CN113454122A

CN113454122A - Combination of binding moieties that bind EGFR, HER2 and HER3

Info

Publication number: CN113454122A
Application number: CN202080014304.7A
Authority: CN
Inventors: 塞西利亚·安娜·威廉明娜·热延; 特里斯坦·路易斯·吉恩·加莱纳; 马克·思罗斯比; 科内利斯·阿德里安·德克吕夫
Original assignee: Merus BV
Current assignee: Merus BV
Priority date: 2019-02-14
Filing date: 2020-02-13
Publication date: 2021-09-28

Abstract

The present invention provides a composition comprising two or more binding moieties, wherein each of said binding moieties comprises a variable domain that binds to the extracellular portion of EGFR; and wherein 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 HER 3. The invention also relates to devices and methods for generating the compositions and treating an individual with the compositions.

Description

Combination of binding moieties that bind EGFR, HER2 and HER3

The present invention relates to the field of binding moieties such as antibodies, in particular to the field of therapeutic binding moieties. The binding moieties may be used in the treatment of humans. More specifically the invention relates to compositions comprising two or more multispecific binding moieties, preferably multispecific antibodies. The binding moieties bind EGFR, HER2 and HER 3. A single host cell can produce multiple binding moieties.

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

EGFR is present on the cell surface and is activated by binding of its specific ligands, including epidermal growth factor and transforming growth factor alpha (TGF α). Upon activation by its growth factor ligand, the receptor undergoes a transition from the inactive primary monomeric form to the active homodimer. In addition to forming homodimers upon ligand binding, EGFR can be paired with another member of the ErbB receptor family, such as HER2, to produce an activated heterodimer. There is also evidence suggesting: dimers form in the absence of ligand binding and clusters consisting of activated EGFRs form upon ligand binding.

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

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

Although there has been some success with EGFR-targeted therapies, most have been associated with the development of treatment resistance over time. One of the ways that EGFR-positive tumors can escape this targeted therapy is through signaling through another receptor dimer. For example, increased signaling by the EGFR/HER3 dimer due to increased HER3 expression or regulatory protein expression (heregulin expression) is correlated with EGFR-targeted drug resistance in, for example, lung and head and neck cancers. In addition to the induction of therapy resistance, certain side effects of EGFR-targeted antibodies have been observed. One example is the development of skin rashes associated with EGFR inhibition or anti-EGFR biotherapy. In extreme cases, such rashes may lead to a reduction in the treatment cycle and/or to a premature termination of the treatment.

A wide variety of activation patterns for signaling of this EGF receptor family have been identified. Among these are ligand-dependent (ligand dependent) and ligand-independent (ligand independent) activation of signal transduction. That is, overexpressed HER2 is able to generate oncogenic signaling through HER2/HER3 heterodimer in the absence of HER3 ligand (Junttila, Akita et al 2009). HER2 activity may be inhibited by HER2 specific antibodies. Such HER 2-specific antibodies can be used, for example, for HER2 positivity (HER 2)⁺) In the treatment of tumors. The problems with such treatments are: often tumors escape the HER2 specific therapy and continue to grow even in the presence of the inhibitory antibody. What has been observed is: HER2 positive tumors, such as breast, ovarian, cervical and gastric tumors, can escape treatment by exhibiting selective overgrowth of a sub-population of tumor cells that are up-regulated for HER3 expression (Ocana, Vera-Badillo et al 2013) and/or HER3 ligand expression (Wilson, fridlyland et al 2012). Also, activating mutations located in the HER3 receptor have been identified.

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

Disclosure of Invention

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

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

wherein 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 HER 3.

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

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

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

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

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

providing a cell comprising

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

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

-a nucleic acid encoding a polypeptide comprising a heavy chain which, together with the common light chain, forms a variable domain which binds to the extracellular portion of HER 3; and

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

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

Further provided is a cell including:

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

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

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

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

-a nucleic acid encoding a polypeptide comprising a heavy chain which, together with a common light chain, forms a variable domain which binds to the extracellular portion of HER 3; and

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

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

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

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

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

Detailed Description

The term EGFR as used herein means that protein encoded by the epidermal growth factor receptor gene (EGFR) in humans. The protein is known as a number of alternative names, including: Erb-B2 receptor tyrosine kinase 1; Proto-Oncogene (Proto-Oncogene) C-ErbB-1; ERBB 1; and HER 1. The database accession number relating to this human EGFR protein and the gene encoding it is (GenBank NM _ 005228.3). The accession number is given primarily to provide further means to identify the EGFR protein as a target, and the actual sequence of the EGFR protein bound by the antibody may vary, for example, due to mutations within the encoding gene (such as those occurring in certain cancers), and so forth. When reference is made herein to EGFR, reference is made to human EGFR, unless otherwise indicated. Due to sequence and tertiary structural similarity between orthologues (orthologues) of EGFR in humans and other mammals, the EGFR variable domain may, but need not, also bind such orthologues. This variable domain that binds EGFR can bind EGFR as well as its various variants, such as those expressed on certain EGFR-positive tumors.

The variable domain of an antibody or binding portion of the invention that binds EGFR preferably binds domain I or domain III of EGFR. The structure of the EGFR protein has been described in Ferguson (2008: Annu Rev Biophys.2008; 37: 353-373. doi: 10.1146/Annu Rev. biophyss.37.032807.125829), among other documents. The domains of human EGFR are depicted in figure 1 of the above-mentioned Ferguson reference data. The variable domains that bind EGFR of the embodiments of the invention disclosed herein preferably bind domain III of EGFR. The antibody preferably inhibits EGF-induced proliferation of BxPC-3 cells (ATCC CRL-1687) or BxPC-3-luc2 cells (Perkin Elmer 125058).

The term HER2 as used herein means that protein encoded by the ERBB-2 gene in humans. Alternative names for the gene or protein include: CD 340; ErbB-2; HER-2/neu; MLN 19; NEU; NGL; TKR 1. This ERBB-2 gene is often referred to as HER2 (derived from human epidermal growth factor receptor 2). When reference is made herein to HER2, this is referred to as human HER 2. An antibody comprising a variable domain that binds HER2 binds human HER 2. Due to sequence and tertiary structural similarity between orthologs of HER2 in humans and other mammals, the HER2 variable domain may, but need not, also bind such orthologs. The database accession numbers relating to the human HER2 protein and the gene encoding it are (NP-001005862.1, NP-004439.2, NC-000017.10, NT-010783.15). Such accession numbers are given primarily to provide further means to identify HER2 as the target, the actual sequence of HER2 protein bound by the antibody may vary, for example, because of mutations within the encoding gene (such as those occurring in certain cancers), and so on. The HER2 variable domain can bind to HER2 as well as its various variants, such as those expressed on certain HER2 positive tumor cells.

