Anti-egfr and her3 bispecific antibody-drug conjugates and uses thereof

Abstract

Provided are bispecific antibody-drug conjugates targeting EGFR and HER3, pharmaceutical compositions comprising the conjugates, their use for treating or preventing diseases or conditions associated with EGFR and / or HER3 activity, particularly EGFR and / or HER3-positive tumors, and their use in the preparation of pharmaceuticals for treating or preventing diseases or conditions associated with EGFR and / or HER3 activity, particularly EGFR and / or HER3-positive tumors.

Description

Anti-EGFR and HER3 bispecific antibody-drug conjugates and uses thereof Technical Field

[0001] The present invention relates to an anti-EGFR and HER3 bispecific antibody-drug conjugate (ADC), a pharmaceutical composition comprising the ADC, the use of the ADC for treating or preventing diseases or conditions associated with EGFR and / or HER3 activity, in particular EGFR and / or HER3-positive tumors, and the use of the ADC for preparing a medicament for treating or preventing diseases or conditions associated with EGFR and / or HER3 activity, in particular EGFR and / or HER3-positive tumors. Background Art

[0002] The human epidermal growth factor receptor (EGFR, also known as ErbB1, HER1) family has four members, EGFR, HER2, HER3 and HER4. Deregulation of each member through mutation, amplification and overexpression plays an important role in tumorigenesis and tumor metastasis. Overexpression is associated with the development of a variety of tumors. Blocking EGFR signaling by blocking the EGFR binding site on the receptor extracellular domain or by inhibiting intracellular tyrosine kinase activity can prevent the growth of tumors expressing EGFR and improve the patient's condition.

[0003] HER3 (encoded by ErbB3) is a member of the HER protein receptor family. Initially thought to lack kinase activity, recent studies have shown that HER3 can bind ATP and promote phosphorylation of its intracellular domain, but its kinase activity is relatively weak. Studies have shown that HER3 plays a crucial role in HER2 / HER3 heterodimerization and subsequent activation of the PI3K / AKT cascade, leading to its widespread interest and research in recent years. HER3 is overexpressed in various cancers, including gastric, breast, colorectal, and lung cancers, and HER3 expression is associated with poor survival after surgery. Studies have suggested that MET amplification may induce resistance to EGFR-targeted therapies by maintaining HER3-mediated PI3K / AKT signaling. Furthermore, HER3 is not only expressed in a variety of cancers but also has the potential to mediate resistance to targeted therapies, making HER3 a promising and attractive therapeutic target.

[0004] A bispecific antibody-drug conjugate (ADC) (BL-B01D1) that simultaneously targets EGFR and HER3 has emerged in the prior art and has demonstrated encouraging clinical efficacy in advanced solid tumors. However, the activity of this bispecific antibody-drug conjugate differs significantly between its anti-EGFR and anti-HER3 arms, resulting in a potential for stronger endocytosis-killing activity in tumors with high EGFR expression and weaker endocytosis-killing activity in tumors with low EGFR expression but high HER3 expression. This limitation limits its efficacy in patients with tumors that have low EGFR and high HER3 expression, reducing the potential population coverage and undermining the full potential of bispecific ADCs. Furthermore, this bispecific antibody-drug conjugate exhibits off-target toxicity, particularly severe hematologic toxicity. Severe hematologic toxicity can be distressing for patients and limits the ability to further increase the clinical dosage of this bispecific antibody-drug conjugate, resulting in an inability to further enhance its efficacy and, at the same time, failing to fully realize the advantages of dual EGFR and HER3 targeting.

[0005] Therefore, there is a need to develop more bispecific antibodies and bispecific antibody-drug conjugates targeting EGFR and HER3 that can overcome the above-mentioned shortcomings and thus better treat or prevent diseases or conditions associated with EGFR and / or HER3 activity, especially EGFR and / or HER3-positive tumors or cancers.

