Combination of binding moieties that bind to EGFR, her2 and her3

Abstract

To provide combinations of binding moieties targeting various members of an EGF receptor family and methods of making them.SOLUTION: The invention provides a composition comprising two or more binding moieties, where each of the binding moieties comprises a variable domain that binds to an extracellular moiety of EGFR, where a first binding moiety comprises a variable domain that binds to an extracellular moiety of HER2, and a second binding moiety comprises a variable domain that binds to an extracellular moiety of HER3. The invention also provides means and a method for producing the composition and for treating a subject using the composition.SELECTED DRAWING: None

Description

[Technical field]

[0001] 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 therapy. More particularly, the present invention relates to compositions comprising two or more multispecific binding moieties, preferably compositions comprising multispecific antibodies. The binding moieties bind to EGFR, HER2 and HER3. A single host cell can produce multiple binding moieties. [Background technology]

[0002] The epidermal growth factor (EGF) receptor (EGFR) is the prototypic cell surface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands, which currently comprises four closely related receptor tyrosine kinases: EGFR, HER2 (ErbB-2 / c-neu), HER3 (ErbB-3), and HER4 (ErbB-4).

[0003] 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 ligand, the receptor transitions from an inactive, mostly monomeric form to an active homodimer. In addition to forming homodimers after ligand binding, EGFR can pair with other members of the ErbB receptor family, such as HER2, to generate activated heterodimers. There is also evidence suggesting that dimers form in the absence of ligand binding and that clusters of activated EGFR form after ligand binding.

[0004] EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase (PTK) activity, which induces several signaling cascades that lead to cell proliferation and differentiation. The kinase domain of EGFR can cross-phosphorylate tyrosine residues of other receptors with which it is complexed, and thus activate itself.

[0005] 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. Summary of the Invention [Problem to be solved by the invention]

[0006] Although EGFR-targeted therapy has been somewhat successful, most cases are associated with the development of treatment resistance over time. One way that EGFR-positive tumors can escape targeted therapy is by signaling through dimers of another receptor. Increased signaling through EGFR / HER3 dimers, for example due to increased HER3 expression or heregulin expression, is associated with EGFR-targeted drug resistance, for example, in lung and head and neck cancers. Apart from causing treatment resistance, several side effects of EGFR-targeted antibodies have been observed. One example is the development of skin rashes associated with EGFR inhibition or anti-EGFR biological therapy. In extreme cases, such rashes can lead to reduced treatment cycles and / or premature termination of treatment.

[0007] Various modes of signal transduction of the EGF receptor family have been identified. Among these are ligand-dependent and ligand-independent activation of signal transduction. Overexpressed HER2 can generate oncogenic signal transduction through HER2 / HER3 heterodimers even in the absence of HER3 ligands (Junttila, Akita et al., 2009). HER2 activity can be inhibited by HER2-specific antibodies. Such HER2-specific antibodies are used, for example, to treat HER2-positive (HER2+) tumors. The problem with such treatment is that tumors often escape HER2-specific treatment and continue to grow even in the presence of inhibitory antibodies. It has been observed that HER2-positive tumors, such as breast, ovarian, cervical and gastric tumors, can escape treatment by selective proliferation 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). Activating mutations in the HER3 receptor have also been identified.

[0008] Thus, despite the promising results of antibody therapy that specifically targets EGF receptor family members, it has been observed that not all tumors respond or respond well.The present invention provides combinations of binding moieties that target various members of the EGF receptor family and methods for their production.The combinations of the present invention show good efficacy.The combinations can be produced in a cost-effective and efficient manner. [Means for solving the problem]

[0009] The present invention provides a composition comprising two or more binding moieties, Each binding moiety comprises a variable domain that binds to the extracellular portion of EGFR; A composition is provided in which a first of said binding moieties comprises a variable domain that binds to the extracellular portion of HER2 and a second of said binding moieties comprises a variable domain that binds to the extracellular portion of HER3.

[0010] 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. The antibody is preferably a multispecific antibody, preferably a bispecific antibody. Preferably, at least one, more preferably at least two of the antibodies are IgG antibodies. In a preferred embodiment of the present invention, the composition comprises two bispecific antibodies.

[0011] The multispecific antibodies described herein preferably comprise a heavy chain having 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 heavy chain of the HER2 variable domain and the heavy chain of the HER3 variable domain, respectively.

[0012] The present invention also provides a composition as described herein for use in the treatment of cancer. In some embodiments, the cancer is a solid epithelial cancer. Preferably, the composition is used for cancer expressing EGFR, HER2 and / or HER3. The composition is preferably used for pancreatic cancer, colorectal cancer, head and neck cancer, epithelial ovarian cancer, epithelial fallopian tube cancer, epithelial peritoneal cancer, bladder cancer, or prostate cancer. In some embodiments, the cancer treated by use of the composition is an advanced cancer. The composition is preferably used for metastatic cancer. The composition is preferably used for metastatic pancreatic cancer, metastatic colorectal cancer, metastatic head and neck cancer, metastatic epithelial ovarian cancer, metastatic epithelial fallopian tube cancer, metastatic epithelial peritoneal cancer, metastatic bladder cancer, or metastatic prostate cancer. In some embodiments, the composition is preferably used for gastric cancer, lung cancer, breast cancer, or esophageal cancer. Preferably, the composition is used for metastatic gastric cancer, metastatic lung cancer, metastatic breast cancer, or metastatic esophageal cancer.

[0013] The invention further provides a product comprising two or more binding moieties, each comprising a variable domain that binds to the extracellular portion of EGFR, a first said binding moiety comprising a variable domain that binds to the extracellular portion of HER2 and a second said binding moiety comprising 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.

[0014] The present invention further provides a process for producing a composition according to the invention, the process comprising the steps of: Providing a cell, a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, 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 that allows for expression of the encoded heavy and light chains in the cell, and the method further comprises culturing the cell to allow expression of the heavy and light chains, and optionally harvesting the two or more binding moieties.

[0015] Further, a cell comprising: a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, wherein the two or more nucleic acids may or may not be physically linked, and each of the nucleic acids further comprises an expression control sequence that allows expression of the encoded heavy and light chains in the cell.

[0016] In a further aspect, the invention provides a container containing nucleic acid, comprising: a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, wherein, optionally, two or more of said nucleic acids may or may not be physically linked, each of said nucleic acids further comprising expression control sequences that enable expression of the encoded heavy and light chains in a cell.

[0017] The invention further provides compositions comprising binding moieties that specifically bind to the extracellular portion of EGFR and the extracellular portion of HER2.

[0018] The invention also provides compositions comprising binding moieties that specifically bind to the extracellular portion of EGFR and the extracellular portion of HER3.

[0019] The binding moiety is preferably an antibody, preferably an IgG antibody, more preferably a multispecific antibody.

[0020] The term EGFR as used herein refers to the protein encoded by the epidermal growth factor receptor gene (EGFR) in humans. This protein is known by several 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 its encoding gene is (GenBank NM_005228.3). This accession number is provided primarily to provide an additional method of identifying the EGFR protein as a target, and the actual sequence of the EGFR protein bound by an antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers. References made herein to EGFR refer to human EGFR unless otherwise specified. Due to the similarity of sequence and tertiary structure of orthologs of EGFR between humans and other mammals, EGFR variable domains may also bind to such orthologs, but do not necessarily bind to such orthologs. Variable domains that bind EGFR may bind to EGFR and its various variants, such as those expressed on some EGFR-positive tumors.

[0021] The EGFR-binding variable domain of the antibody or binding moiety of the invention preferably binds to domain I or domain III of EGFR. The structure of the EGFR protein is described, inter alia, in Ferguson (2008: Annu Rev Biophys. 2008;37:353-373. doi:10.1146 / annurev.biophysics.37.032807.125829). The domains of human EGFR are set out in FIG. 1 of the Ferguson reference mentioned above. The EGFR-binding variable domain of the embodiments of the invention disclosed herein preferably binds to domain III of EGFR. The antibody preferably inhibits EGF-induced proliferation of BxPC-3 (ATCC CRL-1687) or BxPC-3-luc2 cells (Perkin Elmer 125058).

[0022] The term HER2 as used herein refers to the protein encoded by the ERBB-2 gene in humans. Alternative names for this 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 (human epidermal growth factor receptor 2). When HER2 is mentioned herein, the reference refers to human HER2. Antibodies that contain a variable domain that binds HER2 bind to human HER2. Due to the similarity of sequence and tertiary structure of orthologs of HER2 between humans and other mammals, the HER2 variable domain may, but does not necessarily, bind to such orthologs. 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). This accession number is provided primarily to provide an additional method of identifying HER2 as a target, and the actual sequence of the HER2 protein bound to the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers, etc. The HER2 variable domain can bind to HER2 and various variants thereof, including those expressed by some HER2-positive tumor cells.

[0023] The HER2 protein comprises several domains (see Figure 1 in Landgraf, R Breast Cancer Res. 2007;9(1):202-). The extracellular domains are referred to as domains I-IV. The variable domains of the embodiments of the invention disclosed herein that bind to HER2 preferably bind to domain I or domain IV of HER2, preferably bind to domain IV.

[0024] The term HER3 as used herein 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, the reference refers to human HER3. An antibody comprising a variable domain that binds HER3 binds to human HER3. Due to the similarity of sequence and tertiary structure of orthologs of HER3 between humans and other mammals, the HER3 variable domain may also bind to such orthologs, but does not necessarily bind to such orthologs. Database accession numbers for the human HER3 protein and the gene encoding it are (NP_001005915.1; NP_001973.2, NC_000012.11, NT_029419.12). This accession number is provided primarily to provide an additional method of identifying HER3 as a target, and the actual sequence of the HER3 protein bound to the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers, etc. The HER3 variable domain can bind to HER3 and various variants thereof, such as those expressed by some HER2 positive tumor cells.

[0025] The structure of HER3 is described, inter alia, in Cho et al. (2002; Science 297, 1330-1333: DOI:10.1126 / science.1074611). The human protein has four extracellular domains. The variable domain of the embodiments of the invention disclosed herein that bind to HER3 preferably binds to domain III of HER3. In a preferred embodiment, the affinity (KD) of the variable domain for HER3 positive cells is 2.0 nM or less, more preferably 1.5 nM or less, more preferably 1.39 nM or less, more preferably 0.99 nM or less. In a preferred embodiment, the antibody according to the invention comprises a variable domain that binds to at least one amino acid in domain III of HER3, preferably selected from the group consisting of R426 and amino acid residues located within 11.2 Å of R426 in the native HER3 protein. In a preferred embodiment, the affinity (KD) of the variable domain for HER3 on SK-BR-3 cells is 2.0 nM or less, more preferably 1.5 nM or less, more preferably 1.39 nM or less, preferably 0.99 nM or less. In one embodiment, the affinity (KD) is in the range of 1.39 to 0.59 nM. In a preferred embodiment, the affinity (KD) of the variable domain for HER3 on BT-474 cells is 2.0 nM or less, more preferably 1.5 nM or less, more preferably 1.0 nM or less, more preferably 0.5 nM or less, more preferably 0.31 nM or less, more preferably 0.23 nM or less. In one embodiment, the affinity (KD) is in the range of 0.31 to 0.15 nM. The above affinities are preferably measured using steady-state cell affinity measurements, where cells are incubated at 4°C with radiolabeled antibodies and then cell-bound radioactivity is measured.

