Combinations of binding moieties that bind EGFR, HER2 and HER3

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

[0001] This application is a divisional application of the Chinese patent application with application number 202080014304.7 and invention name “Combination of binding portions that bind to EGFR, HER2 and HER3”. The original application is the PCT international application PCT / NL2020 / 050081 filed on February 13, 2020, which entered the Chinese national phase on August 13, 2021. Technical Field

[0002] The present invention relates to the field of binding moieties such as antibodies, and in particular to the field of therapeutic binding moieties. The binding moieties can be used for human treatment. More specifically the present invention relates to compositions comprising two or more multispecific binding moieties, preferably multispecific antibodies. The binding moieties bind EGFR, HER2 and HER3. A single host cell can generate multiple binding moieties. Background Art

[0003] The epidermal growth factor (EGF) receptor (EGFR) is the prototype cell-surface receptor for members of the EGF-family that contain extracellular protein ligands. The family currently has four closely related receptor tyrosine kinases: EGFR, HER2 (ErbB-2 / c-neu), HER3 (ErbB-3), and HER4 (ErbB-4).

[0004] EGFR is present on the cell surface and is activated by the binding of its specific ligands, including epidermal growth factor and transforming growth factor alpha (TGFα). Upon activation by its growth factor ligands, the receptor undergoes a transition from an inactive, predominantly monomeric form to an active homodimer. In addition to forming homodimers after ligand binding, EGFR can pair with another member of the ErbB receptor family (such as HER2) to produce an activated heterodimer. There is also evidence suggesting that dimers are formed in the absence of ligand binding and that clusters composed of activated EGFRs are formed after ligand binding.

[0005] Dimerization of EGFR stimulates its intrinsic intracellular protein-tyrosine kinase (PTK) activity. This activity induces several signal transduction cascades leading to cell proliferation and differentiation. The kinase domain of EGFR can cross-phosphorylate tyrosine residues of other receptors with which it is complexed, and it itself can be activated in that way.

[0006] Mutations and overexpression involving EGFR have been identified in several types of cancer, and it is the target of an expanding class of anticancer therapies. These include EGFR-targeted small molecules such as gefitinib and erlotinib for lung cancer, and antibodies such as cetuximab and panitumumab for colon and head and neck cancers.

[0007] Although there are some successes with EGFR-targeted therapies, most are associated with the development of treatment resistance over time. One of the ways that EGFR-positive tumors can escape targeted therapies is through signaling through another receptor dimer. For example, increased signaling due to EGFR / HER3 dimers, either due to increased HER3 expression or heregulin expression, is associated with drug resistance to EGFR-targeted drugs in, for example, lung cancer and head and neck cancer. In addition to the induction of treatment resistance, certain side effects of EGFR-targeted antibodies have been observed. One example is the development of a skin rash associated with EGFR inhibition or anti-EGFR biological therapy. In extreme cases, such a rash may lead to a reduction in treatment cycles and / or early termination of treatment.

[0008] Various activation modes of signaling for the EGF receptor family have been identified. Among these are ligand dependent and ligand independent activation of signaling. Even in the absence of HER3 ligand, overexpressed HER2 can generate oncogenic signaling via HER2 / HER3 heterodimers (Junttila, Akita et al. 2009). HER2 activity can be inhibited by HER2-specific antibodies. Such HER2-specific antibodies can, for example, be used in HER2-positive (HER2 +) tumors. A problem with such treatments is that tumors often escape the HER2-specific treatment and continue to grow even in the presence of the inhibitory antibody. It has been observed that HER2-positive tumors, such as breast, ovarian, cervical and gastric tumors, can escape treatment by selective overgrowth of a subpopulation of tumor cells that exhibit upregulated HER3 expression (Ocana, Vera-Badillo et al. 2013) and / or HER3 ligand expression (Wilson, Fridlyand et al. 2012). Again, activating mutations in the HER3 receptor have been identified.

[0009] Therefore, despite the promising results of antibody treatments that specifically target members of the EGF receptor family, it has been observed that not all tumors respond or respond adequately. The present invention provides combinations consisting of binding moieties that target a variety of members of the EGF receptor family and methods for generating them. The combinations of the present invention show good efficacy. Such combinations can be generated in a cost-effective and efficient manner. Summary of the invention

[0010] The present invention provides a composition comprising two or more binding moieties,

[0011] wherein each binding moiety comprises a variable domain that binds to the extracellular portion of EGFR; and

[0012] wherein a first of the binding moieties comprises a variable domain that binds to the extracellular portion of HER2, and a second of the binding moieties comprises a variable domain that binds to the extracellular portion of HER3.

[0013] 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 are IgG antibodies. In a preferred embodiment of the present invention, the composition comprises two bispecific antibodies.

[0014] The multispecific antibodies as described herein preferably include a heavy chain with a CH3 heterodimerization domain. In one embodiment, the CH3 heterodimerization domain of the first and / or 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.

[0015] The present invention also provides a composition as described herein for 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 HER3. The composition is preferably used in 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 advanced cancer. The composition is preferably used in metastatic cancer. The composition is preferably used in 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 in cancers that are gastric cancer, lung cancer, breast cancer or esophageal cancer. Preferably, the composition is used in metastatic gastric cancer, metastatic lung cancer, metastatic breast cancer or metastatic esophageal cancer.

[0016] The present invention further provides a product containing two or more binding moieties, each of which comprises a variable domain that binds to the extracellular portion of EGFR; 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, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.

[0017] The present invention further provides a method for producing a composition according to the present invention, the method comprising:

[0018] Provide cells including the following

[0019] - 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;

[0020] - 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;

[0021] - 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

[0022] - a nucleic acid encoding a polypeptide comprising the common light chain;

[0023] 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 method further comprises culturing the cell to allow expression of the heavy and light chains, and optionally, collecting the two or more binding moieties.

[0024] Further provided is a cell comprising:

[0025] - 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;

[0026] - 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;

[0027] - 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

[0028] - a nucleic acid encoding a polypeptide comprising the common light chain;

[0029] 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.

[0030] In a further aspect, the present invention provides a container comprising nucleic acid, comprising:

[0031] - 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;

[0032] - 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 HER2;

[0033] - 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 HER3; and

[0034] - a nucleic acid encoding a polypeptide comprising the common light chain;

[0035] 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 regulatory sequence to allow expression of the encoded heavy and light chains in the cell.

[0036] The invention further provides a composition comprising a binding moiety that specifically binds to an extracellular portion of EGFR and to an extracellular portion of HER2.

[0037] The present invention also provides a composition comprising a binding moiety that specifically binds to the extracellular portion of EGFR and the extracellular portion of HER3.

[0038] The binding moiety is preferably an antibody, preferably an IgG antibody, more preferably a multispecific antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1

[0040] A schematic illustration 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 four heavy chains have three different variable regions (5, 6 and 7). The heavy chain with the shared variable region (5) has a portion (3) of a heterodimerization domain. The heavy chain with variable regions (6) and (7) has a compatible portion (2) of the heterodimerization domain. The preferred pairing of heterodimerization regions (2) and (3) can guide the formation of a bispecific antibody.

[0041] Figure 2

[0042] Two For the growth factor-dependent cell line BxPC-3-luc2 (Perkin Elmer 125058) and N87 cells (NCI-87 cells ( CRL-5822 TM ))'s proliferation inhibitory effect.

[0043] Two The effect on the proliferation of BxPC-3-luc2 (left panel) and N87 (right panel) cells was tested. The results of the panel screening were compared with a combination of two monospecific antibodies (EGFR binding antibody cetuximab and HER3 monospecific antibody PG3178) or with the EGFRxHER3 binding bispecific antibody PB4522. The cells were grown in the presence of saturating amounts of HRG and EGF. The level of cell growth of the corresponding cells with HRG and EGF but without antibody (basal w / ligand) and the basal level without HRG and EGF and without antibody (w / oligand) are shown.

[0044] The monospecific antibody PG3178 has an IgG1 constant region and two Figure 7 or Figure 8 The variable domain of the heavy chain variable region of MF3178 and Figure 7 The common light chain variable region of sequence 11 in.

[0045] The bispecific antibody PB4522 has an IgG1 constant region and two variable domains. The HER3 variable domain has Figure 7 or Figure 8 The EGFR variable domain has Figure 7 or Figure 8 The light chain variable regions of the two antibodies are identical and have Figure 7 The amino acid sequence of the common light chain variable region of sequence 11 in.

[0046] Figure 3

[0047] A group of ADCC activity of a small group of The ADCC activity of the bispecific antibodies was tested using N87 and a CD16 / NFAT reporter assay. The bispecific antibodies have an IgG1 constant region and two variable domains. The amino acid sequences of the heavy chain variable regions of the variable domains are shown in Figure 7 or Figure 8 The light chain variable regions in these antibodies are identical and have Figure 7 The amino acid sequence of the common light chain variable region of sequence 11 in.

