CN114426578B

CN114426578B - Combinations of binding moieties that bind EGFR, HER2 and HER3

Info

Publication number: CN114426578B
Application number: CN202210129458.1A
Authority: CN
Inventors: 塞西利亚·安娜·威廉明娜·热延; 特里斯坦·路易斯·吉恩·加莱纳; 马克·思罗斯比; 科内利斯·阿德里安·德克吕夫
Original assignee: Merus BV
Current assignee: Merus BV
Priority date: 2019-02-14
Filing date: 2020-02-13
Publication date: 2025-03-21
Anticipated expiration: 2040-02-13
Also published as: CN114426578A

Abstract

The present invention relates to a combination of binding moieties that bind to EGFR, HER2, and HER3, and more specifically to a composition comprising two or more binding moieties, wherein each of the binding moieties comprises a variable domain that binds to the extracellular portion of EGFR; and wherein the first of the binding moieties comprises a variable domain that binds to the extracellular portion of HER2, and the second of the binding moieties comprises a variable domain that binds to the extracellular portion of HER3. The present invention also relates to devices and methods for generating the composition and treating an individual with the composition.

Description

Combinations of binding moieties that bind EGFR, HER2 and HER3

The application is a divisional application of China patent application with the application number 202080014304.7, namely 'combination of binding parts binding EGFR, HER2 and HER 3', and the original application is PCT International application PCT/NL2020/050081 which is filed on 13 days of 02 and 02 in 2020, and enters China national stage on 13 days of 08 in 2021.

Technical Field

The present invention relates to the field of binding moieties (binding moieties) such as antibodies, and in particular to the field of therapeutic binding moieties. The binding moiety may be used in the treatment of humans. More particularly the invention relates to a composition comprising two or more multispecific binding moieties, preferably multispecific antibodies. The binding moiety binds EGFR, HER2 and HER3. A single host cell may produce multiple binding moieties.

Background

The Epidermal Growth Factor (EGF) receptor (EGFR) is a prototype cell surface receptor (prototype cell-surface receptor) that contains members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. This family currently has 4 closely related receptor tyrosine kinases, EGFR, HER2 (ErbB-2/c-neu), HER3 (ErbB-3) and HER4 (ErbB-4).

EGFR is present on the cell surface and is activated by binding of its specific ligands, including epidermal growth factor and transforming growth factor alpha (tgfα). Upon activation by its growth factor ligand, the receptor undergoes a transition from an inactive primary monomeric form to an active homodimer. In addition to forming a homodimer upon ligand binding, EGFR can be paired with another member of the ErbB receptor family, such as HER2, to produce an activated heterodimer (ACTIVATED HETERO-dimer). There is also evidence that dimers are formed without ligand binding and clusters of activated EGFRs are formed after ligand binding.

Dimerization of EGFR stimulates its intrinsic intracellular protein-tyrosine kinase (PTK) activity. This activity induces several signaling cascades (signal transduction cascades) that lead to cell proliferation and differentiation. The kinase domain of EGFR can cross-phosphorylate (cross-phosphorylate) its tyrosine residues with other receptors of the complex and can itself be activated in that way.

Mutations and hypermanifestations involving EGFR have been identified in several types of cancers, and it is a target for an extended type of anti-cancer therapy. These include small molecules such as gefitinib and erlotinib for EGFR targets for lung cancer, and antibodies such as cetuximab and panitumumab for colon and head and neck cancers.

While therapies employing EGFR targets have met with some success, most have been associated with the development of therapeutic resistance over time. One of the ways in which EGFR-positive tumors can escape this target therapy is through signaling through another receptor dimer. For example, increased signaling for EGFR/HER3 dimers due to increased HER3 expression or regulatory protein expression (heregulin expression) has been associated with drug resistance of EGFR targets in, for example, lung cancer and head and neck cancer. In addition to induction of therapeutic resistance, certain side effects of EGFR-target antibodies have been observed. One example is the development of skin rash associated with EGFR inhibition or anti-EGFR biologic therapy. At extreme times, such rash may lead to a decrease in the treatment cycle and/or premature termination of treatment.

A wide variety of activation patterns of signaling of the EGF receptor family have been identified. Among these are ligand-dependent (LIGAND DEPENDENT) and ligand-independent (LIGAND INDEPENDENT) activation of signaling. That is, HER2 that is overexpressed in the absence of HER3 ligand is enabled to transmit HER2/HER3 heterodimers to generate oncogenic signaling (oncogenic signaling) (Junttila, akita et al 2009). HER2 activity may be inhibited by HER2 specific antibodies. Such HER2 specific antibodies may be used, for example, in the treatment of HER2 positive (HER 2 ⁺) tumors. The problem with such treatments is that often tumors escape the HER2 specific treatment and continue to grow even in the presence of the inhibitory antibodies. It has been observed that HER2 positive tumors, such as breast, ovarian, cervical and gastric tumors, can escape treatment by selective overgrowth of a sub-population of tumor cells exhibiting up-regulated HER3 expression (Ocana, vera-balloon et al 2013) and/or HER3 ligand expression (Wilson, FRIDLYAND et al 2012). Also, activating mutations located in the HER3 receptor have been identified.

Thus, despite the promising results of treatment with antibodies specifically targeting EGF receptor family members, it has been observed that not all tumors respond or respond adequately. The present invention provides combinations of binding moieties that target various members of the EGF receptor family and methods for producing the same. The combination of the present invention shows good efficacy. Such combinations may be generated in a cost-effective and efficient manner.

Disclosure of Invention

The present invention provides a composition comprising two or more binding moieties,

Wherein each binding moiety comprises a variable domain that binds to an extracellular portion of EGFR, and

Wherein a first one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER2 and a second one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER 3.

Preferably, at least one of the two or more binding moieties is an antibody. In a preferred embodiment, at least two of the two or more binding moieties are antibodies. Such antibodies are preferably multispecific antibodies, preferably bispecific antibodies. Preferably at least one of such antibodies, and more preferably at least two of them, is an IgG antibody. In a preferred embodiment of the invention, the composition comprises two bispecific antibodies.

Multispecific antibodies as described herein preferably comprise heavy chain bearing CH3 heterodimerization domains. In one embodiment, the CH3 heterodimerization domain of the first and/or the second multispecific antibody is engineered to promote heterodimerization of the heavy chain of the EGFR variable domain with the corresponding heavy chains of the HER2 and HER3 variable domains.

The invention also provides a composition as described herein for use in the treatment of cancer. In embodiments, the cancer is a physical epithelial cancer. Preferably, the composition is used in a cancer that expresses EGFR, HER2 and/or HER 3. The composition is preferably used for pancreatic cancer, colorectal cancer, head and neck cancer, epithelial ovarian cancer, epithelial fallopian tube cancer, epithelial peritoneal cancer, bladder cancer or prostate cancer. In embodiments, the cancer treated by using the composition is an advanced cancer (ADVANCED CANCER). The composition is preferably used for metastatic cancer (METASTATIC CANCER). The composition is preferably used for metastatic pancreatic cancer, metastatic colorectal cancer, metastatic head and neck cancer, metastatic epithelial ovarian cancer, metastatic epithelial fallopian tube cancer, metastatic epithelial peritoneal cancer, metastatic bladder cancer or metastatic prostate cancer. In embodiments, the composition is preferably used for cancers that are gastric, lung, breast or esophageal cancers. Preferably, the composition is used for metastatic gastric cancer, metastatic lung cancer, metastatic breast cancer or metastatic esophageal cancer.

The invention further provides a product comprising two or more binding moieties each comprising a variable domain that binds to an extracellular portion of EGFR, wherein a first one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER2 and a second one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER3 as a combined preparation for simultaneous, separate or sequential use in treating cancer.

The invention further provides a method for producing a composition according to the invention, the method comprising:

providing a cell comprising

-A nucleic acid encoding a polypeptide comprising a heavy chain that forms, together with a common light chain, a variable domain that binds to the extracellular portion of EGFR;

-a nucleic acid encoding a polypeptide comprising a heavy chain forming, together with the common light chain, a variable domain that binds to the extracellular portion of HER 2;

A nucleic acid encoding a polypeptide comprising a heavy chain forming together with the common light chain a variable domain binding to the extracellular portion of HER3, and

-A nucleic acid encoding a polypeptide comprising the common light chain;

Wherein two or more of the nucleic acids may or may not be physically linked (PHYSICALLY LINKED), and wherein each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in the cell, and wherein the method further comprises culturing the cell to allow expression of the heavy and light chains, and optionally, collecting the two or more binding moieties.

Further provided is an cell comprising:

-A nucleic acid encoding a polypeptide comprising the common light chain;

wherein two or more of the nucleic acids may or may not be physically linked, and wherein each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in the cell.

In a further aspect, the invention provides a container comprising nucleic acid, comprising:

-a nucleic acid encoding a polypeptide comprising a heavy chain forming, together with a common light chain, a variable domain that binds to the extracellular portion of HER 2;

A nucleic acid encoding a polypeptide comprising a heavy chain forming together with a common light chain a variable domain binding to the extracellular portion of HER3, and

-A nucleic acid encoding a polypeptide comprising the common light chain;

wherein, optionally, two or more of the nucleic acids may or may not be physically linked, and wherein each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in the cell.

The invention further provides a composition comprising a binding moiety that specifically binds to the extracellular portion of EGFR and the extracellular portion of HER 2.

The invention also provides compositions comprising a binding moiety that specifically binds to the extracellular portion of EGFR as well as the extracellular portion of HER 3.

The binding moiety is preferably an antibody, preferably an IgG antibody, more preferably a multispecific antibody.

Drawings

FIG. 1

A schematic representation of an embodiment wherein the composition comprises two bispecific antibodies sharing a common arm. The figure depicts an antibody with a heavy chain (1) and a light chain (4). The 4 heavy chains have 3 different variable regions (5, 6 and 7). The heavy chain with the shared variable region (5) has a portion of a heterodimerization domain (3). The heavy chain with variable region (6) and (7) has a compatible portion of the heterodimerization domain (2). The preferential pairing of heterodimerization regions (2) and (3) can lead to the formation of bispecific antibodies.

FIG. 2

Two kinds of materialsFor the growth factor dependent cell line BxPC-3-luc2 (PERKIN ELMER 125058) and N87 cells (NCI-87 cells ]CRL-5822 ^TM)).

Two kinds of materialsThe effect on BxPC-3-luc2 (left panel) and N87 (right panel)) cell proliferation was tested. The results of panel screening were compared with a combination of two monospecific antibodies (EGFR binding antibody cetuximab and HER3 monospecific antibody PG 3178) or with EGFRxHER binding bispecific antibody PB 4522. The cells are grown in the presence of saturated amounts of HRG and EGF. The level of cell growth (basal value of ligand) of the corresponding cells using HRG and EGF without antibody was also indicated without HRG and EGF and without antibody (ligand (w/oligand)).

Monospecific antibody PG3178 has one IgG1 constant region and two variable domains with the heavy chain variable region of MF3178 in fig. 7 or 8 and the common light chain variable region of sequence 11 in fig. 7.

Bispecific antibody PB4522 has one IgG1 constant region and two variable domains. The HER3 variable domain has the heavy chain variable region of MF3178 in fig. 7 or 8. The EGFR variable domain has the heavy chain variable region of MF4280 in fig. 7 or 8. The light chain variable regions of the two antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence 11 in fig. 7.

FIG. 3

A subgroup ofADCC activity of (a) and (b). A subgroup ofIs tested using N87 and a CD16/NFAT reporter assay (CD 16/NFAT reporter assay). Such bispecific antibodies have one IgG1 constant region and two variable domains. The amino acid sequence of the heavy chain variable region of such a variable domain is shown in fig. 7 or fig. 8. The light chain variable regions in such antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence 11 in fig. 7.

FIG. 4

Various differentIs also disclosed, and their ADCC activity. "-" indicates that no activity was observed.

Each row represents a cell comprising two bispecific antibodiesThe internal codes of such bispecific antibodies are shown in such straight run bispecific 1 (Bispecific 1) and bispecific 2 (Bispecific 2). The heavy chain variable regions of HER2, HER3 and EGFR binding domains are indicated in the straight lines labeled MF a, MF B and MF C. MF numbers 3178 and 2703 combine to form a HER3 binding variable domain with the common light chain. MF numbers 4280, 3755, 4003, 4016 combine with the common light chain to form a variable domain that binds EGFR, while MF numbers 1871, 1847, 1849, and 2032 combine with the common light chain to form a variable domain that binds HER 2. Such bispecific antibodies have one IgG1 constant region and two variable domains. The amino acid sequences of the heavy chain variable regions of the variable domains are shown in FIG. 7 or FIG. 8. The light chain variable regions in these antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence 11 in fig. 7.

FIG. 5

In vivo. BxPC-3-luc2 or N87 cells were injected into the xenograft model on day 0. Comprising bispecific antibody PB4516 XPB 6892 (see FIG. 4)Or control groups were injected on days 1,7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg/kg. Is depicted as involvingResults of (PB 4516 and PB 6892). Vehicle and cetuximab served as control groups.

FIG. 6

Comprising bispecific antibodies PB4516 and PB6892In vivo experiments in various PDX models.