[0006] Most existing ADC conjugation methods still rely on random conjugation, including cysteine ​​and lysine conjugation. This conjugation approach can result in heterogeneous ADC product distribution. For example, a cysteine-conjugated ADC with an average DAR value of 4 may contain multiple components ranging from DAR0 to DAR8. Even within components with the same DAR value, there may be differences in conjugation at different sites. These components often have distinct properties. For example, DAR0 components competitively bind to the target, while DAR8 components, due to the high concentration of conjugated hydrophobic drugs, are prone to aggregation and thus more easily cleared from the body. The varying efficacy and pharmacokinetic characteristics of these components complicate PK / PD analysis of heterogeneous ADC mixtures and require sophisticated production processes to produce relatively stable ADC products.

[0007] Therefore, novel antibody modification technologies are needed. The antibodies obtained based on this technology can achieve ADCs with greater stability and efficacy when constructing ADCs.

[0008] Summary of the Invention

[0009] The present invention is based on bispecific antibodies and bispecific antibody-drug conjugates targeting EGFR and HER3 developed by the inventors, which have stronger stability and stronger anti-tumor activity than the existing technologies.

[0010] In some embodiments, the present invention specifically targets bispecific antibodies targeting EGFR and HER3, wherein the constant region of the antibody or antigen-binding fragment thereof undergoes cysteine ​​modification, such that one or more amino acids in the heavy or light chain of the antibody or antigen-binding fragment thereof are mutated to cysteine. The bispecific antibodies of the present invention have cysteine ​​mutations at relatively hidden sites in the constant region, thereby conferring improved stability and / or hydrophilicity to antibody-drug conjugates containing the same.

[0011] Therefore, the bispecific antibody-drug conjugate targeting EGFR and HER3 of the present invention is particularly suitable for treating or preventing diseases or disorders associated with EGFR and / or HER3 activity, especially EGFR and / or HER3 positive tumors.

[0012] In one aspect, the present invention provides a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof, comprising

[0013] a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and

[0014] heavy chain constant region and light chain constant region, wherein

[0015] The bispecific antibody against EGFR and HER3 comprises one or more mutations to cysteine, for example, one or more mutations selected from the following mutations: mutation of position 118 of the heavy chain constant region to cysteine, mutation of position 239 of the heavy chain constant region to cysteine, mutation of position 160 of the light chain constant region to cysteine, and mutation of position 166 of the light chain constant region to cysteine.

[0016] In another aspect, the present invention provides a bispecific antibody-drug conjugate having formula (I), a stereoisomer or a pharmaceutically acceptable salt or solvate thereof: Ab-(LD) n (Ⅰ)

[0017] in,

[0018] Ab is the anti-EGFR and HER3 bispecific antibody or its antigen-binding fragment according to the present invention,

[0019] L is a linker,

[0020] D is a cytotoxic compound,

[0021] n represents the number of connections, and n is a natural number selected from 1-15.

[0022] In yet another aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody-drug conjugate of the present invention, its stereoisomers or pharmaceutically acceptable salts or solvates, and a pharmaceutically acceptable carrier or excipient.

[0023] In another aspect, the present invention provides use of the bispecific antibody-drug conjugate of the present invention, its stereoisomers, or pharmaceutically acceptable salts or solvates in the preparation of a medicament for treating or preventing a disease or condition associated with EGFR and / or HER3 activity.

[0024] In another aspect, the present invention provides the bispecific antibody-drug conjugate of the present invention, its stereoisomers or pharmaceutically acceptable salts or solvates, for use in therapy, such as for treating or preventing diseases or conditions associated with EGFR and / or HER3 activity.

[0025] In another aspect, the present invention provides use of the bispecific antibody-drug conjugate of the present invention, its stereoisomers, or pharmaceutically acceptable salts or solvates for treating or preventing diseases or conditions associated with EGFR and / or HER3 activity.

[0026] In another aspect, the present invention provides a method for treating or preventing a disease or condition associated with EGFR and / or HER3 activity, the method comprising administering to a subject an effective amount of the bispecific antibody-drug conjugate of the present invention, its stereoisomers, or pharmaceutically acceptable salts or solvates thereof.

