[00516] In some of these modalities, the immune cells are a) CD8+ T cells, CD4+ T cells and macrophages, b) CD8+ T cells, CD4+ T cells, NK cells, c) CD8+ T cells, CD4+ T cells, CD45+ cells, d) CD8+ T cells, CD4+ T cells, NK cells and macrophages, e) CD8+ T cells, CD4+ T cells, NK cells and macrophages, for example, ACOD1+ macrophages, f) CD8+ T cells, CD4+ T cells, NK cells, CD45+ cells, g) CD8+ T cells, CD4+ T cells, NK cells, CD45+ cells and macrophages, for example, ACOD1+ macrophages, h) CD8+ T cells, ACOD1+ macrophages (M1 macrophages) and cells B,i) CD8+ T cells, CD4+ T cells and dendritic cells, wherein the dendritic cells are characterized by the expression of CD1c, CD14, CD16, CD141, CD11c and CD123, or j) CD8+ T cells and Acod1+ macrophages (M1 macrophages).

[00517] In some of these preferred modalities, the immune cells are a) CD8+ T cells, CD4+ T cells and macrophages, or b) CD8+ T cells, CD4+ T cells, NK cells and macrophages, or c) CD8+ T cells, ACOD1+ macrophages (M1 macrophages) and B cells.

[00518] According to some other modalities, immune cells are CD45+ cells and CD8+ T cells, CD45+ cells and CD4+ T cells, CD45+ cells and macrophages (such as M1 macrophages or M2 macrophages), CD45+ cells and NK cells, CD45+ cells and B cells, CD45+ cells and dendritic cells, CD45+ cells and gamma delta T cells, CD45+ cells and iNKT cells, CD8+ T cells and CD4+ T cells, CD8+ T cells and macrophages (such as M1 macrophages or M2 macrophages), CD8+ T cells and NK cells, CD8+ T cells and B cells, CD8+ T cells and dendritic cells, CD8+ T cells and gamma delta T cells, CD8+ T cells and iNKT cells, CD4+ T cells and macrophages (such as M1 macrophages or macrophages (M2), CD4+ T cells and NK cells, CD4+ T cells and B cells, CD4+ T cells and dendritic cells, CD4+ T cells and Gamma delta T cells, CD4+ T cells and iNKT cells, macrophages (such as M1 or M2 macrophages) and NK cells, macrophages (such as M1 or M2 macrophages) and B cells, macrophages (such as M1 or M2 macrophages) and dendritic cells, macrophages (such as M1 or M2 macrophages) and gamma delta T cells, macrophages (such as M1 or M2 macrophages) and iNKT cells, B cells and dendritic cells, B cells and gamma delta T cells, B cells and iNKT cells, dendritic cells and gamma delta T cells, dendritic cells and iNKT cells gamma delta T and iNKT cells.

[00519] The supplied isolated antibody or antigen-binding fragment is preferably also an antibody according to any of aspects 16, 17 or 18 or a combination thereof.

Aspect 16 - Tertiary lymphoid structures forming CCR8 antibody.

[00520] According to a 16th aspect, which may or may not be the same as aspects 6, 7, 8, 9, 10, 11, 12, 13, 14 and/or 15, an isolated antibody or antigen-binding fragment thereof is provided that binds specifically to CCR8, wherein the antibody or antigen-binding fragment induces the formation of tertiary lymphoid structures. Tertiary lymphoid structures are intra-tumoral structures characterized by increased expression of LTta, LTtb, Cxcr5 and its ligand Cxcl13. Tertiary lymphoid structures have been described as major drivers of an antitumor effect in humans. Without being limited by theory, the inventors believe that the formation of these substructures may contribute to the altered pattern in immune cells and an antitumor effect of the inventive antibodies, for example, in humans. As described in Example 12.8, chronic treatment with the antibodies of the invention consistently increased expression levels for LTta, LTtb, as well as Cxcr5 and its ligand Cxcl13.

ANTIBODIES DEFINED BY SEQUENCE

[00521] Antibodies according to aspects 17 and 18 below were obtained using a method according to aspect 3 or 4. The resulting antibodies according to aspects 17 and 18 bind specifically to a polypeptide comprising the sulfated TRD of a human chemokine receptor, for example, wherein at least 50% of the tyrosine residues have been sulfated. As described earlier herein, the method influences the specific structural characteristics of the HCDR3 of the antibodies obtained and may also influence functional characteristics, such as modulation of G protein-independent signaling or internalization behavior.

[00522] Where CDRs, variable heavy chains, variable light chains, heavy chains or light chains are described, the modular nature of antibodies normally allows their combination, but also combination with various functional features, as described elsewhere here. ASPECT 17 - ANTI-HUMAN CCR8 ANTIBODY (SEQ DEFINED)

[00523] In accordance with a 17th aspect, which may or may not be the same as aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and/or 16, an isolated anti-CCR8 antibody or its antigen-binding fragment is provided.

[00524] Preferably, the antibodies according to the present aspect bind (a) to a polypeptide isolate according to SEQ ID NO:43 and/or SEQ ID NO:46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and (b) to a polypeptide isolate according to SEQ ID NO:44 and/or SEQ ID NO:47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and the antibodies are therefore cross-reactive for human CCR8 and cynomolgus. In addition, the antibodies are preferably characterized by properties that make them particularly suitable for therapy, for example, they are antibodies according to at least one or more of aspects 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[00525] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises at least one, two, and preferably three, four, five, or six CDR sequences with at least 90%, 95%, 98%, or 100% sequence identity with any of the following: a) SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:263, or SEQ ID NO:264 (TPP-16966), b) SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:281, or SEQ ID NO:282 (TPP-17575), c) SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:299 or SEQ ID NO:300 (TPP-17576),d) SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314,SEQ ID NO:316, SEQ ID NO:317 or SEQ ID NO:318 (TPP-17577),e) SEQ ID NO:330, SEQ ID NO:331, SEQ ID NO:332,SEQ ID NO:334, SEQ ID NO:335 or SEQ ID NO:336 (TPP-17578),f) SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:350,SEQ ID NO:352, SEQ ID NO:353 or SEQ ID NO:354 (TPP-17579),g) SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368,SEQ ID NO:370, SEQ ID NO:371 or SEQ ID NO:372 (TPP-17580),h) SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386,SEQ ID NO:388, SEQ ID NO:389 or SEQ ID NO:390 (TPP-17581) NO:425 or SEQ ID NO:426 (TPP-18206) NO:461 or SEQ ID NO:462 (TPP-19546),m) SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476,SEQ ID NO:478, SEQ ID NO:479 or SEQ ID NO:480 (TPP-20950),n) SEQ ID NO:492, SEQ ID NO:493, SEQ ID NO:494,SEQ ID NO:496, SEQ ID NO:497 or SEQ ID NO:498 (TPP-20955),o) SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512,SEQ ID NO:514, SEQ ID NO:515 or SEQ ID NO:516 (TPP-20965),p) SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530,SEQ ID NO:532, SEQ ID NO:533 or SEQ ID NO:534 (TPP-21045),q) SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548,SEQ ID NO:550, SEQ ID NO:551 or SEQ ID NO:552 (TPP-21047), r) SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566,SEQ ID NO:568, SEQ ID NO:569 or SEQ ID NO:570 (TPP-21181),s) SEQ ID NO:582, SEQ ID NO:583, SEQ ID NO:584,SEQ ID NO:586, SEQ ID NO:587 or SEQ ID NO:588 (TPP-21183),t) SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602,SEQ ID NO:604, SEQ ID NO:605 or SEQ ID NO:606 (TPP-21360),u) SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620,SEQ ID NO:622, SEQ ID NO:623 or SEQ ID NO:624 (TPP-23411),v) SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663,SEQ ID NO:665, SEQ ID NO:666 or SEQ ID NO:667 (TPP-29596),w) SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683,SEQ ID NO:685, SEQ ID NO:686 or SEQ ID NO:687 (TPP-29597),x) SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:705,SEQ ID NO:707, SEQ ID NO:708 or SEQ ID NO:709 (TPP-18429) NO:728 or SEQ ID NO:729 (TPP-18430),z) SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745,SEQ ID NO:747, SEQ ID NO:748 or SEQ ID NO:749 (TPP-18432) NO:768 or SEQ ID NO:769 (TPP-18433),bb) SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785,SEQ ID NO:787, SEQ ID NO:788 or SEQ ID NO:789 (TPP-18436),cc) SEQ ID NO:803, SEQ ID NO:804, SEQ ID NO:805,SEQ ID NO:807, SEQ ID NO:808 or SEQ ID NO:809 (TPP-19571),dd) SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 829,SEQ ID NO: 831, SEQ ID NO: 832 or SEQ ID NO: 833 (TPP-27477),ee) SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849,SEQ ID NO: 851, SEQ ID NO: 852 or SEQ ID NO: 853 (TPP-27478),ff) SEQ ID NO: 867, SEQ ID NO: 868, SEQ ID NO: 869,SEQ ID NO: 871, SEQ ID NO: 872 or SEQ ID NO: 873 (TPP-27479), gg) SEQ ID NO: 887, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 891, SEQ ID NO: 892 or SEQ ID NO: 893 (TPP-27480),hh) SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909,SEQ ID NO: 911, SEQ ID NO: 912 or SEQ ID NO: 913 (TPP-29367) 948, SEQ ID NO: 949, SEQ ID NO: 951, SEQ ID NO: 952 or SEQ ID NO: 953 (TPP-29369).

[00526] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises an HCDR3 sequence with at least 90%, 95%, 98%, or 100% sequence identity with any of SEQ ID NO:260 (TPP-16966), SEQ ID NO:278 (TPP-17575), SEQ ID NO:296 (TPP-17576), SEQ ID NO:314 (TPP-17577), SEQ ID NO:332 (TPP-17578), SEQ ID NO:350 (TPP-17579), SEQ ID NO:368 (TPP-17580), SEQ ID NO:386 (TPP-17581), SEQ ID NO:404 (TPP-18205), SEQ ID NO:422 (TPP-18206), SEQ ID NO:440 (TPP-18207), SEQ ID NO:458 (TPP-19546), SEQ ID NO:476 (TPP-20950), SEQ ID NO:494(TPP-20955), SEQ ID NO:512 (TPP-20965), SEQ ID NO:530 (TPP-21045), SEQ ID NO:548 (TPP-21047), SEQ ID NO:566 (TPP-21181), SEQ ID NO:584 (TPP-21183), SEQ ID NO:602 (21360), or SEQ ID NO:620 (TPP-23411), SEQ ID NO:663 (TPP-29596), SEQ ID NO:683 (TPP-29597), SEQ ID NO:705 (TPP-18429), SEQ ID NO:725 (TPP-18430), SEQ ID NO:745 (TPP-18432), SEQ ID NO:765 (TPP-18433), SEQ ID NO:785 (TPP-18436), SEQ ID NO:805 (TPP-19571), SEQ ID NO:829 (TPP-27477), SEQ ID NO:849 (TPP-27478), SEQ ID NO:869 (TPP-27479), SEQ ID NO:889 (TPP-27480), SEQ ID NO:909 (TPP - 29367), SEQ ID NO:929 (TPP-29368), or SEQ ID NO:949 (TPP- 29369).

[00527] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises at least one, two, and preferably three, four, five, or six CDR sequences according to a) SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:263, and SEQ ID NO:264 (TPP-16966), b) SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:281, and SEQ ID NO:282 (TPP-17575), c) SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:299, and SEQ ID NO:300 (TPP-17576) NO:335 and SEQ ID NO:336 (TPP-17578),f) SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:350,SEQ ID NO:352, SEQ ID NO:353 and SEQ ID NO:354 (TPP-17579),g) SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368,SEQ ID NO:370, SEQ ID NO:371 and SEQ ID NO:372 (TPP-17580),h) SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386,SEQ ID NO:388, SEQ ID NO:389 and SEQ ID NO:390 (TPP-17581),i) SEQ ID NO:402, SEQ ID NO:403, SEQ ID NO:404,SEQ ID NO:406, SEQ ID NO:407 and SEQ ID NO:408 (TPP-18205),j) SEQ ID NO:420, SEQ ID NO:421, SEQ ID NO:422,SEQ ID NO:424, SEQ ID NO:425 and SEQ ID NO:426 (TPP-18206),k) SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440,SEQ ID NO:442, SEQ ID NO:443 and SEQ ID NO:444 (TPP-18207),l) SEQ ID NO:456, SEQ ID NO:457, SEQ ID NO:458,SEQ ID NO:460, SEQ ID NO:461 and SEQ ID NO:462 (TPP-19546),m) SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476,SEQ ID NO:478, SEQ ID NO:479 and SEQ ID NO:480 (TPP-20950), h) SEQ ID NO:492, SEQ ID NO:493, SEQ ID NO:494,SEQ ID NO:496, SEQ ID NO:497 and SEQ ID NO:498 (TPP-20955),o) SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512,SEQ ID NO:514, SEQ ID NO:515 and SEQ ID NO:516 (TPP-20965),p) SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530,SEQ ID NO:532, SEQ ID NO:533 and SEQ ID NO:534 (TPP-21045) NO:569 and SEQ ID NO:570 (TPP-21181) NO:605 or SEQ ID NO:606 (TPP-21360),u) SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620,SEQ ID NO:622, SEQ ID NO:623 or SEQ ID NO:624 (TPP-23411),v) SEQ ID NO:661, SEQ ID NO:662, SEQ ID NO:663,SEQ ID NO:665, SEQ ID NO:666 or SEQ ID NO:667 (TPP-29596), w) SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683,SEQ ID NO:685, SEQ ID NO:686 or SEQ ID NO:687 (TPP-29597), NO:705,SEQ ID NO:707, SEQ ID NO:708 or SEQ ID NO:709 (TPP-18429),y) SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725,SEQ ID NO:727, SEQ ID NO:728 or SEQ ID NO:729 (TPP-18430),z) SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745,SEQ ID NO:747, SEQ ID NO:748 or SEQ ID NO:749 (TPP-18432),aa) SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765,SEQ ID NO:767, SEQ ID NO:768 or SEQ ID NO:769 (TPP-18433),bb) SEQ ID NO:783, SEQ ID NO:784, SEQ ID NO:785,SEQ ID NO:787, SEQ ID NO:788 or SEQ ID NO:789 (TPP-18436), cc) SEQ ID NO:803, SEQ ID NO:804, SEQ ID NO:805, SEQ ID NO:807, SEQ ID NO:808 or SEQ ID NO:809 (TPP-19571),dd) SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ ID NO: 831, SEQ ID NO: 832 or SEQ ID NO: 833 (TPP-27477),ee) SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, SEQ ID NO: 851, SEQ ID NO: 852 or SEQ ID NO: 853 (TPP-27478),ff) SEQ ID NO: 867, SEQ ID NO: 868, SEQ ID NO: 869, SEQ ID NO: 871, SEQ ID NO: 872 or SEQ ID NO: 873 (TPP-27479) 889, SEQ ID NO: 891, SEQ ID NO: 892 or SEQ ID NO: 893 (TPP-27480),hh) SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO: 911, SEQ ID NO: 912 or SEQ ID NO: 913 (TPP-29367),ii) SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 929, SEQ ID NO: 931, SEQ ID NO: 932 or SEQ ID NO: 933 (TPP-29368), and/orjj) SEQ ID NO: 947, SEQ ID NO: 948, SEQ ID NO: 949, SEQ ID NO: 951, SEQ ID NO: 952 or SEQ ID NO: 953 (TPP-29369), optionally in which up to one, two, three, four, or five mutations have been introduced into at least one CDR. Preferably, HCDR3 comprises or is designed to comprise at least one or more histidine residues as described elsewhere herein.

[00528] For example, tyrosine can be exchanged for a positively charged amino acid, such as histidine, and vice versa. For example, a positively charged amino acid can be exchanged for a different positively charged amino acid, a negatively charged amino acid can be exchanged for a different negatively charged amino acid, a polar amino acid can be exchanged for a different polar amino acid, an uncharged polar amino acid can be exchanged for a different uncharged polar amino acid, a small amino acid can be exchanged for a different small amino acid, an amphiphatic amino acid can be exchanged for a different amphiphatic amino acid, an aromatic amino acid can be exchanged for a different aromatic amino acid. As understood by a person skilled in the art, in particular, those amino acid exchanges are possible that do not alter the specific interaction between the antibody and the sulfated CCR8 TRD.

[00529] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises a variable heavy chain sequence and/or a variable light chain sequence with at least 90%, 95%, 98% or 100% sequence identity with: a) a variable heavy chain sequence according to SEQ ID NO:257 and/or a variable light chain sequence according to SEQ ID NO:261 (TPP-16966), b) a variable heavy chain sequence according to SEQ ID NO:275 and/or a variable light chain sequence according to SEQ ID NO:279 (TPP-17575), c) a variable heavy chain sequence according to SEQ ID NO:293 and/or a variable light chain sequence according to SEQ ID NO:297 (TPP-17576), d) a variable heavy chain sequence according to SEQ ID NO:311 and/or a variable light chain sequence according to SEQ ID NO:315 (TPP-17577),e) a variable heavy chain sequence according to SEQ ID NO:329 and/or a variable light chain sequence according to SEQ ID NO:333 (TPP-17578),f) a variable heavy chain sequence according to SEQ ID NO:347 and/or a variable light chain sequence according to SEQ ID NO:351 (TPP-17579),g) a variable heavy chain sequence according to SEQ ID NO:365 and/or a variable light chain sequence according to SEQ ID NO:369 (TPP-17580),h) a variable heavy chain sequence according to SEQ ID NO:383 and/or a variable light chain sequence according to SEQ ID NO:387 (TPP-17581),i) a variable heavy chain sequence according to SEQ ID NO:401 and/or a variable light chain sequence according to SEQ ID NO:405 (TPP-18205),j) a variable heavy chain sequence according to SEQ ID NO:419 and/or a variable light chain sequence according to SEQ ID NO:423 (TPP-18206),k) a variable heavy chain sequence according to SEQ ID NO:437 and/or a variable light chain sequence according to SEQ ID NO:441 (TPP-18207),l) a variable heavy chain sequence according to SEQ ID NO:455 and/or a variable light chain sequence according to SEQ ID NO:459 (TPP-19546),m) a variable heavy chain sequence according to SEQ ID NO:473 and/or a variable light chain sequence according to SEQ ID NO:477 (TPP-20950),n) a variable heavy chain sequence according to SEQ ID NO:491 and/or a variable light chain sequence according to SEQ ID NO:495 (TPP-20955),o) a variable heavy chain sequence according to SEQ ID NO:509 and/or a variable light chain sequence according to SEQ ID NO:513 (TPP-20965),p) a variable heavy chain sequence according to SEQ ID NO:527 and/or a variable light chain sequence according to SEQ ID NO:531 (TPP-21045),q) a variable heavy chain sequence according to SEQ ID NO:545 and/or a variable light chain sequence according to SEQ ID NO:549 (TPP-21047),r) a variable heavy chain sequence according to SEQ ID NO:563 and/or a variable light chain sequence according to SEQ ID NO:567 (TPP-21181),s) a variable heavy chain sequence according to SEQ ID NO:581 and/or a variable light chain sequence according to SEQ ID NO:585 (TPP-21183), t) a variable heavy chain sequence according to SEQ ID NO:599 and/or a variable light chain sequence according to SEQ ID NO:603 (TPP-21360), u) a variable heavy chain sequence according to SEQ ID NO:617 and/or a variable light chain sequence according to SEQ ID NO:621 (TPP-23411), v) a variable heavy chain sequence according to SEQ ID NO:660 and/or a variable light chain sequence according to SEQ ID NO:664 (TPP-29596), w) a variable heavy chain sequence according to SEQ ID NO:680 and/or a variable light chain sequence according to SEQ ID NO:684 (TPP-29597), x) a variable heavy chain sequence according to SEQ ID NO:702 and/or a variable light chain sequence according to SEQ ID NO:706 (TPP-18429),y) a variable heavy chain sequence according to SEQ ID NO:722 and/or a variable light chain sequence according to SEQ ID NO:726 (TPP-18430),z) a variable heavy chain sequence according to SEQ ID NO:742 and/or a variable light chain sequence according to SEQ ID NO:746 (TPP-18432),aa) a variable heavy chain sequence according to SEQ ID NO:762 and/or a variable light chain sequence according to SEQ ID NO:766 (TPP-18433),bb) a variable heavy chain sequence according to SEQ ID NO:782 and/or a variable light chain sequence according to SEQ ID NO:786 (TPP-18436),cc) a variable heavy chain sequence according to SEQ ID NO:802 and/or a variable light chain sequence according to SEQ ID NO:806 (TPP-19571),dd) a variable heavy chain sequence according to SEQ ID NO:826 and/or a variable light chain sequence according to SEQ ID NO:830 (TPP-27477),ee) a variable heavy chain sequence according to SEQ ID NO:846 and/or a variable light chain sequence according to SEQ ID NO:850 (TPP-27478),ff) a variable heavy chain sequence according to SEQ ID NO:866 and/or a variable light chain sequence according to SEQ ID NO:870 (TPP-27479),gg) a variable heavy chain sequence according to SEQ ID NO:886 and/or a variable light chain sequence according to SEQ ID NO:890 (TPP-27480),hh) a variable heavy chain sequence according to SEQ ID NO:906 and/or a variable light chain sequence according to SEQ ID NO:910 (TPP-29367), ii) a variable heavy chain sequence according to SEQ ID NO:926 and/or a variable light chain sequence according to SEQ ID NO:930 (TPP-29368), or jj) a variable heavy chain sequence according to SEQ ID NO:946 and/or a variable light chain sequence according to SEQ ID NO:950 (TPP-29369).

[00530] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises: a) a variable heavy chain sequence according to SEQ ID NO:257 and/or a variable light chain sequence according to SEQ ID NO:261 (TPP-16966), b) a variable heavy chain sequence according to SEQ ID NO:275 and/or a variable light chain sequence according to SEQ ID NO:279 (TPP-17575), c) a variable heavy chain sequence according to SEQ ID NO:293 and/or a variable light chain sequence according to SEQ ID NO:297 (TPP-17576), d) a variable heavy chain sequence according to SEQ ID NO:311 and/or a variable light chain sequence according to SEQ ID NO:315 (TPP-17577), e) a variable heavy chain sequence according to SEQ ID NO:329 and/or a variable light chain sequence according to SEQ ID NO:333 (TPP-17578),f) a variable heavy chain sequence according to SEQ ID NO:347 and/or a variable light chain sequence according to SEQ ID NO:351 (TPP-17579),g) a variable heavy chain sequence according to SEQ ID NO:365 and/or a variable light chain sequence according to SEQ ID NO:369 (TPP-17580),h) a variable heavy chain sequence according to SEQ ID NO:383 and/or a variable light chain sequence according to SEQ ID NO:387 (TPP-17581),i) a variable heavy chain sequence according to SEQ ID NO:401 and/or a variable light chain sequence according to SEQ ID NO:405 (TPP-18205),j) a variable heavy chain sequence according to SEQ ID NO:419 and/or a variable light chain sequence according to SEQ ID NO:423 (TPP-18206),k) a variable heavy chain sequence according to SEQ ID NO:437 and/or a variable light chain sequence according to SEQ ID NO:441 (TPP-18207),l) a variable heavy chain sequence according to SEQ ID NO:455 and/or a variable light chain sequence according to SEQ ID NO:459 (TPP-19546),m) a variable heavy chain sequence according to SEQ ID NO:473 and/or a variable light chain sequence according to SEQ ID NO:477 (TPP-20950),n) a variable heavy chain sequence according to SEQ ID NO:491 and/or a variable light chain sequence according to SEQ ID NO:495 (TPP-20955),o) a variable heavy chain sequence according to SEQ ID NO:509 and/or a variable light chain sequence according to SEQ ID NO:513 (TPP-20965),p) a variable heavy chain sequence according to SEQ ID NO:527 and/or a variable light chain sequence according to SEQ ID NO:531 (TPP-21045),q) a variable heavy chain sequence according to SEQ ID NO:545 and/or a variable light chain sequence according to SEQ ID NO:549 (TPP-21047),r) a variable heavy chain sequence according to SEQ ID NO:563 and/or a variable light chain sequence according to SEQ ID NO:567 (TPP-21181),s) a variable heavy chain sequence according to SEQ ID NO:581 and/or a variable light chain sequence according to SEQ ID NO:585 (TPP-21183),t) a variable heavy chain sequence according to SEQ ID NO:599 and/or a variable light chain sequence according to SEQ ID NO:603 (TPP-21360),u) a variable heavy chain sequence according to SEQ ID NO:617 and/or a variable light chain sequence according to SEQ ID NO:621 (TPP-23411),v) a variable heavy chain sequence according to SEQ ID NO:660 and/or a variable light chain sequence according to SEQ ID NO:664 (TPP-29596),w) a variable heavy chain sequence according to SEQ ID NO:680 and/or a variable light chain sequence according to SEQ ID NO:684 (TPP-29597),x) a variable heavy chain sequence according to SEQ ID NO:702 and/or a variable light chain sequence according to SEQ ID NO:706 (TPP-18429),y) a variable heavy chain sequence according to SEQ ID NO:722 and/or a variable light chain sequence according to SEQ ID NO:726 (TPP-18430),z) a variable heavy chain sequence according to SEQ ID NO:742 and/or a variable light chain sequence according to SEQ ID NO:746 (TPP-18432),aa) a variable heavy chain sequence according to SEQ ID NO:762 and/or a variable light chain sequence according to SEQ ID NO:766 (TPP-18433),bb) a variable heavy chain sequence according to SEQ ID NO:782 and/or a variable light chain sequence according to SEQ ID NO:786 (TPP-18436),cc) a variable heavy chain sequence according to SEQ ID NO:802 and/or a variable light chain sequence according to SEQ ID NO:806 (TPP-19571),dd) a variable heavy chain sequence according to SEQ ID NO:826 and/or a variable light chain sequence according to SEQ ID NO:830 (TPP-27477),ee) a variable heavy chain sequence according to SEQ ID NO:846 and/or a variable light chain sequence according to SEQ ID NO:850 (TPP-27478),ff) a variable heavy chain sequence according to SEQ ID NO:866 and/or a variable light chain sequence according to SEQ ID NO:870 (TPP-27479),gg) a variable heavy chain sequence according to SEQ ID NO:886 and/or a variable light chain sequence according to SEQ ID NO:890 (TPP-27480),hh) a variable heavy chain sequence according to SEQ ID NO:906 and/or a variable light chain sequence according to SEQ ID NO:910 (TPP-29367),11) a variable heavy chain sequence according to SEQ ID NO:926 and/or a variable light chain sequence according to SEQ ID NO:930 (TPP-29368), or a variable heavy chain sequence according to SEQ ID NO:946 and/or a variable light chain sequence according to SEQ ID NO:950 (TPP-29369).

[00531] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain sequence and/or a light chain sequence with at least 90%, 95%, 98% or 100% sequence identity with a) a heavy chain according to SEQ ID NO:273 and a light chain according to SEQ ID NO:274 (TPP-16966), b) a heavy chain according to SEQ ID NO:291 and a light chain according to SEQ ID NO:292 (TPP-17575), c) a heavy chain according to SEQ ID NO:309 and a light chain according to SEQ ID NO:310 (TPP-17576), d) a heavy chain according to SEQ ID NO:327 and a light chain according to SEQ ID NO:328 (TPP-17577),e) a heavy chain according to SEQ ID NO:345 and a light chain according to SEQ ID NO:346 (TPP-17578),f) a heavy chain according to SEQ ID NO:363 and a light chain according to SEQ ID NO:364 (TPP-17579),g) a heavy chain according to SEQ ID NO:381 and a light chain according to SEQ ID NO:382 (TPP-17580),h) a heavy chain according to SEQ ID NO:399 and a light chain according to SEQ ID NO:400 (TPP-17581),i) a heavy chain according to SEQ ID NO:417 and a light chain according to SEQ ID NO:418 (TPP-18205),j) a heavy chain according to SEQ ID NO:435 and a light chain according to with SEQ ID NO:436 (TPP-18206),k) a heavy chain according to SEQ ID NO:453 and a light chain according to SEQ ID NO:454 (TPP-18207),l) a heavy chain according to SEQ ID NO:471 and a light chain according to SEQ ID NO:472 (TPP-19546),m) a heavy chain according to SEQ ID NO:489 and a light chain according to SEQ ID NO:490 (TPP-20950),n) a heavy chain according to SEQ ID NO:507 and a light chain according to SEQ ID NO:508 (TPP-20955),o) a heavy chain according to SEQ ID NO:525 and a light chain according to SEQ ID NO:526 (TPP-20965),p) a heavy chain according to SEQ ID NO:543 and a light chain according to SEQ ID NO:544 (TPP-21045), q) a heavy chain according to SEQ ID NO:561 and a light chain according to SEQ ID NO:562 (TPP-21047), r) a heavy chain according to SEQ ID NO:579 and a light chain according to SEQ ID NO:580 (TPP-21181), s) a heavy chain according to SEQ ID NO:597 and a light chain according to SEQ ID NO:598 (TPP-21183), t) a heavy chain according to SEQ ID NO:615 and a light chain according to SEQ ID NO:616 (TPP-21360), u) a heavy chain according to SEQ ID NO:633 and a light chain according to SEQ ID NO:634 (TPP-23411), v) a heavy chain according to SEQ ID NO:676 and a light chain according to SEQ ID NO:677 (TPP-29596),w) a heavy chain according to SEQ ID NO:696 and a light chain according to SEQ ID NO:697 (TPP-29597),x) a heavy chain according to SEQ ID NO:718 and a light chain according to SEQ ID NO:719 (TPP-18429),y) a heavy chain according to SEQ ID NO:738 and a light chain according to SEQ ID NO:739 (TPP-18430),z) a heavy chain according to SEQ ID NO:758 and a light chain according to SEQ ID NO:759 (TPP-18432),aa) a heavy chain according to SEQ ID NO:778 and a light chain according to SEQ ID NO:779 (TPP-18433),bb) a heavy chain according to SEQ ID NO:798 and a light chain according to SEQ ID NO:799 (TPP-18436),cc) a heavy chain according to SEQ ID NO:818 and a light chain according to SEQ ID NO:819 (TPP-19571),dd) a heavy chain according to SEQ ID NO:842 and a light chain according to SEQ ID NO:843 (TPP-27477),ee) a heavy chain according to SEQ ID NO:862 and a light chain according to SEQ ID NO:863 (TPP-27478),ff) a heavy chain according to SEQ ID NO:882 and a light chain according to SEQ ID NO:883 (TPP-27479),gg) a heavy chain according to SEQ ID NO:902 and a light chain according to SEQ ID NO:903 (TPP-27480),hh) a heavy chain according to SEQ ID NO:922 and a light chain according to SEQ ID NO:923 (TPP-29367),11) a heavy chain according to SEQ ID NO:942 and a light chain according to SEQ ID NO:943 (TPP-29368), orjj) a heavy chain according to SEQ ID NO:962 and a light chain according to SEQ ID NO:963 (TPP-29369).

[00532] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises: a) a heavy chain according to SEQ ID NO:273 and a light chain according to SEQ ID NO:274 (TPP-16966), b) a heavy chain according to SEQ ID NO:291 and a light chain according to SEQ ID NO:292 (TPP-17575), c) a heavy chain according to SEQ ID NO:309 and a light chain according to SEQ ID NO:310 (TPP-17576), d) a heavy chain according to SEQ ID NO:327 and a light chain according to SEQ ID NO:328 (TPP-17577), e) a heavy chain according to SEQ ID NO:345 and a light chain according to SEQ ID NO:346 (TPP-17578),f) a heavy chain according to SEQ ID NO:363 and a light chain according to SEQ ID NO:364 (TPP-17579),g) a heavy chain according to SEQ ID NO:381 and a light chain according to SEQ ID NO:382 (TPP-17580),h) a heavy chain according to SEQ ID NO:399 and a light chain according to SEQ ID NO:400 (TPP-17581),i) a heavy chain according to SEQ ID NO:417 and a light chain according to SEQ ID NO:418 (TPP-18205),j) a heavy chain according to SEQ ID NO:435 and a light chain according to SEQ ID NO:436 (TPP-18206),k) a heavy chain according to SEQ ID NO:453 and a light chain according to SEQ ID NO:454 (TPP-18207),l) a heavy chain according to SEQ ID NO:471 and a light chain according to SEQ ID NO:472 (TPP-19546),m) a heavy chain according to SEQ ID NO:489 and a light chain according to SEQ ID NO:490 (TPP-20950),n) a heavy chain according to SEQ ID NO:507 and a light chain according to SEQ ID NO:508 (TPP-20955),o) a heavy chain according to SEQ ID NO:525 and a light chain according to SEQ ID NO:526 (TPP-20965),p) a heavy chain according to SEQ ID NO:543 and a light chain according to SEQ ID NO:544 (TPP-21045),q) a heavy chain according to the r) a heavy chain according to SEQ ID NO:579 and a light chain according to SEQ ID NO:580 (TPP-21181), s) a heavy chain according to SEQ ID NO:597 and a light chain according to SEQ ID NO:598 (TPP-21183), t) a heavy chain according to SEQ ID NO:615 and a light chain according to SEQ ID NO:616 (TPP-21360), u) a heavy chain according to SEQ ID NO:633 and a light chain according to SEQ ID NO:634 (TPP-23411), v) a heavy chain according to SEQ ID NO:676 and a light chain according to SEQ ID NO:677 (TPP-29596),w) a heavy chain according to SEQ ID NO:696 and a light chain according to SEQ ID NO:697 (TPP-29597),x) a heavy chain according to SEQ ID NO:718 and a light chain according to SEQ ID NO:719 (TPP-18429),y) a heavy chain according to SEQ ID NO:738 and a light chain according to SEQ ID NO:739 (TPP-18430),z) a heavy chain according to SEQ ID NO:758 and a light chain according to SEQ ID NO:759 (TPP-18432),aa) a heavy chain according to SEQ ID NO:778 and a light chain according to SEQ ID NO:779 (TPP-18433),bb) a heavy chain according to SEQ ID NO:798 and a light chain according to SEQ ID NO:799 (TPP-18436),cc) a heavy chain according to SEQ ID NO:818 and a light chain according to SEQ ID NO:819 (TPP-19571),dd) a heavy chain according to SEQ ID NO:842 and a light chain according to SEQ ID NO:843 (TPP-27477),ee) a heavy chain according to SEQ ID NO:862 and a light chain according to SEQ ID NO:863 (TPP-27478),ff) a heavy chain according to SEQ ID NO:882 and a light chain according to SEQ ID NO:883 (TPP-27479),gg) a heavy chain according to SEQ ID NO:902 and a light chain according to SEQ ID NO:903 (TPP-27480),hh) a heavy chain according with SEQ ID NO:922 and a light chain according to SEQ ID NO:923 (TPP-29367),11) a heavy chain according to SEQ ID NO:942 and a light chain according to SEQ ID NO:943 (TPP-29368), orjj) a heavy chain according to SEQ ID NO:962 and a light chain according to SEQ ID NO:963 (TPP-29369).

[00533] Several antibodies comprising random permutations of the CDRs of the anti-CCR8 antibodies provided with the sequence listings are also provided in accordance with the current aspect.

[00534] Preferably, the antibody or antigen-binding fragment according to the current aspect is non-fucosylated, as described elsewhere herein. Preferably, the antibody or antigen-binding fragment according to the current aspect induces ADCC and/or ADCP, as described elsewhere herein. Preferably, the antibody or antigen-binding fragment according to the current aspect is a non-internalizing or low-internalizing antibody, as described elsewhere herein.

ASPECT 18 - CCR8 ANTIMUrine ANTIBODY (SEQ DEFINED)

[00535] According to an 18th aspect, which may or may not be the same as aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and/or 16, an isolated anti-CCR8 antibody or its antigen-binding fragment is provided. Preferably, the antibodies according to the present aspect bind to murine CCR8, in particular to a polypeptide isolated according to SEQ ID NO:45 and/or SEQ ID NO:48, in which at least two or all of Y3, Y14 and Y15 have been sulfated. The CDRs of the antibodies according to the present aspect are preferably CDRs derived from humans. Furthermore, antibodies are characterized by properties that make them particularly suitable for therapy; for example, they are antibodies according to at least one or more of aspects 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In short, antibodies according to the present aspect can be used as surrogate antibodies that recognize murine CCR8 and possess superior therapeutic properties as described, for example, in Example 12.

[00536] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, five, or six CDR sequences with at least 90%, 95%, 98%, or 100% sequence identity with any of the following: a) SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:207, and SEQ ID NO:208 (TPP-14095), b) SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:221, or SEQ ID NO:222 (TPP-14099), c) SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:235 and SEQ ID NO:236 (TPP-15285), ord) SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:249 and SEQ ID NO:250 (TPP-15286).

[00537] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises an HCDR3 sequence with at least 90%, 95%, 98%, or 100% sequence identity with any of the SEQ ID NO:204, SEQ ID NO:218, SEQ ID NO:232, or SEQ ID NO:246.

[00538] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises at least one, two, and preferably three, four, five, or six CDR sequences according to a) SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:207, and SEQ ID NO:208 (TPP-14095), b) SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:221, or SEQ ID NO:222 (TPP-14099), c) SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:235, and SEQ ID NO:236 (TPP-15285), or) SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:249 and SEQ ID NO:250 (TPP-15286), optionally wherein up to one, two, three, four or five mutations have been introduced into at least one CDR.

[00539] For example, tyrosine can be exchanged for a positively charged amino acid, such as histidine, and vice versa. For example, a positively charged amino acid can be exchanged for a different positively charged amino acid, a negatively charged amino acid can be exchanged for a different negatively charged amino acid, a polar amino acid can be exchanged for a different polar amino acid, an uncharged polar amino acid can be exchanged for a different uncharged polar amino acid, a small amino acid can be exchanged for a different small amino acid, an amphiphatic amino acid can be exchanged for a different amphiphatic amino acid, an aromatic amino acid can be exchanged for a different aromatic amino acid. As understood by a person skilled in the art, in particular, those amino acid exchanges that do not alter the specific interaction between the antibody and the sulfated CCR8 TRD are possible. Amino acid exchanges that introduce histidine are preferred.

[00540] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises a variable heavy chain sequence and/or a variable light chain sequence with at least 90%, 95%, 98% or 100% sequence identity with a) a variable heavy chain sequence according to SEQ ID NO:201 and a variable light chain sequence according to SEQ ID NO:205 (TPP-14095), b) a variable heavy chain sequence according to SEQ ID NO:215 and a variable light chain sequence according to SEQ ID NO:219 (TPP-14099), c) a variable heavy chain sequence according to SEQ ID NO:229 and a variable light chain sequence according to SEQ ID NO:233 (TPP-15285), or d) a variable heavy chain sequence according to SEQ ID NO:243 and a variable light chain sequence according to SEQ ID NO:247 (TPP-15286).

[00541] According to some preferred embodiments, the anti-CCR8 antibody or antigen-binding fragment thereof comprises e) a variable heavy chain sequence according to SEQ ID NO:201 and a variable light chain sequence according to SEQ ID NO:205 (TPP-14095), f) a variable heavy chain sequence according to SEQ ID NO:215 and a variable light chain sequence according to SEQ ID NO:219 (TPP-14099), g) a variable heavy chain sequence according to SEQ ID NO:229 and a variable light chain sequence according to SEQ ID NO:233 (TPP-15285), or h) a variable heavy chain sequence according to SEQ ID NO:243 and a variable light chain sequence according to SEQ ID NO:247 (TPP-15286).

[00542] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain sequence and/or a light chain sequence with at least 90%, 95%, 98% or 100% sequence identity with a) a heavy chain according to SEQ ID NO:211 and/or a light chain according to SEQ ID NO:212 (TPP-14095), b) a heavy chain according to SEQ ID NO:225 and/or a light chain according to SEQ ID NO:226 (TPP-14099), c) a heavy chain according to SEQ ID NO:239 and/or a light chain according to SEQ ID NO:240 (TPP-15285), or d) a heavy chain according to SEQ ID NO:253 and/or a light chain according to SEQ ID NO:254 (TPP-15286).

[00543] According to some preferred embodiments, the isolated anti-CCR8 antibody or antigen-binding fragment thereof comprises a) a heavy chain according to SEQ ID NO:211 and/or a light chain according to SEQ ID NO:212 (TPP-14095), b) a heavy chain according to SEQ ID NO:225 and/or a light chain according to SEQ ID NO:226 (TPP-14099), c) a heavy chain according to SEQ ID NO:239 and/or a light chain according to SEQ ID NO:240 (TPP-15285), or d) a heavy chain according to SEQ ID NO:253 and/or a light chain according to SEQ ID NO:254 (TPP-15286).