The PDX model was injected on day 0, while treatment with antibody or control group was completed on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg/kg.

FIG. 7

The amino acid sequences of the heavy chain variable regions of the various variable domains indicated by the MF numbers are referred to by sequence numbers 1-6, while the CDRs and light chain variable regions are referred to by sequence numbers 7-12.

FIG. 8

The amino acid sequences of the various MFs mentioned herein. FR1-4 means architecture regions 1-4.CDR1-3 means complementarity determining regions 1-3.TT is tetanus toxoid (tetanus toxoid).

FIG. 9

A) HER3 crystal structure (pdb#4p59) showing residues Arg 426 located in grey spheres and at a distance from Arg 426Residues within the radius located in the black sphere b) residues Arg 426 shown in gray and at a distance from Arg 426Remote residues within the radius shown in black, c) residues in region Arg 426 that are light gray and surrounding residues (all marked) that are dark gray. The specification drawings and analysis were made using Yasara (www.yasara.org).

Detailed Description

The term EGFR as used herein means that protein encoded by the epidermal growth factor receptor gene (EGFR) in humans. The protein is known to have many aliases, including Erb-B2 receptor tyrosine kinase 1, protooncogene C-ErbB-1, ERBB1, and HER1. The database accession number relating to this human EGFR protein and the gene encoding it is (GenBank NM-005228.3). The accession number is given primarily to provide a further means to identify the EGFR protein as a target, the actual sequence of the EGFR protein bound by the antibody may vary, for example, because of mutations within the coding gene (such as those occurring in certain cancers), and so forth. Unless otherwise indicated, when referring to EGFR herein, reference is made to human EGFR. Because of the sequence and tertiary structural similarity between orthologs (orthologues) of EGFR in humans and other mammals, the EGFR variable domain may also bind such orthologs, but not necessarily. This variable domain that binds EGFR can bind EGFR as well as its diverse variants, such as those that are manifested on certain EGFR-positive tumors.

The variable domain that binds EGFR of an antibody or binding portion of the invention preferably binds domain I or domain III of EGFR. The structure of the EGFR protein has been described in Ferguson (2008:Annu Rev Biophys.2008;37:353-373.Doi:10.1146/annurev. Biohys.37.032817.125829), among other things. The domain of human EGFR is depicted in figure 1 of the Ferguson reference data mentioned above. The variable domain that binds EGFR of embodiments of the invention disclosed herein preferably binds domain III of EGFR. The antibody preferably inhibits EGF-induced proliferation of BxPC-3 cells (ATCC CRL-1687) or BxPC-3-luc2 cells (PERKIN ELMER 125058).

The term HER2 as used herein means that protein encoded by the ERBB-2 gene in humans. Alternative names for the gene or protein include CD340, erbB-2, HER-2/neu, MLN 19, NEU, NGL, TKR1. This ERBB-2 gene is often referred to as HER2 (derived from HER 2). When referring to HER2 herein, reference is made to human HER2. An antibody comprising a variable domain that binds HER2 binds human HER2. Because of sequence and tertiary structural similarity between orthologs of HER2 in humans and other mammals, the HER2 variable domain may also bind such orthologs, but not necessarily so. The accession numbers of databases relating to the human HER2 protein and the gene encoding it are (NP-001005862.1, NP-004439.2, NC-000017.10, NT-010783.15). Such accession numbers are given primarily to provide further means to identify HER2 as a target, the actual sequence of HER2 protein bound by the antibody may vary, for example, because of mutations within the coding gene (such as those occurring in certain cancers), and so forth. The HER2 variable domain can bind HER2 as well as its diverse variants, such as those that are represented on certain HER2 positive tumor cells.

The HER2 protein contains several domains (see FIG. 1 for reference to Landgraf, R Briest Cancer Res.2007;9 (1): 202-). The extracellular domain is called domain I-IV. The variable domain of embodiments of the invention disclosed herein that binds HER2 preferably binds to domain I or domain IV, preferably domain IV, of HER 2.

The term HER3 as used herein means that protein encoded by the ERBB3 gene in humans. The substitution names for the gene or protein are LCCS2, MDA-BF-1;c-ErbB-3;c-ErbB3, erbB3-S, p180-ErbB3, p45-sErbB3, and p85-sErbB3. When referring to HER3 herein, reference is made to human HER3. An antibody comprising a variable domain that binds HER3 binds human HER3. Because of sequence and tertiary structural similarity between orthologs of HER3 in humans and other mammals, the HER3 variable domain may also bind such orthologs, but not necessarily so. The accession numbers of databases relating to the human HER3 protein and the gene encoding it are (NP-001005915.1, NP-001973.2, NC-000012.11, NT-029419.12). Such accession numbers are given primarily to provide further means to identify HER3 as a target, the actual sequence of HER3 protein bound by an antibody may vary, for example, because of mutations within the coding gene (such as those occurring in certain cancers), and so forth. The HER3 variable domain can bind HER3 as well as its diverse variants, such as those that are represented on certain HER2 positive tumor cells.

The structure of HER3 has been described in Cho et al (2002;Science 297,1330-1333: DOI: 10.1126/science.1074611), among others. The human protein has 4 extracellular domains. The variable domain that binds HER3 of embodiments of the invention disclosed herein preferably binds domain III of HER 3. In a preferred embodiment, the affinity (KD) of a variable domain for a HER3 positive cell is less than or equal to 2.0nM, more preferably less than or equal to 1.5nM, even more preferably less than or equal to 1.39nM, and even more preferably less than or equal to 0.99nM. In a preferred embodiment, an antibody according to the invention preferably comprises at least one of domain III, wherein the variable domain binds to HER3, selected from the group consisting of R426 included in the native HER3 protein and is located at a distance from R426Amino acids in the group consisting of amino acid residues within. In a preferred embodiment, the variable domain has an affinity (KD) for HER3 located on SK-BR-3 cells of less than or equal to 2.0nM, more preferably less than or equal to 1.5nM, still more preferably less than or equal to 1.39nM, and still more preferably less than or equal to 0.99nM. In one embodiment, the affinity (KD) falls within the range of 1.39-0.59 nM. In a preferred embodiment, the variable domain has an affinity (KD) for HER3 located on BT-474 cells of less than or equal to 2.0nM, more preferably less than or equal to 1.5nM, still more preferably less than or equal to 1.0nM, still more preferably less than or equal to 0.5nM, still more preferably less than or equal to 0.31nM, still more preferably less than or equal to 0.23nM. In one embodiment, the affinity (KD) falls within the range of 0.31-0.15 nM. The affinities mentioned above are preferably measured when using steady state cell affinity measurement (STEADY STATE CELL AFFINITY measurements), wherein cells are incubated with radiolabeled antibody (radioactively labeled antibody) at 4 ℃, after which the cell-bound radioactivity (cell-bound radioactivity) is measured.

The variable domain of at least one amino acid of domain III that binds HER3 preferably binds to a polypeptide selected from the group consisting of R426 included in the native HER3 protein and is located a distance from R426Amino acids in the group consisting of amino acid residues within. Preferably, is located in the native HER3 protein at a distance from R426The amino acid residues within are selected from the group consisting of L423, Y424, N425, G427, G452, R453, Y455, E480, R481, L482, D483, and K485 (see, e.g., FIG. 9 and Table 1). Amino acid residue number is that of protein database (Protein Data Bank, PDB) ID # 4p59. Antibodies that bind to this region of domain III of HER3 exhibit particularly good binding characteristics and they are capable of counteracting an activity of HER3 on HER3 positive cells. The variable domain with HER3 binding characteristics is described in WO 2015/130172, which is incorporated herein by reference. In a preferred embodiment, a bispecific antibody according to the invention is provided, wherein the antibody comprises R426 of domain III, wherein the variable domain binds at least to HER 3. Preferably, the antibody comprises R426 of domain III, the variable domain of which binds at least to HER 3.

In certain embodiments, the composition comprises two or more antibodies, wherein each of the antibodies comprises a variable domain that binds to the extracellular portion of EGFR, and wherein a first one of the antibodies comprises a variable domain that binds to the extracellular portion of HER2 and a second one of the antibodies comprises a variable domain that binds to the extracellular portion of HER 3. In a preferred embodiment, the variable domains of the first and second antibodies that bind to the extracellular portion of EGFR have substantially the same amino acid sequence. In one embodiment, the first and second antibodies comprise domain I of the variable domain binding to EGFR, and the first antibody comprises domain I of the variable domain binding to HER2, and the second antibody comprises domain III of the variable domain binding to HER 3. In another embodiment, the first and second antibodies comprise domain I of the variable domain binding EGFR, and the first antibody comprises domain IV of the variable domain binding HER2, and the second antibody comprises domain III of the variable domain binding HER 3. In a further embodiment, the first and second antibodies comprise domain III of variable domain binding EGFR, and the first antibody comprises domain I of variable domain binding HER2, and the second antibody comprises domain III of variable domain binding HER 3. In a further embodiment, the first and second antibodies comprise domain III of variable domain binding EGFR, and the first antibody comprises domain IV of variable domain binding HER2, and the second antibody comprises domain III of variable domain binding ER 3.

In certain embodiments, a binding moiety is a protein or an aptamer (aptamer). Binding moieties as described herein generally have two or more binding specificities. The binding moiety preferably comprises two or more variable domains of an antibody. The variable domains may be provided in a variety of different ways. A number of fragments containing antibody variable domains are described in "Nelson 2010:MAbs.2010 Jan-Feb;2 (1): 77-83", and include various FAB fragments, scFv fragments, and so-called single domain antibodies such as VHH fragments. Various FAB fragments or single chain Fv fragments are well known today. A single domain antibody is an antibody fragment consisting of a single monomer variable antibody domain. Just like an intact antibody, it can optionally bind to a specific antigen. With a molecular weight of only 12-15kDa, single domain antibodies are much smaller than the common antibodies (150-160 kDa) consisting of two protein heavy and two light chains, and even smaller than Fab fragments (50 kDa, one light and half heavy) and single chain variable fragments (25 kDa, two variable domains, one from the light chain and one from the heavy chain). Single domain fragments were first made from camelid heavy chain antibodies. Similar single domain fragments can now be manufactured artificially and can be derived from other organisms. A variable domain preferably comprises a heavy chain variable region and a light chain variable region. The variable domains are sometimes referred to as V _H/V_L combinations, where V _H represents the variable region of the heavy chain and V _L represents the variable region of the light chain.

The two or more fragments may be linked to create a binding moiety having a number of binding specificities. Ligation is typically accomplished using a ligation peptide comprising 2 or more amino acid residues. The linking moiety may also be part or all of a protein. For example, human serum albumin is sometimes used. The binding moiety as described herein preferably comprises a heavy chain variable region with at least one variable domain with one MF (e.g., as described in fig. 7 or 8) paired with a light chain variable region (e.g., light chain variable region). In a preferred embodiment, the binding moiety comprises two or more such variable domains.

The binding moiety that binds EGFR and HER2 is a different binding moiety than the binding moiety that binds EGFR and HER3. If at least one of the binding moieties is a multispecific antibody, at least one multispecific antibody may bind at least EGF and HER2 or bind at least EGFR and HER3. In a preferred embodiment, the binding moiety comprises a bispecific antibody, wherein the bispecific antibody binds EGFR and HER2 and the other bispecific antibody binds EGFR and HER3.

The term "antibody" as used herein refers to a proteinaceous molecule belonging to the immunoglobulin class of proteins, which contains one or more domains that bind an epitope located on an antigen, wherein such domains are derived from the variable region of an antibody or share sequence homology with the variable region of the antibody. Antibodies are typically composed of basic structural units-each having two heavy chains and two light chains. The antibody for therapeutic use is preferably a natural antibody as close as possible to the individual to be treated (e.g., a human antibody for a human individual). An antibody according to the invention is not limited to any particular type or method for producing it.

Since an antibody typically recognizes an epitope of an antigen, and such an epitope may also be present in other compounds, an antibody according to the invention that "specifically recognizes" an antigen (e.g., EGFR, HER2 or HER 3) may also recognize other compounds if such other compounds contain the same epitope. Thus, the term "specifically recognizes" or "specifically binds" or terms of identical connotation (connotation), in terms of antigen-to-antibody interactions, does not exclude the binding of such antibodies to other compounds containing the same or the same epitope.

A "bispecific antibody" is an antibody as described herein, wherein one variable domain of the antibody binds to a first antigen and two variable domains of the antibody bind to a second antigen, wherein the first and second antigens are not identical. The term "bispecific antibody" also encompasses antibodies in which the chain variable region/light chain variable region (V _H/V_L) combination binds a first epitope located on an antigen, and the second V _H/V_L combination binds a second epitope. The second epitope may be a different epitope located on the same antigen. The term also includes antibodies, wherein V _H is capable of specifically recognizing a first antigen, and V _L paired with V _H in an immunoglobulin variable region is capable of specifically recognizing a second antigen. The V _H/V_L pairing formed will bind either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008/027236, WO 2010/108127 and Schaefer et al (CANCER CELL 20,472-486, october 2011). The bispecific antibody according to the invention is not limited to any particular pattern or method for producing it.

