Bispecific antibody-like proteins and methods of making and using the same

Abstract

An antibody-like protein having an N-terminus and a C-terminus, comprising a Fab region and an Fc region, wherein the Fc region comprises a first Fc domain linked to a second Fc domain by a second hinge, wherein the first Fc domain and the second Fc domain pair to form the Fc region. The second hinge can have the amino acid sequence (Gly-Gly-Gly-Gly-Ser)6. The antibody-like protein can further comprise a scFv domain, and the antibody-like protein has binding affinity for HER2 and HER3.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application Serial No. 63 / 604,077, 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 antibody-like proteins. 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 field of immunotherapy, more specifically to antibody therapeutics, and even more specifically to antibody-like proteins targeting specific epitopes of HER2 and HER3. HER2 and HER3 often form a partnership that promotes cell transformation, ultimately leading to tumorigenesis and metastasis. This application particularly provides antibody-like proteins, immunoconjugates of such antibody-like proteins, methods for preparing antibody-like proteins or immunoconjugates thereof, pharmaceutical compositions comprising antibody-like proteins and / or their immunoconjugates, and methods for treating diseases using such antibody-like proteins or their immunoconjugates, including, for example, cancer.

[0009] In one aspect, this application provides an antibody-like protein having an N-terminus and a C-terminus. In one embodiment, the protein comprises a Fab region and an Fc region. The Fc region comprises a first Fc domain connected to a second Fc domain via a second hinge. The first Fc domain can pair with the second Fc domain to form the Fc region.

[0010] In one embodiment, the second hinge connecting the first Fc domain and the second Fc domain comprises a flexible GS connector having about 10 to about 100, about 20 to about 80, or about 20 to about 40 amino acids. In one embodiment, the second hinge comprises the amino acid sequence ((Gly-Gly-Gly-Gly-Ser)n, where n is an integer of at least 5. In one embodiment, n is 5 to 10, 6 to 12, 5 to 20, or 5 to 15. In one embodiment, n is 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20. In one embodiment, n is 6.

[0011] In one embodiment, the antibody-like protein comprises a light chain and a heavy chain. The light chain comprises a VL domain and a CL domain from the N-terminus to the C-terminus. The heavy chain comprises a VH domain, a CH1 domain, a first hinge, and an Fc region from the N-terminus to the C-terminus. In one embodiment, the VL domain and the VH domain form a Fab region.

[0012] In one embodiment, the light and heavy chains are covalently paired via at least one disulfide bond between the CH1 and CL domains. In one embodiment, the CL domain can be Cκ or Cλ.

[0013] In one embodiment, the first Fc domain comprises a first CH2 domain and a first CH3 domain. In one embodiment, the second Fc domain comprises a second CH2 domain and a second CH3 domain. In one embodiment, the first Fc domain and the second Fc domain are covalently paired by two disulfide bonds between the first hinge and the second hinge.

[0014] Antibody-like proteins can be single-specific, bispecific, or multispecific.

[0015] In one embodiment, the antibody-like protein may further include a first scFv domain having scFv VH and scFv VL domains. The first scFv domain may be linked to the N-terminus of the heavy chain, the C-terminus of the heavy chain, or the N-terminus of the light chain via a linker. In one embodiment, the first scFv domain is linked to the N-terminus of the heavy chain.

[0016] The antibody-like protein may also include a second scFv domain. In one embodiment, the second scFv domain may be tandemly linked to the first scFv domain. In another embodiment, the tandemly linked first and second scFv domains can be connected to the N-terminus of the heavy chain via a linker.

[0017] In one implementation, the first and second scFv structural domains can be the same. In another implementation, the first scFv structural domain can be different from the second scFv structural domain.

[0018] In one implementation, the antibody-like protein can be bispecific. For example, the antibody-like protein can have binding affinity for both HER2 and HER3.

[0019] The Fab region may have binding affinity for HER2, and the first scFv domain may have binding affinity for HER3. In one embodiment, the antibody-like protein may include first and second scFv domains, each with binding affinity for HER3.

[0020] In one embodiment, the Fab region has a binding affinity for HER2 with a KD of about 1 pM to about 100 nM, about 1 pM to about 10 nM, about 2 pM to about 5 nM, or about 3 pM to about 2 nM. In one embodiment, the first or second scFv domain has a binding affinity for HER3 with a KD of about 1 nM to about 1 μM, about 50 nM to about 500 nM, about 60 nM to about 200 nM, about 80 nM to about 300 nM, about 80 nM to about 150 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, or about 100 nM to about 1 μM.

[0021] The Fab region may have binding affinity for HER3, and the first scFv domain may have binding affinity for HER2. In one embodiment, the antibody-like protein may include first and second scFv domains, each with binding affinity for HER2.

[0022] In one embodiment, the Fab region has a binding affinity for HER3 with a KD of about 1 pM to about 1 μM, about 1 nM to about 120 nM, about 50 nM to about 500 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, or about 100 nM to about 1 μM. In one embodiment, the first or second scFv domain has a binding affinity for HER2 with a KD of about 1 pM to about 100 nM, about 1 pM to about 10 nM, about 3 pM to about 5 nM, about 10 pM to about 5 nM, or about 3 pM to about 2 nM.

[0023] In one embodiment, the heavy chain of the antibody-like protein comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6, 10, 18, 36, 40, 44. In one embodiment, the VH domain of the heavy chain comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 22, 26. In one embodiment, the VH domain of the heavy chain comprises CDR-H1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 47, 53; CDR-H2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 48, 54; and CDR-H3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 49, 55.

[0024] In one embodiment, the heavy chain VH domain comprises CDR-H1 having SEQ ID NO: 47, CDR-H2 having SEQ ID NO: 48, and CDR-H3 having SEQ ID NO: 49. In another embodiment, the heavy chain VH domain comprises CDR-H1 having SEQ ID NO: 53, CDR-H2 having SEQ ID NO: 54, and CDR-H3 having SEQ ID NO: 55.

[0025] In one embodiment, the light chain of the antibody-like protein comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, 12, 20, 38, 42, 46. In one embodiment, the light chain VL domain comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24, 28. In one embodiment, the light chain VL domain comprises CDR-L1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 50, 56; CDR-L2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 51, 57; and CDR-L3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 52, 58.