[0027] Compared with the prior art, the bispecific antibody-drug conjugate targeting EGFR and HER3 provided by the present invention has the following advantages:

[0028] (1) It has a strong affinity for both EGFR and HER3, and exhibits endocytosis activity comparable to that of anti-EGFR and anti-HER3 monoclonal antibodies, respectively. This allows the bispecific ADC molecule to have strong endocytosis and killing activity in tumors with different EGFR and HER3 expression levels, overcoming the limitations of traditional ADCs in highly heterogeneous tumors. This can expand the clinical application range of EGFR / HER3 bispecific ADC molecules and achieve stronger efficacy in tumor patients with low EGFR expression and medium / high HER3 expression.

[0029] (2) The use of a site-specific conjugation linker-payload conjugation method can significantly increase the uniformity of ADC molecules and the stability of linker-payloads. At the same time, due to the reduction in the number of linker-payloads on the same molecule, the hydrophobicity of the entire ADC molecule can be significantly reduced, and its in vivo PK will be significantly improved, ultimately leading to stronger drug efficacy. The improvement in linker-payload stability brought about by site-specific conjugation will significantly reduce the nonspecific shedding of small molecule toxins outside the tumor, which is expected to significantly improve the clinical safety of bispecific ADC molecules, especially reducing the toxicity associated with free small molecules, such as hematotoxicity.

[0030] (3) The Fc part of the antibody molecule adopts IgG1LALA design, which destroys its binding ability to FcγR and reduces the non-tumor specific binding of the ADC molecule. It is expected to significantly improve the clinical safety of the bispecific ADC molecule, especially reduce blood toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 shows the molecular structure of Duligo-LC1-NT3.

[0032] FIG2 shows the results of RP-HPLC analysis of Duligo-LC1-NT3.

[0033] FIG3 shows the results of SEC analysis of Duligo-LC1-NT3.

[0034] FIG4 shows the molecular structure of SIB001-DXd.

[0035] FIG5 shows the results of RP-HPLC analysis of SIB001-DXd.

[0036] FIG6 shows the results of SEC analysis of SIB001-DXd.

[0037] FIG7 shows the molecular structure of Patritumab-DXd.

[0038] FIG8 shows the results of RP-HPLC analysis of Patritumab-DXd.

[0039] FIG9 shows the SEC analysis results of Patritumab-DXd.

[0040] FIG10 shows the molecular structure of Duligo-DXd.

[0041] FIG11 shows the results of RP-HPLC analysis of Dulgio-DXd.

[0042] FIG12 shows the results of SEC analysis of Duligo-DXd.

[0043] FIG13 shows the molecular structure of IgG1LALA-NT3 (DAR=8).

[0044] FIG. 14 shows the results of RP-HPLC analysis of IgG1LALA-NT3 (DAR=8).

[0045] FIG. 15 shows the results of SEC analysis of IgG1 LALA-NT3 (DAR=8).

[0046] FIG16 shows the molecular structure of IgG1LALA-NT3 (DAR=4).

[0047] FIG. 17 shows the results of RP-HPLC analysis of IgG1 LALA-NT3 (DAR=4).

[0048] FIG. 18 shows the results of SEC analysis of IgG1 LALA-NT3 (DAR=4).

[0049] FIG19 shows the molecular structure of IgG1LALA-DXd.

[0050] FIG20 shows the results of RP-HPLC analysis of IgG1LALA-DXd.

[0051] FIG21 shows the results of SEC analysis of IgG1LALA-DXd.

[0052] FIG22 shows the stability of Duligo-LC1-NT3 in mouse and monkey plasma, analyzed by RP-HPLC method.

[0053] Figure 23 shows the stability of Duligo-LC1-NT3 in mouse and monkey plasma, analyzed by LC-MS method.

[0054] FIG24 shows the expression detection of EGFR and HER3 in different tumor cell lines.

[0055] FIG. 25 shows the Fab-ZAP endocytosis-killing of Duligotuzumab, Cetuximab, and Patritumab molecules on human pancreatic cancer cells AsPC1.

[0056] FIG26 shows the Fab-ZAP endocytosis-killing effect of Duligotuzumab, Cetuximab, and Patritumab on human gastric cancer cells NUGC4.