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

The term HER3 as used herein means that protein encoded by the ERBB3 gene in humans. Alternative names for this gene or protein are: LCCS 2; MDA-BF-1; c-ErbB-3; c-ErbB 3; ErbB 3-S; p180-ErbB 3; p45-sErbB 3; and p85-sErbB 3. When reference is made herein to HER3, this is referred to as human HER 3. An antibody comprising a variable domain that binds HER3 binds human HER 3. Due to sequence and tertiary structural similarity between orthologs of HER3 in humans and other mammals, the HER3 variable domain may, but need not, also bind such orthologs. The database accession numbers relating to the human HER3 protein and the gene encoding it are (NP-001005915.1, NP-001973.2, NC-000012.11, NT-029419.12). Such accession numbers are given primarily to provide further means to identify HER3 as a target, and the actual sequence of HER3 protein bound by an antibody may vary, for example, because of mutations within the encoding gene (such as those occurring in certain cancers), and so on. The HER3 variable domain can bind to HER3 as well as its various variants, such as those expressed on certain HER2 positive tumor cells.

The structure of HER3 has been described in Cho et al (2002; Science297, 1330-1333: DOI: 10.1126/science.1074611), among others. The human protein has 4 extracellular domains. The variable domain that binds HER3 of the embodiments of the invention disclosed herein preferably binds to domain III of HER 3. In a preferred embodiment, a variable domain has an affinity (KD) for a HER3 positive cell of less than or equal to 2.0nM, more preferablyIs less than or equal to 1.5nM, more preferably less than or equal to 1.39nM, and even more preferably less than or equal to 0.99 nM. In a preferred embodiment, an antibody according to the invention preferably comprises at least one of the domains III of the variable domain binding HER3 selected from the group consisting of R426 included in the native HER3 protein and located at a distance from R426

Amino acid residues within the group. In a preferred embodiment, the affinity (KD) of the variable domain for HER3 on SK-BR-3 cells is less than or equal to 2.0nM, more preferably less than or equal to 1.5nM, more preferably less than or equal to 1.39nM, more preferably less than or equal to 0.99 nM. In one embodiment, the affinity (KD) falls within the range of 1.39-0.59 nM. In a preferred embodiment, the affinity (KD) of the variable domain for HER3 located on BT-474 cells is lower than or equal to 2.0nM, more preferably lower than or equal to 1.5nM, more preferably lower than or equal to 1.0nM, more preferably lower than or equal to 0.5nM, more preferably lower than or equal to 0.31nM, more preferably lower than or equal to 0.23 nM. In one embodiment, the affinity (KD) falls within the range of 0.31-0.15 nM. The above-mentioned affinity is preferably measured when using steady state cell affinity measurements, wherein the cells are incubated at 4 ℃ with a radio-labelled antibody, after which the cell-bound radioactivity is measured.

The variable domain binding to at least one amino acid of domain III of HER3 preferably binds to R426 selected from the group consisting of the natural HER3 protein and is located at a distance from R426

Amino acid residues within the group. Preferably, it is located in the native HER3 protein at a distance from R426

The amino acid residue within is selected from the group consisting of: l423, Y424, N425, G427, G452, R453, Y455, E480, R481, L482, D483, and K485 (see, e.g., fig. 9 and table 1). The amino acid residue number is that possessed by Protein Database (PDB) ID #4P 59. Antibodies binding to this region of domain III of HER3 exhibit particularly good binding characteristics and they are capable of counteracting an activity of HER3 on HER3 positive cells. Variable domains with HER3 binding characteristics are described in WO 2015/130172, which is incorporated herein by reference. In a preferred embodiment, a bispecific antibody according to the invention is provided, wherein the antibody comprises R426 whose variable domain binds at least domain III of HER 3. Preferably, the antibody comprises R426 whose variable domain binds at least domain III of HER 3.

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

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

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

The binding moiety that binds EGFR and HER2 is not the same binding moiety that binds EGFR and HER 3. If at least one of the binding moieties is a multispecific antibody, at least one multispecific antibody may bind at least EGF and HER2 or at least EGFR and HER 3. In a preferred embodiment, the binding moiety comprises a bispecific antibody, wherein the bispecific antibody binds EGFR and HER2 and the other bispecific antibody binds EGFR and HER 3.

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

Since an antibody typically recognizes an epitope of an antigen, and such an epitope may also be present in other compounds, an antibody according to the invention that "specifically recognizes" an antigen (e.g., EGFR, HER2, or HER3) may also recognize other compounds if such other compounds contain the same epitope. Thus, the terms "specifically recognizes" or "specifically binds" or terms having the same connotation (conjugation) do not exclude the binding of such antibodies to other compounds containing the same or identical epitopes in terms of antigen-antibody interaction.

A "bispecific antibody" is an antibody as described herein, wherein one variable domain of the antibody binds to a first antigen and the second variable domain of the antibody binds to a second antigen, wherein the first and second antigens are not the same. The term "bispecific antibody" also encompasses antibodies in which the variable region/light chain variable region (V)_H/V_L) Combined binding of a first epitope located on an antigen, and a second V_H/V_LThe combination binds to the second epitope. The second epitope may be a non-identical epitope located on the same antigen. The term also includes antibodies wherein V_HCapable of specifically recognizing a first antigen, paired with the V in an immunoglobulin variable region_HV of_LCapable of specifically recognizing a second antigen. Formed V_H/V_LThe pairing will bind either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008/027236, WO 2010/108127 and Schaefer et al (Cancer Cell 20, 472-. The bispecific antibody according to the invention is not limited to any particular format or method for generating it.

Bispecific antibodies are an exemplary multispecific antibody. Tri-or more specific antibodies can be made by adding a binding moiety, such as a scFv fragment, to one or more of the heavy chains. It is also possible to add one or more variable domains to the variable region of a normal or bispecific antibody. A cell-produced common light chain and two non-identical heavy chains, each forming a functional variable domain with the common light chain, among other things, a bispecific antibody with two non-identical heavy chains in combination with a light chain. Likewise, a cell producing a common light chain and three or more non-identical heavy chains may form several bispecific antibodies, which together are capable of targeting three or more antigens. It is now possible to establish standard versions of antibodies (i.e. one constant part and two variable domains) and to add further binding domains. As such, multispecific antibodies having one or more single chain fvs with additional binding specificities attached to one or more of the constant or variable domains of the antibody can be made. It is also possible to generate heavy chains with two or more variable regions. The additional heavy chain regions may advantageously be associated with non-identical or common light chain variable regions. A description of such antibodies is provided in US 62/650467, which is incorporated herein by reference.