[0048] Figure 4

[0049] Various Number and specificity of the samples and their ADCC activity. "-" indicates that no activity was observed.

[0050] Each row represents a bispecific antibody containing two The internal codes of such bispecific antibodies are indicated in such rows Bispecific 1 and Bispecific 2. The heavy chain variable regions of the HER2, HER3 and EGFR binding domains are indicated in the rows labeled MF A, MF B and MF C. MF numbers 3178 and 2703 combine with the common light chain to form a HER3 binding variable domain. MF numbers 4280, 3755, 4003, 4016 combine with the common light chain to form a variable domain that binds EGFR, and MF numbers 1871, 1847, 1849 and 2032 combine with the common light chain to form a variable domain that binds HER2. Such bispecific antibodies have an IgG1 constant region and two variable domains. The amino acid sequences of the heavy chain variable regions of these variable domains are indicated. Figure 7 or Figure 8 The light chain variable regions in these antibodies are identical and have Figure 7 The amino acid sequence of the common light chain variable region of sequence 11.

[0051] Figure 5

[0052] In vivo experiments. BxPC-3-luc2 or N87 cells were injected into the xenograft model on day 0. The bispecific antibody PB4516×PB6892 (see Figure 4 )of or control group were injected on days 1, 7, 14, 21, and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. (Results for PB4516 and PB6892). Vehicle and cetuximab served as control groups.

[0053] Figure 6

[0054] The bispecific antibodies PB4516 and PB6892 are included. In vivo experiments in various PDX models.

[0055] PDX models were injected on day 0, and treatment with antibodies or control group was completed on days 1, 7, 14, 21, and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg.

[0056] Figure 7

[0057] The amino acid sequences of the heavy chain variable regions of the various variable domains indicated by MF numbers are shown in SEQ ID NOs: 1-6, while the CDRs and light chain variable regions are shown in SEQ ID NOs: 7-12.

[0058] Figure 8

[0059] The amino acid sequences of various MFs mentioned herein. FR1-4 means framework regions 1-4. CDR1-3 means complementarity determining regions 1-3. TT is tetanus toxoid.

[0060] Fig. 9

[0061] a) HER3 crystal structure (PDB#4P59) showing residue Arg 426 located in a grey sphere and the distance to Arg 426. radius of the residues in the black sphere; b) Residue Arg 426 shown in grey and the distance to Arg 426 Distant residues within the radius are shown in black; c) Residues located in region Arg 426 in light grey and surrounding residues in dark grey (all labeled). The figures and analyses were prepared using Yasara (www.yasara.org). DETAILED DESCRIPTION

[0062] As used herein, the term EGFR means the protein encoded by the epidermal growth factor receptor gene (EGFR) in humans. The protein is known by many aliases, including: Erb-B2 receptor tyrosine kinase 1; Proto-Oncogene C-ErbB-1; ERBB1; and HER1. The database accession number for the human EGFR protein and the gene encoding it is (GenBank NM_005228.3). The accession number is given primarily to provide further methods for identifying the EGFR protein as a target, and the actual sequence of the EGFR protein bound by the antibody may vary, for example, because of mutations within the encoding gene (such as those that occur in certain cancers), and the like. Unless otherwise specified, when EGFR is mentioned herein, it refers to human EGFR. Due to the sequence and tertiary structural similarities between orthologues of EGFR in humans and other mammals, the EGFR variable domain may also bind to such orthologues, but not necessarily. The variable domain that binds EGFR can bind EGFR and its various variants, such as those expressed on certain EGFR-positive tumors.

[0063] The variable domain that binds to EGFR of an antibody or binding portion of the present invention preferably binds to 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.biophys.37.032807.125829) among other references. The domains of human EGFR are described in the Ferguson reference data mentioned above. Figure 1 The variable domains that bind to EGFR of the embodiments of the invention disclosed herein preferably bind to domain III of EGFR. The antibodies preferably inhibit EGF-induced proliferation of BxPC-3 cells (ATCC CRL-1687) or BxPC-3-luc2 cells (Perkin Elmer 125058).

[0064] The term HER2 as used herein means the 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. The ERBB-2 gene is often referred to as HER2 (derived from human epidermal growth factor receptor 2). When HER2 is mentioned herein, human HER2 is referred to. Antibodies comprising variable domains that bind to HER2 bind to human HER2. Due to the sequence and tertiary structural similarities between orthologs of HER2 in humans and other mammals, the HER2 variable domain may also bind to such orthologs, but not necessarily. The database accession numbers for 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 for identifying HER2 as a target, the actual sequence of the HER2 protein bound by the antibody may vary, for example, because of mutations within the encoding gene (such as those that occur in certain cancers), etc. The HER2 variable domain can bind to HER2 and its various variants, such as those expressed on certain HER2-positive tumor cells.

[0065] The HER2 protein contains several domains (see for reference Landgraf, R Breast Cancer Res. 2007; 9(1): 202- Figure 1). The extracellular domains are referred to as domains I-IV. The variable domains that bind to HER2 of the embodiments of the invention disclosed herein preferably bind to HER2 domain I or domain IV, preferably domain IV.

[0066] As used herein, the term HER3 refers to the protein encoded by the ERBB3 gene in humans. Alternative 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 HER3 is mentioned herein, human HER3 is referred to. Antibodies comprising variable domains that bind to HER3 bind to human HER3. Due to the sequence and tertiary structural similarities between orthologs of HER3 in humans and other mammals, the HER3 variable domain may also bind to such orthologs, but not necessarily. The database accession numbers 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 for identifying HER3 as a target, the actual sequence of the HER3 protein bound by an antibody may vary, for example, because of mutations within the encoding gene (such as those that occur in certain cancers), etc. The HER3 variable domain can bind to HER3 as well as its various variants, such as those expressed on certain HER2-positive tumor cells.

[0067] The structure of HER3 has been described in Cho et al. (2002; Science 297, 1330-1333: DOI: 10.1126 / science.1074611), among other references. The human protein has four extracellular domains. The variable domains that bind to HER3 of the embodiments of the invention disclosed herein preferably bind to domain III of HER3. In a preferred embodiment, the affinity (KD) of a variable domain for a HER3-positive cell is less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.39 nM, more preferably less than or equal to 0.99 nM. In a preferred embodiment, an antibody according to the present invention preferably includes a variable domain that binds to domain III of HER3 selected from R426 in the native HER3 protein and a variable domain that is located 100% away from R426. In a preferred embodiment, the variable domain has an affinity (KD) for HER3 on SK-BR-3 cells of less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.39 nM, more preferably less than or equal to 0.99 nM. 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 on BT-474 cells of less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.0 nM, more preferably less than or equal to 0.5 nM, more preferably less than or equal to 0.31 nM, more preferably less than or equal to 0.23 nM. In one embodiment, the affinity (KD) falls within the range of 0.31-0.15 nM. The above mentioned affinities are preferably measured using steady state cell affinity measurements, where cells are incubated at 4°C with a radioactively labeled antibody, after which the cell-bound radioactivity is measured.

[0068] The variable domain that binds to at least one amino acid of domain III of HER3 preferably binds to a variable domain selected from the group consisting of R426 in the native HER3 protein and a variable domain located at a distance from R426. Preferably, the amino acid residue is located in the native HER3 protein and is at a distance of 1:1 from R426. The amino acid residues in are selected from the group consisting of: L423, Y424, N425, G427, G452, R453, Y455, E480, R481, L482, D483 and K485 (see, for example, Figure 9 and Table 1). The amino acid residue numbers are those of 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 able to counteract an activity of HER3 located on HER3-positive cells. Variable domains with HER3 binding characteristics are described in WO 2015 / 130172, which is incorporated herein by reference. In a preferred embodiment, a bispecific antibody according to the present invention is provided, wherein the antibody comprises a variable domain that binds at least R426 of domain III of HER3. Preferably, the antibody comprises a variable domain that binds at least R426 of domain III of HER3.

[0069] In certain embodiments, the composition includes two or more antibodies, wherein each of the antibodies includes a variable domain that binds to the extracellular portion of EGFR; and wherein a first of the antibodies includes a variable domain that binds to the extracellular portion of HER2 and a second of the antibodies includes a variable domain that binds to the extracellular portion of HER3. In a preferred embodiment, the variable domains that bind to the extracellular portion of EGFR of the first and second antibodies have substantially the same amino acid sequence. In one embodiment, the first and second antibodies include a variable domain that binds to domain I of EGFR, and the first antibody includes a variable domain that binds to domain I of HER2, and the second antibody includes a variable domain that binds to domain III of HER3. In another embodiment, the first and second antibodies include a variable domain that binds to domain I of EGFR, and the first antibody includes a variable domain that binds to domain IV of HER2, and the second antibody includes a variable domain that binds to domain III of HER3. In a further embodiment, the first and second antibodies include a variable domain that binds to domain III of EGFR, and the first antibody includes a variable domain that binds to domain I of HER2, and the second antibody includes a variable domain that binds to domain III of HER3. In a further embodiment, the first and second antibodies comprise a variable domain that binds domain III of EGFR, and the first antibody comprises a variable domain that binds domain IV of HER2, and the second antibody comprises a variable domain that binds domain III of ER3.

