Bispecific tetravalent antibody targeting EGFR and her3

Abstract

To provide methods for producing and using a bispecific tetravalent antibody targeting EGFR and HER3 for treating cancer patients.SOLUTION: Provided is a bispecific antibody comprising two sets of heavy and light chains, wherein each set of the heavy chain and the light chain forms a Fab region having a binding specificity to EGFR, the antibody further comprising an scFv domain covalently linked to each set of the heavy and light chains at an N-terminus of the heavy chain, an N-terminus of the light chain, or a C-terminus of the light chain, wherein the scFv domain has a binding specificity to HER3, and the antibody comprises an amino acid sequence having at least 98% sequence identity to a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 63 / 237,033, filed August 25, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] Technical Field The present disclosure relates generally to the field of antibody cancer therapy, and more particularly to bispecific tetravalent antibodies. [Background technology]

[0003] background The human epidermal growth factor receptor (EGFR) family, also known as ErbB1 or HER1, has four members: EGFR, HER2, HER3, and HER4. Deregulation of each family member through mutation, amplification, and overexpression plays an important role in tumorigenesis and metastasis. Overexpression is associated with the development of a wide variety of tumors, including, but not limited to, breast cancer, ovarian cancer, gastric cancer, lung adenocarcinoma, aggressive uterine cancer, and salivary duct carcinoma. In breast cancer, HER2 overexpression occurs in 30% of breast cancer patients, and the underlying mutations and amplification of HER2 result in the production of aberrant growth signals that activate downstream signaling pathways, leading to tumorigenesis. Among HER2-negative breast cancer subtypes, EGFR is overexpressed in at least 50% of triple-negative breast cancers (those negative for estrogen receptor and progesterone receptor and HER2 protein). HER3 is overexpressed in approximately 20-30% of invasive breast cancers. HER3 is the only member of this family that is catalytically inactive and requires dimerization with other members to become activated. For example, HER3 can dimerize with HER2 on the surface of tumor cells, activating PI3K / AKT signaling, which promotes tumor growth and survival.

[0004] Interfering with EGFR signaling, either by blocking the EGFR binding site on the receptor's extracellular domain or by inhibiting the activity of intracellular tyrosine kinases, can prevent the growth of EGFR-expressing tumors and improve patient outcomes. Several anti-EGFR antibodies, including cetuximab, panitumumab, and nimotuzumab, have been approved for the treatment of metastatic colorectal cancer, head and neck squamous cell carcinoma, and glioma (Price and Cohen, 2012; Bode et al., 2012). Trastuzumab (Herceptin) and other agents targeting HER2 have antitumor effects in patients with HER2-expressing breast and gastric cancer. However, trastuzumab is only effective in cancers in which HER2 is overexpressed. Many tumors that initially respond to these therapeutic agents eventually progress due to acquired resistance to the agents, and some patients appear to see limited long-term benefit. In the case of HER2-targeted therapy, resistance can arise through upregulation of HER3 or its ligand, HRG. Moreover, current therapeutic approaches aimed at inhibiting activation of the HER2 / HER3 signaling pathway have failed to provide meaningful clinical benefit (Geuijen et al. 2018; Yu et al. 2019). The present disclosure relates to methods for making and using bispecific tetravalent antibodies targeting EGFR and HER3 to treat cancer patients. Summary of the Invention

[0005] overview The following summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the detailed description that follows.

[0006] The present disclosure provides bispecific tetravalent antibodies that target two members of the EGFR family, i.e., EGFR and HER3, and methods for making and using these antibodies. The bispecific tetravalent antibodies may comprise an immunoglobulin G (IgG) portion having two heavy chains and two light chains, and two scFv portions covalently linked to either the N-terminus of the heavy chain or the N-terminus or C-terminus of the light chain. The IgG portion may have binding specificity for a first member of the EGFR family. The scFv portion may have binding specificity for a second member of the EGFR family. The IgG portion and the two scFv portions are covalently linked to function as a bispecific antibody. Objects and advantages of the present disclosure will become apparent from the following detailed description of preferred embodiments thereof, taken in conjunction with the accompanying drawings.

[0007] In one aspect, the present application provides a bispecific antibody comprising two sets of heavy and light chains. Each set of heavy and light chains forms a Fab region with binding specificity for EGFR. The antibody may further comprise an scFv domain covalently linked to the N-terminus of the heavy chain, the N-terminus of the light chain, or the C-terminus of the light chain. The scFv domain has binding specificity for HER3. In one embodiment, the bispecific antibody comprises an IgG domain. In one embodiment, the bispecific antibody comprises an IgG1 domain.

[0008] In one embodiment, the scFv domain may be linked to the N-terminus of the heavy chain, hi one embodiment, the scFv domain may be linked to the N-terminus or C-terminus of the light chain.

[0009] In one embodiment, the scFv domain is linked to the N-terminus or C-terminus of the light chain, wherein the light chain comprises an amino acid sequence having sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0010] In one embodiment, the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO: 24.

[0011] In one embodiment, the scFv domain is linked to the N-terminus of a heavy chain, wherein the heavy chain comprises an amino acid sequence having sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.

[0012] In one embodiment, the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:22.

[0013] In one embodiment, the heavy chain may comprise three complementarity determining regions (CDRs) having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33. In one embodiment, the heavy chain may comprise three CDRs having the amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39.

[0014] In one embodiment, the light chain may comprise three CDRs having the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. In one embodiment, the light chain may comprise three CDRs having the amino acid sequences of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.

[0015] In one embodiment, the antibody may comprise an IgG constant region, wherein the IgG constant region comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 19.

[0016] In one embodiment, the antibody may comprise a kappa constant region, wherein the kappa constant region comprises an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:20.

[0017] In one embodiment, the scFv domain may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

[0018] In one embodiment, the scFv domain comprises a variable light chain (V L ), where V L has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. L comprises CDRs having amino acid sequences SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48.

[0019] In one embodiment, the scFv domain comprises a variable heavy chain (V H ), wherein VH has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16. H comprises CDRs having amino acid sequences SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45.

[0020] In one embodiment, the scFv domain comprises, from the N-terminus to the C-terminus, V L V H or V H V L In one embodiment, the scFv may have the configuration L and V HIn one embodiment, the disulfide bond may be between vL100 and vH44 (Kabat) of the scFv domain. In one embodiment, the scFv may include the R19S (Kabat) mutation.

[0021] In one embodiment, the scFv domain comprises a VL having an amino acid sequence having sequence identity to SEQ ID NO: 11 and a VL having an amino acid sequence having sequence identity to SEQ ID NO: 12. H In one embodiment, the scFv comprises a V having an amino acid sequence having sequence identity to SEQ ID NO: 13. L and V having an amino acid sequence having sequence identity to SEQ ID NO: 14 H In another embodiment, the scFv comprises a V having an amino acid sequence having sequence identity to SEQ ID NO: 15. L and V having an amino acid sequence having sequence identity to SEQ ID NO: 16 H Includes.