Preferred combinations according to "all aspects"

[00544] The following modalities are particularly preferred modalities of aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 ("all aspects" according to this section).

[00545] In preferred embodiments in all respects, the antibody or antigen-binding fragment, the isolated antibody or antigen-binding fragment thereof, binds specifically to the sulfated tyrosine-rich domain of CCR8. In preferred embodiments in all respects, the antibody or antigen-binding fragment is characterized by an HCDR3 region comprising between 10 and 34% tyrosine and/or between 2 and 20% histidine. In preferred embodiments in all respects, the antibody or antigen-binding fragment is not internalized or is characterized by internalization in a cell with endogenous target expression that is less than 1.5, 2, 3, 4, 5, 6, 7, or 10 times the internalization of the isotype control.

[00546] In preferred embodiments in all respects, the antibody or antigen-binding fragment comprises human (derived) CDRs. For example, the antibody may be a human anti-human CCR8 antibody. In preferred embodiments in all respects, the antibody or antigen-binding fragment is cross-reactive to CCR8 of at least two species, preferably selected from humans, monkeys, macaques (cynomolgus macaques), macaques (rhesus macaques), rodents, mice, rats, horses, cattle, pigs, dogs, cats, and camels, even more preferably selected from humans, cynomolgus macaques, and mice. According to some of these more preferred embodiments, the antibody or antigen-binding fragment is cross-reactive to human and cynomolgus CCR8.

[00547] In preferred embodiments in all respects, the antibody or antigen-binding fragment binds to CCR8 of a first species with a first dissociation constant KD and binds to CCR8 of a second species with a second dissociation constant KD, wherein the first and second dissociation constants are of the same order of magnitude.

[00548] In preferred embodiments in all respects, the antibody or antigen-binding fragment binds with a KD value of < 5E-8 M, < 4E-8 M, < 3E-8 M, < 2E-8 M, < 1E-8 M, < 9E-9 M, < 8E-9 M, < 7E-9 M, < 6E-9 M, < 5E-9 M, < 4E-9 M, < 3E-9 M, < 2.5E-9 M, < 2E-9 M, < 1.5E-9 M, < 1E-9 M, < 9E-10 M, < 8E-10 M, < 7E-10 M, < 6E-10 M, < 5E-10 M, < 4E-10 M, < 3E-10 M, < 2.5E-10 M, < 2E-10 M, < 1.5E-10 M, < 1E-10 M, or < 9E-11 M) to a polypeptide isolate according to SEQ ID NO:43 and/or SEQ ID NO:46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and/or b) to a polypeptide isolate according to SEQ ID NO:44 and/or SEQ ID NO:47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and/or c) to a polypeptide isolate according to SEQ ID NO:45 and/or SEQ ID NO:48, wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

[00549] In preferred embodiments in all respects, the antibody or antigen-binding fragment (specifically) binds with an EC50 of < 15 nM, < 10 nM, < 5 nM, < 1 nM or < 0.6 nM) to human CCR8 and/or to a polypeptide isolate according to SEQ ID NO:43 and/or SEQ ID NO:46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and/or b) to cynomolgus CCR8 and/or to a polypeptide isolate according to SEQ ID NO:44 and/or SEQ ID NO:47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and/or c) to murine CCR8 and/or to a polypeptide isolate according to SEQ ID NO:45 and/or SEQ ID NO:48, wherein at least two or All of Y3, Y14, and Y15 were sulfated, and optionally bind with an EC50 of < 50 nM, < 25 nM, < 15 nM, or < 10 nM to activated human regulatory T cells.

[00550] According to some of these more preferred embodiments, the EC50 of the antibody or antigen-binding fragment for binding to human CCR8 or to an isolated polypeptide according to SEQ ID NO:46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM and the EC50 of the antibody or antigen-binding fragment for binding to cynomolgus CCR8 or to an isolated polypeptide according to SEQ ID NO:47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated, is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

[00551] As described, for example, in Example 10.1.1, the antibodies according to the present invention have excellent affinities for their respective targets. For example, cross-reacting antibodies TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360 and TPP-23411 bound human CCR8 with an EC50 of 4.8 nM, 1.7 nM, 0.8 nM, 0.6 nM, ~0.9 nM or 1.7 nM. Furthermore, TPP-21181, TPP-17578, TPP-19546, TPP-18206, TPP-21360, and TPP-23411 bound cynomolgus CCR8 with an EC50 of 1.8 nM, 1 nM, 0.5 nM, 0.7 nM, ~0.55 nM, or 0.9 nM. Additionally, TPP-17578, TPP-19546, TPP-18206, and TPP-21360 bound human regulatory T cells with an EC50 of 25 nM, 15 nM, 23 nM, or 10 nM. Furthermore, the anti-murine CCR8 antibody TPP-14099 binds murine CCR8-expressing CHO cells with an EC50 of 3 nM and murine iTregs with an EC50 of 13.2 nM, as shown in Table 10.1.1.5.

[00552] In preferred embodiments in all respects, the antibody or antigen-binding fragment, preferably at a concentration of 10 nM, a) does not or does not substantially or to a lesser degree than L263G8 and 433H antibodies block CCL1-induced β-arrestin signaling, and/or b) does not or does not substantially or to a lesser degree than L263G8 and 433H antibodies induce ERK1/2 phosphorylation, and/or c) does not or does not substantially or to a lesser degree than L263G8 and 433H antibodies induce AKT phosphorylation.

[00553] In preferred embodiments of all respects, the antibody or antigen-binding fragment blocks G protein-dependent signaling of the chemokine receptor.

[00554] In preferred embodiments in all respects, the antibody or antigen-binding fragment is non-fucosylated.

[00555] In preferred embodiments in all respects, the antibody or antigen-binding fragment a) binds to the human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM and/or b) binds to the human Fc gamma receptor RIIA (CD32a) with a dissociation constant (KD) less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

[00556] In preferred embodiments in all respects, the antibody or antigen-binding fragment a) induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells, such as human NK cells and/or b) induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells, such as human macrophages.

[00557] In preferred embodiments in all respects, the antibody or antigen-binding fragment induces ADCC and/or ADCP and a) the maximum ADCC-induced depletion of activated human regulatory T cells is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, and/or b) the maximum ADCP-induced depletion of activated human regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, and/or c) the maximum depletion of intratumoral regulatory T cells, in vitro or in an individual, is at least 50%, 60%, 70%, 80%, 90%, 95% or 99%.

[00558] In preferred embodiments in all respects, the EC50 of the antibody or antigen-binding fragmenta) for ADCC-induced depletion of activated human regulatory T cells is less than 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM or 5 pM and/orb) for ADCP-induced depletion of activated human regulatory T cells is less than 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM or 25 pM.

[00559] In preferred embodiments in all respects, an effective dose of the antibody or antigen-binding fragment a) decreases the number of activated or intratumoral regulatory T cells, in vitro or in an individual, to less than 30%, 25%, 20%, 10%, 5% or 1% and/or b) increases the ratio of intratumoral CD8+ T cells to intratumoral Tregs, in vitro or in an individual, to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or higher and/or c) decreases the percentage of regulatory T cells to intratumoral CD4+ T cells, in vitro or in an individual, to < 30%, < 20%, < 10% or < 5 %.

[00560] In preferred embodiments in all respects, the antibody or antigen-binding fragment increases the absolute number of immune cells in a defined tumor volume by at least a factor of 1.5, 2, 2.5, 3, 3.5, or 4, preferably wherein the immune cells are selected from a) CD45+ cells (intratumoral), b) CD8+ T cells (intratumoral), c) CD4+ T cells (intratumoral), d) Macrophages (intratumoral), such as M1 macrophages or M2 macrophages, e) NK cells (intratumoral), f) B cells (intratumoral), g) Dendritic cells (intratumoral), h) Gamma delta T cells (intratumoral), i) iNKT cells (intratumoral), or any combination thereof.

[00561] For example, the cells of the immune system are a) CD8+ T cells, CD4+ T cells and macrophages, or b) CD8+ T cells, CD4+ T cells, NK cells and macrophages, or c) CD8+ T cells, ACOD1+ macrophages (M1 macrophages) and B cells.

[00562] In preferred embodiments in all respects, three or more effective doses of the antibody or antigen-binding fragment in a tumor increase a) the number of intratumoral CD8+ T cells by at least 150%, 200%, 250%, 300%, or 350%, the ratio of intratumoral CD8+ T cells to intratumoral regulatory T cells by at least 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or higher, b) the number of intratumoral macrophages by at least a factor of 2, 3, 4, or 5, c) the number of intratumoral ACOD1+ macrophages (M1 macrophages) by at least a factor of 2, 3, 4, 5, 6, 7, 8, 9 or 10, d) the ratio of ACOD1+ macrophages (M1 macrophages) to MRC1+ macrophages (M2 macrophages) by at least 1, 5, 2, 3, 4, 5, 10 or more, e) the number of intratumoral NK cells by at least 140% or 200%, f) the number of intratumoral CD3+ T cells by at least 150%, 200%, 300% or 400%, g) the number of intratumoral B cells by at least a factor of 2, 5, 10, 20, 30 or 40, h) the number of intratumoral CD45+ T cells by at least 150%, 200% or 300%, and/or i) the number of T cells Intratumoral CD4+ counts of at least 150%, 200%, 300%, or 400%, preferably in tumors characterized by tumor-infiltrating lymphocytes, such as tumor-infiltrating T cells.

[00563] In preferred modalities of all respects, the antibody or antigen-binding fragment induces the formation of tertiary lymphoid structures.

[00564] In preferred embodiments in all respects, the antibody is an IgG antibody, preferably a human IgG1 or a murine IgG2a.

[00565] In preferred embodiments in all respects, the antibody or antigen-binding fragment binds with an EC50 of < 15 nM, < 10 nM, < 5 nM, < 1 nM or < 0.6 nM) to human CCR8 or to a polypeptide isolate according to SEQ ID NO:46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and/or b) to cynomolgus CCR8 or to a polypeptide isolate according to SEQ ID NO:47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and/or c) to murine CCR8 or to a polypeptide isolate according to SEQ ID NO:48, wherein at least two or all of Y3, Y14 and Y15 have been sulfated.

[00566] In preferred embodiments in all respects, the EC50 of the antibody or antigen-binding fragment for binding to human CCR8 or to an isolated polypeptide according to SEQ ID NO:46, in which at least two or all of Y3, Y15 and Y17 have been sulfated, is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM and the EC50 of the antibody or antigen-binding fragment for binding to cynomolgus CCR8 or to an isolated polypeptide according to SEQ ID NO:47, in which at least two or all of Y3, Y15 and Y17 have been sulfated, is below 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM or 0.25 nM.

[00567] In preferred embodiments in all respects, the isolated antibody or antigen-binding fragment binds with an EC50 of < 50 nM, < 25 nM, < 15 nM or < 10 nM to activated human regulatory T cells.

[00568] In preferred embodiments in all respects, the antibody dissociation constant for binding the first isolated non-sulfated polypeptide is greater than 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.25 μM, 1.5 μM, 1.75 μM, 2 μM, 2.25 μM, 2.5 μM, 2.75 μM or 3 μM, or is not detectable. Preferably, the antibody dissociation constant for binding the first isolated non-sulfated polypeptide is greater than 100 nM, 250 nM, 500 nM, 1 μM, 2 μM, or 3 μM, or it is not detectable.

Most preferred combinations of "all aspects"

[00569] According to a preferred embodiment I, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment is not internalized or is characterized by internalization into a cell with endogenous target expression that is less than 1.5, 2, 3, 4, 5, 6, 7 or 10 times the internalization of the isotype control.

[00570] According to a preferred embodiment II, an isolated antibody or antigen-binding fragment thereof is provided, which binds specifically to CCR8, wherein the antibody or fragment is characterized by an HCDR3 region comprising between 10 and 34% tyrosine and/or between 2 and 20% histidine.

[00571] According to a preferred embodiment III, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein the antibody comprises human-derived CDRs.

[00572] According to a preferred embodiment IV, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment is cross-reactive for CCR8 of at least two species, preferably selected from human, cynomolgus and mouse, more preferably wherein the antibody or antigen-binding fragment is cross-reactive for human and cynomolgus CCR8.

[00573] According to a preferred embodiment V, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment a. does not block CCL1-induced β-arrestin signaling and/or b. does not induce ERK1/2 phosphorylation and/or c. does not induce AKT phosphorylation.

[00574] According to a preferred embodiment VI, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to CCR8, wherein the antibody or antigen-binding fragment is non-fucosylated and a. induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells, such as human NK cells, and b. induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells, such as human macrophages, c. wherein the maximum in vitro depletion induced by ADCC and ADCP of target cells expressing human CCR8 is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%.

[00575] According to a preferred embodiment VII, the isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments II to VI, wherein the antibody or antigen-binding fragment is not internalized or is characterized by internalization into a target endogenously expressing cell that is less than 1.5, 2, 3, 4, 5, 6, 7, or 10 times the internalization of the isotype control.

[00576] According to a preferred embodiment VIII, the isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments I, or III to VII, wherein the antibody or antigen-binding fragment is characterized by an HCDR3 region comprising between 10 and 34% tyrosine and/or between 2 and 20% histidine.

[00577] The isolated antibody or antigen-binding fragment is provided, according to a preferred embodiment IX, according to any of the preferred embodiments I to II, or IV to VIII, wherein the antibody comprises human-derived CDRs.

[00578] The isolated antibody or antigen-binding fragment is provided according to a preferred embodiment X, according to any of the preferred embodiments I to III, or V to IX, wherein the antibody or antigen-binding fragment is cross-reactive for CCR8 of at least two species, preferably selected from human, cynomolgus and mouse, more preferably wherein the antibody or antigen-binding fragment is cross-reactive for human and cynomolgus CCR8.

[00579] According to a preferred embodiment XI, the isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments I to X, wherein the antibody or antigen-binding fragment binds the CCR8 of a first species with a first dissociation constant KD and binds the CCR8 of a second species with a second dissociation constant KD, wherein the first and second dissociation constants are of the same order of magnitude.

[00580] According to a preferred embodiment XII, the isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments I to IV, or VI to XI, wherein the antibody or antigen-binding fragment, a. does not block CCL1-induced β-arrestin signaling and/or b. does not induce ERK1/2 phosphorylation and/or c. does not induce AKT phosphorylation.

[00581] According to a preferred embodiment XIII, the isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments I to V, or VII to XII, wherein the antibody or antigen-binding fragment a. induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells, such as human NK cells, and b. induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells, such as human macrophages, and optionally c. wherein the maximal in vitro depletion induced by ADCC and ADCP of target cells expressing human CCR8 is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95% or 99%.

[00582] According to a preferred embodiment XIV, the isolated antibody or antigen-binding fragment is provided according to any of the preferred embodiments I to XIII, wherein the antibody or antigen-binding fragment binds specifically to the sulfated tyrosine-rich domain of CCR8.

[00583] Provided according to a preferred embodiment XV, the isolated antibody or antigen-binding fragment according to any of the preferred embodiments I to XIV, wherein the antibody or antigen-binding fragment binds specifically (i) with an EC50 of < 15 nM, < 10 nM, < 5 nM, < 1 nM or < 0.6 nM to human CCR8 and/or to a polypeptide isolate according to SEQ ID NO:46, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and/or b) to cynomolgus CCR8 and/or to a polypeptide isolate according to SEQ ID NO:47, wherein at least two or all of Y3, Y15 and Y17 have been sulfated and/or c) to murine CCR8 and/or to an isolated polypeptide according to SEQ ID NO:48, in which at least two or all of Y3, Y14 and Y15 have been sulfated, and/or (ii) with EC50 < 50 nM, < 25 nM, < 15 nM or < 10 nM for activated human regulatory T cells.

[00584] According to a preferred embodiment XVI, the isolated antibody or antigen-binding fragment thereof is provided according to any of the preferred embodiments I to XV,a. wherein the antibody or antigen-binding fragment binds to the human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) less than 530 nM, 500 nM, 450 nM, 400 nM, 300 nM or 200 nM and/or b. wherein the antibody or antigen-binding fragment binds to the human Fc gamma receptor RIIA (CD32a) with a dissociation constant (KD) less than 30 μM, 20 μM, 10 μM, 5 μM or 1 μM.

[00585] It is provided, according to a preferred embodiment XVII, the isolated antibody or antigen-binding fragment thereof according to any of the preferred embodiments I to XVI, wherein the antibody or antigen-binding fragment is fucosylated.

[00586] According to a preferred embodiment XVIII, the isolated antibody or antigen-binding fragment thereof is provided according to any of the preferred embodiments I to XVII, wherein the antibody or antigen-binding fragment a. induces antibody-dependent cell-mediated cytotoxicity (ADCC) in target cells expressing human CCR8 via human effector cells, such as human NK cells and/or b. induces antibody-dependent cell-mediated phagocytosis (ADCP) in target cells expressing human CCR8 via human effector cells, such as human macrophages, preferably where c. the maximum ADCC-induced depletion of activated human regulatory T cells is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, and/or d. the maximum ADCP-induced depletion of activated human regulatory T cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, and/or e. The maximum in vitro depletion of activated human regulatory T cells induced by ADCC and ADCP is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 99%.

[00587] Provided according to a preferred embodiment XIX is the isolated antibody or antigen-binding fragment according to any of preferred embodiments I to XVIII, wherein a. the EC50 of the antibody or antigen-binding fragment for ADCC-induced depletion of activated human regulatory T cells is below 100 pM, 50 pM, 25 pM, 12.5 pM, 10 pM or 5 pM and/or b. the EC50 of the antibody or antigen-binding fragment for ADCP-induced depletion of activated human regulatory T cells is below 500 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM or 25 pM.

[00588] The isolated antibody or antigen-binding fragment is provided according to a preferred embodiment XX, according to any of the above preferred embodiments, wherein an effective dose of the antibody or antigen-binding fragment a. decreases the number of activated or intratumoral regulatory T cells, in vitro or in an individual, to less than 50%, 40%, 30%, 25%, 20%, 10%, 5% or 1% and/or b. increases the ratio of intratumoral CD8+ T cells to intratumoral Tregs, in vitro or in an individual, to at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or higher and/or c. Decreases the percentage of regulatory T cells compared to intratumoral CD4+ T cells, in vitro or in an individual, to < 30%, < 20%, < 10% or < 5%.

[00589] It is provided, according to a preferred embodiment XXI, the isolated antibody or antigen-binding fragment according to any previous preferred embodiment, wherein an effective dose of the antibody or antigen-binding fragment induces the formation of tertiary lymphoid structures in a tumor.

[00590] According to a preferred embodiment XXII, the isolated antibody or antigen-binding fragment thereof is provided according to any of the above preferred embodiments, wherein the antibody is an IgG antibody, preferably a human IgG1 or a murine IgG2a, and/or wherein the antigen-binding fragment is an scFv, Fab, Fab' or F(ab')2 fragment.

[00591] A conjugate comprising an antibody or antigen-binding fragment according to any of the preferred embodiments I to XXII is provided, according to a preferred embodiment XXIII, preferably wherein the conjugate comprises a. a radioactive element, b. a cytotoxic agent, such as an auristatin, a maytansinoid, a kinesin spindle protein inhibitor, a nicotinamide phosphoribosyltransferase inhibitor or a pyrrolobenzodiazepine derivative, c. an additional antibody or antigen-binding fragment, or d. a chimeric antigen receptor.

[00592] A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any of preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII is provided, in accordance with preferred embodiment XXIV.

[00593] A pharmaceutical composition according to preferred embodiment XXIV is provided, comprising one or more other therapeutically active compounds, preferably selected from: a. an antibody or compound targeting a checkpoint protein, such as PD1, PD-L1 or CTLA-4, preferably wherein the antibody or compound targeting the checkpoint protein is Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab or Ipilimumab, b. an antibody targeting another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, c. an antibody or small molecule targeting HER2 and/or EGFR,d. the standard of care for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma and esophageal tumor, melanoma, bladder cancer, liver cancer and/or prostate cancer,e. a chemotherapeutic agent, preferably a taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin or gemcitabine,f. a B-cell depleting agent, such as an anti-CD19 antibody or an anti-CD20 antibody and/org. a targeted kinase inhibitor, such as Sorafinib, Regorafenib or MEKi-1.

[00594] An antibody or antigen-binding fragment according to any of the preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII or a pharmaceutical composition according to preferred embodiment XXIV or XXV is provided for use as a medicament.

[00595] The antibody or antigen-binding fragment according to any of preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII or a pharmaceutical composition according to preferred embodiment XXIV or XXV is provided for use in the treatment of a tumor or disease characterized by CCR8-positive cells, such as CCR8-positive regulatory T cells.

[00596] An antibody or antigen-binding fragment according to any of preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII or a pharmaceutical composition according to preferred embodiment XXIV or XXV is provided for simultaneous, separate, or sequential combination use (i) with one or more therapeutically active compounds, preferably selected from a. an antibody or small molecule targeting a checkpoint protein, such as PD1, PD-L1 or CTLA-4, preferably wherein the antibody or small molecule targeting a checkpoint protein is Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab or Ipilimumab, b. an antibody that targets another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1; c. an antibody that targets a protein that is specifically expressed by tumor cells; d. an antibody or small molecule that targets HER2 and/or EGFR; e. the standard of care for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer; f. a chemotherapeutic agent, preferably a taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin or gemcitabine and/or (i) a targeted kinase inhibitor, such as sorafinib, regorafenib or MEKi-1, and/or (ii) with radiation therapy, and/or (iii) with intra-tumoral B cell depletion, in the treatment of a tumor or disease characterized by CCR8-positive cells, such as CCR8-positive regulatory T cells.

[00597] The antibody, fragment, conjugate or pharmaceutical composition is provided, according to a preferred embodiment XXIX, for use according to preferred embodiment XXVII or XXVIII, wherein the tumor is selected from the group of acute lymphoblastic leukemia, T-cell breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, squamous cell carcinoma of the head and neck, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma and prostate cancer, B-cell lymphoma, T-cell lymphoma or any other cancer involving cells expressing CCR8, preferably wherein the tumor is selected from head and neck cancer, breast cancer, Gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, prostate cancer, B-cell lymphoma, and T-cell lymphoma. The antibody, fragment, conjugate, or pharmaceutical composition is provided, according to a preferred embodiment XXX, for use according to preferred embodiments XXVI to XXIX, wherein the use comprises determining: a. the presence or quantity of tumor-infiltrating lymphocytes; b. the presence or quantity of macrophages and/or NK cells; c. the presence or quantity of CCR8-positive or FOXP3-positive regulatory T cells; d. the mutational load of the tumor; e. the cancer staging; f. the presence, level, or activation of interferon-stimulated genes or proteins; g. the expression of CCR8; h. the presence or quantity of complement factor proteins, serpins, and/or MHC components; i. the presence or quantity of cytokines, such as inflammatory or suppressive cytokines, j. the activation of immune gene expression and/or k. the expression of an immune checkpoint (protein), such as PD-(L)1 or CTLA4 expression, l. the presence or quantity of CD19+ B cells infiltrating the tumor, m. the presence or quantity of CD8+ T cells infiltrating the tumor; to predict or monitor treatment success.

[00598] A polynucleotide encoding an antibody or antigen-binding fragment according to any of preferred embodiments I to XXII is provided, according to a preferred embodiment XXXI.

[00599] A vector comprising a polynucleotide according to preferred embodiment XXXII is provided.

[00600] A single cell arranged for the production of an antibody or antigen-binding fragment according to any of the preferred embodiments I to XXII is provided, according to a preferred embodiment XXXIII.

[00601] A method for producing an antibody or antigen-binding fragment according to any of preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII is provided, comprising culturing a cell according to preferred embodiment XXXIII and optionally purifying the antibody or antigen-binding fragment.

[00602] The antibody or antigen-binding fragment according to any of the preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII is provided for use as an in vivo or in vitro diagnostic agent, in accordance with a preferred embodiment XXXV.

[00603] A kit comprising an antibody or antigen-binding fragment according to any of the preferred embodiments I to XXII or a conjugate according to preferred embodiment XXIII or a pharmaceutical composition according to preferred embodiment XXIV or XXV with instructions for use is provided, in accordance with preferred embodiment XXXVI.

ASPECT 19 - ANTIBODY CONJUGATES

[00604] In addition to naked antibodies, several other antibody- or antibody fragment-based conjugates can be designed using the antibodies or antigen-binding fragments described herein. These conjugates can be used for diagnostic, therapeutic, research applications, and various other purposes. For example, antibodies or antigen-binding fragments thereof are provided conjugated with radionuclides, cytotoxic agents, organic compounds, protein toxins, immunomodulators such as cytokines, fluorescent moieties, cells, other antibodies, or antigen-binding fragments thereof.

[00605] The conjugates described herein, for example, antibody-drug conjugates (ADCs), targeted thorium conjugates (TTCs), bispecific antibodies, etc., are of a "modular" nature. Throughout the invention, various specific embodiments of the various "modules" that make up the conjugates are described. As specific, non-limiting examples, specific embodiments of antibodies or fragments thereof, ligands, and cytotoxic and/or cytostatic agents that may compose the ADCs are described. It is intended that all the specific embodiments described can be combined with each other as if each specific combination were explicitly described individually.

[00606] According to aspect 19, a conjugate is provided comprising an antibody or antigen-binding fragment according to aspect 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18 or a combination thereof. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 6. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 7. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 8. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 9. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 10. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 11. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 12. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 13. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 14. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 15. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 16. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 17. For example, the conjugate comprises an antibody or antigen-binding fragment according to aspect 18.

[00607] According to some preferred embodiments, the conjugate comprises (a) a radioactive element, (b) a cytotoxic agent, such as an auristatin, a maytansinoid, a kinesin spindle protein (KSP) inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor or a pyrrolobenzodiazepine derivative, (c) an additional antibody or antigen-binding fragment, (d) a chimeric antigen receptor.

Radioconjugates

[00608] According to some early embodiments of the 19th aspect, the conjugate comprises or is arranged to comprise a radionuclide, such as a beta particle, an alpha particle, or an Auger electron emitter. Suitable beta emitters according to the present invention are, for example, yttrium-90, iodine-131, strontium-89 chloride, lutetium-177, holmium-166, rhenium-186, rhenium-188, copper-67, promethium-149, gold-199, and rhodium-105. Suitable Auger electron emitters according to the present invention are, for example, bromine-77, indium-111, iodine-123, and iodine-125. Suitable alpha emitters according to the present invention are, for example, thorium-227, bismuth-213, radium-223, actinium-225 and astatine-211.

[00609] For example, thorium-227 (227Th) can be effectively complexed with octadentate 3,2-hydroxypyridinone (3,2-HOPO) chelators that are conjugated with antibodies according to the present invention, resulting in highly stable targeted thorium-227 conjugates (TTCs), as described in Example 13. The targeted thorium conjugates (TTCs) comprise three main building blocks. After the decay of β particles of actinium-227, the first building block, the α particle-emitting radionuclide 227Th is purified by ion-exchange chromatography. The second building block is a chelating agent, such as a siderophore-derived chelating agent containing HOPO groups with four 3-hydroxy-N-methyl-2-pyridinone moieties in a symmetrical polyamine scaffold functionalized with a carboxylic acid ligand for bioconjugation. Conjugation with a targeting moiety can be achieved through amide linkage formation with the ε-amino groups of lysine residues. These octadentate 3,2-HOPO chelating agents can be effectively labeled with 227Th, with high yield, purity, and stability under ambient conditions. Compared to the tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating agent, which often requires heating, HOPO chelating agents are superior due to efficient radiolabeling at room temperature and high stability of the complexes formed. The third building block is the targeting portion, which is the chemokine receptor antibody or antigen-binding fragment thereof according to any of aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

ADCs

[00610] According to some second embodiments of the 19th aspect, the conjugate comprises a cytotoxic agent, for example, to form an antibody-drug conjugate (ADC).

[00611] In some preferred embodiments, the cytotoxic agent is an auristatin, a maytansinoid, a kinesin spindle protein (KSP) inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, or a pyrrolobenzodiazepine derivative. Generation of conjugates comprising maytansinoid may occur as described in Chari, Ravi VJ, et al. "Immunoconjugates containing novel maytansinoids: promising anticancer drugs." Cancer research 52.1 (1992): 127131, or EP2424569 B1, both of which are incorporated herein in their entirety. Generation of conjugates comprising kinesin spindle protein (KSP) inhibitors may occur as described in WO2019243159 A1, incorporated herein in its entirety. The generation of conjugates comprising a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor can occur as described in WO2019149637 A1, incorporated herein in its entirety. The generation of conjugates comprising a pyrrolobenzodiazepine can be obtained as described in EP3355935 A1, incorporated herein in its entirety.

[00612] The cytotoxic and/or cytostatic agent of the antichemokine receptor or anti-CCR8 ADC may be any agent known to inhibit growth and/or replication and/or kill cells. Numerous agents with cytotoxic and/or cytostatic properties are known in the literature. Non-limiting examples of classes of cytotoxic and/or cytostatic agents include, by way of example and not limitation, cell cycle modulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, alkylating agents, DNA cross-linking agents, intercalating agents, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, and antimitotic agents.

[00613] The ligands that link the cytotoxic and/or cytostatic agent(s) to the antigen-binding portion of an antichemokine receptor or anti-CCR8 ADC may be long, short, flexible, rigid, hydrophilic, or hydrophobic in nature, or may comprise segments that have different characteristics, such as flexible segments, rigid segments, etc. The ligand may be chemically stable in extracellular environments, for example, chemically stable in the bloodstream, or it may include linkages that are not stable and release the cytotoxic and/or cytostatic agents into the extracellular medium. In some embodiments, the linkages include linkages that are designed to release the cytotoxic and/or cytostatic agents by internalizing the antichemokine receptor or anti-CCR8 ADC within the cell. In some specific embodiments, the ligands include linkers designed to cleave and/or immolate or otherwise break down specifically or non-specifically within cells. A wide variety of ligands useful for linking drugs to antigen-binding moieties, such as antibodies in the context of ADCs, are known in the art. Any of these ligands, as well as other ligands, can be used to link cytotoxic and/or cytostatic agents to the antigen-binding moiety of the antichemokine receptor or anti-CCR8 ADCs, described herein.

[00614] The number of cytotoxic and/or cytostatic agents bound to the antigen-binding portion of an antichemokine or anti-CCR8 ADC receptor (drug-antibody ratio: DAR) may vary and will be limited only by the number of binding sites available in the antigen-binding portion and the number of agents bound to a single ligand. Typically, a ligand will bind a single cytotoxic and/or cytostatic agent to the antigen-binding portion of an antichemokine or anti-CCR8 ADC receptor. In antichemokine or anti-CCR8 ADC receptor modalities that include more than one cytotoxic and/or cytostatic agent, each agent may be the same or different. Provided that the antichemokine or anti-CCR8 ADC receptor does not exhibit unacceptable levels of aggregation under conditions of use and/or storage, antichemokine or anti-CCR8 ADC receptors with DARs of twenty or more are contemplated. In some embodiments, the antichemokine receptor or anti-CCR8 ADCs described herein may have a DAR in the range of about 1-10, 1-8, 1-6 or 1-4. In certain specific embodiments, the antichemokine receptor or anti-CCR8 ADC may have a DAR of 2, 3 or 4. In some embodiments, the antichemokine receptor or anti-CCR8 ADC are composed according to the structural formula (I):[DL-XY]n-Ab (I) or salts thereof, where each “D” represents, independently of the others, a cytotoxic and/or cytostatic agent; each “L” represents, independently of the others, a ligand; “Ab” represents a binding moiety to the antichemokine receptor, for example, an anti-CCR8 antibody according to the present invention; Each "XY" represents a bond formed between an Rx functional group in the ligand and a "complementary" Ry functional group in the antichemokine receptor-binding portion; and n represents the DAR of the antichemokine receptor ADC.

[00615] In a specific exemplary embodiment, the antichemokine receptor ADCs are composed according to structural formula (I) wherein each “D” is equal to and is a cell-permeable auristatin (e.g., dolastatin-10 or MMAE) or a DNA crosslinking agent binding to the minor cell-permeable grooves; each “L” is equal to and is a lysosomal enzyme-cleavable ligand; each “XY” is a linkage formed between a maleimide and a sulfhydryl group; “Ab” is an antibody or fragment thereof comprising six CDRs corresponding to the six CDRs of an antichemokine receptor or CCR8 antibody according to the present invention; and n is 2, 3, or 4. In a specific embodiment, “Ab” is a fully human antibody comprising human-derived CDRs.

[00616] Cytotoxic and cytostatic agents are agents known to inhibit the growth and/or replication and/or death of cells, and in particular, tumor cells or intratumoral Treg cells. These compounds can be used in combination therapy with an antichemokine receptor antibody, such as a CCR8 antibody, or as part of an antichemokine receptor ADC, as described here:

[00617] In some embodiments, the drug portion of the antichemokine receptor or anti-CCR8 ADC is a cytostatic agent selected from radionuclides, alkylating agents, DNA crosslinking agents, DNA intercalating agents (e.g., sulcus linking agents, such as minor sulcus ligands), cell cycle modulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, and antimitotic agents.

[00618] In some embodiments, the antichemokine receptor drug moiety or anti-CCR8 ADC is an alkylating agent selected from (L-Leucine, N—[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanine]-, ethyl ester); AZQ (1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester); BCNU (N,N'-Bis(2-chloroethyl)-N-nitrosourea); busulfan (1,4-butanediol dimethanesulfonate); (carboxyphthalate)platinum; CBDCA (cis-(1,1-cyclobutanedicarboxylate)diaminoplatinum(II))); CCNU (N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea); CHIP (iproplatin; NSC 256927); chlorambucil; chlorozotocin (2-[[[(2-chloroethyl) nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose); cis-platinum (cisplatin); clomesone; cyanomorpholinodoxorubicin; cyclodysone; dianhydrogalactitol (5,6-diepoxyducitol); fluorodopan ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyluracil); hepsulfam; hycantone; indolinobenzodiazepine dimer DGN462; melphalan; methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea); mitomycin C; mitozolamide; nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride); PCNU ((1-(2-chloroethyl)-3-(2,6)-dioxo-3-piperidyl)-1-nitrosourea)); piperazine alkylating agent ((1-(2-chloroethyl)-4-(3-chloropropyl)piperazine dihydrochloride)); piperazinodione; pipobroman (N,N'-bis(3-bromopropionyl)piperazine); porphyromycin (N-methylmitomycin C); spirohydantoin mustard; teroxirone (triglycidyl lysocyanurate); tetraplatinum; thio-tepa (N,N',N'-tri-1,2-ethanediylthiophosphoramide); triethylenemelamine; uracil nitrogen mustard (desmethyldopan); Yoshi-864 (bis(3-mesyloxypropyl)amine hydrochloride).

[00619] In some embodiments, the antichemokine receptor drug moiety or anti-CCR8 ADC is a DNA alkylation-like agent selected from Cisplatin; carboplatin; Nedaplatin; Oxaliplatin; Satraplatin; triplatin tetranitrate; Procarbazine; altretamine; dacarbazine; mitozolomide; temozolomide.

[00620] In some embodiments, the antichemokine receptor drug portion or anti-CCR8 ADC is an alkylating antineoplastic agent selected from Carboquone; Carmustine; Chlornaphazine; Chlorozotocin; Duocarmycin; Evofosfamide; Fotemustine; Glufosfamide; Lomustine; Mannosolfane; Nimustine; Phenantiplatin; Pipobroman; Ranimustine; Semustine; Streptozotocin; ThioTEPA; Treosulfan; Triaziquone; Triethylenemelamine; Triplatin tetranitrate.

[00621] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a DNA replication and repair inhibitor selected from Altretamine; Bleomycin; Dacarbazine; Dactinomycin; mitobronitol; mitomycin; Pingiangmycin; Plicamycin; Procarbazine; Temozolomide; ABT-888 (veliparib); olaparib; KU-59436; AZD-2281; AG-014699; BSI-201; BGP-15; INO-1001; ONO-2231.

[00622] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a cell cycle modulator, such as Paclitaxel; Nab-Paclitaxel; Docetaxel; Vincristine; Vinblastine; ABT-348; AZD-1152; MLN-8054; VX-680; Aurora A-specific kinase inhibitors; Aurora B-specific kinase inhibitors and pan-Aurora kinase inhibitors; AZD-5438; IMC-1040; BMS-032; BMS-387; CVT-2584; flavopiridol; GPC-286199; MCS-5A; PD0332991; PHA-690509; seliciclib (CYC-202, R-roscovitine); ZK-304709; AZD4877, ARRY-520: GSK923295A.

[00623] In some embodiments, the drug portion of the antichemokine receptor or anti-CCR8 ADC is an apoptosis regulator, such as AT-101 ((-)gssypol); G3139 or oblimersen (antisense oligonucleotide targeting Bcl-2); IPI-194; IPI-565; N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazin-1-ylbenzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide); N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl)propyl)amino)- 3-((trifluoromethyl)sulfonyl)benzenesulfonamide; GX-070 (Obatoclax®; 1H-Indole, 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-)); HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; veneto- clax; Agents that target TRAIL or death receptors (e.g., DR4 and DR5), such as ETR2-ST01, GDC0145, HGS-1029, LBY-135, PRO-1762; drugs that target caspases, caspase regulators, BCL-2 family members, death domain proteins, TNF family members, Toll family members, and/or NF-kappa-B proteins.

[00624] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is an angiogenesis inhibitor, such as ABT-869; AEE-788; axitinib (AG-13736); AZD-2171; CP-547632; IM-862; pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034); vatalanib (PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap; vandetanib; ABT-165; ZD-6474; DLL4 inhibitors.

[00625] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a proteasome inhibitor such as Bortezomib; Carfilzomib; Epoxomicin; Ixazomib; Salinosporamide A.

[00626] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a kinase inhibitor, such as Afatinib; Axitinib; Bosutinib; Crizotinib; Dasatinib; Erlotinib; Fostamati-nib; Gefitinib; Ibrutinib; Imatinib; lapatinib; lenvatinib; Mubritinib; Nilotinib; Pazopanib; Pegaptanib; Sorafenib; Sunitinib; SU6656; Vandetanib; Vemurafenib; CEP-701 (lesaurtinib); XL019; INCB018424 (ruxoli- tinib); ARRY-142886 (selemetinib); ARRY-438162 (binimetinib); PD-325901; PD-98059; AP-23573; CCI-779; everolimus; RAD-001; rapamycin; temsirolimus; ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242, PP30, Torin 1; LY294002; XL-147; CAL-120; ONC-21; AEZS-127; ETP-45658; PX-866; GDC-0941; BGT226; BEZ235; XL765.

[00627] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a protein synthesis inhibitor, such as streptomycin; Dihydrostreptomycin; Neomycin; Framycetin; Paromomycin; Ribostamycin; Kanamycin; Amikacin; Arbecacin; Becanamycin; Dibecacin; Tobramycin; Spectinomycin; Hygromycin B; Paromomycin; Gentamicin; Netilmicin; Sisomicin; Isepamycin; Verdamycin; Astromycin; Tetracycline; Doxycycline; Chlortetracycline; Clomocycline; Demeclocycline; Limecycline; Meclocycline; Metacycline; Minocycline; Oxytetracycline; Penimepicilline; Rolitetracycline; Tetracycline; Glycylcyclines; Tigecycline; Oxazolidinone; Eperezolilide; Linezolid; Posizolid; Radazolid; Ranbezolid; Sutezolid; Tedizolid; Peptidyl transferase inhibitors; Chloramphenicol; Azidamphenicol; Thiamphenicol; Florfenicol; Pleuromutilins; Retapamulin; Tiamulin; Valnemulin; Azithromycin; Clarithromycin; Dirithromycin; Erythromycin; Flurithromycin; Josamycin; Midecamycin; Miocamycin; Oleandomycin; Rokitamycin; Roxithromycin; Spiramycin; Troleandomycin; Tylosin; Ketolides; Telithromycin; Cethromycin; Solithromycin; Clindamycin; Lincomin; Pirlimycin; Streptogramins; Pristinamycin; Quinupristin/dalfopristin; Virginiamycin.

[00628] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a histone deacetylase inhibitor, such as Vorinostat; Romidepsin; Chidamide; Panobinostat; Valproic acid; Belinostat; Mocetinostat; Abexinostat; Entinostat; SB939 (pracinostat); Resminostat; Givinostat; Quisinostat; thioureidobutyronitrile (Kevetrin™); CUDC-10; CHR-2845 (tefinostat); CHR-3996; 4SC-202; CG200745; ACY-1215 (rocilinostat); ME-344; sulforaphane.

