EA046752B1 - POLYPEPTIDES INTERFERING THE TRANSMISSION OF WNT SIGNALS IN TUMOR CELLS - Google Patents

Abstract

The invention provides novel constructs of LRP5-binding polypeptides, and more specifically, a novel LRP5-binding single variable domain immunoglobulin, which can inhibit Wnt signaling pathways. The invention also relates to specific sequences of such polypeptides, methods for their production, and methods for their use, including methods for treating diseases such as cancer.

Description

Field of technology to which the invention relates

The present invention relates to novel low-density lipoprotein receptor family protein-related protein 5 (LRP5) binding polypeptides. The invention also relates to nucleic acids encoding such polypeptides; to methods for producing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions containing such polypeptides; and to the uses of such polypeptides or such compositions, particularly for therapeutic purposes in the field of oncology.

Prerequisites for creating the invention

Activation of the Wnt signaling pathway requires the binding of extracellular Wnt ligands to the Frizzled receptor and the LRP5 coreceptor (accession number: UniProtKB -075197/LRP5_HUMAN). There are 19 Wnt proteins and 10 Frizzled receptors in mammalian cells. In the absence of Wnt ligand, cytoplasmic beta-catenin is phosphorylated by a protein complex consisting of the scaffolding proteins Axin and APC, as well as the kinases GSK3beta and CK1a. Subsequent recognition by beta-TrcP ubiquitin ligase leads to ubiquitin-mediated degradation of beta-catenin. In the presence of Wnt ligand, Wnt binding to Frizzled and LRP5 results in the recruitment of the cytoplasmic effector protein Dvl and phosphorylation of the cytoplasmic tail of LRP5, which provides a site for Axin attachment. Sequestration of Axin by LRP5 leads to inactivation of the Axin-APC-GSK3-beta complex and hence to the stabilization and accumulation of intracellular beta-catenin. Thus, the level of beta-catenin in the cytoplasm increases, and beta-catenin translocates to the nucleus, forming complexes with members of the T cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors. The basal transcription machinery and transcriptional coactivators are then recruited, including a protein (CREB-binding protein (CBP)) that binds to the cAMP response element-binding protein (CREB) or its homologue p300, resulting in the expression of various target genes including Axin2, cyclin D1 and c-Myc.

An additional level of ligand-dependent regulation of the Wnt pathway is mediated by the E3 ligase RNF43 and its closely related homolog ZNRF3, as well as the secreted proteins R-spondins (de Lau et al. The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes Dev 2014;28(4):305-16). RNF43 mediates the ubiquitination of the Frizzled/LRP5 receptor complex on the cell surface, leading to its degradation and thereby suppressing the ligand-dependent activity of the Wnt pathway. RNF43 activity is antagonized by members of the R-spondin family (R-spondin ligands 1–4). In the presence of R-spondin ligand, it eliminates RNF43 from the cell surface, allowing the accumulation of the Frizzled/LRP5 complex and increased Wnt signaling in the presence of Wnt ligands.

LRP5 acts as a gatekeeper of ligand-dependent activation of the Wnt signaling pathway and, therefore, can be considered targets for achieving complete blockade of the pathway mediated by all 19 Wnt ligands and 10 Frizzled receptors, and also enhanced by R-spondin ligands. In particular, Wnt ligands can be divided into the classes Wntl and Wnt3a, each of which binds to different epitopes/sites of LRP5 for signal transduction. The extracellular domain of LRP5 contains four beta-propeller repeat elements associated with an EGF-like domain, followed by three type A LDLR repeats. Combining structural and functional analysis of LRP5 indicates that Wnt1 (a Wnt1 class ligand) binds to a fragment of LRP6. which contains beta-propellers 1 and 2, and Wnt3a binds to the fragment that contains beta-propellers 3 and 4.

LRP5 and agents that interfere with LRP5 activity have been described in the context of various indications, including bone lesions, lipid-modulated disorders, Alzheimer's disease, rheumatoid arthritis and insulin-dependent diabetes (see, for example, WO2002/092015, WO2006/102070, WO2009/155055 and WO1998/046743).

Hyperactivation of the Wnt signaling pathway has been further implicated in the pathogenesis of various types of cancer. In some types of cancer, frequent mutations in molecules of downstream signaling pathways promote constitutive activation of the Wnt pathway (eg, APC mutations in colorectal cancer; betacatenin-activating mutation in hepatocellular carcinoma). In contrast, in triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), pancreatic adenocarcinoma, and a subset of colorectal cancers (CRC) and endometrial cancers, activation of the Wnt signaling pathway is driven by a ligand-dependent mechanism (i.e., autocrine/paracrine activation of Wnt), detected by the intracellular accumulation of beta-catenin. In NSCLC, TNBC and pancreatic adenocarcinoma, ligand-dependent Wnt activation is mediated by multiple mechanisms, including increased expression of Wnt ligands and/or LRP5 receptors, or silencing of the negative regulator LRP5 - DKK1 (TNBC: Khramtsov et al. Wnt/beta-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome. Am J Pathol. 2010; 2911-20; cancer patients. Oncol Rep. 2008; 19(1):203-9; Pancreatic cancer: Zhang et al. Canonical signaling of Wnt. Cancer Res. 2013; Ligand-dependent Wnt activation in tumors has been shown to promote tumor growth and resistance to chemotherapy or immunotherapy, and is associated with relapse in preclinical models.

- 1 046752

Some molecules are known from the prior art that bind LRP5 and are capable of modulating the Wnt signaling pathway:

Dickkopf-1 (DKK1) is an inhibitor of LRP5. DKK1 binds to LRP5 and the transmembrane protein Kremen, suppresses Wnt signaling, and leads to rapid internalization of LRP5. DKK1 has been shown to suppress both Wnt1- and Wnt3a-mediated signal transduction.

In vivo treatment with DKK1 has also been shown to result in severe gastrointestinal toxicities. In particular, adenovirus-mediated expression of DKK1 in adult mice was shown to markedly inhibit proliferation in the small and large intestines, which was accompanied by progressive degeneration of intestinal architecture, severe loss of body weight, and mortality from colitis and generalized infection. In particular, LRP5 is expressed in proliferating epithelial cells of the small intestine and is required for intestinal epithelial proliferation, suggesting that inhibition of LRP5 may be toxic to this and other normal tissues (Zhong et al. Lrp5 and Lrp6 play compensatory roles in mouse intestinal development J Cell Biochem 2012; 113(1):31-8. Therefore, there are doubts that LRP5 inhibitors or, in general, any inhibitors of the Wnt signaling pathway (Wnt1 and Wnt3a) can be used for therapeutic purposes, for example, they can be developed as drugs against cancer.

WO 1998/046743 A1 describes an antibody specific for LRP5 and proposes several LRP5 peptides to enhance such an antibody. However, no experimental results related to the development of such an antibody have been described, nor has such an antibody been specifically described.

US9175090 describes methods of inhibiting Wnt signaling in a cancer cell deficient in Apc expression by administering a monoclonal antibody, specifically an IgM antibody, that binds LRP5.

WO2013/109819 describes anti-LRP5 antibodies, particularly antibodies that potentiate Norrin/Fzd4 activity and/or signaling, and their use for the treatment of conditions associated with angiogenesis.

However, none of the LRP5-binding molecules described in the prior art have yet been approved by health authorities for use as a drug to treat any disease. More specifically, such applications require very specific binding properties, the right specificity so that such molecules do not bind, activate or inhibit other targets (e.g., leading to undesirable activation or inhibition of other signaling pathways, or insufficient activation or inhibition in relation to target isoforms), in the case of bi- or multispecific agents - proper balance of two or more binding specificities, proper pharmacokinetic and pharmacodynamic properties, acceptable toxicological profile and, of course, in vivo effectiveness.

In light of the above, there is a need for new therapeutic agents to effectively treat various types of cancers and tumors. Thus, it is an object of the invention to provide such pharmacologically active agents that can be used in the treatment of a number of cancers, including NSCLC and TNBC.

In particular, it is an object of the invention to provide such pharmacologically active agents, compositions and/or methods of treatment that have certain advantages over agents, compositions and/or methods currently used and/or known from the prior art. These advantages include in vivo efficacy, improved therapeutic and pharmacological properties, fewer side effects, as well as other favorable properties such as simplified manufacturing or reduced cost of goods, particularly compared to established drug candidates.

Summary of the invention

In accordance with a first aspect of the invention, the present invention provides a polypeptide that binds to low-density lipoprotein receptor family protein 5 (LRP5)-related protein, wherein the polypeptide comprises a single immunoglobulin variable domain (ISVD) selected from the group consisting of the following LRP5-binding ISVDs ( I)-(IV): (I) ISVD with the following complementarity determining region (CDR) sequences:

- 2 046752

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NOG), (II) ISVD with the following CDR sequences:

CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NOG)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), (III) ISVD with the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and (IV) ISVD with the following CDR sequences:

CDR1: INAMG (SEQ ID NO:10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9).

In this aspect, the polypeptide of the present invention preferably comprises a first ISVD (a) selected from ISVDs (I) and (II) as defined above, and a second ISVD (b) selected from ISVDs (III) and (IV) as defined above. Even more preferably, one or more ISVDs (I)-(IV) are defined as containing the following sequences, respectively:

(I)

AVQLVESGGGLVQPGGSLRLSCAASGRTFFSTYVMGWFRQAPGKEREFVAAISW

SGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASRGTSTPSR

ASGVSRYDYWGQGTLVTVSS (SEQ ID NO:AND), or

EVQLVESGGGLVQPGGSLRLSCVASGRTFFSTYVMGWFRQAPGKEREFVAAISW

SGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASRGTSTPSR

ASGVSRYDYWGQGTLVTVSS (SEQ ID NO:23), (Π)

EVQLVESGGGLVQPGGSLRLSCAASGLTFSRYAVAWFRQAPGKEREFVAAITW

SSGRIDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADRRPRSTGR

SGTGSPSTYDYWGQGTLVTVSSA (SEQ ID NO: 12), (ΠΙ)

AVQLVESGGGLVQPGGSLRLSCAASGSIFRIGAMGWYRQAPGKQRELVAAVSS

GGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNRETGPYGPPK

RDYWGQGTLVTVSS (SEQ ID NO: 13), or

EVQLVESGGGLVQPGGSLRLSCAASGSIFRIGAMGWYRQAPGKQRELVAAVSS GGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNRETGPYGPPK

RDYWGQGTLVTVSSA (SEQ ID NO:22), and (IV)

EVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMGWYRQAPGKQRELVAAVSS

GGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNRETGPYGPPK

RDYWGQGTLVTVSS (SEQ ID NO: 14).

The terms first and second with respect to such ISVDs or domains in general, as used in the present invention, are intended only to indicate that these domains are two different domains (since they at least contain different CDR sequences). Therefore, these terms should not be understood to indicate the exact order or sequence of domains in such a polypeptide chain. In other words, the above ISVDs (a) and (b) can be dissected

- 3 046752 are placed either in the following order (a)-(b) or in the following order (b)-(a) within the polypeptides described in the present invention.

In some embodiments, a polypeptide described in the present invention contains a first ISVD(a) containing the following CDR sequences:

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3), and the second ISVD (b) containing the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9).

In some embodiments, a polypeptide described in the present invention contains a first ISVD(a) containing the following CDR sequences:

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3), and the second ISVD (b) containing the following CDR sequences:

CDR1: INAMG (SEQ ID NO: 10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9).

In some embodiments, a polypeptide described in the present invention contains a first ISVD(a) containing the following CDR sequences:

CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), and the second ISVD (b) containing the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9).

In some embodiments, a polypeptide described in the present invention contains a first ISVD(a) containing the following CDR sequences:

CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), and the second ISVD (b) containing the following CDR sequences:

CDR1: INAMG (SEQ ID NO: 10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9).

Specifically, the ISVDs of the polypeptides described in the present invention (eg, ISVDs containing CDR sequences as defined above) are VHH domains, preferably humanized VHH domains.

In some embodiments, the ISVD(I) of the polypeptides described herein comprises the sequence SEQ ID NO:11 or the sequence SEQ ID NO:23. In some embodiments, the ISVD (II) contains the sequence of SEQ ID NO:12. In some embodiments, the ISVD (III) contains the sequence of SEQ ID NO:13 or the sequence of SEQ ID NO:22. In some embodiments, the ISVD (IV) contains the sequence of SEQ ID NO:14.

Specifically, the polypeptides described in the present invention contain a first ISVD (a) and a second ISVD (b), wherein said first ISVD contains a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO: 23, and said second ISVD contains a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:22. In some embodiments, the first ISVD contains the sequence SEQ ID NO:11 and the second ISVD contains the sequence SEQ ID NO:22. In some embodiments, the first ISVG contains the sequence SEQ ID NO:12 and the second ISVD contains the sequence SEQ ID NO:13. In some embodiments, the first ISVD contains the sequence SEQ ID NO:23, and the second ISVD contains the sequence SEQ ID NO:14. In some embodiments, the first ISVD contains the sequence SEQ ID NO:11 and the second ISVD contains the sequence SEQ ID NO:14. In some embodiments, the first ISVD contains the sequence SEQ ID NO:11 and the second ISVD contains the sequence SEQ ID NO:13. In some embodiments, the first ISVD contains the sequence SEQ ID NO:12 and the second ISVD contains the sequence SEQ ID NO:14. In some embodiments, the first ISVD contains the sequence SEQ ID NO:12 and the second ISVD contains the sequence SEQ ID NO:22. IN

- 4 046752 In some embodiments, the first ISVD contains the sequence SEQ ID NO:23 and the second ISVD contains the sequence SEQ ID NO:22. In some embodiments, the first ISVD contains the sequence SEQ ID NO:23 and the second ISVD contains the sequence SEQ ID NO:13.

In accordance with one aspect, the first ISVD and the second ISVD of the polypeptides described in the present invention are covalently linked by a linker peptide, wherein said linker peptide optionally contains or consists of a third ISVD; for example, albumin-binding ISVD.

In accordance with a further aspect, the polypeptides described in the present invention further contain a half-life increasing component that is covalently linked to the polypeptide and optionally selected from the group consisting of an albumin binding component, such as an albumin binding peptide or an immunoglobulin albumin binding domain, a transferrin binding component, such as an anti-transferrin domain of immunoglobulin, a polyethylene glycol molecule, human serum albumin, and a fragment of human serum albumin. Specifically, the half-life increasing component is an albumin binding component, preferably an albumin binding ISVD, even more preferably an Alb 11 domain containing the following sequence:

EVQLVESGGGLVQPGNSLRLSCAASGTFSSFGMSWVRQAPGKGLEWVS

SISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG

GSLSRSSQGTLVTVSS (SEQ ID NO:21)

In accordance with a preferred aspect, the polypeptides described in the present invention comprise a first (a) and a second (b) LRPS-binding ISVD and a third ISVD (c), for example, an albumin-binding ISVD (c); said first LRP5-binding ISVD (a) selected from the group consisting of ISVDs (I) and (II):

(I) ISVD containing the following CDR sequences:

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3), and (II) ISVD containing the following CDR sequences:

CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), said second ISVD (b) selected from the group consisting of ISVDs (III) and (IV):

(III) ISVD containing the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and (IV) ISVD containing the following CDR sequences:

CDR1: INAMG (SEQ ID NO: 10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and the specified albumin-binding ISVD (c) is defined as containing the following CDR sequences:

CDR1: SFGMS (SEQ ID NO: 15)

CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 16)

CDR3: GGSLSR (SEQ ID NO:17).

For example, the polypeptide contains a first and second ISVD, as defined by the CDR sequences above, and a third ISVD, which is an albumin-binding ISVD, as defined by the CDR sequences above, and which directly or indirectly connects the first and second ISVD. In some embodiments, the first ISVD is covalently linked via a peptide linker to a third ISVD, which is covalently linked to a second ISVD via a peptide linker. The terms first and second, as defined above, do not indicate their position within the polypeptide, therefore, from the N to C terminus of the ISVD sequences within the polypeptide can be located either in the following order ISVD (a)-(c)-(b), ( a)-[linker]-(c)-[linker]-(b), (b)-(c)-(a), (b)-[linker]-(c)-[linker]-(a) .

In some embodiments, the polypeptide comprises a first ISVD containing the following CDR sequences:

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3), the second ISVD containing the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and albumin-binding ISVD (third ISVD) defining

- 5 046752 is reported as containing the following CDR sequences:

CDR1: SFGMS (SEQ ID NO: 15)

CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 16)

CDR3: GGSLSR (SEQ ID NO:17).

In some embodiments, the polypeptide comprises a first ISVD containing the following CDR sequences:

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3), the second ISVD containing the following CDR sequences:

CDR1: INAMG (SEQ ID NO: 10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and the albumin-binding ISVD is defined as containing the following CDR sequences:

CDR1: SFGMS (SEQ ID NO: 15)

CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 16)

CDR3: GGSLSR (SEQ ID NO:17).

In some embodiments, the polypeptide comprises a first ISVD containing the following CDR sequences:

CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), second ISVD with the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and the albumin-binding ISVD is defined as containing the following CDR sequences:

CDR1: SFGMS (SEQ ID NO: 15)

CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 16)

CDR3: GGSLSR (SEQ ID NO:17).

In some embodiments, the polypeptide comprises a first ISVD containing the following CDR sequences:

CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), second ISVD containing the following CDR sequences:

CDR1: INAMG (SEQ ID NO: 10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and the albumin-binding ISVD is defined as containing the following CDR sequences:

CDR1: SFGMS (SEQ ID NO: 15)

CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 16)

CDR3: GGSLSR (SEQ ID NO:17).

In some embodiments, the ISVDs, as defined by their CDR sequences in the above-described polypeptides, are rearranged such that the albumin-binding ISVD directly or indirectly (eg, via a linker peptide) connects the first and second ISVD.

In some embodiments, a polypeptide described in the present invention contains or consists of a sequence selected from SEQ ID NOs: 18, 19 and 20.

In a still further aspect, the present invention provides a polypeptide that binds to LRP5 containing an ISVD that competes for LRP5 binding with a reference ISVD, wherein the reference ISVD has the sequence identified in SEQ ID NO: 11, 12, 13, 14, 22 or 23.

In another aspect, the present invention provides a polypeptide that binds to LRP5 comprising a first ISVD(a) that competes for binding to LRP5 with a first reference ISVD, wherein the first reference ISVD has the sequence identified in SEQ ID NO: 11, 23, or 12, and a second ISVD (b) that competes for binding to LRP5 with a second reference ISVD, wherein the second reference ISVD has the sequence identified in SEQ ID NO: 13, 22, or 14.

In yet another aspect, the present invention provides a polypeptide that binds to LRP5, comprising a first LRP5 binding domain, preferably an ISVD, and a second LRP5 binding domain, preferably an ISVD, wherein the first domain binds a region of LRP5 that is different from the LRP5 region to which it binds second domain. According to a preferred embodiment, the first LRP5 binding domain blocks the Wnt3a binding site in LRP5, and

- 6046752 preferably inhibits Wnt3a-mediated transcription of the target gene, and/or wherein the second LRP5-binding domain blocks the Wnt1 binding site in LRP5, and preferably inhibits Wnt1-mediated transcription of the target gene.

In further aspects, the invention relates to nucleic acid molecules, expression vectors, host cells, and production methods used to make the inventive polypeptide. The nucleic acid molecules encoding the polypeptides of the invention, in isolated form, can be used to construct appropriate expression vectors, which can then be transferred by transfection into host cells used for the biopharmaceutical production of the polypeptides of the invention. Such manufacturing methods typically include the steps of culturing a host cell under conditions allowing expression of the polypeptide, isolating the polypeptide, and purifying it by methods known in the art.

Further aspects, embodiments, uses and methods in which the polypeptides of the invention are used will become clear from the following detailed description of the invention and the appended claims.

The invention provides new molecules that enable more effective treatment of various types of cancer, such as TNBC, CRC and NSCLC, with fewer side effects. The polypeptides of the invention provide an unexpected therapeutic effect (ie, potency) in the treatment of cancer patients in that they are capable of inducing tumor regression leading to pathological complete response (pCR). This in turn is expected to lead to significant improvements in progression-free survival and overall survival, especially in cases of large unmet medical need such as breast cancer. Thus, the polypeptides of the invention provide new therapeutic options for the treatment of a number of cancer types, particularly those exhibiting dysregulated Wnt signaling and beta-catenin accumulation.

Moreover, the polypeptides of the invention are easy to produce, have high stability and low antigenicity, and also open up a number of options regarding routes of administration other than injection and infusion.

Brief description of the figures

In fig. Figure 1 shows a schematic representation of biparatope polypeptides that antagonize Wnt1 and Wnt3a signaling. They consist of three domains, two of which bind to different epitopes of LRP5 (Wnt1 and Wnt3a blocker) and one half-life prolonging domain (human serum albumin binding domain).