[0022] In one embodiment, the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 18, and the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 17.

[0023] In one embodiment, the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 22, and the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 21.

[0024] In one embodiment, the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 18, and the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 23.

[0025] In one embodiment, the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:25, and the antibody may comprise an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:24.

[0026] In another aspect, the application provides an isolated nucleic acid encoding a bispecific antibody disclosed herein.

[0027] In a further aspect, the present application provides an expression vector comprising an isolated nucleic acid encoding a bispecific antibody disclosed herein. In one embodiment, the expression vector may be expressible intracellularly.

[0028] In a further aspect, the present application provides a host cell comprising a nucleic acid disclosed herein.

[0029] In a further aspect, the present application provides a method of producing a bispecific antibody disclosed herein, the method comprising culturing a host cell disclosed herein such that the bispecific antibody is produced.

[0030] In a further aspect, the present application provides an immunoconjugate comprising the bispecific antibody and a cytotoxic agent, wherein the cytotoxic agent comprises a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioisotope.

[0031] In a further aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may comprise a radioisotope, a radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof. In one embodiment, the pharmaceutical composition may comprise an immunoconjugate and a pharmaceutically acceptable carrier.

[0032] In a further aspect, the present application provides a method of treating a subject suffering from cancer. In one embodiment, the method may comprise administering an effective amount of a bispecific antibody to a subject. In one embodiment, the cancer may comprise cells expressing EGFR, HER3, or both. In one embodiment, the cancer may comprise breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small lung cell cancer, small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, kidney cancer, gastric cancer, liver cancer, bladder cancer, cervical cancer, brain cancer, lymphoma, leukemia, or myeloma.

[0033] In one embodiment, the method further comprises co-administering an effective amount of a therapeutic agent.

[0034] In one embodiment, the therapeutic agent may include an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. In one embodiment, the therapeutic agent may include capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, derivatives or combinations thereof.

[0035] In one embodiment, the subject is a human.

[0036] In a further aspect, the present application provides a solution comprising an effective concentration of the bispecific antibody. In one embodiment, the solution is plasma of a subject. [Brief explanation of the drawings]

[0037] The above and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, in which: The present disclosure may be described with additional specificity and detail through the use of the accompanying drawings, with the understanding that these drawings illustrate only some embodiments arranged in accordance with the present disclosure and, therefore, should not be considered as limiting the scope of the disclosure. [Figure 1] FIG. 1 shows the configuration of a bispecific tetravalent antibody targeting EGFR and HER3, i.e., an EGFRxHER3 bispecific antibody. [Figure 2] FIG. 2 shows the thermal stability data of the EGFRxHER3 bispecific antibody as measured by dynamic light scattering. [Figure 3] FIG. 3 shows biolayer interferometry sensorgrams for the binding of EGFR×HER3 bispecific antibodies to human EGFR. [Figure 4] FIG. 4 shows biolayer interferometry sensorgrams for the binding of EGFR×HER3 bispecific antibodies to human HER3. [Figure 5] FIG. 5 shows the effect of EGFR×HER3 bispecific antibodies on the growth of FaDu tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description The present disclosure provides bispecific tetravalent antibodies with superior therapeutic properties or therapeutic efficacy compared to currently known anti-EGFR antibodies. In one embodiment, the antibodies target members of the EGFR family, including, but not limited to, EGFR and HER3. These bispecific tetravalent antibodies simultaneously inhibit different receptor-mediated oncogenic signaling pathways and may therefore overcome resistance to EGFR inhibitor therapy or monoclonal antibody therapy.

[0039] The present disclosure provides, inter alia, isolated antibodies or antigen-binding fragments; humanized antibodies or antigen-binding fragments; methods for making such antibodies or antigen-binding fragments; monoclonal and / or recombinant, monospecific antibodies, multispecific antibodies; antibody-drug conjugates and / or immunoconjugates composed of such antibodies or antigen-binding fragments; pharmaceutical compositions comprising antibodies, monoclonal and / or recombinant, monospecific antibodies, multispecific antibodies, antibody-drug conjugates and / or immunoconjugates; methods for making the antibodies and compositions; and methods for treating cancer using the antibodies and compositions disclosed herein. Specifically, the present disclosure provides a group of bispecific tetravalent antibodies having binding specificities for human EGFR and HER3, also known as EGFRxHER3 bispecific antibodies (Figure 1), wherein the isolated antibodies comprise an amino acid sequence having identity to a sequence selected from SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.

[0040] The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, and antibody fragments (e.g., Fab, F(ab'), and Fv), so long as they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal, polyclonal, chimeric, single-chain, multispecific, or multipotency antibodies, human, and humanized antibodies, and active fragments thereof. Examples of active fragments of known antigen-binding molecules include Fab, F(ab'), scFv, and Fv fragments, as well as the products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the foregoing antibodies and fragments.

[0041] The term "Fv" refers to the minimum antibody fragment containing one intact site that recognizes and binds antigen. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, these six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) is capable of recognizing and binding antigen, although with lower affinity than the complete binding site.

[0042] In some embodiments, antibodies can include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a binding site and immunospecifically bind to an antigen. A typical antibody refers to a heterotetrameric protein that typically contains two heavy (H) chains and two light (L) chains. Each heavy chain is composed of a heavy-chain variable domain (abbreviated as VH) and a heavy-chain constant domain. Each light chain is composed of a light-chain variable domain (abbreviated as VL) and a light-chain constant domain. The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. The VH and VL regions can be further subdivided into hypervariable complementarity-determining regions (CDRs) and highly conserved regions called framework regions (FRs). Each variable domain (either VH or VL) typically consists of three CDRs and four FRs arranged in the following order: That is, from the amino terminus to the carboxy terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Within the variable regions of the light and heavy chains are binding regions that interact with an antigen.

[0043] Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4, and IgA-1 and IgA-2. The heavy chain constant domains corresponding to these different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0044] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567). By "recombinant" is meant that the antibodies are produced using recombinant nucleic acid techniques in exogenous host cells.

[0045] Monoclonal antibodies can be produced using a variety of methods, including but not limited to, murine hybridomas, phage display, recombinant DNA, molecular cloning of antibodies directly from primary B cells, and antibody discovery methods (see Siegel. Transfus. Clin. Biol. 2002; Tiller. New Biotechnol. 2011; Seeber et al. PLOS One. 2014). Monoclonal antibodies may include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0046] As used herein, the term "multispecific, multivalent" antibody refers to an antibody having at least two binding sites, each having binding affinity for an epitope of an antigen. As used herein, the term "bispecific, tetravalent antibody" refers to an antibody having four antigen-binding sites specific for two different antigens. For example, the antibody disclosed herein is bispecific, tetravalent for EGFR and HER3.

