Bispecific tetravalent antibodies targeting her2 and her3

Abstract

When the Fab region has binding affinity for HER2 and the scFv domain has binding affinity for HER3, or vice versa, the anti-HER2 x HER3 bispecific tetravalent antibody is configured to have two heavy chains and two light chains and two scFv domains linked to the N-terminus of each heavy chain by a linker.

Description

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Application Serial No. 63 / 604,087, filed November 29, 2023, pursuant to 35 USC 119(e), the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to the field of antibody therapy technology for treating cancer, and more specifically to bispecific antibodies. Background Technology

[0003] The human epidermal growth factor receptor (EGFR, also known as ErbB1 or HER1) family has four members: EGFR, HER2, HER3, and HER4. Dysregulation of each member through mutation, amplification, and overexpression plays a crucial role in tumorigenesis and metastasis in various cancers. Disrupting EGFR family signaling by blocking binding sites on the receptor's extracellular domain or by inhibiting intracellular tyrosine kinase activity can prevent the growth of tumors expressing the EGFR family and improve patient outcomes. For example, HER2 overexpression occurs in 30% of breast cancer patients, indicating increased disease recurrence and poor prognosis. HER2 overexpression is also known to occur in stomach cancer, ovarian and gastric cancer, lung adenocarcinoma, invasive uterine cancer, and salivary gland duct carcinoma. HER2 mutations have been found in non-small cell lung cancer. Potential HER2 mutations and amplifications generate aberrant growth signals that activate downstream signaling pathways, leading to tumorigenesis. Specifically, HER2 dimers with HER3 on the surface of tumor cells, activating PI3K / AKT signaling and promoting tumor growth and survival.

[0004] Several therapeutic antibodies and small molecule inhibitors targeting EGFR and HER2 have been approved for cancer treatment (Arteaga et al., 2012). Examples of therapeutic anti-HER2 antibodies include trastuzumab and pertuzumab, which are approved for the treatment of several forms of cancer, including breast and gastric cancer (https: / / www.herceptin.com / hcp / treating-HER2-cancer.html). In particular, monoclonal antibodies targeting EGFR or HER2 have shown good clinical responses in colorectal cancer (Price et al., 2014), head and neck squamous cell carcinoma (Cohen, 2014), breast cancer, and gastric cancer (Arteaga et al., 2012).

[0005] Trastuzumab (Herceptin) and other HER2-targeting agents exert antitumor efficacy in patients with HER2-expressing breast and gastric cancer. Trastuzumab is a monoclonal antibody that binds to HER2, which enhances the activity of p27, a protein that inhibits cell proliferation. Trastuzumab is effective only against cancers that overexpress HER2. For all HER2-positive breast cancer patients receiving concurrent chemotherapy, one year of trastuzumab therapy is recommended; there is no additional benefit beyond 12 months. Pertuzumab is another monoclonal antibody that inhibits the dimerization of HER2 with other receptors such as HER3 and is an FDA-approved therapeutic agent that can be used in combination with trastuzumab and docetaxel (a chemotherapy agent) to treat metastatic HER2-positive breast cancer (Durkee et al., 2016).

[0006] Despite these successes, long-term benefits appear to be limited in some patients. Many tumors that initially respond to these therapies eventually progress due to acquired resistance. The development of resistance reduces the efficacy of these treatments. For example, resistance to HER2-targeted therapies can occur via the upregulation of HER3 or its ligand HRG. These findings suggest that current therapeutic approaches aimed at inhibiting HER2 / HER3 signaling pathway activation have failed to provide meaningful clinical benefit (Geuijen et al., 2018; Yu et al., 2019).

[0007] In summary, currently approved antibody therapies targeting HER2 and / or HER3 all suffer from low treatment response rates or the development of treatment resistance in patients. Better treatment methods for these cancers are urgently needed. Summary of the Invention

[0008] This application generally relates to the technical field of antibody therapeutics, and more specifically to bispecific antibodies targeting HER2 and HER3 specific epitopes. HER2 and HER3 often form a partnership that promotes cell transformation, ultimately leading to tumorigenesis and metastasis. This application particularly provides bispecific tetravalent antibodies. This application also provides pharmaceutical compositions comprising a bispecific tetravalent antibody or its immunoconjugate, methods for treating diseases such as cancer using the antibody or its immunoconjugate, methods for preparing the antibody or its immunoconjugate, and solutions containing the antibody or its immunoconjugate.

[0009] This application provides a bispecific tetravalent antibody. In one embodiment, the bispecific antibody has binding affinity for HER2 and HER3. The antibody has a heavy chain (HC) containing a heavy chain variable (VH) domain, a light chain (LC) containing a light chain variable (VL) domain, and an scFv domain having both a scFv light chain variable (VL) domain and a scFv heavy chain variable (VH) domain. In one embodiment, the VL and VH domains form a Fab region. In one embodiment, the heavy chain has an N-terminus and a C-terminus. In one embodiment, each scFv domain is connected to the N-terminus of each heavy chain via a linker.

[0010] In one aspect, this application provides a bispecific antibody with binding specificity to HER2 and HER3, wherein the Fab region has binding affinity for HER2 and the scFv domain has binding affinity for HER3.

[0011] In one embodiment, the Fab region has a binding affinity for HER2 with a KD of about 100 pM to about 50 nM, about 500 pM to about 10 nM, about 800 pM to about 5 nM, about 1 nM to about 5 nM, about 1 nM to about 3 nM, about 1 nM to about 10 nM, about 800 pM to about 8 nM, or about 1 nM to about 5 nM. In one embodiment, the Fab region has a binding affinity for HER2 with a KD of at least 500 pM, 800 pM, or 1 nM. In one embodiment, the scFv domain has a binding affinity for HER3 with a KD of about 1 nM to about 1 mM, 50 nM to about 500 nM, 80 nM to about 300 nm, 100 nM to about 200 nM, or 100 nM to about 500 nM. In one embodiment, the Fab region has binding affinity for HER2 with a KD of about 0.1 nM to about 10 nM, and the scFv domain has binding affinity for HER3 with a KD of about 80 nM to about 200 nM. In one embodiment, the antibody has binding affinity for HER3 that is at least about 50, 75, 80, 90, 100, 120, or 150 times greater than its binding affinity for HER2.

[0012] In one embodiment, the Fab region comprises a VH domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In one embodiment, the VL domain has an amino acid sequence identity of at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12.

[0013] In one embodiment, the VH domain comprises CDR-H1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 25, CDR-H2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 26, and CDR-H3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 27. In one embodiment, the VL domain comprises CDR-L1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 28, CDR-L2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 29, and CDR-L3 having sequence identity with SEQ ID NO: 30.

[0014] In one embodiment, the amino acid sequence of the scFv VH domain has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14. In one embodiment, the amino acid sequence of the scFv VL domain has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16.

[0015] In one embodiment, the scFv VH domain includes CDR-H1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 31, CDR-H2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 32, and CDR-H3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 33. In one embodiment, the scFv VL domain includes CDR-L1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 34, CDR-L2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 35, and CDR-L3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 36.