Figures 2A/2B show the binding of three biparatopic LRP5-selective VHH extended half-life constructs, i.e., particularly preferred polypeptides of the present invention, to human LRP5 (FIG. 2A) and human LRP6 (FIG. 2B), which are overexpressed in cells. HEK293 lines, compared to a negative control consisting of a nontargeting binding component (a VHH construct that binds to a bacterial protein that is not expressed in HEK293 cells) and compared to an LRP5/6 cross-reacting binding component, MOR08168IgG1LALA 6475 scfV. The biparatopic LRP5 specific VHH extended half-life constructs of the present invention bind only to human LRP5 without detectable binding to human LRP6, unlike the LRP5/6 cross-reactive binding moiety.

Figures 3A/3B show the overall DKK1 competition of three biparatopic LRP5-selective VHH extended half-life constructs, i.e., particularly preferred polypeptides of the present invention, for binding to both human LRP5 (FIG. 3A) and murine LRP5 (FIG. 3B), which are overexpressed in HEK293 cell lines, as detected by FACS-based DKK1 competition assay.

Figures 4A/4B show complete inhibition of the Wnt1 (FIG. 4A) and Wnt3a (FIG. 4B) pathways by three biparatopic LRP5-selective VHH extended half-life constructs, i.e., particularly preferred polypeptides of the present invention.

In fig. Figure 5 shows inhibition of Wnt signaling in cancer cells, as detected by inhibition of relative Axin2 mRNA expression (right panel), and cell proliferation (left panel), assessed by the decrease in percentage (%) of viable cells, after treatment with biparatope LRP5-selective VHH half-life extended constructs, that is, a particularly preferred polypeptide of the present invention (at a final concentration of 1 μM).

Figures 6A/6B show the in vivo efficacy of biparatopic LRP5-selective VHH extended half-life constructs (F012900082 in FIG. 6A and F012900141 in FIG. 6B), i.e., particularly preferred polypeptides of the present invention, in Wnt tumor models ( xenograft model MMTV-Wnt1).

In fig. 7 shows inhibition of the Wnt signaling pathway in tumors treated with biparatope LRP5-selective VHH constructs with extended half-life, i.e.

- 7 046752 particularly preferred polypeptides according to the present invention, as detected by a decrease in the expression of Axin2/RNF43/Notum mRNA compared to the control group.

In fig. Figure 8 shows that LRP5 is expressed at a higher level than LRP6 in human monocytic-derived dendritic cells (left panel). In addition, the suppressive effect of Wnt3a on T cell activation, as measured by interferon gamma release, is strongly suppressed by treatment with biparatope LRP5-selective VHH extended half-life constructs (center and right panels), i.e., the preferred peptides of the present invention . Each symbol represents a different dendritic cell (DC) donor. Data shown are normalized to TNF-alpha levels in untreated controls and each symbol represents a unique donor pair for DCs and T cells.

In fig. Figure 9 shows the selective inhibition of RNF43 mutant CRC organoid proliferation (RNF43 mut1 and RNF43 mut2) compared with the lack of significant effect on RNF43 wild type CRC organoid proliferation (RNF43 WT), assessed by the decrease in the percentage (%) of viable cells, after treatment with biparatope LRP5 -selective VHH design with an extended half-life, that is, a particularly preferred polypeptide according to the present invention. IC values of ₅₀ nM inhibition of RNF43 mutant CRC organoid proliferation are also described.

In fig. 10 shows selective inhibition of Wnt signaling in RNF43 mutant CRC organoids (RNF43 mut1 and RNF43 mut2) compared with no significant effect in RNF43 wild-type CRC organoid (RNF43 WT), as detected by inhibition of relative hAxin2 mRNA expression, following biparatope treatment LRP5-selective VHH design with extended half-life, that is, a particularly preferred polypeptide according to the present invention (at a final concentration of 62 nM).

Detailed Description of the Invention

Definitions

The foregoing and other aspects and embodiments of the invention will become apparent from the following description, in which:

a) Unless otherwise stated or defined, all terms used have their common meaning in the art as would be apparent to one skilled in the art. For example, reference is made to standard textbooks such as Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vol. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, Genes IV, Oxford University Press, New York, (1990), Roitt et al., Immunology (2nd ed.), Gower Medical Publishing, London, New York (1989), and documents cited here general level of technology. In addition, unless otherwise indicated, all methods, steps, techniques and manipulations, the specific details of which are not described, can be and have been performed in a manner known per se as will be apparent to one skilled in the art. By way of example, we again refer to the standard manuals, the general state of the art documents cited here, and further referenced documents.

b) Unless otherwise specified, the terms immunoglobulin and immunoglobulin sequence - whether used to refer to the heavy chain of an antibody or a conventional 4-chain antibody - are used as general terms covering full-length antibodies, their individual chains, and any parts, domains or fragments (including, but not limited to, antigen binding domains or fragments such as VHH domains or VH/VL domains, respectively). In addition, as used herein, the term sequence (for example, as part of terms such as immunoglobulin sequence, antibody sequence, (individual) variable domain sequence, VHH sequence or protein sequence) should be generally understood to include both the corresponding amino acid sequence and encoding it nucleic acid sequences or nucleotide sequences, unless the context requires a more limited interpretation;

c) The term domain (of a polypeptide or protein) hereinafter denotes a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. Typically, domains are responsible for distinct functional properties of proteins, and in many cases can be added, removed, or transferred to another protein without loss of function of the protein residue and/or domain.

d) The term immunoglobulin domain is used hereinafter to refer to a globular region in an antibody chain (such as, for example, a conventional 4-chain antibody chain or an antibody heavy chain) or a polypeptide that essentially consists of such a globular region. Immunoglobulin domains are distinguished by the fact that they retain the immunoglobulin fold characteristic of antibody molecules, which is a 2-layer sandwich of approximately seven antiparallel betanites stacked in two beta sheets, optionally stabilized by a conservative disulfide bond.

e) The term immunoglobulin variable domain hereinafter refers to an immunoglobulin domain consisting essentially of four framework regions, which are referred to herein and in prior art documents as framework region 1 or FR1; frame section 2 or FR2; frame

- 8 046752 section 3 or FR3; and frame section 4 or FR4, respectively; these framework regions are interspersed with three complementarity determining regions or CDRs, referred to herein and in prior art documents as complementarity determining region 1 or CDR1; complementarity determining region 2 or CDR2; and complementarity determining region 3 or CDR3, respectively. Thus, the general structure or sequence of the immunoglobulin variable domain can be displayed as follows: FR1-CDR1-FR2-CDR2-FR3CDR3-FR4. It is the immunoglobulin variable domain(s) that gives the antibody specificity for the antigen, since it contains the antigen-binding site.

e) The term single immunoglobulin variable domain (or ISVD) hereinafter refers to an immunoglobulin variable domain that is capable of specifically binding to an epitope of an antigen without being paired with an additional immunoglobulin variable domain. One example of ISVD in the context of this invention are domain antibodies such as VH and VL ISVDs (VH domains and VL domains). Another important example of ISVDs are the VHH domains (or simply VHH) of camelids, which are defined below.

In light of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD, or

IgE; known in the art) or a Fab fragment, an F(ab') ₂ fragment, an Fv fragment such as a disulfide-linked Fv or scFv fragment, or a diabody (all known in the art) derived from a similar conventional 4- chain antibody will generally not be considered an ISVD, since in these cases binding to the corresponding antigen epitope does not typically involve just one (individual) immunoglobulin domain, but requires a pair of (combined) immunoglobulin domains, such as the mild and severe variable domains chains, i.e., pairs of VH-VL immunoglobulin domains that jointly bind to the epitope of the corresponding antigen.

e1) VHH domains, also known as VHH, VHH domains, VHH antibody fragments, and VHH antibodies, were originally described as the antigen-binding immunoglobulin (variable) domains of heavy chain antibodies (i.e., antibodies that do not have light chains; Hamers-Casterman S, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: Naturally occurring antibodies devoid of light chains; Nature 363, 446-448 (1993)). The term VHH domain was chosen to distinguish such variable domains from the heavy chain variable domains found in conventional 4-chain antibodies (hereinafter referred to as VH domains or VH domains) and from the light chain variable domains found in conventional 4-chain antibodies. chain antibodies (which are hereinafter referred to as VL domains or VL domains). VHH domains are capable of specifically binding to an epitope without an additional antigen-binding domain (unlike the VH or VL domains of a conventional 4-chain antibody, in which the VL and VH domains recognize the epitope together). VHH domains are small, stable and efficient antigen recognition units formed by a distinct immunoglobulin domain.

In the context of the present invention, the terms VHH domain, VHH, VHH domain, VHH antibody fragment, VHH antibody, as well as Nanobody® and Nanobody® domain (Nanobody is a trademark of Ablynx N.V.; Ghent; Belgium) are used interchangeably and refer to ISVD representatives (having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to an epitope without requiring the presence of a second immunoglobulin variable domain), which can also be distinguished from VH domains by so-called distinctive residues according to definition in eg WO2009/109635, FIG. 1.

The amino acid residues of the VHH domain are numbered according to the general VH domain numbering system developed by Kabat et al. (Sequence of proteins of immunological interest, US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to the VHH domains of Camelidae. , shown, for example, in Fig. 2 from Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999). According to this numbering system

FR1 consists of amino acid residues at positions 1-30,

CDR1 consists of amino acid residues at positions 31-35,

FR2 consists of amino acid residues at positions 36-49,

CDR2 consists of amino acid residues at positions 50-65,

FR3 consists of amino acid residues at positions 66-94,

CDR3 consists of amino acid residues at positions 95-102,

FR4 consists of amino acid residues at positions 103-113. However, it should be noted - and this is well known for VH domains and for VHH domains - that the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues provided by the Kabat numbering (namely, one or more positions according to Kabat numbers in the real sequence may not be occupied, or the real sequence may contain more amino acid residues than provided by the Kabat system). This means that in general the Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

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Alternative methods for numbering amino acid residues of VH domains are known in the art, which can be similarly applied to VHH domains. However, unless otherwise indicated, the specification, claims and figures use the Kabat numbering applied to the VHH domains as described above.

The total number of amino acid residues in the VHH domain is typically in the range of 110 to 120, often between 112 and 115. It should be noted, however, that shorter and longer sequences may be suitable for the purposes described herein.

Further structural characteristics and functional properties of VHH domains and polypeptides containing them can be summarized as follows:

VHH domains (designed by nature to functionally bind antigen without the presence of or interaction with a light chain variable domain) are capable of functioning as a separate, relatively small functional antigen-binding entity, domain, or polypeptide. This distinguishes the VHH domains from the VH and VL domains of a conventional 4-chain antibody, which on their own are generally not suitable for practical use as individual antigen-binding proteins or ISVDs, but need to be combined in some form to provide functional antigen-binding unit (as, for example, in conventional antibody fragments such as Fab fragments; in scFvs consisting of a VH domain covalently linked to a VL domain).

Because of these unique properties, the use of VHH domains - alone or as part of a larger polypeptide - has a number of significant advantages over the use of conventional VH and VL domains, scFvs or conventional antibody fragments (such as Fab- or F(ab') ₂ -fragments):

Only one domain is required for high-affinity and highly selective antigen binding, so there is no need to have two separate domains and also to ensure the correct spatial conformation and configuration of these two domains (i.e., using special linkers, as in the case of scFv) .

V HH domains can be expressed from a single gene and do not require post-translational folding or modifications;

V HH domains can be easily incorporated into multivalent and multispecific formats (as discussed below);

V HH domains are highly soluble and do not tend to aggregate (like the mouse antigen binding domains described by Ward et al., Nature 341: 544-546 (1989));

V HH domains are highly resistant to temperature, pH, proteases and other denaturing agents or conditions and can therefore be manufactured, stored or transported without the use of refrigeration, thereby saving money, time and environmental impact;

V HH domains are relatively simple and cheap to produce, even on a production scale. For example, VHH domains and polypeptides containing them can be produced by microbial culture (eg, as described below) without using mammalian expression systems required, for example, for conventional antibody fragments;

VHH domains, compared to conventional 4-chain antibodies and their antigen-binding fragments, are relatively small (approximately 15 kDa, or 10 times smaller than conventional IgG), and therefore penetrate tissue better and can be administered in higher doses than conventional 4-chain antibodies and their antigen-binding fragments;

VHH domains can exhibit so-called cavity binding properties (among other things, due to their CDR3 loop being longer than that of conventional VH domains), and therefore also have access to targets and epitopes that are inaccessible to conventional 4-chain antibodies and their antigen-binding fragments.

Methods for producing VHH domains that bind to a specific antigen or epitope have been described previously, for example, in WO2006/040153 and WO2006/122786. This document also details that camelid VHH domains can be humanized by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues located at the same positions in the VH domain of a conventional 4-chain human antibody. The humanized VHH domain may contain one or more fully human framework sequences, and in an even more specific embodiment, may contain human framework sequences derived from DP-29, DP-47, DP-51, or portions thereof, optionally combined with JH sequences such as JH5.

e2) Domain antibodies, also known as Dab, Domain Antibodies, and dAbs (where the terms Domain Antibodies and dAbs are used as trademarks by the GlaxoSmithKline group of companies), have been described, for example, in Ward E.S. et al.: Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli; Nature 341: 544-546 (1989); Holt L.J. etc.: Domain antibodies: proteins for therapy; TRENDS in Biotechnology 21(11): 484-490 (2003); and also in WO2003/002609.

Domain antibodies essentially correspond to the VH or VL domains of non-mammalians

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Camelids, in particular, human 4-chain antibodies. To be able to bind an epitope as a separate antigen-binding domain, i.e., without being paired with the corresponding VL- or VH-domain, special selection for such antigen-binding properties is necessary, for example, using sequence libraries of individual human VH- or VL- domains.

Domain antibodies, like VHH, have a molecular weight of about 13 to about 16 kDa and, if derived from fully human sequences, do not require humanization for, for example, therapeutic use in humans. As with VHH domains, they are well expressed in prokaryotic expression systems, resulting in a significant reduction in overall production costs.

Domain antibodies, like VHH domains, can be affinity matured by introducing changes into the amino acid sequence of one or more CDRs that result in improved affinity of the resulting ISVD for its corresponding antigen compared to the corresponding parent molecule. Affinity-matured ISVD molecules of the invention can be prepared by methods known in the art, for example those described in Marks et al., 1992, Biotechnology 10:779-783, or Barbas et al., 1994, Proc. Nat. Acad. Sci USA 91: 3809-3813; Shier et al. 1995 Gene 169:147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154(7): 3310-9; and Hawkins et al., 1992, J. Mol. Biol. 226(3): 889 896; K. S. Johnson and R. E. Hawkins, Affinity maturation of antibodies using phage display, Oxford University Press 1996.

e3) In addition, one skilled in the art will also appreciate that one or more of the above CDRs can be grafted onto other scaffolds, including, but not limited to, scaffolds of human origin or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR transplantation are known in the art.

g) The terms epitope and antigenic determinant, which may be used interchangeably, refer to a portion of a macromolecule, such as a polypeptide, recognized by antigen-binding molecules, such as conventional antibodies or polypeptides of the invention, and more particularly by the antigen-binding region of these molecules. Epitopes define the minimal immunoglobulin binding site and are therefore the target of immunoglobulin specificity.

The portion of an antigen-binding molecule (such as a conventional antibody or polypeptide of the invention) that recognizes an epitope is called a paratope.

h) The term biparatopic (antigen-)binding molecule or biparatopic polypeptide hereinafter refers to a polypeptide comprising a first ISVD and a second ISVD as defined herein, wherein the two variable domains are capable of binding to two different epitopes of the same antigen, one monospecific immunoglobulin, such as, for example, a conventional antibody or a single ISVD, does not normally bind to both of these epitopes simultaneously. The biparatope polypeptides of the invention are composed of variable domains with different epitope specificities and do not contain mutually complementary pairs of variable domains that bind to the same epitope. Therefore, they do not compete with each other for binding to LRP5.

i) A polypeptide (such as an immunoglobulin, an antibody, an ISVD, a polypeptide of the invention, or an antigen-binding molecule or fragment thereof in the general sense) capable of binding, binding to, specifically binding to, or specifically binding to, having affinity for and/or specificity for a particular epitope , antigen or protein (or at least one part, fragment or epitope thereof), is called a polypeptide, or a polypeptide directed against the specified epitope, antigen or protein, or is a binding molecule for such epitope, antigen or protein.

k) In general, the term specificity refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecule or antigen-binding protein (such as an immunoglobulin, antibody, ISVD or polypeptide of the invention) can bind. The specificity of an antigen binding protein can be determined based on its affinity and/or avidity. Affinity, as represented by the equilibrium dissociation constant of antigen and antigen-binding protein (KD), is a measure of the binding strength of the epitope and the antigen-binding region of the antigen-binding protein: the lower the KD value, the greater the binding strength of the epitope and the antigen-binding molecule (alternatively, affinity can be expressed as the affinity constant (KA), which is 1/KD). As one skilled in the art will appreciate (eg, based on further information herein), affinity can be determined in a per se known manner, depending on the particular antigen of interest. Avidity is a measure of the strength of binding of an antigen-binding molecule (such as an immunoglobulin, antibody, ISVD or polypeptide of the invention) to the particular antigen in question. Avidity depends both on the affinity of the epitope and its antigen-binding site on the antigen-binding molecule, and on the number of corresponding applicable binding sites on the antigen-binding molecule.

Typically, antigen-binding proteins (such as the polypeptides of the invention) have a binding dissociation constant (KD) of 10E-5 to 10E-14 mol/liter (M) or less, preferably 10E-7 to 10E-14 mol/liter (M) or less, more preferably from 10E-8 to 10E-14 mol/liter, more

- 11 046752 more preferably from 10E-11 to 10E-13 (measured, for example, by Kinexa analysis; known from the prior art), and/or an association constant (K _A ) of at least 10E7 ME-1, preferably at least 10E8 ME-1, more preferably at least 10E9 ME-1, such as at least 10E11 ME-1. Any K _D value above 10E-4 M is generally considered to indicate nonspecific binding. Preferably, the polypeptide of the invention binds to the desired antigen with a K _D of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The specific binding of an antigen binding protein to an antigen or epitope can be determined by any suitable method known per se, including, for example, the assays described herein, the Scatchard assay, and/or competitive binding assays such as radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), and sandwich assays. competition analyses, as well as various variations known per se from the prior art.

k) The term cross-reactive with respect to binding molecules capable of binding to both LRP5 and LRP6 (LRP5/LRP6 cross-reactive) should be understood to mean that such binding molecules can specifically bind to an epitope contained in the LRP5 molecule, and may alternatively specifically bind to an epitope contained in the LRP6 molecule.

l) Amino acid residues are designated by standard three-letter or one-letter amino acid codes well known and accepted in the art. When comparing two amino acid sequences, the term amino acid difference refers to insertions, deletions, or substitutions of a specified number of amino acid residues at a position in a reference sequence compared to a second sequence. In the case of substitution(s), it(-s) is preferably a conservative amino acid substitution(s), that is, an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and that does not affect or significantly affect the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 98/49185, where conservative amino acid substitutions are preferably substitutions in which one amino acid from the following groups (I)-(V) is replaced by another amino acid residue from the same group: ( I) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and GIy; (II) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, GIu and GIn; (III) polar, positively charged residues: His, Arg and Lys; (IV) large aliphatic, non-polar residues: Met, Leu, Ile, VaI and Cys; and (V) aromatic residues: Phe, Tyr and Trp. Particularly preferred are the following conservative amino acid substitutions:

Ala to GIy or to Ser;

Arg to Lys;

Asn to GIn or to His;

Asp on GIu;

Cys to Ser;

GIn to Asn;

GIu on Asp;

GIy on Ala or on Pro;

His to Asn or to GIn;

Ile to Leu or VaI;

Leu on Ile or on VaI;

Lys on Arg, on GIn or on GIu;

Met on Leu, on Tyr or on Ile;

Phe to Met, to Leu or to Tyr;

Ser to Thr;

Thr to Ser;

Trp to Tyr;

Tyr to Trp or to Phe;

VaI on Ile or on Leu.

m) A nucleic acid or polypeptide molecule is considered to be (in) an essentially isolated (form), for example, compared to its natural biological source and/or the reaction medium or culture medium from which it is obtained - after separation of the molecule from the at least one other component with which it is commonly associated in the designated source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule, or at least one contaminant, impurity or minor component. In particular, a nucleic acid or polypeptide molecule is considered to be substantially isolated after it has been purified at least 2-fold, in particular at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more . The nucleic acid or polypeptide molecule, when in substantially isolated form, is preferably substantially homogeneous, as determined by a suitable method, such as a suitable chromatographic method, such as polyacrylamide gel electrophoresis;

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o) Sequence identity, eg, of two ISVD sequences, reflects the percentage of amino acids that are the same in the two sequences. It can be calculated or determined as described in paragraph f) on pages 49 and 50 of WO2008/020079. Sequence similarity reflects the percentage of amino acids that are either identical or conserved amino acid substitutions.