[0047] The term "humanized antibody" refers to a type of engineered antibody whose CDRs are derived from a non-human donor immunoglobulin, and the remaining immunoglobulin-derived portions of the molecule are derived from one or more human immunoglobulins. Additionally, framework support residues may be modified to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (See, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989); Hodgson et al., Bio / Technology, 9:421 (1991)).

[0048] The terms "antigen- or epitope-binding portion or fragment," "variable domain," "variable region," "variable region sequence," or "binding domain" refer to a fragment of an antibody that is capable of binding to an antigen (such as EGFR and HER3 in the present application). An antigen-binding fragment (Fab) is the region of an antibody (the Fab region) that binds to an antigen. These fragments may retain the antigen-binding function of an intact antibody, as well as other functions. Examples of binding fragments include, but are not limited to, a single-chain Fv fragment (scFv) consisting of the variable light (VL) and variable heavy (VH) domains of a single arm of an antibody linked in the form of a single polypeptide chain by a synthetic linker, or a Fab fragment, which is a monovalent fragment consisting of the VL, constant light (CL), VH, and constant heavy 1 (CH1) domains.

[0049] Antibody fragments can be even smaller fragments and can consist of as little as a single CDR domain, the CDR3 region from either a VL domain and / or a VH domain (see, e.g., Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are produced using conventional methods known to those skilled in the art. Antibody fragments can be screened for utility using the same techniques as are used for intact antibodies.

[0050] "Antigen- or epitope-binding portions or fragments," "variable regions," "variable region sequences," or "binding domains" can be derived from the antibodies of the present application by several techniques known in the art. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments (each with a single antigen-binding site), and a residual "Fc" fragment (the name reflects its ability to readily crystallize). Pepsin treatment generates an F(ab')2 fragment, which has two antigen-binding sites and still has the ability to cross-link antigen. Appropriate fractions containing the Fab fragments can then be collected and concentrated, for example, by membrane filtration. For further description of general techniques for isolating active fragments of antibodies, see, e.g., Khaw, BA et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986).

[0051] The terms "isolated" or "purified" refer to a biological molecule that is free from at least some of the components with which it is naturally associated. Either "isolated" or "purified," when used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, a purified polypeptide is prepared by at least one purification step. An "isolated" or "purified" antibody refers to an antibody that is substantially free of other antibodies with different antigen-binding specificities.

[0052] The terms "a," "an," and "the," as used herein, are defined to mean "one or more," and include plurals unless the context is inappropriate.

[0053] The terms "polypeptide," "peptide," and "protein," as used herein, are synonymous and are defined to mean a biomolecule composed of amino acids linked by peptide bonds.

[0054] The term "antigen" means an entity or fragment thereof that is capable of inducing an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions thereof that are responsible for antigenicity or antigenic determinants.

[0055] The term "immunogenic" refers to a substance that induces or promotes the production of antibodies, T cells, or other reactive immune cells directed against the immunogenic substance, contributing to an immune response in a human or animal. An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells to an administered immunogenic composition of the present disclosure to alleviate or ameliorate the disorder being treated. An immunogenic response typically involves both the cellular (T cell) and humoral (antibody) arms of the immune response, and antibodies directed against a therapeutic protein (anti-drug antibodies, ADA) can be of the IgM, IgG, IgE, and / or IgA isotypes.

[0056] The terms "specific binding," "binds specifically," or "specific for a particular antigen or epitope" mean that binding is measurably different from nonspecific interactions. Specific binding can be measured, for example, by measuring binding of a molecule relative to binding of a control molecule, which is typically a structurally similar molecule that has no binding activity. For example, specific binding can be measured based on competition with a control molecule that is similar to the target.

[0057] The term "affinity" refers to the measure of the attractive force between two polypeptides, such as antibody / antigen, receptor / ligand, etc. The inherent attractive force between two polypeptides can be expressed as the binding affinity equilibrium dissociation constant (KD) for each interaction. The KD binding affinity constant can be measured, for example, by biolayer interferometry, where KD is the ratio of kD (dissociation rate constant) to kD (association rate constant), KD = kD / kD.

[0058] Specific binding to a particular antigen or epitope is, for example, a KD for the antigen or epitope of at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 The KD of an antibody can be represented by an M or greater, where KD refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a KD relative to a control molecule that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or greater than that of the antigen or epitope.

[0059] Specific binding to a particular antigen or epitope can also be exhibited, for example, by an antibody having a K A or K A for the antigen or epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or greater for the epitope compared to a control, where K A or K A refers to the on-rate for a particular antibody-antigen interaction.

[0060] The present disclosure may be understood more readily by reference to the following detailed description of specific embodiments and examples contained herein. Although the present disclosure has been described with reference to specific details of particular embodiments thereof, it is not intended that such details should be considered limitations on the scope of the disclosure. [Example]

[0061] Example 1: EGFRxHER3 Bispecific Antibody Configurations Cancer-associated gain-of-function mutations alter the HER3 kinase domain, ultimately enhancing allosteric function, providing a structural and mechanistic basis for developing drugs targeting EGFR / HER3 dimerization. Inhibition of EGFR / HER3 signaling can be achieved by using either small molecule drugs or monoclonal antibodies against members of the EGFR family. For example, cetuximab and nimotuzumab are both anti-EGFR antibodies that have proven therapeutically effective in clinical trials. Binding fragments derived from these antibodies can be referred to as therapeutic binding domains because their anti-tumor growth activity has been demonstrated in clinical trials. While progress has been made in combination therapies involving the use of two therapeutic antibodies, there is a need to develop a single, effective bispecific antibody for inhibiting EGFR / HER3 dimerization. Consideration should be given to the precise geometry of a bispecific antibody (i.e., the spacing and relative configuration of the two sets of binding domains) which can significantly affect the properties of the therapeutic agent, for example, in terms of expression potency, stability, antigen binding, or efficacy in inhibiting proliferation or affecting another biological function.

[0062] Figure 1 shows the configurations of six bispecific tetravalent antibodies. Each of these EGFRxHER3 bispecific antibodies comprises an immunoglobulin G (IgG) moiety with two heavy chains and two light chains, and two scFv binding domains covalently linked to the two designated ends of the antibody via linkers such as (Gly-Gly-Gly-Gly-Ser)n linkers, (Gly-Gly-Gly-Ser)n linkers, or (GmS)n linkers. Of this panel of EGFRxHER3 bispecific antibodies, SI-1X6 and SI-1X4 were shown to be characterized by having an anti-EGFR Fab region and an anti-HER3 scFv domain linked to the C-terminus of the heavy chain (HC) (WO2016106157A1, incorporated herein by reference in its entirety). These two antibodies share the same configuration, in which the two binding domains are separated at the two ends of the HC, with at least CH1, CH2, and CH3 present between them. In comparison, SI-1X22, SI-1X24, SI-1X25, and SI-1X26 are constructed with an anti-HER3 scFv domain linked to either end of the LC or to the N-terminus of the HC. As a result, the space between the two binding domains is reduced to CH1 when the scFv domain is linked to the C-terminus of the LC (SI-1X22 and SI-1X26) or to nothing when the scFv domain is linked to the N-terminus of either the HC or LC (SI-1X24 and SI-1X25).