[0026] The variable domain that binds to at least one amino acid in domain III of HER3 preferably binds to an amino acid selected from the group including R426 in the native HER3 protein and an amino acid residue located within 11.2 Å of R426. Preferably, the amino acid residue located within 11.2 Å of R426 in the native HER3 protein is selected from the group consisting of L423, Y424, N425, G427, G452, R453, Y455, E480, R481, L482, D483 and K485 (see, for example, FIG. 9 and Table 1). The numbering of the amino acid residues is ProteinDataBank (PDB) ID number 4P59. Antibodies that bind to this region of domain III of HER3 show particularly good binding properties and can counteract the activity of HER3 against HER3-positive cells. Variable domains with HER3 binding properties are described in WO2015 / 130172, which is incorporated herein by reference. In one preferred embodiment, a bispecific antibody according to the invention is provided, which comprises a variable domain that binds to at least R426 in domain III of HER3. Preferably, the antibody comprises a variable domain that binds to at least R426 in domain III of HER3.

[0027] In some embodiments, a composition comprises two or more antibodies, each of the antibodies comprising a variable domain that binds to an extracellular portion of EGFR, a first of the antibodies comprising a variable domain that binds to an extracellular portion of HER2, and a second of the antibodies comprising a variable domain that binds to an 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 essentially the same amino acid sequence. In one embodiment, the first and second antibodies comprise a variable domain that binds to domain I of EGFR, the first antibody comprises a variable domain that binds to domain I of HER2, and the second antibody comprises a variable domain that binds to domain III of HER3. In another embodiment, the first and second antibodies comprise a variable domain that binds to domain I of EGFR, the first antibody comprises a variable domain that binds to domain IV of HER2, and the second antibody comprises a variable domain that binds to domain III of HER3. In further embodiments, the first and second antibodies comprise a variable domain that binds to domain III of EGFR, the first antibody comprises a variable domain that binds to domain I of HER2, and the second antibody comprises a variable domain that binds to domain III of HER3. In further embodiments, the first and second antibodies comprise a variable domain that binds to domain III of EGFR, the first antibody comprises a variable domain that binds to domain IV of HER2, and the second antibody comprises a variable domain that binds to domain III of HER3.

[0028] In some embodiments, the binding moiety is a protein or an aptamer. The binding moieties described herein typically have two or more binding specificities. The binding moiety preferably comprises two or more variable domains of an antibody. The variable domains can be provided in a variety of ways. Several antibody variable domain-containing fragments are described in Nelson 2010:MAbs.2010 Jan-Feb;2(1):77-83, including various FAB fragments, scFv fragments, and so-called single domain antibodies, e.g., VHH fragments. Various FAB fragments or single chain Fv fragments are now well known. A single domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it can selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than typical antibodies (150-160 kDa) which are composed of two heavy and two light chains, and even smaller than Fab fragments (~50 kDa, half a light 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 generated from camelid heavy chain antibodies. Similar single domain fragments can now be artificially generated and may be derived from other organisms. The variable domain preferably comprises a heavy chain variable region and a light chain variable region. The variable domain is sometimes called a VH / VL combination (VH represents the variable region of the heavy chain and VL represents the variable region of the light chain).

[0029] Two or more fragments can be linked to generate a binding moiety with the same number of binding specificities. Linking is typically performed using a linking peptide that includes two or more amino acid residues. The linking moiety can also be part or all of a protein. For example, human serum albumin may be used. The binding moieties described herein preferably comprise at least one variable domain paired with a heavy chain variable region and a light chain variable region (e.g., a light chain variable region) of a MF, for example as described in Figure 7 or Figure 8. In a preferred embodiment, the binding moiety comprises two or more such variable domains.

[0030] The binding moiety that binds EGFR and HER2 is a different binding moiety than the binding moiety that binds EGFR and HER3. When at least one of the binding moieties is a multispecific antibody, at least one multispecific antibody can bind at least EGFR and HER2 or at least EGFR and HER3. In a preferred embodiment, the binding moiety comprises a bispecific antibody, where one bispecific antibody binds EGFR and HER2 and another bispecific antibody binds EGFR and HER3.

[0031] As used herein, the term "antibody" refers to a protein molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind to an epitope on an antigen, such domains being derived from or sharing sequence homology with the variable region of the antibody. Antibodies are typically composed of a basic structural unit, each having two heavy chains and two light chains. Antibodies for therapeutic use are preferably as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies of a human subject). The antibodies according to the present invention are not limited to any particular format or method of producing them.

[0032] Since an antibody typically recognizes an epitope of an antigen, and such epitopes may also be present in other compounds, an antibody according to the invention that "specifically recognizes" an antigen, for example, EGFR, HER2 or HER3, may similarly recognize other compounds if such compounds contain the same type of epitope. Thus, the terms "specifically recognize" or "specifically bind" or terms having the same meaning with respect to the interaction of an antigen with an antibody do not exclude the binding of the antibody to other compounds that contain the same or the same type of epitope.

[0033] A "bispecific antibody" is an antibody as described herein in which one variable domain of the antibody binds to a first antigen while another variable domain of the antibody binds to a second antigen, the first and second antigens being non-identical. The term "bispecific antibody" also encompasses antibodies in which one heavy chain variable region / light chain variable region (VH / VL) combination binds to a first epitope on an antigen and another VH / VL combination binds to a second epitope. The second epitope may be a different epitope on the same antigen. The term further includes antibodies in which the VH can specifically recognize a first antigen and the VL can be paired with a VH in an immunoglobulin variable region to specifically recognize a second antigen. The resulting VH / VL pair binds to 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 antibodies according to the invention are not limited to any particular bispecific format or method of producing them.

[0034] Bispecific antibodies are an example of multispecific antibodies. Trispecific (and higher) antibodies can be made by adding binding moieties, such as scFv fragments, to one or more heavy chains. It is also possible to add one or more variable domains to the variable region of a common antibody or bispecific antibody. Cells producing a common light chain and two different heavy chains, each of which can form a functional variable domain with the common light chain, produce, among others, bispecific antibodies with two different heavy and light chain combinations. Similarly, cells producing a common light chain and three or more different heavy chains can form several bispecific antibodies that can be combined to target three or more antigens. It is now possible to build on the standard format of an antibody (i.e., a constant part and two variable domains) and add additional binding domains. Thus, multispecific antibodies can be made with one or more single chain Fvs with additional binding specificities that bind to the constant domain or one or more variable domains of the antibody. It is also possible to make heavy chains with two or more variable regions. The additional heavy chain regions can be advantageously associated with different or common light chain variable regions. For a description of such antibodies, see US Pat. No. 62 / 650467, which is incorporated herein by reference.

[0035] As used herein, when referring to nucleic acid or amino acid sequences, "percent (%) identity" is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after aligning the sequences for optimal comparison purposes. Percent sequence identity for comparing nucleic acid sequences is determined using the AlignX application of Vector NTI Program Advance 10.5.2 software using default settings, which use a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc. Acid Res. 22:4673-4680), a swgapdnarnt scoring matrix, a gap open 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 settings, employing a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ 1994), a blosum62mt2 scoring matrix, a gap open penalty of 10, and a gap extension penalty of 0.1.

[0036] The term "common light chain" as used herein refers to a light chain that can be used, for example, in a multispecific antibody. In a bispecific antibody, the two light chains can be common light chains (or VL portions thereof). The two light chains (or VL portions thereof) can be identical or have some amino acid sequence differences, although the binding specificity of the full-length antibody is not affected. The terms "common light chain", "common VL", "single light chain", "single VL", with or without the addition of the term "rearranged", are all used interchangeably herein. "Common" refers to light chains with the same sequence, and also those that are not identical in amino acid sequence but are functionally equivalent. There are many variants of the light chain, and there are mutations (deletions, substitutions, insertions and / or additions) that do not affect the formation of a functional binding region. The light chain of the present invention can also be a light chain as specified herein, with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or combinations thereof. It is within the scope of the definition of common light chain used herein to prepare or find a light chain that is not identical but is still functionally equivalent, for example by introducing and testing conservative amino acid changes, amino acid changes in regions that do not contribute or only partially contribute to the binding specificity when paired with the heavy chain, etc. In some embodiments, a multispecific antibody with three or more variable domains has different heavy chains and light chains with the same light chain variable domains or with some amino acid differences, but 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 the multispecific antibody comprise a common light chain. The common light chain (variable region) for use in the multivalent antibody of the present invention may be a lambda light chain, thus also provided in the context of the present invention, although a kappa light chain is preferred. The common light chain of the present invention may comprise the constant region of a kappa or lambda light chain. It is preferably a constant region of a kappa light chain, and preferably said common light chain is a germline light chain, preferably a rearranged germline human kappa light chain comprising the IgVK1-39 gene segment, such as a rearranged germline human kappa light chain IgVK1-39. * 01 / IGJK1 *01. Examples of amino acid sequences of common light chains are shown in sequences 10, 11, or 12 of FIG.

[0037] The term "full-length IgG" or "full-length antibody" according to the present invention is defined as comprising essentially a complete IgG, but not necessarily having all the functions of a complete IgG. For the avoidance of doubt, a full-length IgG comprises two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which can be broken down into domains designated CH1, CH2, CH3, VH and CL, VL. IgG antibodies bind to antigens via the variable region domains contained in the Fab portion, and after binding can interact with molecules and cells of the immune system via the constant domains, primarily the Fc portion. Full-length antibodies according to the present invention encompass IgG molecules in which mutations may be present that provide desired properties. Full-length IgG should not have a deletion of a substantial portion of any of the regions. However, IgG molecules in which one or several amino acid residues have been deleted without essentially altering the binding properties of the resulting IgG molecule are included in the term "full-length IgG". For example, such IgG molecules may have a deletion of 1 to 10 amino acid residues, preferably in the non-CDR regions, where the deleted amino acids are not essential for the antigen specificity or epitope binding specificity of the IgG. Examples of IgG antibodies are IgG1, IgG2, IgG3 and IgG4 antibodies. In some embodiments of the invention, the IgG is IgG1.

[0038] Preferably, at least one of the two or more binding moieties is an antibody. The antibody can comprise 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 can comprise a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER3.

[0039] The two or more binding moieties preferably comprise two or more antibodies, preferably multispecific antibodies each comprising a variable domain that binds to the extracellular portion of EGFR, a first such antibody comprising a variable domain that binds to the extracellular portion of HER2 and a second such antibody comprising 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 shown diagrammatically in FIG. 1. Two bispecific antibodies are shown, each having two heavy chains (1) and two light chains (4). The two antibodies share a heavy chain with a heavy chain 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 pair with a common light chain (4) to form a functional binding domain. When produced by the same cell, the heavy chains are induced to heterodimerize by the presence of a heterodimerization domain (2,3). A heterodimerization domain has two parts, one part on one heavy chain and a matching part on the other heavy chain. The heterodimerization domain is often in the IgG1 CH3 region. Heterodimerization can be induced by providing the appropriate part to the selected heavy chain.