Bispecific antibodies are an exemplary case of multispecific antibodies. Tri-or more specific antibodies can be made by adding binding moieties (such as scFv fragments) to one or more of the heavy chains. It is also possible to add one or more variable domains to the variable region of a normal or bispecific antibody. A bispecific antibody with two different heavy and light chain combinations, among other things, generates a common light chain and two different heavy chains, each forming a functional variable domain with the common light chain. Likewise, a cell that produces a common light chain and three or more different heavy chains can form several bispecific antibodies that together are capable of targeting three or more antigens. It is now possible to establish standard versions of antibodies (i.e. one constant part and two variable domains) and to add further binding domains. As such, multispecific antibodies can be made that have one or more single-chain Fv's with additional binding specificity attached to one or more of the constant portion or variable domains of the antibody. It is also possible to generate heavy chains with two or more variable regions. The additional heavy chain regions may advantageously be associated with different or common light chain variable regions. For a description of such antibodies, see US 62/650467, which is incorporated herein by reference.

When referring to nucleic acid or amino acid sequences herein, "percent (%) identity" is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after the aligned sequences are used for optimal comparison purposes. The percent sequence identity of the comparative nucleic acid sequences was determined using the alignX application of Vector NTI Program Advance 10.5.2 software using default values using a modified ClustalW algorithm (Thompson, J.D., higgins, D.G., and Gibson T.J. (1994) nuc.acid Res.22:4673-4680), swgapdnarnt scoring matrix (SWGAPDNARNT SCORE MATRIX), a gap opening penalty of 15 (gap opening penalty), and a gap extension penalty of 6.66 (gap extension penalty). The amino acid sequences were aligned using the alignment application of Vector NTI Program Advance 11.5.2 software using default values using a modified ClustalW algorithm (Thompson, j.d., higgins, d.g., and Gibson t.j., 1994), a blosum62mt2 score matrix (blosum 62mt2 score matrix), a gap opening penalty of 10, and a gap extension penalty of 0.1.

The term "common light chain" as used herein means light chains such as those that may be used in a multispecific antibody. In bispecific antibodies, the two light chains can be one common light chain (or V _L portion thereof). The two light chains (or V _L portions thereof) may be identical or have some amino acid sequence differences while the binding specificity of the full-length antibody is unaffected. The terms "common light chain", "common V _L", "single light chain", "single V _L", with or without the addition of the term "rearranged" are used interchangeably herein. "common" means light chains having the same sequence and also that the amino acid sequences are not identical functional equivalents. Many variants of such light chains exist, wherein mutations (deletions, substitutions, insertions and/or additions) are present that do not affect the formation of a functional binding region. The light chain of the present invention may also be a light chain as indicated herein, having from 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. For example, light chains that are not identical but are still functionally equivalent are prepared or found to fall within the definition of common light chains as used herein, e.g., by introducing and testing conservative amino acid changes, changes in amino acids located in regions that do not or only partially contribute to binding specificity when paired with the heavy chain, and so forth. In certain embodiments, multispecific antibodies with 3 or more variable domains have variable domains with different heavy chains and the same light chains or light chains have certain amino acid differences while the binding specificity of the full-length multispecific antibody is unaffected. Such a light chain is advantageously also a common light chain as described herein. In a preferred embodiment, all of the variable domains of a multispecific antibody comprise one common light chain. A common light chain (variable region) for use in the multivalent antibodies of the invention may be a lambda light chain, and this is thus also provided in the context of the invention, but a kappa light chain is preferred. The common light chain of the present invention may comprise a constant region of a kappa light chain or a lambda light chain. It is preferably a constant region of a kappa light chain, preferably wherein the common light chain is a germline light chain, preferably a rearranged germline human kappa light chain comprising IgVK1-39 gene segments, e.g. rearranged germline human kappa light chain IgVK1-39 x 01/IGJK1 x 01. Exemplary common light chain amino acid sequences are shown in fig. 7, sequences 10, 11 or 12.

The term "full length IgG" or "full length antibody" according to the invention is defined to include a substantially complete IgG, but it does not necessarily have all the functions of a complete IgG. For the avoidance of doubt, a full length IgG contains two heavy chains and two light chains. Each chain contains a constant region (C) and a variable region (V) which can be broken down into domains designated CH1, CH2, CH3, V _H, and C _L、V_L. An IgG antibody binds to an antigen via the variable region domains included in the Fab portion, and upon binding can interact with molecules and cells of the immune system through such constant domains (mostly through the Fc portion). The full length antibodies according to the invention encompass IgG molecules in which mutations that provide the desired characteristics may be present. Full length IgG should not have a substantial portion of any of such regions deleted. However, igG molecules in which one or several amino acid residues are deleted without substantially altering the binding characteristics of the resulting IgG molecule are encompassed within the term "full-length IgG". For example, such an IgG molecule may have a deletion of between 1 and 10 amino acid residues, preferably in the non-CDR region, wherein such deleted amino acids are not necessary for the antigen or epitope binding specificity of the IgG. Exemplary IgG antibodies are IgG1, igG2, igG3, and IgG4 antibodies. In certain embodiments of the invention, the IgG is IgG1.

Preferably at least one of the two or more binding moieties is an antibody. The antibody may comprise one variable domain that binds to the extracellular portion of EGFR and one variable domain that binds to the extracellular portion of HER 2. In another embodiment, the antibody comprises a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER 3.

The two or more binding moieties preferably comprise two or more antibodies (preferably multispecific antibodies), each comprising a variable domain that binds to the extracellular portion of EGFR, and wherein a first one of such antibodies comprises a variable domain that binds to the extracellular portion of HER2 and a second one of such antibodies comprises a variable domain that binds to the extracellular portion of HER 3. A preferred exemplary embodiment of a composition comprising two or more multispecific antibodies is a composition comprising two or more bispecific antibodies. A non-limiting example of a composition comprising two bispecific antibodies as described herein is schematically depicted in figure 1. Two bispecific antibodies are depicted each having two heavy chains (1) and two light chains (4). The two antibodies share a heavy chain with a variable region (5). They differ in the variable region of the other heavy chain. One antibody has a heavy chain variable region (6). The other antibody has a heavy chain variable region (7). All heavy chain variable regions can be paired with the common light chain (4) to form a functional binding domain. When produced by the same cell, the heavy chain is directed to heterodimerize by the presence of one heterodimerization domain (2, 3). The heterodimerization domain has two portions, one on one heavy chain and the compatible portion on the other heavy chain. The heterodimerization domain is often located within the IgG1 CH3 region. Heterodimerization may be directed by providing appropriate moieties to the selected heavy chain.

In the present invention, the selected formation of EGFRxHER and EGFRxHER3 bispecific antibodies can be directed by combining a portion of the heterodimerization domain located in the heavy chain forming the EGFR variable domain (3) and a compatible portion located in the heavy chain forming the HER2 and HER3 binding domain (2).

A heavy chain having a heavy chain variable region together with a light chain forming a variable domain that binds an antigen (such as EGFR, HER2 or HER 3) is also referred to herein as an EGFR heavy chain, HER2 heavy chain, and the like. In a preferred embodiment of the invention, the CH 3-region of the heavy chain of a first antibody and/or a second antibody is engineered to promote heterodimerization of an EGFR heavy chain with a HER2 heavy chain and an EGFR heavy chain with a HER3 heavy chain. In a preferred embodiment, the engineering use to promote heterodimerization is previously described at residue DEKK in U.S. Pat. nos. 9,248,182, 9,358,286, 9,248,182 and 9,758,805.

In certain embodiments, binding of such antibodies of the composition to EGFR blocks binding of EGF to EGFR and/or wherein binding of such antibodies of the composition to HER3 blocks binding of neuregulin-1 (nrg) to HER 3. In a preferred embodiment, binding of such antibodies of the composition to EGFR blocks binding of EGF to EGFR, while binding of antibodies of the composition to HER3 blocks binding of neuregulin-1 (NRG) to HER 3.

A variable domain that binds to the extracellular portion of EGFR preferably comprises a heavy chain variable region comprising a CDR1 sequence NYAMN, a CDR2 sequence WINANTGDPTYAQGFTG, and a CDR3 sequence ERFLEWLHFDY, or a variant of the heavy chain variable region comprising substitutions, deletions, and/or insertions of 1,2, or 3 amino acids in the CDRs.

A variable domain that binds to the extracellular portion of HER2 preferably comprises a heavy chain variable region comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGSNKYYADSVKG, and a CDR3 sequence DYYRRTARAGFDY, or a variant of the heavy chain variable region comprising substitutions, deletions, and/or insertions of 1,2, or 3 amino acids in the CDRs.

A variable domain that binds to the extracellular portion of HER3 preferably comprises a heavy chain variable region comprising a CDR1 sequence GYYMH, a CDR2 sequence WINPNSGGTNYAQKFQG, and a CDR3 sequence DHGSRHFWSYWGFDY, or a variant of the heavy chain variable region comprising substitutions, deletions, and/or insertions of 1,2, or 3 amino acids in the CDRs.

In a preferred embodiment, the composition comprises two bispecific antibodies, wherein a first of such bispecific antibodies comprises a variable domain that binds to the extracellular portion of EGFR comprising a heavy chain variable region comprising a CDR1 sequence NYAMN, a CDR2 sequence WINANTGDPTYAQGFTG, and a CDR3 sequence ERFLEWLHFDY, or a variant of the heavy chain variable region comprising a substitution, deletion, and/or insertion of 1, 2, or 3 amino acids in the CDRs. In a preferred embodiment, a first and a second of such bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR comprising a heavy chain variable region comprising a CDR1 sequence NYAMN, a CDR2 sequence WINANTGDPTYAQGFTG, and a CDR3 sequence ERFLEWLHFDY, or a variant of the heavy chain variable region comprising substitutions, deletions, and/or insertions of 1, 2, or 3 amino acids in the CDRs.

In a preferred embodiment, a first and a second of such bispecific antibodies comprise a variable domain that binds to the extracellular portion of HER2, preferably comprising a heavy chain variable region comprising a CDR1 sequence NYAMN, a CDR2 sequence WINANTGDPTYAQGFTG and a CDR3 sequence ERFLEWLHFDY, or a variant comprising 1,2 or 3 amino acids substituted, deleted and/or inserted in said CDRs, and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, preferably comprising a heavy chain variable region comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGSNKYYADSVKG and a CDR3 sequence DYYRRTARAGFDY, or a variant comprising 1,2 or 3 amino acids substituted, deleted and/or inserted in said CDRs, and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, preferably comprising a CDR3 sequence WINPNSGGTNYAQKFQG and a variable domain comprising a CDR3 sequence comprising a CDR3 sequence 5224 and a variant comprising a deletion or insertion of amino acids, or a variant comprising a CDR3 sequence DYYRRTARAGFDY.

In a preferred embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of HER2, the variable domain preferably comprising a heavy chain variable region comprising a CDR1 sequence NYAMN, a CDR2 sequence WINANTGDPTYAQGFTG and a CDR3 sequence ERFLEWLHFDY, or a variant of the heavy chain variable region comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids in said CDRs, and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain preferably comprising a heavy chain variable region comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGSNKYYADSVKG and a CDR3 sequence DYYRRTARAGFDY, or a variant of the heavy chain variable region comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids in said CDRs, and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER3, the variable domain preferably comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGSNKYYADSVKG and a CDR3 sequence DYYRRTARAGFDY, or a variant of the variable domain comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids in said CDRs, a variable domain comprising a CDR 24, a CDR3 sequence and/or a variant of the variable domain comprising substitution, deletion and/or insertion of amino acids in said CDRs 2.

Conservative variations of 1,2 or 3 amino acids from the CDR sequences detailed are allowed, while retaining the same type of binding activity (in type, not necessarily in number). Thus, the heavy chain CDR1, CDR2 and CDR3 sequences preferably contain sequences that deviate from such detailed CDR sequences by no more than 3 (preferably no more than two, more preferably no more than one) amino acids. In certain embodiments, the heavy chain CDR1, CDR2, and CDR3 sequences are identical to such detailed CDR sequences.

In certain embodiments, the EGFR variable domain comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the EGFR V _H region set forth in fig. 7 or 8. Preferably the EGFR V _H region of MF3755 in FIG. 7 or FIG. 8.

In certain embodiments, the EGFR variable domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% (preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%) identical or 100% identical to an amino acid sequence of the EGFR V _H region set forth in fig. 7 or 8. Preferably the EGFR V _H region of MF3755 in FIG. 7 or FIG. 8.

For example, in certain embodiments, the human EGFR-binding heavy chain variable region of the bispecific antibody may have 0-10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions, or a combination of these in sequences of the heavy chain variable region other than the 3 CDR sequences. In certain embodiments, for the indicated amino acid sequences, the heavy chain variable region comprises from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1, and preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination of these.

In certain embodiments, the EGFR variable domain comprises a heavy chain variable region comprising an amino acid sequence from an EGFR V _H region selected from fig. 7 or 8. Preferably the EGFR V _H region of MF3755 in FIG. 7 or FIG. 8.

In some embodiments, the HER2 variable domain comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the HER 2V _H region set forth in fig. 7 or 8 (preferably the HER 2V _H region of MF2032 in fig. 7 or 8).