[0063] Although each domain may exert independent binding specificity, maintaining the two binding domains in closer physical proximity may improve the efficiency of antibody binding to both EGFR and HER3 on the same tumor cells. For example, the proximity of the binding domains may result in a more rigid conformation, in which steric constraints prevent the domains from rearranging in a manner that would allow EGFR and HER3 dimerization. In contrast, a long physical distance between the domains and a flexible region between the binding domains may allow conformational flexibility, which may lead to undesirable receptor dimerization and downstream proliferative signaling. The R19S (Kabat) mutation in the VH of the scFv on the light chain (WO2021092266A1, incorporated herein by reference in its entirety) was used to prevent the light chain component from binding to Protein A during purification. When the anti-HER3 scFv domain was fused to the light chain, the VH / VL pairing within the Fab was stabilized by a disulfide staple (VH 44C / VL 100C, Kabat).

[0064] Example 2: Generation of EGFRxHER3 bispecific antibodies SI-1X22, SI-1X24, SI-1X25, and SI-1X26 were cloned and purified. Genes encoding the antibody heavy and light chains (preceded by Kozak and secretory signal peptides) were cloned into the pTT5 vector using standard molecular biology techniques. Antibodies were expressed by transiently transfecting heavy and light chain expression plasmids in the ExpiCHO system (Thermo Fisher). Briefly, 10 μg of each expression plasmid was added to 1 ml of OptiPRO SFM medium. 1 ml of OptiPRO SFM medium containing 80 μl of Expifectamine CHO reagent was added to the DNA and incubated at room temperature for 2.5 minutes. The resulting mixture was then transferred to 6 × 10 ribosomal RNAi strains in a 125 ml Erlenmeyer flask. 6The cells were added to 25 ml of ExpiCHO cells at 1000 cells / ml and incubated at 37°C, 5% CO2, and 150 rpm. At 24 hours post-transfection, the cells were fed with 8.75 ml of ExpiCHO feed and 150 μl of CHO enhancer and shifted to 32°C, 5% CO2, and 150 rpm. At 48 hours post-transfection, the cells were again fed with 8.75 ml of ExpiCHO feed. Culture supernatants were harvested 9 days post-transfection and centrifuged at 4500 rpm for 1 hour to pellet the cells, then passed through a 0.2 mm filter. Expression titers were quantified using biolayer interferometry on an Octet384 system with a Protein A sensor, and a calibration curve was generated using the purified bispecific antibody protein.

[0065] Protein was purified from the collected supernatant using a 1 ml MabSelect PrismA Protein A column (GE Healthcare). The column was equilibrated with phosphate-buffered saline. The supernatant was then passed through the column at a flow rate of 1 ml / min. The column was washed with 10 ml of PBS. The protein was then eluted by passing 5 ml of 50 mM sodium acetate, pH 3.5 through the column. The eluted protein was immediately neutralized by adding 0.5 ml of 1 M Tris-Cl, pH 8.0.

[0066] Immediately after the first step of Protein A or His-tag purification, proteins were analyzed by analytical SEC using a Waters Acquity UPLC H-Class with an ACQUITY UPLC® Protein BEH SEC 200Å, 4.6 mm x 150 mm, 1.7 μm column. PBS (125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8) was used as the mobile phase, with 10 μg of protein injected and run at 0.3 ml / min for 10 minutes. Proteins were further purified by preparative SEC using a Superdex Increase 10 / 300 GL column in a mobile phase of 25 mM sodium acetate, 125 mM NaCl, pH 5.5, and finally buffer-exchanged to 25 mM sodium acetate, 125 mM NaCl, 10% sucrose, pH 5.5. The final samples contained >95% of the protein of interest (POI) as assessed by analytical SEC and were used for subsequent assays.

[0067] Example 3: Protein Stability Protein stability is an important parameter determined by the difference in free energy between the folded and unfolded states. In protein therapeutics, stability can affect immunogenicity, pharmacokinetics, and even efficacy, and reducing aggregation can help develop therapeutics that are easier to manufacture and safer for patients. Furthermore, expression efficiency and protein yield directly determine the cost of protein therapeutics. If proteins can be expressed more efficiently to achieve higher titers and increased yields of purified protein, production costs can be significantly reduced.

[0068] After transient expression in ExpiCHO cells, the titers of the bispecific antibodies were quantified using biolayer interferometry. As shown in Table 1, the data demonstrate that all proteins were expressed in the ExpiCHO expression system, indicating that they were sufficiently stable and efficiently produced. For antibodies containing nimotuzumab variable regions (SI-1X4 and SI-1X26), the titers were comparable. For antibodies containing cetuximab variable regions (SI-1X6, SI-1X22, SI-1X24, and SI-1X25), the titers were higher than those of the nimotuzumab-based antibodies, with the highest being SI-1X24, which contains an anti-HER3 scFv at the N-terminus of the cetuximab heavy chain.

[0069] Another parameter related to protein stability is the amount of aggregation after the first step of affinity purification. Antibodies with higher stability tend to aggregate less and therefore have a higher %POI (percentage of protein of interest) by analytical size-exclusion chromatography. After Protein A purification, the bispecific antibodies were analyzed by analytical SEC to check for aggregation (see Table 1). Among the antibodies containing the nimotuzumab variable region (SI-1X4 and SI-1X26), SI-1X4, which contains an anti-HER3 scFv at the C-terminus of the nimotuzumab heavy chain, had significantly less aggregation (and therefore a higher %POI). For the cetuximab-based antibodies (SI-1X6, SI-1X22, SI-1X24, and SI-1X25), SI-1X24 (containing an anti-HER3 scFv at the N-terminus of the heavy chain), which had the highest %POI after purification, exhibited the least aggregation.

[0070] Example 4: Thermal Stability Thermal stability is another parameter for assessing the quality of any antibody. Dynamic light scattering was used to compare the thermal stability of EGFR x HER3 bispecific antibodies. In the thermal stability experiment, the radius of the protein (1 mg / ml) was monitored using a Wyatt DynaPro Plate Reader III while the temperature was increased from 25 to 85 °C at 0.5 °C / min. As shown in Figure 2, an increase in particle size suggests protein aggregation or other unfolding events. As an objective measure of thermal stability, the temperature at which the radius exceeded 10 nm was tabulated (Table 1). Among the cetuximab-based antibodies (SI-1X6, SI-1X22, SI-1X24, and SI-1X25), SI-1X24 was the most stable in the assay, with a Tm of 64.75 °C. The other three antibodies in the family (SI-1X6, SI-1X22, and SI-1X25) had similar Tms, ranging from 62 to 63 °C. Therefore, the position of the anti-HER3 scFv in cetuximab-based bispecific antibodies can cause significant differences in thermal stability. As for the nimotuzumab-based antibodies (SI-1X4, SI-1X26), both antibodies unfolded at approximately 61.5°C, indicating that these two molecules have similar thermal stabilities.