[00629] In some embodiments, the antichemokine receptor drug portion or anti-CCR8 ADC is a topoisomerase I inhibitor, such as camptothecin; Various campotothecin derivatives and analogues (e.g., NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC 295500, NSC 249910, NSC 606985, NSC 74028, NSC 17633, NSC 618939, NSC 610457, NSC 610459, NSC 606499, NSC 610456, NSC 364830 and NSC 606497); morpholinisoxorubicin; SN-38.

[00630] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a topoisomerase II inhibitor such as doxorubicin; amonafide (benzisoquinolinedione); m-AMSA (4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide); anthrapyrazole derivative ((NSC 355644); etoposide (VP-16); pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine, 9-methoxy-N,N-dimethyl-5-nitro-, monomethanesulfonate); bisanthrene hydrochloride; daunorubicin; desoxyxorubicin; mitoxantrone; menogaril; N,N-dibenzyl daunomycin; oxanthrazole; rubidazone; teniposide.

[00631] In some embodiments, the drug portion of the antichemokine receptor or anti-CCR8 ADC is a DNA intercalating agent, such as anthramycin; chicamycin A; tomamycin; DC-81; sibiromycin; pyrrolobenzodiazepine derivative; SGD-1882 ((S)-2-(4-aminophenyl)-7-methoxy-8-(3S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin- 5(11aH)-one); SG2000 (SJG-136; (11aS,11a'S)-8,8'-(propane-1,3-di-ylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)).

[00632] In some embodiments, the antichemokine receptor drug portion or anti-CCR8 ADC is an RNA/DNA antimetabolite, such as L-alanosine; 5-azacitidine; 5-fluorouracil; acivicin; aminopterin derivative N-[2-chloro-5[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC 132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid; aminopterin derivative monohydrate of N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acid; antifolate PT523 ((Nα-(4-amino-4-deoxypteroyl)-NY-hemifthaloyl-L-ornithine)); Baker's soluble antifol (NSC 139105); dichloralyl leisone ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone); brequinar; phthorafur ((prodrug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil); 5,6-dihydro-5-azacytidine; methotrexate; methotrexate derivative (acid N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]L-glutamic acid ((N-(phosphonoacetyl)-L-aspartate); pyrazofurin; trimetrexate.

[00633] In some embodiments, the drug portion of the antichemokine receptor or anti-CCR8 ADC is a DNA antimetabolite such as 3-HP; 2'-deoxy-5-fluorouridine; 5-HP; α-TGDR (α-2'-deoxy-6-thioguanosine); aphidicolin glycinate; araC (cytosine arabinoside); 5-aza-2'-deoxycytidine; β-TGDR (β-2'-deoxy-6-thioguanosine); cyclocytidine; guanazole; hydroxyurea; inosine glycodialdehyde; macbecin II; pyrazoloimidazole; thioguanine; thiopurine.

[00634] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a mitochondrial inhibitor, such as pancratistatin; fenpanstatin; rhodamine-123; edelfosine; d-alpha-tocopherol succinate; compound 11β; aspirin; ellipticin; berberine; cerulenin; GX015-070 (Obatoclax®; 1H-Indole, 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-); celastrol (tripterin); metformin; Brilliant Green; ME-344.

[00635] In some embodiments, the antichemokine receptor drug portion or anti-CCR8 ADC is an antimitotic agent, such as allocolchicine; auristatins, such as MMAE (monomethyl auristatin E) and MMAF (monomethyl auristatin F); halicondrin B; cemadotine; colchicine; colchicine derivative (N-benzoyl-desacetylbenzamide); dolastatin-10; dolastatin-15; maytansine; maytansinoids, such as DM1 (N2'-desacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine); rozoxine; paclitaxel; paclitaxel derivative ((2'-N-[3-(dimethylamino)propyl]glutaramate paclitaxel); docetaxel; thiocolchicine; tritylcysteine; vinblastine sulfate; vincristine sulfate.

[00636] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a nuclear export inhibitor, such as calistatin A; delactonmycin; KPT-185 ((Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]prop-2-enoate propan-2-yl); cazusamycin A; leptolstatin; leptofuranin A; leptomycin B; ratjadone; Verdinexor ((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N-pyridin-2-ylprop-2-enehydrazide).

[00637] In some embodiments, the antichemokine receptor or anti-CCR8 ADC drug portion is a hormonal therapeutic product, such as anastrozole; exemestane; arzoxifene; bicalutamide; cetrorelix; degarelix; deslorelin; trilostane; dexamethasone; flutamide; raloxifene; fadrozole; toremifene; fulvestrant; letrozole; formestane; glucocorticoids; doxercalciferol; sevelamer carbonate; lasofoxifene; leuprolide acetate; megesterol; mifepristone; nilutamide; tamoxifen citrate; abarelix; prednisone; finasteride; rilostane; buserelin; luteinizing hormone-releasing hormone (LHRH); histrelin; trilostane or modrastan; fosrelin; goserelin.

[00638] Any of these agents that include, or can be modified to include, an antibody binding site and/or binding fragment can be included in an anti-chemokine receptor ADC, for example, in an anti-CCR8 ADC.

CAR T cells

[00639] According to some third modalities of the 19th aspect, the conjugate is a CAR T cell conjugate designed for chemokine receptor or CCR8 targeting. Recently, CAR T cells have gained attention for their clinical successes and expedited FDA approvals, as per WO2020102240, incorporated herein in its entirety. In the CAR T cell approach, T cells are collected from the patient's blood and then genetically modified to express CARs specific for an antigen present on tumor cells. These modified T cells are then readministered to the same patient. After injection, the CAR T cells recognize the target antigen on the target cells to induce target cell death. T cells expressing chimeric antigen receptors (CAR T cells) thus constitute a novel modality for medical uses, such as tumor treatment. A chimeric antigen receptor (CAR) is a genetically modified receptor that is designed to target a specific antigen, for example, a tumor antigen. This targeting can result in cytotoxicity against the tumor, for example, so that CAR T cells expressing CARs can target and kill tumors through specific tumor antigens.

[00640] According to the present invention, antibodies or antigen-binding fragments provided for CCR8 or chemokine receptor recognition can be used to manipulate CAR T cells for specific recognition of cells expressing CCR8 or cells expressing the respective chemokine receptor. CARs according to the present invention may comprise a) a recognition region, for example, a single-strand fragment variable (scFv) region derived from an anti-CCR8 antibody or anti-chemokine receptor provided for recognition and binding to the CCR8 or chemokine receptor expressed by the target cell, and b) an activation signaling domain, for example, the CD3 chain of T cells, which can serve as an activation signal for T cells in CARs.

[00641] Preferably, the CARs according to the present invention comprise a co-stimulatory domain (e.g., CD137, CD28, or CD134) to achieve prolonged T cell activation in vivo. The addition of a co-stimulatory domain increases the in vivo proliferation and survival of CAR-containing T cells, and initial clinical data have shown that such constructs are promising therapeutic agents in the treatment of diseases such as cancer. According to the present invention, CAR T cells can be used to treat any disease with aberrant local or systemic presence of cells expressing the target chemokine receptor, in particular cells expressing CCR8, such as Treg cells.

Bispecific

[00642] According to some fourth embodiments of the 19th aspect, the conjugate is a bispecific antibody or a multispecific antibody. In some preferred embodiments, the bispecific antibody comprises at least one Fc domain.

[00643] In some preferred embodiments of the 19th aspect, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment according to the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th aspect.

[00644] In some embodiments of the fourth embodiments of the 19th aspect, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment according to the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th aspect and the second binding portion of the bispecific antibody is the same or a different antibody or antigen-binding fragment according to the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th aspect.

[00645] In some embodiments of the fourth embodiments of the 19th aspect, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18 that binds to human CCR8 and the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment that binds to a different chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1. For example, the second binding portion of the bispecific antibody is Mogamulizumab or an antigen-binding fragment of the same.

[00646] In some embodiments of the fourth embodiments of aspect 19, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, preferably binding to CCR8, and the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment that binds to a cell surface protein such as a cell surface protein expressed on immune cells or a tissue- or cell-type-specific antigen. In some of these embodiments, the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment that targets a checkpoint protein, such as an anti-PD1 antibody, an anti-PD-L1 antibody or a CTLA-4 antibody. Antibodies that target the appropriate checkpoint protein include Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, or Ipilimumab. In some other of these modalities, the second binding portion of the bispecific antibody is an antibody directed against HER2, such as Trastuzumab, Pertuzumab, and/or Margetuximab.

[00647] In some embodiments of the fourth embodiments of the 19th aspect, the first binding portion of the bispecific antibody is an antibody or antigen-binding fragment according to the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th aspect, which preferably binds to CCR8, and the second binding portion of the bispecific antibody is an antibody or antigen-binding fragment that binds to a cell surface molecule associated with T cell activation, preferably selected from CD25, CTLA-4, PD-1, LAG3, TIGIT, ICOS and members of the TNF receptor superfamily, 4-1BB, OX-40 and GITR.

[00648] Techniques for preparing bispecific or multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain-light chain pairs with different specificities (see Milstein and Cuello. "Hybrid hybridomas and their use in immunohistochemistry." Nature 305.5934 (1983): 537-540; WO1993008829 A1, and Traunecker, André, Antonio Lanzavecchia, and Klaus Karjalainen. "Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV-infected cells." The EMBO Journal 10.12 (1991): 3655-3659), and chemical conjugation of two different monoclonal antibodies (see Staerz, Uwe D., Osami Kanagawa, and Michael J. Bevan. "Hybrid antibodies can target sites for attack by T cells." Nature 314.6012 (1985): 628-631.). Multispecific antibodies can also be made by crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan, Maureen, Peter F. Davison, and Henry Paulus. "Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments." Science 229,4708 (1985): 81-83.), using leucine zippers to produce bispecific antibodies (see, for example, Kostelny, Sheri A., M. S. Cole, and J. Yun Tso. "Formation of a bispecific antibody by the use of leucine zippers." The Journal of Immunology 148.5 (1992): 1547-1553.), using diacorporeal technology to prepare bispecific antibody fragments (see, for example, Holliger, Philipp, Terence Prospero, and Greg Winter). "Diabodies: small bivalent and bispecific antibody fragments." Proceedings of the National Academy of Sciences 90.14 (1993): 6444-6448.), using single-chain Fv dimers (sFv) (see, for example, Gruber, Meegan, et al. "Efficient tumor cell lysis mediated by a bispecific single-chain antibody expressed in Escherichia coli." The Journal of Immunology 152.11 (1994): 5368-5374.), preparing trispecific antibodies as described (for example, in Tutt, Alison, GT Stevenson, and M. J. Glennie. "Trispecific F(ab')3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells." The Journal of Immunology 147.1 (1991): 60-69.), and by switching controlled Fab branching (cFAE) according to Labrijn, Aran F., et al. "Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange." Proceedings of the National Academy of Sciences 110.13 (2013): 5145-5150.

Combined components for diagnostic and research applications.

[00649] According to some fifth embodiments of the 19th aspect, the conjugate comprises a detectable portion. Examples of detectable portions include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, non-radioactive paramagnetic metal ions, and reactive portions. The detectable substance may be coupled or conjugated directly to the antibody or a fragment thereof or indirectly, for example, through a ligand known in the art or another portion, using techniques known in the art. Examples of enzyme markers include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase, such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, acetylcholinesterase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[00650] Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99mTc.

[00651] The detection of the expression of a chemokine receptor or CCR8 generally involves contacting a biological sample (tumor, cells, tissue or body fluid from an individual) with one or more antibodies or fragments according to the present invention (optionally conjugated to a detectable portion), and detecting whether the sample is positive or not for the chemokine receptor or CCR8, or whether the sample has altered expression (e.g., reduced or increased) compared to a control sample.

ASPECT 20 - PHARMACEUTICAL COMPOSITION

[00652] According to a 20th aspect, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19. Preferably, the pharmaceutical composition comprises an antibody or fragment thereof or a conjugate according to any of the embodiments described herein, or a combination thereof, in a therapeutically effective amount together with a pharmaceutically acceptable carrier, excipient or stabilizer.

[00653] Pharmaceutical compositions can be prepared by mixing the antibody, fragment, or conjugate with the desired degree of purity with optional physiologically acceptable vehicles, excipients, or stabilizers (Remington, Joseph Price. Remington: The science and practice of Pharmacy. Vol. 1. Lippincott Williams & Wilkins, 2006.). Pharmaceutical compositions may be, for example, in the form of lyophilized formulations or aqueous solutions.

Vehicles, excipients, stabilizers

[00654] Acceptable vehicles, excipients or stabilizers are not toxic to receptors at the dosages and concentrations used and include buffers such as phosphate buffer (e.g., PBS), citrate buffer and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptide (e.g., less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as Tween®, Pluronic® or polyethylene glycol (PEG).

Several therapeutically active compounds

[00655] According to the present invention, a pharmaceutical composition may contain more than one active compound, for example, as needed or beneficial for the particular indication to be treated.

[00656] According to some early embodiments of the 20th aspect, the pharmaceutical composition comprises one or more other therapeutically active compounds.

[00657] In some of the first preferred embodiments of aspect 20, the pharmaceutical composition comprises an antibody or antigen-binding fragment according to aspect 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or aspect 18, or a conjugate according to aspect 19 and a) an antibody or small molecule targeting a checkpoint protein, such as PD1, PD-L1 or CTLA-4, and/or b) an antibody targeting another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, and/or c) an antibody that targets a protein that is specifically expressed by tumor cells and/or d) an antibody or small molecule that targets HER2 and/or EGFR, and/or e) the standard of care for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer and/or prostate cancer and/or f) a chemotherapeutic agent, preferably a taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, paclitaxel or gemcitabine and/or g) a targeted kinase inhibitor, such as Sorafinib, Regorafenib or MEKi-1.

Combination with checkpoint inhibitors

[00658] According to some preferred embodiments of the first embodiments of the 20th aspect, the pharmaceutical composition also comprises an antibody or a small molecule that targets a checkpoint protein, such as PD1, PD-L1 or CTLA-4. Suitable checkpoint targeting antibodies include Nivolumab (PD1; human IgG4), Pembrolizumab (PD1; humanized IgG4), Atezolizumab (PD-L1; humanized IgG1), Avelumab (PD-L1; human IgG1), Durvalumab (PD-L1; human IgG1), Cemiplimab, cemiplimab-rwlc (PD-1; human mAb), Dostarlimab (TSR-042) (PD-1; humanized IgG4) or Ipilimumab (CTLA-4; human IgG1).

[00659] In some embodiments, the antibody or small molecule that targets a checkpoint protein targets CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands, or a combination thereof.

Combination with chemokine receptor antibodies

[00660] According to some preferred embodiments B of the first embodiments of aspect 20, the pharmaceutical composition also comprises an antibody or a small molecule that targets another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1. Suitable antibodies targeting another chemokine receptor include antibodies provided according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or Mogamulizumab.

[00661] Combination with antibodies or molecules that target HER2 or EGFR

[00662] According to some preferred embodiments C of the first embodiments of the 20th aspect, the pharmaceutical composition also comprises an antibody or a small molecule that targets HER2 and/or EGFR. Suitable antibodies targeting HER2 are Trastuzumab (HER2; humanized IgG1), Pertuzumab (HER2; humanized IgG1), Ado-trastuzumab emtansine (HER2; humanized IgG1; ADC), [fam-]trastuzumab deruxtecan, famtrastuzumab deruxtecan-nxki (HER2; humanized IgG1 ADC), Sacituzumab govitecan; sacituzumab govitecan-hziy (TROP-2; humanized IgG1 ADC) and/or Margetuximab (HER2; chimeric IgG1). Suitable antibodies that target EGFR are Cetuximab (EGFR; chimeric IgG1), Panitumumab (EGFR; human IgG2), and Necitumumab (EGFR; human IgG1).

Combination with therapeutic antibodies

[00663] According to some embodiments D of the first embodiments of the 20th aspect, the pharmaceutical composition comprises another therapeutic antibody selected from Muromonab-CD3 (CD3; Murine IgG2a), Efalizumab (CD11a; Humanized IgG1), Tositumomab-I131 (CD20; Murine IgG2a), Nebacumab (Endotoxin; Human IgM), Edrecolomab (EpCAM; Murine IgG2a), Catumaxomab (EPCAM/CD3; Mouse/mouse bispecific mAb), Daclizumab (IL-2R; Humanized IgG1), Abciximab (GPIIb/IIIa; Chimeric Fab IgG1), Rituximab (CD20; Chimeric IgG1), Basiliximab (IL-2R; Chimeric IgG1), Palivizumab (RSV; Humanized IgG1), Infliximab (TNF; IgG1 Chimeric), Trastuzumab (HER2; Humanized IgG1), Adalimumab (TNF; Human IgG1), Ibritumomab tiuxetan (CD20; Murine IgG1), Omalizumab (IgE; Humanized IgG1), Cetuximab (EGFR; Chimeric IgG1), Bevacizumab (VEGF; Humanized IgG1), Natalizumab (a4 integrin; Humanized IgG4), Panitumumab (EGFR; Humanized IgG2), Ranibizumab (VEGF; humanized IgG1 Fab), Eculizumab (C5; humanized IgG2/4), Certolizumab pegol (TNF; Humanized Fab, pegylated), Ustekinumab (IL-12/23; human IgG1), Canakinumumab (IL-1β; human IgG1), Golimumab (TNF; human IgG1), Ofatumumab (CD20; human IgG1), Tocilizumab (IL-6R; humanized IgG1), Denosumab (RANK- L; human IgG2), Belimumab (BLyS; human IgG1), Ipilimumab (CTLA-4; human IgG1), Brentuximab vedotin (CD30; chimeric IgG1; ADC), Pertuzumab (HER2; humanized IgG1), Ado-trastuzumab emtansine (HER2; humanized IgG1; ADC), Raxibacumab (B. anthrasis PA; human IgG1), Obinutuzumab (CD20; Glycoengineered humanized IgG1), Siltuximab (IL-6; chimeric IgG1), Ramucirumab (VEGFR2; human IgG1), Vedolizumab (α4β7 integrin; humanized IgG1), Nivolumab (PD1; human IgG4), Pembrolizumab (PD1; humanized IgG4), Blinatumomab (CD19, CD3; murine bispecific tandem scFv), Alemtuzumab (CD52; humanized IgG1), Evolocumab (PCSK9; human IgG2), Idarucizumab (Dabigatran; humanized Fab), Necitumumab (EGFR; Human IgG1), Dinutuximab (GD2; Chimeric IgG1), Secukinumab (IL-17a; Human IgG1), Mepolizumab (IL-5; Humanized IgG1), Alirocumab (PCSK9; Human IgG1), Daratumumab (C D38; human IgG1), Elotuzumab (SLAMF7; humanized IgG1), Ixekizumab (IL-17a; humanized IgG4), Reslizumab (IL-5; humanized IgG4), Olaratumab (PDGFRα; human IgG1), Bezlotoxumab (enterotoxin B of Clostridium difficile; human IgG1), Atezolizumab (PD-L1; humanized IgG1), Obiltoxaximab (B. anthrasis PA; chimeric IgG1), Brodalumab (IL-17R; human IgG2), Dupilumab (IL-4Rα; human IgG4), Inotuzumab ozogamicin (CD22; humanized IgG4; ADC), Guselkumab (IL-23 p19; human IgG1), Sarilumab (IL-6R; human IgG1), Avelumab (PD-L1; human IgG1), Emicizumab (Factor Ixa, Erenumab, erenumab-aooe (CGRP receptor; Human IgG2), Galcanezumab, galcanezumab-gnlm (CGRP; Humanized IgG4), Burosumab, burosumab-twza (FGF23; Human IgG1), Lanadelumab, lanadelumab-flyo (Plasma kallikrelin; Human IgG1), Mogamulizumab, mo- gamulizumab-kpkc (CCR4; Humanized IgG1), tildrakizumab; tildra- kizumab-asmn (IL-23 p19; humanized IgG1), Fremanezumab, fremanezumab-vfrm (CGRP; humanized IgG2), Ravulizumab, ravulizumab- cwvz (C5; Humanized IgG2/4), Cemiplimab, cemiplimab-rwlc (PD-1; Human mAb), Ibalizumab, ibalizumab-uiyk (CD4; Humanized IgG4), Emapalumab, empalumab-lzsg (IFNg; human IgG1), Moxetumomab pasudotox, moxetumomab pasudotox-tdfk (CD22; murine IgG1 dsFv immunotoxin), Caplacizumab, caplacizumab-yhdp (von Willebrand Factor; Humanized Nanobody), Risankizumab, risankizumab-rzaa (IL-23 p19; Humanized IgG1), Polatuzumab vedotin, polatuzumab vedotin-piiq (CD79b; Humanized IgG1 ADC), Romosozumab, romosozumab-aqqg (Sclerostin; Humanized IgG2), Brolucizumab, brolucizumab-dbll (VEGF-A; humanized scFv), Crizanlizumab; crizan-lizumab-tmca (CD62 (also known as P-selectin); Humanized IgG2), Enfortumab vedotin, enfortumab vedotin-ejfv (Nectin-4; Human IgG1 ADC), [fam-]trastuzumab deruxtecan, famtrastuzumab deruxtecan-nxki (HER2; Human IgG1 ADC), Tepro-tumumab, teprotumumab-trbw (IGF-1R; human IgG1), Eptinezumab, eptinezumab-jjmr (CGRP; humanized IgG1), Isatuximab, isatuximab-irfc (CD38; chimeric IgG1), Sacituzumab govitecan; sacituzumab go-vitecan-hziy (TROP-2; Human IgG1 ADC), Inebilizumab (CD19; humanized IgG1), Leronlimab (CCR5; humanized IgG4), Satralizumab (IL-6R; humanized IgG2), Narsoplimab (MASP-2, human IgG4), Tafasitamab (CD19; humanized IgG1), REGNEB3 (Ebola virus; mixture of 3 human IgG1), Naxitamab (GD2; humanized IgG1), Opotuzumab monatox (EpCAM; humanized scFv immunotoxin), Belantamab mafodotin (B cell maturation antigen; Human IgG1 ADC), Margetuximab (HER2; chimeric IgG1), Tanezumab (Nerve growth factor; humanized IgG2), Dostarlimab (TSR-042) (PD-1; humanized IgG4), Teplizumab (CD3; humanized IgG1), Aducanumab (amyloid beta; human IgG1), Sutimlimab (BIVV009) (C1s; humanized IgG4), Evinacumab (Angiopoietin-like 3; Human IgG4).

Combination with cytotoxic or cytostatic agents

[00664] According to some embodiments of the first embodiments of the 20th aspect, the pharmaceutical composition also comprises a cytostatic and/or cytotoxic agent selected from radionuclides, alkylating agents, DNA crosslinking agents, DNA intercalating agents (e.g., groove-binding agents, such as minor groove ligands), cell cycle modulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA anti-metabolites and antimitotic agents, as described according to the second embodiments of the 19th aspect.

[00665] In some preferred embodiments, the cytotoxic agent is an auristatin, a maytansinoid, a kinesin spindle protein (KSP) inhibitor, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, or a pyrrolobenzodiazepine derivative.

ASPECT 21 - MED USE/TREATMENT METHODS

[00666] According to a 21st aspect, the antibody or antigen-binding fragment is provided according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or the pharmaceutical composition according to aspect 20, for use as a medicament.

[00667] The use of the antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or the pharmaceutical composition according to aspect 20, for the manufacture of a medicament, for example, for the treatment of a tumor or a disease involving cells expressing a chemokine receptor and in particular CCR8, is also provided.

[00668] In addition, a method of treating a disease is provided, the method comprising administering an effective dose of the antibody or antigen-binding fragment according to the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th aspect, or a conjugate according to the 19th aspect or the pharmaceutical composition according to the 20th aspect to a patient in need.

[00669] For therapeutic applications, the antibody or antigen-binding fragment or conjugate or pharmaceutical composition according to the invention may be administered to a patient or individual, for example, to a human or non-human individual, in a pharmaceutically acceptable dosage form. For example, administration may occur intravenously as a bolus or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes. The antibodies, fragments, conjugates and pharmaceutical compositions according to the present invention are particularly suitable for administration via intratumoral, peritumoral, intralesional or perilesional routes, to exert local and systemic therapeutic effects.

[00670] Possible routes of administration include parenteral (e.g., intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous), intrapulmonary, and intranasal. In addition, antibodies, fragments, conjugates, and pharmaceutical compositions may be administered by pulse infusion, with, for example, decreasing doses of the antibody, fragment, or conjugate. Preferably, the dosage is administered by injection, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. The amount to be administered may depend on a variety of factors, such as clinical symptoms, patient or individual weight, and whether other medications are administered. A skilled practitioner will recognize that the route of administration will vary depending on the disorder or condition being treated.

[00671] The frequency of antibody dosing can vary from once every 3 to 6 months to weekly, biweekly (BIW), or daily dosing. Similarly, dose levels range from a low fixed mg dose (daily, weekly, biweekly, or monthly, depending on the antibody) to doses of approximately 1 g. Dosing frequency depends on a variety of factors, including target concentration and turnover rate, target biodistribution, antibody half-life, fragments or conjugates, and potential pharmacodynamic effects that may enhance the biological effects of antibodies, fragments, conjugates, and pharmaceutical compositions beyond their presence at pharmacologically relevant levels.

[00672] In some embodiments, the anti-CCR8 antibody is administered at a dose of 1 mg/kg, for example, daily, weekly, every 2 weeks, every 3 weeks or every 4 weeks. In some embodiments, the anti-CCR8 antibody is administered at a dose of 1 to 30 mg/kg, preferably 5 to 10 mg/kg, for example, daily, weekly, every 2 weeks, every 3 weeks or every 4 weeks. In some embodiments, the anti-CCR8 antibody is administered at a dose of 4 to 8 mg/kg, preferably 5 to 6 mg/kg, for example, daily, weekly, every 2 weeks, every 3 weeks or every 4 weeks.

[00673] For the prevention or treatment of disease, the appropriate antibody dosage will depend on the type of disease being treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, prior therapy, the individual's clinical history and response to the antibody variant, and at the discretion of the attending physician or veterinary health professional. The antibody is appropriately administered to the individual at one time or over a series of treatments.

[00674] As shown in Example 12.4.2, it was surprisingly observed that even a single dose can be sufficient to establish a substantial response to treatment.

[00675] According to the present invention, an anti-CCR8 antibody inducing ADCC and/or ADCP is therefore provided for use in the treatment of a tumor, wherein only a single dose of the anti-CCR8 antibody is administered to an individual, so that no additional dose of the same or a different anti-CCR8 antibody is administered to the individual. This method is not only logistically superior and particularly convenient for patients, but also avoids problems with patient adherence.

[00676] When administered intravenously, the pharmaceutical composition comprising the antibodies, fragments or conjugates may be administered by infusion over a period of about 0.5 to about 5 hours. In some embodiments, the infusion may occur over a period of about 0.5 to about 2.5 hours, over a period of about 0.5 to about 2.0 hours, over a period of about 0.5 to about 1.5 hours or over a period of about 1.5 hours, depending on the antibodies, fragments, conjugates and pharmaceutical compositions administered and the amount of antibody, fragment or conjugate administered.

ASPECT 22 - SECOND USE OF MED/TREATMENT METHODS

[00677] Treatment with an anti-CCR8 antibody according to the present invention has shown remarkable efficacy in several syngeneic tumor models. In some cases, administration of an effective dose of the antibody or antigen-binding fragment to a group of diseased individuals leads to aa. a complete response in at least 15%, 20% or 25% of individuals, and/or parab. an overall response rate in at least 40%, 50%, 60% or 70% of individuals.

[00678] According to aspect 22, the antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or the pharmaceutical composition according to aspect 20, is provided for use in the treatment of a tumor or disease characterized by chemokine receptor-positive cells, preferably CCR8-positive cells, such as CCR8-positive regulatory T cells. Treatment may occur as described for aspect 21.

[00679] Preferably, the antibody or antigen-binding fragment according to aspect 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19 or the pharmaceutical composition according to aspect 20 is provided for use in the treatment of a tumor or disease characterized by the involvement of cells expressing the seven-transmembrane receptor or target chemokine receptor.

[00680] Multiple modes of action can be predicted for the antibodies, antigen-binding fragments or conjugates according to the present invention. One mode of action is the conjugation of an inventive antichemokine receptor antibody to a drug in the form of an antibody-drug conjugate (ADC). Another mode of action is the ability of an antibody targeting a chemokine receptor to induce ADCC. A third mode of action lies in the ability of the antibody targeting the chemokine receptor to induce ADCP.

[00681] CCR8 antibodies or antigen-binding fragments, particularly according to aspects 8, 11, 12, 13 and 14, are particularly suitable for an ADCC/ADCP-based approach, for example, because they are characterized by particularly low internalization or are non-internalizing antibodies, thus residing on the surface of target cells expressing CCR8 and causing their efficient death, as demonstrated for activated human Tregs in vitro (Examples 10.3.3 ff, Examples 10.3.4 ff) or in vivo (Examples 12 ff).

[00682] Regulatory T cells (Tregs) promote tumor growth by suppressing the function of effector T cells, including tumor-responsive T cells in the tumor microenvironment. In fact, Tregs are one of the main resistance mechanisms that hinder the effectiveness of immune checkpoint inhibitors (ICIs) in many indications.

[00683] CCR8 is a surface receptor of the GPCR family of chemokine receptors, which is predominantly expressed on activated immunosuppressive Tregs frequently found in tumors. Unlike other approaches targeting these suppressor T cells, targeting CCR8 offers the opportunity to deplete intratumoral Tregs without affecting resting Tregs or other immune cells systemically. Thus, targeting CCR8 may be superior in terms of efficacy and safety. This mode of action can therefore be used in tumors with intratumoral Tregs, but it can also be used in other diseases characterized by an aberrant presence of activated Tregs, i.e., those expressing CCR8.

Specific Indications

[00684] In principle, antibodies, fragments, conjugates and pharmaceutical compositions can be used in the treatment of any cancer involving cells expressing CCR8. For example, the cancer may comprise tumor cells expressing CCR8, such as B-cell lymphoma and T-cell lymphoma. Alternatively, the cancer may comprise intratumoral Tregs expressing CCR8. As shown in Example 11, CCR8 is upregulated in several tumor indications, such as T-cell acute lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, squamous cell carcinoma of the head and neck, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic adenocarcinoma, ovarian or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma, and prostate cancer. According to some preferred embodiments, its use as a medicine is in the treatment of head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer.

[00685] According to some early modalities according to the 22nd aspect, the tumor is selected from adrenal cancer (e.g., adrenocortical carcinoma or pheochromocytoma), bladder cancer (e.g., transitional cell carcinoma, transitional cell-papillary carcinoma), brain cancer (e.g., glioma-astrocytoma, glioma-astrocytoma-glioblastoma, glioma-oligoastrocytoma, glioma-oligodendroglioma), breast cancer (e.g., ADC, ADC-ductal, ADC-ductal-TNBC, ADC-ductal-TPBC, ADC-lobular), colorectal cancer (e.g., ADC), esophageal cancer (e.g., ADC), esophageal cancer (e.g., SCC), gastric cancer (e.g., ADC, diffuse ADC, ADC-intestinal, ADC-intestinal-tubular), cancer of Head and neck cancer (e.g., laryngeal cancer-SCC, SCC, oral cancer-SCC), kidney cancer (e.g., ccRCC, chromophobe, papillary, papillary type I, papillary type II), liver cancer (e.g., HCC), lung cancer (e.g., NSCLC-ADC, NSCLC-ADC-Mixed, NSCLC-SCC, SCLC), mesothelioma (e.g., epithelioid), ovarian cancer (e.g., ADC-cystadenocarcinoma-serous papillary), pancreatic cancer (e.g., ADC-ductal), prostate cancer (e.g., ADC-acinar type), sarcoma (e.g., leiomyosarcoma, undifferentiated liposarcoma, malignant fibrous histiocytoma), skin cancer (e.g., melanoma), testicular cancer (e.g., germ cell tumor-seminoma), thymoma, cancer of Thyroid (e.g., Follicular carcinoma, Papillary carcinoma - Classic variant) or uterine cancer (e.g., Cervical SCC, Keratinizing Cervical SCC, Non-keratinizing Cervical SCC, Endometrial ADC - Endometrioid, Endometrial ADC - Serous Papillary, Endometrial Carcinosarcoma - Malignant Müllerian tumor), B-cell lymphoma and T-cell lymphoma (as per Table 11.1.2).

[00686] According to some second modalities according to the 22nd aspect, the tumor is selected from acute T-cell lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, squamous cell carcinoma of the head and neck, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma and prostate cancer or any other cancer involving chemokine receptor or cells expressing CCR8. Preferably, the tumor is selected from head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer.

Combination Treatment

[00687] According to some third embodiments according to the 22nd aspect, which can be and are suggested to be combined with the embodiments of the first and/or second embodiments according to the 22nd aspect, the use is a simultaneous, separate or sequential combination use with one or more other therapeutically active compounds. Examples for combination treatments are provided in Examples 12.6, 12.6.1, 12.6.2, 12.6.3, 12.6.4, 12.6.5, 12.6.6, 12.6.7, 12.6.8 and 12.6.9, demonstrating the wide applicability of anti-CCR8 antibodies according to the present invention and anti-CCR8 antibodies in general in combination treatment.

[00688] Antichemokine receptor antibodies, for example, CCR8 antibodies, or ADCs, may be used as adjuvants or with other agents or treatments with anticancer properties. When used together, the antibodies, fragments or conjugates and other agent(s) may be formulated together in a single combination pharmaceutical composition or formulation, as described for aspect 20, or may preferably be formulated and administered separately, either in a single coordinated dosing regimen or in different dosing regimens.

Sequential Administration: Combination partner after treatment with anti-CCR8 antibody.

[00689] It has been found, according to the present invention, that administering the anti-CCR8 antibody as a first agent and subsequently administering one or more therapeutically active compounds increased efficacy in an unexpected manner.

[00690] According to a preferred treatment regimen, treatment begins with administration of the anti-CCR8 antibody (e.g., daily, weekly, q2w, q3w, or q4w, as described above), optionally followed by administration of one or more additional therapeutically active compounds. As described in Examples 12.6 ff., initiating administration of the second therapeutic agent or therapy after the initial dose of the anti-CCR8 antibody has been associated with improved efficacy. Without being limited by theory, the time between initial doses should be chosen to allow for intratumoral Treg depletion of at least 30%, 40%, 50%, or 60%. It is suggested that one or more therapeutically active compounds or combination partners be any of those described herein for this purpose. For example, one or more additional therapeutically active compounds may comprise a checkpoint inhibitor (e.g., anti-PD1/anti-PD-L1/anti-CTLA4 antibody), an antibody targeting a protein that is specifically expressed by tumor cells, or a chemotherapeutic agent described herein.

[00691] In a different modality, treatment begins with the administration of a chemotherapeutic agent and is followed by the administration of the anti-CCR8 antibody (e.g., weekly or biweekly schedule).

Other therapeutically active compounds for the treatment of Combination

[00692] Agents administered together with an antichemokine receptor antibody or ADC, for example, with an anti-CCR8 antibody or ADC, may have complementary activities to the antichemokine receptor antibody or ADC, so that the antibodies/ADCs and other agents do not adversely affect each other. The agents that can be used in conjunction with anti-chemokine or anti-CCR8 receptor antibodies or ADCs according to the present invention may be alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (Bispecific T-cell activator) antibodies, antibody-drug conjugates, biological response modifiers, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, viral leukemia oncogene homolog receptor (ErbB2) inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, immunological agents, protein inhibitors. apoptosis inhibitors (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamcin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-type kinase inhibitors (Plk), phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, tyrosine kinase receptor inhibitors, retinoid/deltoid plant alkaloids, small ribonucleic acid (siRNA) inhibitors, topoisomerase inhibitors, ubiquitin ligase inhibitors and the like, as well as combinations of one or more of these agents.

[00693] According to some highly preferred embodiments of the third embodiments of aspect 22, the antibody or antigen-binding fragment according to aspect 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19 or the pharmaceutical composition according to aspect 20 is provided for use in simultaneous, separate or sequential combination(i) with one or more other therapeutically active compounds, preferably selected from(a) an antibody or small molecule targeting a checkpoint protein, such as PD1, PD-L1 or CTLA-4,(b) an antibody targeting another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, c) an antibody directed to a protein that is specifically expressed by tumor cells, d) an antibody or small molecule that targets HER2 and/or EGFR, e) the standard of care for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer and/or prostate cancer and/or f) a chemotherapeutic agent, preferably a taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin or gemcitabine, g) a targeted kinase inhibitor, such as Sorafinib, Regorafenib or MEKi-1 and/or (ii) with radiotherapy, and or (iii) with depletion of intratumoral B cells, for example, in the treatment of a tumor or disease, preferably characterized by chemokine receptor-positive cells, such as CCR8-positive regulatory T cells.

[00694] Suitable taxanes may be selected, for example, from paclitaxel, abraxane, cabazitaxel or docetaxel, or derivatives thereof.

[00695] Modalities in which the anti-CCR8 antibody is combined with doxorubicin, taxanes, cisplatin or carboplatin, modalities in which the anti-CCR8 antibody is combined with trastuzumab or pertuzumab, and modalities in which the anti-CCR8 antibody is combined with a PD-L1 antibody, such as atezolizumab, are particularly suitable for the treatment of breast cancer, such as stage IV breast cancer.

[00696] Modalities in which the anti-CCR8 antibody is combined with cis-/carboplatin, paclitaxel, docetaxel or gemcitabine, modalities in which the anti-CCR8 antibody is combined with avastine, an EGFR inhibitor (such as Tarceva or Iressa), an ALK inhibitor, a ROS inhibitor, a BRAF inhibitor or an NTRK inhibitor, and modalities in which the anti-CCR8 antibody is combined with nivolumab, pembrolizumab or atezolizumab, or a derivative thereof, are particularly suitable for the treatment of NSCLC.

[00697] Modalities in which the anti-CCR8 antibody is combined with nivolumab, pembrolizumab or ipilimumab (CTLA4), anti-IL2 antibody or a derivative thereof, modalities in which the anti-CCR8 antibody is combined with a BRAF inhibitor or MEK inhibitor and modalities in which the anti-CCR8 antibody is combined with darcabazine, paclitaxel, carboplatin or cisplatin are particularly suitable for the treatment of melanoma.

Combination with Checkpoint Inhibitors

[00698] Tumors were found to be particularly responsive to anti-CCR8 antibody therapy if the tumor responded to immune checkpoint inhibition or if the tumor had high or medium immune infiltration and if a substantial number of CD8+ T cells and/or NK cells were present or could be induced, for example, in addition to high CCR8/FoxP3 ratios (as per Example 12.6 ff, as per responsive syngeneic models).

[00699] According to some particularly preferred embodiments of the third embodiment according to aspect 22, one or more therapeutically active compounds comprise an antibody or a small molecule that targets a checkpoint protein, such as PD1, PD-L1 or CTLA-4. Suitable checkpoint targeting antibodies include Nivolumab (PD1; human IgG4), Pembrolizumab (PD1; humanized IgG4), Atezolizumab (PD-L1; humanized IgG1), Avelumab (PD-L1; human IgG1), Durvalumab (PD-L1; human IgG1), Cemiplimab, cemiplimab-rwlc (PD-1; human mAb), Dostarlimab (TSR-042) (PD-1; humanized IgG4) or Ipilimumab (CTLA-4; human IgG1). In some embodiments, the antibody or small molecule targeting a checkpoint protein targets and/or inhibits a checkpoint protein that may be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or a combination thereof. Examples 12.6 et seq. Examples 12.6 et seq. demonstrate that a superior therapeutic effect was obtained by combining an antibody targeting the PD-L1 checkpoint protein and an inventive antibody according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18. Examples 12.6 et seq. demonstrate that this combination of an inventive antibody according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18 and a CTLA4 antibody targeting checkpoint protein also showed excellent efficacy in a tumor model.

Combination with Chemokine Receptor Antibodies

[00700] According to some preferred embodiments of the third embodiments according to aspect 22, one or more therapeutically active compounds comprise an antibody targeting another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1. Suitable antibodies targeting another chemokine receptor include antibodies provided according to aspect 6, for example, a CCR5 antibody, CXCR5 antibody, CCR4 antibody or Mogamulizumab.

Combination with antibodies or molecules that target HER2 or EGFR.