[0016] On the other hand, this application provides a bispecific antibody with binding affinity for HER3 and HER2, wherein the Fab region has binding affinity for HER3 and the scFv domain has binding affinity for HER2.

[0017] In one embodiment, the Fab region has a binding affinity for HER3 with a KD of about 1 pM to about 10 pM, 1 pM to about 5 pM, or 1 pM to about 100 pM. In one embodiment, the scFv domain has a binding affinity for HER2 with a KD of about 1 nM to about 1 mM, 50 nM to about 300 nM, or 1 pM to about 1 mM. In one embodiment, the Fab region has a binding affinity for HER3 with a KD of about 1 pM to about 10 pM, and the scFv domain has a binding affinity for HER2 with a KD of about 100 nM to about 300 nM.

[0018] In one embodiment, the Fab region comprises a VH domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the Fab region comprises a VL domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0019] In one embodiment, the Fab VH domain comprises CDR-H1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 31, CDR-H2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 32, and CDR-H3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 33. In one embodiment, the Fab VL domain comprises CDR-L1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 34, CDR-L2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 35, and CDR-L3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 36.

[0020] In one embodiment, the scFv domain comprises an scFv VH domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In one embodiment, the scFv domain comprises an scFv VL domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12.

[0021] In one embodiment, the scFv VH domain includes CDR-H1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 25, CDR-H2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 26, and CDR-H3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 27. In one embodiment, the scFv VL domain includes CDR-L1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 28, LCDR CDR-L2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 29, and CDR CDR-L3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 30.

[0022] The tetravalent bispecific antibody may comprise a heavy chain having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 2, 6, or 22. In one embodiment, the tetravalent bispecific antibody may comprise a light chain having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 4, 8, or 24.

[0023] In one embodiment, the Fab VH domain comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 10 or 14. In one embodiment, the Fab VL domain comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 12 or 16.

[0024] In one embodiment, the scFv domain comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 18 or 20.

[0025] In one embodiment, the scFv VH comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 10 or 14. In one embodiment, the scFv VL domain comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 12 or 16.

[0026] The linker may comprise a flexible GC linker having about 10 to about 20 amino acids. In one embodiment, the linker comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)m, where m is an integer of at least 3. In one embodiment, m may be 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, m is 4.

[0027] On the other hand, this application provides nucleic acid sequences that encode the antibodies disclosed herein. In one embodiment, the isolated nucleic acid sequence encodes the bispecific tetravalent antibody disclosed herein.

[0028] In another aspect, this application provides an expression vector. In one embodiment, the expression vector comprises a separated nucleic acid sequence encoding a bispecific antibody.

[0029] In another aspect, this application provides a host cell. In one embodiment, the host cell comprises a separated nucleic acid sequence encoding a bispecific antibody. The host cell may be a prokaryotic cell or a eukaryotic cell.

[0030] In another aspect, this application provides immunoconjugates. In one embodiment, the immunoconjugate comprises a bispecific antibody conjugated to a pharmaceutical moiety or a cytotoxic agent. In one embodiment, the bispecific antibody may comprise a Fab region having binding affinity for HER2 and an scFv domain having binding affinity for HER3. In one embodiment, the bispecific antibody may comprise a Fab region having binding affinity for HER3 and an scFv domain having binding affinity for HER2. In one embodiment, the pharmaceutical moiety or cytotoxic agent comprises a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof. In one embodiment, the pharmaceutical moiety or cytotoxic agent may be an imaging agent.

[0031] In another aspect, this application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises a bispecific tetravalent antibody or an immunoconjugate thereof disclosed herein. In one embodiment, the bispecific antibody may comprise a Fab region having binding affinity for HER2 and an scFv domain having binding affinity for HER3. In one embodiment, this application may also comprise an optionally pharmaceutically acceptable carrier.

[0032] In one embodiment, the pharmaceutical composition may further comprise a cytotoxic agent. In one embodiment, the cytotoxic agent may be a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

[0033] In another aspect, this application provides methods for treating or preventing a disease. In one embodiment, the disease is cancer. In one embodiment, the method of treating or preventing cancer in a subject includes the step of administering to the subject a pharmaceutical composition comprising a bispecific antibody or an immunoconjugate thereof. In one embodiment, the method of treating or preventing cancer in a subject includes the step of administering to the subject an effective amount of a bispecific antibody or an immunoconjugate thereof. In one embodiment, the bispecific antibody may comprise a Fab region having binding affinity for HER2 and an scFv domain having binding affinity for HER3.

[0034] In one embodiment, a method of treating or preventing cancer may include co-administering an effective amount of a therapeutic agent. In one embodiment, the therapeutic agent may be an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

[0035] Cancer can be any cancer expressing HER2 and / or HER3. In one implementation, cancer includes cells expressing HER3 or EGFR, including breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, endometrial cancer, epidermal cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, kidney cancer, stomach cancer, liver cancer, bladder cancer, cervical cancer, brain cancer, lymphoma, leukemia, or myeloma.

[0036] In another aspect, this application provides methods for preparing bispecific antibodies and their immunoconjugates. In one embodiment, the method for generating a bispecific antibody includes culturing host cells to express a DNA sequence encoding the bispecific antibody, and purifying the bispecific antibody. In one embodiment, the method for generating an immunoconjugate includes conjugating the bispecific antibody to a cytotoxic agent to provide an immunoconjugate, and purifying the immunoconjugate.

[0037] In another aspect, this application provides a solution containing a bispecific antibody or an immunoconjugate thereof. In one embodiment, the solution contains an effective concentration of a bispecific antibody or an immunoconjugate thereof. In one embodiment, the solution is the plasma of a subject. Attached Figure Description

[0038] The foregoing and other features of this disclosure may become more apparent from the accompanying drawings, the following description, and the appended claims. It should be understood that these drawings depict only a few embodiments arranged according to this disclosure and are therefore not intended to limit the scope of the disclosure, which can be described with further specificity and detail using the drawings, wherein: Figure 1 shows a schematic diagram of a bispecific tetravalent antibody with two light chains and two heavy chains, wherein the heavy and light chains pair to form a Fab region called the first binding domain D1, and the heavy chain monomer contains an scFv binding domain (D2) linked to the N-terminal Fab (D1) (1A). Three anti-HER2×HER3 bispecific tetravalent antibodies and two parental bispecific monospecific antibodies are also shown (1B).

[0039] Figure 2 depicts the VH-VL pairing of trastuzumab and MM-111 Fv, showing the contact between Q39 of VH and Q38 of VL (numbered by Kabat) (2A), the correct pairing with reversed charge orientation (VH+ / VL- vs. VH- / VL+) (2B upper panel), and the incorrect pairing (2B lower panel).

[0040] Figure 3 shows the octet binding affinity of the anti-HER2×HER3 bispecific tetravalent antibodies SI-71X24, SI-71X25 and SI-71X33 for HER2 (top) and HER3 (bottom) (3A), and the octet binding affinity of the parental bivalent antibodies SI-4C12 (trastuzumab) and SI-1C16 (MM-111) for HER2 (top) and HER3 (bottom) (3B).

