CN114426578A - Combinations of binding moieties that bind to EGFR, HER2 and HER3 - Google Patents

Abstract

The present invention relates to a combination of binding moieties that bind EGFR, HER2 and HER3, more specifically 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

Combinations of binding moieties that bind to EGFR, HER2 and HER3

This application is a divisional application of a Chinese patent application entitled "Combination of Binding Moieties Binding EGFR, HER2 and HER3" with application number 202080014304.7. The original application is PCT International Application PCT/NL2020 filed on February 13, 2020 /050081 Applications that entered the Chinese national phase on August 13, 2021.

technical field

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 can be used in human therapy. More specifically the present invention relates to compositions comprising two or more multispecific binding moieties, preferably multispecific antibodies. The binding moiety binds EGFR, HER2 and HER3. A single host cell can produce multiple binding moieties.

Background technique

The epidermal growth factor (EGF) receptor (EGFR) is a prototype cell-surface receptor of a member of the epidermal growth factor family (EGF-family) containing extracellular protein ligands. This family currently has four 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 the binding of its specific ligands, including epidermal growth factor and transforming growth factor alpha (TGF[alpha]). Upon activation by its growth factor ligand, the receptor undergoes a transition from an inactive primary monomeric form to an active homodimer. In addition to forming a homodimer upon ligand binding, EGFR can pair with another member of the ErbB receptor family, such as HER2, to produce an activated hetero-dimer. There is also evidence that dimers are formed without ligand binding and that clusters of activated EGFRs are formed after ligand binding.

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

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

While there has been some success with therapies targeting EGFR, most have been associated with the development of treatment resistance over time. One of the ways EGFR-positive tumors can escape therapy targeting this target is through signaling through another receptor dimer. For example, increased signaling due to EGFR/HER3 dimerization due to increased HER3 expression or heregulin expression is associated with drug resistance to EGFR targets, eg, in lung and head and neck cancers. In addition to induction of treatment resistance, certain side effects of EGFR-targeted antibodies have been observed. An 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 treatment cycles and/or premature termination of treatment.

A wide variety of activation modes of signaling of this family of EGF receptors have been identified. Among these are ligand dependent and ligand independent activation of signaling. Even in the absence of HER3 ligand, overexpressed HER2 is able to generate oncogenic signaling through HER2/HER3 heterodimerization (Junttila, Akita et al. 2009). HER2 activity can be inhibited by HER2-specific antibodies. Such HER2-specific antibodies can be used, for example, in the treatment of HER2-positive (HER2 ⁺ ) tumors. The problem with such treatment is that often tumors escape the HER2-specific treatment and continue to grow even in the presence of the inhibitory antibody. It has been observed that HER2 positive tumors, such as breast, ovarian, cervical and gastric tumors, can be expressed by exhibiting up-regulated HER3 expression (Ocana, Vera-Badillo et al. 2013) and/or HER3 ligand expression (Wilson , Fridlyand et al. 2012) selective overgrowth of subpopulations of tumor cells that escape therapy. Also, activating mutations in the HER3 receptor have been identified.

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

SUMMARY OF THE 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 the first of the binding moieties comprises a variable domain that binds to the extracellular portion of HER2, and the second of the binding moieties comprises a variable domain that binds to the extracellular portion of HER3.

Preferably, at least one of the two or more binding moieties is an antibody. In a preferred embodiment, at least two of the two or more binding moieties are antibodies. Such antibodies are preferably multispecific antibodies, preferably bispecific antibodies. Preferably at least one and more preferably at least both 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 facilitate interaction of the heavy chain of the EGFR variable domain with the HER2 and HER3 variable structures Heterodimerization of the corresponding heavy chains of the domains.

The present invention also provides compositions as described herein for use in the treatment of cancer. In embodiments, the cancer is a physical epithelial cancer. Preferably, the composition is used in a cancer expressing EGFR, HER2 and/or HER3. The composition is preferably used for pancreatic cancer, colorectal cancer, head and neck cancer, epithelial ovarian cancer, epithelial fallopian tube cancer, epithelial peritoneal cancer, bladder cancer or prostate cancer. In embodiments, the cancer treated by using the composition is 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 an embodiment, the composition is preferably used for cancers that are gastric, lung, breast or esophageal cancer. 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, said binding moieties each comprising a variable domain that binds to the extracellular portion of EGFR; wherein a first of said binding moieties comprises binding A variable domain to the extracellular portion of HER2, and a second of said binding moieties includes a variable domain that binds to the extracellular portion of HER3, for simultaneous, separate or sequential use in the treatment of cancer Combination preparations.

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

Provides cells including the following

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

- a nucleic acid encoding a polypeptide comprising a heavy chain which, together with the common light chain, forms a variable domain that binds to the extracellular part of HER2;

- a nucleic acid encoding a polypeptide comprising a heavy chain, which together with the common light chain forms a variable domain that binds to the extracellular portion of HER3; and

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

wherein two or more of the nucleic acids may or may not be physically linked, and wherein each of the nucleic acids further includes expression control sequences to allow the encoded heavy and light chains Expression in the cell, and wherein the method further comprises culturing the cell to allow expression of the heavy and light chains, and optionally, collecting the two or more binding moieties.

Further provided is a cell comprising:

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

- a nucleic acid encoding a polypeptide comprising a heavy chain which, together with the common light chain, forms a variable domain that binds to the extracellular part of HER2;

- a nucleic acid encoding a polypeptide comprising a heavy chain, which together with the common light chain forms a variable domain that binds to the extracellular portion of HER3; and

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

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

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

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

- a nucleic acid encoding a polypeptide comprising a heavy chain which, together with a common light chain, forms a variable domain that binds to the extracellular part of HER2;

- a nucleic acid encoding a polypeptide comprising a heavy chain which, together with a common light chain, forms a variable domain that binds to the extracellular portion of HER3; and

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

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

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

The present invention also provides compositions comprising a binding moiety that specifically binds to the extracellular portion of EGFR as well as to the extracellular portion of HER3.

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

Description of drawings

figure 1

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

figure 2

对于生长因子依赖型细胞株BxPC-3-luc2(Perkin Elmer125058)以及N87细胞(NCI-87细胞(

CRL-5822^TM))的增殖的抑制效应。two

For growth factor-dependent cell lines BxPC-3-luc2 (Perkin Elmer125058) and N87 cells (NCI-87 cells (

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

对于BxPC-3-luc2(左侧图片))以及N87(右侧图片))细胞增殖的效用被测试。小组筛选的结果与一由两种单特异性抗体所构成的组合(EGFR结合抗体西妥昔单抗以及HER3单特异性抗体PG3178)或者与EGFRxHER3结合双特异性抗体PB4522来进行比较。所述细胞被生长在饱和量的HRG和EGF的存在下。使用HRG和EGF而未用抗体的相应的细胞的细胞生长的电平(有配体的基础值(basal w/ligand))此外未用HRG和EGF且无抗体的基础电平(无配体(w/oligand))被表明。two

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

Monospecific antibody PG3178 has one IgGl constant region and two variable domains with heavy chain variable regions of MF3178 in Figure 7 or Figure 8 and a common light chain variable region of sequence 11 in Figure 7 .

The bispecific antibody PB4522 has one IgG1 constant region and two variable domains. The HER3 variable domain has the heavy chain variable region of MF3178 in FIG. 7 or FIG. 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 .

image 3

的ADCC活性。一个小组的

的ADCC活性使用N87以及一个CD16/NFAT报道子分析(CD16/NFAT reporter assay)来被测试。此类双特异性抗体具有一个IgG1恒定区以及两个可变结构域。此类可变结构域的重链可变区的氨基酸序列被表明于图7或图8中。此类抗体中的轻链可变区是相同的并且具有图7中的序列11的共同轻链可变区的氨基酸序列。a group

ADCC activity. a group

The ADCC activity of N87 was tested using N87 as well as a CD16/NFAT reporter assay. Such bispecific antibodies have one IgGl constant region and two variable domains. The amino acid sequences of the heavy chain variable regions of such variable domains are shown in FIG. 7 or FIG. 8 . The light chain variable regions in such antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence 11 in FIG. 7 .

Figure 4

的编号与特异性以及它们的ADCC活性。“-”表示无活性被观察到。Various

number and specificity and their ADCC activity. "-" indicates that no activity was observed.

此类双特异性抗体的内码被表明于此类直行双特异性1(Bispecific 1)和双特异性2(Bispecific 2)中。HER2、HER3和EGFR结合结构域的重链可变区被表明于被标示为MF A、MF B和MF C的直行中。MF编号3178和2703组合以该共同轻链形成一个HER3结合可变结构域。MF编号4280、3755、4003、4016组合以该共同轻链形成一个结合EGFR的可变结构域，而MF编号1871、1847、1849和2032组合以该共同轻链形成一个结合HER2的可变结构域。此类双特异性抗体具有一个IgG1恒定区以及两个可变结构域。该等可变结构域的重链可变区的氨基酸序列被表明于图7或图8中。该等抗体中的轻链可变区是相同的并且具有图7中的序列11的共同轻链可变区的氨基酸序。Each row represents a sample containing two bispecific antibodies.

The inner code of such bispecific antibodies is indicated in such straight line Bispecific 1 (Bispecific 1) and Bispecific 2 (Bispecific 2). The heavy chain variable regions of the HER2, HER3 and EGFR binding domains are indicated in the straight line labeled MFA, MFB and MFC. MF numbers 3178 and 2703 combine to form a HER3 binding variable domain with this common light chain. MF numbers 4280, 3755, 4003, 4016 combine to form an EGFR-binding variable domain with the common light chain, while MF numbers 1871, 1847, 1849, and 2032 combine to form a HER2-binding variable domain with the common light chain . Such bispecific antibodies have one IgGl constant region and two variable domains. The amino acid sequences of the heavy chain variable regions of these variable domains are shown in FIG. 7 or FIG. 8 . The light chain variable regions in these antibodies are identical and have the amino acid sequence of the common light chain variable region of sequence 11 in FIG. 7 .

