Asymmetric antibody-like proteins and methods of making and using thereof

Abstract

Asymmetric antibody-like proteins having binding affinity to EGFR and HER3 are disclosed. In certain embodiments, the antibody-like proteins comprise a Fab region, an Fc region comprising a first Fc domain and a second Fc domain, and one or more single-chain variable fragment (scFv) domains, wherein the Fab region is linked to the first Fc domain and a first scFv domain is linked to the second Fc domain or to the Fab region. In some embodiments, a second scFv domain is tandemly linked to the first scFv domain.

Description

[0001] ASYMMETRIC ANTIBODY-LIKE PROTEINS AND METHODS OF MAKING AND USING THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 749,722, filed January 27, 2025, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] SEQUENCE LISTING

[0004] The sequence listing associated with this application is provided electronically in XML format in compliance with WIPO Standard ST.26. The sequence listing file is named “SIBA136PCT_SequenceListing_Final_012526.xml”, has a file size of approximately 29,000 bytes, and was created on January 25, 2026. The contents of the sequence listing are hereby incorporated by reference in their entirety for all purposes.

[0005] TECHNICAL FIELD

[0006] The present disclosure generally relates to the technical field of antibody therapy for treating cancer, and more particularly relates to bispecific antibody-like proteins targeting both EGFR and HER3 and their drug conjugates.

[0007] BACKGROUND

[0008] Epidermal growth factor receptor (EGFR / ErbB1) is part of a critical growth pathway in cancer cells [1], Treatments with anti -EGFR antibodies such as Cetuximab can result in the inhibition of EGFR signaling which can reduce the rate or inhibit the growth of cancer cells [2], Cetuximab blocks binding of ligands to EGFR, inhibiting receptor phosphorylation and prosurvival signaling at the plasma membrane [1] [3], Antibody binding to EGFR also induces internalization of EGFR, and its degradation, enhancing the signal inhibition derived from blocking ligand binding [4], However, in some cases, cancer cells can develop resistance to Cetuximab by upregulating the expression of other proteins that compensate for the loss of EGFR signaling [5],

[0009] One of these proteins is human epidermal growth factor receptor 3 (HER3 / ErbB3). HER3 can activate compensatory signaling pathways that allow cancer cells to continue growing despite the inhibition of EGFR [5-7], This phenomenon is known as HER3 -dependent compensatory signaling [8-10], Multiple studies have demonstrated the high occurrence of HER3 overexpression in specific cancer types, such as head and neck squamous cell carcinoma (HNSCC), colorectal cancer (CRC), lung cancer, and esophageal cancer. In these malignancies, HER3 plays a critical role in driving the development of resistance to EGFR-targeted therapies, thereby contributing significantly to treatment challenges [5, 11-13], When activated, HER3 can promote resistance to EGFR inhibition by activating the PI3K / AKT and ERK signaling pathways [8, 13-21], For this reason, simultaneous targeting of both EGFR and HER3 has been shown to be an effective strategy to overcome resistance to EGFR-targeted anti-cancer therapy [13, 22-25],Several studies have reported that a combination of anti-EGFR and anti-HER3 therapies can be more effective than those targeting EGFR alone [22, 26], Treatment with a combination of Cetuximab, and the anti-HER3 antibody, MM- 121, has been shown to be more effective than either therapy alone in studies focused on inhibiting the growth of HNSCC cells and preventing the onset of resistance to EGFR inhibition

[0026] , Similar work in CRC revealed that this drug combination blocked EGFR and HER3 activities and inhibited the EGFR-PI3K-AKT-ERK signaling pathways and reduced CRC cell growth more effectively than each antibody administered alone

[0022] , Testing of a recently described bispecific antibody designed to target both EGFR and HER3 reported long-lasting growth suppression in a subcutaneous xenograft head and neck tumor model

[0027] , The 1 to 1 ratio controlled blocking of EGF and HER3 with this bispecific antibody was effective at reducing the number of cells with stem-like properties which inhibited the proliferation and formation of cancer stem cell-initiated spheroids

[0027] , These studies suggest that simultaneous targeting of both EGFR and HER3 may be a promising strategy for overcoming resistance to EGFR inhibitors in cancer treatment and may be particularly useful in patients diagnosed with EGFR wild type driven signaling.

[0010] The increased expression levels of EGFR and HER3 in epithelial tumors present an attractive therapeutic target for cancer, but each protein is independent and dynamically regulated, leading to heterogeneous expression levels in tumors. Efforts to utilize EGFR evaluations by immunohistochemistry (IHC) as biomarkers for response and survival after treatment with EGFR targeting antibodies have had mixed results, and required complex scoring rules to serve as a correlate of response in trials in NSCLC [28, 29].

[0011] However, earlier clinical development of the first-in-class dual action anti -HER3 / EGFR antibody MEHD7945A (Duligotuzumab) was halted due to lack of efficacy. Duligotuzumab is an immunoglobulin (Ig) G1 monoclonal antibody that binds to both EGFR and HER3 and inhibits their activation of EGFR / HER3-mediated neoplastic signaling, and ultimately, EGFR / HER3-dependent tumor cell proliferation. Clinical trials in locally advanced or metastatic epithelial tumors revealed that the drug was well-tolerated with evidence of tumor pharmacodynamic modulation and antitumor activity in patients diagnosed with HNSCC

[0025] , This led to the initiation of Phase II studies in patients with HN SCC and CRC that were not showing improvement with Cetuximab. The overall lack of efficacy in this setting is attributed to the reduced EGFR affinity compared to Cetuximab, which was not overcome with higher affinity HER3 binding by Duligotuzumab

[0030] , The clinical testing of Duligotuzumab in combination with paclitaxel in HNSCC resulted in gastrointestinal toxicity, suggesting that in this setting HER3 was the dose limiting target, rather than EGFR

[0031] ,

[0012] Preclinical testing well indicates that combining anti-EGFR and anti-HER3 therapies can disrupt the intricate signaling interplay between these two receptors, of which HER3 often leads to resistance when targeting EGFR alone. By concurrently inhibiting both EGFR and HER3, synergistic effects may be achieved, resulting in improved treatment responses if the anti-EGFR activity is increased above that of Duligotuzumab and the anti-HER3 activity is not dose limiting.SUMMARY

[0013] The present application generally relates to the technical field of immunotherapy, and more specifically relates to antibody therapeutic agents, and more particularly relates to asymmetric antibody-like proteins against epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) and their immunoconjugates. EGFR and HER3 often form a partnership in promoting cellular transformation that may ultimately lead to tumorigenesis and tumour metastasis. The application provides, among others, antibody-like proteins, the immunoconjugates of such antibody-like proteins, the method of making antibody-like proteins or their immunoconjugates, pharmaceutical compositions including the antibody-like proteins and / or their immunoconjugates, the method of using such antibody-like proteins or their immunoconjugates for treating diseases including, for example, cancers.

[0014] In one aspect, an asymmetric antibody-like protein is provided. In one embodiment, the asymmetric antibody-like protein comprises a Fab region, an Fc region having a first Fc domain and a second Fc domain, and at least one single-chain variable fragment (scFv) domain. The Fab region comprises a light chain and a Fab domain. The Fc region comprises a first Fc domain and a second Fc domain, and the Fab domain is linked to the first Fc domain forming a heavy chain or a part of a heavy chain. In one embodiment, a first scFv domain is linked to the second Fc domain at either its N-terminus or its C-terminus. In one embodiment, a first scFv domain is linked to the Fab domain at its N-terminus. The asymmetric antibody-like protein may be bispecific, trispecific, or multi-specific.

[0015] In certain embodiments, the antibody-like protein further comprises a second scFv domain. The second scFv domain may be tandemly linked to the first scFv domain. The antibody-like protein is tri -specific.

[0016] In some embodiments, the asymmetric antibody-like protein comprises a first heavy chain and a second heavy chain. The first heavy chain comprises the Fab domain and the first Fc domain. The second heavy chain comprises the second Fc domain. In one embodiment, the second heavy chain is truncated with no Fab domain. The first scFv domain is linked to the second Fc domain at its N-terminus or C-terminus. In certain embodiments, the first scFv domain is linked to the second Fc domain at its N-terminus.

[0017] In some embodiments, the Fab region has binding affinity to HER3 and the first scFv domain has binding affinity to EGFR. In other embodiments, the Fab region has binding affinity to EGFR and the first scFv domain has binding affinity to HER3.

[0018] In further embodiments, the antibody-like protein comprises a second scFv domain that is tandemly linked to the first scFv domain. In one embodiment, the first scFv is linked to the second Fc domain at its N-terminus, leading to the structure including, from N-terminus to C-terminus, the second scFv domain, the first scFv domain, and the second Fc domain. In certain embodiments, the Fab region has binding affinity to HER3, and each of the first scFv domain and the second scFv domain has binding affinity to EGFR.

[0019] In some embodiments, the light chain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 or SEQ IDNO: 9. In some embodiments, the first heavy chain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 7. In some embodiments, the second heavy chain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 13.

[0020] In certain embodiments, the Fab region comprises complementarity determining regions (CDRs) having the amino acid sequences set forth in SEQ ID NOs: 37-48. In certain embodiments, the first scFv domain comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 31-36 and 43-48. In embodiments comprising a second scFv domain, the second scFv domain comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 31-36.

[0021] In another aspect, the antibody-like protein comprises a single heavy chain that includes the Fab domain, the first Fc domain, and the second Fc domain. In such embodiments, the first Fc domain is linked to the second Fc domain through a hinge, and the first Fc domain and second Fc domain are configured to pair to form the Fc region. In certain embodiments, the Fab domain comprises a VH domain and a CHI domain, the first Fc domain comprises a first CH2 domain and a first CH3 domain, and the second Fc domain comprises a second CH2 domain and a second CH3 domain, and the heavy chain comprises the VH domain, CHI domain, first CH2 domain, first CH3 domain, second CH2 domain, and second CH3 domain arranged in tandem.

[0022] In certain embodiments, the first scFv domain is linked to the second Fc domain at its C-terminus. In some embodiments, the Fab region has binding affinity to EGFR and the first scFv domain has binding affinity to HER3. In some embodiments, the first scFv domain is linked to the Fab domain at its N-terminus. In some embodiments, the Fab region has binding affinity to HER3 and the first scFv domain has binding affinity to EGFR.

[0023] In certain embodiments, a second scFv domain is tandemly linked to the first scFv domain. In some embodiment, the first scFv domain is linked to the second Fc domain at its C-terminus resulting in the asymmetric antibody-like protein comprising a light chain and a construct having, from N-terminus to C-terminus, the Fab domain, the first Fc domain, the second Fc domain, the first scFv domain, and the second scFv domain linked in tandem. In some such embodiments, the Fab region has binding affinity to EGFR, the first scFv domain has binding affinity to HER3, and the second scFv domain has binding affinity to EGFR.

[0024] In some embodiments, the light chain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 21. In some embodiments, the heavy chain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 23.