[0070] In certain embodiments, a binding moiety is a protein or an aptamer. A binding moiety as described herein generally has two or more binding specificities. The binding moiety preferably includes two or more variable domains of an antibody. The variable domains can be provided in a variety of different ways. Many 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 composed of a single monomer variable antibody domain. Like a complete antibody, it can optionally bind to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than ordinary antibodies (150-160 kDa), which are composed of two heavy protein chains and two light chains, and are even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) 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 produced from camelid heavy chain antibodies. Similar single domain fragments can now be produced artificially and can be derived from other organisms. A variable domain preferably includes both the heavy chain variable region and the light chain variable region. Variable domains are sometimes called V H / V L Combination, where V H represents the variable region of the heavy chain, and V L Represents the variable region of the light chain.

[0071] The two or more fragments can be linked to produce a binding moiety with multiple binding specificities. Linking is usually accomplished using a linker peptide comprising two or more amino acid residues. The linking moiety can 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 having at least one variable domain with a MF (e.g., Figure 7 or Figure 8 In a preferred embodiment, the binding moiety comprises two or more such variable domains.

[0072] The binding moiety that binds to EGFR and HER2 is a different binding moiety from the binding moiety that binds to EGFR and HER3. If at least one of the binding moieties is a multispecific antibody, at least one multispecific antibody can bind to at least EGF and HER2 or to at least EGFR and HER3. In a preferred embodiment, the binding moiety comprises a bispecific antibody, wherein the bispecific antibody binds to EGFR and HER2, and the other bispecific antibody binds to EGFR and HER3.

[0073] 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 to an epitope located on an antigen, wherein such domains are derived from or share sequence homology with the variable regions of an antibody. Antibodies are generally composed of basic structural units - each having two heavy chains and two light chains. Antibodies for therapeutic use are preferably natural antibodies that are as close as possible to the individual to be treated (e.g., human antibodies for human individuals). An antibody according to the present invention is not limited to any particular format or method for generating it.

[0074] Since an antibody generally recognizes an epitope of an antigen, and such an epitope may also exist in other compounds, an antibody according to the present invention that "specifically recognizes" an antigen (e.g., EGFR, HER2 or HER3) may also recognize other compounds if such other compounds contain the same epitope. Therefore, the term "specifically recognizes" or "specifically binds" or terms with the same connotation, with respect to the interaction between an antigen and an antibody, does not exclude the binding of such an antibody to other compounds containing the same or the same epitope.

[0075] 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 wherein the light chain variable region (V H / V L ) combines to bind a first epitope located on the antigen, and a second V H / V L The combination binds to a second epitope. The second epitope may be a different epitope located on the same antigen. The term also includes antibodies where V H Capable of specifically recognizing a first antigen, and paired with the V H V L Can specifically recognize the second antigen. H / V LThe pairing 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 format or method for generating it.

[0076] Bispecific antibodies are an example of multispecific antibodies. Three- or more specific antibodies can be produced by adding a binding moiety (such as an scFv fragment) 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 cell that generates a common light chain and two different heavy chains (each forming a functional variable domain with the common light chain) generates, among other things, a bispecific antibody with two different heavy chains and light chains. Similarly, a cell that generates a common light chain and three or more different heavy chains can form several bispecific antibodies that can target three or more antigens together. It is now possible to establish a standard version of an antibody (i.e., a constant part and two variable domains) and add further binding domains. In this case, a multispecific antibody with one or more single-chain Fvs with additional binding specificities attached to one or more of the constant part or variable domains of the antibody can be produced. 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 hereby incorporated by reference.

[0077] 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 sequences have been aligned for optimal comparison purposes. The percent sequence identity of compared nucleic acid sequences was determined using the AlignX application of Vector NTI Program Advance 10.5.2 software using default values, which use a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc. Acid Res. 22: 4673-4680), a swgapdnarnt score matrix, a gap opening penalty of 15, and a gap extension penalty of 6.66. Amino acid sequences were aligned using the AlignX application of Vector NTI Program Advance 11.5.2 software using default values, which use a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, 1994), the blosum62mt2 score matrix, a gap opening penalty of 10, and a gap extension penalty of 0.1.

[0078] As used herein, the term "common light chain" refers to light chains such as those that can be used in a multispecific antibody. In a bispecific antibody, the two light chains can be a common light chain (or its V L The two light chains (or their V L The terms "common light chain", "common V chain" and "common V chain" may be identical or have certain amino acid sequence differences without affecting the binding specificity of the full-length antibody. L ", "Single light chain", "Single V L", with or without the addition of the term "rearranged", are used interchangeably herein. "Common" means light chains with the same sequence and also means that the amino acid sequences are not identical functional equivalents. Many variants of such light chains exist, in which mutations (deletions, substitutions, insertions and / or additions) that do not affect the formation of a functional binding region are present. The light chain of the present invention may also be a light chain as specified herein, having from 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. For example, making or finding non-identical but still functionally equivalent light chains falls within the definition of a common light chain as used herein, for example, by introducing and testing conserved 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 the like. In certain embodiments, a multispecific antibody with three or more variable domains has variable binding domains. The domains carry non-identical heavy chains and identical light chains or the light chains carry certain amino acid differences while the binding specificity of the full-length multispecific antibody is not affected. Such a light chain is advantageously also a common light chain as described herein. In a preferred embodiment, all variable domains of a multispecific antibody comprise a common light chain. A common light chain (variable region) for use in the multivalent antibody of the present invention may be a λ light chain, and this is therefore also provided in the context of the present invention, but a κ light chain is preferred. The common light chain of the present invention may comprise a constant region of a κ light chain or a λ light chain. It is preferably a constant region of a κ light chain, preferably wherein the common light chain is a germline light chain, preferably a rearranged germline human κ light chain comprising the IgVK1-39 gene segment, such as a rearranged germline human κ light chain IgVK1-39*01 / IGJK1*01. Examples of common light chain amino acid sequences are shown in Figure 7 In sequence 10, 11 or 12.

[0079] The term "full-length IgG" or "full-length antibody" according to the present invention is defined to include a substantially intact 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 regions designated as CH1, CH2, CH3, V H and C L 、V Ldomains. An IgG antibody binds to the antigen via the variable region domains included in the Fab portion, and after binding can interact with molecules and cells of the immune system through such constant domains (mostly through the Fc portion). Full-length antibodies according to the present 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 one of such regions deleted. However, IgG molecules in which one or several amino acid residues are deleted without substantially changing the binding characteristics of the formed IgG molecule are included in the term "full-length IgG". For example, such an IgG molecule may have a deletion of between 1 and 10 amino acid residues, preferably located in the non-CDR region, wherein such deleted amino acids are not essential 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 present invention, the IgG is IgG1.

[0080] Preferably at least one of the two or more binding moieties is an antibody. The antibody may include a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2. In another embodiment, the antibody includes a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER3.

[0081] The two or more binding moieties preferably comprise two or more antibodies (preferably multispecific antibodies), each of such antibodies comprising a variable domain that binds to the extracellular portion of EGFR; and wherein a first of such antibodies comprises a variable domain that binds to the extracellular portion of HER2 and a second of such antibodies comprises a variable domain that binds to the extracellular portion of HER3. A preferred example 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 1In the figure, two bispecific antibodies are depicted as 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 chains are directed to heterodimerize by the presence of a heterodimerization domain (2, 3). The heterodimerization domain has two parts, one part is located on one heavy chain, and the compatible part is located on the other heavy chain. The heterodimerization domain is often located in the IgG1 CH3 region. Heterodimerization can be directed by providing appropriate parts to the selected heavy chain.

[0082] In the present invention, the selected formation of EGFRxHER2 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 (2) located in the heavy chain forming the HER2 and the HER3 binding domains.

[0083] 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 HER3) is also referred to herein as an EGFR heavy chain, a HER2 heavy chain, etc. In a preferred embodiment of the present invention, the CH3-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 used to promote heterodimerization uses the DEKK residue position previously described in U.S. Patents 9,248,182, 9,358,286, 9,248,182 and 9,758,805.

[0084] In certain embodiments, the binding of such an antibody of the composition to EGFR blocks the binding of EGF to EGFR and / or wherein the binding of such an antibody of the composition to HER3 blocks the binding of neuregulin-1 (NRG) to HER3. In a preferred embodiment, the binding of such an antibody of the composition to EGFR blocks the binding of EGF to EGFR, while the binding of the antibody of the composition to HER3 blocks the binding of neuregulin-1 (NRG) to HER3.

[0085] A variable domain that binds to the extracellular part 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 substitution, deletion and / or insertion of 1, 2 or 3 amino acids in said CDRs.

[0086] A variable domain that binds to the extracellular part 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 a substitution, deletion and / or insertion of 1, 2 or 3 amino acids in said CDRs.