[0026] In one embodiment, the light chain VL domain comprises CDR-L1 having SEQ ID NO: 50, CDR-L2 having SEQ ID NO: 51, and CDR-L3 having SEQ ID NO: 52. In another embodiment, the light chain VL domain comprises CDR-L1 having SEQ ID NO: 56, CDR-L2 having SEQ ID NO: 57, and CDR-L3 having SEQ ID NO: 58.

[0027] In one embodiment, the Fab region may include CDR-H1 having SEQ ID NO: 47, CDR-H2 having SEQ ID NO: 48, and CDR-H3 having SEQ ID NO: 49; and CDR-L1 having SEQ ID NO: 50, CDR-L2 having SEQ ID NO: 51, and CDR-L3 having SEQ ID NO: 52.

[0028] In one embodiment, the Fab region may include CDR-H1 having SEQ ID NO: 53, CDR-H2 having SEQ ID NO: 54, and CDR-H3 having SEQ ID NO: 55; and CDR-L1 having SEQ ID NO: 56, CDR-L2 having SEQ ID NO: 57, and CDR-L3 having SEQ ID NO: 58.

[0029] In one embodiment, the antibody-like protein comprises a first scFv domain or a second scFv domain, the amino acid sequence of which has at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 30, 32, or 34.

[0030] In one embodiment, the first or second scFv domain comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 22, 26. In one embodiment, the first or second scFv VH domain comprises CDR-H1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 47, 53, CDR-H2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 48, 54, and CDR-H3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 49, 55.

[0031] In one embodiment, the first or second scFv VH structural domain comprises CDR-H1 having SEQ ID NO: 47, CDR-H2 having SEQ ID NO: 48, and CDR-H3 having SEQ ID NO: 49. In one embodiment, the first or second scFv VH structural domain comprises CDR-H1 having SEQ ID NO: 53, CDR-H2 having SEQ ID NO: 54, and CDR-H3 having SEQ ID NO: 55.

[0032] In one embodiment, the first or second 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: 24, 28. In one embodiment, the first or second scFv VL domain comprises CDR-L1 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 50, 56, CDR-L2 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 51, 57, and CDR-L3 having at least 98%, 99%, or 100% sequence identity with SEQ ID NO: 52, 58.

[0033] In one embodiment, the first or second scFv VL domain includes CDR-L1 having SEQ ID NO: 50, CDR-L2 having SEQ ID NO: 51, and CDR-L3 having SEQ ID NO: 52. In one embodiment, the first or second scFv VL domain includes CDR-L1 having SEQ ID NO: 56, CDR-L2 having SEQ ID NO: 57, and CDR-L3 having SEQ ID NO: 58.

[0034] In one embodiment, the first or second scFv structural domain includes CDR-H1 having SEQ ID NO: 47, CDR-H2 having SEQ ID NO: 48, and CDR-H3 having SEQ ID NO: 49; and CDR-L1 having SEQ ID NO: 50, CDR-L2 having SEQ ID NO: 51, and CDR-L3 having SEQ ID NO: 52.

[0035] In one embodiment, the first or second scFv structural domain includes CDR-H1 having SEQ ID NO: 53, CDR-H2 having SEQ ID NO: 54, and CDR-H3 having SEQ ID NO: 55; and CDR-L1 having SEQ ID NO: 56, CDR-L2 having SEQ ID NO: 57, and CDR-L3 having SEQ ID NO: 58.

[0036] In one embodiment, the linker connecting the first scFv domain and the second scFv domain, or the linker connecting the first scFv domain and the heavy or light chain, may be a flexible GS linker. These two linkers may be different or the same. Each linker may independently have about 10 to about 20, about 15 to about 50, about 20 to about 40, or about 10 to about 50 amino acids. In one embodiment, each linker may independently contain the amino acid sequence ((Gly-Gly-Gly-Gly-Ser)m. In one embodiment, m is an integer of at least 3. In one embodiment, the linkers may be 3 to 5, 2 to 10, or 3 to 8. In one embodiment, m is 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, m is 4.

[0037] In a second aspect, this application provides isolated nucleic acid sequences. In one embodiment, the isolated nucleic acid sequence encodes the antibody-like protein disclosed herein.

[0038] In another aspect, this application provides expression vectors. In one embodiment, the expression vector may comprise an isolated nucleic acid sequence encoding an antibody-like protein disclosed herein.

[0039] In another aspect, this application provides a host cell. In one embodiment, the host cell may contain an isolated nucleic acid sequence encoding an antibody-like protein disclosed herein.

[0040] In another aspect, this application provides immunoconjugates. In one embodiment, the immunoconjugate may comprise an antibody-like protein conjugated to a cytotoxic agent. In one embodiment, the cytotoxic agent may include a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof. In one embodiment, the immunoconjugate may comprise an antibody-like protein conjugated to an imaging agent.

[0041] In another aspect, this application provides pharmaceutical compositions.

[0042] In one embodiment, the pharmaceutical composition may comprise an antibody-like protein disclosed herein. In another embodiment, the pharmaceutical composition may comprise an immunoconjugate disclosed herein.

[0043] In one embodiment, the pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier. 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.

[0044] In another aspect, this application provides methods for treating or preventing cancer in a subject. In one embodiment, the method may include the step of administering to the subject a pharmaceutical composition comprising an antibody-like protein or an immunoconjugate thereof.

[0045] In one embodiment, the method may include co-administering an effective amount of the therapeutic agent. In one embodiment, the therapeutic agent may be an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

[0046] In one implementation, the cancer includes cells expressing HER2 and / or HER3. In one implementation, the cancer includes 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.

[0047] In one implementation, the subject can be a human being.

[0048] In another aspect, this application provides a method for generating protein-like antibodies or immunoconjugates thereof.

[0049] In one embodiment, this application provides a method for preparing an antibody-like protein. In one embodiment, the method may include the steps of culturing host cells to express a DNA sequence encoding the antibody-like protein, and purifying the antibody-like protein.

[0050] In one embodiment, this application provides a method for generating an immunoconjugate derived from an antibody-like protein. In one embodiment, the method may include the step of conjugating the antibody-like protein to a pharmaceutical moiety or a cytotoxic agent. In one embodiment, the pharmaceutical moiety or cytotoxic agent may be 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.

[0051] In another aspect, this application provides a solution. In one embodiment, the solution contains an effective concentration of an antibody-like protein, its immunoconjugate, or a combination thereof. In one embodiment, the solution is the plasma of a subject. Attached Figure Description

[0052] The foregoing and other features of this disclosure will 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 an antibody-like protein consisting of a monovalent Fab as its first binding domain (D1), one or two monovalent scFvs as its second binding domain (D2) and attached to the N-terminus of the heavy chain monomer (1A), and anti-HER2 / HER3 bispecific antibody-like proteins (1B) with various compositions and configurations shown.