[0041] Figure 4 shows the antiproliferative activity of SI-71X24 and SI-71X25 against BT-474 breast cancer cells (4A) and the antiproliferative activity of SI-71X33 against FaDu head and neck cancer cells (4B) in the Alamar-blue proliferation assay, revealing the unexpected proliferative activity of SI-71X24.

[0042] Figure 5 shows the proliferative effect of SI-71X24 on FaDu cells (head and neck cancer) (5A) and Oka-C-1 cells (lung squamous cell carcinoma) (5B) compared with SI-71X33 in the confluence proliferation assay; and the mean gMFI signal of SI-71X25 and SI71X24-bound cells (5C).

[0043] Figure 6 The mean gMFI signal of the antibodies (10 nM SI-71X24, SI-71X25, and anti-CD20 rituximab as an antibody control) is shown in 11 HER2 / HER3-expressing cancer cell lines and 1 HER2 / HER3-negative lung cancer cell line (COR-L279); the bar chart represents the mean gMFI of the anti-Fc AF647 APC channel; the scatter plot represents the replicates (n=4); the titles indicate the tissue source, cell line name, anti-HER2 antibody binding capacity, and anti-HER3 antibody binding capacity. Detailed Implementation

[0044] This disclosure provides a protein with therapeutic properties or efficacy superior to currently known bispecific antibodies or similar antibodies targeting one or two members of the EGFR family. In one embodiment, the antibody is structurally configured to effectively target two members of the EGFR family, HER2 and HER3, with cancer cell proliferation activity as the endpoint. In anti-HER2×HER3 bispecific tetravalent antibodies, an unexpected finding is that alterations to the positions of the HER2 and HER3 binding domains produce opposing effects on cancer cell proliferation. Typically, antigen-binding fragments function independently in multispecific antibodies and contribute to their overall function. For example, parental monospecific antibodies are known to inhibit cancer cell proliferation, and anti-HER2×HER3 bispecific antibodies are expected to bind antiproliferative activity. This application discloses a novel and surprising structure-function relationship that is crucial in the development of highly effective antibodies, such as those described herein. These antibodies exhibit significant therapeutic advantages compared to existing antibodies in the art for treating cancers expressing HER2 / HER3.

[0045] As used herein, the terms “a”, “an”, and “the” are defined as meaning “one or more” and include the plural form unless the context is inappropriate.

[0046] As used herein, the terms “polypeptide,” “peptide,” and “protein” are interchangeable and are defined to refer to a biomolecule composed of amino acids linked by peptide bonds.

[0047] The term "antigen" refers to an entity or segment thereof that can induce an immune response in an organism, particularly animals, and more particularly mammals, including humans. This term includes immunogens and the regions responsible for antigenicity or antigenic determinants.

[0048] The terms “antigen or epitope binding portion or fragment,” “variable domain,” “variable region,” “variable region sequence,” or “binding domain” refer to fragments of antibodies capable of binding to antigens, such as HER2 and HER3 in this application. These fragments may possess the antigen-binding function of the complete antibody as well as additional functions.

[0049] The term "Fv" or "scFv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a tightly bound, non-covalently bound dimer of a heavy chain variable domain and a light chain variable domain. In this configuration, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. These six CDRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind antigens, although with less affinity than the entire binding site. Examples of binding fragments include, but are not limited to, single-chain Fv fragments (scFv) consisting of variable light chain (VL) and variable heavy chain (VH) domains of an antibody single arm linked as a single polypeptide chain by a synthetic linker, or monovalent fragments (Fab fragments) consisting of VL, constant light chain (CL), VH, and constant heavy chain 1 (CH1) domains. Antibody fragments can even be smaller subfractions and can consist of domains as small as a single CDR domain, particularly the CDR3 region from the VL and / or VH domains.

[0050] Antibody fragments are produced using conventional methods known to those skilled in the art. The utility of the antibody fragments can be screened using the same techniques employed for whole antibodies. Purified monoclonal antibodies can be lysed with enzymes such as pepsin and subjected to HPLC gel filtration. Papain digestion of the antibody produces two identical antigen-binding fragments (referred to as “Fab” fragments) and a residual “Fc” fragment, each Fab fragment having a single antigen-binding site; the name Fc fragment reflects its ease of crystallization. Pepsin treatment produces F(ab’)2 fragments, which have two antigen-binding sites and are still capable of cross-linking antigens. Appropriate fractions containing Fab fragments can then be collected and concentrated by methods such as membrane filtration. General techniques for isolating active antibody fragments are further described.

[0051] The term "antibody" is used in the broadest sense to specifically encompass monoclonal and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions with multi-epitope specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), provided they exhibit the desired biological activity. In some embodiments, antibodies can be monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, multispecific or pleiotropic antibodies, human antibodies, and humanized antibodies, as well as their active fragments. Examples of molecularly active fragments that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, including products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the aforementioned antibodies and fragments.

[0052] In some implementations, antibodies may include immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing binding sites that specifically bind antigens. A typical antibody is a heterotetrameric protein that typically comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain consists of a light chain variable domain (abbreviated as VL) and a light chain constant domain. Based on the amino acid sequence of the constant domain, the light chains of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, called κ and λ. The VH and VL regions can be further subdivided into highly variable complementarity-determining region (CDR) domains and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Binding regions that interact with antigens exist within the variable regions of both the light and heavy chains.

[0053] Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4; and IgA1 and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well-known.

[0054] The term "valence" refers to the valence of an antibody, which is the number of antigenic determinants a single antibody molecule can bind to. All antibodies are at least divalent, while "antibody affinity" refers to the tendency of an antibody to bind to a specific epitope on the surface of an antigen, i.e., the strength of the interaction.

[0055] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possible small number of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody formulations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to specificity, monoclonal antibodies have the advantage of being synthesized from hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by the hybridoma method first described by Kohler & Milstein, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Recombinant" means that the antibody is generated using recombinant nucleic acid technology in a heterologous host cell. Monoclonal antibodies can be produced using a variety of methods, including but not limited to mouse hybridoma, phage display, recombinant DNA, molecular cloning of antibodies directly from primary B cells, and antibody discovery methods. Monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, provided they exhibit the desired biological activity.

[0056] The term "humanized antibody" refers to a class of engineered antibodies whose core receptor (CDR) is derived from a non-human donor immunoglobulin, while the remaining immunoglobulin-derived portions of the molecule are derived from one (or more) human immunoglobulins. Additionally, the framework region support residues can be modified to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art.

[0057] The terms "isolated" or "purified" refer to a biomolecule that does not contain at least some of its naturally occurring components. When used to describe the various polypeptides disclosed herein, "isolated" or "purified" means a polypeptide that has been identified and isolated and / or recovered from the cells or cell cultures expressing it. Typically, purified polypeptides are prepared through 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.

[0058] The term "immunogenicity" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells against an immunogenic agent and contributes 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 in response to the administered immunogenic composition of this disclosure to reduce or alleviate the condition to be treated. While an immunogenic response typically includes both cellular (T cell) and humoral (antibody) arms of the immune response, antibodies against therapeutic proteins (antidrug antibodies, ADA) may consist of IgM, IgG, IgE, and / or IgA isotypes.