[00701] According to some preferred embodiments of the third embodiments according to aspect 22, one or more therapeutically active compounds comprise a small molecule or an antibody that targets HER2 and/or EGFR. Suitable antibodies targeting HER2 are Trastuzumab (HER2; humanized IgG1), Pertuzumab (HER2; humanized IgG1), Ado-trastuzumab emtansine (HER2; humanized IgG1; ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki (HER2; humanized IgG1 ADC), Sacituzumab govitecan; sacituzumab go-vitecan-hziy (TROP-2; humanized IgG1 ADC) and/or Margetuximab (HER2; chimeric IgG1). Suitable antibodies that target EGFR are Cetuximab (EGFR; chimeric IgG1), Panitumumab (EGFR; human IgG2), and Necitumumab (EGFR; human IgG1).

Combination with Therapeutic Antibodies

[00702] According to some embodiments of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise an additional therapeutic antibody selected from Muromonab-CD3 (CD3; Murine IgG2a), Efalizumab (CD11a; Humanized IgG1), Abciximab (GPIIb/IIIa; Chimeric Fab IgG1), Rituximab (CD20; Chimeric IgG1), Basiliximab (IL-2R; Chimeric IgG1), Palivizumab (RSV; Humanized IgG1), Infliximab (TNF; Chimeric IgG1), Trastuzumab (HER2; Humanized IgG1), Adalimumab (TNF; Human IgG1), Ibritumomab tiuxetan (CD20; Murine IgG1), Omalizumab (IgE; Humanized IgG1), Cetuximab (EGFR; Chimeric IgG1), Bevacizumab (VEGF; humanized IgG1), Natalizumab (a4 integrin; humanized IgG4), Panitumumab (EGFR; human IgG2), Ranibizumab (VEGF; humanized IgG1 Fab), Eculizumab (C5; humanized IgG2/4), Certolizumab pegol (TNF; humanized Fab, pegylated), Ustekinumab (IL-12/23; Human IgG1), Canakinumab (IL-1β; Human IgG1), Golimumab (TNF; Human IgG1), Ofatumumab (CD20; Human IgG1), Tocilizumab (IL-6R; humanized IgG1), Denosumab (RANK-L; human IgG2), Belimumab (BLyS; human IgG1), Ipilimumab (CTLA-4; human IgG1), Brentuximab vedotin (CD30; chimeric IgG1; Pertuzumab (HER2; Humanized IgG1), Ado-trastuzumab emtansine (HER2; Humanized IgG1; ADC), Raxibacumab (B. anthrasis PA; Human IgG1), Obinutuzumab (CD20; Glycoengineered Humanized IgG1), Siltuximab (IL-6; Chimeric IgG1), Ramucirumab (VEGFR2; Human IgG1)), Vedolizumab (α4β7 integrin; humanized IgG1), Nivolumab (PD1; human IgG4), Pembrolizumab (PD1; humanized IgG4), Blinatumomab (CD19, CD3; murine bispecific tandem scFv), Alemtuzumab (CD52; humanized IgG1), Evolocumab (PCSK9; human IgG2), Idarucizumab (Dabigatran; humanized Fab), Necitumumab (EGFR; human IgG1), Dinutuximab (GD2; chimeric IgG1), Secukinumab (IL-17a; human IgG1), Mepolizumab (IL-5; humanized IgG 1), Alirocumab (PCSK9; human IgG1), Daratumumab (CD38; human IgG1), Elotuzumab (SLAMF7; humanized IgG1), Ixekizumab (IL-17a; Humanized IgG4), Reslizumab (IL-5; humanized IgG4), Olaratumab (PDGFRα; human IgG1), Bezlotoxumab (Clostridium difficile Enterotoxin B; human IgG1), Atezolizumab (PD-L1; humanized IgG1), Obiltoxaximab (B. anthrasis PA; chimeric IgG1), Brodalumab (IL-17R; human IgG2), Dupilumab (IL-4Rα; human IgG4), Inotuzumab ozogamicin (CD22; humanized IgG4; ADC), Guselkumab (IL-23 p19; human IgG1), Sarilumab (IL-6R; human IgG1), Avelumab (PD-L1; human IgG1), Emicizumab (Factor Ixa, X; humanized IgG4, bispecific), Ocrelizumab (CD20; humanized IgG1), Benralizumab (IL-5Rα; humanized IgG1); human), Lanadelumab, lanadelumab-flyo (Plasma kalicrelin; Human IgG1), Mogamulizumab, mogamulizumab-kpkc (CCR4; Humanized IgG1), tildrakizumab; tildrakizumab-asmn (IL-23 p19; humanized IgG1), Fremanezumab, fremanezumab-vfrm (CGRP; humanized IgG2), Ravulizumab, ravulizumab-cwvz (C5; Humanized IgG2/4), Cemiplimab, cemiplimab-rwlc (PD-1; Human mAb), Ibalizumab, iba- lizumab-uiyk (CD4; Humanized IgG4), Emapalumab, empalumab-lzsg (IFNg; human IgG1), Moxetumomab pasudotox, moxetumomab pasudotox-tdfk (CD22; murine IgG1 dsFv immunotoxin), Caplacizumab, caplacizumab-yhdp (von Willebrand Factor; Humanized Nanobody), Risankizumab, risankizumab-rzaa (IL-23 p19; Humanized IgG1), Polatuzumab vedotin, polatuzumab vedotin-piiq (CD79b; Humanized IgG1 ADC), Romosozumab, romosozumab-aqqg (sclerostin; Humanized IgG2), Brolucizumab, brolucizumab-dbll (VEGF-A; humanized scFv), Crizanlizumab; crizanlizumab-tmca (CD62 (also known as P-selectin); Humanized IgG2), Enfortumab vedotin, enfortumab vedotin-ejfv (Nectin-4; human IgG1 ADC), [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki (HER2; humanized IgG1 ADC), Teprotumumab, teprotumumab-trbw (IGF-1R; human IgG1), Eptinezumab, eptinezumab-jjmr (CGRP; humanized IgG1), Isatuximab, isatuximab-irfc (CD38; chimeric IgG1), Sacituzumab govitecan; Sacituzumab govitecan-hziy (TROP-2; humanized IgG1 ADC), Inebilizumab (CD19; humanized IgG1), Leronlimab (CCR5; humanized IgG4), Satralizumab (IL-6R; humanized IgG2), Narsoplimab (MASP-2, human IgG4), Tafasitamab (CD19; humanized IgG1), REGNEB3 (Ebola virus; mixture of 3 human IgG1), Naxitamab (GD2; humanized IgG1), Oportuzumab monatox (EpCAM; humanized scFv immunotoxin), Belantamab mafodotin (B-cell maturation antigen; humanized IgG1 ADC), Margetuximab (HER2; chimeric IgG1), Tanezumab (Nerve growth factor; humanized IgG2), Dostarlimab (TSR-042) (PD-1; humanized IgG4), Teplizumab (CD3; humanized IgG1), Aducanumab (amyloid beta; human IgG1), Sutimlimab (BIVV009) (C1s; humanized IgG4), Evinacumab (Angiopoietin-like 3; Human IgG4).

[00703] Combination treatment with any of these additional therapeutic antibodies is preferred, particularly for a tumor specifically expressing the target of the selected additional therapeutic antibody.

[00704] According to some embodiments and of the third embodiments according to the 22nd aspect, one or more other therapeutically active compounds comprise another therapeutic antibody targeting CD16a, ILDR2, PSMA or Mesothelin.

Combination with cytotoxic or cytostatic agents

[00705] Several chemotherapeutic agents, such as doxorubicin and oxalic acid, induce immunogenic cell death (ICD). However, ICD has been linked to the response to anti-CCR8 antibodies, so combination therapy may add more benefits to the application of monotherapy.

[00706] According to some embodiments of the third embodiments according to aspect 22, one or more therapeutically active compounds comprise a cytostatic agent selected from radionuclides, alkylating agents, DNA crosslinking agents, DNA intercalating agents (e.g., sulcus linking agents, such as minor sulcus ligands), cell cycle modulators, apoptosis regulators, kinase inhibitors, protein synthesis inhibitors, mitochondrial inhibitors, nuclear export inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites and antimitotic agents.

Alkylating Agent

[00707] According to some F1 embodiments of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise an alkylating agent selected from (L-Leucine, N—[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanine]-, ethyl ester); AZQ (1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester); BCNU (N,N'-Bis(2-chloroethyl)-N-nitrosourea); busulfan (1,4-butanediol dimethanesulfonate); (carboxyphthalate)platinum; CBDCA (cis-(1,1-cyclobutanedicarboxylate)diaminoplatinum(II))); CCNU (N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea); CHIP (iproplatin; NSC 256927); chlorambucil; chlorozotocin (2-[[[(2-chloroethyl) nitrosoamino]carbonyl]amino]-2-deoxy-D-glucopyranose); cis-platinum (cisplatin); clomesone; cyanomorpholinodoxorubicin; cyclodysone; dianhydrogalactitol (5,6-diepoxyducitol); fluorodopan ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyluracil); hepsulfam; hycantone; indolinobenzodiazepine dimer DGN462; melphalan; methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea); mitomycin C; mitozolamide; nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride); PCNU ((1-(2-chloroethyl)-3-(2,6)-dioxo-3-piperidyl)-1-nitrosourea)); piperazine alkylator ((1-(2-chloroethyl)-4-(3-chloropropyl)piperazine dihydrochloride)); piperazinodione; pipobroman (N,N'-bis(3-bromopropionyl) piperazine); porphyromycin (N-methylmitomycin C); spirohydantoin mustard; teroxirone (triglycidylisocyanurate); tetraplatinum; thiotepa (N,N',N'-tri-1,2-ethanediylthiophosphoramide); triethylenemelamine; uracil nitrogen mustard (desmethyldopan); Yoshi-864 (bis(3-mesyloxypropyl)amine hydrochloride).

DNA alkylation-like agent

[00708] According to some F2 embodiments of the third embodiments according to the 22nd aspect, one or more therapeutically active compounds comprise a DNA alkylation-like agent selected from Cisplatin; Carboplatin; Nedaplatin; Oxaliplatin; Satraplatin; Triplatin tetranitrate; Procarbazine; Altretamine; Dacarbazine; Mitozolomide; Temozolomide.

Alkylating Antineoplastic Agent

[00709] According to some embodiments F3 of the third embodiments according to the 22nd aspect, one or more therapeutically active compounds comprise an alkylating antineoplastic agent selected from Carboquone; Carmustine; Chlornaphazine; Chlorozotocin; Duocarmycin; Evofosfamide; Fotemustine; Glufosfamide; Lomustine; Mannosolfane; Nimustine; Phenantiplatin; Pipobroman; Ranimustine; Semustine; Streptozotocin; ThioTEPA; Treosulfane; Triaziquone; Triethylenemelamine; Triplatin tetranitrate.

DNA replication and repair inhibitor

[00710] According to some F4 embodiments of the third embodiments according to the 22nd aspect, one or more therapeutically active compounds comprise a DNA replication and repair inhibitor selected from Altretamine; Bleomycin; Dacarbazine; Dactinomycin; mitobronitol; mitomycin; Pingyangmycin; Plicamycin; Procarbazine; Temozolomide; ABT-888 (veliparib); olaparib; KU-59436; AZD-2281; AG-014699; BSI-201; BGP-15; INO-1001; ONO-2231.

Cell cycle modulator

[00711] According to some F5 embodiments of the third embodiments according to the 22nd aspect, one or more other therapeutically active compounds comprise a cell cycle modulator, such as Paclitaxel; Nab-Paclitaxel; Docetaxel; Vincristine; Vinblastine; ABT-348; AZD-1152; MLN-8054; VX-680; Aurora A-specific kinase inhibitors; Aurora B-specific kinase inhibitors and pan-Aurora kinase inhibitors; AZD-5438; IMC-1040; BMS-032; BMS-387; CVT-2584; flavopiridol; GPC-286199; MCS-5A; PD0332991; PHA-690509; seliciclib (CYC-202, R-roscovitine); ZK-304709; AZD4877, ARRY-520: GSK923295A.

Apoptosis regulator

[00712] According to some F6 embodiments of the third embodiments according to aspect 22, one or more therapeutically active compounds comprise an apoptosis regulator, such as AT-101 ((-)gssypol); G3139 or oblimersen (antisense oligonucleotide directed at Bcl-2); IPI-194; IPI-565; N-(4-(4-((4'-chloro(1,1'-biphenyl)-2-yl)methyl)piperazin-1-ylbenzoyl)-4-(((1R)-3-(dimethylamino)-1-((phenylsulfanyl)methyl)propyl)amino)-3-nitrobenzenesulfonamide); N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-en-1-yl)methyl)piperazin-1-yl)benzoyl)-4-(((1R)-3-(morpholin-4-yl)-1-((phenylsulfanyl)methyl)propyl)amino)- 3-((trifluoromethyl)sulfonyl)benzenesulfonamide; GX-070 (Obatoclax®; 1H-Indole, 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-)); HGS1029; GDC-0145; GDC-0152; LCL-161; LBW-242; veneto- clax; Agents that target TRAIL or death receptors (e.g., DR4 and DR5), such as ETR2-ST01, GDC0145, HGS-1029, LBY-135, PRO-1762; drugs that target caspases, caspase regulators, BCL-2 family members, death domain proteins, TNF family members, Toll family members, and/or NF-kappa-B proteins.

Angiogenesis inhibitor

[00713] According to some F7 embodiments of the third embodiments according to the 22nd aspect, one or more other therapeutically active compounds comprise an angiogenesis inhibitor, such as ABT-869; AEE-788; axitinib (AG-13736); AZD-2171; CP-547632; IM-862; pegaptamib; sorafenib; BAY43-9006; pazopanib (GW-786034); vatalanib (PTK-787, ZK-222584); sunitinib; SU-11248; VEGF trap; vandetanib; ABT-165; ZD-6474; DLL4 inhibitors.

Proteasome inhibitor

[00714] According to some F8 embodiments of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise a proteasome inhibitor, such as Bortezomib; Carfilzomib; Epoxomicin; Ixazomib; Salinosporamide A.

Kinase inhibitor

[00715] According to some embodiments of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise a kinase inhibitor such as Afatinib; Axitinib; Bosutinib; Crizotinib; Dasatinib; Erlotinib; Fostamatinib; Gefitinib; Ibrutinib; Imatinib; lapatinib; lenvatinib; Mubritinib; Nilotinib; Pazopanib; Pegaptanib; Sorafenib; Sunitinib; SU6656; Vandetanib; Vemurafenib; CEP-701 (lesaurtinib); XL019; INCB018424 (ruxolitinib); ARRY-142886 (selemetinib); ARRY-438162 (binimetinib); PD-325901; PD-98059; AP-23573; CCI-779; everolimus; RAD-001; rapamycin; temsirolimus; ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242, PP30, Torina 1; LY294002; XL-147; CAL-120; ONC-21; AEZS-127; ETP-45658; PX-866; GDC-0941; BGT226; BEZ235; XL765, Regorafenib and MEKi-1. Protein synthesis inhibitor

[00716] According to some embodiments of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise a protein synthesis inhibitor, such as streptomycin; Dihydrostreptomycin; Neomycin; framycetin; Paromomycin; Ribostamycin; kanamycin; Amikacin; Arbekacin; Becanamycin; Dibekacin; Tobramycin; Pectinomycin; Hygromycin B; Paromomycin; Gentamicin; Netilmycin; Sisomicin; Isepamycin; Verdamycin; Astromycin; Tetracycline; Doxycycline; Chlortetracycline; Clomocycline; Demeclocycline; Limecycline; Meclocycline; Metacycline; Minocycline; Oxytetracycline; Penimepicilcycline; Rolitetracycline; Tetracycline; Glycylcyclines; Tigecycline; Oxazolidinone; Eperezolid; Linezolid; Posizolid; Radazolide; Ranbezolid; Sutezolide; Tedizolid; Peptidyl transferase inhibitors; Chloramphenicol; Azidamphenicol; Thiamphenicol; Florfenicol; Pleuromutilins; Retapamulin; Tiamulin; Valnemulin; Azithromycin; Clarithromycin; Dirithromycin; Erythromycin; Flurithromycin; Josamycin; Midecamycin; Myocamycin; Ole-andomycin; Rokitamycin; Roxithromycin; Spiramycin; Troleandomycin; Tylosin; Ketolides; Telithromycin; Cethromycin; solithromycin; Clindamycin; Lincomycin; Pirlimycin; Streptogramins; Pristinamycin; Quinupristin/dalfopristin; Virginiamycin.

Histone deacetylase inhibitor

[00717] According to some embodiments of the third embodiments according to the 22nd aspect, one or more other therapeutically active compounds comprise a histone deacetylase inhibitor, such as Vorinostat; Romidepsin; Chidamide; Panobinostat; Valproic acid; Belinostat; Mocetinostat; Abexinostat; Entinostat; SB939 (pracinostat); Resminostat; Givinostat; Quisinostat; thioureidobutyronitrile (Kevetrin™); CUDC-10; CHR-2845 (tefinostat); CHR-3996; 4SC-202; CG200745; ACY-1215 (rocilinostat); ME-344; sulforaphane.

Topoisomerase I inhibitor

[00718] According to some embodiments of the third embodiments according to the 22nd aspect, one or more other therapeutically active compounds comprise a topoisomerase I inhibitor such as camptothecin; Various derivatives and analogues of camptothecin (e.g., NSC 100880, NSC 603071, NSC 107124, NSC 643833, NSC 629971, NSC 295500, NSC 249910, NSC 606985, NSC 74028, NSC 17633, NSC 618939, NSC 610457, NSC 610459, NSC 606499, NSC 610456, NSC 364830 and NSC 606497); morpholinisoxorubicin; SN-38.

Topoisomerase II inhibitor

[00719] According to some embodiments of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise a topoisomerase II inhibitor such as doxorubicin; amonafide (benzisoquinolinedione); m-AMSA (4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide); anthrapyrazole derivative ((NSC 355644); etoposide (VP-16); pyrazoloacridine ((pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine, 9-methoxy-N,N-dimethyl-5-nitro-, monomethanesulfonate); bisanthrene hydrochloride; daunorubicin; deoxyxorubicin; mitoxantrone; menogaril; N,N-dibenzyl daunomycin; oxanthrazole; rubidazone; teniposide.

DNA Intercalating Agent

[00720] According to some embodiments of the third embodiments according to aspect 22, one or more therapeutically active compounds comprise a DNA intercalating agent, such as anthramycin; chicamycin A; tomamycin; DC-81; sibiromycin; pyrrolobenzodiazepine derivative; SGD-1882 ((S)-2-(4-aminophenyl)-7-methoxy-8-(3S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one); SG2000 (SJG-136;(11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one)). RNA/DNA antimetabolite

[00721] According to some embodiments of the third embodiments according to aspect 22, one or more therapeutically active compounds comprise an RNA/DNA antimetabolite, such as gemcitabine, L-alanosine; 5-azacitidine; 5-fluorouracil; acivicin; aminopterin derivative N-[2-chloro-5[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC 132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid; aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acid monohydrate; antifolate PT523 ((Nα-(4-amino-4-deoxypteroyl)-NY-hemifthaloyl-L-ornithine)); Baker's soluble antifol (NSC 139105); dichloralyl leisone ((2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone); brequinar; ftorafur ((prodrug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil); 5,6-dihydro-5-azacytidine; methotrexate; methotrexate derivative (acid N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]L-glutamic acid; PALA ((N-(phosphonoacetyl)-L-aspartate); pyrazofurin; trimetrexate. DNA antimetabolite

[00722] According to some embodiments of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise a DNA antimetabolite such as 3-HP; 2'-deoxy-5-fluorouridine; 5-HP; α-TGDR (α-2'-deoxy-6-thioguanosine); aphidicolin glycinate; araC (cytosine arabinoside); 5-aza-2'-deoxycytidine; β-TGDR (β-2'-deoxy-6-thioguanosine); cyclocytidine; guanazole; hydroxyurea; inosine glycodialdehyde; macbecine II; pyrazoloimidazole; thioguanine; thiopurine. Mitochondrial inhibitor

[00723] According to some embodiments F17 of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise a mitochondrial inhibitor, such as pancratistatin; fenpanstatin; rhodamine-123; edelfosine; d-alpha-tocopherol succinate; compound 11β; aspirin; ellipticin; berberine; cerulenin; GX015-070 (Obatoclax®; 1H-Indole, 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-); celastrol (tripterin); metformin; Brilliant Green; ME-344. Antimitotic Agent

[00724] According to some embodiments F18 of the third embodiments according to aspect 22, one or more other therapeutically active compounds comprise an antimitotic agent, such as alocolchicine; auristatins, such as MMAE (monomethyl auristatin E) and MMAF (monomethyl auristatin F); halicondrin B; cemadotin; colchicine; colchicine derivative (N-benzoyl-desacetyl benzamide); dolastatin-10; dolastatin-15; maytansine; maytansinoids, such as DM1 (N2'-desacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine); rozoxin; paclitaxel; Paclitaxel derivative ((2'-N-[3-(dimethylamino)propyl]glutamate paclitaxel); docetaxel; thiocolchicine; tritylcysteine; vinblastine sulfate; vincristine sulfate. Nuclear Export Inhibitor

[00725] According to some embodiments of the third embodiments according to aspect 22, one or more therapeutically active compounds comprise a nuclear export inhibitor, such as calistatin A; delactonmycin; KPT-185 ((Z)-3-[3-[3-methoxy-5-(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]prop-2-enoate); cazusamycin A; leptolstatin; leptofuranin A; leptomycin B; ratjadone; Verdinexor ((Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N-pyridin-2-ylprop-2-enohydrazide). Hormonal Therapists

[00726] According to some embodiments F20 of the third embodiments according to aspect 22, one or more therapeutically active compounds comprise(s) a hormonal therapy such as anastrozole; exemestane; arzoxifene; bicalutamide; cetrorelix; degarelix; deslorelin; trilostane; dexamethasone; flutamide; raloxifene; fadrozole; toremifene; fulvestrant; letrozole; formestane; glucocorticoids; doxercalciferol; sevelamer carbonate; lasofoxifene; leuprolide acetate; megesterol; mifepristone; nilutamide; tamoxifen citrate; abarelix; prednisone; finasteride; rilostane; buserelin; luteinizing hormone-releasing hormone (LHRH); histrelin; trilostane or modrastane; fosrelin; goserelin.

[00727] According to some G embodiments of the third embodiments according to the 22nd aspect, one or more therapeutically active compounds comprise an antibody that targets myeloid suppressor cells.

[00728] According to some H-modalities of the third modalities according to the 22nd aspect, one or more therapeutically active compounds comprise agents that initiate antigen-presenting cells (APCs), which in turn can activate T cells.

[00729] According to some embodiments of the third embodiments according to the 22nd aspect, one or more therapeutically active compounds comprise a BiTE antibody. BiTE antibodies are bispecific antibodies that target T cells to attack cancer cells by simultaneously binding to both cells. The T cell then attacks the target cell, i.e., the Treg. For example, a BiTE approach is complementary to the ADCC-based approach of a nuCCR8 antibody. Standard Therapy

[00730] According to some J modalities of the third modalities according to the 22nd aspect, the combination is a combination with the standard of care for the tumor or disease characterized by positive chemokine receptor, for example, CCR8-positive cells. The standard of care may be the standard of care for any T-cell acute lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), testicular cancer, gastric cancer, squamous cell carcinoma of the head and neck, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, melanoma, thyroid cancer, mesothelioma, sarcoma, and prostate cancer.

[00731] According to some preferred modalities, the standard of care is the standard of care for the treatment of head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer and/or prostate cancer. In case of doubt, the standard of care is defined as in the "S3 Leitlinie" for the respective tumor, e.g. Leitlinie Analkarzinom, Aktinische Keratose und Plattenepithelkarzinom der Haut, Chronische Lymphatische Leukamie (CLL), Endometriumkarzinom, Follikulares Lymphom, Harnblasenkarzinom, Hautkrebs-Pravention, Hepatozellulars Karzinom (HCC), Hodentumoren, Hodgkin-Lymphom, Kolorektales Karzinom, Larynxkarzinom, Lungenkarzinom, Magenkarzinom, Mammakarzinom, Melanom, Mundhoehlenkarzinom, Nierenzellkarzinom, Ovarialkarzinom, Osophaguskarzinom, Palliativmedizin, Pankreaskarzinom, Peniskarzinom, Prostatakarzinom, Psychoonkolo- gie, Supportive Therapie, Zervixkarzinom, in effect as of 26-06-2020, as per https://www.leitlinienprogramm-onkologie.de/leitlinien/, incorporated herein by reference in its entirety, as available on 01-06-2021. Radiotherapy

[00732] According to some K modalities of the third modalities according to the 22nd aspect, which can be and are suggested to be combined with the modalities of the first and/or second modalities according to the 22nd aspect, the use is simultaneous, separate combination or sequential use with radiotherapy.

[00733] For example, radiotherapy may be a therapy involving a radiation regimen as known in the art, for example, comprising external beam X-ray therapy with a typical total dose of 40 Gy fractionated into 15 daily treatments or a total dose of 70 Gy fractionated into 37 daily treatments. For example, radiotherapy may be brachytherapy. Example 12.6.8 shows a combination treatment with an anti-CCR8 antibody and radiotherapy. NK cell therapy

[00734] According to some L modalities of the third modalities according to the 22nd aspect, which can be and are suggested to be combined with the modalities of the first and/or second modalities according to the 22nd aspect, the use is simultaneous, separate combination or sequential use with NK cell therapy. NK cell therapy may, for example, comprise the use of a manipulated NK cell line as described in EP1771471 or may comprise the use of isolated primary NK cells, potentially including modifications such as a chimeric receptor, as per WO2006052534. Targeting cell surface molecules associated with T cell activation

[00735] According to some embodiments of the third embodiments of aspect 22, the antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or the pharmaceutical composition according to aspect 20, is provided for use in simultaneous, separate or sequential combination with one or more other therapeutically active compounds targeting cell surface molecules associated with T cell activation, selected from CD25, CTLA-4, PD-1, LAG3, TIGIT, ICOS and members of the TNF receptor superfamily, 4-1BB, OX-40, and GITR. B cell depletion

[00736] According to some N embodiments of the third embodiments according to the 22nd aspect, which can be and are suggested to be combined with the embodiments of the first and/or second embodiments according to the 22nd aspect, the use is simultaneous, separate combination or sequential use with intratumoral B cell depletion. Intratumoral B cell depletion can occur by administering an antibody or other molecule or any other treatment specifically targeted to intratumoral B cells, preferably CD19+ B cells. Example 12.3.2 shows a treatment in which an anti-CCR8 antibody was administered and intratumoral CD19+ B cells were depleted. Although CD8+ T cell depletion abolished the beneficial effect of treatment with an anti-CCR8 antibody, it was surprisingly found that B cell depletion enhanced the beneficial effect of treatment with an anti-CCR8 antibody. Without limitation, B cell depletion can be achieved using an antibody that targets CD19 or CD20 or another suitable B cell marker. Stratification/prediction and monitoring schemes

[00737] The stratification steps described according to the following modalities are also provided as independent methods, for example, for stratification, diagnosis or monitoring of treatment success for an anti-CCR8 antibody treatment.

[00738] According to some fourth modalities according to the 22nd aspect, which can be and are suggested to be combined with the modalities of the first, second and/or third modalities according to the 22nd aspect, the use comprises determining certain parameters to predict or monitor treatment success, for example, to stratify a group of individuals or patients to predict tumor response or monitor treatment success. For example, the individuals may be human or non-human, such as mice, rodents or cynomolgus.

[00739] According to some modalities A of the fourth modalities according to the 22nd aspect, the use comprises determining: a. the presence or quantity of tumor-infiltrating lymphocytes, b. the presence or quantity of macrophages and/or NK cells, c. the presence or quantity of regulatory T cells positive for CCR8 or positive for FOXP3, d. the mutational load of the tumor, e. the cancer staging, f. the presence, level or activation of genes or proteins stimulated by interferon, g. the CCR8 expression, h. the presence or quantity of complement factor proteins, serpins and/or MHC components, i. the presence or quantity of cytokines, such as inflammatory or suppressive cytokines, j. the activation of immune gene expression, k. the expression of immune checkpoint protein, such as PD-(L)1 or CTLA4 expression, l. The presence or quantity of CD19+ B cells infiltrating the tumor and/or the presence or quantity of CD8+ T cells infiltrating the tumor, to stratify a group of individuals or patients to predict tumor response, or to predict or monitor treatment success.

[00740] According to some A1 modalities of the fourth modalities according to the 22nd aspect, the use comprises determining the presence or quantity of (a) tumor-infiltrating lymphocytes, (b) macrophages and/or NK cells and/or (c) CCR8-positive or FOXP3-positive regulatory T cells. The assessment of the presence or quantity of cell types such as (a) tumor-infiltrating lymphocytes, (b) macrophages and/or NK cells and/or (c) CCR8-positive regulatory T cells may occur using biopsy, such as skin biopsy, surgical biopsy, endoscopic biopsy or needle biopsy, such as fine needle aspiration, core needle biopsy, vacuum-assisted biopsy or image-guided biopsy and subsequent staining. The number or relative quantity of (a) tumor-infiltrating lymphocytes, (b) macrophages and/or NK cells, and/or (c) CCR8-positive regulatory T cells can then be compared to a reference, for example, a reference sample or a reference value. The presence or relative quantity of (a) tumor-infiltrating lymphocytes, (b) macrophages and/or NK cells, and/or (c) CCR8-positive regulatory T cells can be used to predict a favorable response to treatment with an antibody or fragment according to the present invention. Alternatively, a scoring system can be used, for example, to determine the number or relative quantity of tumor-infiltrating lymphocytes, such as the scoring system described in Zhang, Dachuan, et al. "Scoring system for tumor-infiltrating lymphocytes and its prognostic value for gastric cancer." Frontiers in Immunology 10 (2019): 71.

[00741] According to some A2 embodiments of the fourth embodiments according to the 22nd aspect, the use comprises determining the tumor mutational burden to predict and monitor tumor response. Tumor mutational burden (TMB) is a biomarker that measures the number of somatic mutations present in a cancer patient's tumor and is quantified as mutations per megabase (mut/Mb). This metric can be used to stratify patients, for example, to predict or monitor the response to treatment with an antibody or conjugate according to the present invention. There are FDA-approved tests that can be used, for example, with a reference value of >10 mut/Mb. Alternatively, microsatellite instability (MSI) can be used for stratification, which is caused by the failure of the DNA mismatch repair system.

[00742] According to some embodiments A3 of the fourth embodiments according to the 22nd aspect, the use comprises determining cancer staging to predict and monitor tumor response. Cancer staging can be performed as known in the art, for example, using the TNM classification system or FIGO staging, and can be used to stratify patients, for example, to predict or monitor response to treatment with an antibody or conjugate according to the present invention.

[00743] According to some A4 modalities of the fourth modalities according to the 22nd aspect, the use comprises the determination of the presence, level or activation of interferon-stimulated genes or proteins or interferon to predict and monitor tumor response. An interferon-stimulated gene is a gene whose expression is stimulated by interferon, in particular by IFNγ. Suitable interferon-stimulated genes or proteins include, but are not limited to, ACOD1, ACTG1, ACTR2, ACTR3, ADAMTS13, AIF1, AQP4, ASS1, B2M, BST2, C9JQL5, CALCOCO2, CAMK2A, CAMK2B, CAMK2D, CAMK2G, CASP1, CCL1, CCL11, CCL13, CCL14, CCL15, CCL15, CCL14, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL7, CCL8, CD40, CD44, CD58, CDC42EP2, CDC42EP4, CIITA, CITED1, CLDN1, CX3CL1, CXCL16, CYP27B1 DAPK1, DAPK3, EDN1, EPRS, EVL, FCGR1A, FCGR1B, FLNB, GA-PDH, GBP1, GBP2, GBP4, GBP5, GBP6, GCH1, GSN, HCK, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-E, HLA-F, HLA-G, HLA-H, ICAM1, IFI30, IFITM1, IFITM2, IFITM3, IFNG, IFNGR1, IFNGR2, IL12B, IL12RB1 IL23R, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF9, JAK1, JAK2, KIF16B, KIF5B, KYNU, LGALS9, MEFV, MID1, MRC1, MT2A, MYO1C, NCAM1, NMI, NOS2, NUB1, OAS1, OAS2, OAS3, OASL, PDE12, PML, PRKCD, PTAFR, RAB12, RAB20, RAB43, RAB7B, RPL13A, RPS6KB1 RYDEN, SEC61A1 SLC11A1 SLC26A6 SLC30A8 SNCA, SP100, STAR, STAT1, STX4, STX8, STXBP1, STXBP2, STXBP3, STXBP4, SYNCRIP TDGF1, TLR2, TLR3, TLR4, TRIM21, TRIM22, TRIM25, TRIM26, TRIM31, TRIM34, TRIM38, TRIM5, TRIM62, TRIM68, TRIM8, UBD, VAMP3, VCAM1, VIM, VPS26B, WAS, WNT5A, XCL1, XCL2, ZYX.

[00744] According to some A5 modalities of the fourth modalities according to the 22nd aspect, the use comprises determining CCR8 expression to predict and monitor tumor response. CCR8 expression levels can be determined, for example, based on biopsy samples, as known in the art, for example, using IHC, PCR or ELISA-based methods, for example, using an antibody, fragment or conjugate as described herein.

[00745] According to some A6 embodiments of the fourth embodiments according to the 22nd aspect, the use comprises determining the presence, level, or activation of selected genes or proteins of (a) complement factor proteins, (b) serpins, (c) MHC components, or (d) Arg2, or (e) another biomarker described herein (see, for example, Example 12.7.1 or 12.7.2) to predict and monitor tumor response. Suitable complement factor proteins include, without limitation, C1R, C1S, C4A, C5ar2, F12, and MASP2, or their respective species analogues for treatment. Suitable serpins include, without limitation, Serpina1a, Serpina1b, Serpina1d, Serpina3i, or their other species analogues. Suitable MHC components include, without limitation, H2-K2, H2-T24, H2-Q10, H2-Bl, H2-Q1, H2-Q5 and H2-DMb2, or their analogues from other species.

[00746] As described in Example 12.1.2, the inventors discovered, based on RNA-seq genome data from early tumors in syngeneic mouse models, that increased levels of the following genes strongly correlated with tumor response: Eif3j2, Eno1b, Ifi44l, Hist1h2al, Ifi202b, Hmga1b, Amd2, Sycp1, Itln1, Trim34b, Catsperg2, Zfp868, Serpina1b, Prss41, C1rb, Cyld, Ccnb1ip1, Masp2, Acaa1b, C4a, Snord93, Abhd1, Serpina3h, H2-K2, Cd1d2, Hal, Rnf151, Rbm46, Arg2, Mir8099-2, Igsf21, Olfr373, C1s2, Crym, Arv1, Hddc3, Plppr4, Ppp1r11, Rps3a2, Zfp459, Rnd1, Serpina1a, Vcpkmt, Atp10d, Gbp2b, H2-T24, Tlcd2, Ctse, H2-Q10, Cyp2c55, Borcs8, Tpsab1, Trim43b, Cc2d1a, Serpina1d, Cacna1a, Kcnj14, Ttc13, Farsa, Olfr1217, Jaml, H2-Bl, Tnpo2, Rims3, Dock9, Car5b, Atp1a4, H2-Q1, Zfp69, Slpi, Pcdhgb8, Ocel1, Selenbp2, Nsd3, Wt1, Nap1l2, Ranbp9, Gtpbp3, AY761185, Rnaset2a, Serpina3i, Ell2, Gal3st2b, Urb2, F12, Klk1, Ifi214, Cstl1, Agtpbp1, Msh5, Cox18, Zfp330, Ttc37, Klk4, H2-Q5, Cxcl11, Rab39, Pm20d1, Nod2, H2-DMb2. Interestingly, this set is highly enriched for early complement factors, complement regulatory factors such as Serpins, and MHC components. In particular, the presence of high levels of Complement C1/C4 may contribute to Treg lysis. When depletion/consumption of complement factors reduces the effectiveness of Treg depletion, supplementation of the complement system, for example in a combination treatment, may be an option.

[00747] According to some preferred embodiments of the fourth embodiments according to the 22nd aspect, the use comprises determining the presence, level or activation of selected genes or proteins of Eif3j2, Eno1b, Ifi44l, Hist1h2al, Ifi202b, Hmga1b, Amd2, Sycp1, Itln1, Trim34b, Catsperg2, Zfp868, Serpina1b, Prss41, C1rb, Cyld, Ccnb1ip1, Masp2, Acaa1b, C4a, Snord93, Abhd1, Serpina3h, H2-K2, Cd1d2, Hal, Rnf151, Rbm46, Arg2, Mir8099-2, Igsf21, Olfr373, C1s2, Crym, Arv1, Hddc3, Plppr4, Ppp1r11, Rps3a2, Zfp459, Rnd1, Serpina1a, Vcpkmt, Atp10d, Gbp2b, H2-T24, Tlcd2, Ctse, H2-Q10, Cyp2c55, Borcs8, Tpsab1, Trim43b, Cc2d1a, Serpina1d, Cacna1a, Kcnj14, Ttc13, Farsa, Olfr1217, Jaml, H2-Bl, Tnpo2, Rims3, Dock9, Car5b, Atp1a4, H2-Q1, Zfp69, Slpi, Pcdhgb8, Ocel1, Selenbp2, Nsd3, Wt1, Nap1l2, Ranbp9, Gtpbp3, AY761185, Rnaset2a, Serpina3i, Ell2, Gal3st2b, Urb2, F12, Klk1, Ifi214, Cstl1, Agtpbp1, Msh5, Cox18, Zfp330, Ttc37, Klk4, H2-Q5, Cxcl11, Rab39, Pm20d1, Nod2, H2-DMb2 or any combination thereof to predict and monitor tumor response. As immediately understood by the skilled person, for a human individual, the method of stratification, prediction or monitoring comprises determining the presence, level or activation of the human counterparts of the genes or proteins provided.

[00748] According to some A7 modalities of the fourth modalities according to the 22nd aspect, the use comprises the determination of the presence or quantity of inflammatory or suppressive cytokines to predict and monitor tumor response. The assessment of the presence or quantity of inflammatory or suppressive cytokines may occur as known in the art.

[00749] According to some A8 modalities of the fourth modalities according to the 22nd aspect, the use comprises predicting or monitoring tumor response based on the activation of immune gene expression. Measurement of immune gene expression activation may occur using a method known in the art, for example, using gene set enrichment analysis or using a marker-based assay known in the art. In addition, stratification, prediction, or monitoring may occur based on a gene signature characteristic for activated Tregs or immune cells.

[00750] According to some highly preferred A9 modalities of the fourth modalities according to the 22nd aspect, the use comprises stratifying a group of individuals or patients to predict tumor response based on the expression of immune checkpoint protein, such as PD-L1 expression, PD-1 expression or CTLA4 expression, or the use comprises determining the expression levels of immune checkpoint protein, such as PD-L1 expression, PD-1 expression or CTLA4 expression to predict and monitor tumor response.

[00751] Programmed death ligand 1 (PD-L1) is an immune-related biomarker that can be expressed on the surface of many tissue types, including tumor cells. PD-L1 protein expression can be determined using Tumor Ratio Score (TPS) or Combined Positive Score (CPS). According to the present invention, it was surprisingly found that the response to anti-CCR8 antibody (mono)therapy was improved for individuals with an ICI-responsive tumor and, in particular, a tumor with (high) PD-L1 expression (as per Example 12.7). Following this observation, the inventors found that infiltration with FoxP3+ Treg cells was also associated with high PD-L1 expression. The inventors therefore suggest PD-L1 as a surrogate marker for stratification to overcome the problem that CCR8 is difficult to analyze and therefore cannot be easily used to select populations of individuals who could likely benefit from anti-CCR8 antibody treatment. In particularly preferred embodiments, PD-L1 expression is determined based on CPS or TPS. An individual or patient is determined as eligible for treatment with an anti-CCR8 antibody if PD-L1 expression is present or high, for example, as indicated by a CPS > 1, preferably by a CPS > 5, more preferably by a CPS > 10, or as indicated by a TPS > 1%, preferably by a TPS > 10%, more preferably by a TPS > 50%.

ASPECT 23 - OTHER USES AND DIAGNOSTICS

[00752] Anti-chemokine receptor or anti-CCR8 antibodies, fragments and conjugates as described herein can be used for a variety of purposes, such as to aid the purification or immobilization of the chemokine receptor or CCR8-expressing cells, such as activated or intratumoral Tregs, for in vitro, in vivo and ex vivo applications or diagnostics. As a specific example, antibodies can be used in immunoassays to qualitatively and/or quantitatively measure the levels of chemokine receptors or cells expressing chemokine receptors in biological samples; see, for example, Harlow et al., Antibodies: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1988).