[0071] Example 5: Octet conjugation The Sartorius Octet platform applies biolayer interferometry (BLI) as a label-free technique for measuring protein-protein interactions. This is an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized proteins on a biosensor chip and an internal reference layer. Any change in the number of molecules bound to the biosensor chip causes a change in the interference pattern that can be measured in real time. In this method, binding of an antigen in solution to an antibody / Fc-containing protein immobilized on the surface of an anti-human IgG Fc capture (AHC) biosensor chip increases the optical thickness at the biosensor chip, resulting in a wavelength shift Δλ. This wavelength shift Δλ directly reflects changes in the thickness of the biological layer. The interaction of these two molecules is measured in real time, allowing for precise and accurate monitoring of binding specificity, binding and dissociation rates, or concentrations. Unbound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate do not affect the interference pattern.

[0072] Biolayer interferometry (Octet) binding assays were performed on an Octet384 instrument to quantify the binding kinetics of bispecific antibodies against EGFR and HER3. Antibodies were captured onto an anti-human Fc (AHC) sensor chip by loading at 5 μg / ml for 180 seconds. After a 60-second baseline step, a 180-second binding phase was performed using serial dilutions (0–100 nM; 1:2 dilution factor) of His-tagged EGFR (expressed / purified in-house) or HER3 (purchased from Acro Bio) in assay buffer (phosphate-buffered saline containing 0.1% BSA and 0.05% Tween 20), followed by a 300-second dissociation phase in assay buffer. Regeneration was achieved using 10 mM glycine, pH 1.5. Binding curves were globally fitted to a 1:1 model to determine the dissociation constant, K D , and kinetic association and dissociation rates were derived.

[0073] Biolayer interferometry was used to measure the binding kinetics of EGFR x HER3 bispecific antibodies to human EGFR. As shown in Figure 3 and Table 2, the EGFR binding data reveal that all cetuximab-based antibodies (SI-1X6, SI-1X22, SI-1X24, and SI-1X25) had similar KD values ​​ranging from 3 to 6 nM, whereas the nimotuzumab-based antibodies (SI-1X4 and SI-1X26) had weaker affinities with KD values ​​ranging from 11 to 24 nM. The cetuximab-based antibodies had a larger binding response in the assay, again suggesting stronger binding. The difference in EGFR binding within the two families (cetuximab and nimotuzumab) was not significant.

[0074] Biolayer interferometry was used to measure the binding kinetics of EGFR x HER3 bispecific antibodies to human HER3. As shown in Figure 4 and Table 3, the HER3 binding data revealed that all bispecific antibodies (SI-1X4, SI-1X6, SI-1X22, SI-1X24, SI-1X25, and SI-1X26) had similar KD values ​​ranging from 94 to 164 nM. This similarity in HER3 binding makes sense, as the HER3-binding domain in all of these proteins is derived from the same antibody. This result suggests that the anti-HER3 scFv can be placed at any position in cetuximab- and nimotuzumab-based bispecific antibodies without significant differences in in vitro binding.

[0075] Example 6: Inhibition of tumor cell proliferation To evaluate the effect of the EGFR x HER3 bispecific antibody on cell proliferation, a proliferation assay using FaDu cells was performed using Alamar Blue, which was used to quantify proliferation. The hypopharyngeal squamous cell carcinoma line FaDu was purchased from ATCC (Cat. No. HTB-43) and maintained at 37°C and 5% CO in EMEM medium supplemented with 10% fetal bovine serum. FaDu cells were detached from flasks using trypsin and cultured at 1.2 x 10 cells / ml in EMEM medium + 1% FBS. 5The antibody was diluted to 2x the final concentration in EMEM medium + 1% FBS. 50 ml of the cell suspension (6000 cells) was seeded into 60 inner wells of a 96-well tissue culture plate. The outer wells were filled with 300 ml of sterile HO to minimize evaporation in the inner wells. The cells were allowed to adhere for 4 hours at 37°C and 5% CO2. The antibody to be tested was diluted to 2x the final concentration in EMEM medium + 1% FBS. 50 ml of test antibody was added to each well for a total volume of 100 ml per well. Each antibody was tested in triplicate at the following final concentrations: 25 nM, 6.25 nM, 1.563 nM, 0.391 nM, 0.098 nM, 0.024 nM, 0.006 nM, 0.0015 nM, and 0.0004 nM. Each plate contained two antibodies at the above concentrations, tested in triplicate. Six control wells per plate contained cells and medium only. Immediately after the addition of the test compounds, 10 ml of Alamar Blue (Thermo Fisher catalog number DAL1100) was added to three of the medium-only control wells on each plate. The cells were incubated for 2 hours at 37°C and 5% CO2. After the 2-hour incubation, a 110 ml sample was removed from each control well and placed into a black, opaque 96-well plate. The plate was centrifuged at 2000 RPM for 5 minutes to remove any air bubbles. Fluorescence was then measured (excitation = 535 nm, emission = 595 nm) in a Molecular Devices FilterMax F5 microplate reader. The measured control fluorescence values ​​(C 開始時 ) served as a baseline for measuring assay endpoint growth. Plates were returned to 37°C, 5% CO2 for 7 days (168 hours). After incubation, 10 ml of Alamar Blue was added to each test well and three other control (media only) wells. After a 2-hour incubation at 37°C, 5% CO2, fluorescence was measured as described above. Endpoint control fluorescence values ​​(C 終了時 ) and test well fluorescence values ​​(T 終了時 ) was used to calculate the percent of control growth using the following formula: Percent of control growth = ((T 終了時 -C 開始時 ) / (C 終了時 -C開始時 ) x 100

[0076] Data points were analyzed by GraphPad Prism, and inhibition curves were fitted by nonlinear regression [log(inhibitor) vs. response, 4 parameters] to obtain IC 50 Values ​​were calculated. Data for the cetuximab-based proteins are shown in Figure 5A, and data for the nimotuzumab-based proteins are shown in Figure 5B. Fitted parameters for both sets of molecules are shown in Table 4. Cetuximab-based bispecific antibodies (SI-1X6, SI-1X22, SI-1X24, SI-1X25) all inhibited FaDu proliferation equally well, and this was more effective (64-76%) than the cetuximab control antibody (SI-1C6, 60%) and the anti-HER3 control Fc-scFv (SI-1C7, 9%). Nimotuzumab-based bispecific antibodies (SI-1X4, SI-1X26) inhibited proliferation less potently, consistent with nimotuzumab's lower affinity for EGFR. Unexpectedly, the maximum inhibition of SI-1X26 was significantly higher than that of SI-1X4, suggesting that the geometric shape of SI-1X26 allows for more efficient blockage of EGFR and / or HER3 signaling compared to the geometric shape of SI-1X4.