[0057] Figure 27 shows the Fab-ZAP endocytosis-killing of Duligotuzumab, Cetuximab, and Patritumab molecules on Chinese Hamster Ovary cells CHO-HER3

[0058] FIG28 shows the in vitro killing of human breast cancer cells MCF7 by Duligo-DXd and SIB001-DXd.

[0059] FIG29 shows the in vitro killing of human breast cancer cells MDA-MB-453 by Duligo-DXd and SIB001-DXd.

[0060] FIG30 shows the in vitro killing of human colorectal cancer cells GP2D by Duligo-DXd and SIB001-DXd.

[0061] FIG31 shows the in vitro killing of human lung cancer cells H1568 by Duligo-DXd and SIB001-DXd.

[0062] FIG32 shows the in vitro killing of human colorectal cancer cells SW480 overexpressing HER3 by Duligo-DXd and SIB001-DXd.

[0063] FIG33 shows the in vitro killing of human breast cancer cells MDA-MB-453 by Duligo-LC1-NT3 and SIB001-DXd.

[0064] FIG34 shows the in vitro killing of human lung cancer cells HCC95 by Duligo-LC1-NT3 and SIB001-DXd.

[0065] FIG35 shows the in vitro killing of human lung cancer cells H1568 by Duligo-LC1-NT3 and SIB001-DXd.

[0066] FIG36 shows the in vitro killing of human colorectal cancer cells H508 by Duligo-LC1-NT3 and SIB001-DXd.

[0067] FIG37 shows the in vitro killing of human pancreatic cancer cells AsPC1 by Duligo-LC1-NT3 and SIB001-DXd.

[0068] FIG38 shows the in vitro killing of HER3-overexpressing human colorectal cancer cells SW480 by Duligo-LC1-NT3 and SIB001-DXd.

[0069] FIG39 shows the in vitro killing of HER3-overexpressing human lung cancer cells H1703 by Duligo-LC1-NT3 and SIB001-DXd.

[0070] FIG40 shows the affinity of Duligo-LC1-NT3 and SIB001-DXd to EGFR detected by ELISA.

[0071] FIG41 shows the affinity of Duligo-LC1-NT3 and SIB001-DXd for EGFR detected by ELISA.

[0072] FIG42 shows the anti-tumor efficacy of EGFR / HER3-ADCs molecules in the H508 tumor-bearing mouse model.

[0073] FIG43 shows the effects of EGFR / HER3-ADCs on body weight changes in mice.

[0074] FIG44 shows the anti-tumor efficacy of EGFR / HER3-ADCs molecules in the NUGC-4 tumor-bearing mouse model.

[0075] FIG45 shows the changes in body weight of EGFR / HER3-ADCs in the NUGC-4 tumor-bearing mouse model.

[0076] FIG46 shows the anti-tumor efficacy of EGFR / HER3-ADCs molecules in the SW620 tumor-bearing mouse model.

[0077] FIG47 shows the changes in body weight of EGFR / HER3-ADCs in the SW620 tumor-bearing mouse model.

[0078] FIG48 shows the anti-tumor efficacy of EGFR / HER3-ADCs molecules in the ASPC1 tumor-bearing mouse model.

[0079] FIG49 shows the changes in body weight of EGFR / HER3-ADCs in the ASPC1 tumor-bearing mouse model.

[0080] FIG50 shows the anti-tumor efficacy of EGFR / HER3-ADCs molecules in the NCI-H1568 tumor-bearing mouse model.

[0081] FIG51 shows the changes in body weight of EGFR / HER3-ADCs in the NCI-H1568 tumor-bearing mouse model. DETAILED DESCRIPTION

[0082] Certain embodiments will now be described in detail, examples of which are illustrated in the accompanying specific embodiments. Although the embodiments of the present invention will be described, it should be understood that they are not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to encompass all alternatives, modifications, and equivalents, which may be included within the scope of the present invention defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used for the practice of the present invention. The present invention is in no way limited to the methods and materials described. If one or more incorporated documents and similar materials differ from or contradict the present invention, including but not limited to defined terms, term usage, described technology, etc., the present invention shall prevail.