In some embodiments, the HER2 variable domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% (preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%) identical or 100% identical to an amino acid sequence of a HER2V _H region set forth in fig. 7 or 8 (preferably the HER2V _H region of MF2032 in fig. 7 or 8).

For example, in certain embodiments, the heavy chain variable region of the bispecific antibody that binds human HER2 may have 0-10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions, or a combination of these in sequences of the heavy chain variable region other than the 3 CDR sequences. In certain embodiments, for the indicated amino acid sequences, the heavy chain variable region comprises from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1, and preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination of these.

In certain embodiments, the HER2 variable domain comprises a heavy chain variable region comprising an amino acid sequence selected from MF1849 or MF2032 (preferably MF2032 of fig. 7 or 8) of fig. 7 or 8.

In some embodiments, the HER3 variable domain comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 comprising the V _H region of MF3178 of fig. 7 or fig. 8.

In some embodiments, the HER3 variable domain comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% (preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%) identical or 100% identical to the amino acid sequence of MF3178 set forth in fig. 7 or 8.

For example, in certain embodiments, the heavy chain variable region of the bispecific antibody that binds human HER3 may have 0-10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions, or a combination of these in sequences of the heavy chain variable region other than the 3 CDR sequences. In certain embodiments, for the indicated amino acid sequences, the heavy chain variable region comprises from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1, and preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination of these.

In certain embodiments, the HER3 variable domain comprises a heavy chain variable region comprising the amino acid sequence from MF3178 in fig. 7 or 8.

In a preferred embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR, the variable domain comprising a heavy chain variable region comprising an amino acid sequence from an EGFR V _H region selected from fig. 7 or 8, or a variant thereof, preferably the EGFR V _H region of MF3755 of fig. 7 or 8, or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs), and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprising an amino acid sequence selected from MF1849 or MF2032 of fig. 7 or 8, preferably an amino acid sequence of MF2032 of fig. 7 or 8, wherein the variant comprises a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs), and wherein the variable domain comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprises a variable domain comprising an amino acid sequence selected from MF1849 or MF2032 of fig. 7 or 8, preferably an amino acid sequence of MF2032, wherein the variant comprises a substitution, deletion and/or insertion of 1, 2, and/or a variant thereof, wherein the variant comprises a variant of amino acid sequence selected from MF1849 or MF2032, and/or amino acid sequence of said variant thereof is selected from the amino acid sequence of either amino acid sequence of the variant of either amino acid sequence of the antibody.

In a preferred embodiment, a first and a second of the bispecific antibodies comprise a variable domain comprising a heavy chain variable domain comprising an amino acid sequence from MF3755 in fig. 7 or 8 or a variant thereof comprising 1,2 or 3 amino acids (not preferably not located in said CDRs) substituted, deleted and/or inserted, bound to the extracellular portion of HER2, and wherein the second bispecific antibody further comprises a variable domain comprising a heavy chain variable domain comprising an amino acid sequence from MF2032 in fig. 7 or 8 or a variant thereof comprising 1,2 or 3 amino acids (not preferably not located in said CDRs) substituted, deleted and/or inserted, and wherein the second bispecific antibody further comprises a variable domain comprising an amino acid sequence from MF2032 in fig. 7 or 8 or a variant thereof comprising 1,2 or 3 amino acids (not preferably not located in said CDRs) bound to the extracellular portion of HER3, bound to the extracellular portion of HER2, and wherein the variable domain comprises a variable domain comprising a amino acid sequence from MF2032 in fig. 7 or 8 or a variant thereof comprising a substitution, deletion and/or an amino acid sequence not located in said CDRs.

In one embodiment, a first and a second of the bispecific antibodies comprise a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF4280 in fig. 7 or 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs) that binds to the extracellular portion of HER2, and wherein the second bispecific antibody further comprises a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF1849 in fig. 7 or 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs), and wherein the second bispecific antibody further comprises a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF 7 or 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs) that binds to the extracellular portion of HER 3.

In one embodiment, a first and a second of the bispecific antibodies comprise a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF2032 in fig. 7 or 8 or a variant thereof comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs) or comprising an amino acid sequence from MF4280 in fig. 7 or 8, and wherein the second bispecific antibody further comprises a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF2032 in fig. 7 or 8 or a variant thereof comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs) or a variant thereof comprising a variant thereof binding to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising amino acid sequence from MF 7 or 8 or a variant thereof comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs) or a variant thereof.

In one embodiment, a first and a second of the bispecific antibodies comprise a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF1849 in fig. 7 or 8 or a variant thereof comprising substitution, deletion and/or insertion of 1,2 or 3 amino acids (not preferably not located in said CDRs) or comprising an amino acid sequence from MF4003 in fig. 7 or 8, and wherein the second bispecific antibody further comprises a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF1849 in fig. 7 or a variant thereof comprising substitution, deletion and/or insertion of 1,2 or 3 amino acids (not preferably not located in said CDRs) bound to the extracellular portion of HER2, and wherein the second bispecific antibody further comprises a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF 7 or 8 or a variant thereof comprising substitution, deletion and/or insertion of 1,2 or 3 amino acids (not preferably not located in said CDRs) bound to the extracellular portion of HER 3.

In one embodiment, a first and a second of the bispecific antibodies comprise a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF2032 in fig. 7 or 8 or a variant thereof comprising substitution, deletion and/or insertion of 1,2 or 3 amino acids (not preferably not located in said CDRs) or comprising an extracellular portion of MF4003 from fig. 7 or 8, and wherein the second bispecific antibody further comprises a variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF2032 in fig. 7 or 8 or a variant thereof comprising substitution, deletion and/or insertion of 1,2 or 3 amino acids (not preferably not located in said CDRs) or a variant thereof comprising a variant binding to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF 7 or 8 or a variant thereof comprising substitution, deletion and/or insertion of 1,2 or 3 amino acids (not preferably not located in said CDRs) or a variant thereof.

Exemplary EGFR heavy chain variable regions are described in WO 2015/130172 and PCT/NL2018/050537, such patents being incorporated herein by reference. An exemplary HER2 heavy chain variable region is described in WO 2015/130173, which is incorporated herein by reference. Exemplary HER3 heavy chain variable regions are described in WO 2015/130172 and WO 2015/130173, such patents being incorporated herein by reference.

Additional variants of the disclosed amino acid sequences that retain EGFR, HER2, or HER3 binding may be obtained, for example, from phage display libraries containing rearranged human IGKVl-39/IGKJl V _L regions (De Kruif et al Biotechnol bioeng.2010 (106) 741-50), as well as a collection of amino acid substitutions into the V _H region of the amino acid sequence of an EGFR, HER2, or HER 3V _H region disclosed herein, as previously described. Phages encoding Fab regions that bind EGFR, HER2 or HER3 can be selected and analyzed by flow cytometry and sequenced to identify variants with amino acid substitutions, insertions, deletions or additions and retain antigen binding.

The invention further provides a binding moiety that specifically binds to an extracellular portion of EGFR and an extracellular portion of HER 2. The binding moiety preferably comprises one variable domain that binds EGFR and one variable domain that binds HER 2. The variable domain that binds EGFR is preferably an EGFR variable domain as described herein. The HER2 binding variable domain is preferably a HER2 variable domain as described herein. Preferably both the EGFR and the HER2 variable domain are variable domains as described herein.

The invention further provides a binding moiety that specifically binds to an extracellular portion of EGFR and an extracellular portion of HER 3. The binding moiety preferably comprises one variable domain that binds EGFR and one variable domain that binds HER 3. The variable domain that binds EGFR is preferably an EGFR variable domain as described herein. The HER3 binding variable domain is preferably a HER3 variable domain as described herein. Preferably both the EGFR and the HER3 variable domain are variable domains as described herein.

The invention further provides a composition comprising a binding moiety that specifically binds to the extracellular portion of EGFR and the extracellular portion of HER2, and a binding moiety that specifically binds to the extracellular portion of EGFR and the extracellular portion of HER 3.

The binding moiety as described herein is preferably an antibody, preferably a multispecific antibody, preferably a bispecific antibody.

The EGFR variable domain, HER2 variable domain, and light chain variable region (VLs) of the HER3 variable domain of the binding moiety (such as such bispecific antibody) may be the same as the V _L region of the parent EGFR monospecific antibody, the V _L region of the parent HER2 monospecific antibody, and/or the V _L region of the parent HER3 monospecific antibody. Alternative V _L regions may be used for one or more of such V _H/V_L region combinations, provided that such variable domains retain binding to EGFR, HER2, or HER3, respectively.

In certain embodiments, the V _L region of the EGFR variable domain, the HER2 variable domain, and the HER3 variable domain are similar. In certain embodiments, the V _L region located in all of the variable domains of the binding portion is the same.

In certain embodiments, the light chain variable region of 1,2, 3 or more variable domains of the binding moiety of the invention comprises one common light chain variable region. In certain embodiments, the common light chain variable region of 1,2, 3 or more variable domains comprises one reproductive variable region V-segment. In certain embodiments, the light chain variable region of 1,2, 3 or more variable domains comprises kappa light chain V-segment igvκ1-39 x 01.IgV kappa 1-39 is a shorthand for immunoglobulin variable kappa 1-39 genes. The gene is also called kappa variable 1-39, IGKV139 and IGKV1-39. The external identifier for this gene is HGNC:5740;Entrez Gene:28930;Ensembl:ENSG00000242371. The amino acid sequence for this V-region is provided in sequence 10 of fig. 7. The V-regions may also be combined with one of the 5J-regions. Preferred J-regions are jk1 and jk5, and the linked sequences are denoted IGKV1-39/jk1 and IGKV1-39/jk5, alternatively named IgV kappa 1-39/IGJ kappa 1-01 or IgV kappa 1-39/IGJ kappa 5-01 (according to the nomenclature of the IMGT database Global website at imgt.org). In certain embodiments, the light chain variable region of one or both V _H/V_L binding regions comprises kappa light chain igvk1-39×01/igjk1×01 or igvk1-39×01/igjk1×05 (sequence 11 or sequence 12, respectively, of fig. 7).

In certain embodiments, the light chain variable region of 1,2,3 or more variable domains of the binding moiety of the invention comprises an LCDR1 comprising the amino acid sequence QSISY (SEQ ID NO: 7 of FIG. 7), an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 9 of FIG. 7) (i.e., such CDRs according to IMGT, IGKV 1-39). In certain embodiments, the light chain variable region of 1,2,3 or more variable domains of the binding moiety of the invention comprises an LCDR1 comprising the amino acid sequence QSISY (SEQ ID NO: 7 of FIG. 7), an LCDR2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 8 of FIG. 7), and an LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 9 of FIG. 7).

In certain embodiments, 1, 2, 3 or more variable domains of the binding moiety of the invention comprise a light chain variable region comprising an amino acid sequence that is at least 90% (preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%) identical or 100% identical to the amino acid sequence of sequence 11 set forth in fig. 7. In certain embodiments, 1, 2, 3 or more variable domains of the binding moiety of the invention comprise a light chain variable region comprising an amino acid sequence that is at least 90% (preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%) identical or 100% identical to the amino acid sequence of sequence 11 set forth in fig. 7.

For example, in certain embodiments, for sequence 11 in fig. 7 or sequence 12 in fig. 7, the variable light chain of 1, 2, 3 or more variable domains of the binding moiety of the invention may have 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. In certain embodiments, for the indicated amino acid sequences, the light chain variable region of 1, 2, 3 or more variable domains of the binding moiety of the invention comprises from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1 and preferably 0 amino acid insertions, deletions, substitutions, additions or a combination of these.

In other embodiments, the light chain variable region of 1,2, 3 or more variable domains of the binding moiety of the invention comprises the amino acid sequence of sequence 11 in fig. 7 or sequence 12 in fig. 7. In certain embodiments, all of the variable domains of the binding moieties of the invention comprise the same V _L region. In one embodiment, V _L of all variable domains of the binding portion of the invention comprises the amino acid sequence of sequence 11 set forth in figure 7. In one embodiment, V _L of all variable domains of the binding portion of the invention comprises the amino acid sequence set forth in sequence 12 in fig. 7 or sequence 12 in fig. 7.

The multispecific antibodies (such as bispecific antibodies) disclosed herein may be provided in a number of formats. Many different types of multispecific antibodies are known in the art and have been reviewed by Kontermann (Drug Discov Today,2015Jul;20 (7): 838-47; MAbs,2012 Mar-Apr;4 (2): 182-97) and Spiess et al (Alternative molecular formats and therapeutic applications for bispecific antibodies.Mol.Immunol.(2015)http：)dx.doi.org/10.1016/j.molimm.2015.01.003), each of which is incorporated herein by reference. For example, a multispecific antibody version (such as a bispecific antibody version that is not a typical antibody with two variable domains) has at least one variable domain comprising one heavy chain variable region and one light chain variable region. The variable domain may be linked to a single chain Fv-fragment, monomer, a VHH and a Fab-fragment which provide a second binding activity.

In certain embodiments, such multispecific antibodies used in the methods provided herein are generally of the human IgG subtype (e.g., igG1, igG2, igG3, igG4, for example). In certain embodiments, such antibodies are of the human IgG1 subtype. Full length IgG antibodies are favored because of their favorable half-life for low immunogenicity. Such multispecific antibodies may have two different heavy chains comprising one heterodimerization domain. Thus, in certain embodiments, such EGFR/HER2 and EGFR/HER3 bispecific antibodies are full length IgG molecules. In one embodiment, such EGFR/HER2 and EGFR/HER3 bispecific antibodies are full length IgG1 molecules.