[0053] 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 that favors reverse charge orientation (VH+ / VL- vs. VH- / VL+) (2B upper panel), and the incorrect pairing (2B lower panel).

[0054] Figure 3 shows the Octet binding affinity of two monospecific control antibodies, SI-71MM4 (αHER2) and SI-71MM7 (αHER3) (3A); anti-HER2×HER3 bispecific bivalent and trivalent (bivalent against HER3) antibody-like proteins SI-71XM21 and SI-71XM20 (3B); anti-HER2 (D2) / HER3 (D1) bispecific antibody-like proteins with and without mutations for proper pairing (3C); anti-HER2 (D1) / HER3 (D2) bispecific antibody-like proteins with and without mutations for proper pairing (3D); and two monospecific antibodies against HER2 (SI-4C12) and HER3 (SI-1C16) (3E), respectively.

[0055] Figure 4 The Octet binding affinity of SI-71MX21 and SI-71MX21 (HER3) is shown.

[0056] Figure 5 The study demonstrated the antiproliferative effect of anti-HER2 / HER3 monovalent Fab antibody-like protein on BT-474 cells (a breast cancer cell line expressing HER2 and HER3) in an Alamar-blue proliferation assay, indicating the positional effect of the D1 and D2 binding domains in the antibody-like protein.

[0057] Figure 6 illustrates the antiproliferative effect of anti-HER2 / HER3 monovalent Fab antibody-like protein on FaDu cells (a head and neck cancer cell line expressing HER2 and HER3) in the Almar Blue proliferation assay, indicating that SI-71XM40 (6A) and SI-71XM41 (6B) have an improving effect on the anti-HER2 / HER3 bispecificity, while SI-71XM40 (6C) or SI-71XM41 (6D) has no improving effect on the mutation effect for proper pairing.

[0058] Figure 7 The mean geometric mean fluorescence intensity (gMFI) of antibodies or antibody-like proteins (10 ng SI-71MX40, SI-71MX41) binding to 10 cancer cell lines expressing HER2 / HER3 is shown; 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 origin, cell line name, HER2 antibody binding capacity, and HER3 antibody binding capacity. Detailed Implementation

[0059] This disclosure provides antibody-like proteins and their immunoconjugates that possess therapeutic properties or efficacy superior to currently known antibodies targeting one or two members of the EGFR family. In one embodiment, the antibody-like protein is structurally configured to effectively target two members of the EGFR family: HER2 and HER3. The antiproliferative activity of these bispecific antibody-like proteins can be generated by simultaneously blocking or inhibiting oncogenic signaling mediated by different receptors.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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. In general, the 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) can recognize and bind antigens, although its affinity is lower than that of 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.

[0065] The term "VH-VL pairing" refers to the selection of appropriate human lineages during the humanization of the heavy and light chains to form a stable Fv. The mutual orientation of the VH and VL domains should correspond to the orientation observed in the parent antibody.

[0066] 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.

[0067] 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.

[0068] The term "antibody-like protein" refers to an antibody-like protein that can bind specifically to an antigen with high specificity and affinity. Sometimes, in this application, "antibody" and "antibody-like protein" are used interchangeably.

[0069] 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.

[0070] Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these can be further subdivided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; and IgA-1 and IgA-2. 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.

[0071] The term "valence" refers to the number of antigenic determinants that a single antibody molecule can bind to. All natural antibodies are at least divalent, while the term "monovalent" refers to an engineered antibody or antibody-like protein with a single binding domain containing a pair of six hypervariable complementary determinant regions (CDRs) (i.e., three HC-CDRs and three LC-CDRs). In this paper, "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.

[0072] 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 a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Recombinant" means that the antibody is generated in a foreign host cell using recombinant nucleic acid technology. Monoclonal antibodies can be produced using a variety of methods, including but not limited to mouse hybridoma, phage display, recombinant DNA, direct molecular cloning of antibodies from primary B cells, and antibody discovery methods. Monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to a 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] This application suggests that antibody-like proteins potentially offer advantages over any combination therapy, which is generally more toxic than monotherapy. Bispecific agents, such as the bispecific antibody-like proteins 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. Compared to combination therapies using two monoclonal antibodies, bispecific therapeutic agents, such as the antibody-like proteins disclosed herein, are less toxic to patients and / or more effective due to increased binding specificity.

[0081] In one aspect, this application provides a bispecific antibody-like protein 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 an antibody. The Fab region and the scFv domain each independently exhibit binding specificity for different proteins in the EGFR family.

[0082] 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-like protein 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 (SGm)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.

[0083] 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.

[0084] 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.

[0085] Bispecific antibody-like proteins may include immunoglobulin G (IgG) moieties having at least a light chain and a heavy chain. In one embodiment, the antibody-like protein may include two scFv moieties covalently linked to the C or N terminus of the heavy or light chain via a linker such as (Ser-Gly-Gly-Gly-Gly)n linker, (Gly-Gly-Gly-Gly-Ser)n linker, (Gly-Gly-Gly-Ser)n linker, or (GmS)n or (SGm)n linker.

[0086] 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.

[0087] 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).

[0088] 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).

[0089] MCLA-128 (Zeno, zastuzumab) is a bispecific humanized immunoglobulin G1 (IgG1) containing two distinct Fab arms that target the 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.

[0090] 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 in patients. This application discloses anti-HER2 / HER3 antibodies characterized by their conformation and domain composition (see Table 1 and Figure 1). In some embodiments, the antibody-like protein comprises a monovalent Fab for binding HER2 or HER3 as a first binding domain (D1). In some embodiments, one or two scFv domains are linked to the N-terminus of the heavy chain variable domain as a second binding domain (D2). In one embodiment, the antibody-like protein comprises an anti-HER2 / HER3 monovalent Fab antibody having a second Fc linked to the C-terminus of the heavy chain.