[0025] In certain embodiments, the Fab region comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 25-36. In certain embodiments, the first scFv domain comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 25-30 and 43-48. In embodiments comprising a second scFv domain, the second scFv domain comprises CDRs having amino acid sequences set forth in SEQ ID NOs: 25-30.In some embodiments, the first Fc domain and the second Fc domain comprise at least one mutation configured to form a knob-in-hole structure. In certain embodiments, the mutation is a knob-in-hole mutation. In certain embodiments, the mutation is an Fc null mutation.

[0026] In some embodiments, the antibody-like protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23.

[0027] In certain embodiments, the Fab region has binding affinity to HER3 with a dissociation constant (KD) ranging from about 1 pM to about 500 nM, from about 100 nM to about 500 nM, or from about 150 nM to about 300 nM, and the first scFv domain has binding affinity to EGFR with a KD ranging from about 1 pM to about 1 pM. In certain embodiments, the Fab region has binding affinity to HER3 with a KD ranging from about 1 pM to about 1 pM, from about 50 nM to about 350 nM, from about 100 nM to about 500 nM, or from about 150 nM to about 300 nM, and the first scFv domain has binding affinity to EGFR with a KD ranging from about 1 pM to about 100 nM. In certain embodiments, the first scFv domain has binding affinity to HER3 with a KD ranging from about 100 nM to about 1 pM, and the Fab domain has binding affinity to EGFR with a KD ranging from about 1 pM to about 1 pM.

[0028] In embodiments comprising a second scFv domain, the second scFv domain has binding affinity to HER3 with a KD ranging from about 150 nM to about 300 nM, or has binding affinity to EGFR with a KD ranging from about 1 pM to about 1 pM.

[0029] In some embodiments, the first Fc domain is linked to the second Fc domain through a hinge comprising a glycine-serine repeat sequence of the formula (Gly-Gly-Gly-Gly-Ser)n, wherein n is an integer of at least 5, and in certain embodiments n is 6. In some embodiments, the first scFv domain is linked to the Fc region or the Fab region through a linker comprising a glycineserine repeat sequence of the formula (Gly-Gly-Gly-Gly-Ser)m, wherein m is an integer of at least 3.

[0030] In further aspects, isolated nucleic acid sequences encoding the antibody-like proteins are provided, as well as expression vectors comprising such nucleic acid sequences and host cells comprising such nucleic acid sequences.

[0031] In additional aspects, immunoconjugates are provided that comprise the antibody-like proteins conjugated to a cytotoxic agent, wherein the cytotoxic agent comprises a radioisotope, radionuclide, therapeutic agent, chemotherapeutic agent, or a combination thereof.

[0032] Pharmaceutical compositions are also provided that comprise the antibody-like protein or the immunoconjugate, optionally together with a pharmaceutically acceptable carrier, and in some embodiments further comprising a cytotoxic agent.

[0033] Methods are provided for treating or preventing cancer in a subject by administering to the subject a pharmaceutical composition comprising the antibody-like protein or the immunoconjugate. In certain embodiments, the method further comprises co-administering an effective amount of a therapeutic agent comprising an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. In certain embodiments, the cancer comprises cells expressing HER2 or HER3 and includes breast cancer, colorectal cancer, pancreatic cancer, head and neckcancer, melanoma, ovarian cancer, endometrial cancer, epidermal cancer, prostate cancer, nonsmall 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. In some embodiments, the subject is a human.

[0034] Methods are also provided for producing the antibody-like protein, comprising culturing a host cell such that a nucleic acid encoding the antibody-like protein is expressed and purifying the expressed antibody-like protein. Methods are further provided for producing an immunoconjugate by conjugating the antibody-like protein with a cytotoxic moiety.

[0035] In further aspects, solutions are provided that comprise an effective concentration of the antibody-like protein or immunoconjugate, wherein the solution comprises blood plasma in a subject.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The foregoing and other features of this disclosure may become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure may be described with additional specificity and detail through use of the accompanying drawings, in which:

[0038] FIGURE 1 shows diagrams of six representative antibody-like protein embodiments, designated SI-136X1 through SI-136X6, each characterized by having a monoval ent-Fab domain as a first binding domain and either one or two monovalent-scFv as a second binding domain or as second and third binding domains: SI-136X1, SI-136X2, and SI-136X3 are characterized by having three polypeptides (i.e., chain 1 to 3) and an Fc region, whereas SI-136X4, SI-136X5, and SI-136X6 are characterized by having two polypeptides (i.e., chain 1 and 2) and an Fc region formed by two sets of covalently linked CH2CH3 domains; the binding specificity of each Fab domain and scFv domain is indicated; and

[0039] FIGURE 2 shows the FaDu proliferation quantified by normalized area under the curve (AUC), calculated from the time-course measurements for each treatment condition and normalized to the corresponding time 0 value, where Bars show the mean normalized AUC across replicates, representing relative proliferation of the solid tumor cancer cell line FaDu under each indicated treatment and concentration.

[0040] DETAILED DESCRIPTION

[0041] This disclosure provides antibody-like proteins and their immunoconjugates with superior therapeutic properties or efficacies over the currently known antibodies targeting one or two members of EGFR family. In one embodiment, the antibody-like proteins are structurally configured to efficiently target two members of EGFR family, HER2 and HER3. The antiproliferative activities of these bispecific antibody-like proteins may result from blocking or inhibiting different receptor-mediated oncogenic signaling simultaneously.The terms “a”, “an” and “the” as used herein are defined to mean “one or more” and include the plural unless the context is inappropriate.

[0042] The terms “polypeptide”, “peptide”, and “protein”, as used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.

[0043] The term “antigen” refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions thereof responsible for antigenicity or antigenic determinants.

[0044] The terms “antigen- or epitope-binding portion or fragment”, “variable domain”, “variable region”, “variable region sequence”, or “binding domain” refer to fragments of an antibody that are capable of binding to an antigen (such as EGFR and HER3 in this application). These fragments may be capable of the antigen-binding function and additional functions of the intact antibody.

[0045] The terms “Fv” or “scFv” refers to the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can recognize and bind antigen, although at a lower affinity than the entire binding site. Examples of binding fragments include, but are not limited to, a single-chain Fv fragment (scFv) consisting of variable light chain (VL) and variable heavy chain (VH) domains of a single arm of an antibody connected in a single polypeptide chain by a synthetic linker, or a Fab fragment which is a monovalent fragment consisting of VL, constant light (CL), VH and constant heavy 1 (CHI) domain. Antibody fragments can be even smaller sub-fragments and can consist of domains as small as a single CDR domain, in particular the CDR3 regions from either the VL and / or VH domains.

[0046] The term “VH-VL pairing” refers to the selection of suitable human germlines during the humanization process for the heavy and light chains to form a stable Fv. The mutual orientation of the VH and VL domains should correspond to that observed in the parental antibody.

[0047] Antibody fragments are produced using conventional methods known to those skilled in the art. The antibody fragments can be screened for utility using the same techniques employed with intact antibodies. Purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. Papain digestion of antibodies produces two identical antigen binding fragments, called “Fab” fragments, each with a single antigen binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2fragment that has two antigen combining sites and is still capable of cross-linking antigen. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies.The term “antibody” is used in the broadest sense and specifically covers single monoclonal antibodies and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments (e g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, the antibody may be monoclonal, polyclonal, chimeric, single chain, multi-specific or multi-effective, human and humanized antibodies, as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab'), scFv and Fv fragments, including the products of a Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above.

[0048] The term “antibody-like protein” are proteins that are antibody-like and can specifically bind to antigens with high specificity and affinity. Sometimes, in this application, “antibody” and “antibody-like protein” may be used interchangeably.

[0049] In some embodiments, antibody may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site and that immunospecifically bind an antigen. A typical antibody refers to heterotetrameric protein comprising typically of two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain is comprised of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. The VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions (CDR), and more conserved regions called framework regions (FR). Each variable domain (either VH or VL) is typically composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino-terminus to carboxy-terminus. Within the variable regions of the light and heavy chains there are binding regions that interacts with the antigen.

[0050] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0051] The term “valency” refers to the number of antigenic determinants that an individual antibody molecule can bind. The valency of all natural antibodies is at least two, whereas the term “mono-valent” or “monovalent” refers to an engineered antibody or antibody-like protein with a single binding domain comprising the pairing of six hypervariable complementarity determining regions (CDRs), i.e., three HC-CDRs and three LC-CDR. In this context, “antibody affinity”refers to the tendency of an antibody to bind to a specific epitope at the surface of an antigen, i.e., to the strength of the interaction.

[0052] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler & Milstein or may be made by recombinant DNA methods (see, e.g., U. S. Pat. No. 4,816,567). " Recombinant" means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells. Monoclonal antibodies can be produced using various methods, including without limitation, mouse hybridoma, phage display, recombinant DNA, molecular cloning of antibodies directly from primary B cells, and antibody discovery methods. Monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0053] The term “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity. Methods to obtain “humanized antibodies” are well known to those skilled in the art.

[0054] The terms “isolated” or “purified” refer to a biological molecule free from at least some of the components with which it naturally occurs. Either “Isolated" or “purified," when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and / or recovered from a cell or cell culture from which it was expressed. Ordinarily, a purified polypeptide is prepared by at least one purification step. An "isolated” or a “purified” antibody refers to an antibody which is substantially free of other antibodies having different antigenic binding specificity.

[0055] The term “immunogenic” refers to substances which elicit or enhance the production of antibodies, T-cells or other reactive immune cells directed against an immunogenic agent andcontribute to an immune response in humans or animals. An immune response occurs when an individual produces sufficient antibodies, T-cells and other reactive immune cells against administered immunogenic compositions of the present disclosure to moderate or alleviate the disorder to be treated. While the immunogenic response generally includes both cellular (T cell) and humoral (antibody) arms of the immune response, antibodies directed against therapeutic proteins (anti-drug antibodies, ADA) may consist of IgM, IgG, IgE, and / or IgA isotypes.

[0056] The terms "specific binding", "specifically binds to", or “is specific for a particular antigen or an epitope” means that the binding is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0057] The term “affinity” refers to a measure of the attraction between two polypeptides, such as antibody / antigen, receptor / ligand, etc. The intrinsic attraction between two polypeptides can be expressed as the binding affinity equilibrium dissociation constant (KD) of a particular interaction. A KD binding affinity constant can be measured, e.g., by Bio-Layer Interferometry, where KD is the ratio of kdis (the dissociation rate constant) to kon (the association rate constant), as KD = kdis / kon.

[0058] It is considered by the application that the antibody-like proteins potentially have the advantage over any combination therapy, which often has greater toxicity than a single agent treatment. Bispecific agents, such as bispecific antibody-like proteins as disclosed in the application, may act as a single agent targeting the same antigens as the combination therapy does but with the increased efficacy and response rate and reduced toxicity when compared to the combination therapy. In comparison to the combination therapy using two monoclonal antibodies, bispecific therapeutics such as antibody-like proteins disclosed herein can be less toxic to patients and / or more potent due to the increased binding specificity.