Thus, in certain embodiments, such multispecific EGFR/HER2 and EGFR/HER3 antibodies comprise a crystallizable fragment (Fc). The Fc region of such multispecific antibodies is preferably comprised of one human constant region. A constant region or Fc of such a multispecific antibody may contain one or more (preferably no more than 10, preferably no more than 5) amino acid differences to the constant region of a naturally occurring human antibody. For example, in certain embodiments, each Fab-arm of such bispecific antibodies may further comprise an Fc-region comprising modifications that promote the formation of the bispecific antibody, modifications that affect Fc-mediated effector function, and/or other features described herein.

In preferred embodiments, the multispecific (preferably bispecific) full-length IgG antibody has a lower hinge and/or CH2 domain such that the bispecific IgG antibody interaction with an Fc gamma (fcγ) receptor is enhanced. Antibody-dependent cellular cytotoxicity (also referred to as ADCC activity) of an antibody can often be improved when the antibody itself has a low ADCC activity. This can be achieved, for example, by removing fucose residues from the glycosylated portion of the antibody. One technique for enhancing ADCC by nonfucosylation (afucosylation) is described, for example, in Junttila, t.t., k.parsons, et al (2010)."Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer."Cancer Research 70(11)：4481-4489). The multispecific antibodies as described herein are preferably non-fucosylated. Preferably the two multispecific antibodies are nonfucosylated. Other strategies have been reported to achieve ADCC enhancement, including, for example, glycation engineering (Kyowa Hakko/Biowa, glycArt (Roche) and Eureka Therapeutics) and mutation induction (mutagenesis) (Xencor and Macrogenics), all of which seek to improve Fc binding to low affinity activated fcγriiia (low-AFFINITY ACTIVATING FC γriiia) and/or to reduce binding to low affinity inhibited fcγriib (low affinity inhibitory Fc γriib).

Bispecific antibodies are typically produced by cells that express nucleic acids encoding the antibodies. Thus, in certain embodiments, a method for producing a composition comprising a multispecific antibody that binds EGFR and HER2 and a multispecific antibody that binds EGFR and HER3 is provided, the method comprising providing a cell having:

-a nucleic acid encoding a polypeptide comprising a heavy chain forming together with the common light chain a variable domain that binds to the extracellular portion of HER 2;

-A nucleic acid encoding a polypeptide comprising the common light chain;

Wherein two or more of said nucleic acids may or may not be physically linked, and wherein each of the nucleic acids further comprises an expression control sequence to allow expression of said encoded heavy and light chains in the cell, and wherein the method further comprises culturing the cell to allow expression of the heavy and light chains, and optionally, collecting the two or more antibodies. The two or more antibodies may be collected from the cells and/or supernatant.

The level at which such corresponding strands are generated in the cell may be tailored, for example, by selection of appropriate expression control sequences or by selection of the number of copies of the nucleic acid that are introduced, or both. In a preferred embodiment, a collection of cells is provided with the nucleic acid, and an appropriate level of clones exhibiting such corresponding strands are selected. The clones are also typically selected based on the number of antibodies produced. In one embodiment, the method includes providing the nucleic acid to a collection of cells and selecting from the collection a cell having a desired expression ratio of the respective heavy and light chains. In certain embodiments, the two or more binding moieties are antibodies, preferably bispecific antibodies. In certain embodiments, the cells preferably produce a substantially equimolar amount of the two or more binding moieties. In other embodiments, the cell produces more of one binding moiety than the other binding moiety of the two or more binding moieties.

The invention also provides a cell comprising:

-A nucleic acid encoding a polypeptide comprising the common light chain;

The invention further provides a container comprising a nucleic acid comprising:

-A nucleic acid encoding a polypeptide comprising the common light chain;

Wherein two or more of the nucleic acids may or may not be physically linked, and wherein each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in a cell.

The cell that produces the binding moiety is preferably an animal cell, more preferably a mammalian cell, even more preferably a primate cell, most preferably a human cell. A suitable cell is any cell that is capable of including, and preferably producing, the binding moiety as described herein (preferably such multispecific antibodies and preferably such bispecific antibodies).

Suitable cells for antibody production are known in the art and include a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, an NS0 cell, a HEK293 cell, a 293-F cell, or a PER-C6 cell. Various institutions and companies have developed cell lines for the large-scale production of antibodies (e.g., for clinical use). Non-limiting examples of such cell lines are CHO cells, NS0 cells or per.c6 cells. In a particularly preferred embodiment, the cell is a human cell. Preferably, a cell transformed with an adenovirus E1 region or a functional equivalent thereof. In a particularly preferred embodiment, the cell is a CHO cell or a variant thereof. Preferably, a variant that utilizes a Glutamine Synthetase (GS) vector system for expression of an antibody. In a preferred embodiment, the cell is a CHO cell.

In certain embodiments, the cell exhibits 3 distinct heavy chains and at least one light chain. In a preferred embodiment, the cells exhibit "common light chains" as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, together with rearranged human IGKV1 39/IGKJ1 (huV kj1 39) light chains, the corresponding VH region is transfected into an expression vector using methods known in the art for the generation of bispecific IgG (WO 2013/157954, which is incorporated herein by reference). The huV kappa 1 39 was previously shown to be capable of pairing with more than one heavy chain, thus resulting in antibodies with disparate specificities, which facilitate the production of bispecific molecules (WO 2009/157771).

Antibody-producing cells that exhibit one common light chain and an equal number of two heavy chains typically produce 50% bispecific antibodies and 25% of each such monospecific antibody (i.e., having the same combination of heavy and light chains). Several methods have been published to favor the generation of bispecific antibodies over the generation of corresponding monospecific antibodies. This is typically achieved by modifying the constant regions of the heavy chains such that they favor heterodimerization (i.e., dimerization of the heavy chain in combination with another heavy/light chain) over homodimerization (homodimerization). In a preferred embodiment, the bispecific antibodies of the invention comprise two different immunoglobulin heavy chains with compatible heterodimerization domains. A wide variety of compatible heterodimerization domains have been described in the art. Such compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. The art describes various ways in which such heterodimerization of the heavy chain (hetero-dimerization) may be achieved.

A preferred method for generating a multispecific antibody as described herein is disclosed in US 9,248,181 and US 9,358,286. Specifically, the preferred mutations used to generate substantially only bispecific full length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) located in the first CH3 domain (the "KK-variant" heavy chain) and the amino acid substitutions L351D and L368E located in the second domain (the "DE-variant" heavy chain), or vice versa. As previously described, the DE-variant and KK-variant preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE-variant heavy chains (DEDE homodimer) or KK-variant heavy chains (KKKK homodimer) hardly occurs due to strong repulsion between charged residues in the CH3-CH3 interface between identical heavy chains. The introduction of a further heavy chain with a DE-or KK-variant heavy chain allows the production of a further DEKK bispecific molecule. A newly introduced DE-heavy chain (DE ²) may be associated with the existing KK heavy chain. The cells thus produced two bispecific antibodies, one DE ¹ KK and one DE ² KK diabody. If a new KK heavy chain (KK ²) is introduced instead of the new DE heavy chain, bispecific antibodies with a combination of DEKK ¹ and DEKK ² are generated. The level at which different antibodies can be produced by the cell can be adjusted by adjusting the relative manifestation of such HER2 and HER3 chains with respect to each other. The light chain is typically generated sufficiently to reduce the level of a single heavy chain, while the level at which such EGFR chains are generated is typically sufficient to allow for efficient pairing with such HER2 chains, HER3 chains.

Thus, in one embodiment, the heavy chain/light chain combination comprising the variable domain that binds EGFR comprises a DE variant of the heavy chain. In this embodiment, the heavy chain/light chain combination comprising a variable domain that binds HER2 and the heavy chain/light chain combination comprising a variable domain that binds HER3 comprises a KK variant of the heavy chain.

Candidate EGFR/HER2 or EGFR/HER3 IgG bispecific antibodies can be tested for binding using any suitable assay. For example, binding to membrane-expressed EGFR, HER2 or HER 3. This is typically done on a cell that does not normally express the EGFR, HER2 or HER3 or is transformed to express one of EGFR, HER2 or HER 3. Binding of the antibody to the transformed cells, but not to the untransformed cells, indicates specific binding of the antibody. Binding can be assessed by, for example, flow cytometry (according to FACS procedures as previously described in WO 2015/130172, PCT/NL2018/050537, and WO 2015/130173). The corresponding monospecific antibodies can be used together as a control, in addition to an unrelated IgG1 isotype control mAb.

Binding moieties, such as antibodies, can be collected from cells and/or supernatant of a cell culture. Typically, they are collected from supernatant fluid over such producer cells. Binding moieties (such as antibodies) can be purified from the supernatant. Many purification methods are known in the art. Some of the more common methods use affinity purification.

Antibodies produced by a cell may be purified by affinity purification. This is advantageously accomplished by protein a extraction. The eluted antibodies can be tested by ELISA for the presence of specific binding properties (i.e., binding to EGFR, HER2, and HER 3). The antibody preparation can be further analyzed by ion exchange column chromatography. Individual bispecific antibodies can be purified from each other by routine techniques, such as using ion exchange chromatography. The presence of the corresponding bispecific antibodies can also be analyzed by ELISA. Binding of the preparation to HER2 and washing should remove all EGFR/HER3 antibodies. Staining with labeled soluble HER3 does not give a signal, whereas staining with labeled soluble EGFR does. Binding of the preparation to HER3 and washing should remove all EGFR/HER2 antibodies. Staining with labeled soluble HER2 does not give a signal, whereas staining with labeled soluble EGFR does. Binding of the preparation to EGFR and washing should not remove EGFR/HER2 and EGFR/HER3 antibodies. Staining with labeled soluble HER2 furthermore staining with labeled soluble HER3 should give a signal. The level of such corresponding antibodies in a preparation can also be estimated using such an ELISA using appropriate controls with known levels of such mono-bispecific antibodies.

A method for producing a composition comprising two or more bispecific antibodies, the method comprising:

-providing cells with nucleic acids encoding the bispecific antibodies;

-culturing the cells;

-harvest clarification (harvest clarification);

collecting such bispecific antibodies from the culture, and

-Separating the bispecific antibody produced from the half-antibody by ion exchange chromatography (IEX);

The method is characterized in that such bispecific antibodies exhibit similar IEX residence times under the IEX conditions used, preferably a deviation of 10% or less from the average value of the residence times of such individual antibodies. In one embodiment, such antibodies are selected to have an IEX residence time that deviates by 10% or less from the average of the residence times of such individual antibodies under the IEX conditions used. Such antibodies may first be purified from other proteins within the culture. This is usually done by affinity purification, preferably by protein a extraction. Such bispecific antibodies are preferably selected to have half antibodies with residence times outside the range spanned by the residence times of such antibodies. Where a combination of bispecific antibodies is generated and monospecific antibodies are not envisioned, such bispecific antibodies are preferably selected to have a residence time different from the residence time of such monospecific antibodies. Such monospecific antibody residence times in this embodiment preferably fall outside the range spanned by the residence times of such corresponding bispecific antibodies. The cells located in the culture preferably simultaneously express the 3 heavy chains, wherein the heavy chains include a CH3 heterodimerization domain that promotes the formation of EGFR/HER2 and EGFR/HER3 heavy chain heterodimerization. The cells preferably represent one common light chain in fig. 7. Such bispecific antibodies in one embodiment have isoelectric points (isoelectric points, PI) that are similar, and preferably do not differ by more than 0.5 units from the average PI of the at least two bispecific antibodies.

The affinity of EGFR, HER2, and HER3 FABs of candidate EGFR/HER2 or EGFR/HER bispecific antibodies for their targets can be measured by surface plasmon resonance (surface plasmon resonance, SPR) techniques using one BIAcore T100. An anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat.nr.555784) was coupled to the surface of one CM5 sensor chip using free amine chemistry (NHS/EDC). The bsAb is then captured on the sensor chip. Subsequently, recombinant purified antigens human EGFR-Fc, HER2-Fc and HER3-Fc proteins were run on the sensor surface over a range of concentrations to measure the binding and separation rate (on-and off-rates). After each cycle, the sensor surface is regenerated by a pulse of HCl, and the bsAb is captured again. From the obtained sensorgrams (sensorrams), binding and separation rates and affinity values for binding to human EGFR, HER2 and HER3 were determined using BIAevaluation software.

The invention also provides a composition as described herein for use in the treatment of a cancer. In embodiments, the cancer is a physical epithelial cancer. Preferably, the composition is used in a cancer that expresses EGFR, HER2 and/or HER 3. The composition is preferably used for pancreatic cancer, colorectal cancer, head and neck cancer, epithelial ovarian cancer, epithelial fallopian tube cancer, epithelial peritoneal cancer, bladder cancer or prostate cancer. In embodiments, the cancer treated by using the composition is an advanced cancer. The composition is preferably used for metastatic cancer. The composition is preferably used for metastatic pancreatic cancer, metastatic colorectal cancer, metastatic head and neck cancer, metastatic epithelial ovarian cancer, metastatic epithelial fallopian tube cancer, metastatic epithelial peritoneal cancer, metastatic bladder cancer or metastatic prostate cancer. In embodiments, the composition is preferably used for cancers that are gastric, lung, breast or esophageal cancers. Preferably, the composition is used for metastatic gastric cancer, metastatic lung cancer, metastatic breast cancer or metastatic esophageal cancer.