[0083] It will be appreciated that certain features of the present invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0084] I. Definition

[0085] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0086] The term "or" refers to a single element of the listed alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0087] As used herein, the term "about" refers to a value within ±10%, or 5%, 4%, 3%, 2%, or 1% of a given value.

[0088] As used herein, the terms "include", "comprise", "have" and similar expressions do not exclude unrecited elements. These terms also include the situation that the composition consists of only the listed elements.

[0089] As used herein, the term "HER3" (human epidermal growth factor receptor 3, also known as ErbB3) is a receptor protein tyrosine kinase and belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes HER1 (also known as EGFR), HER2, and HER4. HER3 is a transmembrane receptor and consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, and an intracellular protein tyrosine kinase domain (TKD) and a C-terminal phosphorylation domain. HER3 has been found to be overexpressed in several types of cancer (e.g., gastric cancer, breast cancer, colorectal cancer, and lung cancer). The association between the expression of HER2 / HER3 and the progression from non-invasive to invasive stages has been shown.

[0090] As used herein, the term "antibody" is used in its broadest sense and includes immunoglobulins or other types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen, which are proteins or polypeptides that exhibit binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, and the like. Classical antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on the conservative differences in the amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region is used to recognize and bind to the antigen, and the constant region (e.g., Fc fragment) is used to initiate downstream effects, such as antibody-dependent cellular cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions with a higher degree of variation in amino acid composition and arrangement order, which are key positions for antibody binding to antigens and are therefore also referred to as complementary determining regions (CDRs). In this article, the three heavy chain complementary determining regions are referred to as HCDR1, HCDR2 and HCDR3, respectively, and the three light chain complementary determining regions are referred to as LCDR1, LCDR2 and LCDR3, respectively. In a given light chain variable region or heavy chain variable region amino acid sequence, the amino acid sequence of the CDR can be easily determined using a numbering scheme recognized in the art, such as Kabat, Chothia, IMGT, AbM or Contact. For example, in a given light chain variable region or heavy chain variable region amino acid sequence, the amino acid sequence of each CDR can be determined by one of the above-mentioned numbering schemes, or each can be independently determined by any of the above-mentioned numbering schemes. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" covers CDR sequences determined in any of the above-mentioned ways.

[0091] Based on the amino acid sequence of the constant region of its heavy chains, antibodies can be divided into five major classes: IgA, IgD, IgE, IgG, and IgM. These antibody classes are further divided into subclasses based on the size of the hinge region, the location of interchain disulfide bonds, and molecular weight, such as IgG1, IgG2a, IgG2b, and IgG3. Based on the amino acid composition and arrangement of the constant region of its light chains, light chains can be divided into two types: kappa and lambda. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known in the art.

[0092] As used herein, when referring to amino acid positions in antibody domains other than the variable region (e.g., the constant region, e.g., the Fc region), numbering is according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. When position numbers and / or amino acid residues are assigned to a particular antibody isotype, it is intended to apply to the corresponding positions and / or amino acid residues of any other antibody isotype, as known to those skilled in the art.

[0093] General information on human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0094] The amino acid position to be mutated to cysteine ​​is generally indicated by "chain type, mutation position". In this document, unless otherwise specified, LLC represents lambda light chain, LC represents kappa light chain, and HC represents heavy chain. Therefore, "LLC160" refers to EU numbering position 160 of the lambda light chain, for example, the amino acid at this position is mutated to cysteine. When heavy chain amino acid positions are mentioned in the present invention, unless otherwise specified, they refer to amino acid positions numbered according to the IgG1 heavy chain, that is, they cover amino acid positions based on the IgG1 heavy chain numbering, as well as amino acid positions corresponding to the amino acid positions on other heavy chains. For example, when HC118 is mentioned, it refers to EU numbering position 118 of the IgG1 heavy chain, and also covers the position of amino acid 118 of IgG isotypes other than IgG1. When referring to a combination of mutations, the combined mutations are connected by a hyphen (-). “LLC160-LLC166” indicates that the amino acids at positions 160 and 166 (EU numbering) of the lambda light chain are simultaneously mutated to cysteine.