When referring to nucleic acid or amino acid sequences herein, "percent (%) identity" is defined as: after aligning the sequences for optimal comparison purposes, the residues in a candidate sequence are the same percentage as the residues in a selected sequence. Comparison of percent sequence identity of nucleic acid sequences was determined using default values using the AlignX application of Vector NTI Program Advance 10.5.2 software, such default values being determined using a modified ClustalW algorithm (Thompson, J.D., Higgins, D.G., and Gibson T.J (1994) Nuc.acid Res.22: 4673. sup. 4680), swgapdna score matrix (swgapdpnarnt score matrix), a gap opening penalty of 15 (gapopen penalty) and a gap extension penalty of 6.66 (gapextension penalty). Amino acid sequences were aligned using default values using the AlignX application of Vector NTI Program Advance 11.5.2 software using a modified ClustalW algorithm (Thompson, j.d., Higgins, d.g., and Gibson t.j., 1994), a blosum62mt2 scoring matrix (blosum62mt2 score matrix), a gap open penalty of 10, and a gap extension penalty of 0.1.

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

sequences

10, 11, or 12.

The term "full-length IgG" or "full-length antibody" according to the present invention is defined to include an essentially intact IgG, but it does not necessarily have all the functions of an intact IgG. In order to avoidIn question, a full-length IgG contains two heavy chains and two light chains. Each chain contains a constant region (C) and a variable region (V), which can be broken down into the regions designated CH1, CH2, CH3, V_HAnd C_L、V_LThe domain of (a). An IgG antibody binds to antigen via a variable region domain included in the Fab portion and can, upon binding, interact with molecules and cells of the immune system through such constant domains (mostly through the Fc portion). Full length antibodies according to the invention encompass IgG molecules in which mutations providing the desired characteristics may be present. A full-length IgG should not have a deletion of a substantial portion of any of such regions. However, IgG molecules in which one or several amino acid residues are deleted without substantially altering the binding characteristics of the resulting IgG molecule are encompassed by the term "full-length IgG". For example, such an IgG molecule may have a deletion of between 1 and 10 amino acid residues, preferably in the non-CDR regions, wherein such deleted amino acids are not necessary for the antigen or epitope binding specificity of the IgG. Examples of IgG antibodies are IgG1, IgG2, IgG3 and IgG4 antibodies. In certain embodiments of the invention, the IgG is IgG 1.

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

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

In the present invention, the selected formation of EGFRxHER2 and EGFRxHER3 bispecific antibodies can be directed by combining a portion of the heterodimerization domain located in the heavy chain forming the EGFR variable domain (3) and a compatible portion located in the heavy chain forming the HER2 and the HER3 binding domain (2).

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

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

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

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

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

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

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

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

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

In certain embodiments, the EGFR variable domain comprising a heavy chain variable region comprises EGFR V as set forth in fig. 7 or fig. 8_HHCDR1, HCDR2, and HCDR3 of the region. Preferably EGFR V of MF3755 in FIG. 7 or FIG. 8_HAnd (4) a zone.

In certain 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 an EGFR V set forth in fig. 7 or fig. 8_HThe amino acid sequence of the region. Preferably EGFR V of MF3755 in FIG. 7 or FIG. 8_HAnd (4) a zone.

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

In certain embodiments, the EGFR variable domain comprising a heavy chain variable region comprising an amino acid sequence is from an EGFR V selected from figure 7 or figure 8_HAnd (4) a zone. Preferably EGFR V of MF3755 in FIG. 7 or FIG. 8_HAnd (4) a zone.

In some embodiments, the HER2 variable domain comprises a heavy chain variable region comprising HER2V as set forth in figure 7 or figure 8_HRegion (preferably HER2V of MF2032 in FIG. 7 or FIG. 8_HRegion) HCDR1, HCDR2, and HCDR 3.

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 a HER2V set forth in figure 7 or figure 8_HRegion (preferably HER2V of MF2032 in FIG. 7 or FIG. 8_HRegion) of a nucleic acid sequence.

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

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

In some embodiments, the HER3 variable domain comprises a heavy chain variable region comprising the V of MF3178 in figure 7 or figure 8_HHCDR1, HCDR2, and HCDR3 of the region.

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

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

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

In a preferred embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR, the variable domain comprising a heavy chain variable region comprising an amino acid sequence from an EGFR V selected from figure 7 or figure 8_HA region or variant thereof, preferably EGFR V of MF3755 of fig. 7 or fig. 8_HA region, or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain(ii) the domain comprises a heavy chain variable region comprising an amino acid sequence selected from MF1849 or MF2032 of fig. 7 or fig. 8, or a variant thereof, preferably MF2032 of fig. 7 or fig. 8, or a variant thereof, wherein the variant comprises a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs); and wherein the second bispecific antibody further comprises a variable domain which binds to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF3178 in fig. 7 or fig. 8, or a variant thereof, wherein the variant comprises a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs).

In a preferred embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR, which variable domain comprises a heavy chain variable region comprising the amino acid sequence of MF3755 from fig. 7 or fig. 8 or a variant thereof comprising 1, 2 or 3 amino acids (not preferably located in the CDRs) substituted, deleted and/or inserted; and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF2032 in fig. 7 or fig. 8 or a variant thereof comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably located in said CDRs); and wherein the second bispecific antibody further comprises a variable domain which binds to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF3178 in fig. 7 or fig. 8, or a variant thereof, wherein the variant comprises a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs).

In one embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR, which variable domain comprises a heavy chain variable region comprising the amino acid sequence from MF4280 in fig. 7 or fig. 8 or a variant thereof comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain which binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF1849 in fig. 7 or fig. 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs); and wherein the second bispecific antibody further comprises a variable domain which binds to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF3178 in fig. 7 or fig. 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs).

In one embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR, which variable domain comprises a heavy chain variable region comprising the amino acid sequence from MF4280 in fig. 7 or fig. 8 or a variant thereof comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF2032 in fig. 7 or fig. 8 or a variant thereof comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably located in said CDRs); and wherein the second bispecific antibody further comprises a variable domain which binds to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF3178 in fig. 7 or fig. 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs).

In one embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR, which variable domain comprises a heavy chain variable region comprising the amino acid sequence of MF4003 from fig. 7 or fig. 8 or a variant thereof comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain which binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF1849 in fig. 7 or fig. 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs); and wherein the second bispecific antibody further comprises a variable domain which binds to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF3178 in fig. 7 or fig. 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs).

In one embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR, which variable domain comprises a heavy chain variable region comprising the amino acid sequence of MF4003 from fig. 7 or fig. 8 or a variant thereof comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably located in the CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF2032 in fig. 7 or fig. 8 or a variant thereof comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably located in said CDRs); and wherein the second bispecific antibody further comprises a variable domain which binds to the extracellular portion of HER3, the variable domain comprising a heavy chain variable region comprising the amino acid sequence from MF3178 in fig. 7 or fig. 8 or a variant thereof comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably located in said CDRs).

Exemplary EGFR heavy chain variable regions are described in WO 2015/130172 and PCT/NL2018/050537, which are incorporated herein by reference. An exemplary HER2 heavy chain variable region is described in WO2015/130173, which is incorporated herein by reference. Exemplary HER3 heavy chain variable regions are described in WO 2015/130172 and WO2015/130173, which are hereby incorporated by reference.