[0059] The terms "specific binding," "specifically bound to," or "specific to a particular antigen or epitope" indicate that binding is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule to a control molecule (which is typically a similarly structured molecule that does not have binding activity). For instance, specific binding can be determined by competition with a target-like control molecule.

[0060] The term "affinity" refers to a measure of the attractive force between two peptides, such as antibody / antigen, receptor / ligand, etc. The inherent attractive force between two peptides can be expressed as the binding affinity equilibrium dissociation constant (KD) of a specific interaction. The KD binding affinity constant can be measured, for example, by biolayer interferometry, where KD is the ratio of kdis (dissociation rate constant) to kon (association rate constant), such as KD = kdis / kon.

[0061] Specific binding to a particular antigen or epitope can be manifested, for example, by antibodies with a KD 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 alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, at least about 10⁻¹² M, or greater, where KD refers to the equilibrium dissociation constant of the specific antibody-antigen interaction. Typically, the KD of an antibody that specifically binds to an antigen is 20, 50, 100, 500, 1000, 5000, 10000, or more times greater than that of the control molecule relative to the antigen or epitope.

[0062] Furthermore, specific binding to a particular antigen or epitope can be manifested, for example, by an antibody whose KA or Ka is at least 20, 50, 100, 500, 1000, 5000, 10000 or more relative to the control, where KA or Ka refers to the association rate of the specific antibody-antigen interaction.

[0063] This application argues that bispecific antibodies potentially offer advantages over any combination therapy, which is generally more toxic than monotherapy. Bispecific agents, such as the bispecific antibodies disclosed in this application, can function as monotherapies targeting the same antigens as in combination therapies, but with increased efficacy and response rates, and reduced toxicity compared to combination therapies. Bispecific antibody therapeutics are less toxic to patients and / or more effective due to increased binding specificity compared to combination therapies using two monoclonal antibodies.

[0064] In one aspect, this application provides a bispecific tetravalent antibody having an N-terminus and a C-terminus, comprising at least two binding domains, wherein the binding domains include a Fab region and an scFv domain. The scFv domain can be attached to either the N-terminus or the C-terminus of the antibody. The Fab region and the scFv domain each independently exhibit binding specificity for different proteins in the EGFR family.

[0065] In some embodiments, the scFv molecule described herein contains a (GmS)n linker that operatively connects VH and VL regardless of V region orientation (LH or HL). The remaining positions in the bispecific antibody may consist of a human IgG Fc or nonfunctional IgG Fc heavy chain, a VH-CH1-hinge-CH2-CH3, and its corresponding κ or λ light chain VL-CL. These scFv domains are linked to the N-terminus or C-terminus of the IgG heavy chain via the (GmS)n linker, resulting in a ~75 kDa continuous heavy chain monomeric peptide. When co-transfected with a suitable light chain, the final symmetrical bispecific molecule can be purified from human IgG Fc (protein A) and assayed to evaluate functional activity.

[0066] In one implementation, the binding domain that is HER2-specific includes trastuzumab, which is administered as a HER2 inhibitor via intravenous infusion for the treatment of HER2+ breast cancer.

[0067] In one implementation, the binding domain specific for HER3 includes MM-111, a bispecific HER2 and HER3 binding protein. MM-111 is a bispecific antibody fragment scaffolded by human serum albumin (HSA) containing a therapeutic binding to HER3, but its binding to HER2 alone is insufficient to be considered a therapeutic binding. In contrast, trastuzumab contains a single therapeutic binding to HER2.

[0068] Bispecific tetravalent antibodies may comprise immunoglobulin G (IgG) portions having two heavy chains and two light chains, and two scFv portions covalently linked to the C-terminus or N-terminus of the heavy or light chain via adapters such as (Gly-Gly-Gly-Gly-Ser)n adapters, (Gly-Gly-Gly-Ser)n adapters, (GmS)n adapters, or (SGm)n adapters.

[0069] It is known that using a single therapeutic agent presents significant challenges, as the choice of binding site and scaffold structure can affect in vivo binding efficiency and therapeutic efficacy in patients. For example, ALM is a bispecific antibody targeting HER2 / HER3 that exhibits antiproliferative activity against tumor cells in vitro. However, its rapid renal clearance and short circulating half-life make it unsuitable as a candidate drug. These bispecific antibodies can simultaneously inhibit different receptor-mediated oncogenic signaling, thus overcoming resistance to EGFR family inhibitors or monoclonal antibody therapy.

[0070] In one implementation, the binding domain specific for HER2 includes trastuzumab. Trastuzumab inhibits HER2 phosphorylation and thus its downstream signaling pathways (Gijsen et al., 2010; Goel et al., 2015). However, not all patients with HER2 overexpression in their cancer cells can benefit from trastuzumab therapy due to initial or acquired resistance (Luque-Cabal et al., 2016). The exact mechanisms of trastuzumab resistance remain unclear. The HER2 / HER3 heterodimer and its downstream signaling play crucial roles in tumor resistance and metastasis in HER2-positive cancers (Wang et al., 2016; Yang et al., 2017). Trastuzumab inhibits ligand-independent HER3 / HER2 interactions rather than blocking HER2 signaling. In fact, many forms of HER2-amplified metastatic cancers are unresponsive to trastuzumab or eventually develop resistance. These cancers often revert to phosphorylated HER3 and PI3K-Akt-mTOR downstream signaling (Diaz-Serrano et al., 2018).

[0071] HER2-targeting bispecific antibodies have demonstrated significant efficacy in preclinical studies of treating HER2-expressing resistant malignancies. HER2-targeting bispecific antibodies include MM-111, ALM, PB4188, and MCLA-128 (McDonagh et al., 2012; Robinson et al., 2008; Geuijen et al., 2018; and Schram et al., 2022). MM-111 targets the HER2 / HER3 heterodimer, blocking moscin binding and inhibiting downstream signaling pathways. In contrast, trastuzumab alone has no effect on moscin-induced paclitaxel resistance. Compared to the HER3 arm, MM-111 has a higher affinity for the HER2 arm to target HER2-amplified tumors (McDonagh et al., 2012). When MM-111 binds with high affinity to HER2-positive cells, it effectively blocks submaximal ligand-driven proliferation but does not block supermax activation. This is because, compared to trastuzumab, the HER2-binding portion of MM-111 has no sustained effect on tumor cell growth, while trastuzumab binds to different epitopes in the HER2 IV region (Neve et al., 2001). In fact, combination therapy with trastuzumab and MM-111 shows significantly greater activity than either antibody alone (McDonagh et al., 2012).

[0072] MCLA-128 (zastuzumab, Zeno) is a bispecific humanized immunoglobulin G1 (IgG1) containing two distinct Fab arms targeting extracellular domains of HER2 and HER3. Recent clinical studies (Schram et al., 2022) have revealed that Zeno mediates durable clinical responses in a small number of NRG1 fusion-positive cancer patients. Using a single therapeutic agent presents significant challenges because the choice of binding site and scaffold structure can affect in vivo binding efficiency and therapeutic efficacy.