[00753] For example, anti-chemokine receptor antibodies, anti-CCR8 antibodies, or their antigen-binding fragments can be used to detect the presence of tumors expressing the chemokine receptor or CCR8. The presence or level of cells expressing the chemokine receptor or CCR8, or the chemokine receptor or CCR8 released in various biological samples, including serum and tissue biopsy specimens, can be analyzed. Furthermore, anti-chemokine receptor or anti-CCR8 antibodies can be used in various imaging methodologies, such as immunoscintigraphy with a 99Tc-conjugated antibody (or a different isotope). For example, an imaging protocol similar to that described using an anti-PSMA antibody conjugated to 111In can be used to detect cancer (Sodee, D. Bruce, et al. "Preliminary Imaging Results Using In-111 Labeled CYT-356 (Prostascintst) in the Detection of Recurrent Prostate Cancer." Clinical Nuclear Medicine 21.10 (1996): 759-767). Another detection method that can be used is positron emission tomography, for example, by conjugating the antibodies of the invention with a suitable isotope (see Herzog, Hans, et al. "Measurement of pharmacokinetics of yttrium-86 radiopharmaceuticals with PET and radiation dose calculation of analogous yttrium-90 radiotherapeutics." Journal of Nuclear Medicine 34.12 (1993): 2222-2226).

[00754] According to a 23rd aspect, the antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18 is provided, or a conjugate according to aspect 19 or the pharmaceutical composition according to aspect 20 for use in diagnostic or diagnostic applications.

[00755] In some early embodiments of aspect 23, an antibody or antigen-binding fragment is provided according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19 or a pharmaceutical composition according to aspect 20 for use in an in vivo diagnostic method. The use may be for the diagnosis of a tumor or disease characterized by chemokine receptor-positive cells, for example, CCR8-positive cells, such as CCR8-positive regulatory T cells. For example, the tumor or disease characterized by cells positive for chemokine receptors is selected from acute T-cell lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), squamous cell lung cancer (SCLC), testicular cancer, gastric cancer, squamous cell carcinoma of the head and neck, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer or cervical cancer, acute myeloid leukemia, kidney cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma, and prostate cancer. According to some preferred modalities, the use in an in vivo diagnostic method is the use in the diagnosis of head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer, and/or prostate cancer, or any other tumor described herein.

[00756] According to some preferred embodiments, the antibody or antigen-binding fragment according to the 6th, 7th, 8th, 9th, 10th, 11th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th aspect, or a conjugate according to the 19th aspect or the pharmaceutical composition according to the 20th aspect is used for the stratification of individuals or patients for treatment, for example, for the treatment of a disease described herein.

ASPECT 24 - DNA/RNA FOR CCR8 ANTIBODY

[00757] According to a 24th aspect, a polynucleotide is provided that encodes an antibody or antigen-binding fragment according to the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and/or 18th aspect and according to , , , , , , , , , , , and/or 18th aspect. Preferably, the polynucleotide according to this aspect is a polynucleotide as provided according to the sequence listing.

ASPECT 25 - ANTIBODY VECTOR

[00758] According to a 25th aspect, a vector comprising a polynucleotide according to the 24th aspect is provided. Several suitable vector systems are known in the art or described herein.

ASPECT 26 - ANTIBODY-PRODUCING CELL

[00759] According to a 26th aspect, an isolated cell is provided that is disposed for the production of an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18. Preferably, the isolated cell according to the present aspect is a eukaryotic cell, such as a CHO or HEK cell comprising a vector according to aspect 25. In another preferred embodiment, the cell is a mouse myeloma YB2/0 cell.

ASPECT 27 - ANTIBODY PRODUCTION METHOD

[00760] According to a 27th aspect, a method is provided for producing an antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, comprising culturing a cell according to aspect 26 and optionally purifying the antibody or antigen-binding fragment. In some highly preferred embodiments, the method comprises fucosylation of the antibody as described elsewhere herein. In some embodiments, the method comprises purification of the antibody.

[00761] For example, the antibody according to the present invention can be purified by the purification process described below. The described purification process is suitable for a wide range of IgG monoclonal antibodies and is optimized for bioreactor titers up to 8 g/L with an expected collection volume of, for example, 2000 L of a single-use or stainless steel bioreactor. With appropriate modifications, antibody cultures with other titer ranges can be processed. Cell cultures are harvested and clarified by depth filtration and/or loaded filtration. The clarified collection is stored in disposable bags or in a stainless steel storage tank with optional refrigeration. General Process Conditions

[00762] Closed processing and single-use systems are preferred; skids or stainless steel systems are also possible with appropriate cleaning and changeover procedures. Disposable bags are typically made of low or ultra-low density polyethylene as the contact layer. Among others, sodium acetate/acetic acid or Tris/Tris-HCl buffer systems may be used, typically at a total concentration of 50 mM and including 50 mM NaCl, unless otherwise indicated. Buffers and process intermediates from the respective unit operations (e.g., Feed, Eluate, or Flow) are generally filtered in-line to 0.2 μm or with a filtration assembly, using, for example, PES membrane and/or wool filters. These solutions are generally stored in single-use bags or stainless steel containers, under ambient conditions (e.g., 18 to 26 °C) or 2-8 °C. Capture

[00763] Antibody capture is performed using Protein A-based chromatography, using, for example, Cytiva Mab-Select SuRe, MabSelect SuRe LX, Mab Select PrismA, or Purolite A50 resins. Loading is performed after clarification, with or without pre-concentration by ultrafiltration. The loading density is typically 45 to 75 g/L, using a single or variable flow rate to apply the clarified sample to the column (120 to 400 cm/h). Chromatography can be performed in batch mode with one or more columns, or in continuous mode (MCC (multicolumn chromatography)/SMB (simulated moving bed)) with up to 8 columns. Pre-packed or self-packed columns are used with a typical bed height of 20 cm for batch mode. For continuous mode, the contact time of the clarified sample with the resin is 1 to 4 min. Capture is performed in one or more cycles, depending on the antibody titer. Before loading, the column is equilibrated using a Tris buffer (e.g., pH 7); washing is performed with at least two washing steps; high concentration NaCl in the first wash buffer, then low concentration NaCl in the second buffer (e.g., 1st wash: 1 M NaCl, pH 5.5; 2nd wash: 50 mM NaCl, pH 6.0) in acetate buffer. Elution can, for example, be achieved using low pH acetate buffer (e.g., 3.5) containing 0 to 50 mM NaCl in typically < 5 column volumes (CV) with UV-controlled or volume-controlled eluate collection. Eluates can be pooled and thoroughly mixed at this step or after virus inactivation. The affinity column is regenerated with > 3 CV of acetic acid (e.g., 0.1 M, incl. 500 mM NaCl), sanitization (clean-in-place, CIP) occurs with 0.5-1.0 M NaOH for 30 min, re-equilibration with > 4 CV. The column is stored in, for example, 20% EtOH or 2% BnOH.

[00764] Alternatively, affinity capture of the clarified collection can be performed with affinity binders in a cellulose architecture (e.g., Fibro PrismA) using the same loading schemes and buffer system as described above. Regeneration or CIP block can be bypassed for optimal processing time. A contact time of 5 to 20 seconds can be applied to Fibro units of varying sizes (e.g., 0.4 mL to 2.4 L). Typically, a loading capacity of 25 to 35 g/L unit volume can be achieved. A single unit can be used to process a single batch or multiple batches depending on the Fibro unit and bioreactor sizes, as well as the antibody culture titer. Affinity loading materials can be further clarified by loaded filters with a throughput >300 L/m2 prior to loading to maximize lifetime utilization and minimize fouling. Low pH virus inactivation

[00765] The capture eluate is adjusted with HOAc or HCl to pH 3.7 to 3.9 and maintained for 120 to 240 min in the same bag or in a separate bag at >18 °C to inactivate potentially present viruses. Alternatively, viral inactivation is carried out at pH 3.4-3.6 for >30 minutes. The process intermediate is then adjusted for storage or for further processing conditions, for example, to pH 4-7 using 1-2 M Tris/-HCl titrant stock. Polishing 1

[00766] The antibody is further polished by anion exchange chromatography (AEX), typically in the form of membrane capsules or cassettes (e.g., Sartorius Sartobind STIC PA 4 mm or 3M MA ST Polisher) in continuous flow mode, with flow rates < 350 MV/h, to remove impurities and process- and/or product-related particles. The total quantity of a batch can be processed in one or several cycles, depending on the amount of antibody. Before loading, the intermediate is adjusted to a target conductivity of, for example, 8 to 20 mS/cm, and a target pH of, for example, 7.0 for antibodies with basic isoelectric points. For antibodies with an isoelectric point lower than pH 7.0, the conductivity can be 5 to 20 mS/cm, and the pH can be between its isoelectric point and pH 5.0. The membrane is equilibrated with Tris buffer, pH 7.0, or Acetate buffer pH 5-6, and loaded with densities typically between 0.5 and 10 g of antibody/mL of membrane volume (MV). A chase with equilibration buffer can be performed and combined with the collected flow, whose collection criteria are controlled, for example, by UV or volume. The membrane adsorbent can be used once or several times, for which it is regenerated (1 M NaCl for 25 MV) and re-equilibrated (Tris buffer pH 7.0). Polishing 2

[00767] Final polishing can, for example, be achieved by cation exchange chromatography (CEX). In principle, the order of the AEX and CEX unit operations can be reversed. The CEX unit operation is generally performed in ligation and elution mode with flow rates between 100 and 200 cm/h. Pre- or self-packed columns with resins, for example, Cytiva Capto S ImpAct or Capto SP ImpRes, can be used at a bed height of 20 cm. The polishing step is performed in one or more cycles, depending on the amount of antibody. If necessary, the load can be adjusted to achieve conductivities between 5 and 9 mS/cm and pH 4.5 - 7.5 with HOAc, Tris or WFI titrants. The loading is applied at typical densities between 40 and 105 g/L to a CEX column equilibrated with > 5 CV of acetate buffer (pH and conductivity compatible with the CEX loading) and subsequently washed using > 5 CV of the same buffer. The antibody is eluted using an acetate buffer with an appropriate NaCl concentration (> 50 mM; < 500 mM), and the eluate is collected by UV control in typically < 10 CV of eluate volume collected. The column is regenerated using 0.5 to 2 M NaCl in acetate buffer, reequilibrated (> 5 CV with CEX equilibration buffer), sanitized (0.5-1.0 M NaOH for > 30 min), reequilibrated (> 5 CV with CEX equilibration buffer), and finally stored in, for example, 20% EtOH or 2% BnOH. Additional Polishing

[00768] When additional polishing is required to remove residual impurities related to the product or process, mixed-mode chromatography (MMC) can be used in place of or in conjunction with the CEX step. Examples of mixed-mode chromatographic resins are Capto adhere and Capto MMC ImpRes. This unit operation is typically operated in a continuous flow mode, but bonding and elution modes can also be employed. The column can be self-packed or pre-packed with a bed height of 5-20 cm in various diameters and operated at flow rates between 100-500 cm/h in continuous flow mode or 100-220 cm/h in bonding and elution modes.

[00769] The loading is normally adjusted, if necessary, to target conductivities between 3 and 12 mS/cm and pH 4.2–7.5 with HOAc, Tris titrants, or WFI. The loading is applied at typical densities between 75 and 300 g/L on an MMC column equilibrated with acetate buffer > 5 CV (pH and conductivity compatible with the MMC loading). A chase with equilibrated buffer can be performed and combined with the collected flow, the collection criteria of which are controlled, for example, by UV or volume. The MMC column can be used once or several times, depending on the amount of antibody to be polished. The column is regenerated using 0.5 to 2 M NaCl in acetate buffer, re-equilibrated (> 5 CV with equilibrium buffer), sanitized (0.5-1.0 M NaOH for > 30 min) and finally stored in, for example, 20% EtOH or 2% BnOH. Nanofiltration

[00770] Potentially remaining adventitious viruses are removed by PDVF-based nanofiltration, for example, using Planova BioEX with or without a pre-filter. The load is applied at constant pressure (e.g., 2.0–3.4 bar) with a loading density of 1500–5000 g antibody/m² to a pre-equilibrated nanofilter (> 5 L/m² acetate buffer, recipe, for example, compatible with MA AEX or CEX elution conditions). The filter can be chased using the same buffer, and the filter integrity is typically tested after use. In case of integrity test failure, this step can be repeated. Alternatively, adventitious viruses can be removed by cellulose-based nanofiltration with or without a pre-filter. The load is applied at constant pressure (e.g., 0.8–1.2 bar) with a loading density of 500–2000 g antibody/m² to a pre-equilibrated nanofilter (> 5 L/m² acetate buffer, recipe, for example, compatible with MA AEX or CEX elution conditions). Concentration and changing the tampon

[00771] The intermediate is concentrated by ultrafiltration to 15-110 g/L, diafiltered with > 6 diafiltration volumes against a diafiltration buffer (DFB) (e.g., 10 mM histidine, 10 mM methionine, 30 mM arginine, pH 5.3) and, if necessary, further concentrated to 200 g/L. This is typically accomplished using a tangential flow filtration device and a suitable membrane with a cutoff of 30 to 50 kDa (e.g., Millipore Pellicon, Sartorius Hydrosart, Pall Omega) with a loading of typically 300-1000 g antibody/m2. The process is typically controlled by the feed or retentate flow rate (e.g., 2 to 7 L/min/m2), alternatively by the feed pressure (2-4 bar) and by TMP (0.7 to 2 bar). The membrane can be chased by DFB and the retentate mixed diluted with DFB to the target concentration. The TFF system and membranes are sanitized with 0.5 M NaOH and equilibrated with acetate buffer or DFB. The integrity or permeability of the membrane is tested before use. Stabilization

[00772] In an optional stabilization step, an excipient concentrate is added to produce bulk drug substance (BDS). A bioload reduction filtration (e.g., 0.2 μm) is performed using, for example, a pre-equilibrated filter (e.g., excipient concentrate diluted in BDS), which is tested for integrity after use. In case of integrity test failure, the filtration step can be repeated. Loading and Freezing

[00773] The BDS is filled into suitable bags with an LDPE contact layer (e.g., 5 or 10 L) with individually identifying sampling compartments that are preferably aseptically connected and disconnected. The bags are individually packaged with optional vacuum protection (LDPE overwrap bags) and placed in protective wrappers (e.g., RoSS Shells). After optional intermediate storage at 2-8 °C, the shell-less bags are frozen (using a plate or a passive freezer) and subsequently stored at < -30 or < -60 °C, e.g., +/- 5 °C, alternatively at < -25 or < -60 °C, e.g., +/- 5 °C. ASPECT 28 - ANTIGEN-DEFINED ANTIBODY PARTS KIT

[00774] According to aspect 28, a kit is provided comprising the antibody or antigen-binding fragment according to aspects 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and/or 18, or a conjugate according to aspect 19, or the pharmaceutical composition according to aspect 20, with instructions for use.

[00775] The antibodies, fragments, conjugates, or pharmaceutical compositions of the present invention may be supplied in a kit, that is, a packaged combination of reagents in predetermined quantities in one or more containers with instructions. For example, when the antibody, fragment, or conjugate is a therapeutic antibody, fragment, or conjugate, the instructions for use may comprise the package insert. For example, the package insert may comprise information describing an advantageous mode of administration or combination treatment as described herein.

[00776] For example, when the antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides the detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffers (e.g., a block buffer or lysis buffer), and the like. The relative amounts of the various reagents may vary widely to provide reagent solution concentrations that substantially optimize assay sensitivity. In particular, reagents may be supplied as dry powders, usually lyophilized, including excipients which, upon dissolution, will provide a reagent solution with the appropriate concentration.

ASPECT 29 - COMBINATION TREATMENT COMPRISING ANTI-CCR8 ANTIBODY

[00777] According to one aspect, an anti-CCR8 antibody and another therapeutically active compound or therapy are provided for use in the treatment of a tumor, wherein the other therapeutically active compound is a) a chemotherapeutic agent, preferably a taxane, doxorubicin, cisplatin, carboplatin, oxaliplatin or gemcitabine, b) an antibody targeting another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, c) an antibody targeting a protein that is specifically expressed by tumor cells, d) an antibody or small molecule targeting HER2 and/or EGFR, e) the pattern of treatment for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer and/or prostate cancer, f) a targeted kinase inhibitor, such as Sorafinib, Regorafenib or MEKi-1, and/or g) radiotherapy.

[00778] In other words, an anti-CCR8 antibody is provided, preferably an ADCC and/or ADCP-inducing anti-CCR8 antibody as described elsewhere herein, for use in the treatment of a tumor, wherein the use comprises the administration of another therapeutically active compound. or therapy that does not target a checkpoint protein and is selected from: h) a chemotherapeutic agent, preferably a taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine; i) an antibody that targets another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1; j) an antibody that targets a protein that is specifically expressed by tumor cells; k) an antibody or small molecule that targets HER2 and/or EGFR; l) the standard of care for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, tumor esophageal, melanoma, bladder cancer, liver cancer and/or prostate cancer, m) a targeted kinase inhibitor, such as Sorafinib, Regorafenib or MEKi-1, n) radiotherapy and/or o) (a compound or antibody for) depletion of intratumoral B cells, preferably CD19+ B cells.

[00779] The anti-CCR8 antibody may be an inventive anti-CCR8 antibody as described herein or any other therapeutically suitable anti-CCR8 antibody known in the art. The tumor may be any tumor described herein, for example, for aspect 22. Preferably, the tumor or disease is characterized by chemokine receptor-positive cells, such as CCR8-positive cells, such as CCR8-positive regulatory T cells or CCR8-positive tumor cells. Preferably, the tumor is an ICI-responsive tumor. Based on the Examples described herein (e.g., Examples 12.6 et seq., see responding mouse models), it is plausible that, in particular, those tumors that are or were initially responsive to immune checkpoint inhibition will benefit from treatment with anti-CCR8 antibody, alone and in combination. The other therapeutically active compound may be a therapeutically active compound as described elsewhere herein. Suitable taxanes may be selected, for example, from paclitaxel, abraxane, cabazitaxel or docetaxel, or derivatives thereof. According to a preferred embodiment, the use also comprises the administration of a checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody or a CTLA4 antibody, for example, as described elsewhere herein.

ASPECT 30 - Triple combination treatment comprising anti-CCR8 antibody

[00780] According to one aspect, an anti-CCR8 antibody, a checkpoint inhibitor such as an anti-PD-1 antibody, an anti-PD-L1 antibody or a CTLA4 antibody, and another therapeutically active compound or therapy are provided for use in the treatment of a tumor, wherein the additional therapeutically active compound or therapy is preferably a) an antibody targeting another chemokine receptor such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, b) an antibody targeting a protein that is specifically expressed by tumor cells, c) an antibody or small molecule targeting HER2 and/or EGFR, d) the standard of care for any head and neck cancer, cancer of breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer and/or prostate cancer, e) a chemotherapeutic agent, preferably a taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin or gemcitabine, f) a targeted kinase inhibitor, such as Sorafinib, Regorafenib or MEKi-1, and/or g) radiotherapy, h) (a compound or antibody for) depletion of intratumoral B cells, preferably CD19+ B cells.

[00781] In other words, an anti-CCR8 antibody that induces ADCC and/or ADCP is provided for use in the treatment of a tumor, wherein the use comprises the administration of another therapeutically active compound or therapy that does not target a checkpoint protein and is selected from a) a chemotherapeutic agent, preferably a taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin or gemcitabine, b) an antibody that targets another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or CXCR1, c) an antibody that targets a protein that is specifically expressed by tumor cells, d) an antibody or a small molecule targeting HER2 and/or EGFR, e) a targeted kinase inhibitor, such as Sorafinib, Regorafenib or MEKi-1, f) radiotherapy and/or depletion of intratumoral B cells, preferably CD19+ B cells, and the use also includes the administration of a checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody or a CTLA4 antibody, for example, as described elsewhere herein. ASPECT 31 - Sequential combination therapy comprising anti-CCR8 antibody

[00782] According to one aspect, an anti-CCR8 antibody and an additional therapeutically active compound or therapy are provided for use in the treatment of a tumor, wherein a dose of the additional therapeutically active compound or therapy is administered after the first dose of the anti-CCR8 antibody, for example a) after the anti-CCR8 antibody has increased the ratio of intratumoral CD8 cells to Treg cells by at least a factor of 2, 3, 4 or 5 or b) after the anti-CCR8 antibody has depleted at least 40, 45, 50, 55, 60, 65 or 70% of the intratumoral Treg cells.

[00783] In other words, an anti-CCR8 antibody is provided, preferably an anti-CCR8 antibody inducing ADCC and/or ADCP as described elsewhere herein, for use in the treatment of a tumor, wherein the use comprises the administration of another therapeutically active compound or therapy, wherein a dose of the other therapeutically active compound or therapy is administered after the first dose of the anti-CCR8 antibody, preferably a) after the anti-CCR8 antibody has induced an increase in the ratio of intratumoral CD8 cells to Treg cells by at least a factor of 2, 3, 4 or 5 or b) after the anti-CCR8 antibody has depleted at least 40, 45, 50, 55, 60, 65 or 70% of the intratumoral Treg cells.

[00784] According to a preferred treatment regimen, treatment begins with the administration of the first dose of anti-CCR8 antibody (e.g., daily, weekly, q2w, q3w, or q4w as described above), followed by the administration of the additional therapeutically active compound or therapy. Without being limited by theory, the time between the initial dose of anti-CCR8 antibody and the other therapeutically active compound should be chosen to allow for intratumoral Treg depletion of at least 40, 50, or 60%. The anti-CCR8 antibody may be an inventive anti-CCR8 antibody as described herein, but it may also be any other therapeutically suitable anti-CCR8 antibody. The tumor may be any tumor described herein, for example, for aspect 22. Preferably, the tumor or disease is characterized by chemokine receptor-positive cells, such as CCR8-positive regulatory T cells or CCR8-positive tumor cells. Preferably, the tumor is or was initially an ICI-responsive tumor. Since acquired resistance to PD(L)-1 antibodies can be strongly influenced by intratumoral activated regulatory T cells (characterized by CCR8 expression), sequential treatment with an anti-CCR8 antibody combined with a checkpoint inhibitor is beneficial to overcome such resistance. Administration of the additional therapeutically active compound or therapy after the first dose of the anti-CCR8 antibody has been found to substantially improve treatment efficacy, even for extremely challenging tumor models. The time between the first dose of the anti-CCR8 antibody and the dose of the other therapeutically active compound should be sufficient to induce an initial antitumor immune response. According to the data provided here (as per Examples 12.6 ff), this is indicated by an increase in the ratio of intratumoral CD8 cells to T reg cells by at least a factor of 2, 3, 4, or 5, or by (temporary) depletion of at least 40, 45, 50, 55, 60, 65, or 70% of intratumoral Treg cells.

[00785] The additional therapeutically active compound or therapy may be any compound or therapy described elsewhere herein. The therapeutically active compound may or may not be a checkpoint inhibitor, for example, an anti-PD-L1 antibody, an anti-PD1 antibody, or an anti-CTLA4 antibody. Although the second therapeutic agent may be a non-targeted therapeutic product, targeted therapeutic products have been found to be highly preferred for this purpose. Targeted therapeutic products, as defined herein, are therapeutically active compounds that specifically recognize tumor cells rather than all rapidly dividing cells or immune cell populations. While agents targeting all dividing cells or affecting immune cell populations may be used in combination and may improve efficacy (as per Example 12.6.8, 12.6.9), they may interfere with immune cell populations that were necessary for a sustained tumor immune response. This problem was solved by using an antibody that targets a protein specifically expressed by tumor cells. Preferred targeted therapeutic products are antibodies that bind to a protein specifically expressed by tumor cells.

[00786] Preferably, the therapeutically active compound or additional therapy is selected from a) an antibody or small molecule targeting a checkpoint protein, such as PD-(L)1 or CTLA-4, b) a chemotherapeutic agent, preferably a taxane, paclitaxel, doxorubicin, cisplatin, carboplatin, oxaliplatin, or gemcitabine, c) an antibody targeting another chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or CXCR1, d) an antibody targeting a protein specifically expressed by tumor cells, e) an antibody or small molecule targeting HER2 and/or EGFR, f) a targeted kinase inhibitor, such as Sorafinib, Regorafenib or MEKi-1, g) the standard of care for any head and neck cancer, breast cancer, gastric cancer, lung cancer, squamous cell carcinoma, esophageal tumor, melanoma, bladder cancer, liver cancer and/or prostate cancer, h) radiotherapy and/or i) (a compound or antibody for) depletion of intratumoral B cells, preferably CD19+ B cells.

[00787] Suitable taxanes may be selected, for example, from paclitaxel, abraxane, cabazitaxel or docetaxel, or derivatives thereof.

[00788] Preferably, the dose of the additional therapeutically active compound or therapy is the first dose of the additional therapeutically active compound or therapy mentioned. ASPECT 32 - Stratification methods/diagnostic methods

[00789] As described elsewhere herein, the stratification steps described for the fourth modalities according to the 22nd aspect may be used as part of a treatment method according to the 22nd aspect or are provided independently as a) a method for selecting individuals who are likely to benefit from treatment with an anti-CCR8 antibody, b) a diagnostic method for diagnosing a tumor as sensitive to treatment with an anti-CCR8 antibody, or c) a method for monitoring treatment success for treatment with an anti-CCR8 antibody.

[00790] For the present aspect, the antibody may be an antibody according to the present invention, but it may also be any therapeutically suitable anti-CCR8 antibody. Biomarker for anti-CCR8 antibody treatment

[00791] To date, no biomarker is available to predict or monitor treatment success for anti-CCR8 antibodies. Although the demonstrated mode of action suggests that CCR8 itself may serve as a potential biomarker, the inventors found that CCR8 levels are somewhat difficult to assess in a clinical setting or in clinical studies. To address this pressing problem, the inventors generated hypotheses and were able to validate some of them as suitable biomarkers with excellent predictive power, see, for example, Examples 12.7.1 and 12.7.2. The marker expression level is generally determined with an assay or method known in the art, and the level is subsequently compared to a reference value as described elsewhere herein and is subsequently used to predict or monitor treatment response.

[00792] A biomarker is provided for use in a method for diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody, the use comprising: a. determining the level of the biomarker in a tumor or tumor sample comprising regulatory T cells, b. comparing the level of the biomarker with a sample or reference value, and c. diagnosing/stratifying an individual as having a tumor sensitive to treatment with an anti-CCR8 antibody if the level of the biomarker is greater than or equal to a sample or reference value, wherein the biomarker is d. an immune checkpoint protein, preferably PD-1, PD-L1 or CTLA4, and a granzyme or immune cell marker, preferably a marker for lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or a combination thereof, f. a Treg infiltration marker, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9, g. a T-cell marker or cytotoxic T-cell marker preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB, OX-40 or GITR, h. an interferon or interferon-inducible protein preferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2 and TNF alpha, i. a complement factor, j. a serpin and/or k. a molecule derived from a gene according to Table 12.7.2.1, 12.7.2.2 or 12.7.2.3.

[00793] Preferably, the biomarker is an immune checkpoint protein, most preferably PD-1, PD-L1 or CTLA4.

[00794] Also provided is the use of a biomarker to predict or monitor the therapeutic success of a therapy comprising the administration of an anti-CCR8 antibody, the use comprising a. determining the level of the biomarker in a sample, and b. comparing the level of the biomarker with a sample or reference value, wherein the biomarker is c. an immune checkpoint protein, preferably PD-1, PD-L1 or CTLA4, d. a granzyme or immune cell marker, preferably a marker for lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells or a combination thereof. a Treg infiltration marker, preferably selected from FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, CXCL9,f. a T cell marker or cytotoxic T cell marker, preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5 and members of the TNF receptor superfamily, such as 4-1BB, OX-40 or GITR,g. an interferon-inducible protein, preferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2 and TNF alpha,h. a complement factor,i. a serpin and/or a molecule derived from a gene according to Table 12.7.2.1, 12.7.2.2 or 12.7.2.3.

[00795] Preferably, the biomarker is an immune checkpoint protein, most preferably PD-1, PD-L1 or CTLA4.

[00796] In addition, a molecule that binds to a biomarker is also provided for use in a method for diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody, the use comprising determining the level of the biomarker in a tumor or tumor sample using the biomarker-binding molecule, wherein the biomarker is (preferably) a. an immune checkpoint protein, preferably PD-1, PD-L1 or CTLA4, b. a granzyme or immune cell marker, preferably a marker for lymphocytes, effector cells, T cells, cytotoxic T cells, macrophage cells, M1 macrophage cells, M2 macrophage cells, B cells, NK cells or a combination thereof c. a marker of Treg infiltration, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7 or CXCL9, d. a T cell marker or cytotoxic T cell marker preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB, OX-40 or GITR, e. an interferon or interferon-inducible protein preferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2 and TNF alpha, f. a complement factor, g. a serpin and/or h. a molecule derived from a gene according to Table 12.7.2.1, 12.7.2.2 or 12.7.2.3.

[00797] The molecule that binds to the biomarker is preferably an antibody or antigen-binding fragment, or a conjugate thereof. More preferably, the molecule that binds to the biomarker is an anti-PD-1, PD-L1 or CTLA4 antibody, such as Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab or Ipilimumab.

[00798] In addition, the use of a molecule that binds to a biomarker to predict or monitor the therapeutic success of a therapy comprising the administration of an anti-CCR8 antibody is also provided, the use comprising determining the level of the biomarker in a tumor or tumor sample using the molecule that binds to the biomarker, wherein the biomarker is (preferably) a. an immune checkpoint protein, preferably PD-1, PD-L1 or CTLA4, b. a granzyme or immune cell marker, preferably a marker for lymphocytes, effector cells, T cells, cytotoxic T cells, macrophage cells, M1 macrophage cells, M2 macrophage cells, B cells, NK cells or a combination thereof, c. a marker of Treg infiltration, preferably CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7 or CXCL9, d. a T cell marker or cytotoxic T cell marker preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, 4-1BB, OX-40 or GITR, e. an interferon or interferon-inducible protein preferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2 and TNF alpha, f. a complement factor, g. a serpin and/or h. a molecule derived from a gene according to Table 12.7.2.1, 12.7.2.2 or 12.7.2.3, wherein the molecule that binds to the biomarker is preferably an antibody or antigen-binding fragment, or a conjugate thereof. More preferably, the molecule that binds to the biomarker is an anti-checkpoint, anti-PD-1, anti-PD-L1 or anti-CTLA4 antibody, such as Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab or Ipilimumab.

[00799] In preferred embodiments of the present aspect, the biomarker is a marker of immune cells and the molecule that binds to a biomarker is preferably an antibody that binds to a marker for lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells or a combination thereof.

[00800] In preferred embodiments of the present aspect, the biomarker-binding molecule is a molecule that binds to a Treg infiltration marker, preferably an antibody that binds to CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, or CXCL9.

[00801] In preferred embodiments of the present aspect, the molecule that binds to the biomarker is an antibody that binds to a T-cell marker preferably selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, and members of the TNF receptor superfamily, including 4-1BB, OX-40, or GITR.

[00802] In preferred embodiments of the present aspect, the biomarker-binding molecule is an antibody that binds to an interferon or interferon-inducible protein, preferably selected from INF gamma, IL10, IL12p70, IL1beta, IL2, and TNF alpha. Immune checkpoint protein as a biomarker for anti-CCR8 antibody treatment.

[00803] According to the present invention, it has been surprisingly found that the response to (mono)therapy with anti-CCR8 antibody was improved for individuals who responded at least initially to ICI treatment and, in particular, to treatment with anti-PD-(L)1 or CTLA4 antibodies, see, for example, Table 12.7.1. Since PD-L1 expression is a predictor of responsiveness to anti-PD-L1 antibody treatment, the inventors hypothesized that PD-L1 expression and checkpoint protein expression per se may also be suitable for directly predicting the response to anti-CCR8 antibody treatment. This hypothesis can be confirmed by correlation data between PD-L1 expression and response to anti-CCR8 antibody treatment (Table 12.8.1). PD-L1 expression can be determined as known in the art, for example, without limitation, using tumor proportion score (TPS), combined positive score (CPS), or mRNA expression. The inventors, therefore, suggest an immunological checkpoint marker, and particularly PD-L1, as a surrogate marker for stratification to overcome the problem that CCR8 is difficult to analyze and therefore difficult to implement for selecting patient populations that could likely benefit from treatment with anti-CCR8 antibodies.

[00804] Consequently, a molecule is provided that binds to an immune checkpoint protein for use in a method for diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody, the method comprising: a. determining the expression level of the immune checkpoint protein in a tumor (sample), b. comparing the expression level of the immune checkpoint protein with a sample or reference value, and c. diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody if the expression level of the immune checkpoint protein is greater than or equal to a sample or reference value.

[00805] In addition, the use of a molecule that binds to an immune checkpoint protein to monitor the success of anti-CCR8 antibody therapy treatment is provided, the method comprising: a. determining the expression level of the immune checkpoint protein in a tumor (sample)/a sample, and b. comparing the expression level of the immune checkpoint protein with a reference sample or value.

[00806] The molecule that binds to an immune checkpoint protein is preferably an antibody. The immune checkpoint protein is preferably PD-1, PD-L1, or CTLA4. The molecule that binds to an immune checkpoint protein is preferably an antibody that binds to PD-1, PD-L1, or CTLA4. For example, the antibody could be PD-L1 28-8, PD-L1 22C3, PD-L1 SP142, PD-L1 SP263, or any other suitable antibody known in the art as complementary diagnostic. In particularly preferred embodiments, the expression level of the checkpoint protein is determined based on the combined positive score (CPS) or the tumor ratio score (TPS). A patient's tumor is diagnosed/stratified as sensitive to treatment with an anti-CCR8 antibody if the expression of the immune checkpoint protein is present or high, for example, as indicated by a CPS > 1, preferably by a CPS > 5, more preferably by a CPS > 10, or as indicated by a TPS > 1%, preferably by a TPS > 10%, more preferably by a TPS > 50%. Thus, in some preferred embodiments, the reference value is CPS = 1, more preferably CPS = 5 or more preferably CPS = 10. In some other or the same preferred embodiments, the reference value is TPS = 1%, more preferably TPS = 10%, or more preferably TPS > 50%.

[00807] In some preferred embodiments, PD-L1 expression is determined by a CPS > 1, preferably by a CPS > 5, more preferably by a CPS > 10, or by a TPS > 1%, preferably by a TPS > 10%, more preferably by a TPS > 50%. Treg infiltration marker as a biomarker for anti-CCR8 antibody treatment.

[00808] A molecule is provided that binds to a Treg infiltration marker for use in a method for diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody, the method comprising: a. determining the level of expression of the Treg infiltration marker in a tumor (sample), b. comparing the level of expression of the Treg infiltration marker with a sample or reference value, and c. diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody if the level of the Treg infiltration marker is greater than or equal to a sample or reference value.

[00809] The Treg infiltration marker is a marker highly expressed on activated Tregs. Preferably, the Treg infiltration marker is selected from CCR8, FOXP3, ICOS, CCR4, TIGIT, P2RY10, CD80, TNFRSF9, CD3(G), SLAMF1, IL7R, IL2RB, CTLA4, CD5, ITK, IL2RA, LAX1, IKZF3, GBP5, CXCR6, SIRPG, CD2, CSF2RB, SLAMF7, CXCL9, as per Table 11.1.1, Table 12.8.1. The Treg infiltration marker may be different from CCR8. The molecule that binds to a Treg infiltration marker is preferably an antibody, for example, an antibody that binds to one or more Treg infiltration markers.

[00810] The Treg infiltration marker described herein can also be used as a biomarker to monitor treatment success, for example, as described elsewhere herein. A treatment is considered successful if a (temporary) depletion of Treg of at least 40% or 50% is achieved, as indicated, for example, by a decrease in the Treg infiltration marker by a factor of 2. Immune cell marker as a biomarker for anti-CCR8 antibody treatment

[00811] A molecule is provided that binds to an immune cell marker for use in a method for diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody, the method comprising: a. determining the level of expression of the immune cell marker in a tumor (sample); b. comparing the level of expression of the immune cell marker with a sample or reference value; and c. diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody if the level of the immune cell marker is greater than or equal to a sample or reference value.

[00812] The immune cell marker may be a marker for lymphocytes, effector cells, T cells, cytotoxic T cells, macrophages, M1 macrophages, M2 macrophages, B cells, NK cells, or a combination thereof. Suitable markers for these immune cell populations are described throughout this invention. For example, suitable T cell markers include CD3 (e.g., CD3E, D, and/or G). For example, suitable cytotoxic T cell markers include CD8 (e.g., CD8A and/or B). For example, suitable macrophage cell markers include MS4A7. For example, suitable M1 macrophage cell markers include Acod1. For example, suitable M2 macrophage cell markers include Mrc1. For example, suitable B cell markers include CD19, CD20, CD22, and/or MSA41.

[00813] The immune cell marker described herein may also be used as a biomarker to monitor treatment success, for example, as described elsewhere herein. A treatment is considered successful if there is a (temporary) increase in immune cells, for example, of at least 50%, as indicated by an increase in the immune cell infiltration marker by a factor of at least 2. T cell marker as a biomarker for anti-CCR8 antibody treatment

[00814] A molecule is provided that binds to a T-cell marker for use in a method for diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody, the method comprising: a. determining the level of T-cell marker expression in a tumor (sample); b. comparing the level of T-cell marker expression with a sample or reference value; and c. diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody if the T-cell marker level is greater than or equal to a sample or reference value.

[00815] The T-cell marker can be a marker highly expressed on T cells, such as activated T cells. Preferably, the T-cell marker is selected from CD3, CD4, CD8, CD25, CXCR3, CCR5, and members of the TNF receptor superfamily, including 4-1BB, OX-40, or GITR. The molecule that binds to the T-cell marker is preferably an antibody, for example, an antibody that binds to one or more of the T-cell markers described herein.

[00816] The T-cell marker described herein may also be used as a biomarker to monitor treatment success, alone or in combination with a Treg infiltration marker, for example, as described elsewhere herein. A treatment is considered successful if a (temporary) increase in T cells occurs, for example, of at least 50%, as indicated by an increase in the T-cell marker by a factor of at least 2. Interferon or interferon-inducible protein as a biomarker for anti-CCR8 antibody treatment.

[00817] An interferon or interferon-inducible protein is provided for use in a method for diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody or for monitoring treatment success, the method comprising: a. determining the level of interferon or interferon-inducible protein in a tumor (sample), b. comparing the level of interferon or interferon-inducible protein with a sample or reference value, and c. diagnosing/stratifying an individual as having a tumor that is sensitive to treatment with an anti-CCR8 antibody if the level of interferon or interferon-inducible protein is greater than or equal to a sample or reference value.