[0059] In one aspect, the application provides a bispecific antibody-like proteins having a N terminal and a C terminal, comprising at least two binding domains, wherein the binding domain comprises a Fab region and a scFv domain. The scFv domain may be attached to either the N terminal or the C terminal of the antibody. The Fab region and the scFv domain each independently have a binding specificity to different proteins in the EGFR family.

[0060] In some embodiments, scFv molecules described herein contain a linker of (GmS)nthat operably links the VH and VL, regardless of the V-region orientation (L-H or H-L). The remaining positions in the bispecific antibody-like protein may consist of a human IgG Fc or IgG null Fc heavy chain, VH-CH1-Hinge-CH2-CH3, and its corresponding kappa or lambda light chain, VL-CL. Those scFv domains were genetically linked through a linker of (SGm)nto either N-terminal or C-terminal of IgG heavy chain, resulting in a contiguous ~ 75 kDa heavy chain monomer peptide. When co-transfected with the appropriate light chain, the final symmetric bispecific molecule can be purified through the human IgG Fc (Protein A) and assayed to assess functional activity.In one embodiment, the binding domain having the binding specificity to HER3 comprises MM-111, a bispecific HER2 and HER3 binding protein. MM-111 is a human serum albumin protein (HSA)-backed bispecific antibody fragment comprises one therapeutic binding to HER3, but its binding to HER2 alone is not sufficient to be considered as a therapeutic binding.

[0061] The bispecific antibody-like protein may include an immunoglobulin G (IgG) moiety having at least a light chain and a heavy chain. In one embodiment, the antibody-like protein may include two scFv moieties being covalently connected to either C or N terminals of the heavy or light chains via a linker, such as (Ser-Gly-Gly-Gly-Gly)n linkers, (Gly-Gly-Gly-Gly-Ser)n linkers, (Gly-Gly-Gly-Ser)n linkers, or (GmS)nor (SGm)nlinkers.

[0062] HER2 -targeted bispecific antibodies exhibit significant efficiency in preclinical studies for treating drug-resistant HER2-expressing malignant tumours. HER2 -targeted bispecific antibodies include MM-111, ALM, PB4188, and MCLA-128 (32-35). MM-111 targets the HER2 / HER3 heterodimer, blocks heregulin binding, and inhibits downstream signaling pathways. In contrast, Trastuzumab alone had no effect on heregulin-induced paclitaxel resistance. MM-111 has a higher-affinity HER2 arm to target HER2-amplified tumors as compared with its HER3 arm (32). Upon its binding to HER2-positive cells with high avidity, MM-111 can effectively block submaximal ligand-driven proliferation but not supramaximal activation. This is because the HER2 binding moiety of MM- 111 does not have any sustained effect on tumor cell growth when compared with that of Trastuzumab, which binds to a different epitope in the region IV of HER2 (36). Indeed, the combination therapy of Trastuzumab and MM-111 shows significantly greater activity than either antibody used alone (32).

[0063] MCLA-128 (Zenocutuzumab, Zeno) is a bispecific humanized immunoglobulin G1 (IgG1) containing two different Fab arms targeting the extracellular domains of EGFR and HER3. A recent clinical study (35) reveals that Zeno mediates durable clinical responses in a small group of patients with NRG1 fusion-positive cancers.

[0064] The present disclosure may be understood more readily by reference to the following detailed description of specific embodiments and examples included herein. Although the present disclosure has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the disclosure.

[0065] EXAMPLES

[0066] Example 1. Anti-EGFRxHER3 bispecific antibody-like proteins

[0067] EGFR and HER3 are key members of the HER family of receptor tyrosine kinases, implicated in cancer cell survival and proliferation. Bispecific antibodies are an emerging strategy to selectively target these receptors, and with potentially optimized efficacy, providing enhanced therapeutic benefits while reducing off-target effects. Having a single therapeutic agent poses significant challenges due to the selection of binding moieties and the backbone structure that may affect the binding efficiency in vivo and the therapeutic efficacy in patients.The present application disclosed anti-EGFRxHER3 antibody-like proteins characterized by their configurations and domain compositions (S1-136X1 through S1-136X6, see Table 1 and Figure 1):

[0068] SI-136X1

[0069] In the embodiment of monovalent binding to EGFR and HER3 represented by SI-136X1, EGFR is bound by a single Cetuximab scFv and HER3 is bound by a single MM-111 Fab. The two heavy chain monomers are asymmetric but form an Fc with knob mutations (T366W) attached to the Cetuximab scFv and hole mutations (T366S, L238A, Y407V) attached to the MM-111 VH with CHI, CH2, and CH3. The single light chain comprises MM-111 VL with a constant kappa region for pairing with MM-111 VL to form the MM-111 Fab. Cetuximab scFv is oriented as, from N-terminus to C-terminus, VL to VH.

[0070] The specific binding domain orientation and affinities allow for access to differential epitopes and expression levels of EGFR and HER3 on the surface of target cells of different oncology indications.

[0071] SI-136X2

[0072] In the embodiment of monovalent binding to EGFR and HER3 represented by SL136X2, HER3 is bound by MM-111 scFv and EGFR is bound by a single Nimotuzumab Fab. The two heavy chain monomers are asymmetric but form an Fc with knob mutations (T366W) attached to the MM-111 scFv and hole mutations (T366S, L238A, Y407V) attached to the Nimotuzumab VH with CHI, CH2, and CH3. The single light chain comprises Nimotuzumab VL with a constant kappa region for pairing with Nimotuzumab VH to form the Nimotuzumab Fab. MM-111 scFv is oriented as, from N-terminus to C-terminus, VL to VH.

[0073] The specific binding domain orientation and affinities allow for access to differential epitopes and expression levels of EGFR and HER3 on the surface of target cells of different oncology indications. Alternative target binder format (Fab vs scFv) and orientation, compared to SI- 136X2, emphasize potential differences in target binding MOA and alternative therapeutic applications.

[0074] SI-136X3

[0075] In the embodiment of bivalent and monovalent binding to EGFR and HER3 represented by SI-136X3, EGFR is bound by tandem Cetuximab scFvs and HER3 is bound by a single MM-111 Fab. To enhance the avidity binding of the 1 plus 1 molecule, bivalent binding of EGFR was designed. The two heavy chain monomers are asymmetric but form an Fc with knob mutations (T366W) attached to the tandem Cetuximab scFvs and hole mutations (T366S, L238A, Y407V) attached to the MM-111 VH with CHI, CH2, and CH3. The single light chain comprises MM-111 VL with a constant kappa region for pairing with MM-111 VL to form the MM-111 Fab. The two Cetuximab scFv are oriented in tandem as, from N-terminus to C-terminus, the first VL to VH followed by second VL to VH.

[0076] The binding domain orientation and affinities still allow for access to differential epitopes and expression levels of EGFR and HER3 on the surface of target cells. Beyond the utilization of bispecific targeting, the bivalent nature of the tandem EGFR binding domains in SI-136X3 takesadvantage of binder avidity effects that could increase the therapeutic index of the molecule on select oncology indications.

[0077] SI-136X4

[0078] In the embodiment of monovalent binding to EGFR and HER3 represented by SI- 136X4, EGFR is bound by Cetuximab Fab and HER3 is bound by a MM-111 scFv. Cetuximab heavy chain Fc is attached at the C-terminus by a 30 residue GS linker followed by an IgGl CH2 and CH3 whereby the pre-linker and post-linker CH3s dimerize to mimic a canonical IgGl Fc dimer. C-terminal to the IgGl Fc regions is MM-111 scFv oriented with the VL C-terminal to the VH. Cetuximab VL with a constant kappa region is the accompanying light chain to complete the Cetuximab Fab.

[0079] SI-136X5

[0080] In the embodiment of monovalent binding to EGFR and HER3 represented by SI- 136X5, HER3 is bound by Patritumab Fab and EGFR is bound by a Panitumumab scFv. Patritumab heavy chain Fc is attached at the C-terminus by a 30 residue GS linker followed by an IgGl CH2 and CH3 whereby the pre-linker and post-linker CH3s dimerize to mimic a canonical IgGl Fc dimer. N-terminal to the Patritumab VH is the Panitumumab scFv oriented with the VL N-terminal to the VH. Patritumab VL with a constant kappa region is the accompanying light chain to complete the Patritumab Fab.

[0081] The monovalent binders for EGFR and HER3 are arranged on opposing ends of the single chain Fc domain to maximize steric accessibility to their respective targets. Combined with tuned affinities and targeting specific epitopes, this allows for specificity to select oncological indications based on EGFR and HER3 expression.

[0082] ST-136X6

[0083] In the embodiment of monovalent and bivalent binding to EGFR and HER3 represented by SI-136X6, EGFR is bound by Cetuximab Fab and HER3 is bound by tandem MM-111 scFvs. To enhance the avidity binding of the 1 plus 1 molecule, biparatopic binding of HER3 was designed. Cetuximab heavy chain Fc is attached at the C-terminus by a 30 residue GS linker followed by an IgGl CH2 and CH3 whereby the pre-linker and post-linker CH3s dimerize to mimic a canonical IgGl Fc dimer. C-terminal to the IgGl Fc regions is the MM-111 scFv N-terminal to the Panitumumab scFv. The MM-111 and Patritumab scFvs are oriented with the VL C-terminal to the VH. Cetuximab VL with a constant kappa region is the accompanying light chain to complete the Cetuximab Fab.

[0084] The monovalent binders for EGFR and HER3 are arranged on opposing ends of the single chain Fc domain to maximize steric accessibility to their respective targets. Additionally, the tandem HER3 binding domains at the C-terminal of the single chain Fc target different epitopes of HER3 and takes advantage of binder avidity effects that could increase the therapeutic index of the molecule on distinct oncology indications.

[0085] Common modifications

[0086] The inherent nature of the IgGl backbone, allowing for a wider range (and plurality of distribution) of domain steric conformations, further contributes to the unique therapeuticcharacteristics of this molecule. The IgGl effector functions maintain reduced binding of the Fc domain to Fc gamma receptors within the single-chain Fc backbone. The IgGl backbone contains mutations that limit effector function by reducing binding of the Fc domain to Fc gamma receptors. The principal residues mutated that mediate this reduced activity are L234A, L235A, K322A. These mutations do not affect any cysteine residues nor site-specifically inserted cysteines in the kappa constant domain or CH3 domain. Therefore, the coupling of any type of combination of linker-drug compounds to each of six antibody-like proteins is expected to result in a drug-antibody ratio (DAR) in the range of 6-10.

[0087] Example 2. Generation of anti-EGFRxHER3 antibody-like proteins

[0088] Genes encoding antibody heavy and light chains (preceded by Kozak and secretory signal peptide) were cloned into pTT5 vector using standard molecular biology techniques. As shown in Table 1 and Figure 1, SI-136xl, SI-136X2, and SI-136X3 are characterized by having two heavy chain monomers (i.e., HC-1 and HC-2) and one light chain, whereas SI-136x4, SI-136X5, and SI-136X6 are characterized by having one heavy chain and one light chain.