The invention further provides two or more binding moieties for use in the treatment of cancer, each comprising a variable domain that binds to an extracellular portion of EGFR, wherein a first one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER2 and a second one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER 3. Also provided is a product comprising two or more binding moieties, each comprising a variable domain that binds to an extracellular portion of EGFR, wherein a first one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER2 and a second one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER3, for use in treating cancer as a combination for simultaneous, separate or sequential use.

The cancer treated by embodiments of the present invention is preferably a cancer as indicated elsewhere herein. The cancer preferably comprises cells with EGFR-mutations that render the cells resistant to treatment with Tyrosine Kinase Inhibitors (TKIs). In certain embodiments, the cancer comprises cells with EGFR R521K polymorphism. The cancer to be treated with the treatment methods of an invention described herein is preferably gastric cancer, lung cancer or esophageal cancer. In a further embodiment, the invention provides a method for treating an individual having cancer or at risk of recurrence, exacerbation of cancer, the method comprising administering to the individual in need of such treatment two or more binding moieties each comprising a variable domain that binds to an extracellular portion of EGFR, wherein a first one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER2 and a second one of the binding moieties comprises a variable domain that binds to an extracellular portion of HER 3.

As used herein, the terms "individual" and "patient" are used interchangeably and refer to a mammal, such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc. (e.g., a patient with a cancer, such as a human patient).

The terms "treatment", and "treatment", as used herein, refer to any type of intervention or procedure performed on the subject or administration of an active agent or a combination of active agents to the subject with the aim of reversing, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicator associated with a disease.

As used herein, "effective treatment" or "positive therapeutic response" means a treatment that produces a beneficial effect, such as an improvement in at least one symptom of a disease or disorder (e.g., cancer). A beneficial effect may be in the form of an improvement over baseline, including an improvement to a measurement or observation made prior to initiation of therapy according to the present methods. For example, a beneficial effect may be in the form of slowing, stabilizing, halting or reversing the progression of a cancer in an individual at any clinical stage, as evidenced by the reduction or elimination of a clinical or diagnostic symptom of the disease or a marker of cancer. Effective treatment may, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent metastasis of a tumor, slow or arrest tumor growth, and/or prevent or delay tumor recurrence or progression.

The term "effective amount" or "therapeutically effective amount" means an amount of an agent or combination of agents that provides the desired biological, therapeutic, and/or prophylactic result. The result may be a reduction, improvement, alleviation, diminishment, delay and/or diminishment of one or more of the signs, symptoms or causes of a disease, or any other desired alteration of a biological system. In certain embodiments, an effective amount is an amount sufficient to delay tumor progression. In certain embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. An effective amount of the medicament or composition may (i) reduce the number of cancer cells, (ii) reduce the size of the tumor, (iii) inhibit, delay, slow down and may stop infiltration of cancer cells into peripheral organs to some extent, (iv) inhibit tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay the occurrence and/or recurrence of the tumor, and/or (vii) alleviate one or more symptoms associated with the cancer to some extent. In one embodiment, an "effective amount" is the amount of a composition of the invention to achieve a reduction in a cancer (e.g., a reduction in the number of cancer cells) or to slow the progression of a cancer. An effective shu amount of the combination therapy is administered according to such methods described herein in an "effective regimen" (which means a combination of the binding moieties as indicated herein), wherein the order of administration and frequency of dosage is sufficient to achieve treatment.

As used herein, the terms "synergy", "therapeutic synergy (therapeutic synergy)" and "synergistic effect (SYNERGISTIC EFFECT)" mean a phenomenon in which treatment of a patient with a combination of the binding moieties as indicated herein (e.g., a composition comprising binding moieties that bind EGFR and HER2 and a binding moiety that binds EGFR and HER 3) reveals a therapeutically superior outcome over that achieved by the individual components of the combination when used alone (see, e.g., t.h.corbett et al, 1982,Cancer Treatment Reports,66,1187). In this case, a therapeutically advantageous outcome includes one or more of (a) an increase in therapeutic response that is greater than the sum of the individual effects of each binding moiety itself at the same dose as in the combination, (b) a decrease in the dose of one or more agents in the combination without a decrease in therapeutic efficacy, (c) a decrease in the incidence of adverse events while receiving a therapeutic benefit that is equal to or greater than the therapeutic benefit of each agent for monotherapy at the same dose as in the combination, (d) a decrease in dose limiting toxicity while receiving a therapeutic benefit that is greater than the therapeutic benefit of the monotherapy of each agent, (e) a delay or minimization of induction of drug resistance.

In a xenograft model (xenograft models), a combination at its maximum tolerated dose, where each of such components would be present at a dose that does not normally exceed its respective maximum tolerated dose, exhibits therapeutic synergy when the reduction in tumor growth achieved by administration of the combination is greater than the magnitude of the reduction in tumor growth when the optimal components are administered alone. The synergy of a pharmaceutical combination can be determined, for example, according to the Combination Index (CI) theorem of Chou-Talalay (Chou et al, adv. Enzyme Regul.1984;22:27-55;Chou,Cancer Res.2010;70 (2): 440-446).

The invention further provides a composition of the invention for use in the treatment of cancer. The embodiments used are preferably for the treatment of a gastric cancer, colorectal cancer, colon cancer, gastroesophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, bright cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, melanoma, such as these. In one embodiment, the embodiment treats a cancer that is gastric cancer, lung cancer, or esophageal cancer. The use is preferably for the treatment of cancer, which is gastric cancer.

An invention described herein is useful in the treatment of a cancer, preferably a cancer that is tested for the presence of EGFR, HER2 and/or HER3 on cell membranes. This can be done by routine methods and is typically analyzed by immunohistochemistry.

The cancer preferably exhibits HER2. The cancer preferably also exhibits EGFR or HER3. The cancer preferably expresses EGFR. The cancer preferably also exhibits HER2 or HER3. The cancer preferably exhibits HER3. The cancer preferably also exhibits EGFR or HER2. In certain embodiments, the cells of the cancer and/or stromal cells (stroma cells) in the cancer treated by the invention disclosed herein exhibit an EGFR ligand, a HER3 ligand, or both. Expression of the ligand and receptor may thus provide a growth stimulus to the cells of the cancer. A combination of the invention is particularly suitable for the treatment of cancer comprising such cells.

In the treatment of the invention, expression of one of EGFR, HER2 and HER3 may delay escape of at least some tumors. Tumors that are targeted using a monospecific therapy may escape treatment by beginning to express another of EGFR, HER2 or HER3 or by expressing a ligand for a receptor.

Such cells, if they are present, can also be attacked by the binding moieties of the invention and thus can be removed before they grow up and diversify themselves. In one embodiment, the cancer is tested for the presence of a mutated EGFR. Many EGFR-positive tumors have a genetic mutation that renders the cells resistant to treatment with tyrosine kinase inhibitors.

The compositions of the invention are suitable for treating cancers with EGFR-mutations that render the cancer cells resistant to treatment with Tyrosine Kinase Inhibitors (TKIs). In one embodiment, the cancer comprises cells with EGFR 521K polymorphism. In certain embodiments, the cancer is known to be resistant to first generation TKI inhibitors (such as gefitinib and erlotinib).

Cancer treatments as indicated herein may be combined with a further cancer treatment. Such treatment may include further binding moieties such as an antibody and/or a cytostatic drug (cytostatic drug) or protein kinase inhibitor. The protein kinase inhibitor is preferably an inhibitor that is not an EGFR or HER3 tyrosine kinase inhibitor. Non-limiting examples of such further treatments include radiation therapy, chemotherapy, surgery, vascular growth inhibition therapy, and hyperthermia.

The compositions of the invention may be suitable for use in the treatment of a pair of cancers that are resistant to EGFR inhibition, wherein EGFR resistance is a result of HER2 and/or HER3 hypermanifestations.

The compositions of the invention may be suitable for use in the treatment of a pair of cancers that are resistant to HER2 inhibition, wherein HER2 resistance is a result of excessive manifestations of EGFR and/or HER 3.

The compositions of the invention may be suitable for use in the treatment of a pair of cancers that are resistant to HER3 inhibition, wherein HER3 resistance is a result of excessive manifestations of EGFR and/or HER 2.

TerminologyBy antibody, binding moiety, composition or product as described herein is meant the presence of more than one, and typically 10 or less, non-identical antibodies or binding moieties in a preparation, including the presence of a bispecific.One exemplary embodiment of (a) includes a combination of two types of double a combination of specific antibodies.

The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2, wherein the EFGR variable domain comprises a heavy chain variable domain comprising CDRs comprising heavy chain variable domain MF3755, MF4280, MF4003 or MF4016 in FIG. 8, or one of the heavy chain variable domain comprises a variant that replaces, deletes and/or inserts 1, 2 or 3 amino acids in said CDRs, and wherein the HER2 variable domain comprises a CDRs comprising heavy chain variable domain MF2032 or MF1849, or one of the heavy chain variable domain comprises a variant that replaces, deletes and/or inserts 1, 2 or 3 amino acids in said CDRs.

Also provided is a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2, wherein the EFGR variable domain comprises a heavy chain variable domain comprising CDRs comprising heavy chain variable domain MF3755 of fig. 8, or a variant of the heavy chain variable domain comprising 1,2, or 3 amino acids substituted, deleted, and/or inserted in said CDRs, and wherein the HER2 variable domain comprises a heavy chain variable domain comprising CDRs comprising heavy chain variable domain MF2032, or a variant of the heavy chain variable domain comprising 1,2, or 3 amino acids substituted, deleted, and/or inserted in said CDRs.

The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2, wherein the EFGR variable domain comprises a heavy chain variable domain comprising the amino acid sequence of heavy chain variable domain MF3755, MF4280, MF4003 or MF4016 in FIG. 8, or a variant of the heavy chain variable domain comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not in said CDRs), and wherein the HER2 variable domain comprises a heavy chain variable domain comprising the amino acid sequence of heavy chain variable domain MF2032 or MF1849, or a variant of the heavy chain variable domain comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not in said CDRs).

The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2, wherein the EFGR variable domain comprises a heavy chain variable domain comprising the amino acid sequence of heavy chain variable domain MF3755 in FIG. 8, or a variant of the heavy chain variable domain comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs), and wherein the HER2 variable domain comprises a heavy chain variable domain comprising the amino acid sequence of heavy chain variable domain MF2032, or a variant of the heavy chain variable domain comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs).

Also provided is a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2, wherein the EFGR variable domain comprises a heavy chain variable domain comprising CDRs comprising heavy chain variable domain MF3755 of fig. 8, or a variant of the heavy chain variable domain comprising 1,2, or 3 amino acids substituted, deleted, and/or inserted in said CDRs, and wherein the HER2 variable domain comprises a heavy chain variable domain comprising CDRs comprising heavy chain variable domain MF1849, or a variant of the heavy chain variable domain comprising 1,2, or 3 amino acids substituted, deleted, and/or inserted in said CDRs.

The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2, wherein the EFGR variable domain comprises a heavy chain variable domain comprising the amino acid sequence of heavy chain variable domain MF3755 in FIG. 8, or a variant of the heavy chain variable domain comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs), and wherein the HER2 variable domain comprises a heavy chain variable domain comprising the amino acid sequence of heavy chain variable domain MF1849, or a variant of the heavy chain variable domain comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs).

For the purposes of clarity and a brief description of the invention, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the invention may include embodiments having all or some of the described features in combination.

Examples

Cell strain

Hek293 cells and NCI-87 cells ]CRL-5822 ^TM), bxPC-3 (ATCC CRL-1687), bxPC-3-luc2, and CHO-K1 were maintained in growth medium supplemented with 10% heat inactivated Fetal Bovine Serum (FBS).

Bispecific antibody production

Bispecific antibodies were generated using the DEKK CH technology described above for efficient heterodimerization and formation of bispecific antibodies. As previously described (WO 2013/157954 A1), the CH3 technology utilizes charge-based point mutations located in the CH3 region to allow efficient pairing of two different heavy chain molecules.

One VH gene was transfected into one of two non-identical constructs of IgG1 vector. Depending on the binding partner, the VH is cloned into an IgG1 framework comprising a CH3 variant with heterodimerized variant "DE" or an IgG1 framework comprising a complementary CH3 heterodimerized variant "KK". In the case of a bi-or multispecific antibody in which two or more antibodies share a heavy chain, the shared chain preferably has the CH3 heterodimerization variant "DE" (also referred to as the DE-heavy chain), while the two or more unique heavy chains have the CH3 heterodimerization variant "KK" (also referred to as the KK-heavy chain).