[00818] Interferon or interferon-inducible proteins are preferably selected from IFN gamma, IL10, IL12p70, IL1beta, IL2, and TNF alpha. According to the present invention, the correlation of CCR8 mRNA expression with the inflammation marker IFN gamma was found to be significant for 50 tumor indications, as shown in Table 11.1.2. From this, it can be concluded that IFN gamma levels may be suitable as biomarkers, as described herein. This was confirmed, for example, in Example 12.6.2, where IFN gamma and TNF alpha levels correlated with treatment response. In a different experiment (Example 12.6.6), increased levels of IFN gamma, IL-1b, and IL-2 were associated with improved efficacy. Suitable interferon-stimulated genes or proteins include, but are not limited to, ACOD1, ACTG1, ACTR2, ACTR3, ADAMTS13, AIF1, AQP4, ASS1, B2M, BST2, C9JQL5, CALCOCO2, CAMK2A, CAMK2B, CAMK2D, CAMK2G, CASP1, CCL1, CCL11, CCL13, CCL14, CCL15, CCL15, CCL14, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL7, CCL8, CD40, CD44, CD58, CDC42EP2, CDC42EP4, CIITA, CITED1, CLDN1,CX3CL1, CXCL16, CYP27B1 DAPK1, DAPK3, EDN1, EPRS, EVL, FCGR1A, FCGR1B, FLNB, GAPDH, GBP1, GBP2, GBP4, GBP5, GBP6, GCH1, GSN, HCK, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-E, HLA-F, HLA- G, HLA-H, ICAM1, IFI30, IFITM1, IFITM2, IFITM3, IFNG, IFNGR1, IFNGR2, IL12B, IL12RB1 IL23R, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF9, JAK1, JAK2, KIF16B, KIF5B, KYNU, LGALS9, MEFV, MID1, MRC1, MT2A, MYO1C, NCAM1, NMI, NOS2, NUB1, OAS1, OAS2, OAS3, OASL, PDE12, PML, PRKCD, PTAFR, RAB12, RAB20, RAB43, RAB7B, RPL13A, RPS6KB1 RYDEN, SEC61A1 SLC11A1 SLC26A6 SLC30A8 SNCA, SP100, STAR, STAT1, STX4, STX8, STXBP1, STXBP2, STXBP3, STXBP4, SYNCRIP TDGF1, TLR2, TLR3, TLR4, TRIM21, TRIM22, TRIM25, TRIM26, TRIM31, TRIM34, TRIM38, TRIM5, TRIM62, TRIM68, TRIM8, UBD, VAMP3, VCAM1, VIM, VPS26B, WAS, WNT5A, XCL1, XCL2, ZYX.EXAMPLES Example 1: Alignment of CC chemokine receptors and CXC chemokine receptors

[00819] Human CC and CXC chemokine receptor sequences were retrieved from Uniprot and aligned with Clustal Omega. The results were imported into Jalview for visualization (Fig. 1). Negative amino acid residues E and D (gray), as well as tyrosine residues (Y, dark gray) and cysteine residues (C, light gray) are highlighted. While the N-terminal domains deviate in the sequence, they are characterized by a comparatively high number of negatively charged amino acids and tyrosine residues. Example 2: Alignment of human, cynomolgus, and murine CCR8

[00820] Human CCR8, cynomolgus CCR8, and murine CCR8 sequences were retrieved from Uniprot and aligned with Clustal Omega. The results were imported into Jalview for visualization and sequence identity calculation, see Fig. 2a. Although pairwise alignment of human and cynomolgus CCR8 yields a sequence identity of 94.37%, the percentages are considerably lower between mouse and human CCR8 (percent ID = 70.99), as well as between mouse and cynomolgus CCR8 (percent ID = 71.55). Substantial differences can be observed in the N-term region (TRD) and the C-term region.

[00821] Sequence homology between the cynomolgus TRD and the human TRD is 68%, while sequence homology between the murine TRD and the human TRD is 52%. However, the acid-sulfated tyrosine TRD shows a negatively charged cluster that was successfully used to generate cross-reacting antibodies, see Example 6. At least the MDYT and YYPD motifs were found between species. Example 3: Evaluation of antibodies from the previous technique for specific binding to CCR8

[00822] FACS experiments were performed using cell lines expressing CCR8 to evaluate several antibodies from the previous technique that are commercially sold as CCR8-binding due to their specificity and staining for CCR8. The mouse T-lymphoma cell line BW5147.4 expresses murine CCR8 and the human cutaneous T-lymphoma cell line, HuT78, expresses low levels of human CCR8. Prior art antibodies to chemokine receptors, and in particular to CCR8, often suffer from low specificity, as described, for example, by Xing et al., Appl IHC Mol Morphol (2015): "We were unable to evaluate CCR8 expression by immunohistochemistry due to lack of specificity of available antibodies in our experience and as previously published by others." evaluate the attraction of CD4+CCR8+ cells by CCL18 because of the lack of specificity of all commercially available anti-CCR8 Abs, which did not allow cell sorting, at least in our hands.", or Pease, Biochem J (2011): "The latter has been problematic due to the lack of a seemingly reliable antibody against human CCR8. undetectable using a commercially available antibody, although the cells evidently expressed functional CCR8, as assessed by CCL1-driven actin polymerization." Chenivesse, Cécile, et al. “Pulmonary CCL18 recruits human regulatory T cells.” The Journal of Immunology 189.1 (2012): 128-137.;

[00823] Table 3.1 lists three commercially available antibodies analyzed that did not specifically stain CCR8 on the inventors' hands (as per Fig. 3). Mouse IgG2B clone #191704 (MAB1429, Systems &D) did not show specific binding to human CCR8, and this is also in agreement with the observations described in Example 6 of WO2007044756. Abcam E77 showed very weak signal and high background (data not shown). MM0068-4G19 (Abcam), 191704 (R&D Systems), 4G19 (Biozol (Genetex)), and 11n24 and 10k39 (both from antikoerper-online.de) did not show specific binding to human CCR8 on the inventors' hands. Table 3.1: Selected prior art antibodies, as per Fig. 3.

[00824] The inventors found only two reliable monoclonal antibodies in the prior art that specifically bound to human CCR8: Clone 433H developed by ICOS and initially described in WO2007044756 and L263G8 supplied and sold by Biolegend. Both antibodies were generated using cells that overexpress human CCR8 as an immunogen. L263G8 is not cross-reactive to murine CCR8 and did not bind to murine CCR8 in BW5147.3 cells or HEK 293T cells transfected with mCCR8.

[00825] The mouse monoclonal IgG2b, K-cell anti-mouse CCR8 SA214G2 (Biolegend Catalog No. 150302) stained positive for BW5147.4 cells expressing murine CCR8. This antibody was generated from cells transfected with murine CCR8.

[00826] In summary, antibodies that specifically recognize human CC chemokine receptors are rare for some members of this target class. The low rates are consistent with the results of other commercially available antibodies sold for human chemokine receptors. Example 4: Antigens for CC and CXC chemokine receptors

[00827] Given that the literature on tyrosine sulfation is incomplete and partially contradictory, for example, between species, the inventors used various available tools, literature research and their own experiments to predict sulfation sites.

[00828] Table 4.1 summarizes the resulting polypeptides suitable as antigens for obtaining enhanced antibodies for the respective chemokine receptors. These antigens were successfully used for antibody generation, for example, as shown in the subsequent Examples, as antigens or for panning off-target. The underlined sulfation sites were confirmed with an E cutoff value of 55 applying the algorithm according to Monigatti F. et al. (Monigatti, Flavio, et al. "The Sulfinator: predicting tyrosine sulfation sites in protein sequences." Bioinformatics 18.5 (2002): 769-770). None of the sequences had been previously included in the training set for the hidden Markov models.

[00829] The sulfation of tyrosines in parentheses can be omitted. For human CCR3 and human CCR8, additional sulfation sites were predicted for Y172 and Y353, respectively. For monkey CCR3 and monkey CCR2, additional sulfation sites were predicted for Y172 and Y188, respectively. The sulfation sites shown in bold have been previously described in the literature (as per Liu, Justin, et al. "Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease." American Journal of Respiratory Cell and Molecular Biology 38.6 (2008): 738-743; Millard; Christopher J., et al. "Structural basis of receptor sulfotyrosine recognition by a CC chemokine: the N-terminal region of CCR3 bound to CCL11/eotaxin-1." Structure 22.11 (2014): 1571-1581). Peptide design

[00830] The ON term for CCR8 is formed by amino acids 1 to 35 within the full-length human and cynomolgus CCR8 protein and by amino acids 1 to 33 for the full-length mouse CCR8 protein. The ON term consists of the TRD (amino acids 1 to 24 for human and cynomolgus CCR8 and amino acids 1 to 22 for mouse CCR8) and the LID domain (amino acids 26 to 35 for human and cynomolgus CCR8 and amino acids 24 to 33 for mouse CCR8), which are connected by a cysteine. For the synthesis of polypeptides comprising the N term, the cysteine was replaced by a serine to avoid peptide aggregation. Table 5.1 shows the respective IDs of the polypeptides that were used for the generation of anti-CCR8 antibodies by phage display as described in Example 6. To test whether post-translational protein modifications were causing difficulties for antibody generation, peptides comprising TRD and/or N-term sequences were used, which were unmodified or sulfated. Sulfation in the human cynomolgus and TRD of CCR8 was introduced in at least 50% of the tyrosines, for example, at positions 3, 15, and 17 of the respective peptides. For the mouse TRD and N-term peptides, sulfations were introduced in the tyrosines at positions 3, 14, and 15 of the respective peptides (as per Table 6.1). In addition, the peptides comprising the LID were manipulated by the introduction of a C-terminal biotinylation that was linked via a TTDS (trioxatridecanosuccinamic acid) ligand. Other markers and ligands can also be used. The peptides for the TRD or N-term were modified by the introduction of an N-terminal biotinylation linked through a TTDS-Lysine ligand. Again, other known ligands and/or markers can also be used to facilitate peptide immobilization. Table 4.1: Polypeptides for antigen or off-target panning for antibody generation for chemokine receptors. At least one sulfation at one of the listed tyrosine positions of the polypeptide is required according to the invention, but sulfations at two, three or more sites are also possible and may be beneficial. X can be any amino acid or no amino acid and, preferably, is an amino acid other than cysteine, such as serine. Table 5.1: Polypeptides synthesized for CCR8 as a target

[00831] To determine if post-translational protein modifications were causing difficulties in antibody generation, peptides comprising TRD and/or N-term sequences that were unmodified or sulfated were used. Sulfation of the cynomolgus and human CCR8 TRD was introduced in at least 50% of the tyrosines, for example, at positions 3, 15, and 17 of the respective peptides. For mouse TRD and N peptides, sulfations were introduced in the tyrosines at positions 3, 14, and 15 of the respective peptides (as per Table 6.1). In addition, peptides comprising the LID were manipulated by the introduction of a C-terminal biotinylation that was linked via a TTDS (trioxatridecanesuccinamic acid) ligand. Other markers and ligands can also be used. The peptides for the term TRD or N were modified by the introduction of an N-terminal biotinylation linked via a TTDS-Lysine ligand. Again, other known ligands and/or markers can also be used to facilitate peptide immobilization. Peptide synthesis

[00832] Peptides can be obtained from different commercial sources and can be prepared as is known in the art, for example, using standard solid-phase peptide synthesis (SPPS) chemistry. Efficient synthesis of sulfated peptides is technically challenging because tyrosine-sulfated peptides are stable under basic conditions, while sulfotyrosines undergo desulfation under acidic conditions, making the stepwise incorporation of FmocTyr(SO3Na)OH into the growing peptide inadequate. Global sulfation of peptides is possible, for example, using sulfur trioxide-pyridine, but does not allow the specific sulfation of selected tyrosines. However, Fmoc-based SSPS can nevertheless be used for the synthesis of sulfated peptides. For this purpose, orthogonally protected tyrosine derivatives such as fluorosulfated tyrosine, azidomethyl-protected tyrosine, or neopentyl-protected tyrosine can be used for incorporation. The protected tyrosine can then be selectively and quantitatively unmasked.

[00833] Chen et al., Angew Chem, 2016, reported a one-step synthesis of Fmoc-fluorosulfated tyrosine. An efficient Fmoc solid-phase peptide synthesis strategy is then introduced to incorporate the fluorosulfated tyrosine residue into peptides of interest. Standard simultaneous peptide-resin cleavage and removal of acid-unstable side-chain protecting groups provide crude fluorosulfated tyrosine-containing peptides. Basic ethylene glycol, serving as both solvent and reagent, transforms the fluorosulfated tyrosine peptides into sulfotyrosine peptides in high yield.

[00834] The resulting peptides can be further purified as known in the art, for example, by HPLC, for example, on a BEH C-18 column (Waters) or Kinetex C-18 (Phenomenex) and can be analyzed by mass spectrometry (IonSpray and Positive Ion Detector), for example, for quality control.

[00835] In the literature, two disulfide bridges within the full-length human CCR8 protein have been described to connect extracellular loop 1 (ECL1) and extracellular loop 2 (ECL2) via cysteines at positions 106 and 183. An additional disulfide bridge between cysteines 25 and 272 is assumed to link the 7th transmembrane helix with the N-terminus. However, peptides according to Example 6 are not expected to form any disulfide bridges between cysteines. Example 6: Display of phages and analysis of anti-human CCR8 antibodies

[00836] A fully human antibody phage display library (bioinvent n-CoDeR Fab lambda library) was used to isolate human monoclonal antibodies by selection against soluble biotinylated peptides. The peptides were supplied by Pepscan. Peptide synthesis was performed as described in Example 5. The following protocol was applied to the panning procedure. Table 6.1: List of peptides including modifications used for antibody selection by phage display and ELISA analysis.

[00837] Streptavidin-coupled M-280 Dynabeads (Invitrogen™) were coated for one hour at room temperature (RT) with the biotinylated peptide (1 tube) and the off-target biotinylated peptide (3 tubes), respectively. The Dynabeads were washed and subsequently blocked for 1 hour at room temperature with end-to-end rotation.

[00838] For off-target ligand depletion, the blocked phage library was added to the off-target loaded, blocked Dynabeads and incubated for 10 min at room temperature with end-to-end rotation. This depletion step was repeated 2 times. The depleted phage library was added to the blocked target loaded Dynabeads and incubated for 60 min at RT with end-to-end rotation.

[00839] After thorough washing (3x in blocking buffer (PBS-T, 3% milk powder) and 9x in PBS-T (150 mM NaCl; 8 mM Na2HPO4; 1.5 mM KH2PO4; adjusted to pH = 7.4-7.6, 0.05% Tween-20)), Dynabeads with Fab phages that bind specifically to the coated target were used directly to infect the HB101 strain of E. coli. Subsequently, the phages were amplified in the E. coli HB101 strain using the M13KO7 Helper Phage (Invitrogen).

[00840] In subsequent selection cycles, the target concentration was decreased to increase the selection pressure for high-affinity ligands. During library panning, two different selection strategies were performed (Fig. 4). The strategies were designed to identify antibodies exhibiting binding activity to the N-terminal region of human CCR8 comprising the TRD and/or the N-terminal region of cynomolgus CCR8 comprising the TRD. All peptides used as antigens or off-target were sulfated at least once, as per Table 4.1 and Table 6.1.

[00841] For both strategies, a depletion step was included using the N-terminal region of biotinylated human CCR4 comprising TRD (TPP-14829, SEQ ID NO:22, with Y22 being sulfated) as the off-target peptide.

[00842] Strategy I comprised a first panning cycle on the sulfated human N-terminal CCR8 peptide (TPP-14827, SEQ ID NO:46, with Y3, Y15, Y17 being sulfated) followed by 3 cycles on the same peptide (TPP-14827, SEQ ID NO:46 with Y3, Y15, Y17 being sulfated) or on the sulfated N-terminal cynomolgus CCR8 peptide (TPP-14828, SEQ ID NO:47 with Y3, Y15, Y17 being sulfated).

[00843] Strategy II comprised a first round of panning on the N-terminal CCR8 sulfated peptide of cynomolgus (TPP-14828, SEQ ID NO:47 with Y3, Y15, Y17 being sulfated) followed by 3 cycles on the same peptide (TPP-14828, SEQ ID NO:47 with Y3, Y15, Y17 being sulfated) or three cycles on the N-terminal human CCR8 sulfated peptide (TPP-14827, SEQ ID NO:46 with Y3, Y15, Y17 being sulfated), as represented in Fig. 4.

[00844] For an initial qualitative assessment, for each set of clones, monoclonal culture and expression of 88 randomly selected Fab-on-phage clones was performed, and the clones were subsequently tested for binding to the respective target previously used for selection. In addition, binding to cells expressing human CCR8 and cynomolgus was determined by flow cytometry (FACS).

[00845] A specific ligand is defined in the particular context as a molecule showing (i) in the ELISA assay a signal intensity for the target that is at least 10 times greater than the signal intensity for the target, (ii) in the FACS assay a signal for cell lines expressing CCR8 that is at least 2 times greater than the signal for a control cell line that does not express CCR8, and (iii) in the FACS assay a signal in the control cell line that is less than 10,000.

[00846] Plasmid-DNA from 12 pools with significant hit rates in at least one of the two assays was subjected to gene III removal, and the resulting soluble Fabs were selected in a high-throughput ELISA and FACS analysis. Example 7: High-throughput analysis for human anti-CCR8 antibodies

[00847] From the 12 pools of clones generated during phage display, 17,000 different sFab clones were selected in a high-throughput ELISA (HTS-ELISA) analysis to determine their binding to the peptides that were previously used for phage display, namely (i) TPP-14827, SEQ ID NO:46, with Y3, Y15, Y17 being sulfated, (ii) TPP-14828, SEQ ID NO:47 with Y3, Y15, Y17 being sulfated and (iii) the respective off-target peptide TPP-14829, SEQ ID NO:22, with Y22 being sulfated. For ELISA analysis, the peptides were immobilized on streptavidin-coated plates (Greiner bio-one 781997) at a concentration of 0.1 μg/ml at 4°C in coating buffer (Carbonat-Basis, Candor 121125). After washing the plates 3 times with 60 μl of 0.05% Tween PBS and blocking with 50 μl of Smart Block® (Candor 113500) for 1 h at 20°C, 10 μl of sFab samples were added to the plates and incubated at 20°C for 1 h. After subsequent washing with 60 μl of 0.05% Tween PBS three times, 20 μl of an anti-c-Myc HRP antibody were added and incubated for 1 h at 20°C, followed by subsequent washing three times with 60 μl of 0.05% Tween PBS and the addition of 20 μl of Amplex Red solution (Invitrogen A12222, 1:1000 in 50 mM NaP buffer pH 7.6 with 1:10000 of 30% H2O2). After a final incubation for 20 min at 20°C, the signal was determined using an emission wavelength of 595 nm and an excitation wavelength of 530 nm. For analysis, the signal-to-background ratio of the single sFab clones was used, while the background is defined by the average value of 48 wells in each plate containing no sFab culture, but the respective sample medium.

[00848] In total, 2193 different sFab clones showed significant binding to the N-terminal and human cynomolgus sulfated peptides TPP-14827 (SEQ ID NO:46, with Y3, Y15, Y17 being sulfated) and TPP-14828 (SEQ ID NO:47 with Y3, Y15, Y17 being sulfated), as indicated by a signal-to-background ratio greater than 5, although they did not demonstrate any significant binding to off-target TPP-14829 (SEQ ID NO:22 with Y22 being sulfated).

[00849] These sFab clones were reformatted into full-length human IgG1 antibodies and applied to FACS analysis in cell lines expressing human CCR8 and cynomolgus.

[00850] Based on binding to both cynomolgus and human CCR8-expressing cells, and not demonstrating any significant nonspecific binding to a non-CCR8-expressing parental cell line, ten antibody clones were selected for further production and characterization. These initial hits were TPP-17575 to TPP-17581 and TPP-18205 to TPP-18207; the CDRs are shown in Table 7.1. The antibodies were also manually optimized for applicability in therapy to produce the antibody set shown in Table 7.2a and Table 7.2b, as well as sequence listing.

[00851] In addition, to obtain the TPP-27495 antibody, the TPP-23411 sequences were manipulated to encompass the YTE M252Y, S254T, and T256E mutations. TPP-27495 is 70% non-fucosylated.

[00852] In addition, to obtain the TPP-27496 antibody, the TPP-23411 sequences were manipulated to encompass the LS M428L and N434S mutations. TPP-27496 is 70% non-fucosylated. Example 8: Generation of anti-murine CCR8 antibodies Phage display and antibody analysis

[00853] A fully human antibody phage display library (BioInvent n-CoDeR lambda Fab library) was used to isolate human monoclonal antibodies recognizing murine CCR8 by selection against soluble biotinylated peptides.

[00854] The peptides were supplied by Pepscan. Peptide synthesis was performed as described elsewhere here. The following protocol was applied to the panning procedure.

[00855] Streptavidin-coupled M-280 Dynabeads (Invitrogen™) were coated for one hour at room temperature (RT) with the biotinylated peptide (1 tube) and the biotinylated peptide outside the target (3 tubes), respectively. The Dynabeads were washed and subsequently blocked for 1 hour at room temperature with end-to-end rotation. For depletion of off-target ligands, the blocked phage library was added to the blocked off-target loaded Dynabeads and incubated for 10 minutes at room temperature with end-to-end rotation. This depletion step was repeated twice. The depleted phage library was added to the Dynabeads loaded with the blocked target and incubated for 60 minutes at RT with end-to-end rotation.

[00856] After thorough washing (3x in blocking buffer (PBS-T, 3% milk powder) and 9x in PBS (150 mM NaCl; 8 mM Na2HPO4; 1.5 mM KH2PO4; adjusted to pH = 7.4-7.6) with 0.05% Tween-20), Dynabeads with Fab phages that bind specifically to the coated target were used directly to infect the HB101 strain of E. coli. Subsequently, the phages were amplified into the E. coli HB101 strain using M13KO7 Helper Phage (Invitrogen™).

[00857] In subsequent selection cycles, the target concentration was decreased to increase the selection pressure for high-affinity ligands. Table 7.1: CDRs for specific human anti-human CCR8 candidate antibodies obtained using the method according to the present invention. Table 7.2 a: CDRs for specific human anti-human CCR8 candidate antibodies according to the present invention with a superior therapeutic profile. Table 7.2 b: CDRs for candidate anti-human CCR8 antibodies derived from TPP-23411. Table 7.3: CDRs for specific antimurine CCR8 candidate antibodies derived with a mixed approach. Table 8.1: List of peptides used for antibody selection by phage and ELISA analysis. Table 9.1: Structural analysis of HCDR3 for specific human anti-human CCR8 candidate antibodies yielded a method according to the present invention. Table 9.2: Structural analysis of HCDR3 for specific human anti-human CCR8 candidate antibodies with a superior therapeutic profile, e.g., low internalization.

[00858] For the generation of antibodies that bind to mouse CCR8, a selection strategy was designed to identify antibodies that exhibit binding activity to the mouse CCR8 tyrosine-rich domain (TRD) in the N-terminal region. For positive selection on mouse CCR8 TRD peptides, a mixture containing 50% normal peptide and 50% sulfated peptide (SEQ ID NO:45 non-sulfated (TPP-13792) or with Y3, Y14, Y15 being sulfated (TPP-13793)) was used. A depletion step was included using a biotinylated off-target peptide being the human CCR4 TRD within the N-terminal region. Furthermore, the depletion step in human CCR4 was conducted in a mixture of sulfated and non-sulfated peptide (SEQ ID NO: 19 non-sulfated (TPP-13799) or with sulfated Y19 and Y22 (TPP-13800)).

[00859] The panning strategy was to conduct four panning cycles in total, each in the specified mixture of sulfated and non-sulfated peptides, see Fig. 5.

[00860] For each clone pool, monoclonal culture and expression of 88 randomly selected Fab phage clones was performed, and the clones were subsequently tested for binding to the respective target used previously for selection. However, separate ELISA measurements were performed for the sulfated and non-sulfated peptides.

[00861] In addition, binding to cells expressing human, mouse and cynomolgus CCR8 was determined by flow cytometry (FACS).

[00862] A specific ligand is defined in the particular context as a molecule showing (i) in the ELISA assay, a signal intensity for the target that is at least 10 times greater than the signal intensity for the target, and (ii) in the FACS assay, a signal for cell lines expressing CCR8 that is at least 10 times greater than the signal for a control cell line that does not express CCR8.

[00863] Within these measurements, the antibodies TPP-14095 and TPP-14099 were identified demonstrating binding to mouse CCR8-expressing cells, but no binding to control cells, human CCR8-expressing control cells, or cynomolgus CCR8-expressing control cells. Furthermore, the antibodies were found to bind to the sulfated TRD peptide, but not to the non-sulfated peptide. Example 9: Structural characterization of CCR8 antibodies

[00864] Of the six CDR loops, the H3 loop shows the greatest structural diversity and is located in the center of the binding site. It also acquires the most mutations through affinity maturation and has, on average, the highest number of contacts with the antigen. Therefore, it plays a crucial role in antigen binding.

[00865] After analysis of the specific structure of the antibodies obtained in the previous Examples, it was surprisingly found that the composition of HCDR3 was structurally different from the usual human HCDR3 domains. More specifically, the HCDR3 domain of the human anti-human CCR8 antibodies had a substantially higher number of tyrosine residues (~20% for the initial set of human CCR8-specific ligands, ~21% for the optimized candidates) than would be expected for a random all-human HCDR3 of corresponding length (~10%, as per Zemlin, Michael, et al. "Expressed murine and human HCDR3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures." Journal of molecular biology 334.4 (2003): 733-749.).

[00866] These data suggest a beneficial impact of the presence of tyrosine for specific binding to the sulfated antigen. The optimized antibodies were also characterized by a substantial increase in histidine frequency. For these antibodies, the histidine content averaged 10%. The average occurrence of histidine in HCDR3 of a compatible length is approximately 2% in murine HCDR3 and even lower in human HCDR3, according to Zemlin, Michael, et al. (2003).

[00867] Since the antigens used for selection are characterized by negative charges provided in the form of sulfated tyrosines and negatively charged amino acids, the inventors hypothesized that the occurrence of positive charges in HCDR3 improves binding to the sulfated antigen, but is less essential for binding to the non-sulfated antigen. Although the frequency of positively charged residues (H, K, R) in the HCDR3s of the initially obtained antibody set was approximately 7%, the improved antibodies had on average 15% positive amino acids per HCDR3 (e.g., up to ~31%).

[00868] The antibodies according to the present invention are characterized by a tyrosine frequency that is higher than for usual fully human CDR3s. Although these high tyrosine frequencies are more easily obtained with murine CDRs, this may explain why previous attempts to obtain human anti-human antibodies that specifically recognize CC chemokine receptors, such as CCR8, have not been successful. Example 10: Functional characterization of anti-CCR8 antibodies Cell Lines

[00869] Stable HEK 293 and CHO cell lines expressing human, cynomolgus, or murine CCR8 were generated using Inscreenex, as known in the art. The Hut-78 thymoma cell line and the TALL-1 lymphoma cell line were obtained from ATCC and were confirmed to exhibit endogenous CCR8 expression. Since CCR8 expression can vary, expression levels were monitored. Human regulatory T cells

[00870] CD4+ and CD25+ cells sorted by FACS from healthy donor PBMCs were acquired from BioIVT, AllCells, or StemCell Technologies. After thawing and overnight recovery in culture, the cells were activated with anti-CD3 and anti-CD28 beads designed for Miltenyi Treg expansion and supplemented with IL-2 (R&D Systems) as directed by the Treg expansion kit protocol. Typically, after activation of naive PBMCs, CCR8 expression was highest between days 5 and 7, and cells could be re-stimulated 7 to 9 days after primary activation. After re-stimulation, CCR8 expression was highest between days 2 and 4. Example 10.1.1: Evaluation of CCR8 binding affinities from different species. FACS experiments.

[00871] The affinity of antibodies for human, canine, and murine CCR8 and other chemokine receptors was determined by FACS as known in the art. In short, CHO cells were engineered to express the CCR8 of the respective species. Alternatively, activated human Tregs from a human donor were used to determine the affinity of the antibodies for the activated Tregs. EC50 values were determined by FACS. All candidate antibodies had not only a superior affinity for human CCR8 with an EC50 in the low-digit nanomolar range, but also a superior affinity for canine CCR8 by the same order of magnitude. Similar affinities in these species are important to facilitate studies for predicting future safety issues in humans. This is an important issue in the search for therapeutic antibodies in immuno-oncology because mouse models have major limitations in predicting immunological side effects in humans. The EC50 determined for the binding of human Tregs was between 10 and 25 nM for the inventive antibodies shown in Table 10.1.1.1.

[00872] The affinities of inventive antibodies on CHO cells expressing human CCR8, CHO cells expressing cynomolgus CCR8, or activated human Tregs (donor 1) are shown in Figs. 6, 7, and 8, respectively. Table 10.1.1.1: EC50 values of inventive antibodies in nM. FACS staining was performed on CHO cells stably expressing human CCR8 or cynomolgus CCR8, or on activated human CD4+CD25+ T cells. These cells were activated with the Miltenyi Treg expansion kit.

[00873] Antibodies TPP-23411 and TPP-21360 differ from each other by an added lysine residue at the C-term of the heavy chain, i.e., TPP-23411 comprises lysine. TPP-23411 and TPP-21360 were tested in a separate FACS experiment for binding to human and cynomolgus CCR8. The EC50 curves for binding to human CCR8 as well as cynomolgus CCR8 were perfectly aligned between the two antibodies (data not shown, see Table 10.1.1.2 for IC50 values). Table 10.1.1.2: EC50 values of candidates measured in nM. Staining was performed on CHO cells stably expressing human CCR8 or cynomolgus CCR8.

[00874] TPP-21181 and TPP-23411 were tested in a separate FACS experiment for binding to human and cynomolgus CCR8, as shown in Figs. 9, 10. The respective EC50 values are listed in Table 10.1.1.3. Table 10.1.1.3: EC50 values of antibodies measured in nM. Staining was performed on CHO cells stably expressing human CCR8 or cynomolgus CCR8.

[00875] In another FACS experiment, prior art L263G8 (Biolegend) and 433H (ICOS, BD) antibodies were compared with TPP-21360 regarding binding to human and cynomolgus CCR8, as shown in Figs. 11, 12 and Table 10.1.1.4. Prior art antibodies showed only low or substantially no overall binding to cynomolgus CCR8 (low saturation, here MFI << 1000). Only the antibodies according to the present invention specifically recognized human and cynomolgus CCR8 with affinities of the same order of magnitude, i.e., in some cases, even in the subnanomolar range. Table 10.1.1.4: EC50 values of TPP-21360 compared with L263G8 and 433H. Staining was performed on CHO cells stably expressing either human CCR8 or cynomolgus CCR8.

[00876] These results show that the use of sulfated antigen facilitates the generation of cross-reacting antibodies, such as cynomolgus/human, for difficult targets, such as CCR8, which are specific for the intended target. As a proof of concept, activated human Tregs were stained with the TPP-23411 antibody of the invention or the L263G8 antibody of the prior art, as shown in Fig. 13. The inventive antibodies that recognize murine CCR8 have equally excellent affinities for their target, as shown in Table 10.1.1.5. Table 10.1.1.5: EC50 values of TPP-14095 and TPP-14099. FACS staining was performed on CHO cells expressing murine CCR8.

[00877] Table 10.1.1.6 shows the EC50 values for the binding of several additional inventive anti-human CCR8 antibodies to CHO cells transfected with human CCR8 or cynomolgus CCR8, or to Hut78 cells expressing endogenous human CCR8. Interestingly, antibodies with a G50A mutation had a higher binding affinity to CHO cells expressing cynomolgus CCR8. Anti-CCR8 antibodies comprising this structural mutation are therefore preferred. Table 10.1.1.6 EC50 values in M characterizing the binding of inventive anti-human CCR8 antibodies to CHO cells transfected with human CCR8 or cynomolgus CCR8, as described elsewhere herein, or to the HuT78 cell line, which endogenously expresses human CCR8. Example 10.1.2: SPR experiments for characterizing antibody binding affinities for unmodified TRD domains from different species.

[00878] To analyze the affinity of antibodies for their unmodified (i.e., non-sulfated) antigen, surface plasmon resonance (SPR) binding assays were performed on a Biacore T200 instrument at 25°C with 1x HBS-EP+ assay buffer, 1 mg/ml BSA, 300 mM NaCl. IgGs were captured using anti-human Fc IgGs covalently amine-coupled to a CM5 sensor chip. N terminally labeled with His6 of human CCR8 TRD (TPP-19950: MDYTLDLSVTTVTDYYYPDIFSSP, SEQ ID NO:43), cynomolgus CCR8 TRD (TPP-19952: MDYTLDPSMTTMTDYYYP-DSLSSP, SEQ ID NO:44) or murine CCR8 TRD (TPP-19951: MDYTMEPNVTMTDYYPDFFTAP, SEQ ID NO:45) was fused at the C-terminal with human serum albumin (HSA) to generate the CCR8-HSA fusion proteins. The respective CCR8-HSA fusion protein was used as an analyte with a concentration range of 1.56–200 nM in multicycle kinetic mode. The obtained sensorgrams were fitted to a 1:1 Langmuir binding model. For human CCR8 anti-human TPP-23411 and TPP-21360 antibodies, binding was low (160 nM) and binding to cynomolgus CCR8 TRD was even lower, e.g., in the μM range, supporting the importance of sulfation for antibody recognition for the inventive antibodies. Table 10.1.2.1: Binding affinities of inventive antibodies to the fused TRD with His6-labeled HSA from human, cynomolgus, or murine CCR8 determined by SPR. Binding to cynomolgus CCR8 peptide is in the μM range and is an approximation, as the highest concentration tested was 200 nM. HSA alone and isotype controls showed no binding (data not shown). Example 10.1.3: SPR experiments for systematic characterization of binding affinities

[00879] SPR binding assays were performed to systematically evaluate antibody binding to aa) non-sulfated versus sulfated antigen, b) TRD versus N-term, and c) human versus cynomolgus CCR8. SPR binding assays were performed on a Biacore T200 instrument (Cytiva) at 25°C with 1x HBS-EP+ assay buffer (Order No. BR100826, Cytiva). Peptides were used as ligands and were captured using their C-terminal biotin label on a streptavidin-coated sensor chip ("Sensor Chip SA", Order No. BR100398, Cytiva). For this purpose, a terminal lysine (K) was added to the C-term of each peptide for biotinylation. For the N-term of CCR8, the naturally occurring cysteine between the TRD and LID domains was replaced with a serine. Antibodies were used as analytes (TPP-19546, TPP-21181, TPP-23411, hIgG1 isotype control TPP-9809 and mIgG2a isotype control TPP-10748). The analyte concentration in the assay buffer was 0.05–10 nM and the measurement was performed in multicycle kinetic mode. The sensor surface was regenerated with glycine pH 2.0 after each cycle. The obtained sensorgrams were doubly referenced (subtraction of the reference flow cell signal and buffer injection) and fitted to a 1:1 Langmuir binding model to derive kinetic data using the Biacore T200 evaluation software. Table 10.1.3.1: List of peptides used to characterize the antibody epitope. Peptides comprising the TRD (# 1 to 4) or N term (# 5 to 8) sequence of CCR8, derived from cynomolgus (# 1, 2, 5, 6) or human (# 3, 4, 7, 8) CCR8, sulfated (# 2, 4, 6, 8) or non-sulfated (# 1, 3, 5, 7). SO3: sulfate residue. Biot: biotin modification for immobilization.

[00880] For the inventive antibodies analyzed TPP-19546, TPP-21181, and TPP-23411, no binding to the non-sulfated TRD or non-sulfated N-term of CCR8 was detected, regardless of the species. Conversely, excellent affinities with KD values in the pM range were determined for sulfated TRD and sulfated N-term, for example, from cynomolgus and/or human CCR8. In some cases, the KD value for binding to the TRD and the N-term of human CCR8 was substantially the same, i.e., within the margin of error. In other cases, binding to the N-term was slightly improved.

[00881] In summary, these results demonstrate that sulfated tyrosine is the cause of binding. Furthermore, they suggest that sulfated TRD is necessary and sufficient for binding, but that the presence of the complete N term as an epitope may, in some cases, further enhance binding. Finally, these results also demonstrate cross-reactivity for human CCR8 and cynomolgus. Table 10.1.2.3: SPR binding affinities of inventive antibodies for the TRD or N term of cynomolgus or human CCR8. Peptides with odd numbers are characterized by unmodified tyrosines, peptides with even numbers are characterized by sulfated tyrosines (Ys). Isotype controls showed no binding to either peptide (data not shown). 1) mp = multiphase: approximate values. Example 10.2: Link specificity

[00882] FACS analysis was performed as described elsewhere here to determine nonspecific binding to target negative human cell lines. No nonspecific binding of the anti-human CCR8 antibody TPP-23160 was observed to CHO cells with sham transfectants (Fig. 14).

[00883] In addition, HEK cells were transiently transfected with human CCR1 or human CCR4. Off-target binding was low for most inventive antibodies (Figs. 15, 16). A cell panel with cell lines from different tumor tissues was used to characterize nonspecific binding in these tissues (Table 10.2.1). The overall profile was favorable for most antibodies, while a certain degree of polyreactivity could be observed for some antibodies. Overall, the degree of polyreactivity was within an acceptable range for therapeutic applications for all inventive antibodies. However, TPP-20950, TPP-18206, TPP-18429, TPP-18430, and TPP-18433 showed a higher degree of polyreactivity and showed some degree of binding in at least two or three of the seven cell lines shown below. Table 10.2.1: Polyreactivity of inventive antibodies in a cell panel. If applicable, EC50 is shown in M.

[00884] A first ELISA-based experiment was performed to analyze the nonspecific binding to BVP, insulin, or DNA for antibodies TPP-18206, TPP-17578, TPP-19546, and TPP-23411 and two reference antibodies. The nonspecific binding of the inventive antibodies to insulin, DNA, and/or BVP was lower than the nonspecific binding of the first reference antibody (Gantenerumab, Roche) to these antigens and was also lower than the nonspecific binding of the second reference antibody (Remicade, Janssen Biotech) to these antigens. Among the inventive antibodies tested, TPP-23411 showed the lowest polyreactivity for each DNA, BVP, and insulin.

[00885] A second ELISA-based experiment was performed to analyze the nonspecific binding to BVP, insulin, or DNA for antibodies TPP-18206 (wild-type or afuco), TPP-21360 afuco, TPP-23411 (wild-type or afuco), TPP-20955, TPP-21047, and reference antibodies TPP-9809 (isotype control), Gantenerumab (TPP-12151), Lenzilumab (TPP-12166), Remicade (TPP-12160), and Avastin (TPP-17586). The nonspecific binding of the tested inventive antibodies to insulin, DNA, and BVP was lower than the nonspecific binding of the first reference antibody (Gantenerumab, Roche). TPP-20955 and TPP-21047 showed more nonspecific overall binding than the other inventive antibodies (data not shown). Overall, the degree of polyreactivity was within an acceptable range for therapeutic applications for all inventive antibodies, regardless of their afucosylation state.

[00886] A third ELISA-based experiment was performed to analyze nonspecific binding to BVP, insulin, or DNA for TPP-27495 (YTE), TPP-27496 (LS), TPP-18429, TPP-18430, TPP-18432, TPP-18433, TPP-18436, TPP-27479, TPP-27480 antibodies and control antibodies. Overall, the degree of polyreactivity was within an acceptable range for therapeutic applications for all inventive antibodies, regardless of YTE or LS mutations.

[00887] The unwanted binding of inventive CCR8 antibodies to immune cell populations other than human Tregs was analyzed by staining these cell populations, as shown in Fig. 17.

[00888] Fig. 18 shows the upregulation of CCR8 in human Tregs after activation. Example 10.3: Evaluation of ADCC and ADCP induction Example 10.3.1: Antibody afucosylation

[00889] The antibodies according to the present invention were glyco-engineered using GlymaxX technology, as per US8642292, to enhance the interaction between the FC region of the antibodies and the FC receptor expressed by effector cells. In short, the technology is based on the heterologous cytosolic co-expression of the bacterial enzyme GDP-6-deoxy-D-lixo-4-hexulose reductase, which redirects the de novo synthesis pathway of fucose to the sugar-nucleotide GDP-Rhamnose, which cannot be metabolized by eukaryotic cells. This depletes the fucose pool and leads to the production of non-fucosylated antibodies, both in the central (without central fucose) and variable (without antennal fucose) portions of an N-glycan. It also inhibits the fucosylation of O-glycans and protein-O-fucosylation. The removal of fucose from N-glycans in N297 resulted in an increased affinity for FCYRS, as shown in Example 10.3.2. Example 10.3.2: Analysis of binding to the human Fcy receptor based on SPR

[00890] To determine the ability of the inventive antibodies and their afucosylated versions to bind to the respective FC receptors expressed on human effector cells involved in ADCC or ADCP, SPR binding assays were performed on a T200 instrument at 25°C with a CM5 sensor chip and HBS-EP+500 mM NaCl buffer assay. FcyR variants were captured using an amine-coupled anti-His capture antibody (ab), and IgGs were used as analyte at concentrations up to 25 μM. KD values were derived from steady-state affinity analysis or from kinetic data fitted to a 1:1 Langmuir isotherm. Non-fucosylated versions of the antibodies showed increased binding to human and cynomolgus FcgRIIIa, suggesting enhanced ADCC induction and therefore Treg depletion for the inventive antibodies in their non-fucosylated form in both species (Table 10.3.2.1).

[00891] Furthermore, a comparable affinity for both cynomolgus and human FCYRIII is important for modeling the efficacy of ADCC in an animal model of cynomolgus. These model systems are particularly important in tumor immunology because rodent models are often inadequate to reflect the immunological side effects of a therapeutic antibody. The non-fucosylated antibodies according to the present invention have shown highly comparable affinities for the respective FC receptors in cynomolgus and humans. Table 10.3.2.1: SPR analysis of inventive antibody affinities for different FC receptors. Italics: approximation only when the highest concentration was 25 μM and no saturation was reached; ne: not evaluable; nb: no binding; res. bdg.: residual binding, not quantitatively evaluable; KD >25 μM: adjusted value outside the saturation curve. TPP-9809: isotype control. Example 10.3.3: CCR8 antibody-mediated ADCC induction Example 10.3.3.1: Anti-human CCR8 antibody-mediated ADCC induction

[00892] For functional ADCC assays, HEK cells expressing human CCR8 or activated human Tregs were used as target cells and NK92v-GPF-CD16 176V (NantKwest) cells were used as effector cells. In a 96-well tissue culture-treated plate, a co-culture of the respective CCR8-expressing target cells with effector cells in a 4:1 ratio in the presence of various concentrations of therapeutic antibody was incubated in RPMI 1640 with 1% heat-inactivated FBS, 100 U/ml Pen/Strep, 1mN Na-pyruvate, and 1x NEAA. Digitonin was used for maximal lysis, and CytoTox-Glo (Promega) was added to the assay at the end of a 2-hour incubation. Crude luminescence values were determined using the CytoTox-glo Promega program. The Maximum Background Average, MMB = (Target Max) - (Target Spontaneous), is used to calculate the % lysis. % Lysis = (Gross Luminescent - without ab)/MMB x 100.