[0073] This application discloses a bispecific tetravalent antibody against HER2×HER3 characterized by a symmetrical configuration and a tetravalent composition of binding domains.

[0074] Example Example 1: Anti-HER2×HER3 bispecific tetravalent antibody By fusing a single-chain Fv (scFv) binding fragment to the N-terminus of the human IgG1 heavy chain, a bispecific tetravalent antibody simultaneously targeting HER2 domain IV (referred to as HER2 in this application) and HER3 was constructed. Figure 1A This anti-HER2×HER3 antibody has two heavy chains and two light chains, forming two typical bivalent Fab regions, and a pair of scFvs linked to the N-terminus of the heavy chain monomer. SI-71X25 and SI-71X24 are typical anti-HER2×HER3 tetravalent antibodies, possessing similar characteristics to... Figure 1B The structures shown are identical. SI-71X25 and SI-71X24 differ in their binding specificity to Fab and scFv, characterized by the interconversion of Fab regions or scFv portions. SI-71X25 is characterized by two anti-HER2 Fab regions and two anti-HER3 scFv domains, while SI-71X24 is characterized by two anti-HER3 Fab regions and two anti-HER2 scFv domains. Notably, the HER2-binding variable domain originates from trastuzumab (targeting domain IV), while the HER3-binding variable domain originates from MM-111. The two bivalent monospecific antibodies, SI-4C12 and SI-1C16, are internally generated and are equivalent to the two parental antibodies, trastuzumab and MM111, respectively. Figure 1C ).

[0075] Salt Bridge Engineering for Antibody Stabilization The anti-HER2×HER3 bispecific tetravalent antibody may exhibit more high aggregation after initial protein A purification than some other bispecific antibodies with different structures. This aggregation is presumably due to mispairing of the VH and VL domains during expression. Detection of trastuzumab / MM-111 Fv revealed that most VH / VL interface residues are bulky hydrophobic residues. Both trastuzumab and MM-111 Fv contain a contact between Q39 of VH and Q38 of VL (by Kabat number, ...). Figure 2A Mutating these polar but uncharged Gln residues to charged residues can form salt bridges. Importantly, reversing the charge orientation (VH+ / VL- vs. VH- / VL+) favors correct pairing but disadvantages incorrect strand pairing. Figure 2B Discovery Studio was used to predict the stability of all charge mutants to determine which were most likely to stabilize the correct pairings and destabilize the incorrect pairings. For this purpose, SI-71X33 is an optimized version of SI-71X25. Figure 1B ).

[0076] Generation of anti-HER2×HER3 antibodies SI-71X25, SI-71X24, and SI71X33 were designed and cloned, and incorporated into classic IgG1 antibodies fused to the N-terminus of the heavy chain with scFv, thereby forming bispecific bivalent Fab antibodies. Figure 1A (Table 1). Their characteristic is that all binding domains are bivalent. SI-71X25 and SI-71X33 are characterized by having two HER2-resistant Fab regions and two HER3-resistant scFv domains, while SI-71X24 is characterized by having two HER3-resistant Fab regions and two HER2-resistant scFv domains (Table 1). Figure 1B ).

[0077] Antibodies were expressed by transient transfection of heavy and light chain expression plasmids in the ExpiCHO system (Thermo Fisher). Briefly, 5.6 μg of each expression plasmid was taken and brought to a final volume of 2.4 ml using OptiPRO SFM medium containing 33.8 μg of cleaved herring sperm vector DNA. 2.2 ml of OptiPRO SFM medium containing 192 μl of Expifectamine CHO reagent was added to the DNA, and the mixture was incubated at room temperature for 5 minutes. The resulting mixture was then added at a rate of 6 × 10⁶ cells / ml to 60 ml of ExpiCHO cells in a 250 ml Erlenmeyer flask and incubated at 37°C, 5% CO₂, and 150 rpm. 24 hours post-transfection, 21 ml of ExpiCHO feed solution and 360 μl of CHO enhancer were added to the cells, and the mixture was transferred to a 32°C, 5% CO₂, and 150 rpm incubator. 48 hours post-transfection, another 21 ml of ExpiCHO feed solution was added to the cells. Nine days post-transfection, the culture supernatant was harvested, and cells were precipitated by centrifugation at 7500 rpm for 20 min, followed by filtering through a 0.2 mm filter. Expression titers were quantified using a biolayer interferometry assay on an Octet384 system, employing a protein A sensor and a standard curve prepared from purified antibodies.

[0078] The protein was purified from the harvested supernatant using a 5 ml MabSelect PrismA protein A chromatography column (Cytiva). The column was equilibrated with phosphate-buffered saline. The supernatant was then passed through the column at a flow rate of 5 ml / min. The column was washed with 25 ml of PBS. The protein was then eluted by passing 15 ml of 50 mM sodium acetate (pH 3.5) through the column. The eluted protein was immediately neutralized by adding 1 / 10 volume of 1 M sodium acetate (pH 7.0).

[0079] Immediately after the first-step purification of protein A or His tag, the protein was analyzed by analytical SEC using a Waters Acquity UPLC H-Class column with an ACQUITY UPLC® Protein BEHSEC 200Å, 4.6 mm × 150 mm, 1.7 µm column. Using PBS (125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8) as the mobile phase, 15 µg of protein was injected at 0.3 ml / min for 10 min. The protein was further purified by preparative SEC using a Superdex Increase 10 / 300 GL column with a mobile phase of 25 mM sodium acetate, 125 mM NaCl, pH 5.5, and the buffer was finally exchanged to 25 mM sodium acetate, 125 mM NaCl, 10% sucrose, pH 5.5. As assessed by analytical SEC, the final sample contained >95% of the target protein (POI) and was used for subsequent assays.

[0080] Example 2: Characterization of anti-HER2×HER3 antibodies Biological layer interferometry (Octet) binding assays were performed on an Octet 384 instrument to quantify the binding kinetics of antibodies to HER2 and HER3. For affinity assays, antibodies were captured onto the tip of the anti-human Fc (AHC) sensor by loading at 75 nM for 150 seconds. After a 60-second baseline step, association was performed for 180 seconds with His-tagged HER2 or HER3 (internal expression purified) at serial dilutions (0-100 nM for HER2; 0-400 nM for HER3; 1:2 dilution) in assay buffer (1% BSA, 0.05% Tween 20 phosphate-buffered saline), followed by 300 seconds of dissociation in assay buffer. Regeneration was achieved using 10 mM glycine (pH 1.5). The binding curves were globally fitted to a 1:1 model to obtain the dissociation constant KD and the kinetic association and dissociation rates.

[0081] Protein stability is a key parameter defined by the free energy difference between the folded and unfolded states. For protein therapeutics, stability can affect immunogenicity, pharmacokinetics, and even efficacy, and reduced 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. More efficient protein expression to achieve higher titers and increased purified protein yields can significantly reduce manufacturing costs.