Figure 5

的活体内试验。BxPC-3-luc2或N87细胞在第0天被注射至异种移植物模型的内。包括有双特异性抗体PB4516×PB6892(参见图4)的

或对照组在第1、7、14、21和28天被注射。抗体在25mg/kg的剂量下被腹膜内注射。被描绘的是涉及

(PB4516和PB6892)的结果。载体和西妥昔单抗充当对照组。

in vivo experiments. BxPC-3-luc2 or N87 cells were injected into the xenograft model on day 0. including bispecific antibodies PB4516 x PB6892 (see Figure 4)

Or the control group was injected on days 1, 7, 14, 21 and 28. Antibodies were injected intraperitoneally at a dose of 25 mg/kg. is depicted as involving

(PB4516 and PB6892). Vehicle and cetuximab served as controls.

Image 6

在各种不同的PDX模型中的活体内试验。including bispecific antibodies PB4516 and PB6892

In vivo experiments in various PDX models.

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

Figure 7

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

Figure 8

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

Figure 9

半径内的位于黑色球体中的残基；b)以灰色显示的残基Arg 426以及与Arg 426距离在

半径内的以黑色显示的远距残基；c)呈浅灰色的位于区域Arg 426中的残基以及呈深灰色的周围残基(全部被标记)。说明书附图与分析使用Yasara(www.yasara.org)来被制作。a) Crystal structure of HER3 (PDB#4P59) showing residue Arg 426 located in the grey sphere and at a distance from Arg 426

Residues located in black spheres within the radius; b) residues Arg 426 shown in grey and at a distance from Arg 426

Distant residues shown in black within the radius; c) residues located in region Arg 426 in light grey and surrounding residues in dark grey (all marked). Figures and analyses of the specification were prepared using Yasara (www.yasara.org).

Detailed ways

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

The EGFR-binding variable domain of an antibody or binding portion of the invention preferably binds domain I or domain III of EGFR. The structure of this EGFR protein has been described in Ferguson (2008: AnnuRev Biophys. 2008; 37: 353-373. doi: 10.1146/annurev. biophys. 37.032807.125829), among others. The domains of human EGFR are depicted in Figure 1 of the Ferguson reference data mentioned above. The EGFR-binding variable domains of the embodiments of the invention disclosed herein preferably bind to 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 this gene or protein include: CD340; ErbB-2; HER-2/neu; MLN 19; NEU; NGL; TKR1. The ERBB-2 gene is often referred to as HER2 (derived from human epidermal growth factor receptor 2). When reference is made to HER2 herein, the reference is to human HER2. Antibodies comprising variable domains that bind HER2 bind human HER2. Due to the sequence and tertiary structural similarities between human and other mammalian HER2 orthologs, the HER2 variable domains may also bind such orthologs, but not necessarily. The database accession numbers relating to this 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 a further method for identifying HER2 as a target, the actual sequence of the HER2 protein bound by the antibody may vary, for example, due to mutations within the encoding gene (such as occurs in some those in cancer), and so on. The HER2 variable domains can bind HER2 and its various variants, such as those expressed on certain HER2 positive tumor cells.

The HER2 protein contains several domains (see Figure 1 by reference Landgraf, R Breast Cancer Res. 2007;9(1):202-). The extracellular domains are referred to as domains I-IV. The HER2 binding variable domains of embodiments of the invention disclosed herein preferably bind 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: LCCS2; MDA-BF-1; c-ErbB-3; c-ErbB3; ErbB3-S; pl80-ErbB3; p45-sErbB3; When reference is made to HER3 herein, the reference is to human HER3. Antibodies comprising variable domains that bind HER3 bind human HER3. Due to the sequence and tertiary structural similarities between human and other mammalian HER3 orthologs, it is possible, but not necessarily so, that the HER3 variable domains also bind such orthologs. The database accession numbers relating to this 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 a further method for identifying HER3 as a target, the actual sequence of the HER3 protein bound by an antibody may vary, for example, because of mutations within the encoding gene (such as occurs in some those in cancer), and so on. The HER3 variable domains can bind HER3 and its various variants, such as those expressed on certain HER2 positive tumor cells.

内的氨基酸残基所构成的群组中的氨基酸。在一个优选的实施方案中，可变结构域对于位于SK-BR-3细胞上的HER3的亲和力(KD)低于或等于2.0nM，更加优选地是低于或等于1.5nM，更加优选地是低于或等于1.39nM，更加优选地是低于或等于0.99nM。在一个实施方案中，该亲和力(KD)落在1.39-0.59nM的范围的内。在一个优选的实施方案中，可变结构域对于位于BT-474细胞上的HER3的亲和力(KD)低于或等于2.0nM，更加优选地是低于或等于1.5nM，更加优选地是低于或等于1.0nM更加优选地是低于或等于0.5nM，更加优选地是低于或等于0.31nM，更加优选地是低于或等于0.23nM。在一个实施方案中，该亲和力(KD)落在0.31-0.15nM的范围的内。上面所提及的亲和力优选地是当使用稳定状态细胞亲和力测量术(steady state cell affinity measurements)来被测量的，其中细胞在4℃下使用放射性标记的抗体(radioactively labeled antibody)来被培育，在这之后细胞结合的放射性(cell-bound radioactivity)被测量。The structure of HER3 has been described, among others, in Cho et al. (2002; Science 297, 1330-1333: DOI: 10.1126/science. 1074611). This human protein has 4 extracellular domains. The HER3 binding variable domains of the embodiments of the invention disclosed herein preferably bind domain III of HER3. In a preferred embodiment, the affinity (KD) of a variable domain for a HER3 positive cell is less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.39 nM, more preferably less than or equal to 0.99nM. In a preferred embodiment, an antibody according to the invention preferably comprises at least one variable domain binding HER3 domain III selected from R426 included in the native HER3 protein and located at a distance from R426

Amino acids in the group of amino acid residues within. In a preferred embodiment, the affinity (KD) of the variable domains for HER3 located on SK-BR-3 cells is lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, even more preferably less than or equal to 1.39nM, more preferably less than or equal to 0.99nM. In one embodiment, the affinity (KD) falls within the range of 1.39-0.59 nM. In a preferred embodiment, the affinity (KD) of the variable domains for HER3 located on BT-474 cells is lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, even more preferably lower than or equal to 1.0 nM more preferably less than or equal to 0.5 nM, more preferably less than or equal to 0.31 nM, even more preferably less than or equal to 0.23 nM. In one embodiment, the affinity (KD) falls within the range of 0.31-0.15 nM. The affinities mentioned above are preferably measured using steady state cell affinity measurements, wherein cells are incubated at 4°C with a radioactively labeled antibody, at After this cell-bound radioactivity was measured.

内的氨基酸残基所构成的群组中的氨基酸。优选地，坐落在该天然的HER3蛋白质的中而与R426距离

内的该氨基酸残基选自于由下列所构成的群组：L423、Y424、N425、G427、G452、R453、Y455、E480、R481、L482、D483以及K485(参见举例来说图9以及表1)。氨基酸残基编号为蛋白质数据库(Protein Data Bank，PDB)ID#4P59所具有者。结合HER3的结构域III的这个区域的抗体展现出特别良好的结合特征而且它们能够抵消位于HER3阳性细胞上的HER3的一活性。带有HER3结合特征的可变结构域被描述于WO 2015/130172中，该专利案在此被并入本案以作为参考。在一个优选的实施方案中，根据本发明的双特异性抗体被提供，其中该抗体包括可变结构域至少结合HER3的结构域III的R426。优选地，该抗体包括可变结构域至少结合HER3的结构域III的R426。The variable domain that binds at least one amino acid of domain III of HER3 preferably binds to R426 included in the native HER3 protein and is located at a distance from R426

Amino acids in the group of amino acid residues within. Preferably, 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, eg, Figure 9 and Table 1 ). Amino acid residue numbers are those possessed by Protein Data Bank (PDB) ID#4P59. Antibodies that bind to this region of domain III of HER3 exhibit particularly good binding characteristics and they are able to counteract an activity of HER3 located on HER3 positive cells. Variable domains with HER3 binding characteristics are described in WO 2015/130172, which is hereby incorporated by reference. In a preferred embodiment, a bispecific antibody according to the invention is provided, wherein the antibody comprises a variable domain that binds at least R426 of domain III of HER3. Preferably, the antibody comprises a variable domain that binds at least R426 of domain III of HER3.

In certain embodiments, the composition includes two or more antibodies, wherein each of the antibodies includes a variable domain that binds to the extracellular portion of EGFR; and wherein a first of the antibodies A second of the antibodies includes a variable domain that binds to the extracellular portion of HER2 and that includes a variable domain that binds to the extracellular portion of HER3. 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 variable domains that bind EGFR domain I, and the first antibody comprises variable domains that bind HER2 domain I, and the second antibody comprises variable domains The domain binds domain III of HER3. In another embodiment, the first and second antibodies comprise a variable domain that binds EGFR, domain I, and the first antibody comprises a variable domain that binds HER2, domain IV, and the second antibody comprises a variable domain that binds HER2 The variable domain binds domain III of HER3. In a further embodiment, the first and second antibodies comprise variable domains that bind EGFR, domain III, and the first antibody comprises variable domains that bind HER2, domain I, and the second antibody comprises The variable domains bind domain III of HER3. In a further embodiment, the first and second antibodies comprise variable domains that bind to EGFR, domain III, and the first antibody comprises variable domains that bind HER2, domain IV, and the second antibody comprises The variable domains bind domain III of ER3.

In certain embodiments, a binding moiety is a protein or an aptamer. Binding moieties as described herein typically have two or more binding specificities. The binding moiety preferably comprises two or more variable domains of antibodies. Variable domains can be provided in a variety of different ways. A number of fragments containing antibody variable domains are described in "Nelson 2010: MAbs. 2010 Jan-Feb; 2(1): 77-83" and include various FAB fragments, scFv fragments, and fragments such as VHH fragments of so-called single-domain antibodies. Various different FAB fragments or single-chain Fv fragments are known today. A single domain antibody is an antibody fragment composed 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 ordinary antibodies (150-160kDa) composed of two protein heavy chains and two light chains, and even smaller than Fab fragments (~50kDa, a light chain and half of the heavy chain) and a single chain variable fragment (~25 kDa, two variable domains, one from the light chain and one from the heavy chain). Single domain fragments were first produced from camelid heavy chain antibodies. Similar single-domain fragments can now be made artificially 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. Variable domains are sometimes referred to as _VH / _VL combinations, where _VH represents the variable region of the heavy chain and _VL represents the variable region of the light chain.