[0089] The antibody-like proteins were expressed by transiently transfecting the expression plasmids for heavy and light chains in the ExpiCHO system (Thermo Fisher). Briefly, 5.6μg of each expression plasmid was brought to 2.4ml with OptiPRO SFM medium containing 33.8mg sheared herring sperm carrier DNA. 2.2ml of OptiPRO SFM medium containing 192μl Expifectamine CHO reagents were added to the DNA and incubated at room temperature for 5 minutes. The resulting mixture was added to 60ml ExpiCHO cells at 6x106cells / ml in a 250ml Erlenmeyer flask and incubated at 37°C, 5% CO2, 150rpm. Cells were fed with 21ml ExpiCHO feed and 360 pl of CHO enhancer at 24 hours post-transfection and shifted to 32°C, 5% CO2, 150rpm. Cells were fed again at 48 hours post-transfection with 21ml ExpiCHO feed. Culture supernatant was harvested 9 days post-transfection, spun for 20min at 7500rpm to pellet the cells and then passed through a 0.2μm filter. Expression titer was quantitated using biolayer interferometry on an Octet384 system with protein A sensors and a standard curve prepared with purified antibody-like proteins.

[0090] Proteins were purified from the harvested supernatant using a 5-ml MabSelect PrismA protein-A 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 25ml PBS, and proteins were eluted by passing 15ml of 50 mM sodium acetate, pH 3.5 through the column. The eluted proteins were immediately neutralized by addition of 1 / 10thvolume of 1M sodium acetate, pH7.0.

[0091] Immediately after first-step protein-A or His tag purification, the proteins were analyzed by analytical SEC using Waters Acquity UPLC H-Class with ACQUITY UPLC® Protein BEH SEC 200Å, 4.6mm x 150mm, 1.7 μm column. PBS (125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8) was used as mobile phase for 10-minute runs at 0.3 ml / min, injecting 15 pg protein. Proteins were further purified by preparative SEC using Superdex Increase 10 / 300 GL column in mobile phase of 25 mM sodium acetate, 125 mM NaCl, pH 5.5, ultimately to be buffer exchanged into 25 mM sodium acetate, 125 mM NaCl, 10% sucrose, pH 5.5. Final samplescontained >95% protein of interest (POI) as assessed by analytical SEC and were used for subsequent assays.

[0092] Example 3. Stability

[0093] Protein stability is a key parameter defined by the difference in free energy between the folded and unfolded states. For protein therapeutics, stability may impact immunogenicity, pharmacokinetics, and even efficacy, and reduction of aggregation can help to develop therapeutics that are easier to manufacture and safer for patients. In addition, expression efficiency and protein yield directly determine the cost of protein therapeutics. If proteins can be more efficiently expressed to reach higher titers and increased yield of purified protein, manufacturing costs can be reduced significantly. To characterize the structural and functional attributes of the antibodies, size-exclusion chromatography (SEC) serves as a critical tool to evaluate monomeric purity, revealing the presence of potential aggregates that could affect both efficacy and safety.

[0094] After transient expression in ExpiCHO cells, the titer of each bispecific antibody was quantitated using biolayer interferometry. Another parameter related to protein stability is the amount of aggregation after first step affinity purification. Antibodies with higher stability tend to have lower aggregation and higher % of POI by using analytical size-exclusion chromatography.

[0095] All antibodies expressed in the ExpiCHO expression system demonstrated comparable titers, indicating they were stable enough to be efficiently produced. After protein A purification, the bispecific antibodies were analysed by analytical SEC to check for aggregation. Under varying stress conditions, such as temperature and pH shifts, the stability assessments illuminated the robustness of SI-136X1 through SI-136X6. Of six antibodies, SI-136X1 and SI-136X5 displayed high titers with either very low % of POI or very high % of HMW aggregation, whereas SI- 136X4 showed highest POI and lowest % of HMW aggregation, with SI-136X6 being the second best (Table 2). Overall, SI-136X4 and SI-136X6 displayed desirable features of protein purity and stability providing insights into their viability for long-term clinical applications and their potential for conjugation with cytotoxic payloads. SI-136X4 and SI-136X6 were configured to have the same core structure (e.g., inverted CH2CH3) comprising monovalent binding specificity to EGFR and HER3 (MM111-specific), but SI-136X6 is configured to have a scFv domain for binding to a second epitope of EGFR (Panitumumab-specific) (Table 1).

[0096] Example 4. Binding affinity

[0097] The binding affinity of each antibody to EGFR and HER3 was measured using biolayer interferometry (BLI), a highly sensitive and quantitative method. This technique generates kinetic binding parameters, including association and dissociation rates, delivering KD values that provide a nuanced understanding of the antibodies’ specificity and strength of interaction with their respective targets. The dual-binding capability to both EGFR and HER3 was also evaluated, revealing avidity effects that are crucial for functional bridging. These results shed light on the cooperative dynamics of receptor engagement, a feature critical for therapeutic efficacy in complex biological environments and essential for designing ADCs that require efficient target engagement.

[0098] Biolayer interferometry (Octet) binding assays were performed on an Octet384 instrument to quantify binding kinetics of antibody-like proteins to EGFR and HER3. For affinityexperiments, the antibody -like protein can be captured to anti-human Fc (AHC) sensor tips by loading for 150 seconds at 75nM. Binding curves can be globally fit to a 1: 1 model to extract the dissociation constants, KD, and kinetic association and dissociation rates.

[0099] SI-136X4, SI-136X5, and SI-136X6 share the common structural feature of having two inverted CH2CH3 to a Fc region (Figure 1). The three antibodies displayed high affinity to EGFR when compared to their KD values SI-136X1, SI-136X2, and SI-136X3 (Table 3). Of the three antibodies, SI- 136X4 and SI- 136X6 shared the common feature of a monovalent Cetuximab-derived Fab for binding to EGFR, as compared to SI-136X5 whose EGFR binding activity comes from the scFv domain derived from Panitumumab. This difference may explain the difference in the binding affinity of EGFR by the three antibodies. At meantime, SIBA-136X5 and SI-136X6 displayed lowest KD values indicative of highest affinity to HER3 as compared to any of the other four antibodies (Table 3). The difference seems to be correlated with the binding specificity to different epitopes of EGFR between Cetuximab and Panitumumab. Thus, this result of this screening indicates that the HER3 binding affinity of SI- 136X6 may be enhanced due to the close proximity of EGFR and HER3 binding domains.

[0100] Example 5. Anti-cancer cell proliferative activity

[0101] The functional efficacy of these antibodies was assessed through cancer cell proliferation assays using well -characterized cell lines such as FaDu, A431 and HCC827, which represent EGFR and HER3-driven tumour models. The ability of the antibodies to inhibit cancer cell growth was quantified by IC50 values, offering a direct measure of their potency. This data not only validate the therapeutic mechanism of action but also help rank the antibodies for their implications in cancer immune therapy based on their relative effectiveness in vitro.

[0102] FaDu cells (a human solid tumor cell line) were cultured in human serum-containing media to provide endogenous ligands for EGFR and HER3 signalling. Test articles included EGFR / HER3 bispecific antibody -based molecules (including SI-BOO 1, which is a symmetric tetravalent antibody, see the reference of US Patent 15119694), an EGFR-binding IgG control (Cetuximab), and a negative control antibody (Rituximab). Media-only (human serum-containing) wells were included as an additional baseline control. Cell proliferation was monitored over a time course, and for each condition the response was summarized as the area under the curve (AUC) of the proliferation signal. AUC values were normalized to the corresponding timepoint 0 value to generate a normalized AUC metric for comparison across treatments and concentrations. For each condition, replicate measurements were aggregated and the mean normalized AUC was plotted.

[0103] As shown in Figure 2, Rituximab exhibited minimal effect on FaDu proliferation and performed similarly to the media control, consistent with a non-targeting negative control. In contrast, the EGFR / HER3 bispecific SI-BOO 1 demonstrated greater inhibition of FaDu proliferation than the EGFR-binding IgG Cetuximab under the same assay conditions. Additional EGFR / HER3 bispecific test molecules displayed a range of anti-proliferative activities, with responses generally consistent with SI-BOO 1 and, overall, less anti -proliferative activity than Cetuximab alone. These results demonstrate that EGFR / HER3 bispecific antibodies can inhibitproliferation of FaDu cells in ligand-replete conditions and that SI-BOO 1 provides enhanced inhibition relative to an EGFR-only comparator in this assay.

[0104] SI- 136X6, which is capable of monovalent binding to HER3 and bi-epitopic binding to EGFR molecule, showed anti-proliferative activity like SI-BOO 1 while its molecule weight is about half of SI-BOO 1. This finding unveils the superiority over Cetuximab similar to EGFR and HER3 tetravalent bispecific SI-B001. In contrast, SI-136X4, which lacks the second binding to EGFR shows less anti-proliferative activity indicating the contribution of the second EGFR binding domain for overall anti-proliferative activity.

[0105] To further determine the immune-mediated cytotoxic potential of the antibodies, antibody-dependent cellular cytotoxicity (ADCC) activity can be evaluated by employing effector cells, such as NK cells, and EGFR / HER3-expressing cancer cells, and cytotoxicity can be measured using imaging or flow cytometry-based assays. Comparative activity profiles can be generated and quantified by EC50 values, which may highlight differences in immune effector engagement and therapeutic efficacy. These results not only validate the antibodies’ functional properties, such as SI-136X6, but also inform its potential to complement antibody-drug conjugate (ADC) mechanisms by engaging the immune system in tumour cell elimination.

[0106] Example 6. Internalization and lysosomal trafficking

[0107] The data from Example 5 provided a framework for selecting candidates that exhibit optimal characteristics for payload delivery in an ADC format. Antibody internalization can be tracked via fluorescent labelling, enabling precise quantification through flow cytometry or microscopy to elucidate the dynamics of receptor-mediated endocytosis, a property critical for the intracellular delivery of cytotoxic payloads and provide clues about downstream signalling inhibition. Lysosomal trafficking behaviour can be monitored using pH-sensitive dyes to confirm the delivery of antibodies to acidic compartments.

[0108] Each experimental step above is not merely a test of feasibility but a deeper inquiry into the biological and mechanistic significance of SI-136X1 through SI-136X6. Although the lead candidate from this cycle of screening is SI-136X6, the results also weave a comprehensive narrative of its biophysical and functional properties, solidifying its therapeutic potential and positioning them as strong candidates for further development as both standalone biologies and efficacious ADCs.REFERENCES

[0109] Each reference cited herein is incorporated by reference in its entirety. The incorporation of any reference is not an admission that such reference constitutes prior art with respect to the present application.