[0040] In the present invention, the selective formation of EGFRxHER2 and EGFRxHER3 bispecific antibodies can be induced by incorporating a portion of the heterodimerization domain (3) into the heavy chain forming EGFR variable domain and by incorporating an affinity portion (2) into the heavy chain forming the HER2 and HER3 binding domains.

[0041] A heavy chain having a heavy chain variable region that, together with a light chain, forms a variable domain that binds to an antigen, such as EGFR, HER2, or HER3, is also referred to herein as an EGFR heavy chain, a HER2 heavy chain, or the like. In a preferred embodiment of the invention, the CH3 region of the heavy chain of the first and / or second antibody is engineered to promote heterodimerization of the HER2 heavy chain with the EGFR heavy chain, and the HER3 heavy chain with the EGFR heavy chain. In a preferred embodiment, the engineering to promote heterodimerization uses the DEKK residue positions previously described in U.S. Pat. Nos. 9,248,182, 9,358,286; 9,248,182, and 9,758,805.

[0042] In some embodiments, binding of the antibody of the composition to EGFR blocks EGF binding to EGFR and / or binding of the antibody of the composition to HER3 blocks Neuregulin 1 (NRG) binding to HER3. In preferred embodiments, binding of the antibody of the composition to EGFR blocks EGF binding to EGFR and binding of the antibody of the composition to HER3 blocks Neuregulin 1 (NRG) binding to HER3.

[0043] The variable domain that binds to the extracellular portion of EGFR preferably comprises a heavy chain variable region comprising the CDR1 sequence NYAMN, the CDR2 sequence WINANTGDPTYAQGFTG, and the CDR3 sequence ERFLEWLHFDY, or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in the CDRs.

[0044] The variable domain that binds to the extracellular portion of HER2 preferably comprises a heavy chain variable region comprising the CDR1 sequence SYGMH, the CDR2 sequence VISYDGSNKYYADSVKG, and the CDR3 sequence DYYRRTARAGFDY, or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in the CDRs.

[0045] The variable domain that binds to the extracellular portion of HER3 preferably comprises a heavy chain variable region comprising the CDR1 sequence GYYMH, the CDR2 sequence WINPNSGGTNYAQKFQG, and the CDR3 sequence DHGSRHFWSYWGFDY, or a variant thereof comprising substitutions, deletions and / or insertions of 1, 2 or 3 amino acids in the CDRs.

[0046] In a preferred embodiment, the composition comprises two bispecific antibodies, a first said bispecific antibody comprises a variable domain that binds to the extracellular portion of EGFR comprising a heavy chain variable region comprising the CDR1 sequence NYAMN, the CDR2 sequence WINANTGDPTYAQGFTG, and the CDR3 sequence ERFLEWLHFDY, or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in the CDRs. In a preferred embodiment, the first and second said bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR comprising a heavy chain variable region comprising the CDR1 sequence NYAMN, the CDR2 sequence WINANTGDPTYAQGFTG, and the CDR3 sequence ERFLEWLHFDY, or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in the CDRs.

[0047] In a preferred embodiment, said first and second bispecific antibodies comprise a variable domain binding to the extracellular portion of EGFR comprising a heavy chain variable region comprising the CDR1 sequence NYAMN, the CDR2 sequence WINANTGDPTYAQGFTG, and the CDR3 sequence ERFLEWLHFDY, or a variant thereof comprising one, two or three amino acid substitutions, deletions and / or insertions in the CDRs, and said first bispecific antibody further comprises a variable domain binding to the extracellular portion of HER2, which variable domain preferably comprises the CDR1 sequence SYGMH, the CDR2 sequence VISYDGSNKYYADSVK and the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER3, said variable domain preferably comprising a heavy chain variable region comprising the CDR1 sequence GYYMH, the CDR2 sequence WINPNSGGTNYAQKFQG, and the CDR3 sequence DHGSRHFWSYWGFDY, or a variant thereof comprising one, two or three amino acid substitutions, deletions and / or insertions in the CDRs.

[0048] In a preferred embodiment, said first and second bispecific antibodies comprise a variable domain binding to the extracellular portion of EGFR comprising a heavy chain variable region comprising the CDR1 sequence NYAMN, the CDR2 sequence WINANTGDPTYAQGFTG, and the CDR3 sequence ERFLEWLHFDY, or a variant thereof comprising one, two or three amino acid substitutions, deletions and / or insertions in the CDRs, and said first bispecific antibody further comprises a variable domain binding to the extracellular portion of HER2, which variable domain preferably comprises the CDR1 sequence SYGMH, the CDR2 sequence VISYDGSNKYYADSVK and the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER3, said variable domain preferably comprising a heavy chain variable region comprising the CDR1 sequence GYYMH, the CDR2 sequence WINPNSGGTNYAQKFQG, and the CDR3 sequence DHGSRHFWSYWGFDY, or a variant thereof comprising one, two or three amino acid substitutions, deletions and / or insertions in the CDRs.

[0049] Conservative variations of 1, 2, or 3 amino acid residues from the listed CDR sequences are possible while retaining the same binding activity in kind (essentially equivalent, but not necessarily in amount). Thus, the heavy chain CDR1, 2, and 3 sequences preferably contain sequences that deviate from the listed CDR sequences by no more than 3 amino acids, preferably no more than 2, more preferably no more than 1 amino acid. In certain embodiments, the heavy chain CDR1, 2, and 3 sequences are identical to the listed CDR sequences.

[0050] In some embodiments, the EGFR variable domain comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the EGF VH region set forth in Figure 7 or Figure 8, preferably that of MF3755 in Figure 7 or Figure 8.

[0051] In some embodiments, the EGFR variable domain comprises a heavy chain variable region comprising an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical, or 100% identical to the amino acid sequence of the EGFRVH region set forth in Figure 7 or Figure 8, preferably that of MF3755 in Figure 7 or Figure 8.

[0052] For example, in some embodiments, the heavy chain variable region of a bispecific antibody that binds to human EGFR can have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof in the sequence of the heavy chain variable region outside of the three CDR sequences. In some embodiments, the heavy chain variable region comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, preferably 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof relative to the amino acid sequence shown.

[0053] In a particular embodiment, the EGFR variable domain comprises a heavy chain variable region comprising an amino acid sequence from an EGFRVH region selected from Figure 7 or Figure 8, preferably that of MF3755 in Figure 7 or Figure 8.

[0054] In some embodiments, the HER2 variable domain comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the HER2 VH region set forth in FIG. 7 or FIG. 8, preferably that of MF2032 in FIG.

[0055] 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, or 100% identical to the amino acid sequence of the HER2 VH region depicted in Figure 7 or Figure 8, preferably that of MF2032 in Figure 7 or Figure 8.

[0056] For example, in some embodiments, the heavy chain variable region of a bispecific antibody that binds to human HER2 can have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof in the heavy chain variable region sequence outside of the three CDR sequences. In some embodiments, the heavy chain variable region comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, preferably 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof relative to the amino acid sequence shown.

[0057] In certain embodiments, the HER2 variable domain comprises a heavy chain variable region comprising an amino acid sequence selected from MF1849 or MF2032 of Figure 7 or Figure 8. Preferably, it comprises that of MF2032 of Figure 7 or Figure 8.

[0058] In some embodiments, the HER3 variable domain comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the VH region of MF3178 of FIG.

[0059] In some embodiments, the HER3 variable domain comprises a heavy chain variable region comprising an amino acid sequence 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 set forth in MF3178 in Figure 7 or Figure 8.

[0060] For example, in some embodiments, the heavy chain variable region of a bispecific antibody that binds to human HER3 can have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof in the sequence of the heavy chain variable region outside of the three CDR sequences. In some embodiments, the heavy chain variable region comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof relative to the amino acid sequence shown.

[0061] In certain embodiments, the HER3 variable domain comprises a heavy chain variable region comprising the amino acid sequence of MF3178 in FIG.

[0062] In a preferred embodiment, said first and second bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR, comprising a heavy chain variable region comprising an amino acid sequence from an EGFR VH region selected from Figure 7 or Figure 8 or a variant thereof, preferably the amino acid sequence of MF3755 of Figure 7 or Figure 8 or a variant thereof, said variant comprising substitutions, deletions and / or insertions of 1, 2 or 3 amino acids, preferably not in the CDRs, and said first bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, said variable domain being MF1849 or MF2032 of Figure 7 or Figure 8 or a variant thereof, preferably the amino acid sequence of MF3755 of Figure 7 or Figure 8 or a variant thereof, 8 or a variant thereof, wherein the variant preferably comprises one, two or three amino acid substitutions, deletions and / or insertions that are not in the CDRs, and 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 the amino acid sequence of MF3178 of FIG. 7 or FIG. 8 or a variant thereof, wherein the variant preferably comprises one, two or three amino acid substitutions, deletions and / or insertions that are not in the CDRs.

[0063] In a preferred embodiment, said first and second bispecific antibodies comprise a variable domain binding to the extracellular portion of EGFR comprising a heavy chain variable region comprising the amino acid sequence of MF3755 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs, said first bispecific antibody further comprises a variable domain binding to the extracellular portion of HER2, which variable domain comprises a heavy chain variable region comprising the amino acid sequence of MF2032 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs, and said second bispecific antibody further comprises a variable domain binding to the extracellular portion of HER3, which variable domain comprises a heavy chain variable region comprising the amino acid sequence of MF3178 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs.

[0064] In one embodiment, said first and second bispecific antibodies comprise a variable domain binding to the extracellular portion of EGFR comprising a heavy chain variable region comprising the amino acid sequence of MF4280 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs; said first bispecific antibody further comprises a variable domain binding to the extracellular portion of HER2 comprising a heavy chain variable region comprising the amino acid sequence of MF1849 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs; and said second bispecific antibody further comprises a variable domain binding to the extracellular portion of HER3 comprising a heavy chain variable region comprising the amino acid sequence of MF3178 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs.

[0065] In one embodiment, said first and second bispecific antibodies comprise a variable domain binding to the extracellular portion of EGFR comprising a heavy chain variable region comprising the amino acid sequence of MF4280 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs; said first bispecific antibody further comprises a variable domain binding to the extracellular portion of HER2 comprising a heavy chain variable region comprising the amino acid sequence of MF2032 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs; and said second bispecific antibody further comprises a variable domain binding to the extracellular portion of HER3 comprising a heavy chain variable region comprising the amino acid sequence of MF3178 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs.

[0066] In one embodiment, said first and second bispecific antibodies comprise a variable domain binding to the extracellular portion of EGFR comprising a heavy chain variable region comprising the amino acid sequence of MF4003 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs; said first bispecific antibody further comprises a variable domain binding to the extracellular portion of HER2 comprising a heavy chain variable region comprising the amino acid sequence of MF1849 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs; and said second bispecific antibody further comprises a variable domain binding to the extracellular portion of HER3 comprising a heavy chain variable region comprising the amino acid sequence of MF3178 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs.