[0082] Following transient expression in ExpiCHO cells, antibody titers were quantified using biolayer interferometry. All proteins were expressed in the ExpiCHO expression system, indicating sufficient stability for efficient production. Antibody stability and aggregation were immediately assessed by analytical size exclusion chromatography on a Waters UPLC system immediately after the first-step purification of protein A (Table 2). SI-71X24 showed a higher percentage of target protein (POI) after the first-step purification compared to SI-71X25. Interestingly, SI-71X33 exhibited a lower percentage of high-molecular-weight aggregates than its non-salt-bridge variant (SI-71X25). The data suggest that the salt-bridge mutation indeed stabilized the correct pairing of the V domains during expression and initial purification.

[0083] Example 3: Octet binding affinity of anti-HER2×HER3 antibody The affinity of anti-HER2×HER antibodies for HER2 (Table 3) and HER3 (Table 4) was assessed using biolayer interferometry. Figure 3A As shown, SI-71X24 exhibits impaired binding to both HER2 and HER3 compared to SI-71X25 and SI-71X33. Additionally, the KD calculations for SI-71X24 may not be entirely accurate due to its low koff of 1.00E-07 (Table 3). Notably, SI-71X33 shows higher HER2 and HER3 binding responses than its non-salt-bridge variant (SI-71X25). These data support the idea that the salt-bridged stable V-regions are properly paired, thereby increasing the proportion of molecules with functional antigen-binding domains.

[0084] Example 4: Antiproliferative effect of anti-HER2×HER3 antibody Breast cancer cells (BT-474) To test the effect of anti-HER2×HER3 bispecific antibodies on breast cancer cell growth, BT-474 cells expressing HER2 / HER3 were incubated with different concentrations of the test antibody, and proliferation was measured using Almar Blue.

[0085] The breast ductal carcinoma cell line BT-474 was purchased from ATCC (catalog number HTB-20) and maintained at 37°C and 5% CO2 in Hybri-Care medium supplemented with 10% fetal bovine serum. BT-474 cells were isolated from the flasks using trypsin and diluted to 1.2 × 10⁵ cells / ml in medium + 1% FBS. 50 μl of cell suspension (6000 cells) was seeded into 60 wells of a 96-well tissue culture plate. The outer wells were filled with 300 μl of sterile H₂O to minimize evaporation from the inner wells. Cells were allowed to adhere at 37°C and 5% CO2 for 4 hours. The test antibody was diluted to a final concentration of 2X in Hybri-Care medium + 1% FBS. 50 μl of the test antibody was added to each well, for a total volume of 100 μl / well. Each antibody was tested in triplicate at the following final concentrations: 100 nM, 25 nM, 6.25 nM, 1.563 nM, 0.391 nM, 0.098 nM, 0.024 nM, 0.006 nM, and 0.0015 nM. Each plate contained two antibodies, each at concentrations tested in triplicate. Each plate had six control wells containing only cells and culture medium. Immediately after adding the test compound, 10 μl of Almar Blue (Thermo Fisher, catalog number DAL1100) was added to three culture medium-only control wells on each plate. Cells were incubated at 37°C and 5% CO2 for 2 hours. After two hours of incubation, 110 μl of sample was removed from each control well and placed in a black opaque 96-well plate. The plate was centrifuged at 2000 RPM for 5 minutes to remove any air bubbles. Fluorescence (excitation = 535 nm, emission = 595 nm) was then measured on a Molecular Devices FilterMax F5 microplate reader. The measured control fluorescence value (C start) was used as a baseline to measure the endpoint proliferation. The plate was returned to 37°C and 5% CO2 for 7 days (168 hours). After incubation, 10 μl of Almar Blue was added to each test well and three other control wells (culture medium only). After incubation at 37°C and 5% CO2 for 2 hours, fluorescence was measured as described above. The control proliferation % was calculated using the endpoint control fluorescence value (C end) and the test well fluorescence value (T end) using the following formula: Control proliferation % = ((T) 结束 -C 开始 ) / (C 结束 -C 开始 ) 100 The results showed that SI-71X25 exerted an anti-proliferative effect, while SI-71X24 exerted a pro-proliferative activity. Figure 4AThis finding was unexpected, as their parental antibodies, trastuzumab and MM-111, are both known inhibitors of cancer cell proliferation and tumor growth. To confirm the antiproliferative activity of SI-71X25, SI-71X33 was tested together with its parental antibodies, trastuzumab and MM-111. Figure 4B (See below for details).

[0086] Head and neck cancer cells (FaDu) The proliferative activity of SI-71X33 and its parent antibodies SI-4C12 (anti-HER2 mAb) and SI-1C16 (anti-HER3 mAb) was analyzed using a protocol measuring confluence percentage. The results showed that SI-71X33 exerted anti-proliferative activity against FaDu head and neck cancer cells. Figure 4B Data points were analyzed using GraphPad Prism, and inhibition curves were fitted using nonlinear regression [log(inhibitor) vs. response, 4 parameters], with IC50 values ​​calculated (Table 5). If SI-71X25 is considered an effective inhibitor for treating BT-474 breast cancer cells, then SI-71X33 is an optimized effective inhibitor for FaDu cells.

[0087] Impact on other cancers To evaluate the binding activity of SI-71X25, SI-71X33, and SI-71X24 to HER2 / HER3-positive cancers, a group of cancer cell lines were used for indications, including breast cancer (SK-BR-3 and BT-474), pancreatic cancer (CFPAC-1), gastric cancer (NUGC-4), lung cancer (NCI-H358), endometrial cancer (Ishikawa), liver cancer (HepG2), epidermal cancer (A-431), head and neck cancer (FaDu), another form of lung cancer (Oka-C-1), and colon cancer (Colo-320). gMFI APC signal was presumably correlated with antibody expression density per cell. Using HER2 / HER3-negative lung cancer cells COR-L279, the same assay demonstrated no nonspecific binding. Results showed that the optimized SI-71X33 antibody could at least increase binding to SK-BR-3, BT-474, CFPAC-1, and NUGC-4 cells. Figure 6 ).

[0088] SI-71X25 (and SI-71X33) and SI-71X24 are all bispecific tetravalent antibodies against HER2×HER3, characterized by the linkage of the scFv domain to the N-terminus of each Fab domain. Our results show that positional exchange of the HER2 and HER3 binding domains does not affect binding specificity, but has completely opposite effects on proliferative activity, with anti-HER2 / D1 exhibiting proliferative activity. SI-71X25 is characterized by the pairing of anti-HER2 / D1 with anti-HER3 / D2, while SI-71X24 is characterized by the pairing of anti-HER3 / D1 with anti-HER2 / D2. Since HER2 binding originates from both scFv domains, the HER2-specific KD is 0.001 nM. Compared to the KD values ​​of SI-71X25, SI-71X33, and SI-4C12, SI-71X24 exhibits significantly enhanced HER2 binding affinity (Table 3). The significant increase in HER2 binding affinity may be a key reason for the shift in SI-71X24's activity from antiproliferative to proproliferative. High affinity binding levels are sufficient to activate the HER2 signaling pathway, similar to ligand binding to the HER2 receptor. HER2 is a tumor-associated antigen that has been used to develop targeted therapies for various solid tumors, such as breast, ovarian, uterine, gastric, and lung cancer. This discovery of the structure-function relationship could aid in the design and implementation of antibody therapies for treating HER2-expressing cancers.