Additional variants of the disclosed amino acid sequences that retain EGFR, HER2, or HER3 binding may be obtained, for example, from human IGKVl-39/IGKJl V containing rearrangements as previously described_LPhage display libraries of regions (De Kruif et al Biotechnol Bioeng.2010(106)741-50), and one by incorporating amino acid substitutions into one of EGFR, HER2 or HER 3V disclosed herein_HV in the amino acid sequence of the region_HA collection of zones. Phages encoding Fab regions that bind EGFR, HER2, or HER3 can be selected and analyzed by flow cytometry and sequenced to identify variants with amino acid substitutions, insertions, deletions, or additions that retain antigen binding.

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

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

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

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

The EGFR variable domain, HER2 variable domain, and light chain variable domain (VLs) of the HER3 variable domain of the binding moiety (such as a bispecific antibody) may be the same as the V of a parent EGFR monospecific antibody_LV of domain, parent HER2 monospecific antibody_LDomains and/or V identical to a parent HER3 monospecific antibody_LAnd (4) a zone. Alternative V_LThe zones can be used for such V_H/V_LOne or more of the combinations of regions are used so long as such variable domains retain binding to EGFR, HER2, or HER3, respectively.

In certain embodiments, the EGFR variable domain, the HER2 variable domain, and the V of the HER3 variable domain_LThe zones are similar. In certain embodiments, V is located in all variable domains of the binding moiety_LThe zones are identical.

In certain embodiments, the light chain variable regions of 1, 2, 3 or more variable domains of the binding moieties of the invention comprise a common light chain variable region. In certain embodiments, the common light chain variable region of 1, 2, 3 or more variable domains comprises a reproductive variable region V-segment. In certain embodiments, 1, 2, 3 or more variable domain light chain variable regions comprise a kappa light chain V-segment IgV κ 1-39 x 01. IgV kappa 1-39 is a shorthand for the immunoglobulin variable kappa 1-39 gene. The genes are also called kappa variable 1-39, IGKV139, IGKV 1-39. The external identifiers for this gene are: HGNC: 5740; entrez Gene: 28930, respectively; ensembl: ENGG 00000242371. The amino acid sequence for this V-region is provided in sequence 10 of figure 7. The V-region may also be combined with one of the 5J-regions. Preferred J-regions are jk1 and jk5, and the sequences being linked are designated IGKV1-39/jk1 and IGKV1-39/jk5, with the alternative names IgV kappa 1-39X 01/IGJ kappa 1X 01 or IgV kappa 1-39X 01/IGJ kappa 5X 01 (according to the position in img)t. org's name of IMGT database global website). In certain embodiments, one or two V_H/V_LThe light chain variable region of the binding region comprises kappa light chain IgV kappa 1-39X 01/IGJ kappa 1X 01 or IgV kappa 1-39X 01/IGJ kappa 1X 05 (sequence 11 or sequence 12 in FIG. 7, respectively).

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

In certain embodiments, 1, 2, 3 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 of sequence 11 set forth in fig. 7. In certain embodiments, 1, 2, 3 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 of sequence 11 set forth in fig. 7.

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

In other embodiments, the variable region of the light chain of 1, 2, 3 or more variable domains of said binding moiety of the invention comprises the amino acid sequence of seq id No. 11 in fig. 7 or seq id No. 12 in fig. 7. In certain embodiments, all variable domains of the binding moiety of the invention comprise the same V_LAnd (4) a zone. In one embodiment, all variable domains of the binding moiety of the invention are V_LIncluding the amino acid sequence set forth in sequence 11 in fig. 7. In one embodiment, all variable domains of the binding moiety of the invention are V_LIncluding the amino acid sequence of sequence 12 as set forth in fig. 7 or sequence 12 in fig. 7.

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

In certain embodiments, such multispecific antibodies used in the methods provided herein are generally of the human IgG subtype (e.g., IgG1, IgG2, IgG3, IgG4, for example). In certain embodiments, such antibodies are of human IgG1 subtype. Full-length IgG antibodies are preferred because of their favorable half-life and for low immunogenicity. Such multispecific antibodies may have two non-identical heavy chains comprising a heterodimerization domain. Thus, in certain embodiments, such EGFR/HER2 and EGFR/HER3 bispecific antibodies are full length IgG molecules. In one embodiment, such EGFR/HER2 and EGFR/HER3 bispecific antibodies are full length IgG1 molecules.

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

In preferred embodiments, the multispecific (preferably bispecific) full-length IgG antibody has a lower hinge and/or CH2 domain such that the interaction of the bispecific IgG antibody with an Fc gamma (Fc γ) receptor is enhanced. Antibody-dependent cellular cytotoxicity (also referred to as ADCC activity) of an antibody can often be improved when the antibody itself has a low ADCC activity. This can be achieved, for example, by removing fucose residues from the glycosylated portion of the antibody. A technique for enhancing ADCC by afucosylation (afucosylation) is described, for example, In Junttila, t.t., k.parsons, et al (2010), "Superior In vivo Efficacy of Afucosylated Trastuzumab In the Treatment of HER2-Amplified Research Cancer," Cancer Research 70 (11): 4481 and 4489). Multispecific antibodies as described herein are preferably nonfucosylated. Preferably the two multispecific antibodies are afucosylated. Other strategies have been reported to achieve ADCC enhancement, including, for example, saccharification engineering (Kyowa Hakko/Biowa, glycart (roche) and Eureka Therapeutics) and mutagenesis (mutagenesis) (xenogenesis) (xenocor and macrogenetics), all of which seek to improve Fc binding to low affinity activating Fc γ RIIIa (low-affinity activating Fc γ RIIIa) and/or reduce binding to low affinity inhibiting Fc γ RIIb (low affinity inhibiting Fc γ RIIb).

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

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

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

-nucleic acid encoding a polypeptide comprising a heavy chain which, together with the common light chain, forms a variable domain which binds to the extracellular portion of HER 3; and

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

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

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

The present invention also provides a cell comprising:

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

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

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

-nucleic acid encoding a polypeptide comprising a heavy chain which, together with a common light chain, forms a variable domain which binds to the extracellular portion of HER 3; and

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

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

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

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

In certain embodiments, the cell exhibits 3 non-identical heavy chains and at least one light chain. In a preferred embodiment, the cell exhibits a "common light chain" as described herein to reduce the number of non-identical antibody species (combinations of non-identical heavy and light chains). For example, along with rearranged human IGKV 139/IGKJ 1(huV κ 139) light chains, the corresponding VH regions were transformed into expression vectors using methods known in the art for the generation of bispecific iggs (WO2013/157954, incorporated herein by reference). This huV κ 139 was previously shown to be capable of associating with more than one heavy chain, thus leading to antibodies with distinct specificities, which facilitate the production of bispecific molecules (WO 2009/157771).