Target specificity.

The polypeptides of the invention have LRP5 specificity, in the sense that they typically contain ISVDs that specifically bind to LRP5 epitopes. Preferably, the polypeptides of the invention have an affinity and/or avidity for LRP5, especially human LRP5, that is at least 10-fold, preferably at least 100-fold, more preferably at least 1000-fold, even more preferably at least 10,000 times, even more preferably at least 100,000 times or at least 1,000,000 times stronger than their affinity and/or avidity for LRP6, especially human LRP6 (Accession number: UniProtKB 075581/LRP6_HUMAN). Most preferably, the polypeptides do not cross-react with LRP6, particularly human LRP6 (see Example 7.1).

The molecules of the invention will bind to human forms of LRP5, and preferably also to opponents in other species relevant for drug development, ie, cynomolgus and mouse LRP5.

Polypeptides according to the invention.

In the broadest sense, the invention provides new pharmacologically active agents for the treatment of cancer. Exemplary agents of the invention belong to a new class of binding molecules, namely biparatope polypeptides, which contain two or more ISVDs that bind to LRP5 at different epitopes. The term biparatopic is explained above such that biparatopic molecules can be defined as molecules capable of binding to LRP5 at two different epitopes contained in the LRP5 protein.

In one aspect, the polypeptide of the invention comprises a first LRP5 binding domain, and a second LRP5 binding domain, wherein the first domain binds a region of LRP5 that is different from the LRP5 region to which the second domain binds. Such an LRP5-binding domain may be any immunoglobulin domain, for example, any immunoglobulin domain described herein. Preferably, such LRP5-binding domain is an ISVD.

In one embodiment, the first domain blocks the Wnt3a binding site in LRP5, and preferably inhibits Wnt3a-mediated transcription of the target gene, and/or where the second domain blocks the Wnt1 binding site in LRP5, and preferably inhibits WnH-mediated transcription of the target gene.

In other words, the polypeptides of the invention may include:

a first ISVD that is capable of specifically binding to LRP5 via an epitope/such that it leads to inhibition of the Wnt3a signaling pathway, thereby inhibiting Wnt3a-mediated transcription of a target gene, and a second ISVD that is capable of specifically binding to LRP5 via an epitope/such that leads to inhibition of the Wnt1 signaling pathway, as a result of which Wnt1-mediated transcription of the target gene is inhibited.

Due to the two ISVDs present in the polypeptides described above, where the two domains bind to different epitopes (related to Wnt1/Wnt3a signaling), these molecules are biparatope binding molecules. This biparatope binding mode is shown schematically in FIG. 1.

In this regard, it should be noted that it is contemplated that the polypeptides of the invention can bind to one single LRP5 molecule across both of their LRP5 binding domains, as shown in FIG. 1 (intramolecular binding mode). However, other binding modes may also occur.

Finally, it is believed that the polypeptides of the invention are capable of competing with DKK1, a natural ligand of LRP5, and interfering with Wnt1 and Wnt3a signaling for binding to LRP5, thereby inhibiting the Wnt1 signaling pathway as well as the Wnt3a signaling pathway. However, this theory should also not be construed as limiting the scope of the invention.

Specifically, the present invention provides a polypeptide that binds to low-density lipoprotein receptor family protein 5 (LRP5)-related protein, wherein the polypeptide comprises an ISVD selected from the group consisting of the following LRP5-binding ISVDs:

(I) ISVD having the following complementarity determining region (CDR) sequences:

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3), (II) ISVD having the following CDR sequences:

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CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), (III) ISVD having the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and (IV) ISVD having the following CDR sequences:

CDR1: INAMG(SEQ ID NO:10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9).

The polypeptide of the present invention preferably comprises a first ISVD (a) selected from ISVDs (I) and (II) as defined above, and a second ISVD (b) selected from ISVDs (III) and (IV) as defined above. In one embodiment, the first ISVD is ISVD (I) and the second ISVD is ISVD (III). In a further embodiment, the first ISVD is an ISVD (I) and the second ISVD is an ISVD (IV). In another further embodiment, the first ISVD is an ISVD (II) and the second ISVD is an ISVD (III). In another embodiment, the first ISVD is an ISVD (II) and the second ISVD is an ISVD (IV). Preferably, ISVD (I) is further defined as having the sequence identified as SEQ ID NO:11 or SEQ ID NO:23, ISVD (II) is further defined as having the sequence identified as SEQ ID NO:12, ISVD (III) is additionally defined as having the sequence identified as SEQ ID NO:12 is defined as having the sequence identified as SEQ ID NO:13 or SEQ ID NO:22, and/or ISVD (IV) is further defined as having the sequence identified as SEQ ID NO:14.

The use of the terms first and second in relation to such ISVDs is intended only to indicate that these domains are different domains since they contain different CDR sequences and will bind to different epitopes. However, these terms should not be understood to indicate the exact order or sequence of domains in such a polypeptide chain. In other words, the above ISVDs can be arranged either in the following order (I)/(II) - (III)/(IV) or in the following order (III)/(IV) - (I)(II) within such a polypeptide according to invention.

ISVDs typically consist of essentially four framework regions (FR1-FR4, respectively) and three complementarity determining regions (CDR1-CDR3, respectively). To reside in the same polypeptide or polypeptide chain, said first and said second ISVD must be covalently linked, either directly or through a linker peptide (e.g., a linker sequence derived from the hinge region of an antibody heavy chain, a poly-alanine linker sequence, Gly/Ser linkers of different lengths or the like).

Thus, the general structure of the molecules according to the invention can also be represented as follows:

FR(a)1 - CDR(a)1 - FR(a)2 - CDR(a)2 - FR(a)3 - CDR(a)3 - FR(a)4 - [linker peptide] - FR(6 )1 CDR(6)1 - FR(6)2 - CDR(6)2 - FR(6)3 - CDR(6)3 -FR^X where

FR(a) denotes the frame portion of the first ISVD,

FR(6) denotes the frame portion of the second ISVD,

CDR(a) denotes the CDR of the first ISVD,

CDR(6) denotes the CDR of the second ISVD,

[linker peptide] means a linker peptide, which may optionally be present, preferably where the CDRs have the sequences as above (i.e., the CDRs from (a) may be the CDRs from (I) or (II) and the CDRs from (b) may be CDRs from (III) or (IV),).

It should also be taken into account that (a) and (b) can be interchanged, that is, that molecules having the general structure FR(6)1 - CDR(6)1 - FR(6)2 - CDR(6)2 - FR(6)3 - CDR(6)3 - FR(6)4 - [linker peptide] - FR(a)1 - CDR(a)1 - FR(a)2 - CDR(a)2 - FR( a)3 - CDR(a)3 - FR(a)4 may also be covered by the present invention.

The linker peptide may contain an amino acid sequence, for example, having a length of 9 or more amino acids, preferably at least 17 amino acids, such as approximately 20-40 amino acids. The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. Typical linker sequences include, but are not limited to, linker sequences derived from the hinge region of antibody heavy chains, poly-alanine linker sequences, and Gly/Ser linkers of various lengths (for example, (gly _x ser _y ) _z linkers such as (gly ₄ ser) ₃ , (gly4ser) , (glysser)s, _{(gVser) . (gl\ ; ser ; ) ;} gl\ _; ser _; ) ).

Preferably, the polypeptide further comprises an albumin-binding ISVD (e.g., a third ISVD), preferably wherein the albumin-binding ISVD covalently binds a segment of the polypeptide, wherein the segment contains or consists of a first ISVD (e.g., ISVD (I) or (II), as defined

- 14 046752 above), to a polypeptide segment, where the segment contains or consists of a second ISVD (for example, ISVD (III) or (IV), as defined above). In other words, the linker peptide may covalently link said first ISVD to said second ISVD, wherein said linker peptide preferably contains or consists of an additional ISVD, more preferably an albumin-binding ISVD, especially an albumin-binding ISVD as defined herein.

In some embodiments, the linker peptide contains or consists of a third domain, such as, for example, an albumin-binding ISVD such as an Alb11 domain containing the following CDRs:

CDR(Alb11)1: SFGMS (= SEQ ID NO:15)

CDR(Alb11)2: SISGSGSDTLYADSVKG (=SEQ ID NO:16)

CDR(Alb11)3: GGSLSR (= SEQ ID NO:17)

This results in a group of polypeptides according to the invention having the following general structure:

FR(a)1 - CDR(a)1 - FR(a)2 - CDR(a)2 - FR(a)3 - CDR(a)3 - FR(a)4 - [linker peptide] - FR(Alb11 )1CDR(Alb11)1- FR(Alb11)2 - CDR(Alb11)2 -FR(Alb11)3 - CDR(Alb11)3 - FR(Alb11)4 - [linker peptide] FR(6)1 - CDR(6 )1 - FR(6)2 - CDR(6)2 - FR(6)3 - CDR(6)3 - FR(6)4, preferably where the CDRs have the sequences as above (that is, the CDRs from ( a) may be a CDR from (I) or (II) and a CDR from (b) may be a CDR from (III) or (IV).

Again, the order of the three ISVDs (a), (b) and Alb11 is not fixed, on the contrary, the polypeptides with the following order of the above domains are in the following order:

(b) - Alb11 - (a) are also covered by the present invention.

In addition, polypeptides having an Alb11 domain at the N- or C-terminal region of the polypeptide (for example, Alb11 - (a) - (b), Alb11 - (b) - (a), (a) - (b) - Alb11, or (b) - (a) - Alb11) will also be covered by the present invention. In three preferred embodiments, the polypeptides of the invention include ISVDs defined as follows:

First Preferred Embodiment: Polypeptides comprising a first ISVD having the following CDR sequences: CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3), and a second ISVD having the following CDR sequences:

CDR1: INAMG (SEQ ID NO: 10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9).

Second preferred embodiment: Polypeptides comprising a first ISVD having the following CDR sequences:

CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6). and a second ISVD having the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9)

Third preferred embodiment: Polypeptides comprising a first ISVD having the following CDR sequences:

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3) and a second ISVD having the following CDR sequences:

CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9).

Of course, the foregoing embodiments—that is, optionally including linker peptides (e.g., a linker sequence derived from the hinge region of antibody heavy chains, poly-alanine linker sequences, Gly/Ser linkers of varying lengths, or the like) and/or other domains, especially comprising the Alb11 domain, with a different ISVD order, would also apply to these three preferred embodiments.

In a particularly preferred embodiment, the albumin-binding ISVD (eg, Alb11) is located between two LRP5-binding ISVDs (ISVD (a) and (b)). Therefore, such preferred polypeptides may have the structure (a) - Alb11 - (b) or (b) - Alb11 - (a)). Specifically, an albumin-binding ISVD (eg, Alb11) may be covalently linked to, either directly or through a linker peptide (eg, a linker sequence derived from a hinge

- 15 046752 antibody heavy chain regions, poly-alanine linker sequences, Gly/Ser linkers of varying lengths or the like) LRP5-binding ISVD domains (a) and (b) with their N- and C-terminal regions (e.g. the albumin-binding domain region is linked to the ISVD (a) and the C-terminal albumin-binding domain region is linked to the ISVD (b) or vice versa, preferably where the ISVDs contain CDR or full-length ISVD sequences as above (i.e., the CDR/ISVD sequences from (a) ) may be CDR/ISVD sequences from (I) or (II) and the CDR/ISVD sequences from (b) may be CDR/ISVD sequences from (III) or (IV)).

Three particularly preferred embodiments can be represented as follows: First particularly preferred embodiment: Polypeptides comprising a first (LRP5-binding) ISVD having the following CDR sequences:

CDR1: TYVMG (SEQ ID NO:1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3) albumin-binding ISVD having the following CDR sequences:

CDR1: SFGMS (= SEQ ID NO: 15)

CDR2: SISGSGSDTLYADSVKG (=SEQ ID NO:16)

CDR3: GGSLSR (= SEQ ID NO: 17);

and a second (LRP5-binding) ISVD having the following CDR sequences: CDR1: INAMG (SEQ ID NO: 10)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9);

either in the order stated or with the order of the above domains modified.

A second particularly preferred embodiment: Polypeptides comprising a first (LRP5-binding) ISVD having the following CDR sequences:

CDR1: RYAVA (SEQ ID NO:4)

CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5)

CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6); albumin-binding ISVD having the following CDR sequences:

CDR1: SFGMS (= SEQ ID NO: 15)

CDR2: SISGSGSDTLYADSVKG (=SEQ ID NO:16)

CDR3: GGSLSR (= SEQ ID NO: 17);

and a second (LRP5-binding) ISVD having the following CDR sequences: CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9); either in the order stated or with the order of the above domains modified.

A third particularly preferred embodiment: Polypeptides comprising a first (LRP5-binding) ISVD having the following CDR sequences:

CDR1: TYVMG (SEQ ID NO: 1)

CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2)

CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3); albumin-binding ISVD having the following CDR sequences:

CDR1: SFGMS (= SEQ ID NO: 15)

CDR2: SISGSGSDTLYADSVKG (=SEQ ID NO:16)

CDR3: GGSLSR (= SEQ ID NO: 17);

and a second (LRP5-binding) ISVD having the following CDR sequences: CDR1: IGAMG (SEQ ID NO:7)

CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8)

CDR3: ETGPYGPPKRDY (SEQ ID NO:9); either in the order stated or with the order of the above domains modified. The CDR sequences listed above are summarized in Tables IA, IB, and IC:

Table IA

CDR sequences of ISVD interfering with Wnt3a signaling

F0129097A08 F0129093A02 CDR1 TYVMG (SEQ ID NO:1) RYAVA (SEQ ID NO:4)

- 16 046752

CDR2 AISWSGGSTYYAD SVKG (SEQ ID NO:2) AITWSSGRIDYADSV KG (SEQ ID NO:5) CDR3 SRGTSTPSPRASGV SRYDY (SEQ ID NO:3) DRPRPRSTGRSGTGSPS TYDY (SEQ ID NO:6)

Table IB

CDR sequences of ISVD interfering with Wnt1 signaling

F012904 6C1O(E1A,N32G) F012904 6C10 CDR1 IGAMG (SEQ ID NO:7) INAMG (SEQ ID NO: 10) CDR2 AVSSGGSTYYVDSVKG (SEQ ID NO:8) AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3 ETGPYGPPKRDY (SEQ ID NO:9) ETGPYGPPKRDY (SEQ ID NO:9)

IC table

CDR sequence of ISVD binding to serum albumin (Alb11 domain)

Albi 1 domain CDR1 SFGMS (SEQ ID NO: 15) CDR2 SISGSGSDTLYADSVKG (SEQ ID NO: 16) CDR3 GGSLSR (SEQ ID NO:17)

In addition to the CDR sequences as described above, the ISVDs contained in the polypeptides of the invention include immunoglobulin framework region (FR) sequences. These sequences are preferably not immunogenic in humans, and therefore preferably are human or humanized FR sequences. Suitable human or humanized FR sequences are known in the art. Particularly preferred FR sequences can be found in the embodiments shown below, which describe complete ISVDs, and thus CDR sequences, as well as FR sequences.

According to a more specific embodiment, the polypeptides of the invention comprise an ISVD (e.g., an ISVD as defined by the CDR sequences listed in Tables IA-IC forming an ISVD (a), (b), or (c) as defined in embodiments presented above), which are VHH domains, and preferably humanized VHH domains. Specifically, the polypeptides described herein contain a first ISVD (a) that contains CDR sequences from (I) or (II) and a second ISVD (b) that contains CDR sequences from (III) or (IV), and a third ISVD , which is an albumin-binding ISVD (eg, Alb11), the ISVDs of which are VHH domains, and preferably humanized VHH domains.

According to an even more specific embodiment, the polypeptides of the invention comprise a first ISVD(a) selected from the group consisting of ISVD(I) and (II) which have the following sequences:

- 17 046752 (I)

AVQLVESGGGLVQPGGSLRLSCAASGRTFFSTYVMGWFRQAPGKEREFV

AAISWSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYC

AASRGTSTPSPRASGVSRYDYWGQGTLVTVSS (SEQ ID NO: 11), or

EVQLVESGGGLVQPGGSLRLSCVASGRTFFSTYVMGWFRQAPGKEREFV AAISWSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYC AASRGTSTPSRASGVSRYDYWGQGTLVTVSS (SEQ ID NO:23), and (P)

EVQLVESGGGLVQPGGSLRLSCAASGLFTFSRYAVAWFRQAPGKEREFV

AAITWSSGRIDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCA

ADRRPRSTGRSGTGSPSTYDYWGQGTLVTVSSA (SEQ ID NO: 12), and a second ISVD (b) selected from the group consisting of ISVDs (III) and (IV), which have the following sequences:

(III)

AVQLVESGGGLVQPGGSLRLSCAASGSIFRIGAMGWYRQAPGKQRELV

AAVSSGGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYC

NRETGPYGPPKRDYWGQGTLVTVSS (SEQ ID NO: 13), or

EVQLVESGGGLVQPGGSLRLSCAASGSIFRIGAMGWYRQAPGKQRE

VAAVSSGGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYY

NRETGPYGPPKRDYWGQGTLVTVSSA (SEQ ID NO:22), and (IV)

EVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMGWYRQAPGKQRELV

AAVSSGGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYC

NRETGPYGPPKRDYWGQGTLVTVSS (SEQ ID NO: 14).

Preferred embodiments are polypeptides comprising a first ISVD having the amino acid sequence as shown in SEQ ID NO:23 and a second ISVD having the amino acid sequence as shown in SEQ ID NO:14; or a first ISVD having an amino acid sequence as shown in SEQ ID NO:12 and a second ISVD having an amino acid sequence as shown in SEQ ID NO:13; or a first ISVD having an amino acid sequence as shown in SEQ ID NO:11 and a second ISVD having an amino acid sequence as shown in SEQ ID NO:22.

Thus, the above-described embodiments can be schematically represented as isvd(a) - [linker peptide] - isvd^), where isvd denotes the corresponding ISVD, and where the remaining definitions and variations remain the same as above, as above, especially in regarding the presence of optional linker peptides and/or additional domains, especially the Alb11 domain, and also regarding different ISVD orders.

Specifically, an albumin-binding ISVD (eg, Alb11) may be covalently linked to, either directly or through a linker peptide (eg, a linker sequence derived from the hinge region of antibody heavy chains, poly-alanine linker sequences, Gly/Ser linkers of varying lengths, or the like ) LRP5-binding ISVD domains (a) and (b) with their N- and C-terminal regions; for example, the N-terminal region of the albumin-binding domain is linked via a linker peptide to the ISVD (a) and the C-terminal region of the albumin-binding domain is linked via a linker peptide to the ISVD (b) or vice versa, preferably where the ISVDs contain full-length ISVD sequences as above (then that is, the ISVD sequences from (a) may be the ISVD sequences from (I) or (II) and the ISVD sequences from (b) may be the ISVD sequences from (III) or (IV) as defined above).

In some embodiments, the first ISVD is linked to the albumin-binding ISVD via a linker peptide and the second ISVD is linked directly to the albumin-binding ISVD. In some embodiments, the second ISVD is linked to the albumin-binding ISVD via a linker peptide and the first ISVD is linked directly to the albumin-binding ISVD. In some embodiments, both the first and second ISVD are linked to the albumin-binding ISVD via a linker peptide, respectively.

- 18 046752

In accordance with specific embodiments of the invention, said polypeptides may further include a half-life increasing component, wherein said half-life increasing component is covalently linked to said polypeptide and optionally selected from the group consisting of an albumin binding component, such as an albumin binding peptide or an albumin binding domain an immunoglobulin, preferably an albumin-binding ISVD, more preferably an Alb11 domain, a transferrin-binding component such as an anti-transferrin immunoglobulin domain, a polyethylene glycol molecule, serum albumin, preferably human serum albumin, and a fragment of (human) serum albumin.

The sequence of the above Alb11 ISVD is as follows:

EVQLVESGGGLVQPGNSLRLSCAASGTFSSFGMSWVRQAPGKGLE

WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC

T.I.

GGSLSRSSOGTLVTVSS (= Alb 11 domain; = SEQ ID NO: 21)

Further examples of ISVDs binding to human serum albumin are known in the art and are described in more detail, for example, in international patent publications WO2006/122787 and WO2008/028977. Other peptides that bind to human serum albumin are described, for example, in WO2008/068280, WO2009/127691, and WO2011/095545.

Thus, three preferred specific embodiments of the invention are:

First preferred specific embodiment: Polypeptides comprising a first (LRP5-binding) ISVD having the amino acid sequence as shown in SEQ ID NO:23;

an albumin-binding ISVD having the amino acid sequence as shown in SEQ ID NO:21;

a second (LPR5-binding) ISVD having the amino acid sequence as shown in SEQ ID NO:14;

either in the order stated or with the order of the above three domains modified.