[0087] A variable domain that binds to the extracellular part 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 a substitution, deletion and / or insertion of 1, 2 or 3 amino acids in said CDRs.

[0088] 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 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. 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 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.

[0089] 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 substitution, deletion and / or insertion of 1, 2 or 3 amino acids in said CDRs; and wherein the first bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain preferably comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGS NKYYADSVKG and a heavy chain variable region of the 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 part of HER3, the variable domain preferably comprising 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 substitution, deletion and / or insertion of 1, 2 or 3 amino acids in said CDRs.

[0090] 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, 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 first bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain preferably comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGS NKYYADSVKG and a heavy chain variable region of the 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 part of HER3, the variable domain preferably comprising 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 substitution, deletion and / or insertion of 1, 2 or 3 amino acids in said CDRs.

[0091] Conservative variations of 1, 2 or 3 amino acids from the detailed CDR sequences are allowed while retaining the same type of binding activity (in type, not necessarily in quantity). 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.

[0092] In certain embodiments, the EGFR variable domain comprises a heavy chain variable region comprising Figure 7 or Figure 8 EGFR V H Preferably, Figure 7 or Figure 8 EGFR V in MF3755 H district.

[0093] 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%, more preferably at least 98%, more preferably at least 99%) identical to or 100% identical to a heavy chain variable region described in Figure 7 or Figure 8 EGFR V H Preferably Figure 7 or Figure 8 EGFR V in MF3755 H district.

[0094] For example, in certain embodiments, the heavy chain variable region of the bispecific antibody that binds to human EGFR may have 0-10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions, or a combination thereof in the sequence other than the 3 CDR sequences of the heavy chain variable region. In certain embodiments, for the indicated amino acid sequence, the heavy chain variable region includes 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 thereof.

[0095] In certain embodiments, the EGFR variable domain comprises a heavy chain variable region comprising an amino acid sequence selected from Figure 7 or Figure 8 EGFR V H Preferably Figure 7 or Figure 8 EGFR V in MF3755 H district.

[0096] In some embodiments, the HER2 variable domain comprises a heavy chain variable region comprising Figure 7 or Figure 8 HER2 V H Area (preferably Figure 7 or Figure 8 MF2032 HER2 V H region) of HCDR1, HCDR2 and HCDR3.

[0097] 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%, more preferably at least 98%, more preferably at least 99%) identical to or 100% identical to a heavy chain variable region described in Figure 7 or Figure 8HER2 V H Area (preferably Figure 7 or Figure 8 HER2V in MF2032 H region) amino acid sequence.

[0098] For example, in certain embodiments, the heavy chain variable region of the bispecific antibody that binds to human HER2 may have 0-10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions, or a combination of these in the sequence other than the 3 CDR sequences in the heavy chain variable region. In certain embodiments, for the indicated amino acid sequence, the heavy chain variable region includes 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.

[0099] In certain embodiments, the HER2 variable domain comprises a heavy chain variable region comprising a member selected from Figure 7 or Figure 8 MF1849 or MF2032 (preferably Figure 7 or Figure 8 The amino acid sequence of MF2032 in .

[0100] In some embodiments, the HER3 variable domain comprises a heavy chain variable region comprising Figure 7 or Figure 8 The V of MF3178 H The HCDR1, HCDR2 and HCDR3 of the region.

[0101] 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%, more preferably at least 98%, more preferably at least 99%) identical to or 100% identical to the amino acid sequence described in Figure 7 or Figure 8 The amino acid sequence of MF3178 in .

[0102] For example, in certain embodiments, the heavy chain variable region of the bispecific antibody that binds to human HER3 may have 0-10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions, or a combination thereof in the sequence other than the 3 CDR sequences of the heavy chain variable region. In certain embodiments, for the indicated amino acid sequence, the heavy chain variable region includes 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 thereof.

[0103] In certain embodiments, the HER3 variable domain comprises a heavy chain variable region comprising Figure 7 or Figure 8 The amino acid sequence of MF3178 in .

[0104] 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 selected from Figure 7 or Figure 8 EGFR V H region or a variant thereof, preferably Figure 7 or Figure 8 MF3755 EGFR V H region, or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (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 a heavy chain variable region comprising a selected from Figure 7 or Figure 8 The amino acid sequence of MF1849 or MF2032 or a variant thereof, preferably Figure 7 or Figure 8 The amino acid sequence of MF2032 or a variant thereof, wherein the variant comprises a substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF3178 or a variant thereof, wherein the variant comprises substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs).

[0105] 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 Figure 7 or Figure 8 The amino acid sequence of MF3755 in the above-mentioned embodiment or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (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 a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF2032 in or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located 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 comprising a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF3178 or a variant thereof, wherein the variant comprises substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs).

[0106] In one 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 Figure 7 or Figure 8 The amino acid sequence of MF4280 in the above-mentioned embodiment or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF1849 in the above-mentioned embodiment or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located 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 comprising a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF3178 in or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in said CDRs).

[0107] In one 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 Figure 7 or Figure 8 The amino acid sequence of MF4280 in the above-mentioned embodiment or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF2032 in or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located 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 comprising a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF3178 in or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in said CDRs).

[0108] In one 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 Figure 7 or Figure 8 The amino acid sequence of MF4003 in the above-mentioned embodiment or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising Figure 7 or Figure 8 The amino acid sequence of MF1849 in the above-mentioned embodiment or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located 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 comprising a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF3178 in or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in said CDRs).

[0109] In one 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 Figure 7 or Figure 8 The amino acid sequence of MF4003 in the above-mentioned embodiment or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising Figure 7 or Figure 8 The amino acid sequence of MF2032 in or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located 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 comprising a heavy chain variable region comprising from Figure 7 or Figure 8 The amino acid sequence of MF3178 in or a variant thereof comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in said CDRs).

[0110] Exemplary EGFR heavy chain variable regions are described in WO 2015 / 130172 and PCT / NL2018 / 050537, which are hereby incorporated by reference. Exemplary HER2 heavy chain variable regions are described in WO 2015 / 130173, which are hereby incorporated by reference. Exemplary HER3 heavy chain variable regions are described in WO 2015 / 130172 and WO 2015 / 130173, which are hereby incorporated by reference.

[0111] Additional variants of the disclosed amino acid sequences that retain EGFR, HER2 or HER3 binding can be obtained, for example, as previously described, from a human IGKV1-39 / IGKJ1 V containing the rearrangement L Phage display library of the region (DeKruif et al. Biotechnol Bioeng. 2010 (106) 741-50), and a ph ... H The amino acid sequence of the V H Phage 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 that retain antigen binding.

[0112] The present invention further provides a binding moiety that specifically binds to an extracellular portion of EGFR and an extracellular portion of HER2. The binding moiety preferably comprises a variable domain that binds to EGFR and a variable domain that binds to HER2. The variable domain that binds to EGFR is preferably an EGFR variable domain as described herein. The variable domain that binds to HER2 is preferably a HER2 variable domain as described herein. Preferably, both the EGFR and HER2 variable domains are variable domains as described herein.

[0113] The present invention further provides a binding moiety that specifically binds to an extracellular portion of EGFR and an extracellular portion of HER3. The binding moiety preferably comprises a variable domain that binds to EGFR and a variable domain that binds to HER3. The variable domain that binds to EGFR is preferably an EGFR variable domain as described herein. The variable domain that binds to HER3 is preferably a HER3 variable domain as described herein. Preferably, both the EGFR and the HER3 variable domains are variable domains as described herein.

[0114] The present invention further provides a composition comprising: a binding portion that specifically binds to the extracellular portion of EGFR and the extracellular portion of HER2, and a binding portion that specifically binds to the extracellular portion of EGFR and the extracellular portion of HER3.

[0115] The binding moiety as described herein is preferably an antibody, preferably a multispecific antibody, preferably a bispecific antibody.

[0116] The light chain variable regions (VLs) of the EGFR variable domain, HER2 variable domain, and HER3 variable domain of the binding moiety (such as such a bispecific antibody) may be identical to the VLs of the parent EGFR monospecific antibody. L region, V region of the parental HER2 monospecific antibody L region and / or the same as the parental HER3 monospecific antibody L Area. Alternative V L Area can be used for such V H / V L One or more of the variable domain combinations are used as long as such variable domains retain binding to EGFR, HER2 or HER3, respectively.

[0117] In certain embodiments, the V of the EGFR variable domain, the HER2 variable domain, and the HER3 variable domain LIn certain embodiments, the V domains of all variable domains of the binding moiety are similar. L The area is the same.