An antibody-producing cell that exhibits a common light chain and an equivalent number of two heavy chains typically produces 50% bispecific antibodies and 25% of each such monospecific antibody (i.e., having the same heavy chain and light chain combination). Several approaches have been published to favor the generation of bispecific antibodies over the generation of corresponding monospecific antibodies. This is usually achieved by modifying the constant region of the heavy chains such that they favor heterodimerization (i.e. dimerization of a heavy chain in combination with another heavy/light chain) over homodimerization (homodimerization). In a preferred embodiment, the bispecific antibodies of the invention comprise two non-identical immunoglobulin heavy chains with compatible heterodimerization domains. A wide variety of compatible heterodimerization domains have been described in the art. Such compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. The art describes various ways in which such hetero-dimerization (heter-dimerization) of the heavy chain can be achieved.

A preferred method for generating multispecific antibodies as described herein is disclosed in US9,248,181 and US9,358,286. In particular, for generating substantially only bispecificPreferred mutations of the full-length IgG molecule are the amino acid substitutions L351K and T366K (EU numbering) located in the first CH3 domain (the "KK-variant" heavy chain) and the amino acid substitutions L351D and L368E located in the second domain (the "DE-variant" heavy chain), or vice versa. As previously described, the DE-and KK-variants preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of a DE-variant heavy chain (DEDE homodimer) or homodimerization of a KK-variant heavy chain (KKKKKKKK homodimer) hardly occurs due to strong repulsion between charged residues in the CH3-CH3 interfaces located between the same heavy chains. The introduction of a further heavy chain with a DE-or KK-variant heavy chain allows the generation of a further DEKK bispecific molecule. A newly introduced DE-heavy chain (DE)²) May be associated with an existing KK heavy chain. The cell thus produces two bispecific antibodies, one DE¹KK and DE²A KK bivalent antibody. If a new KK heavy chain (KK)²) Instead of the new DE heavy chain being introduced, with DEKK¹And DEKK²Combinatorial bispecific antibodies were generated. The level at which different antibodies can be produced by the cell can be adjusted by adjusting the relative performance of such HER2 and HER3 chains with respect to each other. The light chain is typically generated sufficiently to reduce the level of a single heavy chain, whereas such EGFR chains are typically generated at a level sufficient to allow efficient pairing with such HER2 chains, HER3 chains.

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

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

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

Antibodies produced by a cell can be purified by affinity purification. This is advantageously done by protein a extraction. The eluted antibodies can be tested by ELISA for the presence of specific binding properties (i.e. binding to EGFR, HER2 and HER 3). The antibody preparation may 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 the corresponding bispecific antibody can also be analyzed by ELISA. Binding and washing of the preparation to HER2 should remove all EGFR/HER3 antibodies. Staining with the labeled soluble HER3 gave no signal, whereas staining with the labeled soluble EGFR did. Binding and washing of the preparation to HER3 should remove all EGFR/HER2 antibodies. Staining with the labeled soluble HER2 gave no signal, whereas staining with the labeled soluble EGFR did. Binding and washing of the preparation to EGFR should not remove EGFR/HER2 and EGFR/HER3 antibodies. Staining with labeled soluble HER2 in addition staining with labeled soluble HER3 should provide a signal. With an appropriate control group having known levels of such mono-and bi-specific antibodies, the level of such corresponding antibodies in a preparation can also be estimated using such an ELISA.

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

-providing cells with nucleic acids encoding the bispecific antibodies;

-culturing the cells;

-harvest clarification (harvest clarification);

-collecting such bispecific antibody from the culture; and

-separating the generated bispecific antibody from the half-antibody by ion exchange chromatography (IEX);

the method is characterized in that: such bispecific antibodies exhibit similar IEX retention times under the IEX conditions used, preferably with a mean deviation from the retention times of such individual antibodies of 10% or less. In one embodiment, such antibodies are selected to have an IEX retention time that deviates from the mean value of the retention times of such individual antibodies by 10% or less under the IEX conditions used. Such antibodies may first be purified from other proteins within the culture. This is usually done by affinity purification, preferably by protein a extraction. Such bispecific antibodies are preferably selected to have a half-antibody with a residence time that falls outside the range spanned by the residence times of such antibodies. Where a combination of bispecific antibodies is generated and monospecific antibodies are not desired, such bispecific antibodies are preferably selected to have a retention time that is not the same as that of such monospecific antibodies. Such monospecific antibody retention times in this embodiment preferably fall outside the range spanned by the retention times of such corresponding bispecific antibodies. Cells in this culture preferably simultaneously express the 3 heavy chains, wherein the heavy chains include a CH3 heterodimerization domain formed to promote heterodimerization of the EGFR/HER2 and EGFR/HER3 heavy chains. The cells preferably exhibit a common light chain as in figure 7. Such bispecific antibodies in one embodiment have isoelectric Points (PIs) that are similar, and preferably differ from the average PI of the at least two bispecific antibodies by no more than 0.5 units.

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

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

The invention further provides two or more binding moieties for use in the treatment of cancer, each comprising a variable domain that binds to the extracellular portion of EGFR; wherein 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 HER 3. Also provided is a product containing two or more binding moieties each comprising a variable domain that binds to the extracellular portion of EGFR; wherein 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 as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.

The cancer treated by embodiments of the present invention is preferably a cancer as indicated elsewhere herein. The cancer preferably comprises a cell bearing an EGFR-mutation that renders the cell resistant to treatment with a Tyrosine Kinase Inhibitor (TKI). In certain embodiments, the cancer comprises cells with EGFR R521K polymorphism. The cancer to be treated with a method of treatment of an invention described herein is preferably gastric, lung or esophageal cancer. In a further embodiment, the invention provides a method for treating an individual having cancer or at risk of relapse, exacerbation of cancer, comprising administering to the individual in need of such treatment two or more binding moieties each comprising a variable domain that binds to the extracellular portion of EGFR; wherein 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 HER 3.

As used herein, the terms "subject" and "patient" are used interchangeably and mean a class of mammals, such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, and the like (e.g., a patient with a cancer, such as a human patient).

The terms "treatment," "treating," "treatment," and "treatment," as used herein, refer to any type of intervention or procedure performed on the subject or the administration of an active agent or a combination of active agents to the subject in order to reverse, alleviate, ameliorate, inhibit, or slow or prevent the progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicator associated with a disease.