Second preferred specific embodiment: Polypeptides comprising a first (LPR5-binding) ISVD having the amino acid sequence as shown in SEQ ID NO:12;

an albumin-binding ISVD having the amino acid sequence as shown in SEQ ID NO:21;

a second (LRP5-binding) ISVD having the amino acid sequence as shown in SEQ ID NO:13;

either in the order stated or with the order of the above three domains modified.

Third preferred specific embodiment: Polypeptides comprising a first (LPR5-binding) ISVD having the amino acid sequence as shown in SEQ ID NO:11;

an albumin-binding ISVD having the amino acid sequence as shown in SEQ ID NO:21;

a second (LRP5-binding) ISVD having the amino acid sequence as shown in SEQ ID NO:22;

either in the order stated or with the order of the above three domains modified.

In even more preferred specific embodiments, the albumin-binding ISVD is located between two LRP5-binding ISVDs.

The sequences of the ISVDs listed above are summarized in Tables IIA, IIB, and IIC:

- 19 046752

Table IIA

ISVD sequences that interfere with Wnt3a signaling

VHH ID SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 F012909 7AO8(E1 A,V23A) SEQ ID NO: 11 AVQLVESG GGLVQPGG SLRLSCAA SGRTFS (SEQ ID NO:24) TYVMG (SEQ ID NO:1) WFRQAPGK EREFVA (SEQ ID NO:25) AISWSGG STYYADS VKG (SEQ ID NO:2) RFTISRDNSKNTVY LQMNSLRPEDTAVY YCAA (SEQ ID NO:26) SRGTST PSRASG VSRYD Y(SEQ ID NO:3) WGQGTL VTVSS (SEQ ID NO:27) F012909 3A02 SEQ ID NO: 12 EVQLVESG GGLVQPGG SLRLSCAA SGLTFS (SEQ ID NO:28) RYAVA (SEQ ID NO:4) WFRQAPGK EREFVA (SEQ ID NO:25) AITWSSG RIDYADS VKG (SEQ ID NO:5) RFTISRDNSKNTVY LQMNSLRPEDTAVY YCAA (SEQ ID NO:26) DRRPRS TGRSGT GSPSTY DY (SEQ ID NO:6) WGQGTL VTVSSA (SEQ ID NO:29) F012909 7A08 (SEQ ID NO:23) EVQLVESG GGLVQPGG SLRLSCVA SGRTFS (SEQ ID NO:30) TYVMG (SEQ ID NO:1) WFRQAPGK EREFVA (SEQ ID NO:25) AISWSGG STYYADS VKG (SEQ ID NO:2) RFTISRDNSKNTVY LQMNSLRPEDTAVY YCAA (SEQ ID NO:26) SRGTST PSRASG VSRYD Y (SEQ ID NO:3) WGQGTL VTVSS (SEQ ID NO:27)

Table IIB

ISVD sequences that interfere with Wnt1 signaling

VHH ID SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 F012904 6C10 (E1A,N32G) SEQ ID NO:13 AVQLVE SGGGLV QPGGSL RLSCAA SGSIFR (SEQ ID NO:31) IGAMG (SEQ ID NO:7) WYRQAPG KQRELVA (SEQ ID NO:32) AVSSGG STYYVD SVKG (SEQ ID NO:8) RFTISRDNSK NTVYLQMNS LRPEDTAVYY CNR (SEQ ID NO:33) ETGPY GPPKR DY (SEQ ID NO:9) WGQGTL VTVSS (SEQ ID NO:27) F012904 6C10 SEQ ID NO:14 EVQLVE SGGGLV QPGGSL RLSCAA SGSIFR (SEQ ID NO:34) INAMG (SEQ ID NO:10) WYRQAPG KQRELVA (SEQ ID NO:32) AVSSGG STYYVD SVKG (SEQ ID NO:8) RFTISRDNSK NTVYLQMNS LRPEDTAVYY CNR (SEQ ID NO:33) ETGPY GPPKR DY (SEQ ID NO:9) WGQGTL VTVSS (SEQ ID NO:27) VHH ID SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 F012904 6C10(N32G) SEQ ID NO:22 EVQLVE SGGGLV QPGGSL RLSCAA SGSIFR (SEQ ID NO:34) IGAMG (SEQ ID NO:7) WYRQAPG KQRELVA (SEQ ID NO:32) AVSSGG STYYVD SVKG (SEQ ID NO:8) RFTISRDNSK NTVYLQMNS LRPEDTAVYY CNR (SEQ ID NO:33) ETGPY GPPKR DY (SEQ ID NO:9) WGQGTL VTVSSA (SEQ ID NO:29)

Table IIC

Sequence of ISVDs binding to serum albumin (Alb11 domain)

VHH ID SEQ ID NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Alb 11 SEQ ID NO:21 EVQLVES GGGLVQP GNSLRLS CAASGFT FS (SEQ ID NO:35) SFGMS (SEQ ID NO:15) WVRQAPG KGLEWVS (SEQ ID NO:36) SISGSGS DTLYAD SVKG (SEQ ID NO:16) RFTISRDNAKTTL YLQMNSLRPEDT AVYYCTI (SEQ ID NO:37) GGSLS R (SEQ ID NO:17) SSQGTL VTVSS (SEQ ID NO:38)

In one aspect, the present invention provides a polypeptide that binds to LRP5 containing an ISVD that competes for LRP5 binding with a reference ISVD, wherein the reference ISVD has the sequence identified in SEQ ID NO: 11, 12, 13, 14, 22 or 23. Preferably, ISVD blocks the Wnt1 or Wnt3a binding site in the LRP. Specifically, ISVD inhibits Wnt1-mediated or Wnt3a-mediated target gene transcription.

In another aspect, the present invention provides a polypeptide that binds to LRP5 comprising a first ISVD that competes for binding to LRP5 with a first reference ISVD, wherein the first reference ISVD has the sequence identified in SEQ ID NO: 11, 23 or 12, and a second ISVD that competes for binding to LRP5 with a second reference ISVD, wherein the second reference ISVD has the sequence identified in SEQ ID NO: 13, 22 or 14. Preferably, the first ISVD blocks the Wnt3a binding site in LRP5, particularly inhibits Wnt3a mediated transcription target gene. Alternatively, or in addition to this, a second ISVD

- 20 046752 can block the Wntl binding site in LRP5, in particular inhibit Wntl-mediated transcription of the target gene.

In the present invention, an expression of an ISVD that competes for antigen binding with a reference ISVD (or similar expressions) includes all ISVDs that bind to the same epitope as the reference ISVD or an epitope that overlaps with an epitope of the reference ISVD and inhibits binding of the reference ISVD with antigen (ie, LRP5) to a certain extent. Preferably, such ISVDs bind to the same epitope as the reference ISVD and/or substantially completely inhibit the binding of the reference ISVD (such as any of the above) to an antigen (i.e., LRP5). Regardless of whether a particular ISVD represents an ISVD that competes for binding with a reference ISVD can be easily tested by one skilled in the art if he has knowledge of the reference ISVD and the antigen, even without knowledge of the epitope of the antigen to which the reference ISVD binds, by performing any of the competition assays bindings known in the art. Such competitive binding assays are well known in the art. For example, one skilled in the art may incubate an antigen with a labeled reference ISVD and may compare the amount of antigen-bound labeled reference ISVD with the amount of labeled reference ISVD that binds to the antigen when preincubated with a test ISVD. If this latter amount is reduced (that is, because preincubation allows the test ISVD to block binding sites that it shares with the reference ISVD), then it can be concluded that the test ISVD is competing for antigen binding with the reference ISVD.

As stated above, the (at least two) ISVDs present in the polypeptide of the invention may be connected to each other directly, without the use of a linker, or through a linker. The linker is preferably a linker peptide and, in accordance with the invention, can be selected to allow binding of at least two different ISVDs to each of their target epitopes.

Suitable linkers inter alia depend on the epitopes and, specifically, on the distance between epitopes on the target molecules to which the ISVD will bind. One skilled in the art will understand this based on this description, not necessarily after a limited number of routine experiments.

Thus, suitable linkers may comprise an amino acid sequence, for example having a length of 9 or more amino acids, preferably at least 17 amino acids, for example about 20-40 amino acids. The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. When used for therapeutic purposes, the linker is preferably not immunogenic for the subject to which the polypeptide of the invention is administered.

One useful group of linker sequences are those derived from the hinge region of antibody heavy chains, as described in WO1996/34103 and WO1994/04678. Other examples are poly-alanine linker sequences such as Ala-Ala-Ala.

Further preferred examples of linker sequences are Gly/Ser linkers of various lengths, such as (gly _x ser _y ) _z linkers, including for example (gly ₄ ser) ₃ , (gly ₄ ser) ₅ , (gVser) , (gly3ser) 3 , (gly3ser)5, (gly3ser)7, (g^ser^, (gl\ _; ser _; ),. and (gl\ _; ser _; ) .

Alternatively, or in addition to a polypeptide linker, the at least two ISVDs present in a polypeptide of the invention may be linked to each other by another component, such as another polypeptide, which, in a preferred but non-limiting embodiment, may be an additional ISVD, as already described above. Such a component may either be substantially inactive or may have a biological effect such as enhancing the desired properties of the polypeptide or may impart one or more desirable properties to the polypeptide. As discussed above, a preferred additional polypeptide domain will prolong the half-life of the polypeptide, being such as a (human) serum albumin binding domain, such as the Alb 11 domain.

Thus, according to a further embodiment, the invention specifically includes polypeptides containing any of the following sequences, the exact amino acid sequences being taken from Table 1. III below:

F012900082, having the sequence identified as SEQ ID NO: 18,

F012900135, having the sequence identified as SEQ ID NO: 19, and

F012900141, having the sequence identified as SEQ ID NO: 20.

- 21 046752

Table III

Sequences of three specific embodiments of the polypeptides of the invention

ID SEQ ID NO: Amino acid sequence (CDR sequences are underlined) F01290008 2 SEQ ID NO:18 EVOLVESGGGLVOPGGSLRLSCVASGRTFSTYVMGWFRQAPGKEREFVAAI SWSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAASRG TSTPSRASGVSRYDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGTFSSFGMSWVRQ APGKGLEWVS SISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMGWYRQAPG KQRELVAAVSSGGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVY YCNRETGPYGPPK RDYWGQGTLVTVSS F01290013 5 SEQ ID NO:19 AVOLVESGGGLVOPGGSLRLSCAASGSIFRIGAMGWYROAPGKORELVAA VSSGGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNRETGP YGPPKRDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGGGGSGGGGS GGGGSEVQLVESGGGLVQPGNSLRLSCAASGTFSSFGMSWVRQAPGKGLE GSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI GGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGLTFSRYAVAWFRQAPGKEREFVA AITWSSGRIDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADR RPRSTGRSGTGSPSTY DYWGQGTLVTVSSA F01290014 1 SEQ ID NO:20 APGKGLEWV SSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRIGAMGWYRQAPG KQRELVAAVSSGGSTYYVDSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVY YCNRETGPYGPP KRDYWGOGTLVTVSSA

As previously explained, unless otherwise indicated, the polypeptides of the invention may contain further fragments and/or additional polypeptide domains, unless such additional fragment or domain interferes with their binding to LRP5.

The polypeptides of the invention may further contain modifications, such as glycosyl residues or modified amino acid side chains, and may also be PEGylated to extend the half-life and other properties of such a molecule. Methods and reagents for PEGylation of biparatope ISVD constructs can be found, for example, in WO2011/107507.

The polypeptides of the invention may have their sequence modified at the N-terminus, e.g., by removing one or more N-terminal amino acids, or by substituting, for example, the first N-terminal amino acid (e.g., glutamate to alanine), in order to optimize the molecule for expression in specific expression systems (such as specific vectors or host cells), or for expression as inclusion bodies or in soluble form, or for secretion into the medium or periplasmic space, or for retention in the cell, or for production more homogeneous product. The polypeptides of the invention may have a sequence modified at the C-terminus, e.g. with an additional alanine and/or with additional amino acid substitutions at the C-terminal portion or at other specified positions in any of the framework regions, as explained in, e.g., WO2012/175741 , WO2011/075861 or WO2013/024059, in order to further improve the stability or reduce the immunogenicity of such polypeptides.

In addition, the half-life of the polypeptides of the invention can be extended by adding an albumin domain, ie, by converting them into albumin fusion proteins. Examples of suitable albumin moieties and methods for adding them to binding molecules are described, for example, in WO2001/079271 and WO2003/059934.

Preferably, the polypeptides of the invention have binding values (EC ₅₀ ), measured by the FACS binding assay described in Example 7.1 below, in the range of ^10-6 mol/liter or less, more preferably 10-9 mol/liter or less, even more preferably in the range from 10 ^-10 to 10 ^-13 mol/liter, or have an IC ₅₀ value, as measured by the combined Wnt1 and Wnt3a reporter gene assay described in Example 7.3 below, of 10 ^-9 mol/liter or less, more preferably in the range of 5x10 ' ¹⁰ mol/liter to 10 ^-12 mol/liter.

The polypeptides of the invention enable more effective treatment of a number of cancer types, such as TNBC, CRC and NSCLC. They have improved in vitro characteristics (i.e., higher efficiency of suppression of the Wnt pathway), see, for example, Examples 7 and 8 below, as well as significant tumor growth suppression abilities in vivo, leading to higher effectiveness in vivo compared to LRP6 binding molecules, as shown, for example, in example 9. Additionally, the polypeptides according to the invention have significantly lower gastrointestinal toxicity compared to LRP6 binding molecules described in the prior art, as shown, for example, in example 9.

- 22 046752

In particular, as demonstrated in vivo in a Wnt-driven tumor model, biparatopic LRP5 humanized VHH constructs with extended half-life were able to suppress Wnt signaling and tumor growth in vivo, providing even significant tumor reduction (i.e., suppressing tumor growth more than 100%). Tumor reduction (ie, tumor regression) is a desirable therapeutic effect (ie, efficacy) in the treatment of cancer patients. Moreover, tumor regression leading to pathological complete response (pCR) is a recognized clinical endpoint indicating significant improvement in progression-free survival and overall survival.

In the same in vivo experiments, no significant changes in body weight (<10%) were observed, and the results of histopathological analyzes of gastrointestinal tissues did not show any toxic effect of the above-mentioned polypeptides according to the invention. This is especially surprising in light of the aforementioned in vivo studies of DKK1 expression (i.e., leading to intestinal mucosal ulceration and body weight loss).

In addition, the examples described below demonstrate the ability of a biparatope LRP5 specific VHH extended half-life construct to selectively inhibit the proliferation of cancer cells harboring mutations in the RNF43 gene that are dependent on active Wnt signaling. Specifically, this further demonstrates that the biparatope LRP5 specific VHH extended half-life constructs described herein selectively inhibit Wnt signaling in cancer cells harboring mutations in the RNF43 gene.

Thus, the polypeptides of the invention actually provide new therapeutic options for the treatment of cancers, and in particular in the case of a large unmet medical need, such as, for example, in (triple-negative) breast cancer.

The above-described advantageous effects are further illustrated by the examples below and the comparative data therein.

Moreover, the polypeptides of the invention are easy to manufacture and are more soluble, meaning that they can be stored and/or administered in higher concentrations than conventional antibodies. They are stable at room temperature and remain stable longer even at extreme pH values, so they can be manufactured, stored and/or transported without refrigeration, saving money, time and environmental impact. Due to the above, as well as their low immunogenicity, they further open up a number of possibilities regarding routes of administration other than injection and infusion, as well as regarding administration protocols and the use of specific devices.

Nucleic acids, vectors, host cells.

In further aspects, the invention relates to nucleic acid molecules and expression vectors encoding polypeptides of the invention, as well as host cells expressing them. These nucleic acids, vectors and host cells are useful in the manufacture of the polypeptides of the invention, and additional aspects and embodiments thereof will be described in more detail below in conjunction with the discussion of methods for producing the polypeptides of the invention.

Therapeutic use.

Due to their biological properties, the polypeptides of the invention are suitable for the treatment of diseases characterized by excessive or abnormal cell proliferation, such as cancer and idiopathic pulmonary fibrosis (IPF).

For example, the following cancers, tumors and other proliferative diseases can be treated with the polypeptides of the invention:

Head and neck cancer; Lung cancer, such as, for example, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC); Neoplasms of the mediastinum, such as, for example, neurogenic tumors and mesenchymal tumors; Cancers of the gastrointestinal (GI) tract, such as cancers of the esophagus, stomach, pancreas, liver and bile ducts (including, for example, hepatocellular carcinoma (HCC)), and small and large intestine (including, e.g. ., colorectal cancer); Prostate cancer; Testicular cancer; Gynecological cancers, such as ovarian cancer; Breast cancer, such as, for example, breast carcinoma, hormone receptor-positive breast cancer, Her2-positive breast cancer and triple negative breast cancer; Endocrine system cancer; Soft tissue sarcomas, such as, for example, fibrosarcoma, rhabdomyosarcoma, angiosarcoma, Kaposi's sarcoma; Bone sarcomas such as, for example, myeloma, osteosarcoma, Ewing's tumor, fibrosarcoma, osteochondroma, osteoblastoma and chondroblastoma; Mesotheliomas;

Skin cancer such as, for example, basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma and melanoma; Neoplasms of the central nervous system and brain, such as, for example, astrocytoma, glioblastoma, glioma, neuroblastoma and retinoblastoma; Lymphomas and leukemias, such as, for example, B-cell non-Hodgkin lymphomas (NHL), T-cell non-Hodgkin lymphomas, B-cell chronic lymphocytic leukemia (B-CLL), T-cell chronic lymphocytic leukemia (T-CLL), Hodgkin's disease (HD), large granular lymphocyte leukemia (LGL), chronic myelogenous leukemia (CML), acute myelogenous/myeloid leukemia (AML), acute lymphoma

- 23 046752 chelic/lymphoblastic leukemia (ALL), multiple myeloma (MM), plasmacytoma and myelodysplastic syndromes (MDS); as well as tumors of unknown primary localization.

It is intended that all of the above-mentioned cancers, tumors, neoplasms, etc., characterized by a specific location/site of origin in the body, include both primary tumors and metastatic tumors derived from them.

More particularly, the polypeptides of the invention are useful in the treatment of diseases, especially cancers, in which abnormal cell proliferation is caused by or participates in abnormal (activated) Wnt signaling.

Therefore, the polypeptides of the invention are particularly useful for the treatment of solid tumors, more particularly for the treatment of cancers of the lung, liver, colon, brain, thyroid, pancreas, breast, ovary and prostate, and even more particularly for the treatment of non-small cell lung cancer ( NSCLC), triple negative breast cancer (TNBC) and colorectal cancer (CRC). In particular, the polypeptides of the invention may be used to treat patients with locally advanced or metastatic TNBC, patients with metastatic NSCLC, or locally advanced or metastatic CRC, as a single agent or in combination, to prolong progression-free survival (PFS) and overall survival (OS). . In addition, the polypeptides of the invention can be used as neoadjuvant therapy for patients with breast cancer to achieve pathological complete response (pCR; defined as the absence of residual invasive cancer and cancer in situ on histopathological evaluation of a completely excised breast specimen and samples from all regional lymph nodes after completion of neoadjuvant systemic therapy).

The polypeptides of the invention can be used in therapeutic protocols in the context of first line, second line or any further line of treatment.

The polypeptides of the invention can be used for the prevention, short-term or long-term treatment of the above-mentioned diseases, optionally in combination with radiation therapy and/or surgery.

Also, the polypeptides of the invention are particularly useful for the treatment of other diseases caused by abnormal cell proliferation involving the Wnt signaling pathway, such as idiopathic pulmonary fibrosis (IPF). (Konigshoff et al. Functional Wnt signaling is increased in idiopathic pulmonary fibrosis. PLoS One 2008;3(5):e2142; Lam et al. Wnt coreceptor Lrp5 is a driver of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2014; 190(2):185-95).

In addition, the polypeptides of the invention are particularly useful for the treatment of retinopathy, especially for the treatment of diabetic retinopathy due to abnormal activation of Wnt in cells of the inner retina, which causes increased abnormal formation of retinal blood vessels, which leads to the development and progression of diabetic retinopathy (Chen Y. et al. Activation of the Wnt pathway plays a pathogenic role in diabetic retinopathy in humans and animal models The Am J Pathol 2009; 175(6):2676-85. Gao et al. Elevated LRP6 levels correlate with vascular endothelial growth factor. in the vitreous of proliferative diabetic retinopathy Mol Vis. 2015;21:665-72).