The two or more fragments can be ligated to produce binding moieties with a number of binding specificities. Linking is usually accomplished using a linking peptide comprising 2 or more amino acid residues. The linking moiety can also be part or all of a protein. For example, human serum albumin is sometimes used. Binding moieties as described herein preferably comprise at least one variable domain heavy chain variable region with one MF (eg, as described in Figure 7 or Figure 8) paired with a light chain variable region (eg light chain variable region). In a preferred embodiment, the binding moiety comprises two or more such variable domains.

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

The term "antibody" as used herein refers to a proteinaceous molecule belonging to the immunoglobulin class of proteins containing one or more domains that bind an epitope located on an antigen, wherein such domains are derived from a The variable region of an antibody or the sequence homology that shares the variable region of the antibody. Antibodies are usually made up of basic building blocks - each with two heavy chains and two light chains. Antibodies for therapeutic use are preferably native antibodies that are as close as possible to the individual to be treated (eg, human antibodies for human subjects). An antibody according to the present invention is not limited to any particular format or method used to generate 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 (eg, EGFR, HER2 or HER3) may also Identify other compounds if such other compounds contain the same epitope. Thus, the terms "specifically recognizes" or "specifically binds" or terms with the same connotation, with respect to antigen-antibody interaction, do not exclude that such pairs of antibodies contain the same or the same epitope combination of other compounds.

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

Bispecific antibodies are an example of multispecific antibodies. Tri- or more specific antibodies can be made by adding binding moieties, such as scFv fragments, to one or more of the heavy chains. It is also possible to add one or more variable domains to the variable regions of normal or bispecific antibodies. A cell production that produces a common light chain and two non-identical heavy chains (each forming a functional variable domain with the common light chain), one with two non-identical heavy chains, among other things. A bispecific antibody with a chain combined with a light chain. Likewise, a cell that produces a common light chain and three or more different heavy chains can form several bispecific antibodies that together are capable of targeting three or more antigens. It is now possible to create standard versions of antibodies (ie one constant part and two variable domains) and to add further binding domains. As such, multispecific antibodies with one or more single-chain Fvs with additional binding specificities attached to one or more of the constant portion or variable domains of the antibody can be made out. It is also possible to generate heavy chains with two or more variable regions. Additional heavy chain regions may advantageously be associated with non-identical or common light chain variable regions. For a description of such antibodies, see US 62/650467, which is hereby incorporated by reference.

When referring to nucleic acid or amino acid sequences herein, "percent (%) identity" is defined as the difference between residues in a candidate sequence and residues in a selected sequence after aligning the sequences for optimal comparison purposes for the same percentage. The percent sequence identity of the compared nucleic acid sequences was determined using the AlignX application of the Vector NTI Program Advance 10.5.2 software using default values using a modified ClustalW algorithm (Thompson, J.D., Higgins, D.G., and Gibson). T.J. (1994) Nuc. Acid Res. 22:4673-4680), swgapdnarnt score matrix, a gap opening penalty of 15 and a gap extension penalty of 6.66 penalty). Amino acid sequences were aligned using the AlignX application of the Vector NTIProgram Advance 11.5.2 software using default values using a modified ClustalW algorithm (Thompson, J.D., Higgins, D.G., and Gibson T.J., 1994), blosum62mt2 score matrix (blosum62mt2 score matrix), a gap opening penalty of 10 and a gap extension penalty of 0.1.

The term "common light chain" as used herein means light chains such as those that can be used in a multispecific antibody. In a bispecific antibody, the two light chains may be a common light chain (or _VL portion thereof). The two light chains (or _VL portions thereof) may be identical or have some amino acid sequence differences without affecting the binding specificity of the full-length antibody. The terms "common light chain", "common _VL ", "single light chain", "single _VL ", with or without the addition of the term "rearranged", are used interchangeably herein. "Common" means that the light chains have 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) do not affect the formation of a functional binding region. The light chain of the present invention may also be a light chain as specified herein, having from 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. For example, making or finding light chains that are not identical but still functionally equivalent falls within the scope of the definition of a common light chain as used herein, e.g., by introducing and testing conserved amino acid changes, located in Amino acid changes within 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 The binding specificity of the full-length multispecific antibody was not affected. Such a light chain is advantageously also a common light chain as described herein. In a preferred embodiment, all variable domains of a multispecific antibody comprise a common light chain. A common light chain (variable region) for use in the multivalent antibodies of the present invention may be a lambda light chain, which is thus also provided in the context of the present 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 a segment of the IgVK1-39 gene, For example rearranged germline human kappa light chain IgVK1-39*01/IGJK1*01. Exemplary common light chain amino acid sequences are shown in Sequences 10, 11 or 12 of Figure 7.

The term "full-length IgG" or "full-length antibody" according to the present invention is defined to include a substantially intact IgG, but which does not necessarily have all the functions of an intact IgG. For the avoidance of doubt, a full-length IgG contains two heavy chains and two light chains. Each chain contains constant (C) and variable (V) regions, which can be broken down into domains assigned as CH1, CH2, CH3, _VH and _CL , _VL . An IgG antibody binds to antigen via variable region domains included in the Fab portion, and after binding can interact with molecules and cells of the immune system through such constant domains (mostly through the Fc portion) effect. Full-length antibodies according to the present invention encompass IgG molecules in which mutations that provide the desired characteristics may be present. Full-length IgG should not have deletions of substantial portions of any of these regions. However, IgG molecules in which one or several amino acid residues have been deleted without substantially changing 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 a non-CDR region, wherein such deleted amino acid binds the antigen or epitope of the IgG Not required for specificity. Exemplary of IgG antibodies are IgGl, IgG2, IgG3 and IgG4 antibodies. In certain embodiments of the invention, the IgG is IgGl.

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 HER2. 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 HER3.

The two or more binding moieties preferably comprise two or more antibodies (preferably multispecific antibodies) each comprising a variable domain that binds to the extracellular portion of EGFR; and wherein A first of such antibodies includes a variable domain that binds to the extracellular portion of HER2 and a second of such antibodies includes a variable domain that binds to the extracellular portion of HER3. A preferred example of a composition comprising two or more multispecific antibodies is a composition comprising two or more bispecific antibodies. A non-limiting example of a composition comprising two bispecific antibodies as described herein is schematically depicted in FIG. 1 . The 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. An antibody has a heavy chain variable region (6). Another antibody has a heavy chain variable region (7). All heavy chain variable regions can pair with this common light chain (4) to form a functional binding domain. When produced by the same cell, the heavy chain is directed to heterodimerize by the presence of a heterodimerization domain (2,3). The heterodimerization domain has two parts, one part on one heavy chain and a compatible part on the other heavy chain. The heterodimerization domain is often located within the IgG1 CH3 region. Heterodimerization can be directed by providing suitable moieties to the selected heavy chain.

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

A heavy chain having the variable region of the heavy chain together with the light chain to form a variable domain that binds an antigen (such as EGFR, HER2 or HER3) is also referred to herein as an EGFR heavy chain, a HER2 heavy chain, and the like. In a preferred embodiment of the invention, the CH3-regions of the heavy chains of a primary antibody and/or a secondary antibody are engineered to facilitate an EGFR heavy chain with a HER2 heavy chain and an EGFR heavy chain with Heterodimerization of a HER3 heavy chain. In a preferred embodiment, engineering for promoting heterodimerization uses previously described in U.S. Patent Nos. 9,248,182, 9,358,286, 9,248,182, and 9,758,805 DEKK residue positions in No.

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

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 one of the heavy chain variable regions is included in Variants of substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs.

A variable domain that binds to the extracellular portion of HER2 preferably comprises a heavy chain variable region comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGSNKYYADSVKG and a CDR3 sequence DYYRRTARAGFDY, or one of the heavy chain variable regions is included in Variants of substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs.

A variable domain that binds to the extracellular portion of HER3 preferably comprises a heavy chain variable region comprising a CDR1 sequence GYYMH, a CDR2 sequence WINPNSGGTNYAQKFQG and a CDR3 sequence DHGSRHFWSYWGFDY, or one of the heavy chain variable regions is included in Variants of substitution, deletion and/or insertion of 1, 2 or 3 amino acids in the CDRs.

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

In a preferred embodiment, a first and a second of such bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR comprising a CDR1 sequence NYAMN, a CDR2 sequence WINANTGDPTYAQGFTG and a heavy chain variable region of the CDR3 sequence ERFLEWLHFDY, or a variant of the heavy chain variable region comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids in said CDRs; and wherein the second The specific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain preferably comprising a heavy chain variable region comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGSNKYYADSVKG and a CDR3 sequence DYYRRTARAGFDY, or One of the heavy chain variable regions comprises variants with substitutions, deletions and/or insertions of 1, 2 or 3 amino acids in said CDRs; and wherein the second bispecific antibody further comprises a variable domain that binds to The extracellular portion of HER3, the variable domain preferably comprises a heavy chain variable region comprising a CDR1 sequence GYYMH, a CDR2 sequence WINPNSGGTNYAQKFQG and a CDR3 sequence DHGSRHFWSYWGFDY, or one of the heavy chain variable regions is included in the Variants with substitutions, deletions and/or insertions of 1, 2 or 3 amino acids in the CDRs.