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

The term "effective amount" or "therapeutically effective amount" means an amount of an agent or a combination of agents that provides the desired biological, therapeutic and/or prophylactic result. The result can be a reduction, amelioration, palliation, reduction, delay, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In certain embodiments, an effective amount is an amount sufficient to delay tumor development. In certain embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. The effective amount of the drug or composition may be: (i) reducing the number of cancer cells; (ii) reducing the size of the tumor; (iii) inhibit, retard, slow and may stop to some extent the infiltration of cancer cells into peripheral organs; (iv) inhibiting tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the occurrence and/or recurrence of a tumor; and/or (vii) relieve to some extent one or more symptoms associated with the cancer. In one example, an "effective amount" is an amount of a composition of the invention to achieve a reduction in a cancer (e.g., a reduction in the number of cancer cells) or to slow the progression of a cancer. An effective amount of the combination therapy is administered in accordance with such methods described herein in an "effective regimen" (which means a combination of the binding moieties as indicated herein), wherein the order and frequency of administration is sufficient to effect treatment.

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

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

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

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

The cancer preferably exhibits HER 2. The cancer preferably also exhibits EGFR or HER 3. The cancer preferably exhibits EGFR. The cancer preferably also exhibits HER2 or HER 3. The cancer preferably exhibits HER 3. The cancer preferably also exhibits EGFR or HER 2. In certain embodiments, cells of a cancer and/or stromal cells (stromal cells) in the cancer treated by the invention disclosed herein exhibit an EGFR ligand, a HER3 ligand, or both. Expression of the ligand and receptor thus provides a growth stimulus to the cells of the cancer. A combination of the invention is particularly suitable for the treatment of cancer comprising such cells.

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

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

The compositions of the invention are suitable for treating cancers with EGFR-mutations that render the cancer cells resistant to treatment with Tyrosine Kinase Inhibitors (TKIs). In one embodiment, the cancer comprises cells with EGFR R521K polymorphism. In certain embodiments, the cancer is known to be resistant to first generation TKI inhibitors (such as gefitinib and erlotinib).

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

The compositions of the invention may be suitable for use in the treatment of cancers that are resistant to EGFR inhibition as a result of over-expression of HER2 and/or HER 3.

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

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

Term(s) for

By the context of an antibody, binding moiety, composition or product as described herein is meant the presence of more than one, and typically 10 or fewer, non-identical antibodies or binding moieties in a preparation, including the presence of a dual specificity.

An exemplary embodiment of (a) includes a combination of two bispecific antibodies.

The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER 2; wherein the EFGR variable domain comprises a heavy chain variable region comprising the CDRs of heavy chain variable regions MF3755, MF4280, MF4003 or MF4016 in figure 8, or a variant of the heavy chain variable region comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids in said CDRs, and wherein the HER2 variable domain comprises a heavy chain variable region comprising the CDRs of heavy chain variable regions MF2032 or MF1849, or a variant of the heavy chain variable region comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids in said CDRs.

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 HER 2; wherein the EFGR variable domain comprises the CDRs of heavy chain variable region MF3755 in figure 8, or a variant of the heavy chain variable region comprising 1, 2 or 3 amino acids substituted, deleted and/or inserted in said CDRs, and wherein the HER2 variable domain comprises the CDRs of heavy chain variable region MF2032, or a variant of the heavy chain variable region comprising 1, 2 or 3 amino acids substituted, deleted and/or inserted in said CDRs.

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

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

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

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

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

Drawings

FIG. 1 shows a schematic view of a

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

FIG. 2

Two kinds of

For the growth factor-dependent cell lines BxPC-3-luc2(Perkin Elmer 125058) and N87 cells (NCI-87 cells: (NCI-87 cells))

CRL-5822^TM) Inhibitory effect on proliferation of plants).

Two kinds of

The efficacy of cell proliferation was tested for BxPC-3-luc2 (left panel)) and N87 (right panel)). The results of the panel screen were compared with a combination of two monospecific antibodies (EGFR binding antibody cetuximab and HER3 monospecific antibody PG3178) or with EGFRxHER3 binding bispecific antibody PB 4522. The cells were grown in the presence of saturating amounts of HRG and EGF. The level of cell growth of the corresponding cells using HRG and EGF without antibody (basal with ligand (basal w/ligand)) and furthermore the basal level without HRG and EGF without antibody (ligand-free (w/ligand)) is indicated.

Monospecific antibody PG3178 has one IgG1 constant region and two common light chain variable regions with the variable domain of the heavy chain variable region of MF3178 in fig. 7 or fig. 8 and sequence 11 in fig. 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 fig. 7 or fig. 8. The light chain variable regions of the two antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence 11 in fig. 7.

FIG. 3

Of a small group

ADCC activity of (1). Of a small group

ADCC activity of (D) was tested using N87 and a CD16/NFAT reporter assay (CD16/NFAT reporter assay). Such bispecific antibodies have one IgG1 constant region and two variable domains. Such asThe amino acid sequence of the heavy chain variable region of the variable domain is illustrated in figure 7 or figure 8. The light chain variable regions in such antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence 11 in fig. 7.

FIG. 4

A variety of different

Number and specificity of (a) and their ADCC activity. "-" indicates no activity was observed.

Each row represents a panel comprising two bispecific antibodies

The internal code of such Bispecific antibodies is indicated in such straightforward Bispecific 1(Bispecific 1) and Bispecific 2(Bispecific 2). The heavy chain variable regions of HER2, HER3, and EGFR binding domains are indicated in the direct lines labeled MF a, MF B, and MF

C. MF numbers

3178 and 2703 combine to form a HER3 binding variable domain with the common light chain. MF nos. 4280, 3755, 4003, 4016 combine to form a variable domain that binds EGFR with the common light chain, while MF nos. 1871, 1847, 1849, and 2032 combine to form a variable domain that binds HER2 with the common light chain. Such bispecific antibodies have one IgG1 constant region and two variable domains. The amino acid sequences of the heavy chain variable regions of the variable domains are indicated 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 fig. 7.

FIG. 5

In vivo test of (1). BxPC-3-luc2 or N87 cells were injected into the xenograft model on day 0. Comprising the bispecific antibody PB4516 XPB 6892 (see FIG. 4)

Or the control group is in1. Injections were given on

days

7, 14, 21 and 28. The antibody was injected intraperitoneally at a dose of 25 mg/kg. Is depicted as relating to

(PB4516 and PB 6892). Vehicle and cetuximab served as control groups.

FIG. 6

Comprising bispecific antibodies PB4516 and PB6892

In vivo testing in various different PDX models.

The PDX model was injected on day 0, while treatment with antibody or control was completed on

days

1, 7, 14, 21 and 28. The antibody was injected intraperitoneally at a dose of 25 mg/kg.

FIG. 7

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

FIG. 8

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

FIG. 9

a) HER3 crystal structure (PDB #4P59) showing residue Arg 426 located in gray sphere and at a distance from Arg 426

Residues located in the black sphere within the radius; b) residue Arg 426 shown in gray and at a distance from Arg 426

Distant residues within the radius shown in black; c) residues located in region Arg 426 in light grey and residues surrounding it in dark grey (all labelled). The drawings and analysis of the specification were made using Yasara (www.yasara.org).