[00893] For antibody candidates TPP-19546 (top panel) and TPP-21360 (bottom panel), fucosylation increased ADCC-induced cytotoxicity to ~50 and ~70%, respectively, in HEK cells expressing human CCR8 (as shown in Fig. 19).

[00894] In activated human Tregs with approximately 85% CCR8 expression, ADCC-derived cytotoxicity resulting from non-fucosylated TPP-21360 or TPP-23411 was as high as 59% or 52% (as per Fig. 20, Table 10.3.3.1.3). For non-fucosylated TPP-21360, the mean EC50 % cytotoxicity for activated human Tregs with approximately 85% CCR8 expression using NK92v as effector cells was ~20 pM. Tables 10.3.3.1.1 to 10.3.3.1.6 show the results for different inventive antibodies and/or different donors. Table 10.3.3.1.1: Summary of the ADCC assay: EC50 and Max response using activated Tregs as target cells and NK92v cell line as effector cells. Table 10.3.3.1.2: Summary of the ADCC assay: EC50 and Max response using activated Tregs as target cells and NK92v cell line as effector cells. All TPPs used in this assay were non-fucosylated. Table 10.3.3.1.3: Summary of the ADCC assay: EC50 and Max response using activated Tregs as target cells and NK92v cell line as effector cells. The activated human Treg used in this study had approximately 85% CCR8 expression. Table 10.3.3.1.4: Summary of the ADCC assay: EC50 and Max response using activated Tregs as target cells and NK92v cell line as effector cells. The activated human Treg used in this study had only about 31% CCR8 expression. Table 10.3.3.1.5: ADCC assays: EC50 and Max response using HEK cells expressing human CCR8 (> 95% CCR8) as target cells and NK92v cell line as effector cells. Table 10.3.3.1.6: ADCC assays: EC50 and Max response using HEK cells expressing human CCR8 (> 95% CCR8) as target cells and NK92v cell line as effector cells. Example 10.3.3.2: Induction of ADCC mediated by anti-mouse CCR8 antibodies (surrogate antibodies)

[00895] To compare the behavior of the anti-human/cyno-human CCR8 antibodies of the invention with the anti-murine human CCR8 surrogate antibodies of the invention, the latter were also characterized for ADCC induction. The functional ADCC assay for the surrogate antibodies was performed using primary murine NK cells as effector cells and HEK293 cells expressing murine CCR8 and BW 5417.3 (data not shown) as target cells. Ultralow IgG-containing medium was used during co-culture to avoid nonspecific binding of CCR8 antibodies to serum IgGs. Furthermore, to more closely reflect the in vivo situation, target cells were first pre-incubated with surrogate CCR8 antibodies before effector cells were added, allowing for better FCYRIII clustering on effector cells, which is the initial step in the ADCC mode of action. Effector and target cells were co-cultured at a 10:1 ratio in RPMI 1640 with 1% IgG Ultra-low FBS One Shot medium for 20 hours using 96-well U-bottom plates. The antimurine CCR8 surrogate antibody TPP-15285 and the isotype control antibody TPP-10748 were used at various concentration steps. Cytotoxicity was determined using the CytoTox-Glo™ Cytotoxicity Assay (Promega) (% Cytotoxicity = [(Experimental Value - Control without antibody / (Maximum target cell lysis - Spontaneous target cell lysis)] x 100%). Experimental values were determined by co-culturing target cells with effector cells after the addition of surrogate CCR8 antibodies. Control values were determined by co-culturing target cells with effector cells without the addition of surrogate antibodies. Maximum lysis values were determined for target cells where digitonin was added at the beginning of the assay. Spontaneous lysis values were determined by measuring the spontaneous lysis of target cells in assay medium without the addition of any other reagents. 92% to 97% of target cells showed murine CCR8 expression (determined by FACS). ADCC-related cytotoxicity for TPP-15285 was ~39% and the mean EC50 was ~111. pM (Table 10.3.3.2.1). The higher concentration of inventive antibody resulted in a hook effect, further supporting the suggested mode of action based on ADCC. The optimized functional ADCC assay thus confirmed that ADCC is a relevant mode of action for the in vivo efficacy of the inventive CCR8 surrogate antibodies and, in this way, confirms the suitability of the anti-murine CCR8 antibody TPP-15285 as a surrogate antibody for anti-human CCR8 antibodies. Table 10.3.3.2.1: Summary of the ADCC assay using fucosylated (wild-type) CCR8 surrogate antibodies. Table 10.3.3.2.2: ADCC assays showing cytotoxicity in % induced by the wild-type CCR8 surrogate antibody TPP-15285 (4 replicates). Example 10.3.4: Induction of ADCP mediated by anti-CCR8 antibodies. Example 10.3.4.1: Induction of ADCP mediated by anti-human CCR8 antibodies.

[00896] To generate macrophage effector cells (M2c), CD14+ cells were negatively selected (Miltenyi) from healthy donor PBMCs (AllCells). M2c macrophage differentiation was performed according to an eight-day differentiation protocol provided by StemCell Tech (see Fig. 21). On the day of the assay, M2c was stained for expression of CD16, CD32, CD64, CD163, CD206, CD11b, CD80, and CD14 using FACS. Target cells were HEK cells expressing human CCR8 or activated human Treg cells. In short, target cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher) according to the manufacturer's instructions. Target cells were incubated in a co-culture in 96-well plates with different concentrations of antibodies and effector cells in a target-to-effector ratio of 4:1. In these assays, anti-CD47 (positive control) was added at 1 μg/ml; this provides an enhanced response as it blocks the "don't eat me" signal. After 3–4 hours of incubation, samples were processed using a MACSQuant or Attune flow cytometer. The % of phagocytosis was determined by double-positive CD206+CFSE+ cells. CD206 is a marker expressed on macrophages.

[00897] M1 macrophages were generated similarly and from the same source material (fresh PBMCs). In summary, CD14+ cells were isolated by negative selection and cultured for four days in serum-free medium (StemCell Tech Immnocult) with 50 ng/mL of M-CSF. However, on day 5, M1 macrophages were polarized by the addition of 10 ng/mL of LPS + 50 ng/mL of IFN-γ, while M2c macrophages were polarized with the addition of 10 ng/mL of IL-10.

[00898] Fig. 22 shows the induction of phagocytosis in HEK cells expressing human CCR8 as target cells with in vitro differentiated M2c macrophages as effector cells. Both wild-type and non-fucosylated versions of the inventive antibodies TPP-19546, TPP-21360, and TPP-23411 induced ADCP. Figs. 23 and 24 and Tables 10.3.4.1.1 to 10.3.4.1.7 show the induction of ADCP as characterized by EC50 and maximal response for multiple inventive antibodies, multiple donors, and different macrophage populations. For all antibodies, a substantial degree of ADCP was observed. Table 10.3.4.1.1: Summary of the ADCP assay: EC50 and Max response using activated Tregs as target cells and M2c macrophages as effector cells. All TPPs used in this assay were non-fucosylated. TPP-9809 is the isotype control. For the human Treg donor, 1212 CCR8 was expressed by 40% of the cells. Table 10.3.4.1.2: Summary of the ADCP assay: EC50 and Max response using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 is the isotype control. For the human Treg donor 1163, CCR8 was expressed by 67% of the cells. Table 10.3.4.1.3: Summary of the ADCP assay: EC50 and Max response using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 is the isotype control. For the human Treg donor, 1163 CCR8 was expressed by 67% of the cells. Table 10.3.4.1.4: Summary of the ADCP assay: EC50 and Max response using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 is the isotype control. For the human Treg donor 1212, CCR8 was expressed by 40% of the cells. Table 10.3.4.1.5: Summary of the ADCP assay: EC50 and Max response using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 is the isotype control. For the human Treg donor 1212, CCR8 was expressed by 40% of the cells. Table 10.3.4.1.6: Summary of the ADCP assay: EC50 and Max response using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 is the isotype control. Table 10.3.4.1.7: Summary of the ADCP assay: EC50 and Max response using activated Tregs as target cells and M2c macrophages as effector cells. TPP-9809 is the isotype control. Table 10.3.4.1.7: Summary of the Incucyte live imaging ADCP assay: EC50 and Max response using macrophages as effector cells and HEK cells expressing human CCR8 as target cells in a 4:1 ratio. Human CCR8 expression was 80%. TPP-9809 is the isotype control. M2c macrophages were seeded in 96-well ultralow fixation plates. After the addition of therapeutic antibodies at various concentrations, target cells labeled with pHrodo red dye (Sartorius #4649) were added and the plates were incubated at 37°C and 5% CO2. Phase contrast and red fluorescence images were automatically acquired and analyzed every 30 min for 12 hours using a 20x objective on an Incucyte S3 (4 images/well in triplicate). ADCP induction varied for the tested wild-type therapeutic antibodies. Example 10.3.4.2: Induction of ADCP mediated by antimurine CCR8 antibodies (surrogate antibodies)

[00899] To compare the behavior of the inventive anti-human/human CCR8 antibodies with the anti-murine human CCR8 surrogate antibodies of the invention, the latter were similarly characterized for ADCP induction. For the functional ADCP assay, murine primary bone marrow-derived M2 macrophages were used as effector cells and murine CCR8-expressing cells HEK293 and BW 5417.3 (data not shown) as target cells. To generate the effector cells, murine bone marrow-derived macrophages were seeded in a 24-well plate and polarized into M2 macrophages by the addition of 20 ng/ml M-CSF, 0.05 μg/ml IL-4, and 0.05 μg/ml IL-13. Target cells were labeled with carboxyfluorescein succinimidyl ester dye (CFSE, ThermoFisher) and added to effector cells in a 2:1 effector-to-target ratio. Finally, the surrogate antimurine CCR8 antibody TPP-15285 and the isotype control TPP-10748 were added at various concentrations. After 2 hours of co-culture, cells were stained for the murine M2 macrophage marker F4/80, and the % phagocytosis was determined using flow cytometry by measuring the proportion of double-positive F4/80+ and CFSE+ macrophages. 92% to 97% of target cells showed murine CCR8 expression (as determined by FACS). Phagocytosis induced by TPP-15285 was ~11% and the mean EC50 was ~402 pM (Table 10.3.4.2.1). The higher antibody concentration resulted in a hook effect, further supporting the suggested mode of action. The optimized functional ADCP assay further confirmed that ADCP is a relevant mode of action for the in vivo efficacy of the inventive CCR8 surrogate antibodies and thus confirms the suitability of the anti-murine CCR8 antibody TPP-15285 as a surrogate antibody for anti-human CCR8 antibodies. Table 10.3.4.2.1: Summary of the ADCP assay using fucosylated (wild-type) CCR8 surrogate antibodies. Example 10.4: Modulation of CCR8 signaling by CCR8 antibodies

[00900] CCL1 is a specific ligand for CCR8. Upon binding to CCR8, CCL1 can induce calcium (Ca) influx, chemotaxis, and – via β-arrestin signaling – receptor internalization, the latter possibly being independent of G protein signaling. There are at least 6 different ways an antibody can modulate a GPCR: 1. An inverse agonist, upon binding, calms the potential endogenous activation of the GPCR. For CCR8, endogenous activation can be, for example, ~10%. 2. A polarized antagonist for β-arrestin or G protein blocks G protein-dependent signaling or β-arrestin signaling and has no effect on the respective other pathway. 3. A neutral antagonist can block both G protein and β-arrestin signaling pathways. 4. A full agonist activates both G protein and β-arrestin signaling pathways. 5. A polarized agonist for β-arrestin or G protein activates G protein or β-arrestin signaling pathways, but not both.6. A dimerization-promoting agonist crosslinks two GPCRs, for example, with different antibody arms, resulting in multiple signaling activities.

[00901] For an ADCC and ADCP-based approach to deplete intratumoral Tregs, it would be ideal to exclude any potential signaling effects that an antibody might have on CCR8. To assess this, the inventors used the Ca flux assay as a readout for G protein-dependent signaling and β-arrestin activation, and phospho signaling as a readout for G protein-independent signaling pathways. Phospho Erk1/2 is involved in the MAP kinase pathway and regulates a variety of cellular responses, while phospho AKT is well known to be associated with the PI3 kinase pathway and has been shown in CCR8 to be involved in promoting chemotaxis. The AKT pathway is also involved in promoting cell survival and growth, and recently, CCL1 has been shown to promote the survival of cells expressing CCR8 (Barsheshet, Yiftah, et al. "CCR8+ FOXp3+ Treg cells as master drivers of immune regulation." Proceedings of the National Academy of Sciences 114.23 (2017): 6086-6091). Example 10.4.1: DiscoverX assay for monitoring β-arrestin signaling

[00902] In response to a stimulus, i.e., the binding of a ligand—such as CCL1 to CCR8—GPCRs can activate G protein-independent signaling, such as β-arrestin signaling. This can result in chemokine receptor internalization (Fox, James M., et al. "Structure/Function Relationships of CCR8 Agonists and Antagonists. Amino-terminal extension of CCL1 by a single amino acid generates a partial agonist." Journal of Biological Chemistry 281.48 (2006): 36652-36661). The β-arrestin assay was acquired from DiscoverX. In summary, CCR8 is fused within the frame with a small ProLink™ enzyme donor fragment (PK) and co-expressed in cells that stably express a β-arrestin fusion protein and the larger N-terminal deletion mutant of β-galactosidase (called the acceptor enzyme or EA). Activation of CCR8 stimulates the binding of β-arrestin to PK-labeled CCR8 and forces the complementation of the two enzymatic fragments, resulting in the formation of an active β-galactosidase enzyme. This interaction leads to an increase in enzymatic activity that can be measured using PathHunter chemiluminescent detection reagents.

[00903] In a first experiment, the antibody or CCL1 was incubated with CHO cells co-expressing human CCR8 labeled with ProLink and EA for 90 min before adding the detection agent. Neither the antibodies from the prior technique nor the inventive antibodies induced β-arrestin signaling (Fig. 25).

[00904] In a subsequent experiment, cells were stimulated with CCL1 at EC80 to activate β-arrestin signaling, and either inventive antibodies or prior art antibodies were added to assess their ability to block activated β-arrestin signaling. Surprisingly, β-arrestin signaling was blocked by both the prior art L263G8 and 433H antibodies, while the inventive antibody TP-23411 showed no significant effect on β-arrestin signaling, at least up to a concentration of 100 nM (Fig. 26). Table 10.4.1.1: IC50 values for inhibition of CCL1-induced β-arrestin signaling of TPP-23411 and prior art L263G8 and 433H antibodies measured in nM. Staining was performed on CHO cells stably expressing ProLink-labeled human CCR8. No IC50 could be determined for the inventive antibody, suggesting that TPP-23411 does not block CCL1-induced β-arrestin signaling. Example 10.4.2: Phospho ELISA

[00905] Phospho AKT signaling promotes survival and growth, while phospho Erk1/2 signaling regulates a variety of cellular responses. Phospho Erk1/2 and phospho AKT antibodies were purchased from CellSignaling, and ELISA assays were performed according to the manufacturer's protocol. CHO cells expressing human CCR8 or activated human Tregs were treated with CCL1, TPP-23411, Biolegend L263G8, or BD 433H antibody, or remained untreated for the negative control. CCL1 served as the positive control. Interestingly, the antibodies from the previous Biolegend L263G8 technique and the BD 433H antibody induced a significant increase in phosphorylated Erk1/2 levels (Figs. 27, 28), as well as phosphorylated AKT levels (Figs. 29, 30), for example, after 15 minutes in activated human Tregs, whereas this was not the case for inventive antibodies.

[00906] In summary, both antibodies from the previous technique are agonistic for G protein-independent pathways, such as AKT phosphorylation or ERK1/2 phosphorylation. Example 10.4.3: Calcium flow assay

[00907] Measuring intracellular calcium mobilization is a robust assay that can be performed in a high-throughput manner to study the effect of compounds or antibodies on potential drug targets, such as CCR8. To this end, the activity of receptors that signal through Ca2+ release can be monitored using the fluorescence-based FLIPR calcium assay (Molecular Devices, Sunnyvale, CA). In short, human CCR8-expressing CHO cells were seeded in assay plates and incubated overnight. The cells were then loaded with Ca+-sensitive dye BUV396/496.

[00908] For the antagonistic function measurement, monitoring of the Ca flow signal using FLIPR Tera began in parallel with the addition of antibodies or reference compound (MC148) at different concentrations to the cells. After 1 min of incubation of the cells with the antibodies or reference compound, the CCL1 agonist was added and the calcium flow signal was recorded for a further 3 min using FLIPR Tetra. To measure the agonistic function, antibodies/reference compound were added in parallel with monitoring of the Ca flow signal using FLIPR for 3 min.

[00909] Tables 10.4.3.1 to 10.4.3.4 show the results of the calcium flux assay for anti-murine or anti-human CCR8 antibodies. No IC50 could be determined for TPP-14099 and TPP-21047. IC50 for other inventive antibodies varied. In general, most inventive antibodies and also the antibodies from the prior art effectively blocked CCL1-induced G protein-dependent signaling. Table 10.4.3.1: Ca flux assay. Murine CCR8-expressing CHO cells were used to test the ability of antibodies to block CCL1-induced G protein-dependent Ca signaling. CCL1 at 40 ng/ml was used for induction. Both TPP-14095 and TPP-14099 bind to mouse CCR8. No agonist effect on G protein-dependent Ca signaling was observed for the inventive antibodies or the prior art antibodies (data not shown). Table 10.4.3.2: Ca flux assay. CHO cells expressing human CCR8 were used to test the ability of antibodies to block CCL1-induced G protein-dependent Ca signaling. CCL1 was used at a concentration of 80 ng/ml. Table 10.4.3.3: Ca flux assay. CHO cells expressing human CCR8 were used to test the ability of antibodies to block CCL1-induced G protein-dependent Ca signaling. CCL1 was used at a concentration of 60 ng/ml. Table 10.4.3.4: Ca flux assay. CHO cells expressing human CCR8 were used to test the ability of antibodies to block CCL1-induced G protein-dependent Ca signaling. CCL1 was used at a concentration of 60 ng/ml. Example 10.5: Internalization of inventive antibodies

[00910] To evaluate CCR8 internalization, imaging technology was used to visualize this process. The specific anti-CCR8 antibodies TPP-21360 and TPP-23411 and the corresponding isotype control antibody TPP-5657 were labeled with a fluorescent dye. In addition, the commercial antibodies L263G8 and 433H, as well as the murine antibody SA214G2, were also conjugated with the permanent dye BODIPY. These antibodies were conjugated with lysine with a two to six molar excess of BODIPY® FL dye (ester, D2184, ThermoFisher) at pH 8.3. After conjugation, the reaction mixture was purified by chromatography (PD10 1-2.5 ml desalting column; GE Healthcare) to remove excess dye and adjust the pH value. Next, the protein solution was concentrated (VIVASPIN 500, Fa. Sartorius stedim biotec). The determination of the antibody dye load was performed with a spectrophotometer (NanoDrop) and calculated using the formula D:P = A dye ε protein : (A 280 -0.16A dye) ε dye. The dye load of the target-specific antibodies TPP-21360 and TPP-23411 and the isotype control antibody were found to be in a similar range (dye load/antibody: 4.0 and 4.1). Commercial antibodies produce dye loads/antibody of 3.8 and 3.9. Conjugation can negatively impact antibody affinity; therefore, testing conjugated antibodies in a cell binding assay (FACS) is essential to ensure that labeling has not altered binding to CCR8 (CD198) (as per Fig. 31a, Fig. 32a, Fig. 34a, b). The conjugated antibodies were then ready for use in internalization assays performed with the human CCR8-positive cell line HuT78 and the murine CCR8-positive cell line BW5147.3. Prior to treatment, CCR8-expressing cell lines (2 x 10⁴/well) were seeded in 100 μl of medium in a 96-MTP (CellCarrier Ultra, PerkinElmer). After 18 h of incubation at 37°C/5% CO₂, the medium was changed and the labeled antibody was added at various concentrations (2.5, 1 μg/ml; triplicated). The same treatment protocol was applied to the labeled isotype control antibody (negative control). Additionally, the parental CHO-K1 cell line (CCR8-negative cells) was similarly treated, serving as a counter analysis (specificity control). The internalization study with target-specific antibodies was performed kinetically. Images were taken after different time points (0 h, 0.5 h, 2 h, 6 h, and 24 h), and internalization efficacy was determined by measuring the total internalized fluorescence intensity per cell. Measurement was performed using the Operetta CLS (PerkinElmer), and image data analysis was performed using the Harmony high-content analysis software (PerkinElmer).

[00911] Figs. 31 and 32b show substantial internalization of commercial anti-human CCR8 antibodies 433H and L263G8 in HuT78 cells and commercial anti-murine CCR8 antibody SA214G2 in BW5147 cells, while Fig. 33 demonstrates low or no internalization of the inventive antibodies TPP-21360 and TPP-23411, which is substantially comparable to the isotype control. Table 10.5.1: Summary of internalization data for inventive antibodies and prior art antibodies. nD: not tested.

[00912] To confirm that low internalization was indeed a characteristic that characterizes all inventive anti-CCR8 antibodies obtained with a sulfated peptide antigen as described herein, the inventive antibodies listed in Table 10.5.2 (see also Fig. 89) were analyzed regarding their internalization behavior with vesicle area/cell as the readout. An anti-CD71 antibody with known substantial internalization was used as a positive control. In fact, all inventive anti-human CCR8 antibodies tested were non-internalizing antibodies, i.e., antibodies that did not exhibit any substantial degree of internalization. Interestingly, TPP-17577, the only anti-human CCR8 antibody tested without histidine within HCDR3, showed slightly higher internalization than all tested anti-human CCR8 antibodies comprising histidine within HCDR3. Table 10.5.2: Summary of internalization data for inventive antibodies. Not tested. Example 10.6.1: FcRn affinity chromatography of anti-CCR8 antibodies

[00913] The neonatal Fc receptor (FcRn) is linked to the regulation of the metabolic processing of IgG antibodies in vivo. Binding to FcRn in the acidic environment of vascular endothelial cell endosomes protects IgGs from lysosomal degradation. The release of IgGs from FcRn into the bloodstream occurs at physiological pH 7.4. Thus, FcRn binds to IgGs at pH < 6.5 and does not bind at physiological pH 7.4. The interaction between IgGs and FcRn has an impact on the pharmacokinetics of IgGs in vivo. Characterizing the pH-dependent binding of IgG to FcRn, mimicking the antibody recycling interaction via immobilized FcRn, may reveal properties of IgG that contribute to its pharmacokinetics.

[00914] FcRn affinity runs were performed on an Akta Pure 25 chromatography system at 25°C. An FcRn column (Roche, Order No. 8128057001; pre-packed with approximately 1 mL of resin; binding capacity: > 100 μg IgG) was equilibrated with running buffer A: 20 mM MES/HCl pH 5.5 140 mM NaCl. 30 μL of each antibody (1 mg/mL in equilibration buffer) were applied to the column. A linear pH gradient was generated by increasing the percentage of running buffer from 20 mM Tris B/HCl pH 8.8 to 140 mM NaCl as follows: 0 min - 20% B, 10 min - 20% B, 80 min - 100% B, 90 min - 100% B, 93 min - 20% B, 103 min - 20% B. The flow rate was 0.5 mL/min. The pH value at the elution point was monitored and read from chromatograms generated with Unicorn 7.1.

[00915] As can be seen in Table 10.6.1.1, the prior art antibody Ustekinumab served as a control for standard pharmacokinetics and the prior art antibody Briakinumab served as a control for rapid pharmacokinetics (as per Schoch, Angela, et al. "Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics." Proceedings of the National Academy of Sciences 112.19 (2015): 5997-6002.). TPP-17577, TPP-18205, TPP-27495, TPP-21047, and TPP-27496 show an elution pH greater than pH 7.5 and can be considered as exhibiting FcRn binding also at pH 7.4 to pH 7.5. The measured pH can be used to predict the clearance and half-life of an antibody. Table 10.6.1.1: FcRn affinity chromatography for different antibodies: pH at elution. Two pH values indicate a double peak. Example 10.6.2: FcRn binding analysis based on SPR

[00916] To evaluate the binding affinity of anti-CCR8 antibodies for FcRn, binding was investigated using SPR at pH 6.0 and pH 7.4. Binding assays were performed on a Biacore T200 instrument at 25°C with a CM5 sensor chip and PBST assay buffer. For FcRn binding assays, human, mouse, or cynomolgus FcRn was amine-coupled to the surface of the CM5 sensor chip (~300 RU) and IgGs were injected at concentrations ranging from 15.6 to 2000 nM in PBST, pH 6. Regeneration was performed with PBST pH 7.4. In another experiment at pH 7.4 only, IgGs were tested at a concentration of 2 μM, therefore, only qualitative assessment of binding was performed. The KD values were derived from a steady-state affinity analysis as well as from kinetic analysis by fitting to a 1:1 linkage isotherm.

[00917] As can be seen in Table 10.6.2.1, and as described previously, Ustekinumab served as a positive control and Briakinumab as a negative control as published in Schoch et al, 2015. These two antibodies do not differ significantly in their affinity for FcRn, but in their binding response to FcRn at pH 7.4. Thus, it can be assumed that antibodies that exhibit a binding response at pH 7.4 may have an accelerated half-life in vivo. Example 10.7: Heparin affinity chromatography of anti-CCR8 antibodies

[00918] For inventive antibodies that recognize human CCR8, charge-mediated interaction is highly likely because the antibodies recognize a charged antigen tyrosine motif and have a certain structural composition, for example, in relation to their HCDR3. Charge-mediated interactions are, however, a driving force for IgG degradation beyond FcRn binding at pH 7.4. Binding to a negatively charged glycocalyx of monocytes or macrophages can lead to pinocytosis and subsequent proteolytic degradation. Kraft et al. showed that antibody behavior in vivo can be predicted more efficiently and accurately by conducting heparin affinity chromatography together with FcRn chromatography (Kraft, Thomas E., et al. "Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis." MAbs. Vol. 12. No. 1. Taylor & Francis, 2020.). Table 10.6.2.1: SPR analysis of inventive antibody affinities for different FCRN receptors. Small binding response is defined as a response < 30 RU, large binding response as > 30 RU.

[00919] High affinity, high level of interaction, and therefore high retention times may predict a shorter half-life of the tested antibodies. Heparin affinity cycles were performed on an Akta Pure 25 chromatography system at 25°C. A heparin (Tosoh) column was equilibrated with running buffer A: 50 mM Tris, pH 7.4. 50 μL of each antibody (1 mg/mL in 20 mM Histidine pH 5.5) was applied to the column. A linear salt gradient was generated by increasing the percentage of running buffer B: 20 mM Tris pH 7.4 1M NaCl as follows: 2 min after injection - 0% B, 0 to 55% B over 16.5 minutes, 100% over 0.5 minutes, 100% B for four minutes. The flow rate was 0.8 mL/min. The elution time was monitored and read from chromatograms generated with Unicorn 7.1.

[00920] As can be seen in Table 10.7.1, and as described previously, Ustekinumab served as a positive control and Briakinumab as a negative control. Most of the inventive antibodies tested have a retention time of around 15 to 18 min. Therefore, these antibodies may have a reduced half-life if electrostatic interactions contribute in vivo. Table 10.7.1: Heparin affinity chromatography for different antibodies: pH at elution. Example 10.8: Toxicological studies and CCR8 depletion in Macaca fascicularis

[00921] Macaca fascicularis was treated with 50 mg/kg of TPP-23411 for four weeks. The animals showed no signs of toxicity. One week after the last treatment, thymus tissue (which has the highest levels of CCR8 among healthy tissues) was analyzed using RNA-seq. The CCR8 mRNA level was significantly reduced in animals treated with anti-CCR8 antibody. No significant reduction in CCR8 levels was found at lower doses of TPP-23411. Example 11: Therapeutic applicability of the target

[00922] For therapeutic use of a given target in immuno-oncology, specific target expression is of utmost importance to minimize systemic side effects. The target must be specific to the target tissue or cells, for example, to intra-tumoral Tregs, and must not be expressed in healthy tissues. Furthermore, increased target expression in a given tumor tissue may point to the medical use of that indication. For example, CCR8 is also expressed in B-cell lymphoma and T-cell lymphoma tumor cell lines.

[00923] Systemic removal of Treg cells can evoke and enhance not only tumor immunity but also autoimmunity, as evidenced by patients suffering from X-linked polyendocrinopathy-enteropathy immune dysregulation syndrome (IPEX). In this disease, patients lack Tregs due to a genetic alteration and die within the first 2 years of life due to systemic autoimmunity if not treated with appropriate immunosuppression. The high specificity of a target intended for the removal or suppression of intratumoral Tregs is therefore considered very important to avoid side effects of a therapeutic antibody, such as those observed for antibodies targeting CCR4. Example 11.1: Specificity of the CCR8 target

[00924] In an initial analysis, the inventors evaluated CCR8 mRNA expression in different human tissues and cell types. Significant CCR8 mRNA expression was observed only in activated regulatory T cells, as well as in tumor-infiltrating lymphocytes (Fig. 35).

[00925] In a second analysis, the inventors evaluated CCR8 mRNA expression in 50 different tumor indications. The indications with the highest CCR8 expression are, for example, breast cancer, lung adenocarcinoma (ADC), testicular cancer, stomach cancer and squamous cell carcinoma (SCC), head and neck malignancies, as well as esophageal tumors, but high expression was also found in colorectal cancer, ovarian cancer and cervical cancer. In all indications, except pancreatic adenocarcinoma and melanoma, expression was found to be higher in the tumor compared to the corresponding normal tissue (Fig. 36).

[00926] In a third analysis, the inventors evaluated the co-expression of CCR8 mRNA with FOXP3 mRNA in various tumor indications. Co-expression with the Treg marker FOXP3 is important to demonstrate that CCR8 is indeed primarily expressed in Tregs, as per Table 11.1.1. IHC staining confirmed these findings, see Fig. 37. Table 11.1.1: CCR8 and the T-cell defining regulatory marker FOXP3 are the most strongly correlated genes in all 9588 TCGA tumor samples (excluding Thymoma as an indication), pointing to these genes being co-expressed in the same cell type. The table shows the 25 genes (out of 35,021 genes) most strongly correlated with CCR8 and FOXP3 mRNA expression levels and their respective Pearson correlation coefficients, r.

[00927] Furthermore, the correlation of CCR8 mRNA with the pan-T cell marker CD3, cytotoxic T cell marker CD8, macrophage marker MS4A7, general inflammation marker IFNγ, and B cell markers CD19, CD20, CD22 was evaluated for 50 tumor indications, as shown in Table 11.1.2. Interestingly, for almost all indications, the correlation was highly significant, suggesting increased CCR8 expression following immune cell infiltration into the tumor per se, and T reg infiltration in particular. From this, it can be expected that immune cell infiltration, and in particular T cell infiltration, will be equally valuable biomarkers for patient stratification to identify patients who are more likely to benefit from anti-CCR8 therapy. Table 11.1.2: Pearson correlation coefficients, r, between CCR8 log2 mRNA levels and Treg marker FOXP3, pan-T cell marker CD3, cytotoxic T cell marker CD8, macrophage marker MS4A7, general inflammation marker IFNγ, or B cell markers CD19, CD20, CD22. The results suggest a highly significant co-expression of CCR8 with immune cells and, in particular, Treg infiltration in tumors in 48 of the 50 TCGA indications. Therefore, it can be expected that, for example, T cell infiltration will constitute a valuable stratification biomarker for identifying patients likely to benefit from anti-CCR8 therapy. Example 11.2: Specificity of CCR8 for intratumoral Tregs compared to effector T cells

[00928] There are several issues to consider regarding Treg depletion for cancer immunotherapy in humans. Activated Tregs and T effector cells most often share the expression of the same types of cell surface molecules, including CD25 and CTLA-4, making selective depletion of Tregs without also affecting T effector cells by antibodies specific to these molecules difficult.

[00929] Foxp3+CD25+CD4+ Treg cells in tumors (tumor-infiltrating or intratumoral Tregs) express higher levels of cell surface molecules associated with T cell activation, such as CD25, CTLA-4, PD-1, LAG3, TIGIT, ICOS, and members of the TNF receptor superfamily, including 4-1BB, OX-40, and GITR, compared to Tregs in lymphoid or non-lymphoid tissues or in the blood. Furthermore, tumor-infiltrating Tregs express high levels of specific chemokine receptors, including CCR4 and CCR8.

[00930] Sorted and unsorted CD4+ CD25+ PBMCs were activated with anti-CD3 and anti-CD28 + IL2 beads for 6 days. FACS analysis showed preferential expression of the CCR8 protein on stimulated Tregs (CD4+CD25+FoxP3+CD127dim), see Fig. 38. Alternative targets OX40, GITR, and CD25, but not CCR8, are significantly expressed on stimulated CD8+ Teff cells and CD4+ T cells (CD4+CD25+Foxp3-). CCR4 shows fairly specific Treg expression, but no specificity for intratumoral Tregs. These findings are consistent with the observation that CCR4 antibodies have shown immunological side effects, which have been attributed to systemic Treg depletion. In terms of specificity, CCR8 appears to be superior in supporting specific intratumoral Treg depletion.

[00931] To confirm the results, raw data from patients with NSCLC, CRC, and liver cancer were retrieved from Guo, Xinyi, et al. "Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing." Nature medicine 24.7 (2018): 978-985, Zhang, Yuanyuan, et al. "Deep single-cell RNA sequencing data of individual T cells from treatment-naive colorectal cancer patients." Scientific data 6.1 (2019): 1-15, and Zheng, Chunhong, et al. "Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing." Cell 169.7 (2017): 1342-1356, respectively, and used for analysis of CCR8 specificity, see Fig 39. In fact, for these tumors, CCR8 mRNA expression is largely restricted to regulatory T cells (light gray boxes) residing in tumor tissue, while it is largely absent in regulatory T cells from normal tissues, as well as from CD4 helper and CD8 cytotoxic T cells (medium and dark gray boxes, respectively).

[00932] FACS analysis of human tumor lysate samples confirmed that the CCR8 protein is specifically expressed in human tumor-infiltrating Tregs in non-small cell lung cancer (NSCLC), colorectal carcinoma (CRC), renal cell carcinoma (RCC), and lymph nodes (LNs, data not shown), Fig. 40. Up to 90% of tumor-infiltrated Tregs are positive for CCR8, for example, in NSCLC, 40 to 90% in stage III+ and 40 to 70% in stage I/II, in CRC 20 to 40% in stage III+ and in RCC 40 to 50% in stage III+. Based on these data, it is assumed that stratification according to staging leads to an additional benefit for patients treated with an anti-CCR8 antibody. Example 12: In vivo experiments Example 12.1.1: Surrogate antibodies and syngene tumor models

[00933] In mouse experiments, surrogate antibodies that bind to mouse CCR8 were used to demonstrate antitumor efficacy and to characterize the mode of action, i.e., Treg depletion within the tumor depending on the potency of ADCC (antibody-dependent cellular cytotoxicity) and ADCP (antibody-dependent cellular phagocytosis). Table 12.1.1.1: List of mouse CCR8 surrogate antibodies used for in vivo studies. ADCC was determined using murine CCR8-overexpressing HEK293 cells as target cells and NK92 cells as effector cells. ADCC activity is measured by lactate dehydrogenase (LDH) release.

[00934] The anti-murine CCR8 antibody TPP-14099 binds to murine CCR8-expressing CHO cells with an EC50 of 3 nM and murine iTregs with an EC50 of 13.2 nM, as determined by FACS (data not shown).

[00935] Different syngeneic tumor models were used to model tumors with a high degree of immune infiltration, as well as tumors with a low degree of immune cell infiltration. Furthermore, the models used diverge in their responses to treatment with known checkpoint inhibitors, for example, treatment with anti-CTLA4, anti-PD1, or anti-PD-L1 antibodies. An overview of the tumor models tested with efficacy data for the respective inventive anti-CCR8 antibody is shown in Table 12.1.1.2. Notably, efficacy was demonstrated in several syngeneic tumor models.

[00936] In summary, a suspension containing generally 500,000 to 1,000,000 tumor cells per 100 μl of PBS, medium, or a 50% Matrigel mixture was injected subcutaneously into the flank of immunocompetent female mice (e.g., Balb/c, C57Bl6). At palpable tumor sizes of approximately 60–100 mm³ (generally 100 mm³), CCR8 antibody administration began with intraperitoneal injection of typically 10 mg/kg in a volume of 5–10 ml/kg. Lower doses were tested in dose titration studies. Non-binding antibodies of the respective isotype served as controls. Groups comprised n = 10 mice. Therapy was applied twice weekly, typically on a q3/4d schedule, and tumor size and body weight were measured three times weekly. Tumor growth data are plotted as average volume over time.

[00937] Tumor and tissue samples were taken from identically treated satellite animals (n = 5 per group) 24 hours after the second antibody treatment and were analyzed by flow cytometry after single-cell dissociation and erythrocyte lysis with particular focus on changes in absolute numbers of T cells (Tregs) per 100 mg of tissue. The markers used to determine activated Tregs were CD45+CD4+CD25+FoxP3+. Other FACS markers included CD8, NKp46, 4-1BB, F4/80, CD11c, Gr1, and MHCII. Table 12.1.1.2: Overview of different tumor models with parameters to characterize treatment outcome. Efficacy was measured by Treg depletion, overall response rate, and tumor-control ratio based on final tumor volumes of anti-CCR8 surrogate antibodies in syngeneic mouse tumor models. 1 Treg depletion was calculated as the percentage difference between intratumoral Tregs in the isotype control and intratumoral Tregs in tumors treated with the therapeutic antibody. 2 Overall response rate (ORR) and complete response (CR) are based on an adaptation of the RECIST criteria to rapid tumor growth in a mouse model (CR: complete responder (<10% of initial tumor volume); PR: partial responder (maximum 300% of initial tumor volume); NR: non-responder (>300% of initial tumor volume)). 3 T/C refers to the treatment tumor volume/control tumor volume at the end of the study. nd: not determined. 4 Averages from several studies in this tumor model.

[00938] For each syngeneic mouse tumor model, 10 tumors from both the isotype control group and the anti-CCR8 treatment group were collected at the end of the treatment cycle, 24h after the last treatment (biweekly treatment until tumors in either group reached a mean volume of 3000 mm3), and genome-wide mRNA expression was measured using RNA-seq. Of the 17 models evaluated using RNA-seq 13, the models showed elevated CD8 mRNA levels in the anti-CCR8 treatment group compared to the control group with fold alterations as shown in Table 12.1.1.3. For the CT26, H22, RM1, MBT2, and Hepa1-6 models, the increase in CD8 levels was significant according to a T-test statistic indicating significant increases in cytotoxic T cell infiltration at the end of the treatment cycle. These data strongly suggest that CD8+ cells were involved in enforcing the tumor response. Table 12.1.1.3: Changes in CD8 mRNA levels induced by anti-CCR8 treatment. Example 12.1.2: Characterization of a syngeneic model for biomarker identification

[00939] Based on genome-wide RNA-seq data from early tumors in syngeneic models, increased levels of the following genes were strongly correlated with tumor response: Eif3j2, Eno1b, Ifi44l, Hist1h2al, Ifi202b, Hmga1b, Amd2, Sycp1, Itln1, Trim34b, Catsperg2, Zfp868, Serpina1b, Prss41, C1rb, Cyld, Ccnb1ip1, Masp2, Acaa1b, C4a, Snord93, Abhd1, Serpina3h, H2-K2, Cd1d2, Hal, Rnf151, Rbm46, Arg2, Mir8099-2, Igsf21, Olfr373, C1s2, Crym, Arv1, Hddc3, Plppr4, Ppp1r11, Rps3a2, Zfp459, Rnd1, Serpina1a, Vcpkmt, Atp10d, Gbp2b, H2-T24, Tlcd2, Ctse, H2-Q10, Cyp2c55, Borcs8, Tpsab1, Trim43b, Cc2d1a, Serpina1d, Cacna1a, Kcnj14, Ttc13, Farsa, Olfr1217, Jaml, H2-Bl, Tnpo2, Rims3, Dock9, Car5b, Atp1a4, H2-Q1, Zfp69, Slpi, Pcdhgb8, Ocel1, Selenbp2, Nsd3, Wt1, Nap1l2, Ranbp9, Gtpbp3, AY761185, Rnaset2a, Serpina3i, Ell2, Gal3st2b, Urb2, F12, Klk1, Ifi214, Cstl1, Agtpbp1, Msh5, Cox18, Zfp330, Ttc37, Klk4, H2-Q5, Cxcl11, Rab39, Pm20d1, Nod2, H2-DMb2. Interestingly, this set is highly enriched for early complement factors, complement regulatory factors such as Serpins, and MHC components. These markers, or a combination thereof, can be used for stratification or to predict or monitor tumor response. In particular, the presence of high levels of Complement C1/C4 may contribute to Treg lysis. When depletion/consumption of complement factors reduces the effectiveness of Treg depletion, supplementation of the complement system, for example, in a combination therapy, may be an option.