In a preferred embodiment, a first and a second of the bispecific antibodies comprise a variable domain that binds to the extracellular portion of EGFR comprising a CDR1 sequence NYAMN, a CDR2 sequence WINANTGDPTYAQGFTG and a heavy chain variable region of the CDR3 sequence ERFLEWLHFDY, or a variant of the heavy chain variable region comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids in said CDRs; and wherein the second The specific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the variable domain preferably comprising a heavy chain variable region comprising a CDR1 sequence SYGMH, a CDR2 sequence VISYDGSNKYYADSVKG and a CDR3 sequence DYYRRTARAGFDY, or One of the heavy chain variable regions comprises variants with substitutions, deletions and/or insertions of 1, 2 or 3 amino acids in the CDRs; and wherein the second bispecific antibody further comprises a variable domain that binds to HER3 The extracellular portion of the variable domain preferably comprises a heavy chain variable region comprising a CDR1 sequence GYYMH, a CDR2 sequence WINPNSGGTNYAQKFQG and a CDR3 sequence DHGSRHFWSYWGFDY, or one of the heavy chain variable regions is included in the CDRs Variants in which 1, 2 or 3 amino acids are substituted, deleted and/or inserted.

Conserved variations of 1, 2 or 3 amino acids from the CDR sequences being detailed are allowed while retaining the same type of binding activity (in type, not necessarily in quantity). Thus, the heavy chain CDRl, 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 CDRl, CDR2 and CDR3 sequences are identical to such detailed CDR sequences.

In certain embodiments, the EGFR variable domain comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the EGFR _VH region illustrated in Figure 7 or Figure 8. Preferably the EGFR _VH region of MF3755 in Figure 7 or Figure 8.

In certain embodiments, the EGFR variable domain comprises a heavy chain variable region comprising an amino acid sequence of at least 90% (preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, still more Preferably at least 99%) identical or 100% identical to an amino acid sequence of the EGFR _VH region illustrated in Figure 7 or Figure 8. Preferably the EGFR _VH region of MF3755 in Figure 7 or Figure 8.

For example, in certain embodiments, the human EGFR-binding heavy chain variable region of the bispecific antibody may have 0-10 sequences in the heavy chain variable region other than the 3 CDR sequences (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. 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 insertions, deletions, substitutions, additions or of these a combination.

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

In some embodiments, the HER2 variable domain comprises a heavy chain variable region comprising the HER2 _VH region illustrated in Figure 7 or Figure 8 (preferably the HER2 _VH of MF2032 in Figure 7 or Figure 8 region) of HCDR1, HCDR2 and HCDR3.

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

For example, in certain embodiments, the human HER2-binding heavy chain variable region of the bispecific antibody may have 0-10 sequences in the heavy chain variable region other than the 3 CDR sequences (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. 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 insertions, deletions, substitutions, additions or of these a combination.

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

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

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

For example, in certain embodiments, the human HER3-binding heavy chain variable region of the bispecific antibody may have 0-10 sequences in the heavy chain variable region other than the 3 CDR sequences (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. 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 insertions, deletions, substitutions, additions or of these a combination.

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 The region comprises an amino acid sequence from an EGFR _VH region selected from Figure 7 or Figure 8 or a variant thereof, preferably the EGFR _VH region of MF3755 of Figure 7 or Figure 8, or it comprises substitution, deletion and/or or insert a variant of 1, 2 or 3 amino acids (not preferably not located in said CDRs); and wherein the second bispecific antibody further comprises a variable domain that binds to the extracellular portion of HER2, the The variable domain comprises a heavy chain variable region comprising an amino acid sequence selected from MF1849 or MF2032 of Figure 7 or Figure 8 or a variant thereof, preferably the amino acid sequence of MF2032 of Figure 7 or Figure 8 or a variant thereof. Variants, wherein the variant comprises substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably not located in said CDRs); and wherein the second bispecific antibody further comprises a binding to HER3 The variable domain of the extracellular portion of the variable domain comprising a heavy chain variable region comprising the amino acid sequence of MF3178 from Figure 7 or Figure 8 or a variant thereof, wherein the variant comprises substitutions, deletions and /or insertion of 1, 2 or 3 amino acids (not preferably not 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, the variable domain comprising a heavy chain variable The region comprises the amino acid sequence of MF3755 from Figure 7 or Figure 8 or a variant thereof comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably not located in said CDRs); and wherein The second bispecific antibody also 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 of MF2032 from Figure 7 or Figure 8 or comprising Substitute, delete and/or insert a variant of 1, 2 or 3 amino acids (not preferably not located in said CDRs); and wherein the second bispecific antibody further comprises a binding to the extracellular portion of HER3 A variable domain comprising a heavy chain variable region comprising the amino acid sequence of MF3178 from Figure 7 or Figure 8 or a variant thereof, wherein the variant comprises substitution, deletion and/or insertion 1, 2 or 3 amino acids (not preferably not in the 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, the variable domain comprising a heavy chain variable region comprising The amino acid sequence of MF4280 from Figure 7 or Figure 8 or a variant thereof comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably not located in said CDRs); and wherein the second The specific antibody also includes a variable domain that binds to the extracellular portion of HER2, the variable domain includes a heavy chain variable region that includes the amino acid sequence of MF1849 from Figure 7 or Figure 8 or that includes substitutions, deletions and/or insertion of 1, 2 or 3 amino acid variants (not preferably not located in said CDRs); and wherein the second bispecific antibody further comprises a variable structure that binds to the extracellular portion of HER3 domain comprising a heavy chain variable region comprising the amino acid sequence of MF3178 from Figure 7 or Figure 8 or comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably no variants located in the 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, the variable domain comprising a heavy chain variable region comprising The amino acid sequence of MF4280 from Figure 7 or Figure 8 or a variant thereof comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably not located in said CDRs); and wherein the second The specific antibody also includes a variable domain that binds to the extracellular portion of HER2, the variable domain includes a heavy chain variable region that includes the amino acid sequence of MF2032 from Figure 7 or Figure 8 or includes substitutions, deletions and/or insertion of 1, 2 or 3 amino acid variants (not preferably not located in said CDRs); and wherein the second bispecific antibody further comprises a variable structure that binds to the extracellular portion of HER3 domain comprising a heavy chain variable region comprising the amino acid sequence of MF3178 from Figure 7 or Figure 8 or comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably no variants located in the 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, the variable domain comprising a heavy chain variable region comprising The amino acid sequence of MF4003 from Figure 7 or Figure 8 or a variant thereof comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs); and wherein the second The specific antibody also includes a variable domain that binds to the extracellular portion of HER2, the variable domain includes a heavy chain variable region that includes the amino acid sequence of MF1849 from Figure 7 or Figure 8 or that includes substitutions, deletions and/or insertion of 1, 2 or 3 amino acid variants (not preferably not located in said CDRs); and wherein the second bispecific antibody further comprises a variable structure that binds to the extracellular portion of HER3 domain comprising a heavy chain variable region comprising the amino acid sequence of MF3178 from Figure 7 or Figure 8 or comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably no variants located in the 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, the variable domain comprising a heavy chain variable region comprising The amino acid sequence of MF4003 from Figure 7 or Figure 8 or a variant thereof comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably not located in said CDRs); and wherein the second The specific antibody also includes a variable domain that binds to the extracellular portion of HER2, the variable domain includes a heavy chain variable region that includes the amino acid sequence of MF2032 from Figure 7 or Figure 8 or includes substitutions, deletions and/or insertion of 1, 2 or 3 amino acid variants (not preferably not located in said CDRs); and wherein the second bispecific antibody further comprises a variable structure that binds to the extracellular portion of HER3 domain comprising a heavy chain variable region comprising the amino acid sequence of MF3178 from Figure 7 or Figure 8 or comprising substitutions, deletions and/or insertions of 1, 2 or 3 amino acids (not preferably no variants located in the CDRs).

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

Additional variants of the disclosed amino acid sequences that retain EGFR, HER2, or HER3 binding can be obtained, for example, from a phage display library (DeKruif) containing rearranged human IGKV1-39/IGKJ1 _VL regions, as previously described. et al. Biotechnol Bioeng. 2010(106) 741-50), and a set consisting of _VH regions incorporating amino acid substitutions into the amino acid sequence of an EGFR, HER2 or HER3 _VH region disclosed herein. Phage encoding Fab regions that bind EGFR, HER2 or HER3 can be selected and analyzed by flow cytometry and sequenced to identify variants with amino acid substitutions, insertions, deletions or additions that retain antigen binding .

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

The present invention further provides a binding moiety that specifically binds an extracellular portion of EGFR and an extracellular portion of HER3. The binding moiety preferably includes an EGFR-binding variable domain and a HER3-binding variable domain. The EGFR-binding variable domain is preferably an EGFR variable domain as described herein. The HER3 binding variable domain is preferably a HER3 variable domain as described herein. Preferably both the EGFR and the HER3 variable 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 HER3 part.

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

The EGFR variable domains, the HER2 variable domains, and the light chain variable domains (VLs) of the HER3 variable domains of the binding moiety, such as the bispecific antibody, may be the same V domains as the parental EGFR monospecific antibody. The _L region, the _VL region of the parental HER2 monospecific antibody and/or the same as the _VL region of the parental HER3 monospecific antibody. Alternative _VL regions can be used in one or more of such _VH / _VL region combinations, so long as such variable domains retain binding to EGFR, HER2 or HER3, respectively.

In certain embodiments, the _VL regions of the EGFR variable domain, the HER2 variable domain, and the HER3 variable domain are similar. In certain embodiments, the _VL regions located in all variable domains of the binding moiety are the same.

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, the light chain variable region of 1, 2, 3 or more variable domains comprises a kappa light chain V-segment IgVκ1-39*01. IgVκ1-39 is the abbreviation for Immunoglobulin Variable κ1-39 Gene. This gene is also known as kappa variable 1-39, IGKV139, IGKV1-39. The external identifiers for this gene are: HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. The amino acid sequence for this V-region is provided in SEQ ID NO: 10 of Figure 7 . The V-regions can also be combined into one of the five J-regions. The preferred J-regions are jk1 and jk5, and the linked sequences are denoted IGKV1-39/jk1 and IGKV1-39/jk5, alternatively IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5 *01 (named after the IMGT database global site at imgt.org). In certain embodiments, the light chain variable region of one or both of the _VH / _VL binding regions comprises a kappa light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ1*05 (respectively is sequence 11 or sequence 12 of Figure 7).