Examples

Cell line

Hek293 cells, NCI-87 cells: (

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

Production of bispecific antibodies

Bispecific antibodies were generated using the DEKK CH3 technique described above for efficient heterodimerization and formation of bispecific antibodies. As previously described (WO2013/157954 a1), the CH3 technique utilizes charge-based point mutations in the CH3 region to allow efficient pairing of two non-identical heavy chain molecules.

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

Adherent Hek293 cells were cultured in 6-well plates to an 80% confluence. The cells were transiently transfected with DNA-FUGENE mixture and further cultured. 7 days after transfection, the supernatant was harvested and the medium was refreshed. At 14 days after transfection, the supernatant was combined and filtered through (0.22. mu.M. sterile supernatant was stored at 4 ℃ C.) suspension-adjusted Hek293 cells were cultured in T125 flasks (T125 flashes) on an oscillator plate until a 3.0X 10 cell count was reached⁶Density of individual cells/mL. The cells are in a range of 0.3-0.5X 10⁶Viable cells/mL were seeded into each well of a 24 deep well culture dish. The cells were transiently transfected with individual sterile DNA: PEl-MIX and further cultured. 7 days after transfection, the supernatant was harvested and filtered through 0.22. mu.M. The sterile supernatant was stored at 4 ℃.

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

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

Stable cell line clones co-expressing both bispecific antibodies were generated by plating such wells in semi-solid medium. The plated cells were allowed to grow for 7-10 days. Two rounds of single cell cloning were performed by seeding and picking a single colony. Oligoronics are produced from a single cell by fed-batch fermentation (fed-batch fermentation).

Determination of antibody Titers

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

Two sandwich-type elisas (sandwich elisas) were subsequently developed to assay and quantify two bispecific molecules, allowing the estimation of the ratio between the two bispecific. For the determination of the EGFRxHER2 bispecific, the EGFR-Fc (R & D Systems, 344-ER) antigen was coated on top of such wells and determined using ErbB2-Fc (R & D Systems, 1129-ER). For the determination of EGFRxHER3 bispecific, EGFR-Fc antigen was coated on such wells and determined using ErbB3-Fc (R & D Systems, 348-RB).

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

Cation exchange Chromatography (CIEX)

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

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

BxPC-3-luc2 and N87 growth inhibition assay

The antibody compositions were pooled two by two over a range of concentrations of total antibody (tested under. such antibodies were pooled according to equal amounts of weight/weight HRG and EGF were added to the culture at 0.1ng/mL EGF and 10ng/mL HRG for BxPC3-luc2 cells, or 0.1ng/mL EGF and 1ng/mL HRG for N87 cells.

The antibody was diluted in chemically defined starvation medium (CDS: RPMI 1640 medium containing 80U of penicillin and 80. mu.g of streptomycin per mL, 0.05% (w/v) BSA and 10. mu.g/mL holo-transferrin), and 50. mu.L of the diluted antibody was added to a 96 well black well clear bottom plate (Costar) of such wells. Ligands were added (50. mu.L per well of one stock solution containing 40ng/mL or 4ng/mL HRG diluted in CDS and 400ng/mL EGF: R & D systems, cat. nr.396-HB and 236-EG). The plates were left at room temperature for 1 hour and then placed in a container in a 37 ℃ cell culture incubator for 3 days (N87 cells) or 4 days (BxPC-3-luc2 cells). On day 4, Alamar blue (Alamar blue) (Invitrogen, # DAL1100) was added (20 μ L/well) and fluorescence was measured after 6 hours (N87 cells) or 4 hours (BxPC-3-luc2 cells) of incubation with Alamar blue (at 37 ℃) on a Biotek Synergy 2 multi-functional microplate analyzer with an excitation of 560nm and a readout of 590 nm. Fluorescence values were normalized for uninhibited growth (no antibody, but two ligands added).

A variety of different

ADCC Activity of (2)

ADCC Reporter Bioassay (ADCC Reporter Bioassay) (Promega) was used. Two different cell lines were tested: an EGFR-expressing pancreatic cancer cell line BxPC3 and a gastric cancer N87 cell line.

The bioassay uses engineered Jurkat cells that either stably express Fc γ RIIIa receptor V158 (high affinity) variants, and an NFAT responsive element that drives the expression of firefly luciferase, a measure of Fc γ R activation. The assay has been compared to conventional assays by using data obtained from bioassays using this ADCC reporter⁵¹The Cr release analysis was verified by comparison, and both analyses gave similar results. Such ADCC assays were performed using the Promega ADCC bioassay kit using 384 white well plates. In this experimental setup, BxPC3 cells and N87 cells were plated 20-24 hours prior to the assay at a density of 1000 cells/well in 30 μ L assay medium (RPMI with 4% low IgG serum). Every other day, the medium was removed. Secondly, the first step is to carry out the first,

a series of dilutions of (c) and a comparator antibody (comparator antibody) cetuximab were prepared in duplicate. 10 μ L of these antibody dilutions were added to such wells. Control wells without antibody were also included (basal). From the starting concentration of such antibodies, 5-fold serial dilutions were generated to provide a dose-response curve. Finally, 5 μ L of ADCC bioassay effector cells (ADCC Bioassayeffector cells) (15000 cells/well, V158) were added. The cells were incubated at 37 ℃ for 6 hours. Next, 15. mu.L of BIO-Glo luciferase substrate was added and luminescence was measured in a disk reader after 5 minutes. The resulting data are shown in fig. 3. Cetuximab shows ADCC activity against BxPC3 and N87 cells. Various different antibodies of the oligo strain also showed ADCC activity on BxPC3 and/or N87 cellsAnd (4) sex.

Testing including bispecific antibodies PB4516 and PB6892

About it toIn BxPC-3- Effect of growth of luc2 tumor (implanted in situ) and N87 tumor (gastric cells implanted in abdomen side)

8-10 week old CB17 SCID female mice were implanted in situ in the pancreas at the start of the study to fit within 20 μ L of 1 × 10⁶BxPC-3-luc2 tumor cells. Mice were anesthetized and laid down from the right side to expose the left side, and a 0.5cm incision was made on the left flank area. Pancreas and spleen were removed from abdomen and placed in a volume of 20. mu.L at 1X 10⁶Individual tumor cells were injected into the subcapsular space of the pancreatic tail. One week after implantation, Bioluminescence (BLI) data was generated. For the left view of BLI imaging (once or twice weekly), all mice received an intraperitoneal injection (i.p. injections) of fluorescein (Luciferin) (D-fluorescein-EF potassium salt, Cat. # E6552, Promega) at 150mg/kg before 15 minutes of the imaging. Outliers animals (outlier animals) -according to BLI/tumor volume-were removed, while such mice were randomly distributed in groups of 7 mice per group. On day 8 of the experiment, treatment was initiated.