[0067] In one embodiment, said first and second bispecific antibodies comprise a variable domain binding to the extracellular portion of EGFR comprising a heavy chain variable region comprising the amino acid sequence of MF4003 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs; said first bispecific antibody further comprises a variable domain binding to the extracellular portion of HER2 comprising a heavy chain variable region comprising the amino acid sequence of MF2032 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs; and said second bispecific antibody further comprises a variable domain binding to the extracellular portion of HER3 comprising a heavy chain variable region comprising the amino acid sequence of MF3178 of Figure 7 or Figure 8 or a variant thereof, preferably a variant comprising one, two or three amino acid substitutions, deletions and / or insertions that are not present in the CDRs.

[0068] Exemplary EGFR heavy chain variable regions are described in WO 2015 / 130172 and PCT / NL2018 / 050537, which are incorporated herein by reference. Exemplary HER2 heavy chain variable regions are described in WO 2015 / 130173, which are incorporated herein by reference. Exemplary HER3 heavy chain variable regions are described in WO 2015 / 130172 and WO 2015 / 130173, which are incorporated herein by reference.

[0069] Further variants of the disclosed amino acid sequences that retain EGFR, HER2 or HER3 binding can be obtained, for example, from rearranged human IGKV1-39 / IGKJ1 VL regions (DeKruif et al., Biotechnol Bioeng. 2010(106)741-50) and phage display libraries that contain collections of VH regions that incorporate amino acid substitutions in the amino acid sequences of the EGFR, HER2 or HER3 VH regions disclosed herein, as described above. Phages that encode Fab regions that bind EGFR, HER2 or HER3 can be selected, analyzed by flow cytometry, and sequenced to identify variants that retain antigen binding with amino acid substitutions, insertions, deletions, or additions.

[0070] The present invention further provides a binding moiety which specifically binds to the extracellular portion of EGFR and the extracellular portion of HER2. The binding moiety preferably comprises a variable domain which binds to EGFR and a variable domain which binds to HER2. The variable domain which binds to EGFR is preferably an EGFR variable domain as described herein. The variable domain which binds to HER2 is preferably a HER2 variable domain as described herein. Preferably, both the EGFR variable domain and the HER2 variable domain are variable domains as described herein.

[0071] The present invention further provides a binding moiety which specifically binds to the extracellular portion of EGFR and the extracellular portion of HER3. The binding moiety preferably comprises a variable domain which binds to EGFR and a variable domain which binds to HER3. The variable domain which binds to EGFR is preferably an EGFR variable domain as described herein. The variable domain which binds to HER3 is preferably a HER3 variable domain as described herein. Preferably, both the EGFR variable domain and the HER3 variable domain are variable domains as described herein.

[0072] The invention further provides compositions comprising a binding moiety that specifically binds to the extracellular portion of EGFR and the extracellular portion of HER2, and a binding moiety that specifically binds to the extracellular portion of EGFR and the extracellular portion of HER3.

[0073] The binding moieties described herein are preferably antibodies, preferably multispecific antibodies, preferably bispecific antibodies.

[0074] The light chain variable regions (VL) of the EGFR variable domain, the HER2 variable domain and the HER3 variable domain of a binding moiety such as a bispecific antibody can be the same as the VL region of a parent EGFR monospecific antibody, the VL region of a parent HER2 monospecific antibody and / or the same as that of a parent HER3 monospecific antibody. Alternative VL regions can be used in one or more VH / VL region combinations, so long as the variable domains retain binding to EGFR, HER2 or HER3, respectively.

[0075] In some embodiments, the VL regions of the EGFR, HER2 and HER3 variable domains are similar, hi certain embodiments, all of the VL regions of the variable domains of the binding moiety are identical.

[0076] In certain embodiments, the light chain variable regions of one, two, or more than two variable domains of the binding moieties of the invention comprise a common light chain variable region. In some embodiments, the common light chain variable region of the one, two, or more than two variable domains comprises a germline variable region V-segment. In certain embodiments, the light chain variable region of the one, two, or more than two variable domains comprises a kappa light chain V-segment IgVκ1-39. *01. IgVκ1-39 is the short form for the immunoglobulin variable kappa 1-39 gene. This gene is also called immunoglobulin kappa variable 1-39, IGKV139; IGKV1-39. The external Ids for this genetic element are HGNC:5740, Entrez Gene:28930, Ensembl:ENSG00000242371. The amino acid sequence of the V region is shown in sequence 10 of FIG. 7. The V region can be combined with one of five J regions. The preferred J regions are jk1 and jk5, with the combined sequences shown as IGKV1-39 / jk1 and IGKV1-39 / jk5. Alternative names include IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01 (nomenclature according to the worldwide web of the IMGT database at imgt.org). In a specific embodiment, the light chain variable region of one or both of the VH / VL joining regions is the kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01, or IgVκ1-39 * 01 / IGJκ1 * 05 (sequence 11 or sequence 12 in Figure 7, respectively).

[0077] In some embodiments, the light chain variable region of one, two, or more than one variable domain of a binding moiety of the invention comprises an LCDR1 comprising the amino acid sequence QSISSY (sequence 7 in Figure 7), an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSTP (sequence 9 in Figure 7) (i.e., the CDRs of IGKV1-39 according to IMGT). In some embodiments, the light chain variable region of one, two, or more than one variable domain of a binding moiety of the invention comprises an LCDR1 comprising the amino acid sequence QSISSY (sequence 7 in Figure 7), an LCDR2 comprising the amino acid sequence AASLQS (sequence 8 in Figure 7), and an LCDR3 comprising the amino acid sequence QQSYSTP (sequence 9 in Figure 7).

[0078] In some embodiments, one, two, 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 the amino acid sequence set forth in sequence 11 of Figure 7. In some embodiments, one, two, 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 the amino acid sequence set forth in sequence 11 of Figure 7.

[0079] For example, in some embodiments the variable light chains of one, two or more variable domains of a binding moiety of the invention may have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof with respect to sequence 11 of Figure 7 or sequence 12 of Figure 7. In some embodiments the light chain variable region of one, two or more variable domains of a binding moiety of the invention comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof with respect to the amino acid sequence shown.

[0080] In other embodiments, the light chain variable regions of one, two or more variable domains of a binding moiety of the invention comprise the amino acid sequence of sequence 11 of Figure 7 or sequence 12 of Figure 7. In a particular embodiment, all variable domains of a binding moiety of the invention comprise the same VL region. In one embodiment, the VL of all variable domains of a binding moiety of the invention comprise the amino acid sequence set forth in sequence 11 of Figure 7. In one embodiment, the VL of all variable domains of a binding moiety of the invention comprise the amino acid sequence set forth in sequence 12 of Figure 7 or sequence 12 of Figure 7.

[0081] Multispecific antibodies, such as the bispecific antibodies disclosed herein, can be provided in multiple 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; MAb; 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, multispecific antibody formats, such as non-standard antibody bispecific antibody formats with two variable domains, have at least one variable domain that includes a heavy chain variable region and a light chain variable region. This variable domain can be linked to single chain Fv fragments, monobodies, VHHs, and Fab fragments that provide a second binding activity.

[0082] In some embodiments, the multispecific antibodies used in the methods provided herein are generally of the human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the antibodies are of the human IgG1 subclass. Full-length IgG antibodies are preferred due to their advantageous half-life and low immunogenicity. Such multispecific antibodies may have two different heavy chains that contain heterodimerization domains. Thus, in certain embodiments, the EGFR / HER2 bispecific antibodies and the EGFR / HER3 bispecific antibodies are full-length IgG molecules. In one embodiment, the EGFR / HER2 bispecific antibodies and the EGFR / HER3 bispecific antibodies are full-length IgG1 molecules.

[0083] Thus, in certain embodiments, the multispecific EGFR / HER2 and EGFR / HER3 antibodies comprise a fragment crystallizable (Fc). The Fc region of the multispecific antibody preferably consists of a human constant region. The constant region or Fc of the multispecific antibody may contain one or more, preferably no more than 10, preferably no more than 5 amino acid differences from the constant region of a naturally occurring human antibody. For example, in certain embodiments, each Fab arm of the bispecific antibody may further comprise an Fc region that includes modifications that facilitate bispecific antibody formation, modifications that affect Fc-mediated effector functions, and / or other features described herein.

[0084] In a preferred embodiment, the multispecific, preferably bispecific, full-length IgG antibody has a lower hinge and / or CH2 domain so that the interaction of the bispecific IgG antibody with the Fc gamma (Fcγ) receptor is enhanced. In cases where the antibody itself has low ADCC activity, the antibody-dependent cellular cytotoxicity, also called ADCC activity, of the antibody can often be improved. This is achieved, for example, by removing fucose residues from the glycosylated portion of the antibody. One technique for enhancing ADCC by defucosylation 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 described herein are preferably defucosylated. Preferably, both multispecific antibodies are defucosylated. 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 aimed at improving Fc binding to the low affinity activating FcγRIIIa and / or decreasing binding to the low affinity inhibitory FcγRIIb.

[0085] Bispecific antibodies are typically produced by cells expressing nucleic acid encoding the antibodies. Thus, in some embodiments, a method is provided for producing a composition comprising a multispecific antibody that binds EGFR and HER2 and a multispecific antibody that binds EGFR and HER3, the method comprising: a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, wherein the two or more nucleic acids may or may not be physically linked, each of the nucleic acids further comprising expression control sequences allowing expression of the encoded heavy and light chains in the cell, and the method further comprising culturing the cell to allow expression of the heavy and light chains, and optionally harvesting the two or more antibodies. The two or more antibodies may be harvested from the cell and / or the supernatant.

[0086] The level at which each chain is produced in the cell can be adjusted, for example, by selecting the appropriate expression control sequence or by selecting the number of introduced copies of the nucleic acid, or both. In a preferred embodiment, a population of cells is provided with the nucleic acid and clones expressing appropriate levels of each chain are selected. Clones are typically also selected based on the amount of antibody produced. In one embodiment, the method includes providing a population of cells containing the nucleic acid and selecting cells from the population that have a desired expression ratio of each of the heavy and light chains. In some embodiments, the two or more binding moieties are antibodies, preferably bispecific antibodies. In some embodiments, the cells preferably produce essentially equimolar amounts of the two or more binding moieties. In other embodiments, the cells produce more of one binding moiety than another of the two or more binding moieties.

[0087] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, wherein the two or more nucleic acids may or may not be physically linked, and each of the nucleic acids further comprises an expression control sequence that allows expression of the encoded heavy and light chains in the cell.

[0088] The present invention further comprises: a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, The two or more nucleic acids may or may not be physically linked, and each of the nucleic acids further comprises a container comprising expression control sequences that enable expression of the encoded heavy and light chains in the cell.

[0089] The cell producing the binding moiety is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, most preferably a human cell. A suitable cell is any cell that contains and is preferably capable of producing a binding moiety as described herein, preferably a multispecific antibody, preferably a bispecific antibody.