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[0141] Cetuximab: https: / / www.ema.europa.eu / en / documents / scientific-discussion / erbitux-epar- scientific-discussion_en.pdf

[0142] Panitumumab:

[0143] https: / / www.ncbi. nlm.nih.gov / pmc / articles / PMC6763619 / #:~:text=Panitumumab%20bin ds%20EGFR%20with%20an,whether%20this%20characteristic%20is%20favorable Nimotuzumab:https: / / www.nature. com / articles / s41598-019-57279-w / tables / l Trastuzumab: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6244757 / Pertuzumab:https: / / www.tga.gov.au / sites / default / files / auspar-pertuzumab-131001.pdf Patritumab: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5058629 /

[0144] MM-121: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3478453 / MM-111:https: / / pubmed.ncbi.nlm.nih.gov / 22248472 /

[0145] 2in1: https: / / ars.els-cdn.com / content / image / l-s2.0-S1535610811003515-mmcl.pdf SI-1X6.3(C3): US15 / 119,694. https: / / cancerres.aacrjournals.org / content / 64 / 7_Supplement / 163.4.short https: / / aacrjournals.org / cancerdiscovery / article / 12 / 5 / 1233 / 694554 / Zenocutuzumab-a-HER2 / HER3-Bispecific-Antibody-IsTABLES

[0146] Table 1. Configuration of anti-EGFRxHER3 bispecific antibody-like proteins: S1-136X1, Sl-136X2, and SI- 136X3 are configured to have two asymmetric heavy chain monomers (Chain 1 and 3) paired with one light chain (Chain 2); and SI-136X4, SI-136X5, and SI-136X6 are configured to have one monovalent-Fab (Chain 1) paired with one light chain (Chain 2) and characterized by having a single chain Fc region containing inverted CH2-CH3 repeats linked by two disulfide bridges (see FIGURE 1).

[0147] Heavy Chain (HC) Light Chain (LC) Protein Chain N-scFv Fab Fc C-scFv Fab Chain (VH-CH1) (VL-Ck)

[0148] SI-136X1 1 - HER31CH2-CH3 - HER312

[0149] 3 EGFR1- CH2-CH3 - - - SI- 136X2 1 - EGFR2CH2-CH3 - EGFR22

[0150] 3 HER31- CH2-CH3 - - - SI- 136X3 1 - HER31CH2-CH3 - HER312

[0151] 3 scFv-scFv - CH2-CH3 - - - EGFR / EGFR1

[0152] SI- 136X4 1 - EGFR1Inverted HER31EGFR12 CH2-CH3

[0153] SI- 136X5 1 EGFR3HER32Inverted - HER322 CH2-CH3

[0154] SI- 136X6 1 - EGFR1Inverted scFv-scFv EGFR12

[0155]

[0156] CH2-CH3 HER3 / EGFR*

[0157] 1GFR*: EGFR1(Cetuximab); EGFR2(Nimotuzumab); EGFR3(Panitumumab);

[0158] HER3*: HER31(MM111); HER32(Patritumab);

[0159] HER3 / EGFR*: EGFR (Cetuximab) + HER3 (MM-111) + EGFR (Panitumumab).Table 2. Expression and purification properties of anti-EGFRxHER3 bispecific antibody-like proteins from 30 mL ExpiCHO cell expression runs.

[0160] Protein Titer Post proA Purification

[0161] ID POI% HMW% LMW% POI% HMW% LMW% SI-136X1 148 69.30 12.71 17.99 41.28 3.64 55.08 SI- 136X2 99 57.45 31.28 11.27 80.03 19.97 0.00 SI- 136X3 71 73.11 12.10 14.79 88.09 1.58 10.33 SI- 136X4 105 75.23 24.77 0.00 99.68 0.32 0.00 SI- 136X5 180 73.77 26.23 0.00 89.80 10.20 0.00

[0162]

[0163] SI- 136X6 79 80.09 19.91 0.00 97.26 2.74 0.00

[0164] Table 3. Binding properties of anti-EGFRxHER3 bispecific antibody-like proteins to EGFR and HER3 ligands measured by BLI.

[0165] EGFR HER3

[0166] Protein Affinity kon koff Affinity kon koff ID (M) (M-1s-1) (s1) (M) (M-1s-1) (s1) SI-136X1 2.96E-09 8.36E+05 2.48E-03 5.17E-08 8.33E+05 4.31E-02 SI- 136X2 2.01E-08 1.93E+05 3.88E-03 8.64E-08 4.83E+05 4.17E-02 SI- 136X3 3.42E-09 6.27E+05 2.14E-03 5.09E-08 8.26E+05 4.21E-02 SI- 136X4 1.14E-09 1.24E+06 1.42E-03 6.20E-08 5.43E+05 3.37E-02 SI- 136X5 2.27E-09 7.05E+05 1.60E-03 2.35E-08 1.13E+05 2.65E-03

[0167]

[0168] SI- 136X6 2.04E-09 7.91E+05 1.61E-03 2.79E-08 5.12E+05 1.43E-02SEQUENCE LISTING

[0169] Chain 1 Chain 2 Chain 3 SEQ ID AA NT AA NT AA NT SI-136X1 1 2 3 4 5 6 SI- 136X2 7 8 9 10 11 12 SI- 136X3 1 2 3 4 13 14 SI- 136X4 15 16 17 18 n / a n / a SI- 136X5 19 20 21 22 n / a n / a

[0170]

[0171] SI- 136X6 23 24 17 18 n / a n / a

[0172] Domain Protein SEQ ID NO.

[0173] CDR1 CDR2 CDR3

[0174] Panitumumab VH 25 26 27

[0175] Panitumumab VL 28 29 30

[0176] Cetuximab VH 31 32 33

[0177] Cetuximab VL 34 35 36

[0178] Nimotuzumab VH 37 38 39

[0179] Nimotuzumab VL 40 41 42

[0180] Anti-HER3 VH (MM-111) 43 44 45

[0181] Anti-HER3 VL (MM-111) 46 47 48

[0182] Patritumab VH 49 50 51

[0183]

[0184] Patritumab VL 52 53 54

[0185] Domain Protein

[0186] SEQ ID NO.

[0187] human IgGl 55

[0188]

[0189] human Kappa 56

[0190] > Seq ID 1: SI-136X1 / SI-136X3 chain 1 amino acid sequence QVQLQE S GGGLVKPGGS LRL S CAAS G FT FS SYWMS WVRQAPGKGLE WVANINRDGSAS YYVDSV KGRFTISRDDAKNSLYLQMDSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG

[0191] > Seq ID 2: SI-136X1 / SI-136X3 chain 1 nucleotide sequenceCAGGTGCAATTGCAGGAGTCGGGGGGAGGCCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTTAGTAGTTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAA GGGGCTGGAGTGGGTGGCCAACATAAACCGCGATGGAAGTGCGAGTTACTATGTGGACTCTGTG AAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACTCACTGTATCTGCAAATGGACAGCC TGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGTGGGGTGGGCTACTTCGATCT CTGGGGCCGTGGCACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGC CGCGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC AAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCA GGTCAGCCTGTCCTGCGCTGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCGTCAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACAGATTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAG

[0192] > Seq ID 3: SI- 136X1 / SI-136X3 chain 2 amino acid sequence QSALTQPASVSGS PGQS I T I SCTGTSSDVGGYNFVSWYQQHPGKAPKLMI YDVSDRPSGVSDRF SGSKSGNTASLI I SGLQADDEADYYCSSYGSSSTHVI FGGGTKVTVLGQPKAAPSVTLFPPSSE E L QANKAT L VC L I S D FY P GAVT VAWKAD S S P VKAGVE T T T P S KQS NNK YAAS S YL S L T PE QWKS HRSYSCQVTHEGSTVEKTVAPTECS

[0193] > Seq ID 4: SI- 136X1 / SI-136X3 chain 2 nucleotide sequence CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCT GCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTACCAACAACACCCAGG CAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGGGTGTCTGATCGCTTC TCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCCAGGCTGACGACGAGG CTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTTCGGCGGAGGGACCAA GGTGACCGTCCTAGGCCAACCGAAAGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAG GAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGA CAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAA ACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCC CACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTA CAGAATGTTCATGA> Seq ID 5: S I- 136X1 chain 3 amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASES ISGIPSRFSG SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSQ VQLKQSGPGLVQPSQSLS ITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTS RLS INKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGGGGDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRWSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG

[0194] > Seq ID 6: S I- 136X1 chain 3 nucleotide sequence GATATCCTACTAACTCAAAGTCCAGTGATCCTGTCTGTGTCTCCTGGCGAGAGAGTGTCCTTCT CCTGTAGAGCTTCTCAGTCCATCGGCACAAACATCCACTGGTACCAGCAACGGACCAACGGCTC CCCTAGACTGCTGATCAAGTACGCCTCTGAGTCTATCTCCGGCATCCCCAGCCGGTTTAGCGGC TCTGGCAGCGGAACCGACTTCACCCTGTCCATCAACTCCGTGGAATCCGAGGATATCGCCGACT ACTACTGCCAGCAGAACAATAATTGGCCTACCACCTTCGGCGCTGGAACCAAGCTGGAACTGAA AGGAGGAGGAGGGTCTGGAGGAGGCGGAAGTGGAGGCGGAGGATCTGGCGGAGGAGGAAGCCAA GTGCAACTAAAGCAAAGTGGTCCAGGTCTCGTGCAGCCTTCTCAGTCTCTGTCCATCACCTGTA CAGTCTCTGGCTTCTCCCTGACCAACTACGGCGTGCACTGGGTGCGGCAGAGCCCTGGCAAGGG CCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAACACCGATTATAATACACCCTTTACCTCC AGAC T G T C T AT C AAC AAG GAG AAC T C CAAAT C T C AAG T G T T C T T C AAGAT GAAC AG C C T G GAG T CCAACGACACCGCTATCTACTACTGCGCCAGAGCTCTGACCTACTACGACTACGAGTTCGCCTA CTGGGGTCAGGGCACCCTGGTGACCGTCTCGAGTGGTGGCGGTGGTGACAAGACCCACACCTGT CCACCTTGCCCCGCCCCTGAAGCTGCCGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTA AGGACACCCTGATGATCTCTCGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGA AGATCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCAAGAGAAGAACAGTACAACTCCACTTACAGAGTTGTGTCCGTGCTGACCGTGCTGCATCAAG ACTGGCTGAACGGCAAAGAATACAAGTGCGCCGTGAGTAACAAGGCTCTGCCTGCCCCTATCGA GAAAACCATCTCTAAGGCTAAGGGACAACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCC CGGGACGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTGAAAGGCTTCTACCCCTCTG ACATCGCCGTGGAATGGGAGTCGAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGT GCTGGACAGCGACGGATCTTTCTTCCTGTACTCCAAGCTGACCGTCGACAAGTCTAGATGGCAG CAGGGCAACGTGTTCTCCTGCTCTGTGATGCACGAGGCTCTGCACAATCATTACACCCAGAAAA GCTTGTCCCTATCGCCAGGTTGA

[0195] > Seq ID 7: S I- 136X2 chain 1 amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYI YWVRQAPGQGLEWIGGINPTSGGSNFNEKF KTRVT I TADES S T TAYMELS SLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVS SASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLS LSPG