Finally, since it has been shown that inhibition of Wntl/Wnt3a signaling pathways can also affect dendritic cells (DCs) and dendritic cell function, the polypeptides of the invention may be useful for the treatment of immune and infectious diseases, as well as for influencing the tumor microenvironment in various cancers. diseases from those listed above. Tumors actively suppress antitumor immunity, and DCs play an important role in the mechanism of cancer evasion from the immune system. In particular, studies have shown that Wnt ligands in the tumor microenvironment are also able to initiate paracrine signaling in immune cells and regulate host antitumor immunity (Hong et al. beta-catenin promotes regulatory T-cell responses in tumors by inducing vitamin A metabolism in dendritic cells Cancer Res 2015;75(4):656-65.

The above also covers the use of the polypeptides of the invention in various methods of treating the above diseases by administering a therapeutically effective dose to a patient in need thereof, as well as the use of these polypeptides for the manufacture of medicaments for the treatment of such diseases, as well as pharmaceutical compositions comprising such polypeptides of the invention , as well as the formulation and/or manufacture of medicinal products comprising such polypeptides according to the invention, etc. and so on.

Combinations with other active substances.

The polypeptides of the invention can be used alone or in combination with other pharmacologically active substances, such as advanced or standard of care agents, such as, for example, cytostatic or cytotoxic agents, cell proliferation inhibitors, antiangiogenic agents, steroids, immunomodulators/checkpoint inhibitors and so on.

Cytostatic and/or cytotoxic active substances that can be administered in combination with the compounds of the invention include, but are not limited to, hormones, hormone analogs and antihormones, aromatase inhibitors, LHRH agonists and antagonists, growth factor inhibitors (growth factors such as e.g. , platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),

- 24 046752 vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factors (IGF), human epidermal growth factor (HER, e.g. HER2, HER3, HER4) and hepatocyte growth factor (HGF)), wherein the inhibitors are, for example, anti-growth factor antibodies, anti-growth factor receptor antibodies and tyrosine kinase inhibitors, such as, for example, cetuximab, gefitinib, afatinib, nintedanib, imatinib, lapatinib, bosutinib and trastuzumab; antimetabolites (e.g. antifolates such as methotrexate, raltitrexed, pyrimidine analogs such as 5-fluorouracil (5-FU), capecitabine and gemcitabine, purine and adenosine analogs such as mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine (ara C), fludarabine); antitumor antibiotics (eg, anthracyclines); platinum derivatives (eg, cisplatin, oxaliplatin, carboplatin); alkylating agents (eg estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas such as, for example, carmustine and lomustine, thiotepa); antimitotic agents (eg, Vinca alkaloids, such as, for example, vinblastine, vindesine, vinorelbine and vincristine; as well as taxanes, such as paclitaxel, docetaxel); angiogenesis inhibitors, tubulin inhibitors; DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g. epipodophyllotoxins such as etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone), serine/threonine kinase inhibitors (e.g. PDK1 inhibitors, Raf inhibitors, A inhibitors -Raf, B-Raf inhibitors, C-Raf inhibitors, mTOR inhibitors, mTORC1/2 inhibitors, PI3K inhibitors, PI3Ka inhibitors, dual mTOR/PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors (such as volasertib), CDK inhibitors, Aurora kinase inhibitors), tyrosine kinase inhibitors (e.g., PTK2/FAK inhibitors), protein-protein interaction inhibitors, MEK inhibitors, ERK inhibitors, FLT3 inhibitors, BRD4 inhibitors, IGF-1R inhibitors, TRAILR2 agonists, Bcl-xL inhibitors, Bcl-2 inhibitors , Bcl-2/BclxL inhibitors, ErbB receptor inhibitors, BCR-ABL inhibitors, ABL inhibitors, Src inhibitors, rapamycin analogs (eg, everolimus, temsirolimus, ridaforolimus, sirolimus), androgen synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDAC inhibitors , ANG1/2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, immunotherapeutic agents such as immune checkpoint inhibitors (eg, CTLA4, PD1, PD-L1, LAG3 and TIM3 binding molecules/immunoglobulins such as ipilimumab, nivolumab, pembrolizumab) , cancer vaccines such as traditional cancer vaccine (cellular vaccines, e.g. Sipuleucel-T for prostate cancer), personalized neoantigen vaccines and oncolytic viruses, as well as various chemotherapeutic agents such as amifostine, anagrelide, clodronate, filgrastin, interferon , interferon-alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer.

Particularly preferred are methods of treatment comprising the use of polypeptides according to the invention in combination with a drug selected from the group consisting of:

(I) anti-VEGF antibodies (bevacizumab and other antiangiogenic agents), with or without combination with chemotherapy (including doxorubicin/cyclophosphamide combination and/or neoadjuvant capecitabine/docetaxel combination; taxane/platinum protocol for first and later lines of treatment) for patients with breast cancer;

(II) EGFR TKI for EGFR mutant NSCLC, or crizotinib for ALC rearrangement NSCLC, with or without combination with chemotherapy (cytotoxic platinum combination therapy, including gemcitabine/cisplatin as first-line therapy; docetaxel or pemetrexed as second-line therapy for patients with lung cancer;

(III) anti-EGFR antibodies (cetuximab and panitumumab for wild-type KRAS tumors), with or without combination with chemotherapy (including irinotecan), combination with anti-VEGF antibodies (bevacizumab and other antiangiogenic agents) or combination with regorafenib, e.g. treatment of patients with colorectal cancer.

(IV) immunotherapeutic agents, including anti-PD-1 agents such as pembrolizumab and nivolumab, anti-PD-L1 agents, anti-CTLA4 agents, anti-BTLA agents, anti-LAG3 agents and anti-TIM3 agents such as anti-PDL1 antibodies, etc. ., for example, for the treatment of patients with breast, lung and colorectal cancer.

(V) chemotherapeutic agents, such as platinum-based antineoplastic drugs, either in combination with the FOLFOX chemotherapy protocol, including folinic acid, 5'fluorouracil and oxaliplatin, or in combination with the FOLFOXIRI chemotherapy protocol, including folinic acid, 5'-fluorouracil , oxaliplatin and irinotecan, for example, for the treatment of patients with breast cancer and colorectal cancer.

If two or more substances or modes of action are used as part of a combination chemotherapy protocol, they may be administered by the same route or by different routes, at substantially the same time (i.e., simultaneously, in parallel) or at different times (eg. ., sequentially, in a row, alternately, in order or in any other type of alternating mode).

If the substances or principles are administered simultaneously by the same route, they can be administered as different pharmaceutical preparations or compositions or as part of a combination pharmaceutical

- 25 046752 ceutical preparation or composition. In addition, if two or more substances or principles are used as part of a combination chemotherapy protocol, then each substance or principle can be administered in the same amount and according to the same protocol as used for a single application of the compound or principle, and such combination use may be synergistic. effect or not have it. However, if the combined use of two or more active substances results in a synergistic effect, then the amount of one, some, or all of the substances or principles administered can be reduced while maintaining the desired therapeutic effect. This may be useful for, for example, avoiding, limiting or reducing any undesirable side effects associated with the use of one or more substances or principles in normal quantities, while maintaining the desired pharmacological or therapeutic effect.

The above describes the manufacture and methods of making the polypeptides of the invention for combination use with the above combination partners. Also covered are the manufacture and methods of making the above combination partners for combination use with the polypeptides of the invention. Thus, the invention provides, for example, methods of using or preparing for the use of an immunomodulator/checkpoint inhibitor, such as an anti-PD1 antibody, such as pembrolizumab or nivolumab, for administration in combination with a polypeptide according to the invention, and more particularly for administration as part of a combination therapeutic protocol with a polypeptide according to the invention.

Moreover, the invention also covers kits that include at least one polypeptide of the invention and one or more other components selected from the group consisting of other drugs used to treat diseases and disorders as described above, as well as devices as described above below.

Pharmaceutical compositions, methods of administration, dosages.

It will be clear to one skilled in the art that the above-described methods of treating a disease involve the manufacture of a medicament for the treatment of said disease. Thus, the invention also relates to pharmaceutical compositions for the treatment of the above-mentioned diseases, such compositions containing at least one polypeptide according to the invention.

The polypeptides of the invention and/or compositions containing them can be administered to a patient in need thereof by any suitable route, depending on the particular pharmaceutical preparation or composition used. Thus, the polypeptides of the invention and/or compositions containing them can be administered, for example, intravenously (i.v.), subcutaneously (s.c.), intramuscularly (i.m.), intraperitoneally (i.p.), via skin, orally, sublingually (e.g. as a sublingual tablet, spray or drops placed under the tongue and absorbed through mucous membranes into the sublingual capillary network), (intra-)nasal (e.g. as a nasal spray and/or aerosol) , superficially, by suppository, inhalation or any other suitable method in an effective amount or dosage.

The polypeptides of the invention and/or compositions containing them are administered according to a treatment protocol suitable for treating and/or ameliorating the disease, disorder or condition being treated or ameliorated. The physician will generally be able to determine an appropriate treatment protocol depending on factors such as the disease, disorder or condition being treated or ameliorated, the severity of the disease, the severity of its symptoms, the particular polypeptide of the invention being used, the particular route of administration and the pharmaceutical agent being used. or composition, age, gender, weight, diet, general condition of the patient and similar factors well known to the physician. Typically, the treatment protocol involves administering one or more polypeptides of the invention, or one or more compositions containing them, in therapeutically effective amounts or dosages.

Generally, to treat and/or alleviate the diseases, disorders and conditions mentioned herein, depending on the particular disease, disorder or condition being treated, the strength of the particular polypeptide of the invention used, the particular route of administration used and the particular pharmaceutical preparation or composition of the polypeptides of the invention in generally administered in an amount of 0.005 to 20.0 mg per kilogram of body weight and dose, preferably 0.05 to 10.0 mg/kg/dose, even more preferably 0.5 to 10 mg/kg/dose, or continuously (eg, by infusion), or, more preferably, in separate doses (such as, taken twice a week, once a week, once a month; see below), but these may vary, especially in depending on the above parameters. Therefore, in some cases, a dose less than the minimum dose indicated here may be sufficient, while in other cases it may exceed the upper limit. When administering large quantities, it is recommended to divide them into a number of smaller doses taken throughout the day.

Depending on the particular polypeptide of the invention, as well as its particular pharmacokinetic and other properties, it may be administered once daily, every second, third, fourth, fifth or sixth day, once a week, once a month, or the like. The administration protocol may involve long-term weekly treatment. By long-term we mean a duration of at least two

- 26 046752 weeks, preferably months or years.

The effectiveness of the polypeptides of the invention, as well as compositions containing them, can be tested using any suitable known per se in vitro assay, cellular assay, in vivo assay and/or animal model, or any combination thereof, depending on the particular disease in question. Suitable assays and animal models will be apparent to one skilled in the art and include, by way of example, the assays and animal models used in the examples below.

Preferably, the polypeptides of the invention perform better than conventional antibodies known in the art in at least one of these assays or models, and preferably in one or more in vivo models.

Drugs.

For pharmaceutical use, the polypeptides of the invention may be formulated into a pharmaceutical preparation comprising (I) at least one polypeptide of the invention and (II) at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and/or stabilizer, and (III) optionally one or more additional pharmacologically active polypeptides or compounds. By pharmaceutically acceptable is meant a material that does not exhibit any biological or other undesirable effects when administered to an individual and does not interact detrimentally with any of the other components of the pharmaceutical composition (such as, for example, a pharmaceutically active ingredient) that contains the corresponding material. Specific examples can be found in standard textbooks such as, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, USA (1990). For example, the polypeptides of the invention can be formulated and administered by any method known per se for conventional antibodies and antibody fragments, as well as other pharmaceutically active proteins. Thus, according to a further embodiment, the invention relates to a pharmaceutical composition or preparation comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and/or stabilizer, and optionally one or more additional pharmacologically active substances.

Pharmaceutical preparations for parenteral administration, such as intravenous, intramuscular, subcutaneous injection or intravenous infusion, may, for example, be sterile solutions, suspensions, dispersions, emulsions or powders that contain the active substance and are suitable, optionally after a further dissolution or dilution step, for injections or infusions. Suitable carriers or diluents for such preparations include, by way of example and not limitation, sterile water and pharmaceutically acceptable aqueous buffers and solutions such as physiological phosphate saline, Ringer's solution, dextrose solution and Hanks solution; oils with water; glycerol; ethanol; glycols such as propylene glycol, as well as mineral oils, animal oils and vegetable oils, for example peanut oil, soybean oil and suitable mixtures thereof.

Solutions of the polypeptides of the invention may also contain a preservative to prevent the growth of microorganisms, such as antibacterial and antifungal agents, for example, p-hydroxybenzoates, parabens, chlorobutanol, phenol, sorbic acid, thiomersal, ethylenediaminetetraacetic acid (and its alkali metal salts), etc. .P. In many cases it is preferable to include isotonic agents such as sugars, buffers or sodium chloride. Optionally, emulsifiers and/or dispersants may be used. Proper fluidity can be maintained, for example, by the formation of liposomes, maintaining the desired particle size in the case of dispersions and the use of surfactants. Other substances that delay absorption, such as aluminum monostearate and gelatin, can also be added. Solutions can be filled into injection vials, ampoules, infusion bottles, etc.

In any case, the final dosage form must be sterile, liquid and stable under the conditions of production and storage. Sterile injectable solutions are prepared by incorporating the active substance into the required amount of an appropriate diluent, optionally with various other ingredients from the above, followed by sterilizing filtration. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred manufacturing methods are vacuum drying and freeze-drying methods to obtain a powder of the active substance and any desired additional ingredient from their solution, which has previously undergone sterilization filtration.

Typically, aqueous solutions or suspensions are preferred. In general, suitable preparations for therapeutic proteins such as the polypeptides of the invention are buffered solutions of the proteins, such as solutions containing the protein at a suitable concentration (such as 0.001 to 400 mg/ml, preferably 0.005 to 200 mg/ml, more preferably 0.01 to 200 mg/ml, more preferably 1.0 to 100 mg/ml, such as 1.0 mg/ml (IV administration) or 100 mg/ml (SC administration ) and an aqueous buffer such as:

phosphate saline solution, pH 7.4, other phosphate buffers, pH 6.2-8.2,

- 27 046752 acetate buffers, pH 3.2-7.5, preferably pH 4.8-5.5, histidine buffers, pH 5.5-7.0, succinate buffers, pH 3.2-6.6, and citrate buffers, pH 2.1-6.2, and optionally salts (e.g. NaCl) and/or sugars (e.g. sucrose and trehalose) and/or other polyols (e.g. mannitol and glycerol) to provide isotonicity of the solution.

Preferred buffered protein solutions are solutions containing approximately 0.05 mg/ml of the polypeptide of the invention dissolved in 25 mM phosphate buffer, pH 6.5, adjusted to isotonicity with 220 mM trehalose. Additionally, such solutions may contain other agents such as detergents, eg 0.02% Tween-20 or Tween-80. Preparations for subcutaneous use may contain high concentrations of the polypeptide of the invention, such as up to 100 mg/ml or even higher than 100 mg/ml. However, one skilled in the art will appreciate that the above ingredients and amounts are only one preferred option.

The specialist will be immediately obvious, or easily deduced from the above, their alternatives and options.

Also, compared with conventional antibodies or antibody fragments, one of the great advantages of using the polypeptides of the invention is that they can be easily administered by routes other than parenteral administration and can be easily formulated for such administration. For example, as described in international patent application WO2004/041867, such polypeptides can be formulated for oral, intranasal, intrapulmonary and transdermal administration.

In another aspect of the invention, the polypeptide of the invention can be used in combination with a device used to administer the polypeptide, such as a syringe, pen, micropump, or other device.

Production and purification methods.

The invention also provides methods for producing a polypeptide of the invention, such methods generally comprising the steps of:

culturing host cells containing a nucleic acid encoding a polypeptide according to the invention (hereinafter: nucleic acid according to the invention) under conditions allowing expression of the polypeptide according to the invention; and isolating or isolating the polypeptide expressed by the host cells from the culture; and optionally further purification and/or modification and/or formulation of the polypeptide according to the invention.

The nucleic acid according to the invention may, for example, be a DNA molecule containing coding sequences as well as regulatory sequences and optionally natural or artificial introns, or may be a cDNA molecule. It may have its original codons or optimized codons specifically adapted for expression in the intended host cell or organism. In one embodiment, the nucleic acid of the invention is in substantially isolated form as defined above.

The nucleic acid according to the invention may also be in the form of, present in and/or be part of a vector, such as, for example, a plasmid, cosmid or YAC, which again may be in an essentially isolated form. The vector may in fact be an expression vector, ie, a vector capable of expressing the polypeptide in vitro and/or in vivo (eg, in a suitable host cell, host organism and/or expression system). Such an expression vector typically contains at least one nucleic acid of the invention operably linked to one or more suitable regulatory elements, such as promoter(s), enhancer(s), terminator(s), and the like. Specific examples of such regulatory elements and other elements, such as insertion factors, selection markers, signal or leader sequences, reporter genes, and the like, useful or necessary for the expression of polypeptides according to the invention are described, for example, on pages 131-133 WO2006/040153.

The nucleic acids of the invention can be manufactured or obtained by a per se known method (eg, automated DNA synthesis and/or recombinant DNA technology) based on the amino acid sequence information of the polypeptides of the invention provided herein.

In another embodiment, the invention relates to a host or host cell expressing or capable of expressing a polypeptide of the invention; and/or which contains a nucleic acid encoding a polypeptide according to the invention. According to a particularly preferred embodiment, said host cells are bacterial cells, yeast cells, fungal cells or mammalian cells.

For industrial scale production, the preferred heterologous hosts for

- 28 046752 (industrial) production of ISVD polypeptides and protein drugs containing them include strains of E. coli, Pichia pastoris and S. cerevisiae, which are suitable for large-scale expression, production and fermentation, and in particular for large-scale (bio-)pharmaceutical expression, production and fermentation.

The polypeptides of the invention, produced in a cell as described above, can be produced either intracellularly (eg, in the cytosol, periplasm or inclusion bodies) followed by isolation from host cells and optional further purification; or they may be produced extracellularly (secreted into the host cell culture medium), followed by isolation from the culture medium and optional further purification.

Additional methods and reagents used for recombinant production of polypeptides, such as suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, and the like. known from the prior art. Likewise, those skilled in the art will know protein isolation and purification techniques suitable for the method of producing a polypeptide according to the invention.

Production of the polypeptides of the invention by fermentation in convenient recombinant host organisms such as E. coli and yeast is cost effective compared to conventional antibodies, which typically require expensive mammalian cell culture equipment. Moreover, high expression levels are achievable, and the yield of the polypeptides according to the invention is in the range of 1-10 g/l (E. coli) and up to 10 g/l (yeast) or more.

Examples

Example 1: Immunization of llamas with LRP5 to induce a humoral immune response.

To immunize llamas, it was necessary to develop and implement several protocols to identify the VHH domains that bind LRP5: First, llamas were immunized with recombinant extracellular domains of the LRP5 protein (mouse). However, functional evaluation of the above recombinant LRP5 protein showed that the recombinant protein was not properly folded. Therefore, more work was needed to develop suitable antigens for immunization. As a workaround, llamas were immunized with HEK293 cells stably transfected with human LRP5 or cynomolgus LRP5. However, even then only very low expression of human or cyno LRP5 could be achieved, both with transient and stable transfection, and also with the use of different cell lines (HEK293 and CHO). Thus, more work is needed to achieve sufficient expression of LRP5. Ultimately, and after some unsuccessful trial and error, this was achieved by developing a protocol involving stable co-transfection of HEK293 cells using MesDC-2, a chaperone that should increase exogenous LRP5 expression. But even then, that is, after co-expression of MesDC-2 throughout the establishment of a stably transfected LRP5 cell line, protein expression was repeatedly observed. In contrast, significantly higher and more stable expression levels were achieved when human LRP6 was coexpressed with MesDC-2 in HEK293 cells. These findings are consistent with published data showing the intrinsic upregulation of human LRP5 as a very poorly expressed protein (Fleury et al., Protein Expr Purif. 2010 Mar; 70(1):39-47). This has led to the problem that LRP5 expression may be lost during immunization and selection. To overcome this additional problem, the passage number of LRP5-expressing cells was limited as much as possible and additional cell sorting was performed to enrich for LRP5-expressing cells.

Llamas were further immunized with DNA encoding LRP5, with and without the hMesDC-2 chaperone at opposite ends. Several llamas were administered additional stimuli in an attempt to enhance the cross-species cross-reactive immune response to increase the chances of identifying anti-human LRP5 VHH domains cross-reactive with murine and cyno LRP5.

Immunized blood (PBL) samples were collected at regular intervals, serological responses were determined, and total RNA was prepared from isolated PBLs. After immunization with the recombinant protein, a low serological response to LRP5 was observed; in contrast, an average LRP5 immune response was observed for llamas immunized with DNA. An extremely low immune response was observed for cell immunization. Additionally, synthetic libraries were also examined. However, in the end it was possible to achieve a variety of repertoires to continue in the following stages, as outlined in Example 2.