[0077] table [Table 1]

[0078] [Table 2]

[0079] [Table 3]

[0080] [Table 4]

[0081] Sequence Listing JPEG2025172738000005.jpg84150JPEG2025172738000006.jpg112134JPEG2025172738000007.jpg28134JPEG2025172738000008.jpg54137>seq 1 Cetuximab VL amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK >seq 2 Cetuximab VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >seq 3 Humanized Cetuximab VL amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVL >seq 4 Humanized Cetuximab VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >seq 5 Panitumumab VL amino acid sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK >seq 6 Panitumumab VH amino acid sequence QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS >seq 7 Nimotuzumab VL amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQIT >seq 8 Nimotuzumab VH amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSS >seq 9 Necitumumab VL amino acid sequence EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIK >seq 10 Necitumumab VH amino acid sequence QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS >seq 11 MM-111's HER3 VL amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >seq 12 MM-111's HER3 VH amino acid sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >seq 13 Patritumab VL amino acid sequence DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIK >seq 14 Patritumab VH amino acid sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS >seq 15 Seribantumab VL amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVVSWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSGNTASLTISGL QTEDEADYYCCSYAGSSIFVIFGGGTKVTVL >seq 16 Seribantumab VH amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGKGLEWVSSISSSGGWTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGLKMATIFDYWGQGTLVTVSS >seq 17 SI-1X22 light chain amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGCGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >seq 18 SI-1X22, SI-1X25 heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKCLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >seq 19 human IgG1 amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >seq 20 human Kappa amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >seq 21 cetuximab light chain amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >seq 22 SI-1X24 heavy chain amino acid sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >seq 23 SI-1X25 light chain amino acid sequence QVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGCGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >seq 24 SI-1X26 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGCGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >seq 25 SI-1X26 heavy chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQCLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >seq 26 SI-1X4 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >seq 27 SI-1X4 heavy chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >seq 28 SI-1X6 heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >seq 29 cetuximab heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >seq 30 SI-1C7 amino acid sequence EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >seq 31 cetuximab CDR-H1 amino acid sequence NYGVH >seq 32 cetuximab CDR-H2 amino acid sequence VIWSGGNTDYNTPFTS >seq 33 cetuximab CDR-H3 amino acid sequence ALTYYDYEFAY >seq 34 cetuximab CDR-L1 amino acid sequence RASQSIGTNIH >seq 35 cetuximab CDR-L2 amino acid sequence YASESIS >seq 36 cetuximab CDR-L3 amino acid sequence QQNNNWPTT >seq 37 nimotuzumab CDR-H1 amino acid sequence NYYIY >seq 38 nimotuzumab CDR-H2 amino acid sequence GINPTSGGSNFNEKFKT >seq 39 nimotuzumab CDR-H3 amino acid sequence QGLWFDSDGRGFDF >seq 40 nimotuzumab CDR-L1 amino acid sequence RSSQNIVHSNGNTYLD >seq 41 nimotuzumab CDR-L2 amino acid sequence KVSNRFS >seq 42 nimotuzumab CDR-L3 amino acid sequence FQYSHVPWT >seq 43 anti-HER3 CDR-H1 amino acid sequence SYWMS >seq 44 anti-HER3 CDR-H2 amino acid sequence NINRDGSASYYVDSVKG >seq 45 anti-HER3 CDR-H3 amino acid sequence DRGVGYFDL >seq 46 anti-HER3 CDR-L1 amino acid sequence TGTSSDVGGYNFVS >seq 47 anti-HER3 CDR-L2 amino acid sequence DVSDRPS >seq 48 anti-HER3 CDR-L3 amino acid sequence SSYGSSSTHVI >seq 101 Cetuximab VL nucleotide sequence GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA >seq 102 Cetuximab VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACTATGATTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >seq 103 Humanized Cetuximab VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGACAGTTCTT >seq 104 Humanized Cetuximab VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACGACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >seq 105 Panitumumab VL nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATCAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTTCTGTCAACACTTTGATCATCTCCCGCTCGCTTTCGGCGGAGGGACCAAGGTGGAAATTAAA >seq 106 Panitumumab VH nucleotide sequence CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCGTCAGCAGTGGTGATTACTACTGGACCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGACACATCTATTACAGTGGGAACACCAATTATAACCCCTCCCTCAAGAGCCGACTCACCATATCAATTGACACGTCCAAGACTCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCATTTATTACTGTGTGCGAGATCGAGTGACTGGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGC >seq 107 Nimotuzumab VL nucleotide sequence GATATTCAAATGACTCAATCTCCTTCTTCTCTTTCTGCTTCTGTTGGTGATCGTGTTACTATTACTTGTCGTTCTTCTCAAAATATTGTTCATTCTAATGGTAATACTTATCTTGATTGGTATCAACAAACTCCTGGTAAAGCTCCTAAACTTCTTATTTATAAAGTTTCTAATCGTTTTTCTGGTGTTCCTTCTCGTTTTTCTGGTTCTGGTTCTGGTACTGATTTTACTTTTACTATTTCTTCTCTTCAACCTGAAGATATTGCTACTTATTATTGTTTTCAATATTCTCATGTTCCTTGGACTTTTGGTCAAGGTACTAAACTTCAAATTACT >seq 108 Nimotuzumab VH nucleotide sequence CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACTACATCTACTGGGTGCGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGGCATCAACCCCACCAGCGGCGGCAGCAACTTCAACGAGAAGTTCAAGACCCGGGTGACCATCACCGCCGACGAGAGCAGCACCACCGCCTACATGGAGCTGAGCAGCCTGCGGAGCGAGGACACCGCCTTCTACTTCTGCACCCGGCAGGGCCTGTGGTTCGACAGCGACGGCCGGGGCTTCGACTTCTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC >seq 109 Necitumumab VL nucleotide sequence GAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGGGCCACCCTGAGCTGCCGGGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGGCTGCTGATCTACGACGCCAGCAACCGGGCCACCGGCATCCCCGCCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCACCAGTACGGCAGCACCCCCCTGACCTTCGGCGGCGGCACCAAGGCCGAGATCAAG >seq 110 Necitumumab VH nucleotide sequence CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCGGCGACTACTACTGGAGCTGGATTCGGCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACATCTACTACAGCGGCAGCACCGACTACAACCCCAGCCTGAAGAGCCGGGTGACCATGAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGGTGAACAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGGGTGAGCATCTTCGGCGTGGGCACCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC >seq 111 MM-111's HER3 VL nucleotide sequence CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTA >seq 112 MM-111's HER3 VH nucleotide sequence CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGTTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCGCGATGGAAGTGCGAGTTACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGTGGGGTGGGCTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCGAGC >seq 113 Patritumab VL nucleotide sequence GACATCGAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAGGTCCAGCCAGAGTGTTTTATACAGTTCCAGCAATAGAAACTACTTAGCTTGGTACCAGCAGAATCCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTCGCACATTCGGACAAGGGACCAAAGTGGAGATCAAG >seq 114 Patritumab VH nucleotide sequence CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCGGTAGAGACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGATAAATGGACTTGGTATTTTGACTTATGGGGCAGAGGGACACTGGTCACCGTCTCTTCA >seq 115 Seribantumab VL nucleotide sequence CAAAGCGCTCTGACTCAACCCGCATCTGTTTCAGGCTCTCCAGGGCAATCTATCACAATCTCCTGTACCGGCACCTCTAGCGACGTCGGAAGCTACAACGTTGTCTCTTGGTATCAACAGCACCCAGGAAAAGCACCCAAGCTGATAATTTACGAGGTATCCCAGCGTCCCAGCGGAGTGAGCAACAGATTTTCAGGTTCCAAATCAGGTAATACAGCAAGTCTGACCATCTCCGGTCTTCAGACTGAGGACGAGGCTGACTACTATTGCTGTTCCTACGCCGGCAGCTCTATTTTCGTCATTTTTGGTGGCGGGACAAAAGTGACCGTGCTG >seq 116 Seribantumab VH nucleotide sequence GAAGTGCAGTTGCTTGAGAGTGGAGGCGGACTTGTCCAGCCCGGTGGGTCACTGCGGCTGTCTTGCGCTGCCTCCGGTTTTACCTTCAGTCACTATGTGATGGCATGGGTGCGGCAGGCCCCTGGTAAGGGCCTGGAGTGGGTCTCTTCCATTTCTAGTTCAGGTGGGTGGACCTTGTACGCCGACAGTGTGAAGGGACGGTTCACTATCTCACGGGACAACTCAAAGAACACACTCTACTTGCAAATGAATAGTCTCAGGGCCGAGGATACAGCCGTGTATTACTGCACACGCGGTCTGAAGATGGCTACAATCTTCGACTACTGGGGTCAGGGGACTCTGGTGACAGTCAGCTCT > seq 117 SI-1X22 light chain nucleotide sequence >seq 118 SI-1X22, SI-1X25 heavy chain nucleotide sequence >seq 119 human IgG1 nucleotide sequence GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT >seq 120 human Kappa nucleotide sequence CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >seq 121 cetuximab light chain nucleotide sequence GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >seq 122 SI-1X24 heavy chain nucleotide sequence >seq 123 SI-1X25 light chain nucleotide sequence >seq 124 SI-1X26 light chain nucleotide sequence >seq 125 SI-1X26 heavy chain nucleotide sequence >seq 126 SI-1X4 light chain nucleotide sequence GATATTCAAATGACTCAATCTCCTTCTTCTCTTTCTGCTTCTGTTGGTGATCGTGTTACTATTACTTGTCGTTCTTCTCAAAATATTGTTCATTCTAATGGTAATACTTATCTTGATTGGTATCAACAAACTCCTGGTAAAGCTCCTAAACTTCTTATTTATAAAGTTTCTAATCGTTTTTCTGGTGTTCCTTCTCGTTTTTCTGGTTCTGGTTCTGGTACTGATTTTACTTTTACTATTTCTTCTCTTCAACCTGAAGATATTGCTACTTATTATTGTTTTCAATATTCTCATGTTCCTTGGACTTTTGGTCAAGGTACTAAACTTCAAATTACTCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >seq 127 SI-1X4 heavy chain nucleotide sequence >seq 128 SI-1X6 heavy chain nucleotide sequence >seq 129 cetuximab heavy chain nucleotide sequence >seq 130 SI-1C7 amino acid sequence >seq 131 cetuximab CDR-H1 amino acid sequence AACTATGGTGTACAC >seq 132 cetuximab CDR-H2 amino acid sequence GTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCC >seq 133 cetuximab CDR-H3 amino acid sequence GCCCTCACCTACTATGATTACGAGTTTGCTTAC >seq 134 cetuximab CDR-L1 amino acid sequence AGGGCCAGTCAGAGTATTGGCACAAACATACAC >seq 135 cetuximab CDR-L2 amino acid sequence TATGCTTCTGAGTCTATCTCT >seq 136 cetuximab CDR-L3 amino acid sequence CAACAAAATAATAACTGGCCAACCACG >seq 137 nimotuzumab CDR-H1 amino acid sequence AACTACTACATCTAC >seq 138 nimotuzumab CDR-H2 amino acid sequence GGCATCAACCCCACCAGCGGCGGCAGCAACTTCAACGAGAAGTTCAAGACC >seq 139 nimotuzumab CDR-H3 amino acid sequence CAGGGCCTGTGGTTCGACAGCGACGGCCGGGGCTTCGACTTC >seq 140 nimotuzumab CDR-L1 amino acid sequence CGTTCTTCTCAAAATATTGTTCATTCTAATGGTAATACTTATCTTGAT >seq 141 nimotuzumab CDR-L2 amino acid sequence AAAGTTTCTAATCGTTTTTCT >seq 142 nimotuzumab CDR-L3 amino acid sequence TTTCAATATTCTCATGTTCCTTGGACT >seq 143 anti-HER3 CDR-H1 amino acid sequence AGTTATTGGATGAGC >seq 144 anti-HER3 CDR-H2 amino acid sequence AACATAAACCGCGATGGAAGTGCGAGTTACTATGTGGACTCTGTGAAGGGC >seq 145 anti-HER3 CDR-H3 amino acid sequence GATCGTGGGGTGGGCTACTTCGATCTC >seq 146 anti-HER3 CDR-L1 amino acid sequence ACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCC >seq 147 anti-HER3 CDR-L2 amino acid sequence GATGTCAGTGATCGGCCCTCA >seq 148 anti-HER3 CDR-L3 amino acid sequence AGCTCATATGGGAGCAGCAGCACTCATGTGATT References 1. Diaz-Serrano, A. et al. Genomic Profiling of HER2-Positive Gastric Cancer: PI3K / Akt / mTOR Pathway as Predictor of Outcomes in HER2-Positive Advanced Gastric Cancer Treated with Trastuzumab. Oncologist.23, 1092-1102 (2018). 2. Durkee, BY, et al. Cost-Effectiveness of Pertuzumab in Human Epidermal Growth Factor Receptor 2-Positive Metastatic Breast Cancer. Journal of Clinical Oncology. 2016, 34 (9): 902-9. 3. Gijsen, M. et al. HER2 Phosphorylation is Maintained by a PKB Negative Feedback Loop in Response to anti-HER2 Herceptin in Breast Cancer. PLoS Biol.8, e1000563 (2010). 4. Goel, S. & Winer, E. P. POINT: HER2-Targeted Combinations in Advanced HER2-Positive Breast Cancer. Oncology (Williston Park). 29, 797-798, 802 (2015). 5. Luque-Cabal, M. et al. Mechanisms Behind the Resistance to Trastuzumab in HER2-Amplified Breast Cancer and Strategies to Overcome It. Clin. Med. Insights Oncol.10, 21-30 (2016). 6. McDonagh, C. F. et al. Antitumor Activity of a Novel Bispecific Antibody that Targets the ErbB2 / ErbB3 Oncogenic Unit and Inhibits Heregulin-Induced Activation of ErbB3. Mol. Cancer Ther.11, 582-593 (2012). 7. R M Neve 1 , U B Nielsen, D B Kirpotin, M A Poul, J D Marks, C C Benz. Biological effects of anti-ErbB2 single chain antibodies selected for internalizing function Biochem Biophys Res Commun. 2001, 280(1):274-9. 8. M K Robinson 1 , K M Hodge, E Horak, A L Sundberg, M Russeva, C C Shaller, M von Mehren, I Shchaveleva, H H Simmons, J D Marks, G P Adams. Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro Br J Cancer 2008 Nov 4;99(9):1415-25. 9. Wang, Q. et al. The anti-HER3 Antibody in Combination with Trastuzumab Exerts Synergistic Antitumor Activity in HER2-positive Gastric Cancer. Cancer Lett.380, 20-30 (2016). 10. Yang, L. et al. NRG1-dependent Activation of HER3 Induces Primary Resistance to Trastuzumab in HER2-overexpressing Breast Cancer Cells. Int. J. Oncol.51, 1553-1562 (2017). 11. Cetuximab: https: / / www.ema.europa.eu / en / documents / scientific-discussion / erbitux-epar-scientific-discussion_en.pdf 12. Panitumumab: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6763619 / #:~:text=Panitumumab%20binds%20EGFR%20with%20an,whether%20this%20characteristic%20is%20favorable 13. Nimotuzumab: https: / / www.nature.com / articles / s41598-019-57279-w / tables / 1 14. Trastuzumab: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6244757 / 15. Pertuzumab: https: / / www.tga.gov.au / sites / default / files / auspar-pertuzumab-131001.pdf 16. Patritumab: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5058629 / 17. MM-121: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3478453 / 18. MM-111: https: / / pubmed.ncbi.nlm.nih.gov / 22248472 / 19. 2in1: https: / / ars.els-cdn.com / content / image / 1-s2.0-S1535610811003515-mmc1.pdf 20. SI-1X6.3(C3): US15 / 119,694.