[0090] Cells suitable for antibody production are known in the art and include hybridoma cells, Chinese Hamster Ovary (CHO) cells, NS0 cells, HEK293 cells, 293-F cells or PER-C6 cells. Various institutions and companies have developed cell lines for large-scale production of antibodies, for example 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 cells are human cells. Preferably, cells transformed by adenovirus E1 region or functional equivalents thereof. In a particularly preferred embodiment, the cells are CHO cells or variants thereof. Preferably, variants utilizing the glutamine synthetase (GS) vector system for the expression of antibodies. In a preferred embodiment, the cells are CHO cells.

[0091] In some embodiments, the cells express three different heavy chains and at least one light chain. In one preferred embodiment, the cells express a "common light chain" as described herein to reduce the number of different antibody species (different heavy and light chain combinations). For example, each VH region is cloned into an expression vector together with a reshaped human IGKV139 / IGKJ1 (huVκ139) light chain using methods known in the art for the production of bispecific IgG (WO 2013 / 157954; incorporated herein by reference). It has previously been shown that huVκ139 can pair with two or more heavy chains, thereby generating antibodies with diverse specificities and facilitating the generation of bispecific molecules (WO 2009 / 157771).

[0092] Antibody-producing cells expressing a common light chain and equal amounts of two heavy chains typically produce 50% bispecific antibodies and 25% each of monospecific antibodies (i.e., with the same heavy and light chain combination). Several methods have been published that favor the production of bispecific antibodies over the production of each monospecific antibody. This is typically achieved by modifying the constant regions of the heavy chains such that they favor heterodimerization (i.e., dimerization with the heavy chains of other heavy / light chain combinations) over homodimerization. In a preferred embodiment, the bispecific antibody of the present invention comprises two different immunoglobulin heavy chains with compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. The compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. Various methods have been described in the art by which such heterodimerization of heavy chains can be achieved.

[0093] An example of a preferred method for producing the multispecific antibodies described herein is disclosed in US Pat. Nos. 9,248,181 and 9,358,286. Specifically, the preferred mutations for producing essentially only bispecific full-length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK-mutant" heavy chain) and L351D and L368E in the second domain ("DE-mutant" heavy chain), or vice versa. As mentioned above, the DE-mutant and the KK-mutant preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE-mutant heavy chains (DEDE homodimers) or KK-mutant heavy chains (KKKK homodimers) rarely occurs due to the strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains. The introduction of additional heavy chains, either DE-mutant heavy chains or KK-mutant heavy chains, allows the production of additional DEKK bispecific molecules. 2 ) can associate with the existing KK heavy chain. Thus, the cell can express two bispecific antibodies, DE 1 KK Bispecific Antibodies and DE 2 To produce a KK bispecific antibody, a new KK heavy chain (KK) is used instead of the new DE heavy chain. 2 ) is installed, DEKK 1 and DEKK 2 A bispecific antibody is produced having a combination of the HER2 and HER3 chains. The levels at which different antibodies can be produced by a cell can be regulated by regulating the relative expression of the HER2 and HER3 chains relative to each other. The light chain is typically produced at a level sufficient to reduce the level of a single heavy chain, and the level at which the EGFR chain is produced is typically sufficient to allow efficient pairing with the HER2 and HER3 chains.

[0094] Thus, in one embodiment, the heavy / light chain combination comprising a variable domain that binds EGFR comprises a DE mutant of the heavy chain, in this embodiment, the heavy / light chain combination comprising a variable domain that binds HER2 and the heavy / light chain combination comprising a variable domain that binds HER3 comprise a KK mutant of the heavy chain.

[0095] Any suitable assay can be used to test for binding of candidate EGFR / HER2 IgG bispecific antibodies or EGFR / HER3 IgG bispecific antibodies. For example, binding to membrane-expressed EGFR, HER2 or HER3 can be tested. This is typically done with cells that do not normally express EGFR, HER2 or HER3, but have been transformed to express one of EGFR, HER2 or HER3. Binding of the antibody to transformed cells but not to non-transformed cells indicates specific binding of the antibody. Binding can be assessed, for example, by flow cytometry (following the FACS procedures previously described in WO2015 / 130172, PCT / NL2018 / 050537 and WO2015 / 130173). Each monospecific antibody can be used as a control and an irrelevant IgG1 isotype control mAb.

[0096] Binding moieties such as antibodies can be collected from cells and / or cell culture supernatants. Typically, they are collected from the supernatants of production cells. Binding moieties such as antibodies can be purified from the supernatants. Many purification methods are known in the art. Some of the more common methods use affinity purification.

[0097] The antibodies produced by the cells can be purified by affinity purification. This is advantageously carried out by protein A extraction. The presence of specific binding properties of the eluted antibodies (i.e. binding to EGFR, binding to HER2 and HER3) can be tested by ELISA. The antibody preparation can be further analyzed by ion exchange column chromatography. The individual bispecific antibodies can be purified from each other by routine techniques, for example using ion exchange chromatography. The presence of each bispecific antibody can also be analyzed by ELISA. Binding the preparation to HER2 and washing removes all EGFR / HER3 antibodies. Staining with labeled soluble HER3 gives no signal, but staining with labeled soluble EGFR gives a signal. Binding the preparation to HER3 and washing removes all EGFR / HER2 antibodies. Staining with labeled soluble HER2 gives no signal, but staining with labeled soluble EGFR gives a signal. Binding the preparation to EGFR and washing does not remove EGFR / HER2 and EGFR / HER3 antibodies. Staining with labeled soluble HER2 as well as labeled soluble HER3 gives a signal. The levels of each antibody in the preparation can also be estimated using such ELISAs with appropriate controls with known levels of the single bispecific antibodies.

[0098] A method for producing a composition comprising two or more bispecific antibodies comprises the steps of: providing a cell with nucleic acid encoding a bispecific antibody; Culturing the cells; Clarification of the recovered material; harvesting the bispecific antibody from the culture; and separating the produced bispecific antibodies from the half antibodies by ion exchange chromatography (IEX); The method is characterized in that said bispecific antibodies exhibit similar IEX retention times, preferably IEX retention times that deviate by no more than 10% from the average of the retention times of the individual antibodies under the IEX conditions used. In one embodiment, the antibodies are selected to have an IEX retention time that deviates by no more than 10% from the average of the retention times of the individual antibodies under the IEX conditions used. The antibodies may first be purified from other proteins in the culture. This is typically done by affinity purification, preferably by protein A extraction. The bispecific antibodies are preferably selected to have half antibodies with retention times that are outside the range spanned by the retention times of the antibodies. If a combination of bispecific antibodies is produced and monospecific antibodies are not desired, the bispecific antibodies are preferably selected to have retention times that are different from the retention times of the monospecific antibodies. The retention times of the monospecific antibodies in this embodiment are preferably outside the range spanned by the retention times of the respective bispecific antibodies. The cells in the culture preferably express three heavy chains simultaneously, which heavy chains contain a CH3 heterodimerization domain that promotes the formation of EGFR / HER2 and EGFR / HER3 heavy chain heterodimerization. The cells preferably express the common light chain of Figure 7. The bispecific antibody in one embodiment has an isoelectric point (PI) that is similar to the average PI of the at least two bispecific antibodies, and preferably differs by no more than 0.5 units.

[0099] The affinity of the EGFR, HER2 and HER3 FABs of candidate EGFR / HER2 or EGFR / HER bispecific antibodies to the targets can be measured by surface plasmon resonance (SPR) technology using a BIAcore T100. 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 surface. Recombinant purified antigen human EGFR-Fc, HER2-Fc and HER3-Fc proteins are then flowed over the sensor surface in a range of concentrations to measure the on-rates and off-rates. After each cycle, the sensor surface is regenerated by a pulse of HCl and the bsAb is captured again. From the resulting sensorgrams, the on-rates and off-rates and affinity values ​​for binding to human EGFR, HER2 and HER3 are determined using BIAevaluation software.

[0100] The present invention also provides a composition as described herein for use in the treatment of cancer. In some embodiments, the cancer is a solid epithelial cancer. Preferably, the composition is used for cancer expressing EGFR, HER2 and / or HER3. The composition is preferably used for pancreatic cancer, colorectal cancer, head and neck cancer, epithelial ovarian cancer, epithelial fallopian tube cancer, epithelial peritoneal cancer, bladder cancer, or prostate cancer. In some embodiments, the cancer treated by use of the composition is an advanced cancer. The composition is preferably used for metastatic cancer. The composition is preferably used for metastatic pancreatic cancer, metastatic colorectal cancer, metastatic head and neck cancer, metastatic epithelial ovarian cancer, metastatic epithelial fallopian tube cancer, metastatic epithelial peritoneal cancer, metastatic bladder cancer, or metastatic prostate cancer. In some embodiments, the composition is preferably used for gastric cancer, lung cancer, breast cancer, or esophageal cancer. Preferably, the composition is used for metastatic gastric cancer, metastatic lung cancer, metastatic breast cancer, or metastatic esophageal cancer.

[0101] The invention further provides two or more binding moieties, each comprising a variable domain that binds to the extracellular portion of EGFR, a first such binding moiety comprising a variable domain that binds to the extracellular portion of HER2 and a second such binding moiety comprising a variable domain that binds to the extracellular portion of HER3 for use in the treatment of cancer. Also provided is a product comprising two or more binding moieties, each comprising a variable domain that binds to the extracellular portion of EGFR, a first such binding moiety comprising a variable domain that binds to the extracellular portion of HER2 and a second such binding moiety comprising 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.

[0102] The cancers treated by the embodiments of the present invention are preferably those described elsewhere herein. The cancers preferably include cells with EGFR mutations that render the cells resistant to treatment with tyrosine kinase inhibitors (TKIs). In some embodiments, the cancers include cells with EGFR R521K polymorphism. The cancers treated and the methods of treatment of the present invention described herein are preferably gastric cancer, lung cancer or esophageal cancer. In further embodiments, the present invention provides a method of treating a subject with cancer or at risk of recurrence or relapse of cancer, comprising administering to a subject in need thereof two or more binding moieties, each of which comprises a variable domain that binds to an extracellular portion of EGFR, wherein a first of the binding moieties comprises a variable domain that binds to an extracellular portion of HER2, and a second of the binding moieties comprises a variable domain that binds to an extracellular portion of HER3.

[0103] 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, such as a human patient with cancer).

[0104] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or procedure performed or the administration of an active agent or combination of active agents to a subject with the goal of reversing, ameliorating, ameliorating, inhibiting, or slowing or preventing the progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical manifestation associated with a disease.

[0105] As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that results in a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder, such as cancer. A beneficial effect can result in an improved state over a baseline, such as an improvement over a measurement or observation made before the initiation of therapy according to the method. A beneficial effect can result in a state in which the progression of cancer in a subject at any clinical stage is slowed, stabilized, stopped, or reversed, as evidenced, for example, by a reduction or elimination of clinical or diagnostic symptoms of the disease, or a reduction or elimination of a marker of cancer. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent metastasis of tumors, slow or prevent tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0106] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or prophylactic result. The result may be reduction, amelioration, remission, reduction, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, or other desired changes in biology. In some embodiments, an effective amount is an amount sufficient to delay the development of a tumor. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. An effective amount of the drug or composition may (i) reduce the number of cancer cells; (ii) reduce the size of a tumor; (iii) inhibit, prevent, slow, or stop to some extent cancer cell invasion into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor development and / or recurrence; and / or (vii) relieve to some extent one or more symptoms associated with cancer. In one example, an "effective amount" is an amount of a composition of the invention to result in a reduction in cancer (e.g., a reduction in the number of cancer cells) or a delay in the progression of cancer. Effective amounts of the combination therapy are administered in an "effective regimen," which refers to the combination of binding moieties set forth herein, in accordance with the methods described herein, and in a sequence and frequency of administration sufficient to effect treatment.