[0091] Example Example 1: Anti-HER2×HER3 bispecific monovalent Fab antibody Bispecific monovalent Fab antibody-like protein A class of bispecific monovalent Fab antibody-like proteins is characterized by a single heavy chain and a single light chain ( Figure 1A The single heavy chain contains a classic variable heavy chain (VH) domain linked to the CH1 domain of human IgG1, and the variable domains of the heavy chain (VH) and light chain (VL) form a monovalent Fab region, acting as the first binding domain (D1). The second binding domain (D2) can be a monovalent or bivalent scFv linked to the N-terminus of the heavy chain. Another characteristic of this type of antibody-like protein is the connection of two inverse IgG1 Fc elements, including a hinge, a CH2 domain, and a CH3 domain, separated by a 30-residue flexible (GSSSS)6 linker. The second hinge immediately adjacent to the linker has five residues removed from its N-terminus, leaving only two cysteine ​​residues involved in the typical interchain disulfide bond present in the normal human IgG1 molecule. In this way, in the absence of heavy chain dimerization, the second Fc can stabilize the structure of this type of atypical antibody by causing the two Fcs to fold together to form an interchain disulfide bond. Figure 1A This antibody-like protein has a compact structure and can be used to form bispecific antibodies containing monovalent binding specificity (D1) and monovalent or multivalent binding specificity (D2).

[0092] Anti-HER2(IV)×HER3 antibody-like protein To identify effective therapeutic agents for treating cancers expressing HER2 / HER3, eight anti-HER2(IV)×HER3 antibody-like proteins were constructed on a monovalent Fab platform (see Table 1 and...). Figure 1B The sequences encoding the HER2 and HER3 binding domains are derived from trastuzumab (specific to HER2 domain IV) and MM-111 (specific to HER3). The antibody-like protein heavy chain consists of VH, CH1, hinge, CH2, CH3, 30aa linker, hinge, CH3, and CH2 from human IgG1 or IgG2.

[0093] SI-71MM7 and SI71MM4 were prepared as monospecific control antibodies against HER2 / HER3, respectively. SI-71MX21 and SI-71MX20 were prepared to compare the effects of monovalent and bivalent HER3 binding. SI-71MX19 and SI-71MX41 were prepared to test the mutagenic effects of VH-VL pairing, where D1 was HER3-specific and D2 was HER2-specific. Conversely, SI-71MX32 and SI-71MX40 were prepared to test the mutagenic effects of VH-VL pairing, where D1 was HER2-specific and D2 was HER3-specific. From another perspective, SI-71MM4, SI-71XM21, and SI-71XM20 are characterized by having a monovalent HER2-resistant Fab region, while SI-71MM7, SI-71XM19, SI-71MX41, SI-71MX32, and SI-71MX40 are characterized by having a monovalent HER3-resistant Fab region. Figure 1B In general, these anti-HER2 / HER3 antibody-like proteins, characterized by monovalent Fab and single-chain Fc, can be generated by co-transfection with suitable light chains.

[0094] Salt Bridge Engineering for Antibody-like Protein Stabilization The anti-HER2×HER3 bispecific antibody-like protein from the monovalent Fab platform may exhibit higher aggregation after initial protein A purification compared to some other bispecific antibodies or antibody-like proteins with different structures. This aggregation is presumably due to mispairing of the VH and VL domains during expression. Analysis 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 The stability of all charge mutants was predicted using Discovery Studio to determine which were most likely to stabilize correct pairings and destabilize incorrect pairings. For this purpose, SI-71XM41 and SI-71XM40 were prepared to optimize SI-71XM19 and SI-71XM20 (i.e., SI-71XM32), respectively. Figure 1B ).

[0095] Example 2: Generation of anti-HER2×HER3 antibody-like proteins Using standard molecular biology techniques, the genes encoding the antibody heavy and light chains (preceded by Kozak and secretion signal peptides) were cloned into the pTT5 vector. As shown in Table 1 and Figure 1, this type of anti-HER2×HER3 monovalent Fab antibody-like protein contains a heavy chain consisting of a standard variable heavy chain (VH) domain fused to the human IgG1 CH1 domain and two IgG1 Fc elements (hinge, CH2 domain, CH3 domain) separated by a 30-residue flexible (GSSSS)6 linker. The second hinge, immediately adjacent to the linker, has five N-terminal residues removed, leaving only two cysteine ​​residues involved in the typical interchain disulfide bonds present in normal human IgG1 molecules; therefore, this single-chain Fc (scFc) folds correctly and binds to protein A. The N-terminal cysteine ​​in the first hinge participates in the interchain disulfide bond between the light chain and the scFc heavy chain. Co-transfection of the scFc heavy chain with the normal antibody light chain produces monovalent Fab-Fc, which can be purified by protein A affinity chromatography. A single-chain variable fragment (scFv) can be fused to the N-terminus of the scFc heavy chain to prepare a bispecific monovalent Fab antibody-like protein. The resulting antibody-like protein has a monovalent Fab-binding domain, and the other binding domain is monovalent or bivalent depending on whether a single scFv or tandem scFv is fused to the N-terminus. SI-71MM4, SI-71XM21, and SI-71XM20 are generated as exemplary anti-HER2 monovalent Fab antibody-like proteins, characterized by the attachment of an anti-HER3 scFv to the N-terminus of the heavy chain; while SI-71MM7, SI-71MX19, SI-71MX41, SI-71MX32, and SI-71MX40 are generated as exemplary anti-HER3 monovalent Fab antibody-like proteins, characterized by having a HER2-binding scFv domain linked to the heavy chain. Figure 1B ).

[0096] Antibody-like proteins 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 OptiPROSFM medium containing 33.8 μg of cleaved herring sperm vector DNA. 2.2 ml of OptiPROSFM medium containing 120 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 temperature of 32°C, 5% CO₂, and 360 rpm. 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 biolayer interferometry on an Octet384 system, employing a protein A sensor and a standard curve prepared from purified antibody-like proteins.

[0097] 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).

[0098] 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.

[0099] Example 3: Characterization of anti-HER2×HER3 bispecific antibody-like protein Biological layer interferometry (Octet) binding assays were performed on an Octet 384 instrument to quantify the binding kinetics of antibody-like proteins with HER2 and HER3. For affinity assays, antibody-like proteins were captured to the tip of the anti-human Fc (AHC) sensor by loading 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 purification) 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). For affinity assays, biotinylated HER3 was captured to the streptavidin sensor by loading 2 μg / ml for 150 seconds. Following a 60-second baseline step, the antibody protein was associated with serially diluted (0-100 nM) assay protein in assay buffer for 180 seconds, followed by dissociation in assay buffer for 300 seconds. The binding curve was globally fitted to a 1:1 model to obtain the dissociation constant KD and the kinetic association and dissociation rates.

[0100] 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.