[0118] In certain embodiments, the light chain variable regions of one, two, three or more variable domains of the binding portion of the present invention include a common light chain variable region. In certain embodiments, the common light chain variable region of one, two, three or more variable domains includes a germline variable region V-segment. In certain embodiments, the light chain variable region of one, two, three or more variable domains includes a kappa light chain V-segment IgVκ1-39*01. IgVκ1-39 is an abbreviation for the immunoglobulin variable kappa 1-39 gene. The gene is also known as kappa variable 1-39, IGKV139, IGKV1-39. The external identifiers for this gene are: HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. The amino acid sequence of this V-region is provided at Figure 7 The V-region may also be combined with one of the five J-regions. The preferred J-regions are jk1 and jk5, and the linked sequences are represented as IGKV1-39 / jk1 and IGKV1-39 / jk5, with alternative names of IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (according to the nomenclature of the IMGT database global website at imgt.org). In certain embodiments, one or two V H / V L The light chain variable region of the binding region includes a kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ1*05 (respectively Figure 7 Sequence 11 or sequence 12).

[0119] In certain embodiments, the light chain variable region of one, two, three or more variable domains of the binding moiety of the invention comprises: a LCDR1 ( Figure 7 7), a LCDR2 comprising the amino acid sequence AAS, and a LCDR3 comprising the amino acid sequence QQSYSTP ( Figure 7 In certain embodiments, the light chain variable region of one, two, three or more variable domains of a binding moiety of the invention comprises: a LCDR1 ( Figure 7 7), a LCDR2 comprising the amino acid sequence AASSLQS ( Figure 78) and a LCDR3 ( Figure 7 Sequence 9).

[0120] In certain embodiments, one, two, three or more variable domains of a 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 a light chain variable region described in Figure 7 In certain embodiments, one, two, three or more variable domains of a binding moiety of the invention include 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 described in Figure 7 The amino acid sequence of sequence 11 in .

[0121] For example, in certain embodiments, for Figure 7 Sequence 11 or Figure 7 For the sequence 12 in, the variable light chain of 1, 2, 3 or more variable domains of the binding portion of the present invention may have from 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain embodiments, for the amino acid sequence shown, the light chain variable region of 1, 2, 3 or more variable domains of the binding portion of the present 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 thereof.

[0122] In other embodiments, the light chain variable region of one, two, three or more variable domains of the binding moiety of the invention comprises Figure 7 Sequence 11 or Figure 7 In certain embodiments, all variable domains of the binding moiety of the present invention comprise the same V L In one embodiment, the V domains of all variable domains of the binding moiety of the invention are L Including those described in Figure 7 In one embodiment, the V domains of all variable domains of the binding moiety of the present invention are L Including those described in Figure 7Sequence 12 or Figure 7 The amino acid sequence of sequence 12 in .

[0123] The multispecific antibodies disclosed herein (such as bispecific antibodies) can be provided in many formats. Many different formats of multispecific antibodies are known in the art and have been reviewed by Kontermann (Drug Discov Today, 2015 Jul; 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 format (such as a bispecific antibody format that is not a typical antibody with two variable domains) has at least one variable domain including a heavy chain variable region and a light chain variable region. This variable domain can be linked to a single-chain Fv-fragment, monomer, a VHH and a Fab-fragment providing a second binding activity.

[0124] In certain embodiments, such multispecific antibodies used in the methods provided herein are generally human IgG subtypes (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, such antibodies are human IgG1 subtypes. Full-length IgG antibodies are preferred because of their favorable half-life and for the sake of low immunogenicity. Such multispecific antibodies can have two different heavy chains including a 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.

[0125] Thus, in certain embodiments, such multispecific EGFR / HER2 and EGFR / HER3 antibodies include a crystallizable fragment (Fc). The Fc region of such multispecific antibodies is preferably composed of a human constant region. A constant region or Fc of such multispecific antibodies may contain one or more (preferably no more than 10, preferably no more than 5) amino acid differences for the constant region of a naturally occurring human antibody. For example, in certain embodiments, each Fab-arm of such bispecific antibodies may further include an Fc-region including modifications that promote the formation of the bispecific antibody, modifications that affect the effector functions of the Fc-mediator, and / or other features described herein.

[0126] In preferred embodiments, the multispecific (preferably bispecific) full-length IgG antibody has a lower hinge and / or CH2 domain such that the interaction of the bispecific IgG antibody with the Fc gamma (Fcγ) receptor is enhanced. The antibody-dependent cellular cytotoxicity (also known 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. A technique for enhancing ADCC by afucosylation is described, for example, in Junttila, TT, 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 afucosylated. Preferably, the two multispecific antibodies are afucosylated. Other strategies have been reported to achieve ADCC enhancement, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), all of which seek to improve Fc binding to the low-affinity activating FcγRIIIa and / or reduce binding to the low affinity inhibitory FcγRIIb.

[0127] Bispecific antibodies are generally produced by cells expressing 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:

[0128] - 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;

[0129] - 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;

[0130] - 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

[0131] - a nucleic acid encoding a polypeptide comprising the common light chain;

[0132] 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, 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 can be collected from the cell and / or the supernatant.

[0133] The level at which such corresponding chains are produced in cells can be tailored, for example by selecting appropriate expression regulatory sequences or by selecting the number of nucleic acid copies introduced or both. In a preferred embodiment, a collection of cells is provided with the nucleic acid, and a clone that exhibits the appropriate level of such corresponding chains is selected. The clone is also usually 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 a cell with a desired expression ratio of a respective heavy chain and light chain from the collection. In certain embodiments, the two or more binding moieties are antibodies, preferably bispecific antibodies. In certain embodiments, the cell preferably generates substantially equimolar quantities of the two or more binding moieties. In other embodiments, the cell generates one of the two or more binding moieties more than the other binding moiety.

[0134] The present invention also provides a cell comprising:

[0135] - 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;

[0136] - 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;

[0137] - 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

[0138] - a nucleic acid encoding a polypeptide comprising the common light chain;

[0139] Two or more of the nucleic acids may or may not be physically linked, and each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in the cell.

[0140] The present invention further provides a container comprising a nucleic acid, wherein the nucleic acid comprises:

[0141] - 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;

[0142] - 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 HER2;

[0143] - 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 HER3; and

[0144] - a nucleic acid encoding a polypeptide comprising the common light chain;

[0145] Two or more of the nucleic acids may or may not be physically linked, and each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in a cell.

[0146] The cell producing the binding moiety is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, and most preferably a human cell. A suitable cell is any cell that can contain and preferably produces the binding moiety (preferably such multispecific antibodies and preferably such bispecific antibodies) as described herein.

[0147] Suitable cells for antibody production are known in the art and include a hybridoma cell, a Chinese hamster ovary (CHO) cell, a NS0 cell, a HEK293 cell, a 293-F cell or a PER-C6 cell. Various institutions and companies have developed cell lines for 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 by 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 uses a glutamine synthetase (GS) vector system for expression of an antibody. In a preferred embodiment, the cell is a CHO cell.

[0148] In certain embodiments, the cell expresses 3 different heavy chains and at least one light chain. In a preferred embodiment, the cell expresses a "common light chain" as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, together with a rearranged human IGKV1 39 / IGKJ1 (huVκ139) light chain, the corresponding VH region is colonized into the expression vector using methods known in the art for the generation of bispecific IgG (WO2013 / 157954, incorporated herein by reference). The huVκ1 39 was previously shown to be able to pair with more than one heavy chain, resulting in antibodies with different specificities, which promotes the generation of bispecific molecules (WO2009 / 157771).

[0149] An antibody-producing cell that expresses a common light chain and an equal number of two heavy chains typically produces 50% bispecific antibodies and 25% of each such monospecific antibody (i.e., having the same heavy chain light chain combination). Several methods have been published that favor the generation of bispecific antibodies over the generation of corresponding monospecific antibodies. This is typically achieved by modifying the constant region of the heavy chain, so that they favor heterodimerization (i.e., dimerization of a heavy chain with another heavy chain / light chain combination) over homodimerization. In a preferred embodiment, the bispecific antibody of the present invention includes 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 different ways in which such hetero-dimerization of heavy chains can be achieved.

[0150] A preferred method for generating multispecific antibodies as described herein is disclosed in US 9,248,181 and US 9,358,286. Specifically, the preferred mutations for generating IgG molecules having substantially only bispecific full lengths are amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain (the "KK-variant" heavy chain) and amino acid substitutions L351D and L368E 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 (DEDE homodimer) of the DE-variant heavy chain or homodimerization (KKKK homodimer) of the KK-variant heavy chain is due to the strong repulsion between the charged residues in the CH3-CH3 interface between the same heavy chains and hardly occurs. The introduction of an additional heavy chain with a DE- or KK-variant heavy chain allows the generation of an additional DEKK bispecific molecule. 2 ) can be linked to the existing KK heavy chain. The cell thus produces two bispecific antibodies, one DE 1 KK and a DE 2 KK bivalent antibody. If a new KK heavy chain (KK 2 ) rather than the new DE heavy chain being introduced, with DEKK 1 and DEKK 2 The bispecific antibodies of the combination are generated. The levels at which different antibodies can be generated by the cell can be adjusted by adjusting the relative performance of such HER2 chains and HER3 chains relative to each other. The light chain is usually generated sufficiently to reduce the level of a single heavy chain, while the level at which such EGFR chains are generated is usually sufficient to allow effective pairing with such HER2 chains, HER3 chains.