In certain embodiments, the light chain variable region of 1, 2, 3 or more variable domains of the binding moiety of the invention comprises: an LCDR1 comprising the amino acid sequence QSISSY (Fig. 7), an LCDR2 comprising the amino acid sequence AAS and an LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID 9 of Figure 7) (ie such CDRs according to IMGT, IGKV1-39). In certain embodiments, the light chain variable region of one, two, three or more variable domains of a binding moiety of the invention comprises: an LCDR1 comprising the amino acid sequence QSISSY (Figure 7 SEQ ID NO: 7), an LCDR2 comprising the amino acid sequence AASSLQS (SEQ ID NO: 8 of Figure 7), and an LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 9 of Figure 7).

In certain embodiments, 1, 2, 3 or more variable domains of the binding moieties of the invention comprise a light chain variable region comprising an amino acid sequence of at least 90 % (preferably at least 95%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%) identical or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11 in FIG. In certain embodiments, 1, 2, 3 or more variable domains of the binding moieties of the invention comprise a light chain variable region comprising an amino acid sequence of at least 90 % (preferably at least 95%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%) identical or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11 in FIG.

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

In other embodiments, the light chain variable region of 1, 2, 3 or more variable domains of the binding moiety of the invention comprises sequence 11 in FIG. 7 or sequence 11 in FIG. 7 . Amino acid sequence of SEQ ID NO: 12. In certain embodiments, all variable domains of the binding moieties of the invention comprise the same _VL region. In one embodiment, the _VL of the entire variable domain of the binding moiety of the invention comprises the amino acid sequence set forth in SEQ ID NO: 11 in Figure 7 . In one embodiment, the _VL of the entire variable domain of the binding moiety of the invention comprises the amino acid sequence set forth in SEQ ID NO: 12 in Figure 7 or SEQ ID NO: 12 in Figure 7 .

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

In certain embodiments, such multispecific antibodies used in the methods provided herein are generally of the human IgG subtype (eg, eg, IgGl, IgG2, IgG3, IgG4). In certain embodiments, such antibodies are of the human IgGl 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 including a heterodimerization domain. Thus, in certain embodiments, such EGFR/HER2 and EGFR/HER3 bispecific antibodies are full-length IgG molecules. In one embodiment, such EGFR/HER2 and EGFR/HER3 bispecific antibodies are full-length IgGl 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 preferably consists of a human constant region. A constant region or Fc of such a multispecific antibody may contain one or more (preferably no more than 10, preferably no more than 5) amino acid differences with respect to the constant region of a naturally occurring human antibody. For example, in certain embodiments, each Fab-arm of such a bispecific antibody may further comprise an Fc-region comprising modifications that facilitate the formation of the bispecific antibody, affecting Fc-mediated effectors Modifications of function and/or other features described herein.

In preferred embodiments, the multispecific (preferably bispecific) full-length IgG antibody has a lower hinge and/or CH2 domain such that the bispecific IgG antibody interacts with the Fc gamma (Fcy) receptor interaction is enhanced. Antibody-dependent 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 glycated moiety of the antibody. A technique for enhancing ADCC by afucosylation is described, for example, in Junttila, T.T., K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11): 4481-4489). Multispecific antibodies as described herein are preferably afucosylated. Preferably the two multispecific antibodies are afucosylated. Other strategies have been reported to achieve ADCC enhancement, including, for example, glycoengineering (Kyowa Hakko/Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), all of which seek to improve Fc Binding to low-affinity activating FcγRIIIa (low-affinity activating FcγRIIIa) and/or reducing binding to low-affinity inhibitory FcγRIIb (low affinity inhibitory FcγRIIb).

Bispecific antibodies are typically produced by cells expressing the nucleic acid encoding the antibody. 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 composition having the following cells:

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

- a nucleic acid encoding a polypeptide comprising a heavy chain which, together with the common light chain, forms a variable domain that binds to the extracellular part of HER2;

- a nucleic acid encoding a polypeptide comprising a heavy chain, which together with the common light chain forms a variable domain that binds to the extracellular part of HER3; and

- 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 includes an expression control sequence to allow the encoded heavy and light chains to function in the expression in a 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 chains are produced in a cell can be tailored, for example, by selection of appropriate expression control sequences or by selection of the number of nucleic acid copies introduced, or both. In a preferred embodiment, a collection of cells is provided with the nucleic acid, and a clone expressing an appropriate level of such corresponding strands is selected. The colony is also typically selected based on the amount of antibody produced. In one embodiment, the method comprises 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 cells preferably produce substantially equimolar amounts of the two or more binding moieties. In other embodiments, the cell produces more of one of the two or more binding moieties than the other.

The present invention also provides a cell comprising the following:

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

- a nucleic acid encoding a polypeptide comprising a heavy chain which, together with the common light chain, forms a variable domain that binds to the extracellular part of HER2;

- a nucleic acid encoding a polypeptide comprising a heavy chain, which together with the common light chain forms a variable domain that binds to the extracellular part of HER3; and

- 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 includes an expression control sequence to allow the encoded heavy and light chains to function in the expression in cells.

The present invention further 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 that binds to the extracellular part of EGFR;

- a nucleic acid encoding a polypeptide comprising a heavy chain which, together with a common light chain, forms a variable domain that binds to the extracellular part of HER2;

- a nucleic acid encoding a polypeptide comprising a heavy chain that, together with a common light chain, forms a variable domain that binds to the extracellular portion of HER3; and

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

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

The cell from which the binding moiety is produced 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, said binding moiety as described herein, preferably such multispecific antibodies and preferably such bispecific antibodies.

Suitable cells for antibody production are known in the art and include a fusion tumor cell, a Chinese hamster ovary (CHO) cell, an NSO cell, a HEK293 cell, a 293-F cell or a PER-C6 cells. Various institutions and companies have developed cell lines for large-scale production of antibodies (eg, for clinical applications). Non-limiting examples of such cell lines are CHO cells, NSO 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 utilizing the monoglutamine synthase (GS) vector system for expression of an antibody. In a preferred embodiment, the cell is a CHO cell.

In certain embodiments, the cell expresses three different heavy chains and at least one light chain. In a preferred embodiment, the cells express 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 the rearranged human IGKV1 39/IGKJ1 (huVκ139) light chain, the corresponding VH regions used methods known in the art for the generation of bispecific IgG (WO2013/157954, incorporated herein by reference) as reference) into the expression vector. This huVκ1 39 was previously shown to be capable of pairing with more than one heavy chain, thus resulting in antibodies with differing specificities, which facilitated the generation of bispecific molecules (WO2009/157771).

An antibody-producing cell that expresses a common light chain and an equal number of two heavy chains typically produces 50% bispecific antibodies and 25% of each such monospecific antibody (ie, with the same combination of heavy and light chains). Several methods have been published to favor the production of bispecific antibodies over the corresponding monospecific antibodies. This is usually achieved by modifying the constant regions of the heavy chains so that they favor heterodimerization (ie dimerization of the heavy chain combined with another heavy/light chain) over homodimerization Hoodimerization. In a preferred embodiment, the bispecific antibody of the invention comprises 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 of heavy chains can be achieved.

A preferred method for generating multispecific antibodies as described herein is disclosed in US 9,248,181 and US 9,358,286. In particular, preferred mutations to generate substantially only bispecific full-length IgG molecules are the amino acid substitutions L351K and T366K (EU) located in the first CH3 domain (the "KK-variant" heavy chain) numbering) and the amino acid substitutions L351D and L368E in the second domain (the "DE-variant" heavy chain), or vice versa. As previously described, the DE-variant and the KK-variant preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE-variant heavy chains (DEDE homodimers) or homodimerization of KK-variant heavy chains (KKKK homodimers) due to CH3 located between the same heavy chains Strong repulsion between charged residues in the -CH3 interface hardly occurs. Introducing an additional heavy chain with a DE- or KK-variant heavy chain allows the generation of an additional DEKK bispecific molecule. A newly introduced DE ^- heavy chain (DE2) can be linked to the existing KK heavy chain. The cells thus produced two bispecific antibodies, one DE ¹ KK and one DE ² KK diabody. If a new KK heavy chain ( ^KK2 ) is introduced instead of the new DE heavy chain, bispecific antibodies with a combination of ^DEKK1 and ^DEKK2 are 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 usually produced sufficiently to reduce the level of a single heavy chain, while such EGFR chains are usually produced at a level sufficient to allow efficient pairing with such HER2, HER3 chains.

Thus, in one embodiment, a heavy chain/light chain combination comprising an EGFR-binding variable domain comprises a DE variant of the heavy chain. In this embodiment, the heavy chain/light chain combination comprising a HER2 binding variable domain and the heavy chain/light chain combination comprising a HER3 binding variable domain 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 HER3. 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 HER3. Binding of the antibody to transformed cells but not to non-transformed cells indicates specific binding of the antibody. Binding can be assessed eg by flow cytometry (according to the FACS procedure as previously described in WO 2015/130172, PCT/NL2018/050537 and WO 2015/130173). The corresponding monospecific antibodies can be used together as a control, in addition to an unrelated IgG1 isotype control mAb.