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

Cetuximab and oligoclonic significantly reduced BxPC-3 tumor overgrowth (p) in this model<0.05) (fig. 5). Use of

The tumor overgrowth of PB4516 and PB6892 was significantly lower than with cetuximab. Cetuximab was not significantly reducedOvergrowth of N87 cells.

Significantly reduced N87 tumor overgrowth (p) in this model<0.05) (fig. 5).

N87 tumor：

8-12 week old CB17 SCID female mice were inoculated subcutaneously (s.c.) in the flank at the start of the study to 1X 10 in 50% Matrigel (Matrigel)⁷And N87 tumor cells. The cell injection volume was 0.2 mL/mouse. The treatment reaches 150-200mm in tumor³Is started. The antibody was administered once a week for 4 weeks by intraperitoneal injection of mice at 25 mg/kg. Body weight measurements were taken weekly after tumor cell injection, and biweekly from the start to the end of treatment. Tumor growth was monitored every two weeks by caliper measurements (calipers measurements). The end point of the experiment was one at 800mm³The tumor volume of (1) or (60) days, whichever comes first.

Activity of PB11244 and PB4516 in various PDX models

Comprising bispecific antibodies PB11244 and PB4516

Is assessed in a series of PDX models. Testing candidate treatments in a large number of cancer models facilitates the prediction of clinical performance, and factors for patient-selection strategies can be identified.

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

The HER3 variable domain of PB4516 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 MF3755 in fig. 7 or fig. 8.

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

The light chain variable regions in these two antibodies are identical and have the sequence identification numbers in fig. 7: 11(SEQ ID NO: 11).

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

Comprising bispecific antibodies PB4516 and PB11244

Is generated and purified. Such antibodies are represented by a 1: the ratio of 1 was mixed.

Were tested in such models and compared to cetuximab and vehicle (PBS).

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

PB4516 XPB 689225 mg/kg, 5 week dose, intraperitoneal injection

Cetuximab 25mg/kg, dose 5 weeks, intraperitoneal injection

Vehicle (PBS), 5 week dose, intraperitoneally injection

It can be seen that:

significantly reducing the overgrowth of tumor cells in the model. The reduction in overgrowth is equal to cetuximab or more preferably.

TABLE 1 Arg 426 falling in HER3

Residue within radius

Leu 423	L423
		Tyr
424	Y424
		Asn
425	N425
		Gly
427	G427
		Gly
452	G452
		Arg
453	R453
		Tyr
455	Y455
		Glu
480	E480
		Arg 481	R481
Leu
	482	L482
Asp
	483	D483
Lys
	485	K485

Claims

1. A composition comprising two or more binding moieties,

wherein each of the binding moieties comprises a variable domain that binds to the extracellular portion of EGFR; and is

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

3. The composition of claim 1 or claim 2, wherein at least one and preferably at least two of the two or more binding moieties are 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 a heavy chain with a variable domain that binds EGFR with a heavy chain with a variable domain that binds HER2 and/or heterodimerization of a variable domain that binds EGFR with a heavy chain with a variable domain that binds HER 3.

5. The composition of any one of claims 2 to 4, wherein at least one antibody and preferably at least two antibodies of said two or more antibodies are bispecific antibodies.

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

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, preferably domain III, of EGFR.

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

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 HER 3.

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; wherein the variable domain that binds to the extracellular portion of HER2 binds to domain I or domain IV, preferably domain IV, of HER 2; and wherein the variable domain that binds to the extracellular portion of HER3 binds to domain III of HER 3.

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 of domain III of HER 3.

12. The method of any one of claims 9 to 11Composition wherein the variable domain that binds to the extracellular portion of HER3 is for binding to HER3 positive SK-BR-3 cells (

HTB-30^TM) Has an affinity (KD) lower than or equal to 2.0nM, preferably between 2.0 and 0.1 nM.

13. The composition of any one of claims 1 to 12, wherein binding of EGFR by said variable domain that binds EGFR blocks binding of EGF to EGFR and/or wherein binding of HER3 by said variable domain that binds HER3 blocks binding of neuregulin-1 (NRG) to HER 3.

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, CDR2 sequence WINANTGDPTYAQGFTG and CDR3 sequence ERFLEWLHFDY, or a variant of the heavy chain variable region comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs.

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 CDR1 sequence SYGMH, CDR2 sequence VISYDGSNKYYADSVKG and CDR3 sequence DYYRRTARAGFDY, or a variant of the heavy chain variable region comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs.

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, CDR2 sequence WINPNSGGTNYAQKFQG and CDR3 sequence DHGSRHFWSYWGFDY, or a variant of the heavy chain variable region comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs.

17. The composition according to any one of claims 1 to 16,

-wherein the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising the CDR1 sequence NYAMN, CDR2 sequence WINANTGDPTYAQGFTG and CDR3 sequence ERFLEWLHFDY, or a variant of the heavy chain variable region comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs;

-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 of the heavy chain variable region comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs; and

-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 of the heavy chain variable region comprising a substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs.

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

19. A composition according to claim 18 for use in the treatment of cancer, preferably gastric, lung or oesophageal cancer.

20. A pharmaceutical composition comprising the composition of 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; wherein 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, said two or more binding moieties being for use in the treatment of cancer, preferably gastric, lung or esophageal cancer.

22. A product comprising two or more binding moieties, wherein each of said binding moieties comprises a variable domain that binds to the extracellular portion of EGFR; and wherein 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 as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer, preferably gastric, lung or esophageal cancer.

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 carrying an EGFR-mutation rendering 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 EGFR R521K polymorphism.

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

26. A method for treating an individual having cancer or at risk of relapse or exacerbation of cancer, the method comprising administering to an individual in need of said treatment a therapeutically effective amount of two or more binding moieties, wherein each of said binding moieties comprises a variable domain that binds to the extracellular portion of EGFR; and wherein 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 HER 3.

27. A method for producing the composition of any one of claims 1-19, the method comprising:

providing a 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 HER 2;

-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 HER 3; and

-a nucleic acid encoding a polypeptide comprising said common light chain;

wherein optionally two or more of the nucleic acids may be physically linked, and wherein each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in the cell; and

culturing the cell to allow expression of the heavy and light chains;

optionally, the step of (a) is carried out,

recovering the two or more binding moieties.

28. The method of claim 27, comprising providing the nucleic acid to a plurality of cells and selecting cells from a pool that carry a desired expression ratio of the heavy and light chains.

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

30. The method of any one of claims 27 to 29, wherein the cell produces a substantially equimolar amount of the two or more binding moieties.

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

32. A cell comprising:

-a nucleic acid encoding a polypeptide comprising said common light chain;

33. A container comprising nucleic acids, 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 HER 2;

-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 HER 3; and

-a nucleic acid encoding a polypeptide comprising said common light chain;

wherein, optionally, two or more of the nucleic acids may be physically linked, and wherein each of the nucleic acids further comprises an expression control sequence to allow expression of the encoded heavy and light chains in a cell.