[0196] > Seq ID 8: SI- 136X2 chain 1 nucleotide sequence CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCT GCAAGGCCAGCGGCTACACCTTCACCAACTACTACATCTACTGGGTGCGGCAGGCCCCCGGCCA GGGCCTGGAGTGGATCGGCGGCATCAACCCCACCAGCGGCGGCAGCAACTTCAACGAGAAGTTC AAGACCCGGGTGACCATCACCGCCGACGAGAGCAGCACCACCGCCTACATGGAGCTGAGCAGCC TGCGGAGCGAGGACACCGCCTTCTACTTCTGCACCCGGCAGGGCCTGTGGTTCGACAGCGACGG CCGGGGCTTCGACTTCTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGCTAGCACCAAGGGC CCCAGCGTGTTTCCTCTGGCTCCCTCTTCTAAGTCCACCTCGGGCGGCACAGCCGCGCTGGGTT GCCTGGTGAAGGACTACTTCCCTGAACCCGTCACCGTGTCCTGGAACTCCGGCGCCTTAACATC TGGCGTGCACACCTTTCCTGCCGTCCTGCAGAGCTCTGGACTGTACTCTCTTAGTAGCGTGGTG ACAGTGCCTAGCTCATCTCTGGGCACCCAGACCTACATCTGCAACGTCAACCACAAGCCCTCTA ACACCAAGGTGGATAAGCGGGTTGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCACCTTG CCCCGCCCCTGAAGCTGCCGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTAAGGACACC CTGATGATCTCTCGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGAAGATCCTG AGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGAGA AGAACAGTACAACTCCACTTACAGAGTTGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGCTG AACGGCAAAGAATACAAGTGCGCCGTGAGTAACAAGGCTCTGCCTGCCCCTATCGAGAAAACCA TCTCTAAGGCTAAGGGACAACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCCGGGACGA GCTGACCAAGAACCAGGTGTCCCTGTCTTGTGCTGTGAAAGGCTTCTACCCCTCTGACATCGCC GTGGAATGGGAGTCGAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACA GCGACGGATCTTTCTTCCTGGTGTCCAAGCTGACCGTCGACAAGTCTAGATGGCAGCAGGGCAA CGTGTTCTCCTGCTCTGTGATGCACGAGGCTCTGCACAATAGATTCACCCAGAAAAGCTTGTCC CTATCGCCAGGTTGA

[0197] > Seq ID 9: SI- 136X2 chain 2 amino acid sequence

[0198] DI QMTQS PS SLSASVGDRVT ITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLI YKVSNRFSGVP SRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDE QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

[0199] > Seq ID 10: S I-136X2 chain 2 nucleotide sequence GATATTCAAATGACTCAATCTCCTTCTTCTCTTTCTGCTTCTGTTGGTGATCGTGTTACTATTA CTTGTCGTTCTTCTCAAAATATTGTTCATTCTAATGGTAATACTTATCTTGATTGGTATCAACA AACTCCTGGTAAAGCTCCTAAACTTCTTATTTATAAAGTTTCTAATCGTTTTTCTGGTGTTCCT TCTCGTTTTTCTGGTTCTGGTTCTGGTACTGATTTTACTTTTACTATTTCTTCTCTTCAACCTG AAGATATTGCTACTTATTATTGTTTTCAATATTCTCATGTTCCTTGGACTTTTGGTCAAGGTAC TAAACTTCAAATTACTCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAG AAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTTAG

[0200] > Seq ID 11: S I-136X2 chain 3 amino acid sequence

[0201] QVQLQE S GGGLVKPGGS LRL S CAAS G FT FS SYWMS WVRQAPGKGLE WVANINRDGSAS YYVDSV KGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSSGGGGSGGGGS GGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSG VSDRFSGSKSGNTASLI ISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG

[0202] > Seq ID 12: S I-136X2 chain 3 nucleotide sequence CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCCTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTTAGTAGTTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAA GGGGCTGGAGTGGGTGGCCAACATAAACCGCGATGGAAGTGCGAGTTACTATGTGGACTCTGTG AAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACTCACTGTATCTGCAAATGAACAGCC TGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGTGGGGTGGGCTACTTCGATCT CTGGGGCCGTGGCACCCTGGTCACCGTGTCTAGCGGAGGAGGAGGGTCTGGAGGAGGCGGAAGT GGAGGCGGAGGATCTCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGT CGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTTTGTCTCCTGGTA CCAACAACACCCAGGCAAAGCCCCCAAACTCATGATCTATGATGTCAGTGATCGGCCCTCAGGG GTGTCTGATCGCTTCTCCGGCTCCAAGTCTGGCAACACGGCCTCCCTGATCATCTCTGGCCTCC AGGCTGACGACGAGGCTGATTATTACTGCAGCTCATATGGGAGCAGCAGCACTCATGTGATTTT CGGCGGAGGGACCAAGGTGACCGTCCTAGGTGGCGGTGGTGACAAGACCCACACCTGTCCACCT TGCCCCGCCCCTGAAGCTGCCGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTAAGGACA CCCTGATGATCTCTCGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGAAGATCC TGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGA GAAGAACAGTACAACTCCACTTACAGAGTTGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGC TGAACGGCAAAGAATACAAGTGCGCCGTGAGTAACAAGGCTCTGCCTGCCCCTATCGAGAAAAC CATCTCTAAGGCTAAGGGACAACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCCGGGAC GAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTGAAAGGCTTCTACCCCTCTGACATCG CCGTGGAATGGGAGTCGAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGA CAGCGACGGATCTTTCTTCCTGTACTCCAAGCTGACCGTCGACAAGTCTAGATGGCAGCAGGGC AACGTGTTCTCCTGCTCTGTGATGCACGAGGCTCTGCACAATCATTACACCCAGAAAAGCTTGT CCCTATCGCCAGGTTGA> Seq ID 13: S I- 136X3 chain 3 amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASES ISGIPSRFSG SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSQ VQLKQSGPGLVQPSQSLS ITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTS RLS INKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGGGSGGGSEI VLTQSPSTLSVSPGERATFSCRASQS IGTNIHWYQQKPGKPPRLLIKYASES ISGIPDRFSGSG SGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVLGGGGSGGGGSGGGGSGGGGSQVQ LQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRF TITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGGGGDKTHTCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG

[0203] > Seq ID 14: S I- 136X3 chain 3 nucleotide sequence GATATCCTACTAACTCAAAGTCCAGTGATCCTGTCTGTGTCTCCTGGCGAGAGAGTGTCCTTCT CCTGTAGAGCTTCTCAGTCCATCGGCACAAACATCCACTGGTACCAGCAACGGACCAACGGCTC CCCTAGACTGCTGATCAAGTACGCCTCTGAGTCTATCTCCGGCATCCCCAGCCGGTTTAGCGGC TCTGGCAGCGGAACCGACTTCACCCTGTCCATCAACTCCGTGGAATCCGAGGATATCGCCGACT ACTACTGCCAGCAGAACAATAATTGGCCTACCACCTTCGGCGCTGGAACCAAGCTGGAACTGAA AGGAGGAGGAGGGTCTGGAGGAGGCGGAAGTGGAGGCGGAGGATCTGGCGGAGGAGGAAGCCAA GTGCAACTAAAGCAAAGTGGTCCAGGTCTCGTGCAGCCTTCTCAGTCTCTGTCCATCACCTGTA CAGTCTCTGGCTTCTCCCTGACCAACTACGGCGTGCACTGGGTGCGGCAGAGCCCTGGCAAGGG CCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAACACCGATTATAATACACCCTTTACCTCC AGAC T G T C T AT C AAC AAG GAG AAC T C CAAAT C T C AAG T G T T C T T C AAGAT GAAC AG C C T G GAG T CCAACGACACCGCTATCTACTACTGCGCCAGAGCTCTGACCTACTACGACTACGAGTTCGCCTA CTGGGGTCAGGGCACCCTGGTGACCGTCTCGAGTGGCGGAGGAAGTGGTGGCGGTTCTGAGATC GTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTA GAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCG GCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGC TCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACT GCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGACAGTTCTTGGTGG TGGTGGGAGTGGTGGTGGCGGTAGCGGTGGCGGTGGTTCTGGCGGTGGTGGTTCCCAAGTTCAG TTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGT CCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGA ATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTC ACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATG ACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACGACTACGAGTTTGCTTACTGGGG CCAGGGCACCCTGGTCACAGTTTCTTCCGGAGGCGGTGGTGACAAGACCCACACCTGTCCACCT TGCCCCGCCCCTGAAGCTGCCGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTAAGGACA CCCTGATGATCTCTCGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGAAGATCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGA GAAGAACAGTACAACTCCACTTACAGAGTTGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGC TGAACGGCAAAGAATACAAGTGCGCCGTGAGTAACAAGGCTCTGCCTGCCCCTATCGAGAAAAC CATCTCTAAGGCTAAGGGACAACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCCGGGAC GAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTGAAAGGCTTCTACCCCTCTGACATCG CCGTGGAATGGGAGTCGAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGA CAGCGACGGATCTTTCTTCCTGTACTCCAAGCTGACCGTCGACAAGTCTAGATGGCAGCAGGGC AACGTGTTCTCCTGCTCTGTGATGCACGAGGCTCTGCACAATCATTACACCCAGAAAAGCTTGT CCCTATCGCCAGGTTGA

[0204] > Seq ID 15: S I-136X4 chain 1 amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT SRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRD GSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS GGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLM IYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVL

[0205] > Seq ID 16: S I-136X4 chain 1 nucleotide sequence CAAGTGCAACTAAAGCAAAGTGGTCCAGGTCTCGTGCAGCCTTCTCAGTCTCTGTCCATCACCT GTACAGTCTCTGGCTTCTCCCTGACCAACTACGGCGTGCACTGGGTGCGGCAGAGCCCTGGCAA GGGTCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAACACCGATTATAATACACCCTTTACC T C C AGAC T G T C T AT C AAC AAG GAC AAC T C C AAAT C T C AAG T G T T C T T C AAGAT GAAC AG C C T G C AGTCCAACGACACCGCTATCTACTACTGCGCCAGAGCTCTGACCTACTACGACTACGAGTTCGC CTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG ACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA AGCAGCTGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCGCTGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTATACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT GGTGGTGGCGGTTCAGGAGGCGGAGGAAGTGGAGGCGGTGGGTCAGGTGGCGGTGGAAGTGGAG GAGGCGGGTCTGGCGGAGGAGGGTCTGACAAGACCCACACCTGTCCACCTTGCCCCGCCCCTGA AGCTGCAGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATCTCT CGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGAAGATCCTGAGGTGAAGTTCA ACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGAGAAGAACAGTACAA CTCCACTTACAGAGTTGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGCTGAACGGCAAAGAA TACAAGTGCGCTGTGAGTAACAAGGCTCTGCCTGCCCCTATCGAGAAAACCATCTCTAAGGCTA AGGGACAACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCCGGGACGAGCTGACCAAGAA CCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCTGACATCGCCGTGGAATGGGAG TCGAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGATCTT TCTTCCTGTACTCCAAGCTGACCGTCGACAAGTCTAGATGGCAGCAGGGCAACGTGTTCTCCTG CTCTGTGATGCACGAGGCTCTGCACAATCACTACACCCAGAAGTCCCTGTCCCTATCGCCAGGC GGCGGTGGTGGTAGCGGAGGCGGAGGAAGTCAAGTGCAACTACAAGAAAGTGGTGGTGGTCTCG TGAAGCCCGGAGGCTCTCTGCGGCTGTCCTGTGCTGCTTCTGGCTTTACATTCTCCTCTTACTG GATGTCCTGGGTCAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCAACATCAACCGGGAT GGCTCCGCCTCTTATTACGTGGACTCCGTGAAAGGCAGATTCACCATCTCTCGGGACGACGCCA AGAACTCCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGTGC CAGAGATCGCGGCGTGGGCTACTTCGACCTGTGGGGAAGAGGCACTCTGGTGACCGTGTCCAGT GGAGGGGGAGGTTCCGGTGGAGGTGGGTCTGGGGGTGGTGGAAGTCAAAGTGCACTAACTCAAC CAGCAAGTGTGAGCGGATCTCCCGGCCAGTCCATCACCATCTCCTGTACCGGCACATCCAGCGA TGTCGGCGGCTACAACTTCGTGTCTTGGTATCAACAGCACCCTGGCAAGGCCCCTAAGCTGATG ATCTACGACGTGTCCGATAGACCTTCTGGCGTGTCCGACCGGTTTAGCGGTTCCAAGTCCGGCA ACACCGCTTCTCTGATCATCTCTGGCCTGCAAGCCGACGACGAGGCTGACTACTACTGCTCCTC CTACGGCTCCTCTTCTACCCACGTGATCTTCGGAGGAGGTACCAAAGTGACCGTGCTGTGA