Binding moieties, such as antibodies, can be collected from cells and/or supernatants of a cell culture. Typically, they are collected from the supernatant above such producing cells. Binding moieties, such as antibodies, can be purified from this 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 accomplished by protein A extraction. The eluted antibodies can be tested by ELISA for the presence of specific binding properties (ie binding to EGFR, HER2 and HER3). Antibody preparations can be further analyzed by ion exchange column chromatography. Individual bispecific antibodies can be purified from each other by routine techniques, eg, using ion exchange chromatography. The presence of the corresponding bispecific antibodies can also be analyzed by ELISA. Binding of this preparation to HER2 and washing should remove all EGFR/HER3 antibodies. Staining with labeled soluble HER3 did not give a signal, while it did with labeled soluble EGFR. Binding of this preparation to HER3 and washing should remove all EGFR/HER2 antibodies. Staining with labeled soluble HER2 did not give a signal, while it did with labeled soluble EGFR. Binding of this preparation to EGFR and washing should not remove EGFR/HER2 and EGFR/HER3 antibodies. Staining with labeled soluble HER2 in addition to labeled soluble HER3 should provide a signal. Using appropriate controls with known levels of such monobispecific antibodies, levels 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 such bispecific antibodies;

- culturing such cells;

– harvest clarification;

- collecting such bispecific antibodies from cultures; and

- separation of bispecific antibodies and half antibodies produced by ion exchange chromatography (IEX);

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

The affinity of EGFR, HER2 and HER3 FABs of candidate EGFR/HER2 or EGFR/HER bispecific antibodies for their targets can be measured by surface plasmon resonance (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, recombinantly purified antigenic human EGFR-Fc, HER2-Fc and HER3-Fc proteins were run over a range of concentrations on the sensor surface to measure on- and off-rates. After each cycle, the sensor surface was regenerated by a pulse of HCl and the bsAb was captured again. From the obtained sensorgrams, binding and dissociation rates and affinity values for binding to human EGFR, HER2 and HER3 were determined using BIAevaluation software.

The present invention also provides compositions as described herein for the treatment of a cancer. In embodiments, the cancer is a physical epithelial cancer. Preferably, the composition is used in a cancer expressing EGFR, HER2 and/or HER3. The composition is preferably used for pancreatic cancer, colorectal cancer, head and neck cancer, epithelial ovarian cancer, epithelial fallopian tube cancer, epithelial peritoneal cancer, bladder cancer or prostate cancer. In embodiments, the cancer treated by using the composition is 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 an embodiment, the composition is preferably used for cancers that are gastric, lung, breast or esophageal cancer. Preferably, the composition is used for metastatic gastric cancer, metastatic lung cancer, metastatic breast cancer or metastatic esophageal cancer.

The present invention further provides two or more binding moieties for use in the treatment of cancer, said binding moieties each comprising a variable domain that binds to the extracellular portion of EGFR; wherein a first binding moiety of said binding moieties A variable domain that binds to the extracellular portion of HER2 is included, and a second of the binding moieties includes a variable domain that binds to the extracellular portion of HER3. Also provided is a product comprising two or more binding moieties each comprising a variable domain that binds to the extracellular portion of EGFR; wherein a first binding moiety of the binding moieties comprising 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, in the treatment of cancer as simultaneous, separate or Combinations used in sequence.

The cancer to be treated by embodiments of the present invention is preferably a cancer as indicated elsewhere herein. The cancer preferably comprises cells with EGFR-mutations that render the cells resistant to treatment with tyrosine kinase inhibitors (TKIs). In certain embodiments, the cancer comprises cells with EGFR R521K polytype. The cancer to be treated with an inventive method of treatment described herein is preferably gastric, lung or esophageal cancer. In a further embodiment, the present invention provides a method for treating an individual having cancer or at risk for recurrence, progression of cancer, the method comprising administering to the individual in need of such treatment two or more binding moieties each comprising a variable domain that binds to the extracellular portion of EGFR; wherein a first of the binding moieties comprises a variable domain that binds to the extracellular portion of HER2, and The second of the binding moieties includes a variable domain that binds to the extracellular portion of HER3.

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

The terms "treat," "treating," "treatment," as used herein, mean any type of intervention or procedure performed on the individual or administering to the individual a Active agent or a combination of active agents for the purpose of reversing, alleviating, ameliorating, inhibiting or slowing down or preventing the progression, progression, severity or progression of a symptom, complication, condition or biochemical indicator associated with a disease relapse.

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

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, alleviation, reduction, delay and/or alleviation of one or more of the signs, symptoms or etiologies of a disease, or any other desired change in a biological system. In certain embodiments, an effective amount is an amount sufficient to delay tumor development. In certain embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of the drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) to some extent inhibit, delay, slow and can stop the infiltration of cancer cells into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor occurrence and/or recurrence; and/or (vii) alleviate to some extent one or more of the cancer-related symptom. In one example, an "effective amount" is the amount of a composition of the present invention to achieve a reduction in a cancer (eg, a reduction in the number of cancer cells) or slow the progression of a cancer. An effective amount of the combination therapy is administered in an "effective regimen" (which means a combination of the binding moieties as indicated herein) according to such methods described herein, wherein the order of administration and dosage frequency are sufficient to achieve treat.

As used herein, the terms "synergy", "therapeutic synergy" and "synergistic effect" mean a phenomenon in which a patient uses the combination as indicated herein Treatment of a combination of moieties (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 individual components of the combination when used. Results achieved when used alone (see, eg, 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 that is greater than that of each binding moiety by itself at the same dose as it is in the combination The sum of the independent effects; (b) a reduction in dose of one or more agents in the combination without a reduction in therapeutic efficacy; (c) a reduction in the incidence of adverse events while receiving a therapeutic benefit equal to or greater than the monotherapy of each agent at the same dose as it is in the combination; (d) a reduction in dose-limiting toxicity while receiving a treatment that is greater than the monotherapy of each agent Benefit; (e) a delay or minimization of induction of drug resistance.

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

The present invention further provides compositions of the present invention for use in the treatment of cancer. The embodiment used preferably treats - gastric cancer, colorectal cancer, colon cancer, gastroesophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, bright cell sarcoma, salivary gland cancer, Head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, melanoma, such as these. In one embodiment, the embodiment treats a cancer that is gastric, lung or esophageal cancer. This application preferably treats a cancer that 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 the cell membrane. This can be done routinely, and is usually analyzed by immunohistochemistry.

The cancer preferably expresses HER2. The cancer preferably also expresses EGFR or HER3. The cancer preferably expresses EGFR. The cancer preferably also expresses HER2 or HER3. The cancer preferably expresses HER3. The cancer preferably also expresses EGFR or HER2. In certain embodiments, cells of a cancer treated by the invention disclosed herein and/or stromal cells in the cancer express an EGFR ligand, a HER3 ligand, or both. Expression of the ligand and receptor thus provides a growth stimulus to the cancerous cells. A combination of the present invention is particularly suitable for the treatment of cancers comprising such cells.

In the treatment of the present invention, expression of one of EGFR, HER2 and HER3 can delay escape of at least some tumors. A tumor targeted with a monospecific therapy can 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 exist, can also be attacked by the binding moieties of the present invention and thus can be removed before they grow up and diversify themselves. In one embodiment, the cancer is tested for the presence of a mutated EGFR. Many EGFR-positive tumors have genetic mutations that consistently render these cells resistant to treatment with tyrosine kinase inhibitors.

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

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

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

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

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

在如本文所描述的抗体、结合部分、组合物或产物的背景下意指在一制备品中存在多于一种而且通常是10种或更少的不相同抗体或结合部分，包括一双特异性的存在。

的一示范例包括一由两种双特异性抗体所构成的组合。the term

In the context of antibodies, binding moieties, compositions or products as described herein is meant the presence of more than one, and usually 10 or less, different antibodies or binding moieties in a preparation, including a bispecific The presence.

An example 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 HER2; wherein the EFGR variable domain comprises The heavy chain variable region comprises the CDRs of the heavy chain variable region MF3755, MF4280, MF4003 or MF4016 in Figure 8, or one of the heavy chain variable regions comprises substitution, deletion and/or insertion 1, 2 or 3 amino acid variants, and wherein the HER2 variable domain comprises the heavy chain variable region comprising the CDRs of the heavy chain variable region MF2032 or MF1849, or one of the heavy chain variable regions is comprised in the CDRs Variants in which 1, 2 or 3 amino acids are substituted, deleted and/or inserted.

Also provided is a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2; wherein the EFGR variable domain The CDRs comprising the heavy chain variable region comprising the heavy chain variable region MF3755 in Figure 8, or one of the heavy chain variable regions comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids in said CDRs and wherein the HER2 variable domain comprises the CDRs of the heavy chain variable region comprising the heavy chain variable region MF2032, or one of the heavy chain variable regions comprises substitutions, deletions and/or in the CDRs Insertion of 1, 2 or 3 amino acid variants.

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

The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2; wherein the EFGR variable domain comprises The heavy chain variable region includes the amino acid sequence of the heavy chain variable region MF3755 in Figure 8, or the heavy chain variable region includes substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably no be located in the CDRs) variant, and wherein the HER2 variable domain comprises the heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF2032, or the heavy chain variable region comprising substitutions, deletions and/or Or insert variants of 1, 2 or 3 amino acids (not preferably not located in said CDRs).

Also provided is a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2; wherein the EFGR variable domain The CDRs comprising the heavy chain variable region comprising the heavy chain variable region MF3755 in Figure 8, or one of the heavy chain variable regions comprising substitution, deletion and/or insertion of 1, 2 or 3 amino acids in said CDRs and wherein the HER2 variable domain comprises the CDRs of the heavy chain variable region comprising the heavy chain variable region MF1849, or one of the heavy chain variable regions comprises substitutions, deletions and/or in the CDRs Insertion of 1, 2 or 3 amino acid variants.

The invention further provides a binding moiety or bispecific antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of HER2; wherein the EFGR variable domain comprises The heavy chain variable region includes the amino acid sequence of the heavy chain variable region MF3755 in Figure 8, or the heavy chain variable region includes substitution, deletion and/or insertion of 1, 2 or 3 amino acids (not preferably no be located in the CDRs) variant, and wherein the HER2 variable domain comprises the heavy chain variable region comprising the amino acid sequence of the heavy chain variable region MF1849, or the heavy chain variable region comprising substitutions, deletions and/or Or insert variants of 1, 2 or 3 amino acids (not preferably not located in said CDRs).