[0151] Therefore, in one embodiment, the heavy chain / light chain combination comprising a variable domain that binds to EGFR comprises a DE variant of the heavy chain. In this embodiment, the heavy chain / light chain combination comprising a variable domain that binds to HER2 and the heavy chain / light chain combination comprising a variable domain that binds to HER3 comprise a KK variant of the heavy chain.

[0152] Candidate EGFR / HER2 or EGFR / HER3 IgG bispecific antibodies can be tested for binding using any suitable analysis. For example, the binding to membrane-expressed EGFR, HER2 or HER3. This is usually done on a cell that does not usually express the EGFR, HER2 or HER3 or is transformed to express one of EGFR, HER2 or HER3. The binding of the antibody to transformed cells rather than untransformed cells represents the specific binding of the antibody. Binding can be assessed by, for example, flow cytometry (according to the FACS program as previously described in WO 2015 / 130172, PCT / NL2018 / 050537 and WO2015 / 130173). The corresponding monospecific antibodies can be used together as a control group, in addition to an unrelated IgG1 isotype control group mAb.

[0153] Binding moieties (such as antibodies) can be collected from cells and / or supernatants of a cell culture. Typically, they are collected from supernatants of such producing 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.

[0154] Antibodies produced by a cell can 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 HER3). 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. The binding and washing of the preparation to HER2 should remove all EGFR / HER3 antibodies. Staining with the labeled soluble HER3 does not provide a signal, while that with the labeled soluble EGFR does. The binding and washing of the preparation to HER3 should remove all EGFR / HER2 antibodies. Staining with the labeled soluble HER2 does not provide a signal, while that with the labeled soluble EGFR does. The binding of the preparation to EGFR and washing should not remove the EGFR / HER2 and EGFR / HER3 antibodies. Staining with labeled soluble HER2 and staining with labeled soluble HER3 should provide 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- and bi-specific antibodies.

[0155] A method for producing a composition comprising two or more bispecific antibodies, the method comprising:

[0156] - providing cells carrying nucleic acids encoding the bispecific antibodies;

[0157] – cultivate the cells;

[0158] – harvest clarification;

[0159] - collecting such bispecific antibodies from culture; and

[0160] – Separation of the generated bispecific antibody from the half-antibody by ion exchange chromatography (IEX);

[0161] The method is characterized in that: under the IEX conditions used, such bispecific antibodies exhibit similar IEX retention times, preferably with a mean deviation of 10% or less from the retention times of such individual antibodies. In one embodiment, under the IEX conditions used, such antibodies are selected to have an IEX retention time that is 10% or less from the mean deviation of the retention time of such individual antibodies. Such antibodies can first be purified from other proteins in the culture. This is usually accomplished by affinity purification, preferably by protein A extraction. Such bispecific antibodies are preferably selected to have a half antibody with a retention time that falls outside the range spanned by the retention time of such antibodies. In the case where a combination of bispecific antibodies is generated and monospecific antibodies are not desired, such bispecific antibodies are preferably selected to have a retention time that is not the same as the retention time of such monospecific antibodies. Such monospecific antibody retention times in this embodiment preferably fall outside the range spanned by the retention time of such corresponding bispecific antibodies. The cells in the culture preferably express the three heavy chains simultaneously, wherein the heavy chains include a CH3 heterodimerization domain that promotes the formation of heterodimerization of the EGFR / HER2 and EGFR / HER3 heavy chains. The cells preferably express Figure 7 In one embodiment such bispecific antibodies have isoelectric points (PI) that are similar and preferably differ by no more than 0.5 units from the average PI of the at least two bispecific antibodies.

[0162] 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 (SPR) technology using a BIAcore T100. An anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat.Nr.555784) is coupled to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). The bsAb is then captured on the sensor chip. Subsequently, recombinant purified antigen human EGFR-Fc, HER2-Fc and HER3-Fc proteins are run on the sensor surface in a concentration range to measure the 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, the on and off rates and affinity values ​​for binding to human EGFR, HER2 and HER3 were determined using BIAevaluation software.

[0163] The present invention also provides a composition as described herein for 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 HER3. The composition is preferably used in 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 in metastatic cancer. The composition is preferably used in 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 in cancers that are gastric cancer, lung cancer, breast cancer or esophageal cancer. Preferably, the composition is used in metastatic gastric cancer, metastatic lung cancer, metastatic breast cancer or metastatic esophageal cancer.

[0164] The present invention further provides two or more binding moieties for the treatment of cancer, each of which includes a variable domain that binds to the extracellular portion of EGFR; wherein the first binding moiety in the binding moiety includes a variable domain that binds to the extracellular portion of HER2, and the second binding moiety in the binding moiety includes a variable domain that binds to the extracellular portion of HER3. Also provided is a product containing two or more binding moieties, each of which includes a variable domain that binds to the extracellular portion of EGFR; wherein the first binding moiety in the binding moiety includes a variable domain that binds to the extracellular portion of HER2, and the second binding moiety in the binding moiety includes a variable domain that binds to the extracellular portion of HER3, as a combination agent for simultaneous, separate or sequential use in the treatment of cancer.

[0165] The cancer treated by the 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 treated by a method of treatment of the invention described herein is preferably gastric cancer, lung cancer or esophageal cancer. In a further embodiment, the present invention provides a method for treating an individual with cancer or at risk of recurrence or worsening of cancer, the method comprising administering two or more binding moieties to the individual in need of the treatment, each of the binding moieties comprising a variable domain that binds to the extracellular portion of EGFR; wherein the first binding moiety of the binding moiety comprises a variable domain that binds to the extracellular portion of HER2, and the second binding moiety of the binding moiety comprises a variable domain that binds to the extracellular portion of HER3.

[0166] As used herein, the terms "subject" 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).

[0167] The terms "treat," "treating," and "treatment," as used herein, mean any type of intervention or procedure performed on the subject or the administration of an active agent or a combination of active agents to the subject with the purpose of reversing, alleviating, ameliorating, inhibiting or slowing or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical marker associated with a disease.

[0168] As used herein, "effective treatment" or "positive therapeutic response" means a treatment that produces a beneficial effect, such as improvement of at least one symptom of a disease or disorder (e.g., cancer). A beneficial effect can be in the form of an improvement over baseline, including an improvement in a measurement or observation made prior to initiation of therapy according to the methods of the present invention. For example, a beneficial effect can 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 a reduction or elimination of a clinical or diagnostic symptom of the disease or a marker of cancer. Effective treatment can, 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.

[0169] The term "effective amount" or "therapeutically effective amount" means an amount of an agent or a combination of agents that can provide a desired biological, therapeutic and / or preventive result. The result can be a reduction, improvement, alleviation, reduction, delay and / or alleviation of one or more of the signs, symptoms or causes of a disease, or any other desired change in a biological system. In certain embodiments, an effective amount is an amount sufficient to delay tumor development. In certain embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow down and stop the 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 tumors; and / or (vii) alleviate one or more symptoms associated with the cancer to some extent. In one example, an "effective amount" is the amount of the composition of the invention to achieve a reduction in a cancer (e.g., a reduction in the number of cancer cells) or slow the progression of a cancer. An effective 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 the frequency of dosage are sufficient to achieve treatment.

[0170] As used herein, the terms "synergy," "therapeutic synergy," and "synergistic effect" refer to a phenomenon in which treatment of a patient with a combination of the binding moieties described herein (e.g., a composition comprising a binding moiety that binds EGFR and HER2 and a binding moiety that binds EGFR and HER3) exhibits a therapeutically superior result over that achieved when the individual components of the combination are used alone (see, e.g., TH Corbett et al., 1982, Cancer Treatment Reports, 66, 1187). In this context, a therapeutically superior outcome includes one or more of the following: (a) an increase in therapeutic response that is greater than the sum of the independent effects of each binding moiety by itself at the same dose as it is in the combination; (b) a reduction in the dose of one or more agents in the combination without a reduction in therapeutic efficacy; (c) a reduction in the incidence of adverse events while receiving a therapeutic benefit that is equal to or greater than that of the monotherapy of each agent at the same dose as it is in the combination; (d) a reduction in dose-limiting toxicity while receiving a therapeutic benefit that is greater than that of the monotherapy of each agent; (e) a delay or minimization of the induction of drug resistance.

[0171] In xenograft models, a combination used at its maximum tolerated dose (where each of the components will be present at a dose that generally does not exceed its individual maximum tolerated dose) demonstrates therapeutic synergy when the reduction in tumor growth achieved by administration of the combination is greater than the reduction in tumor growth when the best component is administered alone. The synergistic effect of a drug combination can be determined, for example, according to the Chou-Talalay combination index (CI) theorem (Chou et al., Adv. Enzyme Regul. 1984; 22: 27-55; Chou, Cancer Res. 2010; 70(2): 440-446).