Example 2: Isolation of monovalent VHH domains (VHH) binding to LRP5.

Creating a library:

Immediately after collection of immune tissues, total RNA was isolated and the integrity and concentration of RNA were confirmed. From these RNA preparations, cDNA samples were made. Nucleotide sequences encoding VHH were amplified from cDNA samples by one-step RT-PCR. 700 bp amplicons, specifically amplified from the cDNA of IgG2 and IgG3 present in the sample, were isolated from the agarose gel and subsequently used as a template for nested PCR. Then the PCR products were digested with SfiI and BstEII and ligated into the corresponding restriction sites of the pAX50 phagemid vector. Stitched

- 29 046752 mixtures were electroporated into Escherichia coli TG-1. The resulting pool of transformants constituted the genetic diversity of the phage display library.

pAX50 is an expression vector derived from pUC119 containing the ampicillin resistance gene and promoter/ac followed by the pIII protein signal peptide coding sequence in frame with the downstream VHH domain insertion site. In frame with the sequence encoding the VHH domain, the vector encodes the C-terminal Myc, a hexahistidine tag, and the coliphage pIII protein. After infection of a library of E. coli TG-1 clones with a phage helper, the presence of pAX50 allows the production of phage particles in these clones, producing individual VHH domains as a protein fused to the pIII protein.

Selection:

For selection, libraries of VHH-domain phagemids were created and used. Given the very high cross-species homology of the proteins (human and llama LRP5), it was unclear whether the immune response induced in llamas would provide sufficient VHH domain diversity. Therefore, during selection, two synthetic libraries were used in parallel with the immune libraries.

The following different strategies were used during selection:

Modification of the source species (i.e., tools derived from human and mouse LRP5) to enhance the chances of identifying cross-reactive VHH domains of human/mouse LRP5 species, e.g., screening against libraries from llamas immunized with human LRP5 with HEK293 stably expressing mouse LRP5 or the use of HEK293 cell lines expressing both murine and human LRP5 during selection on synthetic libraries (the murine cross-reactivity of such an LRP5 antagonist provides an opportunity to assess efficacy, i.e., tumor growth inhibition, and safety profiles necessary to evaluate the therapeutic window, in the same preclinical models (i.e., xenograft mouse tumor models)).

Solution selections using recombinant LRP5 protein to retain epitopes in their native conformation: An additional obstacle was that recombinant LRP5 protein was found to lose proper folding when directly applied to ELISA binding plates. Therefore, the recombinant protein was biotinylated and, after confirmation of proper folding in functional assays, used for in-solution selection.

Screening using cells overexpressing LRP5 to ensure the natural conformation of the receptors. This unexpectedly turned out to be an important technique, especially necessary to improve the selection of structures that bind to the LRP5 domain of the Wnt3a class, since functional data on the recombinant protein showed the lack of correct folding of the Wnt3a-binding epitope.

Negative selection using LRP6-overexpressing cells to identify LRP5-selective structures.

Example 3: Screening for monovalent VHH.

After selection, clones were grown in 96-well deep-well plates (1 ml volume) and VHH expression was induced by the addition of IPTG. According to standard procedures described, for example, in WO2011/107507, periplasmic extracts of individual clones were prepared and screened for binding to human LRP5. Periplasmic extracts were initially screened by recombinant and LRP5 ELISA binding assay, which is a sensitive, interference-tolerant, and high-throughput assay compared to FACS-based binding assay. Following purification, VHHs identified by ELISA assays were further characterized by FACS binding assay to confirm binding of purified VHHs to the LRP5 receptor in its native conformation.

Good correlation between ELISA and FACS binding assays is generally expected. However, in this case, VHH that binds best to LRP5 in the ELISA assay (ie, with high affinity for the recombinant extracellular domain of LRP5) showed no or very weak binding to human LRP5 in the FACS binding assay. The use of different coating buffers (Dulbecco's phosphate-buffered saline and bicarbonate buffer) and blocking solutions in the ELISA (Marvel and BSA) did not eliminate the observed discrepancy. Instead, it was found that very weak ELISA binders showed high affinity for LRP5 in FACS binding assays using HEK293 cells expressing LRP5. These additional data and experiments made it possible to select high-affinity binders that recognize the natural conformation of the two receptors. Moreover, it was confirmed that these high-affinity binders recognize a conformation-dependent epitope in the LRP5 protein, rather than a linear epitope. These additional unconventional data and experiments made it possible to select therapeutically important LRP5-binding substances that should have high affinity for LRP5 expressed at the plasma membrane in its native conformation.

Therefore, despite (i) the low throughput of FACS binding assays, (ii) the less robust assay design, and (iii) the above-described difficulties arising from loss of recombinant protein expression when passaging cells overexpressing LRP5, these assays are still useful

- 30 046752 was used for subsequent selection and characterization of high-affinity VHH binders. Briefly, cells were incubated with dilutions of purified VHH (1:5 serial dilutions from 1 μM to 1 pM final concentration) for 1.5 h at 4°C on a plate shaker. After washing the cells 5 times with FACS buffer consisting of 1x phosphate-buffered saline (PBS) + 10% fetal bovine serum (FBS) + 0.05% sodium azide, they were incubated for 30 minutes to 1 hour at 4°C with polyclonal a mouse antibody that binds to the VHH framework region and therefore binds to all tested LRP5 binders.

After washing the cells three times with FACS buffer, they were incubated for 30 minutes to 1 hour at 4°C with a labeled secondary antibody (anti-mouse, PE), followed by three washes with FACS buffer. Fluorescence was measured using FACS Array (BD).

Based on FACS binding data and sequence analysis in immune and synthetic libraries, a total of approximately fifty LRP5-selective VHH families/clusters have been identified. Examples of their representatives are shown and defined by their sequence below. VHH was expressed in E. coli and purified. If expression in E. coli was insufficient, VHH was produced in Pichia pastoris. A brief description of VHH expression and purification is given below.

General expression of VHH in E. coli:

The coding sequences were inserted into the expression vector pAX100 and expressed in E. coli as c-Myc proteins with a hexahistidine tag. E. coli TG-1 cells containing the desired VHH constructs were grown (37°C, 250 rpm) in shake flasks in TB medium supplemented with kanamycin and induced by adding 1 mM IPTG for expression. After centrifugation of cell cultures, periplasmic extracts were prepared by freezing-thawing the sediment and resuspending it in PBS.

General expression of VHH in Pichia (P.) pastoris:

The coding sequences were inserted into the pAX159 expression vector and expressed in P. pastoris as c-Myc proteins with a hexahistidine tag. P. pastoris X-33 cells containing the desired VHH constructs were grown (30°C, 250 rpm) in BGCM medium (Buffered Glycerol-Complex Medium; Invitrogen). On the third day, the medium was changed to BMC (Buffered Methanol-Complex Medium, complex medium with buffered methanol; Invitrogen) and the culture was grown further, regularly inducing it by adding 0.5 vol.% methanol (100%). After centrifugation of cell cultures, the supernatant (containing secreted VHH) was collected.

Cleaning VHH:

Hexahistidine-tagged VHH was purified on a Tecan EVO150 by immobilized metal affinity chromatography (RoboColumns 100ul Nickel Sepharose™ 6 FF, Atoll), eluted from the column with 250 mM imidazole and then desalted to PBS. The integrity and purity of VHH was confirmed by SDSPAGE and/or Western blotting with detection with antibodies against Myc and VHH.

Example 4: In vitro characterization of purified monovalent VHH.

After screening, purified VHH with high affinity for LRP5-expressing cells was characterized using a series of functional and biophysical assays described below:

4.1 LRP5 selective binding efficiency, cross-reactivity: FACS-based competition assay with DKK1.

During the characterization of LRP5 selective monovalent VHHs, it was observed that the data obtained in the FACS binding assay did not always correlate with the strength observed in the Wnt1 and Wnt3a gene reporter assays, most likely due to the high dissociation rate of some VHHs. Therefore, there was a need to develop this complementary assay (i.e., FACS for competition with DKK1), which was shown to be more reliable in terms of selectivity and determination of binding strength, including binding to LRP5 and lack of binding to LRP. The goal was to select functional VHHs that selectively bind to LRP5 with a lack of binding to LRP6 detectable at concentrations as low as 1 μM. Therefore, the identified functional VHHs to Wnt1 and Wnt3a characterized FACS to compete with DKK1 as follows:

HEK293 cells stably overexpressing human LRP5 or human LRP6 were used for FACS-based competition analysis of DKK1. Human recombinant DKK1 (rhDKK1 - R&D Systems, part number 5439-DK/CF) was added to the cells at a constant final concentration of 1 nM. Cells were incubated with dilutions of rhDKK1 and LRP5 binder (serial dilutions of purified VHH 1:5) for 1.5 hours at 4°C on a plate shaker. After washing the cells three times with FACS buffer, they were incubated with biotinylated goat anti-human DKK1 antibody (R&D Systems, cat. BAF1096) for 30 minutes at 4°C on a plate shaker. After washing the cells three times with FACS buffer, they were incubated with streptavidin PE (BD Biosciences, cat. 554061) for 30 minutes to 1 hour at 4°C on a plate shaker in the dark. Cells were washed twice with FACS buffer, fluorescence was measured using FACS Array (BD), and mean channel fluorescence (MCF) values were noted.

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It is believed that LRP5-selective VHHs will compete with human DKK1 for binding to HEK293 cells overexpressing human LRP5, but will not compete, or with extremely low efficiency (>1 μM), for binding to HEK293 cells overexpressing human LRP6 (and vice versa the same will apply to LRP6 specific VHH). In contrast, LRP5/LRP6 cross-reactive VHHs will compete with human DKK1 for binding to HEK293 overexpressing human LRP5 as well as for binding to HEK293 overexpressing human LRP6. This experiment demonstrated that LRP5-selective VHHs present compete with human DKK1 for binding to HEK293 cells overexpressing human LRP5 (i.e., a decrease in MCF value with increasing binder concentration, where complete inhibition of DKK1 binding corresponded to values <60 MCF at the highest tested concentration).

4.2 Interspecies cross-reactivity: mouse and cynomolgus macaque.

To determine whether the selected LRP5 panel of selective VHHs was able to bind to LRP5 from mouse and cynomolgus monkey, the following FACS was performed to compete with DKK1:

Serial dilutions of VHH were incubated with HEK293 cells stably expressing mouse LRP5 or macaque LRP5 in the presence of 1 and 0.3 nM hDKK1 (concentration below EC ₅₀ for mouse and macaque, respectively). Binding of DKK1 to cells was detected using a biotinylated anti-DKK1 antibody with streptavidin-PE as a secondary detector as described above. As a result, they were able to demonstrate such cross-reactivity.

4.3 Epitope-specific sorting.

Sorting experiments were performed on the strongest Wnt1 LRP5 signaling blocking selective VHHs to identify different epitope groups. Specifically, individual VHHs were tested for their ability to compete with other biotinylated VHHs (called reference VHHs) for binding to LRP5 receptors using FACS-based assays. Serial dilutions of individual VHHs were incubated on HEK293 cells stably expressing human LRP5 along with 200 pM or 500 pM biotinylated reference VHHs (concentrations below the EC50 value). Binding of biotinylated reference VHH to cells was determined using streptavidin-PE. Competition of VHHs with reference VHHs for binding to LRP5 is manifested by a decrease in fluorescence measured by FACS Array.

As a result of these experiments, Wnt1 blockers and Wnt3 blockers were divided into two and six groups, respectively.

4.4 Wnt1 and Wnt3a reporter gene assays.

The ability of LRP5-selective VHHs to suppress Wnt signaling was tested by functional analysis of Wnt1 and Wnt3a. In this regard it was not possible to use an established protocol, several attempts had to be made to establish biochemical functional assays such as Wnt1/Wnt3a blockade assays - LRP5: To all the difficulties with recombinant LRP5 proteins (see example 1), there is no functional recombinant Wnt1 ligand , not to mention commercially available. Wnt proteins contain many conserved cysteines and are modified with a monounsaturated fatty acid (palmitinoleic acid) attached to a conserved serine. These post-translational modifications are required for efficient Wnt signaling and secretion. Structural analyzes showed that one of the domains containing the lipid palmitinoleic acid is required for binding to the Frizzled receptor, leading to a conformational change that allows the interaction of Wnt ligands with the LRP5 receptor on the cell surface. It turned out that such post-translational modifications are necessary for functional studies with this protein, but at the same time, such lipid post-translational modifications greatly complicate the expression and purification of these proteins (due to poor solubility). Therefore, this became a big obstacle for biochemical analyses.

Therefore, a cell-based functional assay was developed to characterize purified VHH: the beta-lactamase reporter gene Wnt assay. Specifically, to suppress the Wnt1 pathway, CellSensor LEF/TCF-bla FreeStyle 293F cells (Invitrogen, cat. K1677) were transfected with human Wnt1, and clones stably overexpressing human Wnt1 were selected. To test suppression of the Wnt3a pathway, CellSensor LEF/TCF-bla FreeStyle 293F cells were generated stably overexpressing human Wnt3a, and human LRP6 was transiently knocked out using siRNA two days before treatment with LRP5-selective VHHs (SMARTpool, ON-TARGETplus siRNA, Dharmacon , cat. L-003845-0010) in accordance with the manufacturer's instructions.

The CellSensor® LEF/TCF - bla FreeStyle™ 293 cell line contains a beta-lactamase reporter gene under the control of the Wnt-inducible LEF/TCF promoter, which is stably integrated into FreeStyle™ 293 cells (Invitrogen). Thus, expression of Wnt1 or Wnt3a in these cells leads to constitutive expression and hence enzymatic activity of beta-lactamase. Therefore, treatment with functional LRP5 selective VHH should lead to inhibition of the Wnt1 or Wnt3a pathway, which will lead to suppression of beta-lactamase enzymatic activity.

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For the 1E06/ml assay, cells overexpressing Wntl or Wnt3a were seeded into a 384-well tissue culture plate and incubated overnight at 37°C. The next day, serial dilutions of various LRP5 selective VHHs were prepared and added to the cells in the presence of LiCl at a final concentration of 10 nM. As a positive control, DKK1 was added to the cells at a final concentration of 200 nM. Treatment with DKK1 resulted in complete inhibition of the Wnt1 and Wnt3a pathway and therefore complete suppression of beta-lactamase enzymatic activity. Cells were incubated overnight at 37°C. The next day, beta-lactamase enzymatic activity was measured according to the manufacturer's instructions (Invitrogen, cat. K1085). For fluorescence emission, values were obtained at 460 nm and 530 nm using a standard fluorescence plate reader, and the emission ratio at 460/530 nm was plotted against the indicated treatment. Efficiency was calculated relative to the positive control (DKK1 at a final concentration of 200 nM).

Two Wnt1 blockers have been identified. A total of eight Wnt3a blockers showed good activity (IC ₅₀ below 100 nM) and complete effectiveness.

4.5 Wnt1 and Wnt3a phosphorylation assays.

The most potent and effective samples from each group of Wnt1 blockers, as well as the most potent and effective Wnt3a blockers, were subsequently tested in Wnt1- and Wnt3a-dependent LRP5 phosphorylation assays. Phosphorylation assays used Cellsensor LEF/TCF 293F cells from Invitrogen (cat. K1677) co-transfected with expression vectors encoding either Wntl or Wnt3a. Since formation of the Wnt-Frizzled-LRP5 complex results in phosphorylation of LRP5 and subsequent downstream signal transduction, quantitation of phosphorylation can be used to measure such transmission. To obtain LRP5-specific readouts, cells were lysed and immunoprecipitated with an LRP5-selective antibody (directed against the intracellular domain of the receptor). Phosphorylated LRP5 was detected by Western blotting using a polyclonal anti-phospho-LRP6 antibody (Serl490) (Cell Signaling Technology), which cross-reacts with phosphorylated LRP5 protein. A selected panel of purified Wnt1 and Wnt3a blocking VHHs, containing at least one VHH representative from each group, was tested at final concentrations ranging from 10 to 100 nM. Specifically, cells were incubated overnight in the presence of VHH blockers before cell lysis and LRP5 immunoprecipitation. The effectiveness of VHH blocking Wnt1 or Wnt3a in blocking LRP5 phosphorylation was calculated by quantification of Western blot bands compared to the positive control (DKK1, final concentration 1 μM).

4.6 Biophysical characteristics.

LRP5 selective VHH domains were further assessed for expression and purification in E. coli and Pichia pastoris as described in Example 3. In particular, the expression yield of monovalent VHH panel samples was considered acceptable if it was above 0.1 mg/L. Selected LRP5 selective VHHs showed expression in the range of 0.3–22.6 mg/L in E. coli and higher in Pichia pastoris (>1 mg/L). Expression was assessed by SDS-PAGE analysis.

Thermal stability of monovalent LRP5 selective VHHs was determined by fluorescence thermal shift (ATS) assay using a Lightcycler (Roche). VHH was incubated at different pH values in the presence of Sypro Orange using a temperature gradient. Following heat-induced fold unfolding, hydrophobic regions of the proteins are exposed to which Sypro Orange binds, resulting in an increase in fluorescence intensity (Exb/Em = 465/580 nm). The inflection point on the first derivative of the fluorescence intensity curve serves as a measure of the melting temperature (Tm). In all VHH, Tm increased with increasing pH and leveled off at pH 6, which is a typical Tm pattern for VHH. For LRP5 selective VHH blockers Wnt1 and VHH blockers Wnt3a, the average value was greater than 68°C at pH 7.

The potential for aggregation and multimerization of LRP5 selective VHHs was studied using analytical size exclusion chromatography. For this purpose, 8 μg of purified VHH sample at a concentration of 0.5 mg/ml was injected using Dionex Ultimate 3000 equipment into an Agilent SEC-3 column. L-arginine buffer (10 mM phosphate, 300 mM Arg-HCl, pH 6.0) was used as the mobile phase at a flow rate of 1 ml/min. None of the LRP5-selective VHHs had major problems with aggregation during SEC analysis, with profiles indicating more than 95% of the monomers in most samples.

Example 5: Development and characterization of biparatope constructs with extended half-life.

LRP5 selective VHHs for Wnt1 and Wnt3a were used as building blocks to create the biparatope construct depicted in Fig. 1. Gene fusion with VHH binding to serum albumin was used as a method to extend the half-life. Three building blocks (Wnt1-blocker, Wnt3a-blocker and albumin-binding) were connected by a flexible linker. VHH was produced in P. pastoris and purified as described in Example 3. The resulting constructs, i.e. biparatopic LRP5 selective VHH constructs were inserted into the pAX159 expression vector for P.

- 33 046752 pastoris in the form of VHH constructs with a cMyc-hexahistidine tag at the C-terminus, according to standard procedures described, for example, in WO2012/131078. Different orientations of building blocks and different linkers, especially GS linkers, were explored. Based on homology modeling data reflecting increased surface area between potential Wnt1 and Wnt3a binding sites in LRP5, a relatively long GS linker was selected. The best strength results for the combined Wnt1 and Wnt3a reporter gene assay were obtained by inserting the human serum albumin/HSA binding VHH into the middle. 35-GS^uh#P was used, and the VHH blockers Wnt1 and Wnt3a were placed in the preferred order.

To select optimal VHH binders and binder combinations, a library was created in which VHH binding human serum albumin/HSA was placed between LRP5 selective Wnt1-Wnt3a blockers. Specifically, the library used a panel of high-affinity binders with high potency and potency as measured by Wnt1 or Wnt3a assays (reporter gene and phosphorylation assays) to generate biparatope extended half-life constructs designed as shown in FIG. 1. Following expression in Pichiapastoris (as described in Example 3) and subsequent purification, biparatope extended half-life constructs were screened using Wnt1 and Wnt3a reporter gene assays (described in Example 4) in the presence of 30 μM HSA at three dilutions (1 /100, 1/1000, 1/7000) to evaluate effectiveness and relative strength. Overall, good correlation between the Wnt1 and Wnt3a reporter assay data was observed, and high efficiency was measured for a variety of binder formats. A total of ten biparatope LRP5-selective extended half-life constructs were selected for further evaluation, taking into account the efficacy in both reporter assays and the diversity of Wnt1 and Wnt3a blockers. These further evaluation analyzes are described below.

Wnt1 and Wnt3a reporter gene assays:

Wnt1 and Wnt3a reporter gene assays were performed as described in Example 4.4, in the presence of a final HSA concentration of 30 μM. Purified biparatope LRP5 selective constructs were tested at 12 dilutions starting at 2.5 μM.

Most constructs showed high potency—with IC ₅₀ values below 1 nM in Wnt3a and below 5.7 nM in Wnt1 reporter assays, respectively—and full potency in both reporter assays for LRP5-dependent Wnt signaling.

Example 6: Optimization of VHH sequences and VHH constructs.