[00940] The different syngeneic models were also characterized in relation to the absolute number of immune cell subsets and frequency within the total CD45+ immune cells in the tumors. For the models analyzed by flow cytometry (Table 12.1.2.1), B16F10 is characterized by the lowest number of immune cells - and showed the smallest reduction in tumor volume, as shown in Table 12.1.2.1. Thus, immune cell infiltration can be used for stratification to predict the response to treatment with CCR8 antibodies. This hypothesis could be subsequently confirmed using B16Bl6. Table 12.1.2.1: Characterization of the tumor microenvironment by absolute numbers of immune cell subsets and frequency in the total CD45+ immune cells in the tumors of the vehicle group analyzed at an initial time point (24 hours after the second treatment). T/C vol describes the ratio of tumor volume in the group treated with CCR8 antibody (10 mg/kg twice weekly) versus the control group at the end of the study. Example 12.2: Efficacy in tumor-carrying mice CT26

[00941] Treatment with anti-CCR8 antibodies TPP-15285 and TPP-15286 showed strong antitumor efficacy in mice carrying CT26 tumor (Fig. 41). TPP-15285 showed reduced efficacy at the lowest dose of 0.1 mg/kg; other dose-dependent differences were not significant. As expected, an anti-PD-L1 ("PDL1") antibody demonstrated moderate efficacy in the model. Non-binding antibodies of the respective isotypes showed no efficacy.

[00942] The spider plots (tumor growth of individual mice over time) of the corresponding treatment groups (Fig. 42) clearly demonstrated homogeneously strong efficacy with complete responses occurring in all dose groups with both anti-CCR8 antibodies.

[00943] Analysis of Tregs from CT26 tumor samples 24 hours after the second antibody treatment by flow cytometry showed strongly reduced numbers of Tregs in the anti-CCR8 antibody-treated group versus the isotype control group. Treg depletion was clearly dose-dependent in the case of TPP-15285 (Fig. 43). Table 12.2.1: Corresponding data from Fig. 43. Example 12.3: Mode of action studies Example 12.3.1: Mode of action studies of ADCC/ADCP in CT26 tumor-carrying mice

[00944] Aglycosylated antibodies, which can be produced in prokaryotic hosts, show nearly identical antigen-binding affinity, stability at physiological temperature, and persistence in serum in vivo compared to glycosylated counterparts. However, the absence of N-linked glycans negates almost all FCYR-binding affinity and immune effector functions that are essential for clearing antibody-opsonized target cells via ADCC or ADCP. To provide evidence for the ADCC/ADCP-based mode of action, aglycosylated versions of the antibodies were generated as a negative control.

[00945] TPP-18208 and TPP-18209 are glycosylated human IgG1 versions of anti-CCR8 surrogate antibodies and are unable to bind to effector cells, such as NK cells and/or macrophages, via Fc-gamma receptors. These antibodies were tested for antitumor efficacy in mice carrying CT26 tumor and were compared with their respective wild-type/glycosylated counterparts comprising the same sequence (TPP-14095, TPP-14099). In addition, the anti-CCR8 antibodies TPP-15285 and TPP-15286 (glycosylated, mIgG2 isotype) were included in the study.

[00946] Wild-type antibodies showed strong antitumor efficacy, while aglycosylated antibodies showed no efficacy compared to the isotype control (Fig. 44). Spider plots illustrate the results for individual mice in each treatment group (Fig. 45). Consequently, ex vivo tumor analysis by flow cytometry demonstrated Treg depletion only after treatment with wild-type (glycosylated) antibodies, while aglycosylated antibodies showed no Treg depletion compared to the respective unbound isotype control (Fig. 46).

[00947] These results confirmed ADCC and/or ADCP as the predominant mode of action for Treg depletion by anti-CCR8 antibodies in CT26 tumors.Example 12.3.2: Involvement of B cells or T cells and impact of CD8+ cell depletion or CD19+ cell depletion on the antitumor efficacy of anti-CCR8 antibodies

[00948] To test the relevance of the presence of tumor-infiltrating CD8+ T cells or CD19+ B cells on the antitumor efficacy of anti-CCR8 antibodies, the inventors used genetically modified C57BL6 mice expressing the diphtheria toxin receptor (DTR) under the control of the lineage-specific Cd8a+ promoter or the lineage-specific Cd19+ promoter. After diphtheria toxin (DT) injection, CD8+ T cells or CD19+ B cells were quantitatively depleted from both mouse blood (data not shown) and subcutaneously transported syngeneic MC38 tumors (Table 12.3.2.1).

[00949] As expected, depletion of CD8+ T cells completely negated the antitumor effect of the anti-CCR8 treatment, resulting in tumor growth identical to that of the isotype control group (Fig. 87). These results confirmed the dependence of the antitumor efficacy of the inventive anti-CCR8 antibodies on the presence of CD8+ T cells.

[00950] Conversely, and very surprisingly, CD19+ B cell depletion significantly increased the efficacy of TPP15285 (Fig. 88), resulting in a reduction of the T/C ratio from 0.35 to 0.16 (T-test p-value = 0.032). The combination of anti-CCR8 treatment with B cell depletion or B cell depleting agents, such as anti-CD19 antibodies or anti-CD20 antibodies, such as Rituximab, is therefore suggested to further improve cancer treatment in the clinic. Table 12.3.2.1: Depletion of CD8a and CD19 expression in DTR mice treated with diphtheria toxin. Table 12.3.2.2: Treatment groups to evaluate the influence of CD19+ B cell depletion or CD8+ T cell depletion. Example 12.3.3: Time course of increasing CD8/FOXP3 ratios in mice carrying the CT26 syngeneic tumor

[00951] To gain some mechanistic insights into immune cell levels, CD8 and FOXP3 mRNA levels were monitored in mice carrying CT26 syngeneic tumors treated with TPP15285 or isotype control. Ten tumors were collected for RNA-seq analysis directly before the first dose administration, as well as 12h, 24h, 36h, 48h, and 72h after the first dose administration. Similarly, tumors were collected 24h after the administration of a second, third, and fourth dose of TPP15285. All tumors underwent RNA-seq analysis to measure murine Foxp3, CD8a, and CD8b1 mRNA levels. The mean ratios of Cd8a and Cd8b1 to Fopx3 were calculated, and the significance of the differences in ratios between the control and TPP15285-treated groups was assessed using a t-test.

[00952] CD8 to FOXP3 mRNA ratios ranged from 0.9 to approximately 2.0 in the isotype control and early time points after TPP15285 treatment, but then significantly increased in the TPP15285 groups to values of 5.8, 3.9, and 4.4 at 72 h after the 1st dose, 24 h after the 2nd dose, and 24 h after the 3rd dose, respectively, demonstrating significant depletion of regulatory T cells by TPP15285. The ratios fell to a value of 2.6 24 h after the 4th dose administration. Table 12.3.3.1: CD8/FOXP3 ratio of mRNA levels measured at different time points after TPP15285 administration or isotype control. Example 12.4: Efficacy of the CCR8 TPP-15285 antibody in mice carrying the EMT6 tumor. Example 12.4.1: Efficacy of the CCR8 TPP-15285 antibody in mice carrying the EMT6 tumor - different dosage.

[00953] The surrogate anti-CCR8 antibody TPP-15285 showed dose-dependent efficacy in EMT6 tumor-carrying mice, with the strongest effect in the 10 mg/kg dose group. At this dose, TPP-15285 had superior efficacy compared to the anti-CTLA4 antibody (Fig. 47, Fig. 48). Strong efficacy was also observed in the 1 mg/kg dose group, while the 0.1 and 0.01 mg/kg dose groups showed almost no efficacy. Spider plots illustrate these results at an individual mouse level (Fig. 49).

[00954] Analysis of Tregs from EMT6 tumor samples 24 hours after the second antibody treatment by flow cytometry showed strongly reduced numbers of Tregs in the anti-CCR8 antibody-treated group versus the isotype control group. Treg depletion was clearly dose-dependent (Fig. 50). Treg depletion was also observed for anti-CTLA4, as previously described in the literature. Additionally, a strong increase in CD8+ T cells was demonstrated in the tumors sampled at the end of the study for the effective dose groups of 10 mg/kg and 1 mg/kg of TPP-15285, as well as for anti-CTLA4 (Fig. 51). Other changes in immune cell populations are shown in Table 12.4.1.1 and Table 12.4.1.2. Table 12.4.1.1: Relative percentage of immune cell populations determined by FACS analysis of immune cells 24 hours after the second treatment of EMT6 tumor-carrying mice with different doses of CCR8 TPP-15285 antibody, or at the end of the study. Table 1 2.4.1.2: Relative percentage of Tregs, CD8:T Preg ratio or CD4conv:Treg ratio determined by FACS analysis of immune cells 24 hours after the second treatment of EMT6 tumor-carrying mice with different doses of CCR8 TPP-15285 antibody, or at the end of the study. Example 12.4.2: Efficacy of CCR8 TPP-15285 antibody in EMT6 tumor-carrying mice - single or multiple dosing

[00955] The EMT-6 murine breast cancer model was used with TPP-15285 to test the impact of single versus multiple dosing. Four different treatment regimens were tested: single treatment, twice-biw treatment, three times-biw treatment, or four times-biw treatment. Each group was treated with vehicle control or 0.1 mg/kg, 1 mg/kg, or 5 mg/kg of TPP-15285. In addition to the efficacy study, satellite animals were sacrificed for ex vivo phenotyping at 24 hours, 48 hours, and 120 hours after treatments. Antibody administration occurred ip, but other routes may be used.

[00956] Efficacy data for single-dose, double-dose BIW, triple-dose BIW, or quadruple-dose BIW treatment are shown in Fig. 85. Interestingly, single-dose treatment was adequate to achieve excellent treatment efficacy. At the end of the study, there was an increase in the frequency, absolute number, and activation of intratumoral CD8+ T cells, as shown in Table 12.4.2.1. However, at the end of the study, there was no difference in the number and frequency of intratumoral Tregs or CCR8 expression, as shown in Table 12.4.2.2. Furthermore, there was no difference in the number of intratumoral NK cells or macrophages at the end of the study or during the study (data not shown).

[00957] However, over time, there was a dose-dependent reduction in intratumoral Tregs (CD4+CD25+Foxp3+) after the 1st and 2nd treatments, but less pronounced after the 3rd and 4th treatments, as shown in Table 12.4.2.3. This was also observed in CCR8+ Tregs (data not shown). In addition, the absolute number of CD8+ T cells increased over time, as shown in Table 12.4.2.4. Thus, the CD8 to Treg ratios increased after the first dose and remained high until after the fourth dose compared to isotype controls. Characterization of blood samples did not show a substantial decrease in Tregs or an increase in CD8 T cells in the blood in different treatment regimens and over time (data not shown). Furthermore, no changes were observed in NK and macrophages in the blood (data not shown). Table 12.4.2.1: Absolute CD8+ cells per 100 mg tumor as determined by FACS at the end of the study. FACS analysis revealed increased frequency, absolute number, and activation status of cytotoxic T cells. Table 12.4.2.2: Absolute CD4+ CD25+ FoxP3+ Treg cells per 100 mg tumor as determined by FACS at the end of the study. Table 12.4.2.3: Absolute CD4+ CD25+ FoxP3+ Treg cells per 100 mg tumor as determined by FACS in satellite animals. Table 12.4.2.4: Absolute CD8+ cells per 100mg tumor as determined by FACS in satellite animals. Example 12.5: Efficacy of the anti-CCR8 antibody TPP-15285 in mice carrying the F9 tumor.

[00958] The anti-CCR8 surrogate antibody TPP-15285 showed dose-dependent efficacy in F9 tumor-carrying mice with strong effects in the 10 mg/kg and 1 mg/kg dose groups, but reduced efficacy in the 0.4 mg/kg dose group, comparable to the efficacy of the anti-PD-L1 antibody (Fig. 52, Fig. 53). Spider plots illustrate these results on an individual mouse basis (Fig. 54).

[00959] Analysis of Tregs from F9 tumor samples 24 hours after the second antibody treatment was performed by flow cytometry and showed strongly reduced numbers of Tregs in the group treated with anti-CCR8 antibody at a dose of 10 mg/kg versus the isotype control group. Treg depletion was clearly dose-dependent (Fig. 55). Treg depletion was also observed for treatment with anti-PD-L1 antibody. Furthermore, a strong dose-dependent increase in CD8+ T cells in tumors was demonstrated for all TPP-15285 dose groups, as well as for the anti-PD-L1 antibody (Fig. 55). Other effects on various immune cell populations are shown in Fig. 55, Fig. 56 and Table 12.5.1. Table 12.5.1: Effects of anti-CCR8 antibodies or anti-PDL1 antibodies on immune cell populations and their proportions 24 hours after the second treatment. Example 12.6: Efficacy of anti-CCR8 antibodies in combination therapy

[00960] Several experiments were conducted to analyze the efficacy of anti-CCR8 antibodies alone or in combination with antibodies that target the checkpoint, such as anti-PD-L1, anti-PD1, and anti-CTLA4 antibodies. The combination with these checkpoint-targeting molecules was found to provide additional efficacy. Furthermore, other combined treatments were evaluated, for example, with chemotherapeutic agents such as oxaliplatin, doxorubicin, gemcitabine, or radiotherapy. Table 12.6.1 summarizes the results for different combined treatments. Additional benefit was observed if treatment with the second therapeutic agent was initiated only after the start of treatment with the anti-CCR8 antibody. Table 12.6.1: Treatments and results of the combination of anti-CCR8 antibodies.

[00961] These results demonstrate the beneficial impact of combining anti-CCR8 antibody treatment with other therapeutically active agents. Furthermore, the inventors conclude that sequential administration of the therapeutic agents adds further benefits if the anti-CCR8 antibody is administered first and if an additional therapeutic agent is administered only after an initial reduction in Treg cells (e.g., by at least 50%) has occurred, for example, as demonstrated in Examples 12.6.5 and 12.6.7.

[00962] In view of these experiments and results, the inventors are convinced that the triple combination, for example, with an anti-CCR8 antibody, an antibody targeting a checkpoint protein, and a targeted therapeutic agent (small molecules as well as antibodies) provides additional survival benefits due to differences in the modes of action of these treatments. Therefore, the combination of an anti-CCR8 antibody with an antibody targeting the checkpoint and an antibody or agent targeting tumor cells is suggested for the treatment of tumors. In a preferred embodiment, the combination is a combination of a CCR8 antibody, an anti-PD(L)-1 antibody, and paclitaxel. Example 12.6.1: Efficacy of anti-CCR8 antibodies in C38 tumor-bearing mice and combination therapy with anti-PD-L1 antibody

[00963] The anti-CCR8 antibodies TPP-14099 and TPP-15285 surrogate showed strong efficacy in C38 tumor-carrying mice, comparable to the efficacy of the anti-PD-L1 antibody (Fig. 57a). The combination of TPP-15285 with 3 mg/kg of anti-PD-L1 antibody also showed improved efficacy. Both antibodies were formulated separately in phosphate-buffered saline and were administered as a total of four individual intraperitoneal injections twice weekly, i.e., in combination on the same day with a simultaneous treatment initiation.

[00964] Mice were monitored in a survival study for 94 days (Fig. 57b). Notably, 8 of 10 mice treated with the combination of anti-CCR8 antibody and anti-PD-L1 antibody survived to day 94, 4 of 10 mice treated with CCR8 antibody TPP-15285 alone survived to day 94, and none of the mice treated with anti-PD-L1 antibody alone survived to day 94, demonstrating a synergistic efficacy of anti-CCR8 antibody and PD-L1 antibody. C38 is a syngeneic low-infiltration mouse model that uses C38 colon cancer cells for tumor induction. The model is generally considered responsive to treatment with anti-PD-L1 antibody. In view of the available data described here, it is suggested that stratifying patients based on T-cell infiltration and/or response to PD-L1 therapy would provide additional benefits.

[00965] Analysis of Tregs from C38 tumor samples 24 hours after the second antibody treatment by flow cytometry showed strongly reduced numbers of Tregs in the groups treated with anti-CCR8 antibody versus the isotype control group (Fig. 58). The strongest Treg depletion was observed for the combination of TPP-15285 with anti-PD-L1 antibody. Furthermore, the highest CD8+/Treg ratio was observed for the combination of TPP-15285 with anti-PD-L1 antibody. Interestingly, macrophage analysis showed an increase on day 24 of the study (Fig. 59). Example 12.6.2: Efficacy of anti-CCR8 antibodies in mice carrying B16F10-OVA tumor and combination therapy with anti-CTLA4 antibody

[00966] The anti-CCR8 antibody TPP-15285 (10 mg/kg) showed strong efficacy in mice carrying B16F10-OVA comparable to the efficacy of an anti-CTLA4 antibody (10 mg/kg) (Fig. 60). The combination of TPP-15285 with anti-CTLA4 antibody showed synergistic efficacy, correlating with an increase in CD8+ T cells and IFNγ/TNFα levels.

[00967] Both antibodies were formulated in PBS and were administered as a total of three individual intraperitoneal injections twice weekly, in combination on the same day with a simultaneous treatment initiation.

[00968] Analysis of Tregs from B16F10-OVA tumor samples 24 hours after the second antibody treatment (day 12) by flow cytometry showed strongly reduced numbers of Tregs in the CCR8 antibody-treated groups versus the isotype control group (Table 12.6.2.1). The strongest Treg depletion was observed for the combination of TPP-15285 with anti-CTLA4 antibody. In addition, the strongest increase in CD8+ T cells was observed for the combination of TPP-15285 with anti-CTLA4 antibody.

[00969] FACS analysis of B16F10-OVA tumors 24 hours after the second antibody treatment with TPP-15285 + isotype control, anti-CTLA4 antibody + isotype control, or TPP-15285 + anti-CTLA4 antibody demonstrated a decrease in the frequency of intratumoral Tregs and an increase in absolute numbers of CD8+ T cells. Table 12.6.2.1: Intratumoral cell populations determined by FACS and IFNγ determined by ELISA at the end of the study. *mean for n=5 samples. Example 12.6.3: Combination treatment with anti-PD-1 antibody and anti-CCR8 antibody in EMT-6 tumor-carrying mice: synergistic efficacy

[00970] The therapeutic efficacies of the antimurine CCR8 antibody TPP-15285 (1 mg/kg) alone, antimurine PD-1 antibody (CDRs: atezolizumab, 10 mg/kg) alone, or a combination of TPP-15285 (1 mg/kg) and antimurine PD-1 antibody (10 mg/kg) were evaluated in mice carrying the EMT-6 tumor. Both antibodies were formulated in PBS and administered as a total of four individual intraperitoneal injections twice weekly, in combination on the same day with a simultaneous treatment initiation.

[00971] The anti-CCR8 antibody TPP-15285 showed weak efficacy in mice carrying EMT-6 at a low dose of 1 mg/kg, comparable to the weak efficacy of the anti-PD-1 antibody at 10 mg/kg (Fig. 61). The combination of 1 mg/kg of TPP-15285 with 10 mg/kg of anti-PD-1 showed synergistic efficacy.

[00972] Analysis of Tregs from EMT-6 tumor samples 24 hours after the second antibody treatment by flow cytometry showed strongly reduced numbers of Tregs in the CCR8 antibody-treated groups versus the isotype control group or anti-PD-1 monotherapy (Fig. 62). Table 12.6.3.1: Intratumoral cell populations determined by FACS in EMT-6 tumors at the end of the study. * mean for n=5 samples, cells/100mg tumor. Example 12.6.4: Combination treatment with anti-PD-1 antibody and anti-CCR8 antibody in C38 tumor-carrying mice

[00973] The therapeutic efficacies of the antimurine CCR8 antibody TPP-15285 (5 mg/kg) alone, antimurine PD-1 antibody (aPD-1, CDRs: atezolizumab, 5 mg/kg) alone, or a combination of TPP-15285 (5 mg/kg) and antimurine PD-1 antibody (5 mg/kg) were evaluated in mice carrying C38 tumors. Both antibodies were formulated in PBS and administered as a total of four individual intraperitoneal injections twice weekly, in combination on the same day with a simultaneous treatment initiation.

[00974] The anti-CCR8 antibody TPP-15285 showed strong efficacy in C38 tumor-bearing mice that was superior to the efficacy of an anti-PD-1 antibody administered at the same dose (Fig. 63a). After 64 days, the combination of 5 mg/kg of TPP-15285 with 5 mg/kg of anti-PD-1 antibody showed improved survival efficacy with 10/10 complete responders compared to 9/10 for anti-CCR8 monotherapy and 4/10 for anti-PD1 monotherapy (Fig. 63b).

[00975] Treg analysis of C38 tumor samples 24 hours after the second antibody treatment by flow cytometry is shown in Fig. 64, Table 12.6.4.1. Table 12.6.4.1: Intratumoral cell populations determined by FACS in C38 tumors 24 hours after the second antibody treatment. *mean for n=5 samples, cells/100mg tumor. Example 12.6.5: Sequential combination treatment of anti-CCR8 antibody with anti-PD-1 antibody in MB49 tumor-bearing mice

[00976] The therapeutic efficacy of the murine anti-CCR8 antibody TPP-15285 (10 mg/kg) alone, the anti-murine PD-1 antibody (CDRs: atezolizumab, 10 mg/kg) alone, and the combination of TPP-15285 (10 mg/kg) and the anti-PD-1 antibody (10 mg/kg) was evaluated in a study in MB49 tumor-bearing mice. In this study, the first dose of the anti-PD1 antibody was administered only after the anti-CCR8 antibody, that is, after allowing the anti-CCR8 antibody to have an effect on Tregs leading to an increase in the ratio of CD8+ T cells to Tregs. More specifically, treatment with the anti-PD1 antibody was initiated 24 hours after the second treatment with the anti-CCR8 antibody. The anti-CCR8 antibody TPP-15285 showed strong efficacy in mice carrying MB49 tumors, which was also greater than that of the anti-PD1 antibody (Fig. 65). The combination of 10 mg/kg of TPP-15285 with 10 mg/kg of anti-PD-1 also showed improved efficacy.

[00977] FACS analysis of MB49-derived tumor samples at the end of the study showed increased frequency of CD45+ cells, CD8+ cells, and NK cells and reduced frequency of Tregs in the groups treated with anti-CCR8 antibody versus the vehicle control group or anti-PD-1 antibody monotherapy (Fig 66, Table 12.6.5.1).

[00978] MB49 is a syngeneic model infiltrated in medium using bladder cancer cells that respond to immune checkpoint inhibitor (ICI) therapy. In view of these data, it is suggested that stratifying individuals for low immune infiltration and/or response to ICI therapy provides additional benefit for this combination therapy.

[00979] In particular, it was found that initiating combination therapy after anti-CCR8 antibody treatment provides a benefit. Without limiting themselves to theory, the inventors assume that the initial depletion of Tregs by CCR8 antibodies increases antigen-presenting cell activity and tumor T cell initiation, resulting in improved sensitivity to checkpoint inhibitors, such as PD-(L)1, because Tregs as extrinsic tumor cell factors play a role in primary and adaptive resistance to checkpoint inhibitors in cancer patients. Table 12.6.5.1: Intratumoral cell populations determined by FACS in MB49 tumors at the end of the study. *mean for n=5 samples, cells/100mg tumor. Example 12.6.6: Combination treatment comprising anti-CCR8 antibody and chemotherapy in mice carrying the EMT-6 tumor.

[00980] The therapeutic efficacies of the anti-CCR8 antibody TPP-15285 (5 mg/kg, q3/4d ip) alone, Oxaliplatin (5 mg/kg, q4d ip) alone, Doxorubicin (6 mg/kg, iv SD) alone, Docetaxel (10 mg/kg, q2dx5, iv) alone, or a combination of TPP-15285 with Oxaliplatin, Doxorubicin, or Docetaxel were evaluated in mice carrying the EMT-6 tumor. The therapeutics were formulated in PBS, and the combined treatment was initiated on the same day with simultaneous start of treatment.

[00981] Although the combination of anti-CCR8 antibody with each chemotherapeutic agent provided benefits compared to monotherapy with that chemotherapeutic agent, the combination was not superior to anti-CCR8 monotherapy (Fig. 67). The inventors are convinced that sequential administration of the therapeutic agents is beneficial, allowing the anti-CCR8 antibody to effectively deplete Tregs before the chemotherapeutic agent is applied to deplete the rapidly dividing cells.

[00982] Immune cell populations were analyzed at the end of the study, both in the tumor and in the blood by flow cytometry, as shown in Table 12.6.6.1 and Table 12.6.6.2. For combined treatments, the intratumoral CD8/Treg ratio and the frequency of activated CD8+CD25+ T cells in the blood increased compared to each monotherapy and control.

[00983] In addition, IFN gamma, IL10, IL12p70, IL1beta, IL2, and TNF alpha were analyzed. Increased levels of IFN gamma, IL-1b, and IL-2 were observed in both the mono and combination groups. Increased TNF alpha was observed only in the combination groups (data not shown). Table 12.6.6.1: Intratumoral cell populations determined by FACS at the end of the study. *mean for n=5 samples, cells/100 mg tumor, unless otherwise specified. Table 12.6.6.2: Blood immune cell populations determined by FACS at the end of the study. *mean for n=5 samples, cells/100μl of blood. Example 12.6.7: Sequential combination treatment with anti-PD-(L)1 antibody and anti-CCR8 antibody in mice carrying Lewis lung carcinoma.

[00984] The therapeutic efficacies of the anti-CCR8 antibody TPP-15285 (10 mg/kg) alone, anti-PD-1 antibody (aPD-1, CDRs: atezolizumab, 10 mg/kg) alone, anti-PD-L1 antibody (10 mg/kg) alone, anti-CTLA4 antibody alone, TPP-15285 (10 mg/kg) and anti-PD-1 antibody (10 mg/kg) in combination, or TPP-15285 (10 mg/kg) and anti-PD-L1 antibody (10 mg/kg) in combination were evaluated in mice carrying Lewis lung tumor (Fig. 69). The antibodies were formulated in PBS. For the anti-CCR8 antibody, doses were administered on days 11, 14, 18, and 22 after tumor inoculation. For combination therapy, administration of the anti-PD-(L)1 antibody began 24 hours after the second dose of anti-CCR8 antibody. At this time point, FACS analysis showed a 60% depletion of intratumoral Treg cells in the TPP-15285 monogroup compared to the isotype control (data not shown).

[00985] Lewis lung was used as a syngeneic model with a suppressive tumor microenvironment and is known to be unresponsive to anti-PD-(L)1 and anti-CTLA4 treatment. No monotherapy efficacy of TPP-15285 was observed despite effective Treg depletion measured 24 hours after the 2nd treatment. However, combination treatment with anti-murine PD-1 antibody after anti-CCR8 antibody showed improved efficacy and this was also associated with an increased CD8/Treg ratio at the end of the study (Table 12.6.7.1). Table 12.6.7.1: Intratumoral cell populations determined by FACS at the end of the study. *mean for n=5 samples. Example 12.6.8: Combination treatment comprising anti-CCR8 antibody and radiotherapy in mice carrying EMT-6 tumor

[00986] The therapeutic efficacies of the anti-CCR8 antibody TPP-15285 (3 mg/kg) or radiotherapy (RT, 3 x 2 Gy) were evaluated alone or in combination in mice carrying EMT-6 tumor (Fig. 70). The combined treatment was initiated with fractional irradiation of 3 x 2 Gy, followed by 3 mg/kg biw x 2. Radiotherapy alone only slightly retarded tumor growth. The combination of anti-CCR8 antibody and radiotherapy showed only a small improvement in efficacy, but Treg depletion at the end of the study was more pronounced than for each monotherapy (data not shown). Example 12.6.9: Combination treatment comprising anti-CCR8 antibody and administration of PD-1 antibody, PD-L1 antibody or Paclitaxel in mice carrying MBT2 tumor

[00987] The therapeutic efficacies of the inventive anti-CCR8 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, or Paclitaxel were tested alone or in combination in mice carrying MBT2 syngeneic tumors, as shown in Table 12.6.9.1. Each group comprised 10 tumor-carrying mice, and treatment began on day 10 after inoculation, when the tumors reached a size of approximately 100 mm3. Among the single-treatment groups, anti-CCR8 showed the strongest and most significant antitumor effect.

[00988] The antitumor effect of anti-PD-1 antibody, anti-PD-L1 antibody, or Paclitaxel was significantly increased by combining these treatments with anti-CCR8 therapy. The antibodies were formulated in PBS. T/C ratios were analyzed after three weeks of biweekly treatment. Table 12.6.9.1: Treatment groups and T/C ratio relative to isotype control on day 24. Table 12.6.9.2: CD8 mRNA levels increase after anti-CCR8 combination treatments. CD8 mRNA levels were obtained via RNA-seq performed on MBT2 tumors obtained 24h after the end of the treatment cycle. Mean CD8 expression levels were calculated for 10 tumors from each treatment group. Anti-CCR8 treatment alone significantly increased CD8 levels compared to the isotype, while PD1, PDL1, and paclitaxel had no significant effect. CD8 levels were increased by combining anti-CCR8 treatment with the other three treatments. The most significant and strongest increase of more than 8 times was obtained by combining anti-CCR8 with anti-PD1 treatment. Table 12.6.9.3: Tumor sizes on day 24 are significantly reduced by anti-CCR8 treatment and further reduced by the combination of anti-CCR8 with PD1 antibody (CrownVivoPremium, Catalog No. RMP1-14), PDL1 antibody (CrownVivoPremium, Catalog No. CVP034), or Paclitaxel. The greatest and most significant reduction in tumor size compared to the isotype control is achieved by the combination of anti-CCR8 with anti-PD1 antibody. Example 12.7.1: Correlation between the anti-PD-L1 antibody response and the anti-CCR8 antibody response.

[00989] In order to compare the efficacy of anti-CCR8 antibody treatment versus anti-PD-L1 antibody treatment, the results for different syngeneic tumor models were compiled, see Table 12.7.1.1. Surprisingly, a good response to anti-PD-L1 antibody treatment – measured, for example, by T/C volume – was also predictive of a good response to anti-CCR8 antibody treatment. PD-L1 expression is a known predictor for selecting responders to anti-PD-L1 treatment. From these observations, the inventors hypothesized that PD-L1 expression may also be suitable for stratifying individuals in order to identify those likely to benefit from anti-CCR8 antibody treatment. This hypothesis can be retroactively validated by correlating PD-L1 expression levels with tumor volume. Table 12.7.1.1 Comparison between responses to anti-PD-L1 antibody vs. Anti-CCR8 antibody for different syngeneic models. Example 12.7.2: Correlation between antitumor response and mRNA biomarker for disease stratification or control.

[00990] Mean gene expression (RNA-seq) in untreated tumors early (100 to 200 mm3 in size, N = 10 per model), larger untreated tumors (500 and 1000 mm3) or at the end of the study was correlated with T/C ratios of 21 syngeneic mouse models treated with TPP-14099 or TPP-15285 as described elsewhere here.

[00991] The main correlated genes were significantly enriched with T cell and inflammation markers (Pearson correlation values), as shown in Table 12.7.2.1. IFNγ inflammation markers and IFNγ response genes (e.g., Gbp3/4/5/8/9, Cxcl9, Acod1), PDL1 (CD276), complement factors C1, T cell genes such as Klra3/5 and Trav, as well as Treg markers CD25 (IL2RA), FOXP3, and CTLA4 are among the genes most correlated with the response.

[00992] Table 12.7.2.2 shows the change in expression in responder cell lines (T/C < 0.6) versus non-responder cell lines (T/C > 0.6) for tumors not treated early.

[00993] The 100 genes with the greatest expression change between responders and non-responders are listed. IFNγ and IFNγ response genes (e.g., Gbp2/3/4/8/10/11, Cxcl9/11, Ubd), cytotoxic T cell markers (e.g., Granzymes, Prf1), as well as mast cell markers (Tpsab1, Cma1, Tpsb2), and complement factor C1 are among the most predictive response genes.

[00994] Table 12.7.2.3 shows the change in expression in responder cell lines (T/C < 0.6) versus non-responder cell lines (T/C > 0.6) for larger untreated tumors (500 and 1000 mm3).

[00995] Candidate biomarkers are listed in Tables 12.7.2.1, 12.7.2.2, and 12.7.2.3. Patients with high expression of the human counterparts of these genes, or a combination or signature of the human counterparts of these genes, may respond to anti-CCR8 treatment in the clinic. Furthermore, these markers or their combination can also be used to monitor treatment success. Biomarkers derived from genes in italics/bold are particularly preferred. Table 12.7.2.1 Suitable biomarkers correlated with the antitumor response achieved with inventive anti-CCR8 antibodies. These biomarkers were found to be upregulated in early untreated tumors and correlated with treatment response. Biomarkers derived from genes in italics/bold are particularly preferred. The 100 genes with the highest negative correlation coefficient (r values are shown) between expression and T/C ratios are listed. Table 12.7.2.2 Suitable biomarkers correlated with the antitumor response achieved with inventive anti-CCR8 antibodies. Change in expression in responder cell lines (T/C < 0.6) versus non-responder cell lines (T/C > 0.6). The main correlated genes (Pearson) were found to be significantly enriched with T cells and inflammation markers. Table 12.7.2.3 Suitable biomarkers based on genes with major fold changes between responders and non-responders based on large untreated tumors (500 mm3 and 1000 mm3). The expression of granzymes and other immune cell markers is higher in responder models than in non-responder models. Biomarkers derived from genes in italics/bold are particularly preferred. Example 12.8: Altered mRNA expression in syngeneic tumor models after administration of anti-CCR8 antibody.

[00996] For each syngeneic tumor model, 10 mice treated with anti-CCR8 TPP-14099 antibody and 10 mice treated with isotype control were sacrificed 24h after final treatment. Tumors were cut into small pieces and immersed in RNAlater at 4°C. Poly-A mRNA was extracted and cDNA libraries were generated according to the Illumina Hi-seq protocol for subsequent RNA sequencing. Samples were sequenced at a depth of ~40 million reads/samples of 150 bp long pairs.

[00997] Figures 63 to 73 show the impact of treatment with isotype control (TPP-9809) or anti-CCR8 antibody (TPP-14099) on mRNA expression levels of different immune cell markers in different syngeneic tumor models. Treatment increased the inflammation marker lfng, macrophage markers Ms4a7, Acod1 and Mrc1, cytotoxic T cell markers Cd8a and Cd8b1, natural killer (NK) cell marker Ncr1, pan T cell markers Cd3e/d/g and B cell markers Cd19 and Cd22.

[00998] Notably, the inventors observed significant induction of LTta/b, as well as Cxcr5 and its ligand Cxcl13 in the CT26, MBT2, and H22 tumor models, as well as a general upregulation of these genes in several other models, including RM1, Hepa1-6, and 4T1. Without being limited by theory, an induction of tertiary lymphoid structures by the anti-CCR8 antibody TPP-14099 or TPP-15285 may contribute to the antitumor response induced by these antibodies. Table 12.8.1: Anticorrelation of T cell marker, checkpoint proteins, Treg markers, NK cell marker, macrophage marker, B cell marker, and interferon gamma with response to anti-CCR8 antibody treatment. Example 13: Preparation of a Targeted Thorium Conjugate (TTC) comprising anti-CCR8 antibodies

[00999] The invention of WO2016096843 is incorporated herein by reference in its entirety and in particular with regard to the production of the conjugates as described in this Example.

[001000] The conjugation of the chelating agent 3,2-hydroxypyridonone (3,2-HOPO) or any other suitable chelating agent with the antibodies TPP-23411, TPP-21360 or any other anti-CCR8 antibodies described can be carried out as previously described in patent application WO2016096843. In short, to activate the chelating agent, the chelating agent 3,2-HOPO, dissolved in DMA in a 1:1 ratio with 0.1 M MES buffer pH 5.4, NHS and EDC, both dissolved in 0.1 M MES buffer pH 5.4, is mixed in a 1/1/3 ratio. For conjugation with the antibodies, a molar ratio of 7.5/7.5/22.5/1 (chelating agent/NHS/EDC/mAb) of the activated chelating agent can be loaded onto the mAb. After 20 to 60 min, the reaction is quenched with 12% v/v 0.3 M citric acid to adjust the pH to 5.5. The protein concentration is determined by HPLC, integrating the peak area at an absorbance of 280 nm. The solution is then exchanged with a buffer solution containing 30 mM citrate, 50 mg/ml sucrose, 2 mM EDTA, 0.5 mg/ml pABA, and pH 5.5 by constant-volume tangential flow filtration (TFF). At the end of diafiltration, the solution is discharged into a formulation container. The product is formulated with TFF buffer (30 mM citrate, 50 mg/ml sucrose, 2 mM EDTA, 0.5 mg/ml pABA, pH 5.5) and 7% w/v polysorbate 80 to obtain 2.5 mg/ml of the respective CCR8 antibody-chelating conjugate (CCR8-ACCs). CCR8-ACCs can be filtered through a 0.2 μm filter into sterile vials.

[001001] CCR8-ACCs are radiolabeled with thorium-227 as described in WO2016096843. In summary, 5 μl of CCR8-ACCs are mixed with 32 μl of thorium-227 (activity of 3.875 MBq/ml) and 13 μl of citrate buffer, resulting in CCR8-targeted thorium-227 conjugates (CCR8-TTCs) at specific activities of 10 kBq/μg. The sample can be incubated for 60 min at room temperature to allow stable radiolabeling of thorium-227 on the chelating agent 3,2-HOPO. An aliquot of the sample can be analyzed by flash thin-layer chromatography (iTLC). ANTIBODY SEQUENCES

Claims (3)

1. Use of an anti-CCR8 antibody or antigen-binding fragment in simultaneous, separate or sequential combination with one or more additional therapeutically active compounds, characterized in that it is for the manufacture of a medicament, composition, kit or product for inducing antibody-dependent cellular phagocytosis (ADCP) in a target cell in a human patient with a tumor, comprising: (i) an anti-CCR8 antibody or its antigen-binding fragment, wherein said antibody is selected from a. an anti-CCR8 antibody or its antigen-binding fragment comprising six CDR sequences with 100% sequence identity with any of the SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 623 and SEQ ID NO: 624, or b. an isolated anti-CCR8 antibody or its antigen-binding fragment, comprising a variable heavy chain sequence according to SEQ ID NO: 617 and a variable light chain sequence according to SEQ ID NO: 621, or (ii) an isolated anti-CCR8 antibody or its antigen-binding fragment, comprising a heavy chain according to SEQ ID NO: 633 and a light chain according to SEQ ID NO: 634, and (or) one or more additional therapeutically active compounds selected from: a. an antibody targeting a checkpoint protein, wherein said antibody is selected from Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, Ipilimumab, and/or b. a chemotherapeutic agent selected from docetaxel, paclitaxel, doxorubicin, oxaliplatin, or gemcitabine. 2. Use of an anti-CCR8 antibody or its antigen-binding fragment, according to claim 1, characterized in that the tumor is selected from the group of acute T-cell lymphoblastic leukemia, breast cancer, triple-negative breast cancer, triple-positive breast cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), testicular cancer, gastric cancer, squamous cell carcinoma of the head and neck, thymoma, esophageal adenocarcinoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer or cervical cancer, acute myeloid leukemia, renal cancer, bladder cancer, skin cancer, melanoma, thyroid cancer, mesothelioma, sarcoma and prostate cancer, B-cell lymphoma, T-cell lymphoma. 3. Use of an anti-CCR8 antibody or its antigen-binding fragment, according to claim 1, characterized in that the antibody or antigen-binding fragment is afucosylated.