[0206] > Seq ID 17: S I-136X4 / SI-136X6 chain 2 amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSG SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLS S PVTKS FNRGEC

[0207] > Seq ID 18: S I-136X4 / SI-136X6 chain 2 nucleotide sequenceGACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCT CCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTC TCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGC AGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATT ATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG TGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GT TAG

[0208] > Seq ID 19: S I-136X5 chain 1 amino acid sequence

[0209] DI QMTQS PS SLSASVGDRVT I TCQASQDI SNYLNWYQQKPGKAPKLL I YDASNLETGVPSRFSG SGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQ VQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHI YYSGNTNYNPSL KSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSGGGGSGGGGS QVQLQQWGAGLLKP S E T L S LTCAVYGGSFSGYYWSW I RQP PGKGLE W I GEINHSGSTNYNPSLK SRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSS LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0210] > Seq ID 20: S I-136X5 chain 1 nucleotide sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA CTTGCCAGGCGAGTCAGGACATCAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGC CCCTAAACTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGA AGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACAT ATTTCTGTCAACACTTTGATCATCTCCCGCTCGCTTTCGGCGGAGGGACCAAGGTGGAAATTAA AGGAGGAGGAGGGTCTGGAGGAGGCGGAAGTGGAGGCGGAGGATCTGGCGGAGGAGGAAGCCAA GTGC ACTCCAGGAG GTGGGCCGGG TTGGTT AACCGAGCGAGACCCTGAGTCTCACGTGTA CAGTTTCAGGCGGGAGCGTGAGCTCAGGCGACTATTACTGGACATGGATAAGACAGAGTCCTGG AAAGGGCCTGGAGTGGATCGGTCATATTTATTATTCAGGGAACACTAACTATAATCCATCTTTG AAATCCCGATTGACAATCTCTATCGATACCAGTAAAACCCAGTTTAGCCTCAAGCTGTCCAGCG TTACTGCTGCAGACACAGCCATATACTATTGTGTACGCGATCGCGTGACTGGAGCCTTCGACATATGGGGGCAGGGAACGATGGTAACAGTCTCGAGCGGCGGAGGAGGAAGTGGTGGCGGTGGTTCT CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCT GCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAA GGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAG AGTCGAGTCACCATATCGGTAGAGACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGA CCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGATAAATGGACTTGGTATTTTGACTTATG GGGCAGAGGGACACTGGTCACCGTCTCTTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTG GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGA GAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCAGC TGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG TGCGCTGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC AGCCCCGAGAACCACAGGTGTATACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGTGGT GGCGGTTCAGGAGGCGGAGGAAGTGGAGGCGGTGGGTCAGGTGGCGGTGGAAGTGGAGGAGGCG GGTCTGGCGGAGGAGGGTCTGACAAGACCCACACCTGTCCACCTTGCCCCGCCCCTGAAGCTGC AGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATCTCTCGGACA CCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGAAGATCCTGAGGTGAAGTTCAACTGGT ACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGAGAAGAACAGTACAACTCCAC TTACAGAGTTGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGCTGAACGGCAAAGAATACAAG TGCGCTGTGAGTAACAAGGCTCTGCCTGCCCCTATCGAGAAAACCATCTCTAAGGCTAAGGGAC AACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCCGGGACGAGCTGACCAAGAACCAGGT GTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCTGACATCGCCGTGGAATGGGAGTCGAAC GGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGATCTTTCTTCC TGTACTCCAAGCTGACCGTCGACAAGTCTAGATGGCAGCAGGGCAACGTGTTCTCCTGCTCTGT GATGCACGAGGCTCTGCACAATCACTACACCCAGAAGTCCCTGTCCCTATCGCCAGGCTGA

[0211] > Seq ID 21: S I-136X5 chain 2 amino acid sequence DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLS S PVTKS FNRGEC> Seq ID 22: S I-136X5 chain 2 nucleotide sequence GACATCGAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCA ACTGCAGGTCCAGCCAGAGTGTTTTATACAGTTCCAGCAATAGAAACTACTTAGCTTGGTACCA GCAGAATCCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTC CCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGG CTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTCGCACATTCGGACAAGG GACCAAAGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG C C AAAG TAG AG T G GAAG G T G GAT AAC GC C C T C C AAT C G GG T AAC T C C C AGGAGAG T G T C ACAGA GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTAC GAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA GCTTCAACAGGGGAGAGTGTTAG

[0212] > Seq ID 23: S I-136X6 chain 1 amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT SRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSQVQLQESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRD GSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS GGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLM I YDVSDRPSGVS DR FS GS KS GNTAS L 11 S GLQADDEADYYCSSYGSSSTHVI FGGGTKVTVLGG GGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYS GNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK LLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK

[0213] > Seq ID 24: S I-136X6 chain 1 nucleotide sequence CAAGTGCAACTAAAGCAAAGTGGTCCAGGTCTCGTGCAGCCTTCTCAGTCTCTGTCCATCACCT GTACAGTCTCTGGCTTCTCCCTGACCAACTACGGCGTGCACTGGGTGCGGCAGAGCCCTGGCAA GGGTCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAACACCGATTATAATACACCCTTTACC T C C GAC T G T C T AT C AC AAG GAG AAC T C C AAAT C T C AG T G T T C T T C AAGAT GAAG AG C C T G C AGTCCAACGACACCGCTATCTACTACTGCGCCAGAGCTCTGACCTACTACGACTACGAGTTCGC CTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG ACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA AGCAGCTGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCGCTGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA AAGGGCAGCCCCGAGAACCACAGGTGTATACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT GGTGGTGGCGGTTCAGGAGGCGGAGGAAGTGGAGGCGGTGGGTCAGGTGGCGGTGGAAGTGGAG GAGGCGGGTCTGGCGGAGGAGGGTCTGACAAGACCCACACCTGTCCACCTTGCCCCGCCCCTGA AGCTGCAGGCGGCCCTTCCGTTTTTCTGTTCCCCCCTAAGCCTAAGGACACCCTGATGATCTCT CGGACACCCGAAGTGACCTGCGTGGTGGTGGACGTGTCCCATGAAGATCCTGAGGTGAAGTTCA ACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCAAGAGAAGAACAGTACAA CTCCACTTACAGAGTTGTGTCCGTGCTGACCGTGCTGCATCAAGACTGGCTGAACGGCAAAGAA TACAAGTGCGCTGTGAGTAACAAGGCTCTGCCTGCCCCTATCGAGAAAACCATCTCTAAGGCTA AGGGACAACCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCCCGGGACGAGCTGACCAAGAA CCAGGTGTCCCTGACCTGTCTGGTGAAAGGCTTCTACCCCTCTGACATCGCCGTGGAATGGGAG TCGAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGATCTT TCTTCCTGTACTCCAAGCTGACCGTCGACAAGTCTAGATGGCAGCAGGGCAACGTGTTCTCCTG CTCTGTGATGCACGAGGCTCTGCACAATCACTACACCCAGAAGTCCCTGTCCCTATCGCCAGGC GGCGGAGGAGGAAGTGGTGGCGGTGGTTCTCAAGTGCAACTACAAGAAAGTGGTGGTGGTCTCG TGAAGCCCGGAGGCTCTCTGCGGCTGTCCTGTGCTGCTTCTGGCTTTACATTCTCCTCTTACTG GATGTCCTGGGTCAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCAACATCAACCGGGAT GGCTCCGCCTCTTATTACGTGGACTCCGTGAAAGGCAGATTCACCATCTCTCGGGACGACGCCA AGAACTCCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGTGC CAGAGATCGCGGCGTGGGCTACTTCGACCTGTGGGGAAGAGGCACTCTGGTGACCGTGTCCAGT GGAGGGGGAGGTTCCGGTGGAGGTGGGTCTGGGGGTGGTGGAAGTCAAAGTGCACTAACTCAAC CAGCAAGTGTGAGCGGATCTCCCGGCCAGTCCATCACCATCTCCTGTACCGGCACATCCAGCGA TGTCGGCGGCTACAACTTCGTGTCTTGGTATCAACAGCACCCTGGCAAGGCCCCTAAGCTGATG ATCTACGACGTGTCCGATAGACCTTCTGGCGTGTCCGACCGGTTTAGCGGTTCCAAGTCCGGCA ACACCGCTTCTCTGATCATCTCTGGCCTGCAAGCCGACGACGAGGCTGACTACTACTGCTCCTC CTACGGCTCCTCTTCTACCCACGTGATCTTCGGAGGAGGTACCAAAGTGACCGTGCTGGGCGGT GGTGGTTCCGGAGGCGGAGGAAGTCAAGTGCAACTCCAGGAGAGTGGGCCGGGATTGGTTAAAC CGAGCGAGACCCTGAGTCTCACGTGTACAGTTTCAGGCGGGAGCGTGAGCTCAGGCGACTATTACTGGACATGGATAAGACAGAGTCCTGGAAAGGGCCTGGAGTGGATCGGTCATATTTATTATTCA G G GAAC AC T AAC TATAATCCATCTTT GAAAT C C C GAT T GAC AAT CTCTATCGATAC GAG T AAAA CCCAGTTTAGCCTCAAGCTGTCCAGCGTTACTGCTGCAGACACAGCCATATACTATTGTGTACG CGATCGCGTGACTGGAGCCTTCGACATATGGGGGCAGGGAACGATGGTAACAGTCTCTAGTGGA GGAGGAGGGTCTGGAGGAGGCGGAAGTGGAGGCGGAGGATCTGGCGGAGGAGGAAGCGACATCC AGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCA GGCGAGTCAGGACATCAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAA CTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGAT CTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTTCTG TCAACACTTTGATCATCTCCCGCTCGCTTTCGGCGGAGGGACCAAGGTGGAAATTAAATGA