[0101] Following transient expression in ExpiCHO cells, the titers of antibody-like proteins were quantified using a biolayer interferometry method. As shown in Table 2, the data demonstrate that all proteins can be expressed in the ExpiCHO expression system, indicating that they are sufficiently stable and can be produced efficiently. For bispecific monovalent Fab antibody-like proteins, a general trend was observed where decreasing titers corresponded to increasing numbers of binding domains. For example, SI-71MM4 (anti-HER2 Fab only) had a higher titer than SI-71XM21 (anti-HER2 Fab plus an anti-HER3 scFv). SI-71XM20, containing both anti-HER2 Fab and tandem anti-HER3 scFv, had even lower titers. Although the titer of SI-71XM41 (salt-bridge variant) was slightly lower than that of SI-71XM19, the introduction of the salt-bridge mutation did not significantly alter the expression titer.

[0102] Immediately after the first step of protein A purification, the stability and aggregation of antibody-like proteins were assessed by analytical size exclusion chromatography on a Waters UPLC system. An unexpected phenomenon was observed with anti-HER2 / HER3 monovalent Fab antibody-like proteins containing a monovalent anti-HER2 Fab region. As shown in Table 2, SI-71XM20, with a tandem anti-HER3 scFv domain, exhibited a higher percentage of target protein (POI) after the first step of purification than SI-71XM21, which has a single anti-HER3 scFv domain. This higher %POI is attributed to a lower content of high molecular weight (HMW) aggregates. SI-71XM19 showed a higher percentage of HMW than its salt-bridge mutant variant, SI-71XM41. SI-71XM32 contained over 65% HMW. Its salt-bridge mutant variant, SI-71XM40, had a significantly lower percentage of HMW aggregates. The data suggest that the salt-bridge mutation indeed stabilized the correct pairing of the V domains during expression and initial purification.

[0103] Example 4: Octet binding affinity of anti-HER2×HER3 antibody-like proteins Anti-HER2 / HER3 antibody-like proteins share characteristic structural features of monovalent Fab and single-chain Fc regions. Figure 1BUnder these unified characteristics, anti-HER2×HER3 bispecific antibody-like proteins can be divided into two groups based on the position of the HER2 binding domain at D1 (HER2 / D1) and D2 (HER2 / D2). To analyze structure-function relationships, the binding affinity of each antibody to HER2 and HER3 was assessed using biolayer interference. As expected, the anti-HER2 monospecific antibody-like protein SI-71MM4 showed binding specificity for HER2 but not for HER3, while the anti-HER3 monospecific antibody-like protein SI-71MM7 showed binding specificity for HER3 but not for HER2. Figure 3A All anti-HER2×HER3 bispecific antibody-like proteins exhibit binding affinity for both HER2 and HER3. Figure 3B , 3C (See Table 3). To compare the affinity of monovalent and bivalent antibodies for HER2 and HER3, the bivalent monospecific antibodies trastuzumab (SI-4C12) and MM111 (SI-1C16) were used as controls. The results are shown in Table 3. Figure 3E As shown in Table 3.

[0104] To quantify the positional effect of the HER2 or HER3 binding domains, binding affinity KD values ​​are listed in Tables 3A and 3B, respectively. Except for SI-77XM20, HER2 / D1 antibody-like proteins, including SI-77MM4, SI-71XM21, SI-71XM32, SI-71XM40, and SI-4C12, showed binding affinity for HER2, with KD values ​​ranging from 0.967 nM (SI-71MM4) to 1.34 nM (SI-4C12). SI-71MM4 and SI-4C12 are bivalent monospecific controls against HER2. The KD values ​​of SI-71XM21, SI-71XM32, and SI-71XM40 are very close, indicating that HER2 / D1 functions independently in this monovalent Fab bispecific antibody platform, just as it does in the controls. The KD value of SI-71XM20 was 0.003 nM, and this 30-fold increase was attributed to a significant decrease in the KOFF value. On the other hand, these HER2 / D1 antibody-like proteins showed binding affinity for HER3, with KD values ​​ranging from 91.8 nM (SI-71XM20) to 236.7 nM (SI-71XM21). HER3 / D1 antibody-like proteins, including SI-71MM7, SI-71XM19, SI-71XM41, and SI-1C16, showed binding affinity for HER3, with KD values ​​ranging from 147 nM (SI-71XM19) to 364.7 nM (SI-MM7), where SI-71MM7 is a monovalent monospecific control. This indicates that increasing the valence of either HER2 or HER3 increases HER3 binding affinity. The KD values ​​of the HER3 / D1 bispecific antibody-like proteins SI-71XM19 and SI-71XM41 binding to HER2 were 0.93 nM and 1.1 nM, respectively, which are comparable to the KD values ​​of HER2 / D1 antibody-like proteins except SI-77XM20. These data indicate that although the HER3-binding domain derived from MM-111 may exhibit lower affinity in the monovalent Fab structure (SI-71MM7), bivalent antibody-like proteins significantly enhance binding affinity in two ways. First, the bivalent HER3 / D1 in SI-1C16, as well as the bivalent structures of HER3 / D1 and HER2 / D2, can increase binding affinity to KD values ​​of 168 nM and 0.93 nM, respectively. Second, the synergistic effect of HER2 and HER3 binding in the monovalent Fab environment was unexpected but clearly demonstrated in SI-71XM20, which exhibited the lowest KD values ​​for binding to both HER2 and HER3.

[0105] Example 5: Octet binding affinity of anti-HER2×HER3 antibody-like proteins To assess the affinity of SI-71XM21 and SI-71XM20 antibody-like proteins for HER3, a biolayer interferometry method was used. In contrast to the “affinity” assay that uses an AHC sensor to measure the binding of immobilized antibody to HER3 in solution, this assay used a streptavidin (SA) sensor to measure the binding of immobilized biotinylated HER3 to antibody in solution. Therefore, this assay simulates the affinity effect observed when proteins are present on surfaces such as cell membranes. Both SI-71XM20 and SI-71XM21 are HER3 / D2 antibody-like proteins with one or two anti-HER3 scFv domains. SI-71XM21 showed relatively weak affinity for immobilized HER3 (112.8 nM) and a low binding response (0.295 nm), while SI-71XM20 showed excellent affinity for immobilized HER3 (1.7 nM) and an increased binding response (0.881 nm). Figure 4 (and Table 4). This affinity effect overcomes the inherent low affinity of scFv derived from MM-111, allowing it to bind tightly to tumor cells that overexpress HER3, while binding less to cells with lower HER3 surface expression levels.