Attached Hek293 cells were cultured in 6-well plates to a confluence of 80% (confluency). The cells were transiently transfected with a DNA-FUGENE mixture and further cultured. 7 days after transfection, supernatant was harvested and medium was refreshed. The supernatant was pooled and filtered through (0.22. Mu.M. Sterile supernatant was stored at 4 ℃) after transfection, suspension conditioned Hek293 cells were cultured in T125 flasks (T125 flasks) on a shaker dish until a density of 3.0X10. ⁶ cells/mL cells were seeded into each well of a 24 deep well culture dish at a density of 0.3-0.5X10. ⁶ viable cells/mL: PEl-MIX and further cultured 7 days after transfection, supernatant was harvested and filtered through 0.22 μm. Sterile supernatant was stored at 4 ℃.

Production of a pool of stable cell lines co-expressing two bispecific antibodies

CHO cells are transfected with 3 heavy chain constructs and a common light chain construct (lc) in a molar ratio of EGFR heavy chain to HER2 heavy chain to HER3 heavy chain of 2.5:2:1:1. 10 pools (A-J) of stably transfected cells were obtained. ELISA analysis of anti-EGFR, anti-HER 2 and anti-HER 3 antibodies was performed on day 3 and day 6 of the 10 pools. All 3 specificities can be determined in all pools.

Stable cell lines co-expressing two bispecific antibodies were generated by plating such pools into semi-solid medium. The plated cells were allowed to grow for 7-10 days. Two rounds of single cell cloning were performed by seeding and selection of a single colony. Oligoclonics is produced from a single cell by fed-batch fermentation (fed-batch fermentation).

Determination of antibody titres (antibody titers)

Based on total IgG concentration, the supernatant on the cells was diluted 1:4 and 1:50 in PBS. Single antigen ELISAs were first performed to determine the presence of all 3 antibodies. The following antigens were used at 2.5. Mu.g/mL dilution to coat wells of one ELISA plate, recombinant human EGFR-ECD Fc (R & D Systems, 344-ER), recombinant human ErbB2-ECD Fc (R & D Systems, 1129-ER) and recombinant human ErbB3-ECD Fc (R & D Systems, 348-ER).

Two sandwiches ELISAs (sandwich ELISAs) are then developed to determine and quantify the two bispecific molecules, allowing an estimate of the ratio between the two bispecific. With respect to EGFRxHER2 bispecific assays, EGFR-Fc (R & D Systems, 344-ER) antigens were coated on top of such wells and assayed using ErbB2-Fc (R & D Systems, 1129-ER). For EGFRxHER3 bispecific assays, EGFR-Fc antigen was coated on top of such wells and assayed using ErbB3-Fc (R & D Systems, 348-RB).

IgG purification

Purification of IgG was performed using affinity chromatography. Purification was performed using vacuum filtration under sterile conditions. First, the pH of the medium was adjusted to pH 8.0, and the resultant was then incubated with protein ASepharose CL-4B beads (50% v/v) (Pierce) for 2 hours at 25℃on a plate shaker set at 600 rpm. Second, such beads were harvested by vacuum filtration. The beads were washed 2 times with PBS pH 7.4. IgG was eluted using 0.1M citrate buffer at pH 3.0, while IgG fractions were immediately neutralized by Tris pH 8.0. Buffer exchange was performed by centrifugation using Ultracel (Millipore). Such samples were finally in a final buffer of PBS pH 7.4.

Cation exchange Chromatography (CIEX)

CEX-HPLC run was performed using a TSKgel SP-STAT (7 μm particle size, 4.6mM I.D..times. 10cmL,Tosoh 21964) series ion exchange column. Such columns are packed with non-porous resin particles for speed and high resolution analysis of biomolecules as well as separation. The particles in TSKGEL STAT columns contain an open access network of multiple layers of ion exchange groups for loading capacity, while the relatively large particle size makes these columns suitable for use in HPLC and FPLC systems.

TSKgel SP-STAT (7 μm particle size, 4.6mM I.D.times. 10cmL,Tosoh 21964) was equilibrated with buffer A (sodium phosphate buffer, 25mM, pH 6.0), after which the antibody was drained from the column by increasing the salt concentration and running a gradient of buffer B (25 mM sodium phosphate, 1mM NaCl, pH 6.0). The flow rate was set at 0.5mL/min. The injected sample mass for all test samples and control groups (in PBS) was 10. Mu.g, while the injected volume was 10-100. Mu.L. The chromatogram was analyzed for observed peak pattern, residence time, and peak area of the dominant peak based on the 220nm results.

BxPC-3-luc2 and N87 growth inhibition assay

The antibody compositions were added to the cultures at 0.1ng/mL EGF and 10ng/mL HRG for BxPC3-luc2 cells or at 0.1ng/mL EGF and 1ng/mL HRG for N87 cells in a concentration range of total antibodies (the following test. Such antibodies were pooled in pairs according to equal amounts of weight/weight. The ligand-containing Basal value (Basal w/ligand) was a control group without antibody but with the corresponding growth factor. The ligand-containing Basal value (Basal w/o ligand) was a control group without the indicated growth factor and without antibody.

Antibodies were diluted in chemically defined starvation medium (CHEMICALLY DEFINED starvation medium) (CDS: RPMI 1640 medium, containing 80U penicillin and 80. Mu.g streptomycin per milliliter, 0.05% (w/v) BSA, and 10. Mu.g/mL whole iron transferrin (holo-transferrin)), while 50. Mu.L of diluted antibody was added to one of such wells of a 96 well black well clear bottom plate flat plate (Costar). The ligand was added (50. Mu.L of a stock solution per well containing 40ng/mL or 4ng/mL of HRG diluted in CDS with 400ng/mL of EGF: R & D systems, cat. Nr.396-HB and 236-EG). The plates were left at room temperature for 1 hour and then placed in a container located in a 37 ℃ cell culture incubator for 3 days (N87 cells) or 4 days (BxPC-3-luc 2 cells). On day 4, alamarblue (Invitrogen, #dal1100) was added (20 μl/well), while fluorescence was measured after incubation with alamarblue (at 37 ℃) for 6 hours (N87 cells) or 4 hours (BxPC-3-luc 2 cells) using 560nm excitation and 590nm readout on a Biotek Synergy 2 multifunction microdisk analyzer. Fluorescence values were normalized to non-inhibited growth (no antibody, but two ligands were added).

Various differentADCC activity of (a)

ADCC reporter bioassays (ADCC Reporter Bioassay) (Promega) were used. Two different cell lines were tested, EGFR-phenotype pancreatic cancer cell line BxPC3 and gastric cancer N87 cell line.

The bioassay uses engineered Jurkat cells that either stably express the fcyriiia receptor V158 (high affinity) variant, and an NFAT response element that drives expression of firefly luciferase, a measure for fcyr activation. The assay has been validated by comparing the data obtained using this ADCC reporter bioassay with the conventional ⁵¹ Cr release assay, which yields similar results. Such ADCC assays were performed using a Promega ADCC bioassay kit using 384 white well plates. In this experimental setup, bxPC3 cells and N87 cells were plated at a density of 1000 cells/well in 30 μl of assay medium (RPMI with 4% low IgG serum) 20-24 hours prior to the assay. On alternate days, the medium was removed. Secondly, the first step of the method comprises the steps of,Is prepared in duplicate with a comparator antibody (comparator antibody) cetuximab. 10. Mu.L of these antibody dilutions were added to such wells. Control wells without antibody are also included (basal). From the initial concentration of such antibodies, 5-fold serial dilutions were generated to provide a dose-response curve. Finally, 5 μl of ADCC bioassay effector cells (ADCC Bioassay effector cells) (15000 cells/well, V158) were added. The cells were incubated at 37 ℃ for 6 hours. Next, 15. Mu.L of BIO-Glo luciferase matrix was added and luminescence was measured in a disk reader after 5 minutes. The resulting data is shown in fig. 3. Cetuximab shows ADCC activity against BxPC3 and N87 cells. Various oligoantibodies also showed ADCC activity on BxPC3 and/or N87 cells.

The test includes bispecific antibodies PB4516 and PB6892Concerning its effect on the growth of BxPC-3-luc2 tumors (implanted in situ) and N87 tumors (stomach cells implanted in the side wall of the abdomen)

CB17 SCID female mice, 8-10 weeks old, were implanted in situ in the pancreas at the beginning of the study to fit 1X 10 ⁶ BxPC-3-luc2 tumor cells in 20. Mu.L. The mice were anesthetized and laid down from the right side to expose the left side, while a 0.5cm incision was made on the left flank region. The pancreas and spleen were removed from the abdomen, and 1×10 ⁶ tumor cells in 20 μl were injected into the subcapsular space of the pancreatic tail. One week after implantation, bioluminescence (BLI) data was generated. Regarding the left view of BLI imaging (once or twice a week), all mice received an intraperitoneal injection (i.p. injection) of 150mg/kg fluorescein (Luciferin) (D-fluorescein-EF potassium salt, cat.#e6552, promega) before 15 minutes of the imaging. Outlier animals (outlier animals) -removed according to BLI/tumor volume-and such mice were randomly distributed in groups of 7 mice each. On day 8 of the experiment, treatment was started.

Animals located in the antibody-treated group were dosed with 0.3mg/kg of antibody weekly for 4 consecutive weeks (day 0, day 7, day 14 and day 21). On day 0 of the treatment, such animals received twice the loading dose, i.e., 0.6mg/kg of antibody. The final visualization was performed on day 35 or day 40. Only the vehicle and the cetuximab treated group served as control groups.

Cetuximab and oligoclonic significantly reduced BxPC-3 tumor overgrowth in this model (p < 0.05) (fig. 5). UsingTumor overgrowth of PB4516 and PB6892 was significantly lower than with cetuximab. Cetuximab does not significantly reduce overgrowth of N87 cells.Significantly reduced N87 tumor overgrowth in this model (p < 0.05) (FIG. 5).

N87 tumor:

CB17 SCID female mice, 8-12 weeks old, were inoculated subcutaneously (s.c.) in the flank at the beginning of the study to 1 x 10 ⁷ N87 tumor cells in 50% Matrigel (Matrigel). The cell injection volume was 0.2 mL/mouse. Treatment was initiated when the tumor reached an average size of 150-200mm ³. Antibodies were administered once weekly for 4 weeks by intraperitoneal injection of 25mg/kg mice. Body weight measurements were taken weekly after tumor cell injection, and every two weeks from the beginning to the end of treatment. Tumor growth was monitored every two weeks by caliper measurements (caliper measurements). The end point of the experiment was a tumor volume of 800mm ³ or 60 days, whichever was first reached.

Activity of PB11244 and PB4516 in various PDX models

Comprising bispecific antibodies PB11244 and PB4516Is assessed in a series of PDX models. Testing candidate treatments in a large number of cancer models facilitates prediction of clinical performance and can identify factors for patient-selection strategies (factors for patient-selection strategies).

Bispecific antibodies PB4516 and PB11244 have one IgG1 constant region and two variable domains.

The HER3 variable domain of PB4516 has the heavy chain variable region of MF3178 in fig. 7 or 8. The EGFR variable domain has the heavy chain variable region of MF3755 in fig. 7 or 8.

The HER2 variable domain of PB11244 has the heavy chain variable region of MF2032 in fig. 7 or fig. 8. The EGFR variable domain has the heavy chain variable region of MF3755 in fig. 7 or 8.

The light chain variable regions in both antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence identification number 11 (SEQ ID NO: 11) in FIG. 7.

A selection of several gastric, esophageal, and non-small cell lung cancer PDX models was made (fig. 6).

Comprising bispecific antibodies PB4516 and PB11244Is generated and purified. Such antibodies were mixed in a 1:1 ratio.Tested in such models and compared to cetuximab and vehicle (PBS).

Such PDX models were first expanded subcutaneously (s.c.) in donor BALB/c nude mice. Tumors were extracted, cut into small pieces (2-3 mm in diameter) and subcutaneously implanted into new recipient BALB/c nude mice. Tumor recipients were female BALB/c nude mice 6-8 weeks old. Tumor growth was tracked by caliper measurements until the tumor reached an average size of 100-200mm ³. At this stage (noted as day 1), animals were randomly assigned to 3 groups per model. Treatment was initiated on the same day and included:

PB4516 XPB 6892 25mg/kg,5 week dose, intraperitoneal injection

25Mg/kg of cetuximab, 5-week dose, intraperitoneal injection

Vehicle (PBS), 5 week dose, intraperitoneal injection

What can be seen is: Significantly reduces the overgrowth of tumor cells in the model. The reduction in overgrowth is equal to cetuximab or more preferably.

TABLE 1 Arg 426 that falls within HER3Residues within the radius

Sequence listing

<110> The netherlands merchant merles company (MERUS n.v.)