[0172] The present invention further provides the composition of the present invention for use in the treatment of cancer. The used embodiment preferably treats 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 preferably treats a cancer that is gastric cancer.

[0173] An invention described herein is applicable to the treatment of a cancer, preferably a cancer that is tested for the presence of EGFR, HER2 and / or HER3 on the cell membrane. This can be accomplished by routine methods and is usually analyzed by immunohistochemistry.

[0174] The cancer preferably expresses HER2. The cancer preferably also expresses EGFR or HER3. The cancer preferably expresses EGFR. The cancer preferably also expresses HER2 or HER3. The cancer preferably expresses HER3. The cancer preferably also expresses EGFR or HER2. In certain embodiments, the cells of the cancer treated by the invention disclosed herein and / or the stromal cells in the cancer express an EGFR ligand, a HER3 ligand, or both. The expression of the ligand and receptor can therefore provide a growth stimulus to the cells of the cancer. A combination of the present invention is particularly suitable for treating cancers that include such cells.

[0175] In the treatment of the present invention, the expression of one of EGFR, HER2 and HER3 can at least delay the escape of some tumors. Tumors targeted using a monospecific therapy can escape treatment by starting to express another of EGFR, HER2 or HER3 or by expressing a ligand for a receptor.

[0176] Such cells, if they exist, can also be attacked by the binding moieties of the invention, and thus can be removed before they grow and diversify themselves. In one embodiment, the cancer is tested for the presence of a mutated EGFR. Many EGFR-positive tumors have genetic mutations that consistently render the cells resistant to treatment with tyrosine kinase inhibitors.

[0177] 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 R521K polymorphism. In certain embodiments, the cancer is known to be resistant to first generation TKI inhibitors such as gefitinib and erlotinib.

[0178] The cancer treatment 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 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 the further treatment include radiation therapy, chemotherapy, surgery, angiogenesis inhibition therapy, and hyperthermia.

[0179] The compositions of the invention may be suitable for use in the treatment of cancers that are resistant to EGFR inhibition, wherein EGFR resistance is a result of overexpression of HER2 and / or HER3.

[0180] The compositions of the invention may be suitable for use in the treatment of cancers that are resistant to HER2 inhibition, wherein HER2 resistance is a consequence of overexpression of EGFR and / or HER3.

[0181] The compositions of the invention may be suitable for use in the treatment of cancers that are resistant to HER3 inhibition, wherein HER3 resistance is a consequence of overexpression of EGFR and / or HER2.

[0182] the term In the context of antibodies, binding moieties, compositions or products as described herein is meant the presence of more than one and typically 10 or fewer different antibodies or binding moieties in a preparation, including the presence of a bispecific. An example includes a combination of two bispecific antibodies.

[0183] The present 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 EGFR variable domain comprises a heavy chain variable region comprising Figure 8 The HER2 variable domain comprises the CDRs of the heavy chain variable region MF3755, MF4280, MF4003 or MF4016, or one of the heavy chain variable regions comprises a variant of 1, 2 or 3 amino acids substituted, deleted and / or inserted in the CDRs, and wherein the HER2 variable domain comprises the CDRs of the heavy chain variable region MF2032 or MF1849, or one of the heavy chain variable regions comprises a variant of 1, 2 or 3 amino acids substituted, deleted and / or inserted in the CDRs.

[0184] 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 EGFR variable domain comprises a heavy chain variable region comprising Figure 8 The HER2 variable domain comprises the CDRs of the heavy chain variable region MF3755, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids in the CDRs, and wherein the HER2 variable domain comprises the CDRs of the heavy chain variable region MF2032, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids in the CDRs.

[0185] The present 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 EGFR variable domain comprises a heavy chain variable region comprising Figure 8 The amino acid sequence of the heavy chain variable region MF3755, MF4280, MF4003 or MF4016 in the heavy chain variable region, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs), and wherein the HER2 variable domain comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF2032 or MF1849, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs).

[0186] The present 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 EGFR variable domain comprises a heavy chain variable region comprising Figure 8 The amino acid sequence of the heavy chain variable region MF3755 in the embodiment of the present invention, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs), and wherein the HER2 variable domain comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF2032, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs).

[0187] 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 EGFR variable domain comprises a heavy chain variable region comprising Figure 8The HER2 variable domain comprises the CDRs of the heavy chain variable region MF3755, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids in the CDRs, and wherein the HER2 variable domain comprises the CDRs of the heavy chain variable region MF1849, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids in the CDRs.

[0188] The present 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 EGFR variable domain comprises a heavy chain variable region comprising Figure 8 The amino acid sequence of the heavy chain variable region MF3755 in the embodiment of the present invention, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs), and wherein the HER2 variable domain comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF1849, or a variant of the heavy chain variable region comprising substitution, deletion and / or insertion of 1, 2 or 3 amino acids (preferably not located in the CDRs).

[0189] For purposes of clarity and a concise 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 a combination of all or some of the described features.

[0190] Example

[0191] Cell lines

[0192] Hek293 cells, 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).

[0193] Generation of bispecific antibodies

[0194] Bispecific antibodies are produced using the DEKKCH3 technology described above for efficient heterodimerization and formation of bispecific antibodies. As described previously (WO 2013 / 157954 A1), the CH3 technology utilizes charge-based point mutations located in the CH3 region to allow efficient pairing of two non-identical heavy chain molecules.

[0195] A VH gene is cloned into one of two IgG1 vectors of different architectures. Depending on the binding partner, the VH is cloned into an IgG1 architecture comprising a CH3 variant with a heterodimerization variant "DE" or an IgG1 architecture comprising a complementary CH3 heterodimerization variant "KK". In the case of bi- or multi-specific antibodies 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).

[0196] Adherent Hek293 cells were cultured in 6-well culture plates to a confluency of 80%. The cells were briefly transfected with the DNA-FUGENE mixture and further cultured. Seven days after transfection, the supernatants were harvested and the culture medium was renewed. Fourteen days after transfection, the supernatants were combined and filtered through (0.22 μM. Sterile supernatants were stored at 4°C. Suspension-adapted Hek293 cells were cultured in T125 flasks on a shaker plate until a confluency of 3.0×10 6 The density of cells is 0.3-0.5×10 6 The cells were seeded into each well of a 24-deep well culture plate at a density of 10 viable cells / mL. The cells were transiently transfected with individual sterile DNA: PE1-MIX and further cultured. Seven days after transfection, the supernatant was harvested and filtered through 0.22 μM. The sterile supernatant was stored at 4°C.

[0197] Generation of a stable cell line pool co-expressing two bispecific antibodies

[0198] CHO cells were transfected with 3 heavy chain constructs and a common light chain construct at a molar ratio of 2.5:2:1:1 for common light chain construct (cLC):EGFR heavy chain:HER2 heavy chain:HER3 heavy chain. Ten pools (AJ) consisting of stably transfected cells were obtained. ELISA analysis of anti-EGFR, anti-HER2 and anti-HER3 antibodies was performed on the supernatants of the 10 pools at day 3 and day 6. All three specificities could be determined in all pools.

[0199] Stable cell line clones co-expressing two bispecific antibodies were generated by plating such pools in 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 picking of a single colony. Oligoclonics were generated from a single cell by fed-batch fermentation.

[0200] Determination of antibody titers

[0201] Cell supernatants were diluted 1:4 and 1:50 in PBS, depending on the total IgG concentration. Single antigen ELISAs were first performed to determine the presence of all three antibodies. The following antigens were used at a dilution of 2.5 μg / mL to coat the wells of an 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).

[0202] Two sandwich ELISAs were then developed to assay and quantify the two bispecific molecules, allowing estimation of the ratio between the two bispecifics. For the assay of the EGFRxHER2 bispecific, the EGFR-Fc (R&D Systems, 344-ER) antigen was coated on such wells and assayed using ErbB2-Fc (R&D Systems, 1129-ER). For the assay of the EGFRxHER3 bispecific, the EGFR-Fc antigen was coated on such wells and assayed using ErbB3-Fc (R&D Systems, 348-RB).

[0203] IgG purification

[0204] Purification of IgG was performed using affinity chromatography. Purification was performed using vacuum filtration under sterile conditions. First, the pH of the culture medium was adjusted to pH 8.0, and the product was then incubated with Protein A Sepharose CL-4B beads (50% v / v) (Pierce) for 2 hours at 25°C on a plate shaker set at 600 rpm. Secondly, the beads were harvested by vacuum filtration. The beads were washed twice with PBS pH 7.4. IgG was eluted using 0.1M citrate buffer at pH 3.0, and the IgG fraction was immediately neutralized by Tris pH 8.0. Buffer exchange was performed by centrifugation using Ultracel (Millipore). The samples were finally in a final buffer of PBS pH 7.4.