[0089] sheet Table 1: The anti-HER2×HER3 bispecific tetravalent antibody is a homodimer of the antibody monomer, which contains the Fab region and the scFv domain at the C-terminus of the heavy chain. Table 2: Evaluation of expression titer and stability of anti-HER2×HER3 bispecific tetravalent antibody by analytical size exclusion chromatography after first-step protein A purification. Table 3: Binding kinetics (affinity) between anti-HER2×HER3 bispecific tetravalent antibody and HER2 in solution. Table 4: Binding kinetics (affinity) between anti-HER2×HER3 bispecific tetravalent antibody and HER3 in solution. Table 5: Efficacy and efficacy parameters of BT-474 proliferation assay using anti-HER2 / HER3 tetravalent antibody. SI-71X24 is a proliferative agent with an EC50 value. sequence list SIBA077 - HER2×HER3 Symmetrical Sequence List CDRs in the amino acid sequence are underlined. >SEQ ID 01 SI-71X24 heavy chain nucleotide sequence >SEQ ID 02 SI-71X24 Heavy Chain Amino Acid Sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSSGGGGSGGGGQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 03 SI-71X24 Light Chain Nucleotide Sequence CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTAGGCCAACCGAAAGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA >SEQ ID 04 Amino acid sequence of the light chain of SI-71X24 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS >SEQ ID 05 Nucleotide sequence of the heavy chain of SI-71X25 >SEQ ID 06 SI-71X25 Heavy Chain Amino Acid Sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 07 SI-71X25 Light Chain Nucleotide Sequence GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID 08 Amino acid sequence of SI-71X25 light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID 09 Nucleotide sequence of the variable heavy chain (VH) domain of anti-HER2 trastuzumab GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATAT GCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCGAATGAACAGCCTGCTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG >SEQ ID 10 Anti-HER2 trastuzumab variable heavy chain (VH) domain amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS >SEQ ID 11 Nucleotide sequence of the variable light chain (VL) domain of anti-HER2 trastuzumab GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCC TCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA >SEQ ID 12 amino acid sequence of anti-HER2 trastuzumab variable light chain (VL) domain DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK >SEQ ID 13 Anti-HER3 MM-111 Variable Heavy Chain (VH) Domain Nucleotide Sequence CAGGTGCAATTGCAGGAGTCGGGGGGAGGCCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGTTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCGCGATGGAAGTGCGAGTTAC TATGTGGACTCTGTGAAGGGCCGATTCACCATTCCAGAGACGACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGTGGGGTGGGCTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCGAGC >SEQ ID 14 Anti-HER3 MM-111 Variable Heavy Chain (VH) Domain Amino Acid Sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >SEQ ID 15 Anti-HER3 MM-111 Variable Light Chain (VL) Domain Nucleotide Sequence CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTA >SEQ ID 16 Anti-HER3 MM-111 variable light chain (VL) domain amino acid sequence QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >SEQ ID 17 Anti-HER2 trastuzumab scFv nucleotide sequence GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGTGGAGGGTCCGGCGGTGGAGGATCAGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG >SEQ ID 18 Amino acid sequence of anti-HER2 trastuzumab scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS >SEQ ID 19 Anti-HER3 MM-111 scFv nucleotide sequence CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGTTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCGCGATGGAAGTGCGAGTTACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGTGGGGTGGGCTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTGTCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGTTCCGGCGGTGGCGGCTCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTA >SEQ ID 20 Anti-HER3 MM-111 scFv Amino Acid Sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >SEQ ID 21 SI-71X33 Heavy Chain Nucleotide Sequence >SEQ ID 22 Amino acid sequence of the heavy chain of SI-71X33 QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRRAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQEHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVREAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 23 Nucleotide sequence of the light chain of SI-71X33 GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAAAGAAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID 24 SI-71X33 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQRKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID 25 Anti-HER2 trastuzumab CDR-H1 amino acid sequence DTYIH >SEQ ID 26 Anti-HER2 trastuzumab CDR-H2 amino acid sequence RIYPTNGYTRYADSVKG >SEQ ID 27 Anti-HER2 trastuzumab CDR-H3 amino acid sequence WGGDGFYAMDY >SEQ ID 28 Anti-HER2 trastuzumab CDR-L1 amino acid sequence RASQDVNTAVA >SEQ ID 29 Anti-HER2 trastuzumab CDR-L2 amino acid sequence SASFLYS >SEQ ID 30 Anti-HER2 trastuzumab CDR-L3 amino acid sequence QQHYTTPPT >SEQ ID 31 Anti-HER3 MM-111 CDR-H1 amino acid sequence SYWMS >SEQ ID 32 Anti-HER3 MM-111 CDR-H2 amino acid sequence NINRDGSASYYVDSVKG >SEQ ID 33 Anti-HER3 MM-111 CDR-H3 amino acid sequence DRGVGYFDL >SEQ ID 34 Anti-HER3 MM-111 CDR-L1 amino acid sequence TGTSSDVGGYNFVS >SEQ ID 35 Anti-HER3 MM-111 CDR-L2 amino acid sequence DVSDRPS >SEQ ID 36 Anti-HER3 MM-111 CDR-L3 amino acid sequence SSYGSSSTHVI References Each reference cited herein is incorporated herein by reference in its entirety. The inclusion of these references should not be construed as an admission that any material constitutes prior art in this application.

[0090] 1.Diaz-Serrano, A. et al.Genomic Profiling of HER2-Positive GastricCancer:PI3K / Akt / mTOR Pathway as Predictor of Outcomes in HER2-PositiveAdvanced Gastric Cancer Treated with Trastuzumab.Oncologist.23, 1092-1102(2018). 2.Durkee, BY, et al.Cost-Effectiveness of Pertuzumab in HumanEpidermal 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 PKBNegative Feedback Loop in Response to anti-HER2 Herceptin in BreastCancer.PLoS Biol.8, e1000563 (2010). 4.Goel, S. & Winer, E. P. POINT:HER2-Targeted Combinations inAdvanced HER2-Positive Breast Cancer.Oncology (Williston Park).29, 797-798,802 (2015). 5.Luque-Cabal, M. et al.Mechanisms Behind the Resistance toTrastuzumab in HER2-Amplified Breast Cancer and Strategies to OvercomeIt.Clin.Med.Insights Oncol.10, 21-30 (2016). 6.McDonagh, C. F. et al.Antitumor Activity of a Novel BispecificAntibody 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 CBenz. Biological effects of anti-ErbB2 single chain antibodies selected forinternalizing 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 CShaller, M von Mehren, I Shchaveleva, H H Simmons, J D Marks, G PAdams.Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhancestargeting selectivity and induces a therapeutic effect in vitro Br J Cancer2008 Nov 4;99(9):1415-25. 9.Wang, Q. et al.The anti-HER3 Antibody in Combination withTrastuzumab Exerts Synergistic Antitumor Activity in HER2-positive GastricCancer.Cancer Lett.380, 20-30 (2016). 10.Yang, L. et al.NRG1-depstartent Activation of HER3 Induces PrimaryResistance to Trastuzumab in HER2-overexpressing Breast Cancer Cells.Int. J.Oncol.51, 1553-1562 (2017). 11. Cetuximb: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. 21.https: / / cancerres.aacrjournals.org / content / 64 / 7_Supplement / 163.4.short 22.https: / / aacrjournals.org / cancerdiscovery / article / 12 / 5 / 1233 / 694554 / Zenocutuzumab-a-HER2 / HER3-Bispecific-Antibody-Is