Claims

1. A bispecific antibody comprising two sets of heavy and light chains, each set of heavy and light chains forms a Fab region having binding specificity to EGFR; the antibody further comprises an scFv domain covalently linked to each set of heavy and light chains at the N-terminus of the heavy chain, the N-terminus of the light chain, or the C-terminus of the light chain; and A bispecific antibody, wherein the scFv domain has binding specificity for HER3.

2. 2. The bispecific antibody of claim 1, wherein the scFv domain is linked to the N-terminus of the heavy chain and the antibody comprises an amino acid sequence having sequence identity to SEQ ID NO:

22.

3. 2. The bispecific antibody of claim 1, wherein the scFv domain is linked to the N-terminus or C-terminus of the light chain, and wherein the antibody comprises an amino acid sequence having sequence identity to SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO:

24.

4. 2. The bispecific antibody of claim 1, comprising an antigen-binding domain having at least 98% sequence identity to SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO:

24.

5. 2. The bispecific antibody of claim 1, wherein the heavy chain comprises a constant region, and the constant region comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:

19.

6. 2. The bispecific antibody of claim 1, wherein the light chain comprises a kappa constant region, and the kappa constant region comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:

20.

7. 2. The bispecific antibody of claim 1, wherein the scFv domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:

16.

8. 2. The bispecific antibody of claim 1, wherein the scFv domain comprises a variable light chain, and the variable light chain has an amino acid sequence with at least 98% sequence identity to SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:

15.

9. 2. The bispecific antibody of claim 1, wherein the scFv domain comprises a variable heavy chain, and the variable heavy chain has an amino acid sequence with at least 98% sequence identity to SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO:

16.

10. The scFv domain comprises a variable light chain (VL) and a variable heavy chain (VH), and the scFv domain is arranged from the N-terminus to the C-terminus as follows: L V H or V H V L 2. The bispecific antibody of claim 1, having the configuration:

11. The scFv domain is V L and V H The bispecific antibody of claim 10, comprising a disulfide bond between

12. 12. The bispecific antibody of claim 11, wherein the disulfide bond is between vL100 and vH44 (Kabat) of the scFv domain.

13. 2. The bispecific antibody of claim 1, wherein the scFv domain comprises an R19S (Kabat) mutation.

14. 2. The bispecific antibody of claim 1, wherein the antibody comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 17 and SEQ ID NO:

18.

15. 2. The bispecific antibody of claim 1, wherein the antibody comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 21 and SEQ ID NO:

22.

16. 2. The bispecific antibody of claim 1, wherein the antibody comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 18 and SEQ ID NO:

23.

17. 2. The bispecific antibody of claim 1, wherein the antibody comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 24 and SEQ ID NO:

25.

18. 10. An isolated nucleic acid encoding the bispecific antibody of claim 1.

19. 19. An expression vector comprising the isolated nucleic acid of claim 18.

20. 20. The expression vector of claim 19, wherein the vector is expressible in a cell.

21. A host cell comprising the nucleic acid of claim 18.

22. 22. A method for producing a bispecific antibody according to claim 1, comprising culturing a host cell according to claim 21, so that the bispecific antibody is produced.

23. 10. An immunoconjugate comprising the bispecific antibody of claim 1 and a cytotoxic agent, wherein the cytotoxic agent comprises a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioactive isotope.

24. A pharmaceutical composition comprising the bispecific antibody of claim 1 and a pharmaceutically acceptable carrier.

25. 25. The pharmaceutical composition of claim 24, further comprising a radioisotope, radionuclide, toxin, therapeutic agent, chemotherapeutic agent, or a combination thereof.

26. 24. A pharmaceutical composition comprising the immunoconjugate of claim 23 and a pharmaceutically acceptable carrier.

27. A method for treating a subject suffering from cancer, comprising administering to the subject an effective amount of the bispecific antibody of claim 1.

28. 28. The method of claim 27, wherein the cancer comprises cells that express EGFR, HER3, or both, or the cancer comprises breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, kidney cancer, gastric cancer, liver cancer, bladder cancer, cervical cancer, brain cancer, lymphoma, leukemia, or myeloma.

29. 28. The method of claim 27, further comprising co-administering an effective amount of a therapeutic agent.

30. 30. The method of claim 29, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof, and the therapeutic agent comprises capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, derivatives or combinations thereof.

31. 28. The method of claim 27, wherein the subject is a human.

32. 10. A solution comprising an effective concentration of the bispecific antibody of claim 1, wherein the solution is plasma of a subject.

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