[0107] As used herein, the terms "synergy," "therapeutic synergy," and "synergistic effect" refer to the phenomenon whereby treatment of a patient with a combination of binding moieties as provided 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 outcome than that achieved when each of the individual components of the combination is 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 increased therapeutic response greater than the sum of the individual effects of each binding moiety alone at the same dose as the combination; (b) a reduced dose of one or more agents in the combination without a decrease in therapeutic efficacy; (c) a reduced incidence of adverse events while achieving a therapeutic effect greater than that of monotherapy of each agent at the same dose as the combination; (d) a reduced dose-limiting toxicity while achieving a therapeutic effect greater than that of monotherapy of each agent; and (e) a delayed or minimized induction of drug resistance.

[0108] In xenograft models, a combination used at its maximum tolerated dose, where each component is generally present at a dose that does not exceed its individual maximum tolerated dose, demonstrates therapeutic synergy if the tumor growth reduction achieved by administering the combination is greater than the value of the tumor growth reduction of the best component when administered alone.The synergy of drug combinations 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).

[0109] The present invention further provides the composition of the present invention for use in the treatment of cancer.The embodiment used is preferably for treating gastric cancer, colorectal cancer, colon cancer, gastroesophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, melanoma, etc.In one embodiment, the embodiment treats gastric cancer, lung cancer or esophageal cancer.The use is preferably for treating gastric cancer.

[0110] The invention described herein is preferably applied to the treatment of cancers in which the cells are tested for the presence of EGFR, HER2 and / or HER3 on the cell membrane, which can be done by routine methods and is typically analyzed by immunohistochemistry.

[0111] 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 some embodiments, the cells and / or stromal cells of the cancer treated by the invention disclosed herein express EGFR ligands, or HER3 ligands, or both. Thus, expression of the ligands and receptors can provide growth stimuli to the cells of the cancer. The combinations of the present invention are particularly suitable for treating cancers that include such cells.

[0112] Expression of one of EGFR, HER2 and HER3 in the treatment of the invention can delay escape of at least some tumors. Tumors targeted with a monospecific therapy can escape treatment by starting to express another EGFR, HER2 or HER3, or by expressing a ligand for the receptor.

[0113] Such cells, when they occur, are also attacked by the binding moieties of the present invention so that they can be eliminated before they grow and diversify. In one embodiment, the cancer is tested for the presence of mutated EGFR. Many EGFR-positive tumors have mutations in the gene that make the cells resistant to treatment with tyrosine kinase inhibitors.

[0114] The compositions of the present invention are suitable for treating cancers with EGFR mutations that make cancer cells resistant to treatment with tyrosine kinase inhibitors (TKIs).In one embodiment, the cancer comprises cells with EGFR R521K polymorphism.In some embodiments, the cancer is known to be resistant to first generation TKI inhibitors such as gefitinib and erlotinib.

[0115] The cancer treatments presented herein can be combined with additional cancer treatments. Such treatments can include additional binding moieties such as antibodies and / or cytostatic drugs, or protein kinase inhibitors. The protein kinase inhibitors are preferably inhibitors other than EGFR or HER3 tyrosine kinase inhibitors. Non-limiting examples of additional treatments include radiation therapy, chemotherapy, surgery, vascular growth inhibitor therapy, and hyperthermia.

[0116] The compositions of the invention may be suitable for use in the treatment of cancers that are resistant to EGFR inhibition (EGFR resistance being the result of overexpression of HER2 and / or HER3).

[0117] The compositions of the invention may be suitable for use in the treatment of cancers that are resistant to HER2 inhibition (HER2 resistance being the result of overexpression of EGFR and / or HER3).

[0118] The compositions of the invention may be suitable for use in the treatment of cancers that are resistant to HER3 inhibition (HER3 resistance being the result of overexpression of EGFR and / or HER2).

[0119] The term "Oligoclonics" in the context of an antibody, binding moiety, composition or product described herein refers to the presence of multiple, typically up to 10, different antibodies or binding moieties in one preparation, including the presence of bispecifics. Examples of Oligoclonics include the combination of two bispecific antibodies.

[0120] The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2, wherein said EFGR variable domain comprises a heavy chain variable region comprising the CDRs of the heavy chain variable region MF3755, MF4280, MF4003, or MF4016 of Figure 8, or a variant thereof comprising one, two or three amino acid substitutions, deletions and / or insertions in the CDRs, and said HER2 variable domain comprises a heavy chain variable region comprising the CDRs of the heavy chain variable region MF2032 or MF1849, or a variant thereof comprising one, two or three amino acid substitutions, deletions and / or insertions in the CDRs.

[0121] 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 said EFGR variable domain comprises a heavy chain variable region comprising the CDRs of the heavy chain variable region MF3755 of Figure 8 or a variant thereof comprising a substitution, deletion and / or insertion of one, two or three amino acids in the CDRs, and said HER2 variable domain comprises a heavy chain variable region comprising the CDRs of the heavy chain variable region MF2032 or a variant thereof comprising a substitution, deletion and / or insertion of one, two or three amino acids in the CDRs.

[0122] The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2, wherein said EFGR variable domain comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF3755, MF4280, MF4003, or MF4016 of Figure 8, or a variant thereof comprising substitution, deletion and / or insertion of one, two or three amino acids in its CDR, and said 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 thereof comprising substitution, deletion and / or insertion of one, two or three amino acids in its CDR.

[0123] 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 said EFGR variable domain comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF3755 of Figure 8 or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in its CDRs, and said HER2 variable domain comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF2032 or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in its CDRs.

[0124] 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 said EFGR variable domain comprises a heavy chain variable region comprising the CDRs of the heavy chain variable region MF3755 of Figure 8 or a variant thereof comprising a substitution, deletion and / or insertion of one, two or three amino acids in the CDRs, and said HER2 variable domain comprises a heavy chain variable region comprising the CDRs of the heavy chain variable region MF1849 or a variant thereof comprising a substitution, deletion and / or insertion of one, two or three amino acids in the CDRs.

[0125] 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 said EFGR variable domain comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF3755 of Figure 8 or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in its CDRs, and said HER2 variable domain comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF1849 or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in its CDRs.

[0126] Although features are described herein as part of the same or separate embodiments for clarity and conciseness of description, it will be understood that the scope of the invention may include embodiments having all or a partial combination of the described features. [Brief description of the drawings]

[0127] [Figure 1] Schematic diagram of an embodiment in which the composition comprises two bispecific antibodies sharing a common arm. The diagram shows an antibody having a heavy chain (1) and a light chain (4). The four heavy chains have three different variable regions (5, 6, 7). The heavy chains with the shared variable region (5) have a portion of a heterodimerization domain (3). The heavy chains with variable regions (6) and (7) have matching portions of the heterodimerization domain (2). The preferred pairing of the heterodimerization regions (2) and (3) can induce the formation of a bispecific antibody. [Diagram 2]Inhibitory effect of two Oligoclonics® on the proliferation of the growth factor dependent cell lines BxPC-3-luc2 (Perkin Elmer 125058) and N87 cells (NCI-87 cells (ATCC® CRL-5822™)). The two Oligoclonics® were tested for their effect on the proliferation of BxPC-3-luc2 (left panel) and N87 (right panel). The results of the panel screening were compared to the combination of two monospecific antibodies (EGFR binding antibody cetuximab and HER3 monospecific antibody PG3178) or the EGFRxHER3 binding bispecific antibody PB4522. Cells were grown in the presence of saturating amounts of HRG and EGF. The levels of cell proliferation of the respective cells with HRG and EGF and without antibody (basal w / ligand) as well as the basal levels without HRG and EGF and without antibody (w / o ligand) are shown. The monospecific antibody PG3178 has an IgG1 constant region and two variable domains with the heavy chain variable region of MF3178 in Figure 7 or Figure 8 and the common light chain variable region of sequence 11 in Figure 7. The bispecific antibody PB4522 has an IgG1 constant region and two variable domains. The HER3 variable domain has the heavy chain variable region of MF3178 in Figure 7 or Figure 8. The EGFR variable domain has the heavy chain variable region of MF4280 in Figure 7 or Figure 8. The light chain variable regions of both antibodies are the same, with the amino acid sequence of the common light chain variable region of sequence 11 in Figure 7). [Diagram 3] ADCC activity of the Oligoclonics® panel. The ADCC activity of the Oligoclonics® panel was tested using N87 and CD16 / NFAT reporter assays. 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 the antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence 11 in Figure 7. [Figure 4]Numbering and specificity of various Oligoclonics® and their ADCC activity. "-" indicates that no activity was observed. Each row represents an Oligoclonics® containing two bispecific antibodies. The internal codes of the bispecific antibodies are shown in the columns Bispecific 1 and 2. The heavy chain variable regions of the HER2, HER3 and EGFR binding domains are shown in the columns marked MFA, MFB and MFC. MF numbers 3178 and 2703 combine with a common light chain to form a HER3 binding variable domain. MF numbers 4280, 3755, 4003, 4016 combine with a common light chain to form an EGFR binding variable domain, and MF numbers 1871, 1847, 1849 and 2032 combine with a common light chain to form a HER2 binding variable domain. 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 the antibodies are identical and have the amino acid sequence of the consensus light chain variable region of sequence 11 in FIG. [Diagram 5] In vivo testing of Oligoclonics®. BxPC-3-luc2 cells or N87 cells were injected into the xenograft model on day 0. Oligoclonics® containing bispecific antibody PB4516×PB6892 (see FIG. 4) or control was injected on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. Results of Oligoclonics® (PB4516 and PB6892) are shown. Vehicle and cetuximab were used as controls. [Figure 6A] In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 6B]In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 6C] In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 6D] In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 6E] In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 6F] In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 6G] In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 6H]In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 6I] In vivo testing of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 in various PDX models. PDX models were injected on day 0 and treated with antibody or control on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg / kg. [Figure 7] The amino acid sequences of the heavy chain variable regions of the various variable domains indicated by MF numbers (see SEQ ID NOs: 1-6), as well as the CDRs and light chain variable regions (see SEQ ID NOs: 7-12). [Figure 8] Amino acid sequences of the various MFs referred to herein. FR1-4 refers to framework regions 1 to 4. CDR1-3 refers to complementarity determining regions 1 to 3. TT is tetanus toxoid. [Figure 9A] HER3 crystal structure (PDB number 4P59) showing residue Arg426 in a grey sphere and residues within a 11.2 Å radius of Arg426 in a black sphere. Figures and analysis were performed using Yasara (www.yasara.org). [Figure 9B] Residue Arg426 and distant residues within a 11.2 Å radius from Arg426 shown in grey are shown in black. Diagrams and analysis were done using Yasara (www.yasara.org). [Figure 9C] Residues in region Arg426 in light grey and surrounding residues in dark grey (all labelled). Diagrams and analysis were done using Yasara (www.yasara.org). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0128] Working Example cell line Hek293 cells, NCI-87 cells (ATCC® CRL-5822™), 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).