Sequence optimization is a process in which the parent sequence is mutated to make it more similar to the human germline IGHV3-IGHJ consensus sequence. For example, specific amino acids of the framework regions (except for the so-called signature residues) are replaced with their human counterparts so as to preserve the structure, activity and stability of the protein.

These mutations can be divided into the following categories:

1. Standard: Sequence optimization at these positions should not significantly change the stability, activity or affinity of VHH, so they are all changed simultaneously to obtain the base variant.

2. Unique: It is unknown how sequence optimization at these positions will affect VHH stability, activity, or affinity, so they are examined individually based on the base case.

Distinctive residues are known to be critical to the stability, activity, and affinity of VHH and are therefore not altered.

In addition, CDR amino acids that have been experimentally shown to be susceptible to post-translational modifications (PTMs) were modified to inactivate the PTM site without affecting protein structure, activity, or stability. The most common post-translational modifications described for antibodies and VHH are listed in Table. IV below. The sensitivity of VHH to post-translational modifications was analyzed from boost studies using a number of standard conditions, including H ₂ O ₂ treatment to assess methionine oxidation, high temperature, high pH and long-term storage to assess asparagine deamidation and aspartate isomerization. The percentages of oxidation, deamidation and isomerization were measured according to standard procedures and compared with reference samples (VHH, stored at -20°C). Whole protein analysis by reverse phase chromatography (RPC) and peptide mapping by mass spectrometry (MS) were performed to identify potentially sensitive residues. If post-translational modifications were observed in VHH after the stress test, the corresponding amino acid(s) were mutated.

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Table IV

Potential post-translational modifications and potentially triggering motifs

Motive Modification M Met Oxidation N-S/G/H/N/A Deamidation of Asn D-S/G/H Isomerization of Asp N-X-S/T-X (X^P) Asn glycosylation Q/E pyroglutamate

As a result, several mutations were introduced into the above-described constructs, obtaining, among other things, the three constructs shown in Table. III above, selected for further characterization in vitro and in vivo, as shown in further examples.

Example 7: In vitro characterization of three biparatope LRP5 specific VHH constructs with extended half-life; Comparison with LRP6 binding molecules.

Following optimization of the VHH sequences, three biparatope LRP5 selective VHH constructs (see Table III above) with extended half-lives were recombinantly expressed and purified and then characterized using a series of functional and biophysical assays described below.

7.1 FACS Binding Assay.

Binding to human LRP5 and LRP6 was determined on cells by FACS analysis, as shown in Figures 2A and 2B, respectively. Specifically, binding to human LRP5 was tested in HEK293 cells stably overexpressing human LRP5. HEK293 cells stably overexpressing human LRP6 were used to bind to human LRP6. Cells were incubated with dilutions of LRP5 binders (serial dilutions of 1:5 binders corresponding to the final concentrations shown in Figures 2A and 2B) for 1.5 hours at 4°C on a plate shaker. After washing the cells 5 times with FACS buffer consisting of 1x PBS (Invitrogen, art. 141190-094) + 10% FBS (Sigma, art. F7524) + 0.05% sodium azide, they were incubated for 1 hour at 4°C C with a polyclonal mouse antibody that binds to the VHH framework region. After washing the cells three times with FACS buffer, they were incubated for 1 hour at 4°C with a labeled secondary antibody (anti-mouse, PE (115-116-071)) followed by three washes with FACS buffer. Fluorescence was measured using FACS Array (BD). Binding to human LRP5 and LRP6 at the highest concentration tested corresponded to MCF values >3000. The negative control consisted of a nontargeting binder (a VHH construct that binds to a bacterial protein that is not expressed in HEK293 cells). As shown in FIG. 2A, binding to human LRP5 corresponded to values of >3000 MCF at the highest concentrations tested of the three biparatope LRP5 selective VHH extended half-life constructs. In contrast, no binding to human LRP6 was detected (MCF values <300) as shown in FIG. 2B. These data confirm that formatted, sequence-optimized biparatope binding molecules selectively bind to human LRP5 receptors in their native conformation in a cell-based assay system. EC ₅₀ binding values for hLRP5 are given in Table. V below.

Table V

EC ₅₀ binding values for human LRP5 determined by FACS binding assays

FACS-based binding analysis F012900082 F01290135 F012900141 hLRP5, EU so (nM) 0.21 0.042 0.2

The specificity of the LRP5 selective formatted, biparatopic, sequence optimized binding molecules was compared with the previously described LRP6 binding molecules disclosed in WO2011/138391:

WO2011/138391 described multivalent antibodies that bind to LRP6 and inhibit interaction with both Wnt1 and Wnt3 ligand. These multivalent LRP6-binding antibodies are biparatopic LRP6-binding molecules consisting of an IgG antibody as a first receptor binding domain and an scFv fragment as a second receptor binding domain, wherein the IgG antibody and the scFv fragment are connected by a linker. WO2011/138391 reports that all LRP6 binding molecules have approximately equal potency in the Wnt1 and Wnt3a reporter assays (Figure 18 of WO2011/138391). Therefore, any of these multivalent molecules that bind to LRP6 can be selected for comparative experiments. Therefore, as the first connection for

- 35 046752 comparison it was decided to use construct 901 (designated MOR08168IgG1LALA 6475 scfv; also shown in Fig. 27 WO2011/138391).

WO2013/067355 shows derivatives of this construct 901. More specifically, compounds 801T and 802T are described (see information on page 132 of the specification) having two LRP6-binding scFv domains plus a half-life extending moiety. Since, apparently, 801T and 802T have the same strength and biophysical characteristics in vitro, only one of them was taken for the experiments described below - the 802T variant.

The binding affinities of the biparatopic LRP5 specific VHH extended half-life constructs were compared to the MOR08168IgG1LALA 6475 scfv biparatopic LRP6 binding molecule using the FACS binding assays described in Example 3. As discovered in the practice of the present invention and shown in FIG. 2A and FIG. 2B, MOR08168IgG1LALA 6475 scfv biparatopic LRP6 binding molecule binds to both human LRP5 and human LRP6, corresponding to MCF values >3000 at the highest concentration tested; In contrast, the biparatopic LRP5 specific VHH extended half-life constructs of the present invention bind only to human LRP5 without detectable binding to human LRP6 (MCF values <300). These data suggest that the biparatopic LRP5 specific VHH extended half-life constructs have different binding specificities (LRP5 selective binders) when compared to the previously described LRP6 binding molecules described in WO2011/138391, which were found to be LRP5/LRP6 cross-reactive binding moieties when tested in practice of the present invention.

7.2 FACS-based competition analysis of DKK1.

The potency and efficiency of the LRP5 selective VHH constructs were further analyzed using a FACS-based DKK1 competition assay as described in Example 4.1. HEK293 cells stably overexpressing human or mouse LRP5 were incubated with serial dilutions of LRP5 selective VHH constructs (1:5 serial dilutions corresponding to the final concentrations shown in Fig. 3A and Fig. 3B). LRP5-selective VHHs competed with human DKK1 for binding to HEK293 cells overexpressing human LRP5 as well as for binding to HEK293 cells overexpressing murine LRP5, as shown in Figures 3A and 3B, respectively. Complete inhibition of DKK1 binding was achieved at the highest concentrations tested (>1 μM) and this corresponded to MCF values <100. This experiment demonstrated that the presence of LRP5-selective VHHs competes with human DKK1 for binding to HEK293 cells overexpressing human LRP5 as well as cells overexpressing murine LRP5 (i.e., a decrease in MCF value with increasing binder concentration, where complete inhibition of binding DKK1 corresponded to MCF values <100 at the highest concentration tested). Therefore, these LRP5 selective VHHs are murine cross-reactive. The data strengthen the position that formatted, sequence-optimized biparatope binding molecules bind to human/mouse LRP5 receptors in their native conformation.

7.3 Combined Wnt1 and Wnt3a Reporter Gene Assay The potency and potency of formatted, sequence-optimized biparatope LRP5 binding molecules were analyzed using the Wnt1 and Wnt3a reporter gene assay as described in Example 4.4. In fig. 4A, fluorescence ratio value [460/535 nm] designated as Wnt1, which corresponds to activation of the Wnt1 pathway determined based on Wnt1 overexpressing cells; complete inhibition of the Wnt1 pathway corresponds to a fluorescence ratio [460/535 nm] <0.8. In fig. 4B, fluorescence ratio value [460/535 nm] designated as Wnt3a, which corresponds to activation of the Wnt3a pathway determined based on Wnt3a overexpressing cells; fluorescence ratio value [460/535 nm], indicated as the baseline in Fig. 4B corresponds to complete inhibition of the Wnt3a pathway as determined by positive control treatment (DKK1; final concentration 200 nM). As shown in FIG. 4A and 4B, complete inhibition of both Wnt1 and Wnt3a pathways is achieved by treatment with three LRP5 selective, formatted, sequence optimized biparatope binding molecules. In addition, significant strength was also observed, as shown in Table 1 below for the IC ₅₀ values.

Table VI

IC ₅₀ values of Wnt1 and Wnt3a pathway inhibition

Wntl and Wnt3a gene reporter analysis F012900082 F01290135 F012900141 1C ₅₀ (nM) Wntl 0.37 0.37 0.3 1C ₅₀ (nM) Wnt3a <0.1 <0.1 0.05

Example 8: Effect of three biparatope LRP5 selective VHH extended half-life constructs on Wnt signaling and viability of cancer cell lines.

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The ability of biparatopic LRP5 selective VHH constructs with extended half-life to suppress active Wnt signaling was further characterized using cell lines with active Wnt signaling described previously (Bafico et al. An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell 2004 ;6(5):497-506; DeAlmeida et al. The soluble wnt receptor Frizzled8CRD-hFc inhibits the growth of teratocarcinomas in vivo. Cancer Res. 2007;67(11):5371-9); Akiri et al. Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma. Oncogene. 2009; 28(21):2163-72). Briefly, the Wnt signaling-active cancer cell line PA-TU-8988S was seeded in 12-well plates and treated with LRP5 selective VHH constructs at a final concentration of 1 μM overnight. The ability to suppress Wnt signaling was detected by inhibiting the expression of Axin2 mRNA, an endogenous Wnt target gene. qPCR expression analysis was performed using standard RNA techniques:

RNA extraction was performed using the QIAGEN RNeasy Mini Kit according to the QIAGEN protocol; cDNA synthesis - using Superscript VILO cDNA Synthesis Kit (Invitrogen, art. 11754050) and qPCR - using TaqMan Gene Expression Assay with Axin2 TaqMan primers/probes (Hs00610344_m1 AXIN2 FAM, Life Technologies), as well as with eukaryotic 18s endogenous control VIC- MGB (4319413E1307061, Applied Biosystems).

As shown in FIG. 5 (right panel), PA-TU8988S cancer cells treated with the biparatopic LRP5 VHH-selective extended half-life construct showed a significant reduction in relative Axin2 mRNA levels (i.e., normalized to endogenous control) compared to untreated cells or cells treated with a nontargeting VHH construct (negative control). These data demonstrate the ability of a biparatope LRP5 selective VHH construct with an extended half-life to inhibit Wnt signaling in cancer cell lines with active Wnt signaling. In addition, the effect of blockade of Wnt signaling on cell viability was examined in the cancer cell line PA-TU8988S, which was previously reported to depend on active Wnt signaling for proliferation (Jiang et al. Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma Proc Natl Acad Sci USA 2013; 110(31): 12649-54. Cell viability was measured using the Alamar Blue assay (Invitrogen, cat. no. DAL1100) after treatment for days with the biparatope LRP5-specific VHH extended half-life construct (final concentration 1 μM) or the non-targeting VHH construct (negative control) or doxorubicin. (final concentration 1 µM; positive control). As shown in FIG. 5 (left panel), PA-TU8988S cancer cells treated with the biparatope LRP5 selective VHH extended half-life construct showed a significant reduction in the percentage of viable cells (>75% reduction) compared to untreated cells or cells treated with a negative control that had no effect influence on cell viability. The greatest reduction in cell viability corresponded to treatment with a chemotherapeutic agent such as doxorubicin (>80% reduction). These data demonstrate the ability of biparatope LRP5 selective VHH constructs with extended half-life to inhibit the proliferation of cancer cells that are dependent on active Wnt signaling.

Example 9: In Vivo Efficacy.

LRP5 selective biparatope VHH constructs/binding molecules with extended half-life were further characterized in vivo in a Wnt-driven tumor model. Experiments were carried out to determine whether these binding molecules suppress tumor growth in vivo. Transgenic expression of Wnt ligands by the murine mammary tumor virus LTR enhancer (MMTV promoter) in mice results in extensive ductal hyperplasia followed by mammary adenocarcinoma in transgenic (TG) mice up to 6 months of age. These breast tumors are caused by glucocorticoid-induced overexpression of Wnt ligands and have characteristics similar to TNBC tumors, including expression of epithelial and mesenchymal markers (basal-like phenotype) and active Wnt signaling, assessed by the subcellular localization of beta-catenin. In particular, mammary tumors in MMTV-Wnt-1 transgenic mice are dependent on Wnt1. It has been reported that blocking Wnt activity with a soluble Wnt receptor incorporating the cysteine-rich domain (CRD) Frizzled8 fused to a human Fc domain (F8CRDhFc) (DeAlmeida et al. The soluble wnt receptor Frizzled8CRD-hFc inhibits the growth of teratocarcinomas in vivo. Cancer Res. 2007;67(11):5371-9), suppresses tumor growth in vivo. Therefore, tumors isolated from MMTV-Wnt1 transgenic mice were subcutaneously passaged in the form of tumor pieces into athymic mice for 2 to 5 passages before the start of the efficacy experiment. Between days 14 and 21 postimplantation, when tumors had reached a mean volume of approximately 150,300 mm ³ , mice were randomized into groups of 7 and treated with i.v. compounds. LRP5 selective biparatope VHH extended half-life constructs were administered to mice IV three times (F012900082) or twice weekly (F012900141), at the doses shown in FIG. 6A for F012900082 and

- 37 046752 in Fig. 6V for F012900141. Tumor volume (left panels) and body weight (right panels) were monitored during the efficacy trial, and median tumor volumes are shown in Figures 6A and 6B. At the end of the experiment, tumor growth inhibition (TGI) was determined by efficacy. Specifically, TGI was determined for each treatment group compared to the control group (mice treated with histidine buffer - buffer with 20 mM histidine and pH 6.5). In addition, at the end of the efficacy trial, gastrointestinal (GI) histopathological analysis (G&E staining of sections of the GI tract from the duodenum to the rectum) was performed to assess the potential toxicity of LRP5 antagonists. Tumor growth inhibition (TGI), the result of GI histopathological analysis at the end of the in vivo efficacy study, mortality corresponding to the number of mice that had to be sacrificed due to significant loss of body weight (>18% body weight loss compared to the beginning of the efficacy study) , as well as the number of tumor regressions (when the tumor volume at the end of the experiment is less than the tumor volume at the beginning of treatment), for each treatment group are shown in Tables VIIA and VIIB, and regarding the experiments and data are also presented in Figures 6A and 6B, respectively.

Table VIIA

Efficacy of F012900082 in vivo when administered intravenously three times a week

Dose [mg/kg] TGI [%] Regressions [x/7] Mortality [x/7] G istopathological assessment of the gastrointestinal tract Control Histidine buffer - - - - F012900082 50 128 6 0 Without features

The results of this experiment are also shown in Fig. 6A.

Table VIIB

Efficacy of F012900141 in vivo when administered intravenously twice a week

Dose [mg/kg] TGI [%] Regressions [x/7] Mortality [x/7] G istopathological assessment of the gastrointestinal tract Control Histidine buffer - - - - F012900141 50 118 7 0 Without features

Dose [mg/kg] TGI [%] Regressions [x/7] Mortality [x/7] G istopathological assessment of the gastrointestinal tract 15 115 7 0 Without features 5 98 0 0 not def.

The results of this experiment are also shown in Fig. 6B.

As can be understood from Figures 6A-6B and Tables VIIA and VIIB above, treatment with LRP5 selective biparatope VHH constructs with an extended half-life (F012900082 at a dose of 50 mg/kg on a 3 times/week schedule and F012900141 at a dose of 15 and 50 mg/ kg on a 2 p/week schedule) led to tumor regression (that is, tumor growth inhibition (TGI) >100%, which corresponds to tumor reduction; a decrease in tumor volume at the end of the experiment compared to the tumor volume at the beginning of the experiment), with There were no significant changes in body weight (<10%) and no abnormalities were noted on histopathological analysis of the gastrointestinal tract, indicating that treatment was well tolerated.

Tumor shrinkage (ie, tumor regression) is undoubtedly a desirable therapeutic effect (ie, efficacy) in the treatment of cancer patients. Indeed, in clinical trials, treatments that induce tumor regression leading to pathologic complete response (pCR) are positively associated with significant improvements in progression-free survival and overall survival in cases of large unmet medical need, such as in breast cancer. In addition, it is also clinically significant that the treatment is well tolerated with no non-tumor-targeted side effects at effective dosing levels.

As a comparison, it was further examined whether MOR08168IgG1LALA 6475 scfv biparatope LRP6 binding molecule could provide the same beneficial effect. For this purpose we carried out

- 38 046752 the following in vivo tolerability study in mice: Compound MOR08168IgG1LALA 6475 scfv was administered i.v. at a dose of 3 mg/kg twice a week (2qw); this is the same dose and protocol for which in vivo efficacy was found in WO2011/138391 in a xenograft tumor model as described in FIG. 22 of this document. The first treatment with MOR08168IgG1LALA 6475 scfv was carried out on day 1, and starting on day 6, mice showed significant loss of body weight. At day 10, some mice treated with MOR08168IgG1LALA 6475 scfv showed significant body weight loss (>10%). On day 11, the mice were sacrificed, and histopathological analysis of the gastrointestinal tract showed inflammation with erosion in the colon and cecum of the mice. These data indicate that MOR08168IgG1LALA 6475 scfv is not tolerated at the effective dose/protocol. Thus, biparatopic LRP5 selective VHH constructs with extended half-life have an advantage regarding the therapeutic window; that is, they cause tumor regression without significant changes in body weight (<10%) and without features that would be detected by histopathological analysis of the gastrointestinal tract.

Example 10: Inhibition of the Wnt signaling pathway in vivo.

To further evaluate the effect of LRP5 selective biparatope VHH constructs/extended half-life binding molecules on Wnt signaling, tumors were isolated at the end of the efficacy assay described in Example 9. Specifically, tumors were isolated 24 hours after the last injection of compounds (F012900082 at a dose of 50 mg/kg or F012900141 at a dose of 15 mg/kg) or control treatment. Inhibition of Wnt signaling was determined by decreased expression of Axin2 mRNA as well as additional Wnt target genes: RNF43 and Notum in tumors treated with F012900082 and F012900141, respectively, analyzed as described in Example 8. TaqMan primers/probes were used for Notum and Notum assays. RNF43, (Mm01253278_ml Notum FAM and Mm00552558_m1 RNF43 FAM Life Technologies). The fold change in Axin2, RNF43, or Notum mRNA expression compared to the control group is shown in Fig. 7 (left side) for in vivo effectiveness using F012900082 (cf. FIG. 6A) and FIG. 7 (right panel) for in vivo effectiveness using F012900141 (cf. FIG. 6B).

As can be seen from Fig. 7, observed a significant decrease in the expression of Axin2, RNF43 and Notum mRNA in tumors treated with LRP5 selective binding molecules compared to the control group. These results suggest that LRP5 selective binding molecules are able to inhibit tumor growth by suppressing Wnt signaling in tumor cells.

Example 11: Industrial production method.

11.1 Fermentation: Any of the polypeptides listed in Table III above can be expressed in the cytoplasm of various E. coli strains such as W3110, TG1, BL21, BL21(DE3), HMS174, HMS174(DE3), MM294, under the control of an inducible promoter. This promoter can be selected from lacUV5, tac, T7, trp, T5, araB. Culture media are preferably fully specified according to Wilms et al., 2001 (Wilms B., Hauck A., Reuss M., Syldatk C., Mattes R., Siemann M. and Altenbuchner J.: HighCell-Density Fermentation for Production of L-N-Carbamoylase Using an Expression System Based on the Escherichia coli rhaBAD Promoter. Biotechnology and Bioengineering, 73: 95-103 (2001), DeLisa et al., 1999 (DeLisa M. P., Li J. C., Rao G., Weigand W. A. and Bentley W. E.: Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor Biotechnology and Bioengineering, 65: 54-64. (1999)) or equivalent. However, supplementing the medium with amino acids such as isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, or complex components such as soy peptone or yeast extract, may be beneficial. The fermentation process was carried out in a semi-continuous manner. Conditions: Temperature 3040°C, pH 6-7.5, dissolved oxygen more than 20%. After consuming the initial carbon source, the culture is fed with the above (or equivalent) nutrient medium. When 40-90 g/l of dry cell mass accumulates in the fermenter, the culture is induced with an appropriate inducer corresponding to the promoter system used (eg, IPTG, lactose, arabinose). Induction can be carried out as a pulsed full induction or as a partial induction, supplying the appropriate inductor to the fermenter over a long period of time. The production phase must last at least 4 hours. Cells are separated by centrifugation in rotary centrifuges, tube centrifuges or plate separators, and the culture supernatant is drained.