<120> Combinations of binding moieties that bind EGFR, HER2 and HER3

<140> PCT/NL2020/050081

<141> 2020-02-13

<150> EP 19157302.1

<151> 2019-02-14

<150> EP 19178564.1

<151> 2019-06-05

<160> 51

<170> Patent in version 3.5

<210> 1

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> HER3-MF3178

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Gly Ser Arg His Phe Trp Ser Tyr Trp Gly Phe Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 2

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> HER2-MF1849

<400> 2

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Asp Tyr Gly Ser Tyr Ser Ser Tyr Ala Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> EGFR-MF3755

<400> 3

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asn Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Ala Asn Thr Gly Asp Pro Thr Tyr Ala Gln Gly Phe

50 55 60

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Arg Phe Leu Glu Trp Leu His Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 4

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> EGFR-MF4280

<400> 4

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Gly Phe Asp Pro Glu Tyr Gly Lys Thr Phe Phe Ala Gln Asn Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Ala Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Glu Gly Tyr Tyr Glu Thr Thr Thr Tyr Tyr Tyr Asn Leu Phe

100 105 110

Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> EGFR-MF4003

<400> 5

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Ser Phe

20 25 30

Ala Met Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Thr Thr Asn Thr Gly Asp Pro Thr Tyr Ala Gln Gly Phe

50 55 60

Ser Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Tyr Asn Trp Ile Arg Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 6

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> HER2-MF2032

<400> 6

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Tyr Tyr Arg Arg Thr Ala Arg Ala Gly Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 7

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> VL CDR1

<400> 7

Gln Ser Ile Ser Ser Tyr

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> VL CDR2

<400> 8

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 9

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> VL CDR3

<400> 9

Gln Gln Ser Tyr Ser Thr Pro

1 5

<210> 10

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<223> IgVk1-39*01

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro

85 90 95

<210> 11

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Common light chain IgKV.times.39/jk 1

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 12

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Common light chain IgKV1 x 39/jk5

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 13

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> IgG1 CH1

<400> 13

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro

<210> 14

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> IgG1 CH2

<400> 14

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

<210> 15

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> IgG1 CH3 KK

<400> 15

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Lys Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Lys Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 16

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> IgG1 CH3 DE

<400> 16

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Asp Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Glu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 17

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> MF1847 HER2

<400> 17

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Trp Trp His Pro Leu Leu Ser Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> MF1871 HER2

<400> 18

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Arg Tyr Asp Leu Trp Trp Tyr Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> MF4000 EGFR

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Tyr

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val

35 40 45

Ser Phe Ile Ser Ser Ser Ser Glu Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Gly Ala Pro His Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 20

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> MF4290 EGFR

<400> 20

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ile Ser Gly Tyr Thr Leu Thr Glu Leu

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Met

35 40 45

Gly Gly Phe Asp Pro Glu Tyr Gly Glu Thr Phe Phe Ala Gln Gln Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Thr Glu Gly Tyr Tyr Gln Thr Thr Ser Tyr Tyr Tyr Asn Leu Phe

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 21

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> MF4016 EGFR

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Pro Asn Tyr Tyr Gly Ser Gly Ser Pro His Tyr Phe Tyr

100 105 110

Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 22

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> MF2703 HER3

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Phe Ser Gly Ser Asp Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Asn

65 70 75 80

Leu Tyr Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Ser Asp Tyr Ser Ser Gly Trp Tyr Gly Phe Pro Thr Asp

100 105 110

Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 23

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> MF2708

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Thr Lys Tyr Ser Ala Asp Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Glu Gly Trp Ser Phe Asp Ser Ser Gly Tyr Arg Ser Trp Phe

100 105 110

Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 24

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> MF1337 tetanus toxoid

<400> 24

Glu Val Gln Leu Val Glu Thr Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Tyr Ile Phe Thr Lys Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Ser Ala Asn Thr Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Gly Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Ser Leu Phe Lys Thr Glu Thr Ala Pro Tyr Tyr His Phe

100 105 110

Ala Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 25

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> MF3755 EGFR FR1

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr

20 25 30

<210> 26

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> MF3755 EGFR CDR1

<400> 26

Asn Tyr Ala Met Asn

1 5

<210> 27

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> MF3755 EGFR FR2

<400> 27

Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met Gly

1 5 10

<210> 28

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> MF3755 EGFR CDR2

<400> 28

Trp Ile Asn Ala Asn Thr Gly Asp Pro Thr Tyr Ala Gln Gly Phe Thr

1 5 10 15

Gly

<210> 29

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> MF3755 EGFR FR3

<400> 29

Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln

1 5 10 15

Ile Ser Ser Leu Lys Ala Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg

20 25 30

<210> 30

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> MF3755 EGFR CDR3

<400> 30

Glu Arg Phe Leu Glu Trp Leu His Phe Asp Tyr

1 5 10

<210> 31

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> MF3755 EGFR FR4

<400> 31

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 32

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> MF3178 HER3 FR1

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

20 25 30

<210> 33

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> MF3178 HER3 CDR1

<400> 33

Gly Tyr Tyr Met His

1 5

<210> 34

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> MF3178 HER3 FR2

<400> 34

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 35

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> MF3178 HER3 CDR2

<400> 35

Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 36

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> MF3178 HER3 FR3

<400> 36

Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 37

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> MF3178 HER3 CDR3

<400> 37

Asp His Gly Ser Arg His Phe Trp Ser Tyr Trp Gly Phe Asp Tyr

1 5 10 15

<210> 38

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> MF1849 HER2 FR1

<400> 38

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 39

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> MF1849 HER2 CDR1

<400> 39

Ser Tyr Gly Met His

1 5

<210> 40

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> MF1849 HER2 FR2

<400> 40

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10

<210> 41

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> MF1849 HER2 CDR2

<400> 41

Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 42

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> MF1849 HER2 FR3

<400> 42

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys

20 25 30

<210> 43

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> MF1849 HER2 CDR3

<400> 43

Gly Asp Tyr Gly Ser Tyr Ser Ser Tyr Ala Phe Asp Tyr

1 5 10

<210> 44

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> MF2032 HER2 CDR3

<400> 44

Asp Tyr Tyr Arg Arg Thr Ala Arg Ala Gly Phe Asp Tyr

1 5 10

<210> 45

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> MF1337 tetanus toxoid FR1

<400> 45

Glu Val Gln Leu Val Glu Thr Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Asp Tyr Ile Phe Thr

20 25 30

<210> 46

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> MF1337 tetanus toxoid CDR1

<400> 46

Lys Tyr Asp Ile Asn

1 5

<210> 47

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> MF1337 tetanus toxoid FR2

<400> 47

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 48

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> MF1337 tetanus toxoid CDR2

<400> 48

Trp Met Ser Ala Asn Thr Gly Asn Thr Gly Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 49

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> MF1337 tetanus toxoid FR3

<400> 49

Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Thr Ser Gly Asp Thr Ala Val Tyr Phe Cys Ala Arg

20 25 30

<210> 50

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> MF1337 tetanus toxoid CDR3

<400> 50

Ser Ser Leu Phe Lys Thr Glu Thr Ala Pro Tyr Tyr His Phe Ala Leu

1 5 10 15

Asp Val

<210> 51

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> MF1337 tetanus toxoid FR4

<400> 51

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

Claims

1. A composition comprising two antibodies,

wherein each of the antibodies comprises a variable domain that binds to the extracellular portion of EGFR, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 26, 28 and 30, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively; and

The first antibody in the antibodies comprises a variable domain that binds to the extracellular portion of HER2, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 39, 41 and 44, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively; and the second antibody in the antibodies comprises a variable domain that binds to the extracellular portion of HER3, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 33, 35 and 37, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively.

2. The composition of claim 1, wherein at least one of the two antibodies or both antibodies are IgG.

3. A composition according to claim 1 or 2, wherein the CH3 region of the heavy chain of the first and/or second antibody is engineered to promote heterodimerization of a heavy chain with a variable domain that binds to EGFR and a heavy chain with a variable domain that binds to HER2 and/or heterodimerization of a heavy chain with a variable domain that binds to EGFR and a heavy chain with a variable domain that binds to HER3.

4. The composition according to claim 1 or 2, wherein at least one of the two antibodies or both antibodies are bispecific antibodies.

5. The composition of claim 1 or 2, wherein the variable domains of the first and second antibodies that bind to the extracellular portion of EGFR comprise the same heavy chain variable region.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a medicament for the treatment of gastric cancer, lung cancer or esophageal cancer.

7. A pharmaceutical composition comprising the composition according to any one of claims 1 to 5.

8. Use of two antibodies in the preparation of a medicament for the treatment of gastric cancer, lung cancer or esophageal cancer, wherein each of the two antibodies comprises a variable domain that binds to the extracellular portion of EGFR, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 26, 28 and 30, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively; wherein the first antibody in the antibodies comprises a variable domain that binds to the extracellular portion of HER2, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 39, 41 and 44, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, and wherein the second antibody in the antibodies comprises a variable domain that binds to the extracellular portion of HER3, and the variable domain comprises amino acid sequences as shown in SEQ ID NOs: The heavy chain CDR1, CDR2 and CDR3 sequences shown in NOs: 33, 35 and 37 and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 7, 8 and 9, respectively.

9. A product comprising two antibodies, wherein each of the antibodies comprises a variable domain that binds to the extracellular portion of EGFR, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 26, 28 and 30, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively; and wherein the first of the antibodies comprises a variable domain that binds to the extracellular portion of HER2, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 39, 41 and 44, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, and wherein the second of the antibodies comprises a variable domain that binds to the extracellular portion of HER3, and the variable domain comprises amino acid sequences as shown in SEQ ID NOs: The heavy chain CDR1, CDR2 and CDR3 sequences shown in NO:33, 35 and 37 and the light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO:7, 8 and 9, respectively, are used as a combination preparation for simultaneous, separate or sequential use in the treatment of cancers expressing EGFR, HER2 and HER3.

10. The use according to claim 6 or 8, wherein the gastric cancer, lung cancer or esophageal cancer comprises cells with EGFR-mutations, which render the cells resistant to treatment with tyrosine kinase inhibitors (TKIs).

11. The use according to claim 6 or 8, wherein the gastric cancer, lung cancer or esophageal cancer comprises cells with EGFRR521K polymorphism.

12. The use according to claim 6 or 8, wherein the cancer to be treated is gastric cancer.

13. Use of two antibodies in the preparation of a medicament for treating an individual having a cancer that expresses EGFR, HER2 and HER3 or who is at risk of recurrence or worsening of a cancer that expresses EGFR, HER2 and HER3, wherein each of the antibodies comprises a variable domain that binds to the extracellular portion of EGFR, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 26, 28 and 30, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively; and wherein the first of the antibodies comprises a variable domain that binds to the extracellular portion of HER2, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 39, 41 and 44, respectively, and light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 41 and 44, respectively. NO:7, 8 and 9, and the second antibody in the antibody includes a variable domain that binds to the extracellular portion of HER3, and the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences whose amino acid sequences are respectively shown in SEQ ID NO:33, 35 and 37, and light chain CDR1, CDR2 and CDR3 sequences whose amino acid sequences are respectively shown in SEQ ID NO:7, 8 and 9, wherein the cancer is selected from gastric cancer, lung cancer or esophageal cancer.

14. A method for producing a composition according to any one of claims 1 to 5, comprising:

Provide cells including the following

- a nucleic acid encoding a polypeptide comprising a heavy chain, wherein the heavy chain is capable of pairing with a common light chain to form a variable domain that binds to the extracellular portion of EGFR, and wherein the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 26, 28 and 30, respectively;

- a nucleic acid encoding a polypeptide comprising a heavy chain, wherein the heavy chain is capable of pairing with the common light chain to form a variable domain that binds to the extracellular portion of HER2, and wherein the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 39, 41 and 44, respectively;

- a nucleic acid encoding a polypeptide comprising a heavy chain, wherein the heavy chain is capable of pairing with the common light chain to form a variable domain that binds to the extracellular portion of HER3, and wherein the variable domain comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 33, 35 and 37, respectively; and

- a nucleic acid encoding a polypeptide comprising the common light chain, and comprising the light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively;

wherein each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in the cell; and

The cells are cultured to allow expression of the heavy and light chains.

15. The method of claim 14, further comprising recovering the two antibodies.

16. The method of claim 14 or 15, wherein two or more of the nucleic acids are physically linked.

17. The method of claim 14 or 15, comprising providing the nucleic acid to a plurality of cells and selecting cells from the pool with a desired expression ratio of the heavy and light chains.

18. The method of claim 14 or 15, wherein the two antibodies are bispecific antibodies.

19. The method of claim 14 or 15, wherein the cells produce equimolar amounts of the two antibodies.

20. The method of claim 14 or 15, wherein the cell produces more of the first of the two antibodies than the second antibody.

21. A cell comprising:

- a nucleic acid encoding a polypeptide comprising a heavy chain, which together with a common light chain forms a variable domain that binds to the extracellular part of EGFR, and which variable domain comprises the heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 26, 28 and 30, respectively;

- a nucleic acid encoding a polypeptide comprising a heavy chain, which together with the common light chain forms a variable domain that binds to the extracellular part of HER2, and which comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 39, 41 and 44, respectively;

- a nucleic acid encoding a polypeptide comprising a heavy chain, which together with the common light chain forms a variable domain that binds to the extracellular part of HER3, and which comprises heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 33, 35 and 37, respectively; and

wherein two or more of the nucleic acids may or may not be physically linked, and wherein each of the nucleic acids further comprises an expression regulatory sequence to allow expression of the encoded heavy and light chains in the cell, and wherein the cell produces two antibodies, wherein each of the antibodies comprises a variable domain that binds to an extracellular portion of EGFR, and wherein a first of the antibodies comprises a variable domain that binds to an extracellular portion of HER2 and a second of the antibodies comprises a variable domain that binds to an extracellular portion of HER3.