[0129] Generation of bispecific antibodies Bispecific antibodies were generated using the DEKK CH3 technology described above for efficient heterodimerization and bispecific antibody formation, which uses charge-based point mutations in the CH3 region to allow efficient pairing of two different heavy chain molecules as previously described (WO 2013 / 157954).

[0130] The VH genes were cloned into one of two different backbone IgG1 vectors. Depending on the binding partner, the VH was cloned into an IgG1 backbone containing a CH3 variant with the heterodimerization variant "DE" or into an IgG1 backbone containing the complementary CH3 heterodimerization variant "KK". In the case of bispecific or multispecific antibodies, where two or more antibodies share a heavy chain, the shared chain preferably has the CH3 heterodimerization variant "DE" (also called DE heavy chain) and two or more unique heavy chains have the CH3 heterodimerization variant "KK" (also called KK heavy chain).

[0131] Adherent Hek293 cells were cultured in 6-well plates to 80% confluency. Cells were transiently transfected with DNA-FUGENE mix and further cultured. Seven days after transfection, the supernatant was harvested and the medium was refreshed. 14 days after transfection, the supernatants were combined and filtered at 0.22 μM. Sterile supernatants were stored at 4°C. Suspension-adapted Hek293 cells were cultured on a shaker plateau in T125 flasks to a density of 3.0×10e6 cells / ml. Cells were seeded into each well of a 24-deep-well plate at a density of 0.3–0.5×10e6 viable cells / ml. Cells were transiently transfected with individual sterile DNA:PEl-MIX and further cultured. Seven days after transfection, the supernatant was harvested and filtered at 0.22 μM. Sterile supernatants were stored at 4°C.

[0132] Generation of a stable cell line pool co-expressing two bispecific antibodies CHO cells were transfected with the three heavy chain constructs and one common light chain construct at a molar ratio of common light chain construct (cLC):EGFR heavy chain:HER2 heavy chain:HER3 heavy chain=2.5:2:1:1. Ten pools of stably transfected cells were obtained (A-J). ELISA analysis of anti-EGFR, anti-HER2, and anti-HER3 antibodies was performed on day 3 and day 6 supernatants of the ten pools. All three specificities could be detected in all pools.

[0133] Stable cell line clones co-expressing the two bispecific antibodies were generated by plating the pool on semi-solid medium. Plated cells were grown for 7-10 days. Two rounds of single-cell cloning were performed by seeding and selecting single colonies. Oligoclonics were produced from single cells by fed-batch fermentation.

[0134] Measurement of antibody titers Cell supernatants were diluted 1:4 and 1:50 in PBS based on total IgG concentration. Single antigen ELISA was first performed to detect the presence of all three antibodies. The following antigens were used at 2.5 μg / ml dilution to coat the wells of an ELISA plate: recombinant human EGFR-ECDFc (R&D Systems, 344-ER), recombinant human ErbB2-ECDFc (R&D Systems, 1129-ER), and recombinant human ErbB3-ECDFc (R&D Systems, 348-ER).

[0135] Two sandwich ELISAs were then performed to detect and quantify the two bispecific molecules, allowing the estimation of the ratio between the two bispecifics. For detection of EGFRxHER2 bispecifics, EGFR-Fc (R&D Systems, 344-ER) antigen was coated onto the wells and detected with ErbB2-Fc (R&D Systems, 1129-ER). For detection of EGFRxHER3 bispecifics, EGFR-Fc antigen was coated onto the wells and detected with ErbB3-Fc (R&D Systems, 348-RB).

[0136] IgG purification Purification of IgG was performed using affinity chromatography. Purification was performed under sterile conditions using vacuum filtration. First the pH of the medium was adjusted to pH 8.0 and then the product was incubated with Protein A Sepharose CL-4B beads (50% v / v) (Pierce) for 2 h at 25°C and 600 rpm on a shaking platform. The beads were then collected by vacuum filtration. The beads were washed twice with PBS pH 7.4. IgG was eluted with 0.1 M citrate buffer at pH 3.0 and the IgG fraction was immediately neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using Ultracel (Millipore). The sample was finally brought to a final buffer (PBS) at pH 7.4.

[0137] Cation Exchange Chromatography (CIEX) CEX-HPLC chromatography was performed using the TSKgel SP-STAT (particle size 7 μm, 4.6 mm inner diameter × 10 cm length, Tosoh 21964) series of ion exchange columns. The columns are packed with non-porous resin particles for fast and high-resolution analysis and isolation of biomolecules. The particles in the TSKgel STAT columns contain an open-access network of multi-layered ion exchange groups for loading capacity, but the relatively large particle size makes these columns suitable for HPLC and FPLC systems.

[0138] A TSKgel SP-STAT (particle size 7 μm, 4.6 mm inner diameter × 10 cm length, Tosoh 21964) was equilibrated with buffer A (sodium phosphate buffer, 25 mM, pH 6.0), and then the antibody was transferred from the column by increasing the salt concentration and running a gradient of 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 of all test samples and controls (in PBS) was 10 μg, and the injection volume was 10-100 μl. The chromatograms were analyzed for peak pattern, retention time, and peak area of ​​the major peaks observed based on the results at 220 nm.

[0139] BxPC-3-luc2 and N87 growth inhibition assay Antibody compositions were tested at a range of concentrations of all antibodies. Antibodies were pooled in duplicate based on equal weight per weight. HRG and EGF were added to the cultures: for BxPC3-luc2 cells, 0.1 ng / ml EGF and 10 ng / ml HRG, or for N87 cells, 0.1 ng / ml EGF and 1 ng / ml HRG. Basal w / ligand was a control without antibody but with the respective growth factor. Basal w / o ligand was a control without the indicated growth factor and without antibody.

[0140] Antibodies were diluted in synthetic starvation medium (CDS: RPMI 1640 medium containing 80 U penicillin and 80 μg streptomycin / ml, 0.05% (w / v) BSA and 10 μg / ml holotransferrin) and 50 μl of diluted antibodies were added to wells of a 96-well black well clear bottom plate (Costar). Ligand was added (50 μl / well of stock solution containing 40 ng / ml or 4 ng / ml HRG and 400 ng / ml EGF, i.e. R&D systems, cat.nr. 396-HB and 236-EG, diluted in CDS). 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, no. DAL1100) was added (20 μl / well) and after 6 h (N87 cells) or 4 h (BxPC-3-luc2 cells) of incubation with Alamar blue (at 37° C.), fluorescence was measured using 560 nm excitation and 590 nm readout in a Biotek Synergy 2 multimode microplate reader. Fluorescence values ​​were normalized to uninhibited proliferation (no antibody, but both ligands were added).

[0141] ADCC activity of various Oligoclonics® An ADCC reporter bioassay (Promega) was used. Two different cell lines were tested: the EGFR-expressing pancreatic cancer cell line BxPC3 and the gastric cancer cell line N87.

[0142] The bioassay uses engineered Jurkat cells stably expressing either the V158 (high affinity) variant of the FcγRIIIa receptor and an NFAT response element that drives the expression of firefly luciferase, a measure of FcγR activation. The assay was validated by comparing the data obtained with this ADCC reporter bioassay to a standard 51Cr release assay, with both assays yielding similar results. The ADCC assay was performed using a Promega ADCC Bioassay kit using 384 white well plates. In this experimental setup, BxPC3 and N87 cells were plated at a density of 1000 cells / well in 30 μl of assay medium (RPMI with 4% low IgG serum) 20-24 hours prior to the bioassay. The next day, the medium was removed. Then, serial dilutions of Oligoclonics® and the comparison antibody cetuximab were prepared in duplicate. 10 μl of these antibody dilutions were added to the wells. Control wells without antibody were also included (basal). A 5-fold serial dilution was performed from the starting antibody concentration to obtain a dose-response curve. Finally, 5 μL of ADCC Bioassay effector cells (15000 cells / well, V158) were added. The cells were incubated at 37° C. for 6 hours. Then, 15 μL of BIO-Glo luciferase substrate was added and luminescence was detected in a plate reader after 5 minutes. The data obtained are shown in FIG. 3. Cetuximab showed ADCC activity against BxPC3 and N87 cells. Various oligoclonal antibodies also showed ADCC activity against BxPC3 and / or N87 cells.

[0143] Study of the effect of Oligoclonics® containing bispecific antibodies PB4516 and PB6892 on the growth of BxPC-3-luc2 tumors (orthotopically implanted) and N87 tumors (gastric cells implanted in the flank)

[0144] CB17 SCID female mice, 8-10 weeks old at the start of the study, were orthotopically implanted in the pancreas with 1x10e6 BxPC-3-luc2 tumor cells in 20μl. Mice were anesthetized and placed in right lateral recumbency to expose the left side, and a 0.5cm incision was made in the left flank area. The pancreas and spleen were removed, and 1x10e6 tumor cells in 20μl were injected into the subcapsular space of the pancreatic tail. Bioluminescence (BLI) data were generated one week after implantation. For BLI imaging the left lateral view (once or twice weekly), all mice were injected intraperitoneally with 150mg / kg luciferin (D-luciferin-EF potassium salt, catalog number E6552, Promega) 15 minutes prior to imaging all injected mice. Outlier animals were removed based on BLI / tumor volume, and mice were randomly assigned into groups of 7 mice each. Treatment began on day 8 of the study.

[0145] Animals in the antibody treatment groups were administered 0.3 mg / kg of antibody weekly for four consecutive weeks (days 0, 7, 14, and 21). On day 0 of treatment, animals received twice the loading dose of antibody, i.e., 0.6 mg / kg. Final imaging was performed on days 35 or 40. Vehicle-only and cetuximab-treated groups served as controls.

[0146] Cetuximab and oligoclonics significantly reduce the growth of BxPC-3 tumors in the model (p<0.05) (Figure 5). Tumor growth with Oligoclonics® PB4516 and PB6892 was significantly less than with cetuximab. Cetuximab did not significantly reduce the growth of N87 cells. Oligoclonics® significantly reduced the growth of N87 tumors in the model (p<0.05) (Figure 5).

[0147] N87 Tumor: CB17 SCID female mice, 8-12 weeks old at the start of the study, were inoculated in the flank with 1x10e7N87 tumor cells in 50% Matrigel sc. The cell injection volume was 0.2 mL / mouse. Treatment began when tumors reached an average size of 150-200 mm3. Antibody was administered at 25 mg / kg mouse by intraperitoneal injection once a week for 4 weeks. Body weight was measured weekly after tumor cell injection and every other week from the start to the end of treatment. Tumor growth was monitored every other week by caliper measurement. The experimental endpoint was a tumor volume of 800 mm3 or 60 days, whichever came first.