[0106] Example 6: Antiproliferative effect of anti-HER2×HER3 antibody-like protein Breast cancer cells (BT-474) To test the effect of anti-HER2×HER3 bispecific antibody-like protein on breast cancer cell growth, BT-474 cells expressing HER2 and HER3 were incubated with different concentrations of the test antibody-like protein, and proliferation was measured using Almar Blue.

[0107] 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 or antibody-like protein 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 or antibody-like protein 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 or antibody-like proteins, 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 Amaranth 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 was then measured on a Molecular Devices FilterMax F5 microplate reader (excitation = 535 nm, emission = 595 nm). The measured control fluorescence value (Cstart) 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 (Cstart) and the test well fluorescence value (Tstart) using the following formula: Control proliferation % = ((T) 结束 -C 开始 ) / (C 结束 -C 开始 ) 100 Data points were analyzed using GraphPad Prism, and the inhibition curve was fitted using nonlinear regression [log(inhibitor) vs. response, 4 parameters], and the IC50 value was calculated.

[0108] Given the differences in binding affinity of SI-71XM21, SI-71XM20, and SI-71XM19 for HER2 and HER3, their structure and valence influence their function. Specifically, pairing of anti-HER2 / D1 with anti-HER3 / D2 or anti-HER2 / D2 with anti-HER3 / D1 results in varying degrees of effectiveness. In fact, all three antibody-like proteins exhibited antiproliferative activity with double-digit IC50 values ​​and different percentages of efficacy. Moreover, SI-71XM21 demonstrated higher efficacy, consistent with the synergistic effect resulting from HER3 and HER2 binding. Figure 5 (and Table 5).

[0109] Head and neck squamous cell carcinoma (FaDu) HER2 expression in breast cancer may differ from that in head and neck cancer. For example, gene amplification in breast cancer leads to high levels of HER2 overexpression, which is much less common in head and neck cancer, resulting in lower overall expression levels. HER2-targeted therapy is effective in most malignancies, including breast cancer with HER2 overexpression. However, HER2 mutations and gene amplification are relatively rare in head and neck cancer. FaDu, a cell line isolated from hypopharyngeal tumors in patients with squamous cell carcinoma, is widely used to study head and neck cancer cells. A comparative study was conducted to evaluate the effects of SI-71XM41 and SI-71XM40 on the proliferation of HER2 / HER3 double-positive FaDu cells. SI-71XM41 and SI-71XM40 achieved maximum antiproliferative activity at drug concentrations as low as 1 nM and 0.1 nM, respectively, while their parental antibodies MM-111 and trastuzumab showed overlapping activity at approximately 100 nM. Figure 6A and 6B Furthermore, each pair of SI-71XM41 and SI-71XM19 or SI-71XM40 and SI-71XM32 (having the same amino acid sequence as SI-71XM20) exhibited overlapping curves of antiproliferative activity. Figure 6C and 6D This indicates that salt bridge mutations offer little or no advantage in antiproliferative activity. Therefore, SI-71XM20 and SI-71XM40 were constructed based on a monovalent Fab bispecific antibody-like protein platform and optimized for inhibiting cell proliferation in cancer cells expressing HER2 / HER3, such as breast and head and neck cancers.

[0110] Impact on other types of cancer To evaluate the binding activity of SI-71XM41 and SI-71XM40 to other cancer types, a group of cancer cell lines were used for indications including breast cancer (SK-BR-3), pancreatic cancer (CFPAC-1), lung cancer (NCI-H358, Oka-C-1, and COR-L279), endometrial cancer (Ishikawa), liver cancer (HepG2), epidermal cancer (A-431), and colon cancer (Colo-320). The gMFI APC signal for each cell was presumed to correlate with the antibody expression density per cell. Overall, a consistent pattern appeared: the mean gMFI signal for SI-71XM41 was higher than that for SI-71XM40, with trastuzumab's MFI signal in the middle. This pattern was repeated across all indications, although the mean gMFI signals for the two previously characterized cell lines, BT-474 and FaDu, were the highest and lowest, respectively. Therefore, SI-71XM41 and SI-71XM40 are both candidate therapeutic agents for the treatment of these cancer cell lines.