[0205] Cation Exchange Chromatography (CIEX)

[0206] CEX-HPLC chromatography was performed using the TSKgel SP-STAT (7 μm particle size, 4.6 mM ID x 10 cmL, Tosoh 21964) series of ion exchange columns. These columns are packed with non-porous resin particles for the rapid and high resolution analysis and separation of biomolecules. The particles in the TSKgel STAT columns contain an open access network structure consisting of multiple layers of ion exchange groups for loading capacity, and the relatively large particle size makes these columns suitable for use with HPLC and FPLC systems.

[0207] TSKgel SP-STAT (7 μm particle size, 4.6 mM ID×10 cmL, Tosoh 21964) was equilibrated with buffer A (sodium phosphate buffer, 25 mM, pH 6.0), after which the antibody was discharged from the column by increasing the salt concentration and a gradient of running buffer B (25 mM sodium phosphate, 1 mM NaCl, pH 6.0). The flow rate was set at 0.5 mL / min. The injected sample mass for all test samples and controls (in PBS) was 10 μg, and the injection volume was 10-100 μL. The chromatograms were analyzed for the observed peak pattern, retention time, and peak area of ​​the main peak based on the results at 220 nm.

[0208] BxPC-3-luc2 and N87 growth inhibition analysis

[0209] Antibody compositions were tested at a range of total antibody concentrations. The antibodies were pooled in pairs in equal amounts weight / weight. HRG and EGF were added to the cultures at 0.1 ng / mL EGF and 10 ng / mL HRG for BxPC3-luc2 cells or 0.1 ng / mL EGF and 1 ng / mL HRG for N87 cells. Basal w / ligand was a control without antibody but with the corresponding growth factor. Basal w / o ligand was a control without the indicated growth factor and without antibody.

[0210] Antibodies were diluted in chemically defined starvation medium (CDS: RPMI 1640 medium containing 80 U of penicillin and 80 μg of streptomycin per ml, 0.05% (w / v) BSA and 10 μg / mL holo-transferrin) and 50 μL of the diluted antibodies were added to the wells of a 96-well black well clear bottom plate (Costar). Ligands were added (50 μL per well of a stock solution containing 40 ng / mL or 4 ng / mL HRG and 400 ng / mL EGF diluted in CDS: 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 in a 37°C cell culture incubator for 3 days (N87 cells) or 4 days (BxPC-3-luc2 cells). On day 4, Alamar blue (Invitrogen, #DAL1100) was added (20 μL / well) and fluorescence was measured on a Biotek Synergy2 Multi-Mode Microplate Reader using excitation at 560 nm and readout at 590 nm after incubation (at 37°C) with Alamar blue for 6 hours (N87 cells) or 4 hours (BxPC-3-luc2 cells). Fluorescence values ​​were normalized to non-inhibited growth (no antibody, but both ligands were added).

[0211] Various ADCC activity

[0212] ADCC Reporter Bioassay (Promega) was used. Two different cell lines were tested: the EGFR phenotype pancreatic cancer cell line BxPC3 and the gastric cancer N87 cell line.

[0213] The bioassay uses engineered Jurkat cells that either stably express the V158 (high affinity) variant of the FcγRIIIa receptor and a NFAT response element driving the expression of firefly luciferase, a measure of FcγR activation. The assay has been compared to traditional ADCC assays by comparing data obtained using this ADCC reporter bioassay. 51 The ADCC assay was validated by comparison with the Cr release assay, and the two assays gave similar results. This ADCC assay was performed using the 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 assay medium (RPMI with 4% low IgG serum) 20-24 hours before the assay. The next day, the medium was removed. Next, A series of dilutions of and a comparator antibody, cetuximab, were prepared in duplicate. 10 μL of these antibody dilutions were added to the wells. A control well without antibody was also included (basal value). From the starting concentration of the antibody, a 5-fold serial dilution was generated to provide a dose-response curve. Finally, 5 μL of ADCC Bioassay effector cells (15,000 cells / well, V158) were added. The cells were incubated at 37°C for 6 hours. Next, 15 μL of BIO-Glo luciferase substrate was added and luminescence was measured in a plate reader after 5 minutes. The resulting data are shown in Figure 3 Cetuximab showed ADCC activity on BxPC3 and N87 cells. Various oligoclonal antibodies also showed ADCC activity on BxPC3 and / or N87 cells.

[0214] The test included bispecific antibodies PB4516 and PB6892. About it for BxPC-3- Effects on the growth of luc2 tumors (implanted orthotopically) and N87 tumors (gastric cells implanted in the flank)

[0215] 8-10 week old female CB17 SCID mice were orthotopically implanted in the pancreas with 1×10 6 BxPC-3-luc2 tumor cells. The mouse was anesthetized and placed on its right side to expose the left side, and a 0.5 cm incision was made on the left flank area. The pancreas and spleen were removed from the abdomen, and 1×10 6Tumor cells were injected into the subcapsular space of the pancreatic tail. One week after implantation, bioluminescence (BLI) data were generated. For BLI imaging (once or twice a week), all mice received an intraperitoneal injection (ipinjections) of 150 mg / kg Luciferin (D-Luciferin-EF potassium salt, Cat. # E6552, Promega) 15 minutes before the imaging. Outlier animals were removed based on BLI / tumor volume, and such mice were randomly distributed in groups of 7 mice per group. Treatment was started on the 8th day of the experiment.

[0216] Animals in the antibody treatment group were dosed weekly with 0.3 mg / kg of antibody for 4 consecutive weeks (days 0, 7, 14, and 21). On day 0 of the treatment, these animals received a double loading dose of 0.6 mg / kg of antibody. Final imaging was performed on day 35 or 40. Only vehicle and cetuximab-treated groups served as controls.

[0217] Cetuximab and oligoclonic significantly reduced BxPC-3 tumor overgrowth in this model (p<0.05) ( Figure 5 ).use Tumor outgrowth in PB4516 and PB6892 was significantly lower than that in the case of cetuximab. Cetuximab did not significantly reduce the outgrowth of N87 cells. Significantly reduced N87 tumor overgrowth in this model (p<0.05) ( Figure 5 ).

[0218] N87 Tumor :

[0219] CB17 SCID female mice aged 8-12 weeks were inoculated subcutaneously (sc) in the flank with 1×10 7 N87 tumor cells. The cell injection volume was 0.2 mL / mouse. Treatment was performed when the tumor reached 150-200 mm 3 The antibody was administered once a week for 4 weeks by intraperitoneal injection of mice at 25 mg / kg. Body weight was measured once a week after tumor cell injection and every two weeks from the start to the end of treatment. Tumor growth was monitored every two weeks by caliper measurements. The endpoint of the experiment was a tumor of 800 mm 3 of tumor volume or 60 days, whichever comes first.

[0220] Activity of PB11244 and PB4516 in various PDX models

[0221] Including bispecific antibodies PB11244 and PB4516 The activity of is 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.

[0222] The bispecific antibodies PB4516 and PB11244 have an IgG1 constant region and two variable domains.

[0223] The HER3 variable domain of PB4516 has Figure 7 or Figure 8 The EGFR variable domain has Figure 7 or Figure 8 The heavy chain variable region of MF3755 in.

[0224] The HER2 variable domain of PB11244 has Figure 7 or Figure 8 The EGFR variable domain has Figure 7 or Figure 8 The heavy chain variable region of MF3755 in.

[0225] The light chain variable regions in these two antibodies are identical and have Figure 7 The amino acid sequence of the common light chain variable region of sequence identification number: 11 (SEQ ID NO: 11).

[0226] A selection of several gastric cancer, esophageal cancer, and non-small cell lung cancer PDX models were generated (Figure 6).

[0227] The bispecific antibodies PB4516 and PB11244 are included. These antibodies were mixed in a 1:1 ratio. It was tested in this model and compared with cetuximab and vehicle (PBS).

[0228] This type of PDX model is first expanded subcutaneously (sc) in donor BALB / c nude mice. The tumor is extracted, cut into small pieces (2-3 mm in diameter) and implanted subcutaneously into new recipient BALB / c nude mice. Tumor recipients are 6-8 week old female BALB / c nude mice. Tumor growth is tracked by caliper measurement until the tumor reaches 100-200 mm3 The average size of the . During this period (denoted as Day 1), animals were randomly assigned to one of three groups per model. Treatments were initiated on the same day and included:

[0229] – PB4516×PB6892 25 mg / kg, 5-week dose, intraperitoneal injection

[0230] – Cetuximab 25 mg / kg, 5 weeks of dosing, intraperitoneal injection

[0231] – Vehicle (PBS), 5-week dosing, i.p.

[0232] It can be seen that: Significantly reduced the overgrowth of tumor cells in the model. The reduction in overgrowth was equal to or better than that of cetuximab.

[0233] Table 1. Arg 426 in HER3 Residues within the radius

[0234]

[0235] Sequence Listing <110> MERUS NV <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> PatentIn 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 IgKV1*39 / jk1 <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*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, the method 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 said nucleic acids further comprises an expression control sequence to allow expression of said encoded heavy and light chains in said cell; as well as 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 - 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 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.

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