[0148] Activity of Oligoclonics® PB11244 and PB4516 in various PDX models The activity of Oligoclonics®, including bispecific antibodies PB11244 and PB4516, was evaluated in a series of PDX models. Testing candidate therapeutics in multiple cancer models can facilitate prediction of clinical efficacy and identify factors for patient selection strategies.

[0149] The bispecific antibodies PB4516 and PB11244 have an IgG1 constant region and two variable domains.

[0150] The HER3 variable domain of PB4516 has the heavy chain variable region of MF3178 of Figure 7 or Figure 8. The EGFR variable domain has the heavy chain variable region of MF3755 of Figure 7 or Figure 8.

[0151] The HER2 variable domain of PB11244 has the heavy chain variable region of MF2032 of Figure 7 or Figure 8. The EGFR variable domain has the heavy chain variable region of MF3755 of Figure 7 or Figure 8.

[0152] The light chain variable regions of both antibodies were identical and shared the amino acid sequence of the common light chain variable region of SEQ ID NO: 11 in FIG. 7.

[0153] Several gastric, esophageal, and non-small cell lung cancer PDX models were selected (Figure 6).

[0154] Oligoclonics® containing bispecific antibodies PB4516 and PB11244 were generated and purified. These antibodies were mixed in a 1:1 ratio. The Oligoclonics® were tested in the model and compared to cetuximab and vehicle (PBS).

[0155] PDX models were first spread subcutaneously (sc) in donor BALB / c nude mice. Tumors were excised, cut into small pieces (2-3 mm in diameter), and subcutaneously implanted into new acceptor BALB / c nude mice. Tumor recipients were 6-8 week-old female BALB / c nude mice. Tumor growth was monitored by caliper measurement until the tumors reached an average size of 100-200 mm3. At this stage, designated as day 1, animals were randomized into three groups for each model. Treatment was initiated on the same day, PB4516 × PB6892 25 mg / kg, 5 doses per week, intraperitoneal injection Cetuximab 25 mg / kg, 5 times weekly, intraperitoneal injection Vehicle (PBS), five weekly doses, intraperitoneal injections included.

[0156] It can be seen that Oligoclonics® significantly reduced tumor cell proliferation in the model, the reduction in proliferation being equal to or better than cetuximab.

[0157] [Table 1]

Claims

1. A composition comprising two or more binding moieties, each of said binding moieties comprises a variable domain that binds to an extracellular portion of EGFR; A composition, wherein a first said binding moiety comprises a variable domain that binds to an extracellular portion of HER2 and a second said binding moiety comprises a variable domain that binds to an extracellular portion of HER3.

2. The composition of claim 1 , wherein at least one, and preferably at least two, of the two or more binding moieties is an antibody.

3. 3. The composition of claim 1 or claim 2, wherein at least one, preferably at least two of the two or more binding moieties is an IgG.

4. The composition of claim 2 or claim 3, wherein the CH3 region of the heavy chain of the first and / or second antibody is engineered to promote heterodimerization of the heavy chain with an EGFR-binding variable domain having a heavy chain having a HER2-binding variable domain, and / or heterodimerization of the heavy chain with an EGFR-binding variable domain having a heavy chain having a HER3-binding variable domain.

5. The composition of any one of claims 2 to 4, wherein at least one, preferably at least two, of the two or more antibodies is a bispecific antibody.

6. 6. The composition of any one of claims 2 to 5, wherein the variable domains that bind the extracellular portion of EGFR of the first and second antibodies comprise substantially the same heavy chain variable region.

7. 7. The composition of any one of claims 2 to 6, wherein the variable domain that binds to the extracellular portion of EGFR binds to domain I or domain III of EGFR, preferably domain III.

8. The composition according to any one of claims 2 to 7, wherein the variable domain that binds to the extracellular part of HER2 binds to domain I or domain IV of HER2, preferably domain IV.

9. The composition of any one of claims 2 to 8, wherein the variable domain that binds to the extracellular portion of HER3 binds to domain III of HER3.

10. 10. The composition of any one of claims 6 to 9, wherein the variable domain that binds to the extracellular portion of EGFR binds to domain I or domain III, preferably domain III, of EGFR, the variable domain that binds to the extracellular portion of HER2 binds to domain I or domain IV, preferably domain IV, of HER2, and the variable domain that binds to the extracellular portion of HER3 binds to domain III of HER3.

11. 11. The composition of claim 9 or claim 10, wherein the variable domain that binds to the extracellular portion of HER3 binds to at least R426 in domain III of HER3.

12. 12. The composition of any one of claims 9 to 11, wherein the affinity (KD) of the variable domain binding to the extracellular portion of HER3 for binding to HER3 positive SK-BR-3 cells (ATCC® HTB-30™) is 2.0 nM or less, preferably 2.0 to 0.1 nM.

13. 13. The composition of any one of claims 1 to 12, wherein binding of the variable domain that binds EGFR to EGFR blocks binding of EGF to EGFR and / or binding of the variable domain that binds HER3 to HER3 blocks binding of neuregulin 1 (NRG) to HER3.

14. 14. The composition of any one of claims 1 to 13, wherein the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising the CDR1 sequence NYAMN, the CDR2 sequence WINANTGDPTYAQGFTG, and the CDR3 sequence ERFLEWLHFDY, or a variant thereof comprising substitutions, deletions and / or insertions of 1, 2 or 3 amino acids in the CDRs.

15. 15. The composition of any one of claims 1 to 14, wherein the variable domain that binds to the extracellular portion of HER2 comprises a heavy chain variable region comprising the CDR1 sequence SYGMH, the CDR2 sequence VISYDGSNKYYADSVKG, and the CDR3 sequence DYYRRTARAGFDY, or a variant thereof comprising substitutions, deletions and / or insertions of 1, 2 or 3 amino acids in the CDRs.

16. 16. The composition of any one of claims 1 to 15, wherein the variable domain that binds to the extracellular portion of HER3 comprises a heavy chain variable region comprising the CDR1 sequence GYYMH, the CDR2 sequence WINPNSGGTNYAQKFQG, and the CDR3 sequence DHGSRHFWSYWGFDY, or a variant thereof comprising substitutions, deletions and / or insertions of 1, 2 or 3 amino acids in the CDRs.

17. the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising the CDR1 sequence NYAMN, the CDR2 sequence WINANTGDPTYAQGFTG, and the CDR3 sequence ERFLEWLHFDY, or a variant thereof comprising substitutions, deletions and / or insertions of 1, 2 or 3 amino acids in the CDRs; the variable domain that binds to the extracellular portion of HER2 comprises a heavy chain variable region comprising the CDR1 sequence SYGMH, the CDR2 sequence VISYDGSNKYYADSVKG, and the CDR3 sequence DYYRRTARAGFDY, or a variant thereof comprising substitutions, deletions and / or insertions of one, two or three amino acids in the CDRs; The variable domain that binds to the extracellular portion of HER3 comprises a heavy chain variable region comprising the CDR1 sequence GYYMH, the CDR2 sequence WINPNSGGTNYAQKFQG, and the CDR3 sequence DHGSRHFWSYWGFDY, or a variant thereof comprising one, two or three amino acid substitutions, deletions and / or insertions in the CDRs; The composition according to any one of claims 1 to 16.

18. 18. A composition according to any one of claims 1 to 17 for use in therapy.

19. 19. The composition of claim 18 for use in the treatment of cancer, preferably gastric, lung or esophageal cancer.

20. A pharmaceutical composition comprising a composition according to any one of claims 1 to 17.

21. Two or more binding moieties, each comprising a variable domain that binds to the extracellular portion of EGFR; a first said binding moiety comprising a variable domain that binds to the extracellular portion of HER2 and a second said binding moiety comprising a variable domain that binds to the extracellular portion of HER3 for use in the treatment of cancer, preferably gastric cancer, lung cancer or esophageal cancer.

22. 1. A product comprising two or more binding moieties, each of said binding moieties comprising a variable domain that binds to the extracellular portion of EGFR, a first said binding moiety comprising a variable domain that binds to the extracellular portion of HER2 and a second said binding moiety comprising 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, preferably gastric, lung or esophageal cancer.

23. 23. The composition, pharmaceutical composition, binding moiety or product for use according to any one of claims 18 to 22, wherein the cancer comprises cells with an EGFR mutation that renders the cells resistant to treatment with a tyrosine kinase inhibitor (TKI).

24. The composition, pharmaceutical composition, binding moiety or product for use according to any one of claims 18 to 23, wherein the cancer comprises cells with the EGFR R521K polymorphism.

25. 25. A composition, binding moiety or product for use according to any one of claims 1 to 24, wherein the cancer is gastric cancer.

26. 1. A method of treating a subject having cancer or at risk of recurrence or relapse of cancer, comprising administering to a subject in need thereof a therapeutically effective amount of two or more binding moieties, each of said binding moieties comprising a variable domain that binds to an extracellular portion of EGFR, a first of said binding moieties comprising a variable domain that binds to an extracellular portion of HER2, and a second of said binding moieties comprising a variable domain that binds to an extracellular portion of HER3.

27. A method for producing a composition according to any one of claims 1 to 19, comprising the steps of: Providing a cell, the cell comprising: a nucleic acid encoding a polypeptide comprising a heavy chain capable of pairing with a common light chain to form a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain capable of pairing with said common light chain to form a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain capable of pairing with said common light chain to form a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, providing a cell, wherein optionally two or more of said nucleic acids are physically linked, each of said nucleic acids further comprising expression control sequences enabling expression of said encoded heavy and light chains in said cell; Culturing the cells to allow expression of the heavy and light chains, and optionally and recovering said two or more binding moieties.

28. 28. The method of claim 27, comprising providing said nucleic acid to a plurality of cells and selecting cells from said collection having a desired ratio of expression of said heavy and light chains.

29. 29. The method of claim 27 or claim 28, wherein the two or more binding moieties are antibodies, preferably bispecific antibodies.

30. 30. The method of any one of claims 27 to 29, wherein the cells produce essentially equimolar amounts of the two or more binding moieties.

31. 31. The method of any one of claims 27 to 30, wherein the cell produces more of a first binding moiety of the two or more binding moieties than a second binding moiety.

32. A cell, a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain that, together with said common light chain, forms a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, A cell, wherein two or more of said nucleic acids may or may not be physically linked, and each of said nucleic acids further comprises expression control sequences enabling expression of said encoded heavy and light chains in said cell.

33. A container containing nucleic acid, a nucleic acid encoding a polypeptide comprising a heavy chain capable of pairing with a common light chain to form a variable domain that binds to the extracellular portion of EGFR; a nucleic acid encoding a polypeptide comprising a heavy chain capable of pairing with a common light chain to form a variable domain that binds to the extracellular portion of HER2; a nucleic acid encoding a polypeptide comprising a heavy chain capable of pairing with a common light chain to form a variable domain that binds to the extracellular portion of HER3; and a nucleic acid encoding a polypeptide comprising the common light chain, Optionally, two or more of said nucleic acids may be physically linked, each of said nucleic acids further comprising expression control sequences enabling expression of said encoded heavy and light chains in a cell.

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