[0111] sheet Table 1: Domain composition of bispecific monovalent Fab antibody-like proteins for binding HER2 or HER3, comprising a first binding domain (D1, monovalent), a second binding domain (D2, monovalent or bivalent) containing one or two scFvs connected to the N-terminus of the heavy chain variable domain, and a single-chain Fc region containing an inverted CH2-CH3 repeat sequence bridged by two disulfide bonds (see Figure 1). Table 2: Expression titers of antibody-like proteins targeting HER2 and HER3, and stability assessment of antibody-like proteins after purification of protein A in the first step by analytical size exclusion chromatography. Table 3A: Binding kinetics (affinity) of anti-HER2×HER3 antibody-like protein with HER2 in solution. Table 3B: Binding kinetics (affinity) of anti-HER2 / HER3 antibody-like proteins with HER3 in solution. Table 4: Binding kinetics (affinity) between anti-HER2×HER3 monovalent Fab antibody-like protein and HER3 in solution. Table 5: Efficacy and efficacy parameters of BT-474 proliferation assay using anti-HER2×HER3 bispecific antibody-like protein. sequence list SIBA075 - HER2×HER3 Asymmetric Sequence Listing CDRs in the amino acid sequence are underlined. >SEQ ID 01 SI-71MM4 heavy chain nucleotide sequence >SEQ ID 02 SI-71MM4 Heavy Chain Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 03 SI-71MM4 Light Chain Nucleotide Sequence GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID 04 Amino acid sequence of the light chain of SI-71MM4 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID 05 Nucleotide sequence of the heavy chain of SI-71XM21 >SEQ ID 06 SI-71XM21 heavy chain amino acid sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 07 Light chain nucleotide sequence of SI-71XM21 GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID 08 Amino acid sequence of the light chain of SI-71XM21 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID 09 Nucleotide sequence of the heavy chain of SI-71XM20 >SEQ ID 10 SI-71XM20 heavy chain amino acid sequence >SEQ ID 11 SI-71XM20 light chain nucleotide sequence GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID 12 SI-71XM20 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID 13 SI-71MM7 heavy chain nucleotide sequence >SEQ ID 14 SI-71MM heavy chain amino acid sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 15 SI-71MM light chain nucleotide sequence CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTAGGCCAACCGAAAGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA >SEQ ID 16 Amino acid sequence of the light chain of SI-71MM7 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS >SEQ ID 17 Nucleotide sequence of the heavy chain of SI-71XM19 >SEQ ID 18 SI-71XM19 heavy chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSSGGGGSGGGGQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 19 Light chain nucleotide sequence of SI-71XM19 CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTAGGCCAACCGAAAGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA >SEQ ID 20 Amino acid sequence of the light chain of SI-71XM19 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS >SEQ ID 21 Nucleotide sequence of the variable heavy chain (VH) domain of anti-HER-2 trastuzumab GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATAT GCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCGAATGAACAGCCTGCTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG >SEQ ID 22 Anti-HER2 trastuzumab variable heavy chain (VH) domain amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS >SEQ ID 23 nucleotide sequence of anti-HER2 trastuzumab variable light chain (VL) domain GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCC TCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA >SEQ ID 24 Anti-HER2 trastuzumab variable light chain (VL) domain amino acid sequence DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK >SEQ ID 25 Anti-HER3 MM-111 Variable Heavy Chain (VH) Domain Nucleotide Sequence CAGGTGCAATTGCAGGAGTCGGGGGGAGGCCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGTTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCGCGATGGAAGTGCGAGTTAC TATGTGGACTCTGTGAAGGGCCGATTCACCATTCCAGAGACGACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGTGGGGTGGGCTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCGAGC >SEQ ID 26 Anti-HER3 MM-111 Variable Heavy Chain (VH) Domain Amino Acid Sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >SEQ ID 27 Anti-HER3 MM-111 Variable Light Chain (VL) Domain Nucleotide Sequence CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTA >SEQ ID 28 Amino acid sequence of the variable light chain (VL) domain of anti-HER3 MM-111 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >SEQ ID 29 Nucleotide sequence of anti-HER2 trastuzumab scFv GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGTGGAGGGTCCGGCGGTGGAGGATCAGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGAGGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG >SEQ ID 30 Amino acid sequence of anti-HER2 trastuzumab scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS >SEQ ID 31 Anti-HER3 MM-111 scFv nucleotide sequence CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGTTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAACCGCGATGGAAGTGCGAGTTACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGTGGGGTGGGCTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTGTCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGTTCCGGCGGTGGCGGCTCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTA >SEQ ID 32 Amino acid sequence of anti-HER3 MM-111 scFv QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >SEQ ID 33 Anti-HER3 MM-111 scFv Nucleotide Sequence CAAGTGCAACTACAAGAAAGTGGTGGTGGTCTCGTGAAGCCCGGAGGCTCTCTGCGGCTGTCCTGTGCTGCTTCTGGCTTTACATTCTCCTCTTACTGGATGTCCTGGGTCAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCAACATCAACCGGGATGGCTCCGCCTCTTATTACGTGGACTCCGTGAAAGGCAGATTCACCATCTCTCGGGACGACGCCAAGAACTCCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCAGAGATCGCGGCGTGGGCTACTTCGACCTGTGGGGACGGGGCACCCTGGTGACCGTGTCCAGCGGTGGCGGCGGCAGTGGAGGCGGCGGCTCTGGCGGCGGGGGCTCCCAAAGTGCACTAACTCAACCAGCAAGTGTGAGCGGATCTCCCGGCCAGTCCATCACCATCTCCTGTACCGGCACATCCAGCGATGTCGGCGGCTACAACTTCGTGTCTTGGTATCAACAGCACCCTGGCAAGGCCCCTAAGCTGATGATCTACGACGTGTCCGATAGACCTTCTGGCGTGTCCGACCGGTTTAGCGGTTCCAAGTCCGGCAACACCGCTTCTCTGATCATCTCTGGCCTGCAGGCCGACGACGAGGCTGACTACTACTGCTCCTCCTACGGCTCCTCTTCTACCCACGTGATCTTCGGCGGAGGCACCAAAGTGACCGTGCTG >SEQ ID 34 Anti-HER3 MM-111 scFv Amino Acid Sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL >SEQ ID 35 SI-71XM32 heavy chain nucleotide sequence >SEQ ID 36 SI-71XM32 heavy chain amino acid sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLSGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 38 Light chain nucleotide sequence of SI-71XM32 GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID 38 Amino acid sequence of the light chain of SI-71XM32 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID 39 Nucleotide sequence of the heavy chain of SI-71XM40 >SEQ ID 40 SI-71XM40 heavy chain amino acid sequence QVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRRAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQEHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLSGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVREAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 41 Light chain nucleotide sequence of SI-71XM40 GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAAAGAAAACCAGGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGCTCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTACTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >SEQ ID 42 Amino acid sequence of the light chain of SI-71XM40 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQRKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >SEQ ID 43 Nucleotide sequence of the heavy chain of SI-71XM41 >SEQ ID 44 SI-71XM41 heavy chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQRKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVREAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSSGGGGSGGGGQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRRAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >SEQ ID 45 SI-71XM41 light chain nucleotide sequence CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAAGAGCACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAAGGTGACCGTCCTAGGCCAACCGAAAGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA >SEQ ID 46 Amino acid sequence of the light chain of SI-71XM41 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQEHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS >SEQ ID 47 Amino acid sequence of CDR-H1 of anti-HER2 trastuzumab DTYIH >SEQ ID 48 Anti-HER2 trastuzumab CDR-H2 amino acid sequence RIYPTNGYTRYADSVKG >SEQ ID 49 Anti-HER2 trastuzumab CDR-H3 amino acid sequence WGGDGFYAMDY >SEQ ID 50 Anti-HER2 trastuzumab CDR-L1 amino acid sequence RASQDVNTAVA >SEQ ID 51 anti-HER2 trastuzumab CDR-L2 amino acid sequence SASFLYS >SEQ ID 52 anti-HER2 trastuzumab CDR-L3 amino acid sequence QQHYTTPPT >SEQ ID 53 Anti-HER3 MM-111 CDR-H1 amino acid sequence SYWMS >SEQ ID 54 Anti-HER3 MM-111 CDR-H2 amino acid sequence NINRDGSASYYVDSVKG >SEQ ID 55 Anti-HER3 MM-111 CDR-H3 amino acid sequence DRGVGYFDL >SEQ ID 56 Anti-HER3 MM-111 CDR-L1 amino acid sequence TGTSSDVGGYNFVS >SEQ ID 57 Anti-HER3 MM-111 CDR-L2 amino acid sequence DVSDRPS >SEQ ID 58 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.