[0095] As used herein, an "antigen-binding fragment" of an antibody refers to an amino acid fragment of an antibody molecule that participates in antigen-specific binding, such as Fab, Fab', and F(ab')2. Those skilled in the art know how to obtain these antigen-binding fragments. For example, a classical antibody molecule can be digested with papain to obtain a Fab fragment, and with pepsin to obtain a F(ab')2. A Fab' fragment can be formed by treating the F(ab')2 with a reducing agent to break the disulfide bonds within the hinge region.

[0096] As used herein, the term "single-chain antibody (scFv)" refers to a single-chain antibody consisting of the variable regions of the heavy and light chains of an antibody linked by a short peptide. Properly folded, the variable regions from the heavy and light chains interact through non-covalent bonds to form the Fv fragment, allowing the scFv to retain its affinity for the antigen.

[0097] As used herein, the term "single domain antibody (sdAb)", or also referred to as "VHH antibody", refers to an antibody molecule with antigen binding ability, including a heavy chain variable region but no light chain. Structurally, a single domain antibody can also be considered a fragment of a classic four-chain antibody molecule. Single domain antibodies were first discovered in camelids, and subsequently, researchers discovered more single domain antibodies with antigen binding ability by screening antibody libraries (such as phage display libraries). Single domain antibodies have some advantages over ordinary antibody molecules (e.g., classic antibody molecules), such as but not limited to: a smaller molecular weight, which makes it easy to reach tissues or parts that are difficult for ordinary antibody molecules to reach when used in the human body, or the ability to access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; more stable, and able to withstand, for example, changes in temperature and pH, as well as the effects of denaturants and proteases.

[0098] As used herein, the term "Fc fragment" refers to the handle region of the Y-shaped classical antibody molecule, i.e., a crystallizable fragment (Fc), including the second and third constant domains (CH2 and CH3 domains) of the heavy chain. The antibody Fc region can be obtained by hydrolyzing the antibody molecule with a proteolytic enzyme (such as papain). In some instances, the Fc region may include a hinge, CH2, and CH3. When the Fc region includes a hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment may be from IgG, IgM, IgD, IgE, or IgA. In some instances, the Fc region is from IgG1, IgG2, IgG3, or IgG4. "Fc fragment" also includes variant Fc fragments from natural Fc fragments that have been modified but still retain their effector functions. "Variant Fc fragment" comprises an amino acid sequence having at least one amino acid change in the amino acid sequence of a natural Fc fragment. In some instances, the variant Fc fragment has at least one amino acid substitution compared to the parent Fc fragment (native Fc fragment), for example, about 1 to about 10 amino acids are substituted in the parent Fc fragment, and preferably about 1 to about 5 amino acid substitutions. In some instances, the variant Fc fragment Fc region has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the parent Fc fragment. The effector functions of the "Fc fragment" may include binding to Fc receptors, Clq binding and complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediating phagocytosis, and the like.

[0099] As used herein, the term "mouse antibody" refers to an antibody whose variable region and constant region (if any) are derived from mouse or rat immunoglobulin sequences. Mouse antibodies can be conveniently obtained by immunizing mice or rats with the corresponding antigen and isolating the target antibody therefrom. Alternatively, after immunizing mice or rats with the corresponding antigen, cells expressing the target antibody (such as B cells) are isolated and cultured to obtain the target antibody. Alternatively, after immunizing mice or rats with the corresponding antigen, cells expressing the target antibody are isolated and cultured, and the cells are fused with immortalized cells such as myeloma cells to obtain hybridoma cells. Cultivating hybridoma cells can obtain the target antibody (such as monoclonal antibody) for a long time and in large quantities. In some embodiments, the "mouse antibody" is a mouse antibody. "Humanized antibody" refers to a chimeric antibody obtained by artificially modifying a non-human antibody, i.e., an antibody whose variable region and constant region (if any) are not derived from human immunoglobulins, so that it contains the amino acid sequence of a human antibody. A humanized antibody may contain the constant region and / or framework region of a human antibody. Humanized antibodies can be obtained through genetic engineering, for example, by replacing the constant regions of a mouse antibody with those of a human antibody and / or replacing the framework regions of a mouse antibody with those of a human antibody. Such humanization generally does not affect the binding specificity of the original antibody to the corresponding antigen, and therefore such antigens are also included within the scope of the present invention.