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

Example

cell line

CRL-5822^TM)、BxPC-3(ATCC CRL-1687)、BxPC-3-luc2以及CHO-K1被维持于补充有10％热失活的胎牛血清(FBS)的生长培养基中。Hek293 cells, NCI-87 cells (

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

Generation of bispecific antibodies

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

A VH gene was transfected into one of two IgG1 vectors of different architectures. Depending on the binding partner, the VH is cloned to an IgG1 framework that includes a CH3 variant with a heterodimerization variant "DE" or a complementary CH3 heterodimerization variant "KK" " in the IgG1 framework. 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 unique heavy chains have the CH3 heterodimerization variant "KK" (also referred to as the KK-heavy chain).

Adherent Hek293 cells were cultured in 6-well culture dishes to a confluency of 80%. The cells were briefly transfected with DNA-FUGENE mixture and further cultured. Seven days after transfection, the supernatant was harvested and the medium was refreshed. Fourteen days after transfection, supernatants were pooled and filtered through (0.22 μM. Sterile supernatants were stored at 4°C. Suspension-conditioned Hek293 cells were cultured in T125 on a shaker plate. culture flasks ( ^T125 flasks) up to a density of 3.0 x 106 cells/mL. Cells were seeded into a 24 deep well plate at a density of 0.3-0.5 x ¹⁰⁶ viable cells/mL Inside each well. The cells were briefly transfected with individual sterile DNA: PE1-MIX and further cultured. Seven days after transfection, the supernatant was harvested and filtered through 0.22 μM. Sterile The supernatant was stored at 4°C.

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

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

Stable cell line clones co-expressing the two bispecific antibodies were generated by plating such pools in semi-solid medium. Plated cells were allowed to grow for 7-10 days. Two rounds of single cell colonization were performed by seeding and selection of a single colony. Oligoclonics are produced from a single cell by fed-batch fermentation.

Determination of antibody titers

Cell supernatants were diluted 1:4 and 1:50 in PBS depending on the total IgG concentration. Single antigen ELISAs were first performed to determine the presence of all 3 antibodies. The following antigens were used at 2.5 μg/mL dilutions 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 ELISAs were developed in succession to assay and quantify the two bispecific molecules, allowing estimation of the ratio between the two bispecifics. For the determination of EGFRxHER2 bispecificity, EGFR-Fc (R&D Systems, 344-ER) antigen was coated on such wells and assayed using ErbB2-Fc (R&D Systems, 1129-ER). For the determination of EGFRxHER3 bispecificity, EGFR-Fc antigen was coated on such wells and assayed using ErbB3-Fc (R&D Systems, 348-RB).

IgG purification

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

Cation Exchange Chromatography (CIEX)

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

TSKgel SP-STAT (7 μm particle size, 4.6 mM I.D x 10 cmL, Tosoh 21964) was equilibrated using buffer A (sodium phosphate buffer, 25 mM, pH 6.0), after which the antibody was subjected to increasing salt concentration and running buffer B (25 mM sodium phosphate, 1 mM NaCl, pH 6.0) was discharged from the column. The flow rate was set at 0.5 mL/min. The injected sample mass was 10 μg and the injection volume was 10-100 μL for all test samples as well as the control group (in PBS). Chromatograms were analyzed based on the 220 nm results for observed peak morphology, retention time, and peak area of major peaks.

Growth inhibition analysis of BxPC-3-luc2 and N87

Antibody compositions were tested at a range of concentrations of total antibody (0. Such antibodies were pooled pairwise according to equal amounts of weight/weight. HRG and EGF at 0.1 ng/mL EGF versus 10 ng/mL for BxPC3-luc2 cells was added to the culture at 0.1 ng/mL of EGF with 1 ng/mL of HRG for N87 cells. Basal w/ligand with ligand was A control group without antibody but with the corresponding growth factor. Basal w/o ligand without ligand is a control group without indicated growth factor and without antibody.

Antibodies were diluted in chemically defined starvation medium (CDS: RPMI 1640 medium containing 80 U of penicillin per milliliter and 80 μg of streptomycin, 0.05% (w/v) BSA and 10 μg/mL) holo-transferrin), and 50 μL of the diluted antibody was added to such wells in a 96-well black-well clear-bottom plate (Costar). Ligand was added (50 μL per well of a stock solution containing 40 ng/mL or 4 ng/mL HRG and 400 ng/mL EGF diluted in CDS: R&D systems, cat.nr. 396-HB and 236- EG). Plates were left at room temperature for 1 hour and then placed in a container in a 37°C cell culture incubator for 3 days (N87 cells) or 4 days (BxPC-3-luc2 cells). On day 4, Alamar blue (Invitrogen, #DAL1100) was added (20 μL/well), and fluorescence was observed in incubations with Alamar blue (at 37°C) for 6 hours (N87 cells) or 4 hours (BxPC-3-luc2 cells) were then measured on a Biotek Synergy2 multifunction microplate analyzer using excitation at 560 nm and readout at 590 nm. Fluorescence values were normalized to uninhibited growth (no antibody, but both ligands added).

的ADCC活性 Various

ADCC activity

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

的一系列稀释以及一比较器抗体(comparator antibody)西妥昔单抗以两次重复来被制备。10μL的这些抗体稀释物被加入至此类井中。没有抗体的对照组井还被包括(基础值)。从此类抗体的起始浓度，5倍系列稀释物被产生，以提供剂量-反应曲线。最后，5μL的ADCC生物分析效应子细胞(ADCC Bioassay effector cells)(15000个细胞/井，V158)被加入。所述细胞在37℃下被培育历时6小时。其次，15μL的BIO-Glo荧光素酶基质被加入，并且在5分钟之后发光在一个盘读取机内被测定。所得到的数据被显示于图3中。西妥昔单抗显示对于BxPC3以及N87细胞的ADCC活性。各种不同的寡株抗体在BxPC3和/或N87细胞上也显示出ADCC活性。This bioassay uses engineered Jurkat cells that either stably express the FcγRIIIa receptor V158 (high affinity) variant, and one that drives the expression of firefly luciferase, which is a measure of FcγR activation NFAT response elements. This assay has been validated by comparing the data obtained using this ADCC reporter bioassay with a conventional ⁵¹ Cr release assay, both assays yielding similar results. Such ADCC assays were performed using the Promega ADCC Bioassay Kit using a 384 white well plate. In this experimental setup, BxPC3 cells and N87 cells were dosed at a density of 1000 cells/well in 30 μL of assay medium (RPMI with 4% low IgG serum) 20-24 hours prior to the assay was plated. The next day, the medium was removed. Second,

Serial dilutions of and a comparator antibody, cetuximab, were prepared in duplicate. 10 μL of these antibody dilutions were added to such wells. Control wells without antibody were also included (basal value). From starting concentrations of such antibodies, 5-fold serial dilutions were generated to provide dose-response curves. Finally, 5 μL of ADCC Bioassay effector cells (15000 cells/well, V158) were added. The cells were incubated at 37°C for 6 hours. Next, 15 [mu]L of BIO-Glo luciferase substrate was added and luminescence was measured in a disc reader after 5 minutes. The resulting data are shown in Figure 3 . Cetuximab showed ADCC activity on BxPC3 as well as N87 cells. Various oligoclonal antibodies also showed ADCC activity on BxPC3 and/or N87 cells.

有关它对于BxPC-3- luc2肿瘤(被原位植入)以及N87肿瘤(被植入于腹脇部内的胃细胞)的生长的作用 Tests include bispecific antibodies PB4516 and PB6892

Regarding its effect on the growth of BxPC-3- luc2 tumors (implanted in situ) and N87 tumors (stomach cells implanted in the flank)

8-10 week old CB17 SCID female mice were orthotopically implanted in the pancreas at the start of the study with ¹ x 106 BxPC-3-luc2 tumor cells in 20 [mu]L. Mice were anesthetized and laid down from the right side to expose the left side, and a 0.5 cm incision was made on the left flank area. The pancreas and spleen were removed from the abdomen, and 1×10 ⁶ tumor cells in 20 μL were injected into the subcapsular space of the pancreatic tail. Bioluminescence (BLI) data were generated one week after implantation. For BLI imaging (once or twice a week) left side view, all mice received 150 mg/kg Luciferin (D-luciferin-EF) 15 minutes before the imaging Potassium salt, Cat. #E6552, Promega) intraperitoneal injections (ipinjections). Outlier animals - based on BLI/tumor volume - were removed and such mice were randomly distributed into groups of 7 mice per group. On day 8 of the experiment, treatment was started.

Animals in the antibody treatment group were dosed weekly with 0.3 mg/kg of antibody for 4 consecutive weeks (Day 0, Day 7, Day 14 and Day 21). On day 0 of this treatment, such animals received twice the loading dose, ie, 0.6 mg/kg of antibody. Final imaging was performed on day 35 or 40. Vehicle-only and cetuximab-treated groups served as controls.

PB4516和PB6892的肿瘤过度生长明显地低于使用西妥昔单抗。西妥昔单抗没有显着地降低N87细胞的过度生长。

显着地减低该模型中的N87肿瘤过度生长(p<0.05)(图5)。Cetuximab and oligoclonic significantly reduced BxPC-3 tumor overgrowth in this model (p<0.05) (Figure 5). use

Tumor overgrowth was significantly lower with PB4516 and PB6892 than with cetuximab. Cetuximab did not significantly reduce N87 cell overgrowth.

N87 tumor overgrowth in this model was significantly reduced (p<0.05) (Figure 5).

N87 tumors :

8-12 week old CB17 SCID female mice were inoculated subcutaneously (sc) in the flank with ¹ x 107 N87 tumor cells in 50% Matrigel at the start of the study. The cell injection volume was 0.2 mL/mouse. Treatment was started when tumors reached an average size of 150-200 ^mm3 . Antibodies were administered weekly for 4 weeks by intraperitoneal injection in mice at 25 mg/kg. Body weight measurements were taken weekly after tumor cell injection and every two weeks from the start to the end of treatment. Tumor growth was monitored biweekly by caliper measurements. The endpoint of the experiment was a tumor volume of 800 ^mm3 or 60 days, whichever came first.