[0112] 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-dependent Activation of HER3 Induces PrimaryResistance 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. 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. An antibody-like protein having an N-terminus and a C-terminus, comprising a Fab region and an Fc region, wherein the Fc region comprises a first Fc domain connected to a second Fc domain via a second hinge, wherein the first Fc domain and the second Fc domain pair to form the Fc region.

2. The antibody-like protein according to claim 1, comprising: The light chain, from the N-terminus to the C-terminus, comprises a VL domain and a CL domain. The heavy chain, from the N-terminus to the C-terminus, comprises a VH domain, a CH1 domain, a first hinge, and the Fc region, wherein the first Fc domain comprises a first CH2 domain and a first CH3 domain, and wherein the second Fc domain comprises a second CH2 domain and a second CH3 domain. The VL and VH domains form the Fab region. The light chain and the heavy chain are covalently paired via at least one disulfide bond between the CH1 domain and the CL domain.

3. The antibody-like protein according to claim 2, wherein the first Fc domain and the second Fc domain are paired through two disulfide bonds between the first hinge and the second hinge to form the Fc region.

4. The antibody-like protein according to claim 2, further comprising a first scFv domain having scFv VH and scFv VL domains, wherein the antibody-like protein has binding affinity for HER2 and HER3.

5. The antibody-like protein of claim 4, wherein the first scFv domain is connected to the N-terminus of the heavy chain via a linker.

6. The antibody-like protein according to claim 4, further comprising a second scFv domain connected in series with the first scFv domain.

7. The antibody-like protein of claim 6, wherein the first and second scFv domains are identical.

8. The antibody-like protein of claim 7, wherein the Fab region has binding affinity for HER2 and each scFv domain has binding affinity for HER3.

9. The antibody-like protein of claim 4, wherein the Fab region has binding affinity for HER2, and the first scFv domain has binding affinity for HER3.

10. The antibody-like protein of claim 9, wherein the Fab region has binding affinity for HER2 with a KD of about 1 pM to about 100 nM, and wherein the first scFv domain has binding affinity for HER3 with a KD of about 1 pM to about 1 μM.

11. The antibody-like protein of claim 4, wherein the Fab region has binding affinity for HER3 and the scFv domain has binding affinity for HER2.

12. The antibody-like protein of claim 11, wherein the Fab region has binding affinity for HER3 with a KD of about 1 pM to about 1 μM, and wherein the first scFv domain has binding affinity for HER2 with a KD of about 1 pM to about 100 nM.

13. The antibody-like protein of claim 2, wherein the heavy chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 6, 10, 18, 36, 40 or 44.

14. The antibody-like protein of claim 2, wherein the VH domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 22 or 26.

15. The antibody-like protein of claim 2, wherein the light chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 8, 12, 20, 38, 42 or 46.

16. The antibody-like protein of claim 2, wherein the VL domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 24 or 28.

17. The antibody-like protein of claim 2, wherein the Fab region comprises CDR-H1 having SEQ ID NO: 47, CDR-H2 having SEQ ID NO: 48, and CDR-H3 having SEQ ID NO: 49; and CDR-L1 having SEQ ID NO: 50, CDR-L2 having SEQ ID NO: 51, and CDR-L3 having SEQ ID NO:

52.

18. The antibody-like protein of claim 2, wherein the Fab region comprises CDR-H1 having SEQ ID NO: 47, CDR-H2 having SEQ ID NO: 48, and CDR-H3 having SEQ ID NO: 49; and CDR-L1 having SEQ ID NO: 50, CDR-L2 having SEQ ID NO: 51, and CDR-L3 having SEQ ID NO:

52.

19. The antibody-like protein of claim 4, wherein the first scFv domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 30, 32 or 34.

20. The antibody-like protein of claim 4, wherein the first scFv VH domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 22 or 26, and the first scFv VL domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 24 or 28.

21. The antibody-like protein according to claim 4 or 6, wherein the first scFv domain or the second scFv domain comprises CDR-H1 having SEQ ID NO: 47, CDR-H2 having SEQ ID NO: 48, and CDR-H3 having SEQ ID NO: 49; and CDR-L1 having SEQ ID NO: 50, CDR-L2 having SEQ ID NO: 51, and CDR-L3 having SEQ ID NO:

52.

22. The antibody-like protein according to claim 4 or 6, wherein the first scFv domain or the second scFv domain comprises CDR-H1 having SEQ ID NO: 53, CDR-H2 having SEQ ID NO: 54, and CDR-H3 having SEQ ID NO: 55; and CDR-L1 having SEQ ID NO: 56, CDR-L2 having SEQ ID NO: 57, and CDR-L3 having SEQ ID NO:

58.

23. The antibody-like protein of claim 1, wherein the second hinge comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)n, and wherein n is an integer of at least 5.

24. The antibody-like protein according to claim 23, wherein n is 6.

25. The antibody-like protein of claim 5, wherein the linker comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)m, and wherein m is an integer of at least 3.

26. An isolated nucleic acid sequence encoding the antibody-like protein of claim 1.

27. An expression vector comprising the isolated nucleic acid sequence of claim 26.

28. A host cell comprising the isolated nucleic acid sequence of claim 26.

29. An immunoconjugate comprising the antibody-like protein of claim 1 and a cytotoxic agent, wherein the cytotoxic agent comprises a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

30. A pharmaceutical composition comprising the antibody-like protein of claim 1 or the immunoconjugate of claim 29, and optionally a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 30, further comprising a cytotoxic agent, wherein the cytotoxic agent comprises a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

32. A method of treating or preventing cancer in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the antibody-like protein of claim 1 or the immunoconjugate of claim 29.

33. The method of claim 32, 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.

34. The method of claim 33, wherein the cancer comprises cells expressing HER2 or HER3, 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.

35. The method of claim 32, wherein the subject is a human being.

36. A method for producing the antibody-like protein of claim 1, comprising: Culture host cells to express the DNA sequence encoding the antibody-like protein of claim 1, and The antibody-like protein was purified.

37. A method of producing the immunoconjugate of claim 29, comprising conjugating the antibody-like protein of claim 1 to a cytotoxic portion, wherein the cytotoxic portion comprises a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

38. A solution comprising an effective concentration of the antibody-like protein of claim 1 or the immunoconjugate of claim 29, wherein the solution is plasma of a subject.

Images