11.2 Purification: The E. coli cell mass is resuspended in 6-8 times the amount of lysis buffer (phosphate or Tris buffer, pH 7-8.5). Cell lysis is preferably accomplished by high-pressure homogenization followed by removal of cell debris by centrifugation in rotary, tubular, or disc separator centrifuges. The supernatant containing the target protein is optionally filtered through a 0.22-10 µm filter and separated by cation exchange chromatography (e.g. Toyopearl MegaCap® II SP-550EC, Toyopearl GigaCap S-650M, SP Sepharose BB, SP Sepharose FF or S HyperCel™ ) at pH 7-8.5. Elution is carried out with a linearly increasing gradient of NaCl at pH 7-8.5. Fractions containing the target protein are pooled and then incubated with 5-10 mM DTT to prevent dimerization or aggregation mediated by free cysteine residues

- 39 046752 kami. After further addition of 0.8-1 M ammonium sulfate or 2-3 M NaCl, the solution is separated by hydrophilic chromatography (e.g. Phenyl Sepharose HP, Phenyl Sepharose FF, Butyl Sepharose HP, Butyl Sephrose FF, Butyl Toyopearl 650 (S,M, C), Phenyl Toyopearl 650 (S,M,C)) at pH 7-8.5. Elution is carried out with a linearly decreasing gradient of ammonium sulfate or NaCl at pH 7-8.5 in the presence of 5 mM DTT. Fractions containing the target protein with a minimum purity of 90% are pooled and desalted by diafiltration in the presence of 5 mM DTT, followed by concentration to approximately 5 mg/ml. Then refolding is performed by diluting the protein solution 1:5-1:20 with 50 mM Tris, 150 mM NaCl, 4 mM cystamine, 10 mM CHAPS at pH 8.5 to a final protein concentration of 0.25-1 mg/ml. The refolding solution is incubated with stirring for 12-36 hours at room temperature and then separated by cation exchange chromatography (e.g. SP Sepharose FF, SP Sepharose HP, Toyopearl SP-650 (S, M, C)) at pH 7-8, 5. Elution is carried out with a linearly increasing gradient of NaCl at pH 7-8.5. Fractions containing monomeric target protein are pooled and incorporated into 25 mM sodium phosphate and 220 mM endotoxin-free trehalose at pH 7.5 by diafiltration. The solution is sterilized by filtration and stored at 2-8°C.

Example 12: Pharmaceutical preparation for subcutaneous administration.

Any of the above-described biparatope polypeptide constructs according to the invention can be selected for the production of a pharmaceutical preparation for subcutaneous administration with the following composition:

Drug substance: 100 mg/ml (from 1 to 3 nmol/ml)

Acetate buffer: 25 mM

Trehalose: 220 mM

Tween-20: 0.02%

The medicinal substance is added to the solution of the above composition, sterilized and stored at 2-8°C.

Example 13: Pharmaceutical use in humans.

The solution prepared in Example 11.2 is administered to a patient in need, such as a person with cancer sensitive to Wnt signaling inhibitors, by intravenous infusion (at a dose of 100 to 200 mg) at intervals of two to four weeks.

Example 14: Effect of inhibition of Wnt3a signaling on the release of pro-inflammatory cytokines by dendritic cells in an ex-vivo assay.

PBMCs were taken from healthy donors with their informed consent. Monocyte-derived human dendritic cells (Mo-DCs) were generated as follows: PBMCs were cultured in X-VIVO medium supplemented with 50 ng/ml GM-CSF and 50 ng/ml IL-4. After culturing for 24 hours, the supernatant was carefully removed and replaced with X-VIVO medium supplemented with the same GM-CSF and IL-4. On the fourth day, an aliquot of cells was selected for FACS analysis of LRP5 and LRP6 expression, and the supernatant of the remaining cells was carefully removed and replaced with X-VIVO medium in the presence of LPS alone or in combination with human Wnt3a or with human Wnt3a and LRP5 selective binding molecules. The next day, supernatants were collected and subjected to TNF-alpha ELISA according to the manufacturer's instructions.

FACS analysis of differentiated dendritic cells (DCs) was performed as described in Example 7.1 using the following antibodies:

LRP5 specific monoclonal antibody: mouse IgG1 anti-human LRP5 - clone 1E9 (Sigma No. WH0004041M1) at serial dilutions of 1:100 from 1 mg/ml to 10 μg/ml.

LRP6 specific monoclonal antibody: mouse IgG2a anti-human LRP6 (R&D no. Mab1505); at serial dilutions of 1:50 from 500 µg/ml to 10 µg/ml).

Control: mouse IgG2a isotype control (Dako No. X0943); at serial dilutions of 1:10.

Secondary labeled polyclonal antibody: goat anti-mouse IgG/Fc - PE (Dianova No. 115115-164); dilution 1:100.

As shown in FIG. 8 (left panel), FACS analysis of differentiated DCs revealed high LRP5 expression (high PE-A values) compared to controls. Expression of LRP6 was also detected, but less pronounced compared to LRP5 (PE-A values of LRP6 <PE-A values of LRP5).

As previously reported (Oderup et al. Canonical and noncanonical Wnt proteins program dendritic cell responses for tolerance. J Immunol. 2013; 190(12): 6126-344), and as shown in FIG. 8, Wnt3a directly inhibits the secretion of proinflammatory cytokines (i.e., TNF-α release) by differentiated DCs. Wnt3a-induced suppression of TNF-alpha release from DCs was restored upon delivery of LRP5-selective binding molecules.

These data suggest that formatted, biparatopic, sequence-optimized binding molecules are capable of restoring TNF-alpha secretion by

- 40 046752

Wnt3a-treated dendritic cells, thereby suppressing the inhibitory effect of Wnt on dendritic cells.

Of note, blocking the Wnt pathway in dendritic cells of the tumor microenvironment represents a potential therapeutic approach to disrupt tumor-mediated immune suppression and complement antitumor immunity.

To examine the effect of DCs on T cells (effector T cells), Wnt3a-pretreated DCs, with or without LRP5-selective binding molecules, were co-cultured with T cells isolated from PBMCs as described previously (Oderup et al. Canonical and noncanonical Wnt proteins program dendritic cell responses for tolerance. J Immunol. 2013; 190(12): 6126-34. After 3 days of co-culture of DCT cells, the supernatants were collected and subjected to IFN-γ analysis using the ELISA method according to the manufacturer's instructions.

IFN-gamma secretion is a marker of T cell activation. As shown in FIG. 8 (central and right panels), Wnt3a-mediated suppression of DCs leads to reduced secretion of IFN-γ by T cells (suppression of T-cell function), which is restored by treatment with molecules that selectively bind LRP5.

In summary, these data indicate that molecules that selectively bind LRP5 suppress the inhibitory effects of Wnt on dendritic cells, leading to restoration of T cell function.

Chronic activation/stimulation of T cells is known to cause terminal differentiation leading to an exhausted T cell phenotype, a progressive loss of T cell function. It is therefore envisaged that the effects of LRP5-selective binding molecules on T cells, mediated by DC activation, may be limited by T cell exhaustion. Therefore, it is expected that combination therapy, in which the administration of molecules that selectively bind LRP5 is combined with the administration of an immune checkpoint inhibitor that blocks T cell exhaustion, should help activate and maintain T cell function, thereby altering the tumor microenvironment and maintaining the therapeutic effect of the molecules according to the invention.

Example 15: Effect of three biparatope LRP5 selective VHH extended half-life constructs on Wnt signaling and viability in RNF43 mutant CRC organoids.

The ability of biparatopic LRP5 selective VHH extended half-life constructs to selectively inhibit proliferation (as measured by inhibition of cell viability), and active Wnt signaling (as measured by reducing Axin2 mRNA levels) was characterized using RNF43 mutant colorectal cancer (CRC) organoids. CRC organoids were obtained as previously described (van de Wetering et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 2015;161(4):933–45). Briefly, to perform the cell viability assay, three patient-derived CRC organoids (RNF43 mut1, RNF43 mut2, and RNF43 WT) were each filtered through a 40-μm filter and seeded into 384-well plates (BME-coated). Specifically, ∼500 organoids were resuspended in a total volume of 40 μl of medium (with 5% BME) per well. The culture medium did not contain Wnt ligands. Compounds were dispensed using a Tecan D300 Digital Dispenser. Organoids were exposed to 8-point serial dilutions of LRP5 selective VHH construct or control compound and one fixed concentration of staurosporine (2 μM). Fresh compounds were added on day 0 and day 3 after organoid seeding. After 5 days, the CellTiter-Glo 3D Cell Viability Assay (Promega) was used to determine the cell viability of organoids obtained from CRC patients. The CellTiter-Glo 3D Cell Viability Assay is a homogeneous method for determining the number of viable cells in 3D culture based on the quantification of ATP present, which is a marker for the presence of metabolically active cells. In each experiment, treatments with the biparatope LRP5 selective VHH extended half-life construct and control compounds were performed in triplicate per organoid line. As internal controls, three organoids obtained from CRC patients were also treated with DMSO and Staurosporine and the corresponding cell viability data were used as reference: DMSO values as 100% survival and Staurosporine values as 0% survival. DMSO was added to all wells to normalize to the highest concentration (0.1% for DMSO in Staurosporine).

To analyze Axin2 mRNA levels using quantitative real-time PCR, each of three organoids obtained from CRC patients (RNF43 mut1, RNF43 mut2 and RNF43 WT) was filtered through a 40 μm filter and plated in 384-well plates (coated using BME). Specifically, ∼500 organoids were resuspended in a total volume of 40 μl of medium (with 5% BME) per well. The culture medium did not contain Wnt ligands. Compounds were dispensed using a Tecan D300 Digital Dispenser. Organoids were exposed to the biparatope LRP5 selective VHH extended half-life construct at a final concentration of 62 nM or 1 μM control compound). Compounds were added on day 0 and 24 hours after treatment, organoids were collected for qRT-PCR analysis and Axin2 mRNA levels were determined as a biomarker of inoculation.

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Claims (37)

inhibition of active Wnt signaling. As shown in FIG. 9, RNF43 mutant CRC organoids (RNF43 mutl, RNF43 mut2) treated with the biparatope LRP5 selective VHH construct with extended half-life showed a significant reduction in the percentage of viable cells (>50% reduction) compared to cells treated with the non-targeting VHH construct ( control), which had no effect on cell viability. RNF43 wild-type CRC organoids (RNF43 WT) treated with the LRP5-selective VHH extended half-life construct showed no effect on cell viability compared to cells treated with the control compound. These data demonstrate the ability of biparatope LRP5 selective VHH constructs with extended half-life to selectively inhibit the proliferation of cancer cells harboring mutations in the RNF43 gene that are dependent on active Wnt signaling. As shown in FIG. 10, RNF43 mutant CRC organoids (RNF43 mutl, RNF43 mut2) treated with the biparatope LRP5 selective VHH extended half-life construct (final concentration 62 nM) showed a significant reduction in relative Axin2 mRNA levels (i.e., normalized to endogenous control) by compared to cells treated with control (final concentration 1 µM). RNF43 wild-type CRC organoids (RNF43 WT) treated with the biparatope LRP5 selective VHH extended half-life construct (final concentration 62 nM) showed no significant effect on Axin2 mRNA levels compared to cells treated with control compound (final concentration 1 μM ). These data demonstrate the ability of a biparatope LRP5 selective VHH construct with an extended half-life to selectively inhibit Wnt signaling in cancer cells harboring mutations in the RNF43 gene. CLAIM 1. A polypeptide that binds to low-density lipoprotein receptor family protein 5 (LRP5), wherein the polypeptide contains a first immunoglobulin variable domain (ISVD) and a second ISVD, wherein the first ISVD is (I) an ISVD containing the following region sequences, complementarity determining factor (CDR): CDR1: TYVMG (SEQ ID NO:1) CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2) CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3), and a second ISVD selected from (III) ISVD containing the following CDR sequences: CDR1: IGAMG (SEQ ID NO:7) CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and (IV) ISVD containing the following CDR sequences: CDR1: INAMG(SEQ ID NO:10) CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3: ETGPYGPPKRDY (SEQ ID NO:9). 2. The polypeptide of claim 1, wherein the first ISVD is an ISVD (I) and the second ISVD is an ISVD (III). 3. The polypeptide of claim 1, wherein the first ISVD is an ISVD (I) and the second ISVD is an ISVD (IV). 4. A polypeptide that binds to low-density lipoprotein receptor family protein 5 (LRP5), wherein the polypeptide comprises a first immunoglobulin variable domain (ISVD) and a second ISVD, wherein the first ISVD is a (II) ISVD containing the following CDR sequences: CDR1: RYAVA (SEQ ID NO:4) CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5) CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), and a second ISVD selected from: (III) ISVD containing the following CDR sequences: CDR1: IGAMG (SEQ ID NO:7) CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and (IV) ISVD containing the following CDR sequences: CDR1: INAMG(SEQ ID NO:10) CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3: ETGPYGPPKRDY (SEQ ID NO:9). 5. The polypeptide of claim 4, wherein the first ISVD is an ISVD (II) and the second ISVD is an ISVD (III). 6. The polypeptide of claim 4, wherein the first ISVD is an ISVD (II) and the second ISVD is - 42 046752 is ISVD (IV). 7. A polypeptide according to any one of claims 1 to 6, wherein said ISVDs are VHH domains, preferably humanized VHH domains. 8. Polypeptide according to claim 1 or claim 4, where ISVD(I) contains the sequence SEQ ID NO: 11 or the sequence SEQ ID NO:23, ISVD (II) contains the sequence SEQ ID NO:12 ISVD (III) contains the sequence SEQ ID NO: 13 or the sequence SEQ ID NO:22, and/or ISVD (IV) contains the sequence SEQ ID NO:14. 9. The polypeptide of claim 8, comprising a first ISVD and a second ISVD, wherein said first ISVD contains the sequence of SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:23, and said second ISVD contains the sequence of SEQ ID NO:13 , SEQ ID NO:14 or SEQ ID NO:22. 10. The polypeptide of claim 9, wherein said first ISVD contains the sequence of SEQ ID NO:11 and said second ISVD contains the sequence of SEQ ID NO:22. 11. The polypeptide of claim 9, wherein said first ISVD contains the sequence of SEQ ID NO:12 and said second ISVD contains the sequence of SEQ ID NO:13. 12. The polypeptide of claim 9, wherein said first ISVD contains the sequence of SEQ ID NO:23, and said second ISVD contains the sequence of SEQ ID NO:14. 13. A polypeptide according to any one of claims 1 to 12, wherein said first ISVD and said second ISVD are covalently linked by a linker peptide, wherein said linker peptide optionally contains or consists of a third ISVD. 14. The polypeptide according to any one of claims 1 to 13, wherein said polypeptide further comprises a half-life increasing component, wherein said half-life increasing component is covalently linked to said polypeptide and optionally selected from the group consisting of an albumin-binding component, such as an albumin-binding component a peptide or albumin-binding domain of an immunoglobulin, a transferrin-binding component such as an anti-transferrin domain of an immunoglobulin, a polyethylene glycol molecule, human serum albumin and a fragment of human serum albumin. 15. The polypeptide of claim 14, wherein said half-life increasing component is an albumin binding component, preferably an albumin binding ISVD, more preferably an Alb11 domain comprising the sequence of SEQ ID NO:21. 16. A polypeptide containing first and second LRP5-binding ISVDs and one albumin-binding ISVD, said first LRP5-binding ISVD is (I) an ISVD containing the following CDR sequences: CDR1: TYVMG (SEQ ID NO:1) CDR2: AISWSGGSTYYADSVKG (SEQ ID NO:2) CDR3: SRGTSTPSPRASGVSRYDY (SEQ ID NO:3) said second ISVD selected from the group consisting of ISVDs (III) and (IV): (III) ISVD containing the following CDR sequences: CDR1: IGAMG (SEQ ID NO:7) CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and (IV) ISVD containing the following CDR sequences: CDR1: INAMG(SEQ ID NO:10) CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3: ETGPYGPPKRDY (SEQ ID NO:9), the indicated albumin-binding ISVD is defined as containing the following CDR sequences: CDR1: SFGMS (SEQ ID NO: 15) CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 16) CDR3: GGSLSR (SEQ ID NO:17). 17. The polypeptide of claim 16, wherein the first ISVD is an ISVD (I) and the second ISVD is an ISVD (III). 18. The polypeptide of claim 16, wherein the first ISVD is an ISVD (I) and the second ISVD is an ISVD (IV). 19. A polypeptide containing first and second LRP5-binding ISVDs and one albumin-binding ISVD, said first LRP5-binding ISVD is (II) an ISVD containing the following CDR sequences: - 43 046752 CDR1: RYAVA (SEQ ID NO:4) CDR2: AITWSSGRIDYADSVKG (SEQ ID NO:5) CDR3: DRRPRSTGRSGTGSPSTYDY (SEQ ID NO:6), said second ISVD selected from the group consisting of ISVDs (III) and (IV): (III) ISVD containing the following CDR sequences: CDR1: IGAMG (SEQ ID NO:7) CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3: ETGPYGPPKRDY (SEQ ID NO:9), and (IV) ISVD containing the following CDR sequences: CDR1: INAMG (SEQ ID NO: 10) CDR2: AVSSGGSTYYVDSVKG (SEQ ID NO:8) CDR3: ETGPYGPPKRDY (SEQ ID NO:9), the indicated albumin-binding ISVD is defined as containing the following CDR sequences: CDR1: SFGMS (SEQ ID NO: 15) CDR2: SISGSGSDTLYADSVKG (SEQ ID NO: 16) CDR3: GGSLSR (SEQ ID NO:17). 20. The polypeptide of claim 19, wherein the first ISVD is an ISVD (II) and the second ISVD is an ISVD (III). 21. The polypeptide of claim 19, wherein the first ISVD is an ISVD (II) and the second ISVD is an ISVD (IV). 22. A polypeptide that binds to LRP5, containing or consisting of a sequence selected from SEQ ID NO: 18, 19 and 20. 23. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 22. 24. An expression vector containing the nucleic acid according to claim 23. 25. A host cell carrying the expression vector according to claim 24. 26. A method for preparing a polypeptide according to any one of claims 1 to 22, which includes the stage of culturing a host cell according to claim 25 under conditions that allow expression of the polypeptide according to any one of claims. 1-21; and restoring said polypeptide. 27. The method of claim 26, which further includes the step of purifying said polypeptide. 28. A pharmaceutical composition for treating, preventing or alleviating a disease caused by abnormal Wnt signaling, comprising (I) as an active component, a polypeptide according to any one of claims 1 to 22, and (II) a pharmaceutically acceptable carrier. 29. Use of a polypeptide according to any one of claims 1 to 22 as a medicament in a method of treating, preventing or alleviating a disease caused by abnormal Wnt signaling in a human or animal. 30. Use of a polypeptide according to any one of claims 1 to 22 for the treatment of malignancy caused by abnormal Wnt signaling. 31. Use according to claim 30 for the treatment of breast cancer, lung cancer, specifically non-small cell lung carcinoma (NSCLC), pancreatic cancer, colorectal cancer, sarcomas, ovarian cancer or hepatocellular carcinoma. 32. Use according to claim 31 in combination with a chemotherapeutic agent, a therapeutically active compound that inhibits angiogenesis, a signal transduction pathway inhibitor, an EGFR inhibitor, an immunomodulator, an immune checkpoint inhibitor, or a hormone therapy agent. 33. Use of a polypeptide according to any one of claims 1 to 22 for the treatment of triple negative breast cancer (TNBC). 34. The use of a therapeutic agent selected from the group consisting of chemotherapeutic agents, therapeutically active compounds that inhibit angiogenesis, signal transduction pathway inhibitors, EGFR inhibitors, immunomodulators, immune checkpoint inhibitors and hormone therapy agents in combination with a polypeptide according to any one of paragraphs. .122 for the treatment, prevention or attenuation of disease caused by abnormal Wnt signaling. 35. A method of treating a malignancy or idiopathic lung disease or retinopathy caused by abnormal Wnt signaling in a patient in need thereof, comprising administering to said patient an effective amount of a polypeptide of any one of claims 1 to 22, or a pharmaceutical composition of claim 28 . 36. Use of a polypeptide according to any one of claims 1 to 22 for modifying the tumor microenvironment by inhibiting Wnt1- and Wnt3a-mediated transcription of a target gene in dendritic cells. 37. Use according to claim 36, where said use is use in conditions in -