PB11244和PB4516在各种不同的PDX模型中的活性

Activity of PB11244 and PB4516 in various PDX models

的活性在一系列的PDX模型中被评定。在大量数目的癌症模型中测试候选治疗促进了临床性能的预测，并且可以鉴定出用于病患-选择策略的因素(factors for patient-selection strategies)。including bispecific antibodies PB11244 and PB4516

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

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

The HER3 variable domain of PB4516 has the heavy chain variable domain of MF3178 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 HER2 variable domain of PB11244 has the heavy chain variable domain 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 amino acid sequence of the common light chain variable region of SEQ ID NO: 11 (SEQ ID NO: 11) in Figure 7 .

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

被生成与纯化。此类抗体呈一为1：1的比例被混合。

在此类模型中被测试并且与西妥昔单抗和载体(PBS)来做比较。including bispecific antibodies PB4516 and PB11244

generated and purified. Such antibodies were mixed in a 1:1 ratio.

was tested in this model and compared to cetuximab and vehicle (PBS).

Such PDX models were first expanded subcutaneously (sc) 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 tracked by caliper measurements until tumors reached an average size of 100-200 ^mm3 . At this stage (indicated as day 1), animals were randomly assigned to 3 groups per model. Treatment is initiated on the same day and includes:

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

– Cetuximab 25 mg/kg, 5-week dose, ip

– Vehicle (PBS), 5-week dose, intraperitoneal injection

显着地降低模型中的肿瘤细胞的过度生长。过度生长的降低等于西妥昔单抗或者更优选。It can be seen that:

Significantly reduces tumor cell overgrowth in the model. The reduction in overgrowth is equal to cetuximab or more preferably.

半径内的残基 Table 1. Arg 426 that falls in HER3

residues within the radius

sequence listing

<110> MERUS N.V.

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

<140> PCT/NL2020/050081

<141> 2020-02-13

<150> EP 19157302.1

<151> 2019-02-14

<150> EP 19178564.1

<151> 2019-06-05

<160> 51

<170> PatentIn Version 3.5

<210> 1

<211> 124

<212> PRT

<213> Artificial sequences

<220>

<223> HER3-MF3178

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120

<210> 2

<211> 122

<212> PRT

<213> Artificial sequences

<220>

<223> HER2-MF1849

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 3

<211> 120

<212> PRT

<213> Artificial sequences

<220>

<223> EGFR-MF3755

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 4

<211> 125

<212> PRT

<213> Artificial sequences

<220>

<223> EGFR-MF4280

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 5

<211> 119

<212> PRT

<213> Artificial sequences

<220>

<223> EGFR-MF4003

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 6

<211> 122

<212> PRT

<213> Artificial sequences

<220>

<223> HER2-MF2032

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 7

<211> 6

<212> PRT

<213> Artificial sequences

<220>

<223> VL CDR1

<400> 7

Gln Ser Ile Ser Ser Tyr

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial sequences

<220>

<223> VL CDR2

<400> 8

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 9

<211> 7

<212> PRT

<213> Artificial sequences

<220>

<223> VL CDR3

<400> 9

Gln Gln Ser Tyr Ser Thr Pro

1 5

<210> 10

<211> 95

<212> PRT

<213> Artificial sequences

<220>

<223> IgVk1-39*01

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

<210> 11

<211> 107

<212> PRT

<213> Artificial sequences

<220>

<223> Common light chain IgKV1*39/jk1

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 12

<211> 108

<212> PRT

<213> Artificial sequences

<220>

<223> Common light chain IgKV1*39/jk5

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210> 13

<211> 113

<212> PRT

<213> Artificial sequences

<220>

<223> IgG1 CH1

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Pro

<210> 14

<211> 110

<212> PRT

<213> Artificial sequences

<220>

<223> IgG1 CH2

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 15

<211> 107

<212> PRT

<213> Artificial sequences

<220>

<223> IgG1 CH3 KK

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 16

<211> 107

<212> PRT

<213> Artificial sequences

<220>

<223> IgG1 CH3 DE

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 17

<211> 121

<212> PRT

<213> Artificial sequences

<220>

<223> MF1847 HER2

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 121

<212> PRT

<213> Artificial sequences

<220>

<223> MF1871 HER2

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 119

<212> PRT

<213> Artificial sequences

<220>

<223> MF4000 EGFR

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 20

<211> 125

<212> PRT

<213> Artificial sequences

<220>

<223> MF4290 EGFR

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 21

<211> 126

<212> PRT

<213> Artificial sequences

<220>

<223> MF4016 EGFR

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 22

<211> 127

<212> PRT

<213> Artificial sequences

<220>

<223> MF2703 HER3

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 23

<211> 125

<212> PRT

<213> Artificial sequences

<220>

<223> MF2708

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 24

<211> 127

<212> PRT

<213> Artificial sequences

<220>

<223> MF1337 Tetanus Toxoid

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

<210> 25

<211> 30

<212> PRT

<213> Artificial sequences

<220>

<223> MF3755 EGFR FR1

<400> 25

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

1 5 10 15

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

20 25 30

<210> 26

<211> 5

<212> PRT

<213> Artificial sequences

<220>

<223> MF3755 EGFR CDR1

<400> 26

Asn Tyr Ala Met Asn

1 5

<210> 27

<211> 14

<212> PRT

<213> Artificial sequences

<220>

<223> MF3755 EGFR FR2

<400> 27

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

1 5 10

<210> 28

<211> 17

<212> PRT

<213> Artificial sequences

<220>

<223> MF3755 EGFR CDR2

<400> 28

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

1 5 10 15

Gly

<210> 29

<211> 32

<212> PRT

<213> Artificial sequences

<220>

<223> MF3755 EGFR FR3

<400> 29

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

1 5 10 15

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

20 25 30

<210> 30

<211> 11

<212> PRT

<213> Artificial sequences

<220>

<223> MF3755 EGFR CDR3

<400> 30

Glu Arg Phe Leu Glu Trp Leu His Phe Asp Tyr

1 5 10

<210> 31

<211> 11

<212> PRT

<213> Artificial sequences

<220>

<223> MF3755 EGFR FR4

<400> 31

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 32

<211> 30

<212> PRT

<213> Artificial sequences

<220>

<223> MF3178 HER3 FR1

<400> 32

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

1 5 10 15

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

20 25 30

<210> 33

<211> 5

<212> PRT

<213> Artificial sequences

<220>

<223> MF3178 HER3 CDR1

<400> 33

Gly Tyr Tyr Met His

1 5

<210> 34

<211> 14

<212> PRT

<213> Artificial sequences

<220>

<223> MF3178 HER3 FR2

<400> 34

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

1 5 10

<210> 35

<211> 17

<212> PRT

<213> Artificial sequences

<220>

<223> MF3178 HER3 CDR2

<400> 35

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

1 5 10 15

Gly

<210> 36

<211> 32

<212> PRT

<213> Artificial sequences

<220>

<223> MF3178 HER3 FR3

<400> 36

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

1 5 10 15

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

20 25 30

<210> 37

<211> 15

<212> PRT

<213> Artificial sequences

<220>

<223> MF3178 HER3 CDR3

<400> 37

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

1 5 10 15

<210> 38

<211> 30

<212> PRT

<213> Artificial sequences

<220>

<223> MF1849 HER2 FR1

<400> 38

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

1 5 10 15

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

20 25 30

<210> 39

<211> 5

<212> PRT

<213> Artificial sequences

<220>

<223> MF1849 HER2 CDR1

<400> 39

Ser Tyr Gly Met His

1 5

<210> 40

<211> 14

<212> PRT

<213> Artificial sequences

<220>

<223> MF1849 HER2 FR2

<400> 40

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

1 5 10

<210> 41

<211> 17

<212> PRT

<213> Artificial sequences

<220>

<223> MF1849 HER2 CDR2

<400> 41

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

1 5 10 15

Gly

<210> 42

<211> 32

<212> PRT

<213> Artificial sequences

<220>

<223> MF1849 HER2 FR3

<400> 42

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

1 5 10 15

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

20 25 30

<210> 43

<211> 13

<212> PRT

<213> Artificial sequences

<220>

<223> MF1849 HER2 CDR3

<400> 43

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

1 5 10

<210> 44

<211> 13

<212> PRT

<213> Artificial sequences

<220>

<223> MF2032 HER2 CDR3

<400> 44

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

1 5 10

<210> 45

<211> 30

<212> PRT

<213> Artificial sequences

<220>

<223> MF1337 Tetanus toxoid FR1

<400> 45

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

1 5 10 15

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

20 25 30

<210> 46

<211> 5

<212> PRT

<213> Artificial sequences

<220>

<223> MF1337 Tetanus Toxoid CDR1

<400> 46

Lys Tyr Asp Ile Asn

1 5

<210> 47

<211> 14

<212> PRT

<213> Artificial sequences

<220>

<223> MF1337 Tetanus Toxoid FR2

<400> 47

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

1 5 10

<210> 48

<211> 17

<212> PRT

<213> Artificial sequences

<220>

<223> MF1337 Tetanus Toxoid CDR2

<400> 48

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

1 5 10 15

Gly

<210> 49

<211> 32

<212> PRT

<213> Artificial sequences

<220>

<223> MF1337 Tetanus Toxoid FR3

<400> 49

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

1 5 10 15

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

20 25 30

<210> 50

<211> 18

<212> PRT

<213> Artificial sequences

<220>

<223> MF1337 Tetanus Toxoid CDR3

<400> 50

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

1 5 10 15

Asp Val

<210> 51

<211> 11

<212> PRT

<213> Artificial sequences

<220>

<223> MF1337 Tetanus Toxoid FR4

<400> 51

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

Claims (33)

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

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.

2. The composition of claim 1, wherein at least one binding moiety, and preferably at least two binding moieties, of said two or more binding moieties are antibodies.

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 composition of any one of claims 9 to 11, wherein the variable domain that binds to the extracellular portion of HER3 is for binding to HER3 positive SK-BR-3 cells (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 a 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 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.

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 said 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 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 said 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.

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 an extracellular portion of EGFR;

-a nucleic acid encoding a polypeptide comprising a heavy chain capable of pairing with a common light chain to form a variable domain that binds to the extracellular portion of 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.

Images