Claims

1. A bispecific antibody with binding affinity for HER2 and HER3, comprising: Heavy chains (HC) having variable (VH) structural domains, wherein the heavy chains have N-termini and C-termini. Light chains (LC) with variable (VL) structural domains. The VL and VH domains form a Fab region, and The scFv domain has a variable (VL) domain for light chains and a variable (VH) domain for heavy chains, wherein each scFv domain is connected to the N end of each heavy chain via a connector.

2. The bispecific antibody according to claim 1, wherein the Fab region has binding affinity for HER2 and the scFv domain has binding affinity for HER3.

3. The bispecific antibody according to claim 2, wherein the Fab region has binding affinity for HER2 with a KD of about 0.5 nM to about 10 nM, and wherein the scFv domain has binding affinity for HER3 with a KD of about 100 nM to about 200 nM.

4. The bispecific antibody according to claim 2, wherein the Fab region comprises a VH domain having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 10, and a VL domain having at least 98% sequence identity with the amino acid sequence of SEQ ID NO:

12.

5. The bispecific antibody according to claim 2, wherein the VH domain comprises CDR-H1 with sequence identity to SEQ ID NO: 25, CDR-H2 with sequence identity to SEQ ID NO: 26, and CDR-H3 with sequence identity to SEQ ID NO: 27, and wherein the VL domain comprises CDR-L1 with sequence identity to SEQ ID NO: 28, CDR-L2 with sequence identity to SEQ ID NO: 29, and CDR-L3 with sequence identity to SEQ ID NO:

30.

6. The bispecific antibody according to claim 2, wherein the scFv domain comprises an scFv VH domain having at least 98% sequence identity with SEQ ID NO: 14, and an scFv VL domain having at least 98% sequence identity with SEQ ID NO:

16.

7. The bispecific antibody according to claim 2, wherein the scFv VH domain comprises CDR-H1 with sequence identity to SEQ ID NO: 31, CDR-H2 with sequence identity to SEQ ID NO: 32, and CDR-H3 with sequence identity to SEQ ID NO: 33, and wherein the scFv VL domain comprises CDR-L1 with sequence identity to SEQ ID NO: 34, CDR-L2 with sequence identity to SEQ ID NO: 35, and CDR-L3 with sequence identity to SEQ ID NO:

36.

8. The bispecific antibody according to claim 1, wherein the Fab region has binding affinity for HER3 and the scFv domain has binding affinity for HER2.

9. The bispecific antibody according to claim 8, wherein the Fab region has binding affinity for HER3 with a KD of about 1 pM to about 10 pM, and wherein the scFv domain has binding affinity for HER2 with a KD of about 100 nM to about 300 nM.

10. The bispecific antibody according to claim 8, wherein the Fab region comprises a VH domain having at least 98% sequence identity with SEQ ID NO: 14, and a VL domain having at least 98% sequence identity with SEQ ID NO:

16.

11. The bispecific antibody according to claim 8, wherein the VH domain comprises CDR-H1 having sequence identity with SEQ ID NO: 31, CDR-H2 having sequence identity with SEQ ID NO: 32, and CDR-H3 having sequence identity with SEQ ID NO: 33, and wherein the VL domain comprises CDR-L1 having sequence identity with SEQ ID NO: 34, CDR-L2 having sequence identity with SEQ ID NO: 35, and CDR-L3 having sequence identity with SEQ ID NO:

36.

12. The bispecific antibody according to claim 8, wherein the scFv domain comprises an scFv VH domain having at least 98% sequence identity with SEQ ID NO: 10, and an scFv VL domain having at least 98% sequence identity with SEQ ID NO:

12.

13. The bispecific antibody according to claim 8, wherein the scFv VH domain comprises CDR-H1 with sequence identity to SEQ ID NO: 25, CDR-H2 with sequence identity to SEQ ID NO: 26, and CDR-H3 with sequence identity to SEQ ID NO: 27, and wherein the scFv VL domain comprises CDR-L1 with sequence identity to SEQ ID NO: 28, LCDR CDR-L2 with sequence identity to SEQ ID NO: 29, and CDR CDR-L3 with sequence identity to SEQ ID NO:

30.

14. The bispecific antibody according to claim 1, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 2, 6 or 22.

15. The bispecific antibody according to claim 1, wherein the light chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 4, 8 or 24.

16. The bispecific antibody according to claim 1, wherein the VH domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 10 or 14, and wherein the VL domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 12 or 16.

17. The bispecific antibody according to claim 1, wherein the scFv domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 18 or 20.

18. The bispecific antibody according to claim 1, wherein the scFv VH domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 10 or 14, and wherein the scFv VL domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 12 or 16.

19. The bispecific antibody of claim 1, wherein the linker comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)m, and wherein m is an integer of at least 3.

20. An isolated nucleic acid sequence encoding the bispecific antibody of claim 1.

21. An expression vector comprising the isolated nucleic acid sequence of claim 20.

22. A host cell comprising the isolated nucleic acid sequence of claim 20.

23. A pharmaceutical composition comprising the bispecific antibody of claim 2 and optionally a pharmaceutically acceptable carrier.

24. The pharmaceutical composition of claim 23, further comprising a cytotoxic agent, wherein the cytotoxic agent comprises a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

25. An immunoconjugate comprising the bispecific antibody of claim 2 conjugated to a cytotoxic agent, wherein the cytotoxic agent comprises a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

26. A pharmaceutical composition comprising the immunoconjugate of claim 25 and optionally a pharmaceutically acceptable carrier.

27. A method of treating or preventing cancer in a subject, the method comprising administering to the subject an effective amount of the bispecific antibody of claim 2 or the immunoconjugate of claim 25.

28. The method of claim 27, further comprising co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

29. The method of claim 27, wherein the cancer comprises cells expressing HER3 or EGFR, and wherein the cancer comprises breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, endometrial cancer, epidermal 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.

30. A method for generating the bispecific antibody of claim 1, comprising: Culture host cells to express the DNA sequence encoding the bispecific antibody of claim 1, and The bispecific antibody was purified.

31. A method for producing the immunoconjugate of claim 25, comprising: The bispecific antibody of claim 2 is conjugated with a cytotoxic agent to provide the immunoconjugate, and The immunoconjugate was purified.

32. A solution comprising an effective concentration of the bispecific antibody of claim 1 or the immunoconjugate of claim 25, wherein the solution is plasma of a subject.

Images