[0214] > SEQ ID 25: panitumumab CDR-H1 amino acid sequence

[0215] GGSVSSGDY

[0216] > SEQ ID 26: panitumumab CDR-H2 amino acid sequence YYSGNTNYNPSLKS

[0217] > SEQ ID 27: panitumumab CDR-H3 amino acid sequence

[0218] DRVTGAFDI

[0219] > SEQ ID 28: panitumumab CDR-L1 amino acid sequence QASQDISNYLN

[0220] > SEQ ID 29: panitumumab CDR-L2 amino acid sequence

[0221] DASNLET

[0222] > SEQ ID 30: panitumumab CDR-L3 amino acid sequence

[0223] QHFDHLPLA

[0224] > Seq ID 31: cetuximab CDR-H1 amino acid sequence

[0225] NYGVH

[0226] > Seq ID 32: cetuximab CDR-H2 amino acid sequence VI SGGNTDYNTPFTS

[0227] > Seq ID 33: cetuximab CDR-H3 amino acid sequence

[0228] ALTYYDYEFAY

[0229] > Seq ID 34: cetuximab CDR-L1 amino acid sequence

[0230] RASQSIGTNIH

[0231] > Seq ID 35: cetuximab CDR-L2 amino acid sequence

[0232] YASES IS

[0233] > Seq ID 36: cetuximab CDR-L3 amino acid sequence

[0234] QQNNNWPTT> Seq ID 37: nimotuzumab CDR-H1 amino acid sequence NYYIY

[0235] > Seq ID 38: nimotuzumab CDR-H2 amino acid sequence GINPTSGGSNFNEKFKT

[0236] > Seq ID 39: nimotuzumab CDR-H3 amino acid sequence QGLWFDSDGRGFDF

[0237] > Seq ID 40: nimotuzumab CDR-L1 amino acid sequence RSSQNIVHSNGNTYLD

[0238] > Seq ID 41: nimotuzumab CDR-L2 amino acid sequence KVSNRFS

[0239] > Seq ID 42: nimotuzumab CDR-L3 amino acid sequence FQYSHVPWT

[0240] > Seq ID 43: anti-HER3 CDR-H1 amino acid sequence SYWMS

[0241] > Seq ID 44: anti-HER3 CDR-H2 amino acid sequence N I NRDGS AS YYVD S VKG

[0242] > Seq ID 45: anti-HER3 CDR-H3 amino acid sequence DRGVGYFDL

[0243] > Seq ID 46: anti-HER3 CDR-L1 amino acid sequence TGTSSDVGGYNFVS

[0244] > Seq ID 47: anti-HER3 CDR-L2 amino acid sequence DVSDRPS

[0245] > Seq ID 48: anti-HER3 CDR-L3 amino acid sequence SSYGSSSTHVI

[0246] > Seq ID 49: patritumab CDR-H1 amino acid sequence GGSFSGYY

[0247] > Seq ID 50: patritumab CDR-H2 amino acid sequence EINHSGSTNYNPSLKS

[0248] > Seq ID 51: patritumab CDR-H3 amino acid sequence ARDKWTWYFDL

[0249] > Seq ID 52: patritumab CDR-L1 amino acid sequenceRSSQSVLYSSSNRNYLA

[0250] > Seq ID 53: patritumab CDR-L2 amino acid sequence

[0251] WASTRES

[0252] > Seq ID 54: patritumab CDR-L3 amino acid sequence

[0253] QQYYSTPRT

[0254] > Seq ID 55: human IgGl amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG

[0255] > Seq ID 56: human Kappa amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

SIBA136USPASYMMETRIC ANTIBODY-LIKE PROTEINS AND METHODS OF MAKING AND USING THEREOFCLAIMSWhat is claimed is:

1. An asymmetric antibody -like protein having a binding affinity to EGFR and HER3, comprisesa Fab region comprising a light chain and a Fab domain,a Fc region comprising a first Fc domain and a second Fc domain, wherein the Fab domain is linked to the first Fc domain, anda first scFv domain, linked to the second Fc domain or the Fab domain.

2. The asymmetric antibody-like protein of Claim 1, further comprising a second scFv domain linked in tandem to the first scFv dom in.

3. The asymmetric antibody-like protein of Claim 1, comprisinga first heavy chain comprising the Fab domain and the first Fc domain, anda second heavy chain comprising the second Fc domain, wherein the first scFv domain is linked to the second Fc domain.

4. The asymmetric antibody-like protein of Claim 3, wherein the first scFv domain is linked to the second Fc domain at its N-terminus.

5. The asymmetric antibody-like protein of Claim 3, wherein the Fab region has a binding affinity to HER3, and the first scFv domain has a binding affinity to EGFR.

6. The asymmetric antibody-like protein of Claim 3, wherein the Fab region has a binding affinity to EGFR, and the first scFv domain has a binding affinity to HER3.

7. The asymmetric antibody -like protein of Claim 3, further comprising a second scFv domain, wherein the second scFv domain is tandem linked to the first scFv domain.

8. The asymmetric antibody-like protein of Claim 7, wherein the Fab region has a binding affinity to HER3, and the first and the second scFv domain each has a binding affinity to EGFR.

9. The asymmetric antibody-like protein of Claim 3, wherein the light chain comprises amino acid sequences having at least 98% of sequence identity to SEQ ID NO: 3, or 9.

10. The asymmetric antibody-like protein of Claim 3, wherein the first heavy chain comprises amino acid sequence having at least 98% of sequence identity to SEQ ID NO: 1, or 7.

11. The asymmetric antibody-like protein of Claim 3, wherein the second heavy chain comprises amino acid sequence having at least 98% of sequence identity to SEQ ID NO: 5, 11, or 13.SIBA136USP12. The asymmetric antibody-like protein of Claim 3, wherein the Fab region comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48.

13. The asymmetric antibody-like protein of Claim 3, wherein the first scFv domain comprises CDRs having the amino acid sequences set forth in SEQ ID NO: 31, 32, 33, 34, 35, 36, 43, 44, 45, 46, 47, and 48.

14. The asymmetric antibody-like protein of Claim 7, wherein the second scFv domain comprises CDRs having amino acid sequences set forth in SEQ ID NO: 31, 32, 33, 34, 35, and 36.

15. The asymmetric antibody-like protein of Claim 1, comprisinga heavy chain, comprising the Fab domain, the first Fc domain, and the second Fc domain, and wherein the first Fc domain and the second Fc domain are configured to pair to form the Fc region.

16. The asymmetric antibody-like protein of Claim 15, wherein the first scFv domain is linked to the second Fc domain at its C-terminus.

17. The asymmetric antibody-like protein of Claim 15, wherein the Fab region has a binding affinity to EGFR, and the first scFv domain has a binding affinity to HER3.

18. The asymmetric antibody-like protein of Claim 15, wherein the first scFv domain is linked to the Fab domain at its N-terminus.

19. The asymmetric antibody-like protein of Claim 15, wherein the Fab region has a binding affinity to HER3, and the first scFv domain has a binding affinity to EGFR.

20. The asymmetric antibody-like protein of Claim 15, further comprising a second scFv domain, wherein the second scFv domain is linked to the first scFv domain in tandem.

21. The asymmetric antibody-like protein of Claim 20, wherein the Fab region has a binding affinity to EGFR, the first scFv domain has a binding affinity to HER3, and the second scFv domain has a binding affinity to EGFR.

22. The asymmetric antibody -like protein of Claim 15, wherein the light chain comprises an amino acid sequence having at least 98% of sequence identity to SEQ ID NO: 17, or 21.

23. The asymmetric antibody-like protein of Claim 15, wherein the heavy chain comprises an amino acid sequence having at least 98% of sequence identity to SEQ ID NO: 15, 19, or 23.

24. The asymmetric antibody-like protein of Claim 15, the Fab region comprises CDRs having amino acid sequences set forth in SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

25. The asymmetric antibody-like protein of Claim 15, the first scFv domain comprises CDRs having amino acid sequences set forth in SEQ ID NO: 25, 26, 27, 28, 29, 30, 43, 44, 45, 46, 47, and 48.SIBA136USP26. The asymmetric antibody-like protein of Claim 20, the second scFv comprises CDRs having amino acid sequences set forth in SEQ ID NO: 25, 26, 27, 28, 29, and 30.

27. The asymmetric antibody-like protein of Claim 1, wherein the first Fc domain and second Fc domain comprise at least one mutation to form a knob-in-hole structure.

28. The asymmetric antibody -like protein of Claim 1, comprising an amino acid sequence having at least 98% of sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23.

29. The asymmetric antibody-like protein of Claim 1, wherein the first Fc domain is linked to the second Fc domain through a hinge, wherein the hinge comprises an amino acid sequence ((Gly-Gly-Gly-Gly-Ser)n, and wherein n is an integer of at least 5.

30. The asymmetric antibody-like protein of Claim 1, wherein the first scFv domain is linked to the Fc region or the Fab region through a linker, wherein the linker comprises an amino acid sequence (Gly-Gly-Gly-Gly-Ser)m, and wherein m is an integer of at least 3.

31. An isolated nucleic acid sequence encoding the asymmetric antibody-like protein of Claim 1.

32. An expression vector comprising the isolated nucleic acid sequences of Claim 31.

33. A host cell comprising the isolated nucleic acid sequence of Claim 31.

34. An immunoconjugate comprising the asymmetric antibody-like protein of Claim 1 and a cytotoxic moiety, wherein the cytotoxic moiety is derived from a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

35. A pharmaceutical composition, comprising the asymmetric antibody -like protein of Claim 1 or the immunoconjugate of Claim 34, and optionally a pharmaceutically acceptable carrier.

36. The pharmaceutical composition of Claim 35, further comprises a cytotoxic agent, wherein the cytotoxic agent comprises a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

37. A method for treating or preventing cancer in a subject, comprising administering to the subject a pharmaceutical composition comprising the asymmetric antibody-like protein of Claim 1 or the immunoconjugate of Claim 34.

38. The method of Claim 37, further comprises co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent comprises an antibody, a chemotherapy agent, an enzyme, or a combination thereof.

39. The method of Claim 37, 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, nasopharyngealSIBA136USPcancer, kidney cancer, gastric cancer, liver cancer, bladder cancer, cervical cancer, brain cancer, lymphoma, leukaemia, or myeloma.

40. The method of Claim 37, wherein the subject is a human.

41. A method for producing the asymmetric antibody-like protein of Claim 1, comprising culturing a host cell such that the DNA sequence encoding the asymmetric antibody-like protein of Claim 1 is expressed, and purifying said asymmetric antibody-like protein.

42. A method for producing the immunoconjugate of Claim 34, comprising conjugating the asymmetric antibody-like protein of Claim 1 with a cytotoxic agent, wherein the cytotoxic agent comprises a radioisotope, a radionuclide, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

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