[0100] As used herein, the term "monoclonal antibody" refers to an antibody that is homogeneous and directed only against a specific antigenic epitope. Compared to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous character of the antibody and is not to be construed as requiring the antibody to be produced by any particular method. The monoclonal antibodies of the present invention can be produced by hybridoma methods or recombinant DNA methods well known in the art, or obtained by screening methods described elsewhere herein.

[0101] As used herein, the term "purification tag" refers to an amino acid sequence that facilitates the separation of a polypeptide or protein of interest from a cell culture or supernatant expressing the polypeptide or protein of interest. Examples include, but are not limited to, His6 tags, Flag tags, MBP tags, GST tags, SUMO tags, and the like.

[0102] For antibodies or antigen-binding fragments thereof, "binding," "directed against," or "specifically binds" means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher binding affinity for another molecule (e.g., an antigen) relative to other molecules present in the environment. A molecule can bind to, be directed against, or specifically bind to more than one molecule. For example, a bispecific antibody can have a higher binding affinity for two different antigens relative to other molecules. The binding affinity of an antibody for an antigen can be measured by a number of parameters, such as the EC50 value or KD value of the antibody binding to the antigen.

[0103] EC 50 Concentration for 50% of maximal effect refers to the concentration that produces 50% of the maximal effect. In enzyme-linked immunosorbent assays (ELISAs), when used to indicate the binding ability of an antibody molecule to its corresponding antigen, it refers to the antibody concentration that produces half the maximum detection signal (e.g., colorimetric or fluorescence intensity). The lower the EC50 value, the greater the binding affinity for the antigen.

[0104] The KD value can also be used to measure the binding affinity between an antibody and its antigen. The KD value is the equilibrium dissociation constant between the antibody and its antigen, i.e., the ratio of koff / kon. Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody.

[0105] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers composed of amino acid residues. Full-length proteins and fragments thereof are encompassed within this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and similar modifications. In addition, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions, and substitutions (which are generally conservative in practice), as long as the protein retains the desired activity. These modifications may be purposeful, such as through site-directed mutagenesis, or may be accidental, such as through mutations in the host producing the protein or errors due to PCR amplification.

[0106] As used herein, the term "functional variant" refers to a variant molecule obtained by introducing one or more amino acid insertions, deletions or substitutions into a parent protein molecule or a parent protein molecule (e.g., a natural protein molecule), which still retains at least part of the function of the parent protein molecule (especially the function of interest, such as the ability to bind to the corresponding antigen). For example, a functional variant of an antibody molecule may retain at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the antigen binding ability of its parent molecule, or even have a higher binding ability than the parent molecule. In some embodiments, a functional variant of an antibody molecule may retain at least 80%, 85%, 90%, 95% or even 100% or more of the antigen binding affinity of its parent molecule. For an antibody molecule or its antigen-binding fragment, a functional variant typically includes amino acid changes in the variable region framework sequence and / or constant region, but does not exclude the possibility of making one or a few amino acid changes to the CDR region sequence.

[0107] As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0108] As used herein, the term "vector" refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (e.g., a nucleic acid, a plasmid, or a virus, etc.). A vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of the polynucleotide of interest (such as a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used in colorimetric analysis, such as β-galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0109] As used herein, the term "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical to the original parent cell (in terms of morphology or genomic DNA complement) due to natural, accidental, or deliberate mutations. Host cells can be isolated cells or cell lines, and also include cells transfected in vivo with the nucleic acid molecules or expression vectors provided herein.

[0110] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also called "sequence consistency") refers to the amount of consistency between two amino acid or nucleotide sequences (such as a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of consistency between two amino acid or nucleotide sequences, a sequence alignment is performed and gaps (if any) are introduced. If the amino acid residues or bases in the two sequences are the same at a certain comparison position, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the comparison window to obtain sequence consistency. In other algorithms, the number of gaps and / or gap lengths are also taken into account. Commonly used sequence comparison algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purpose of the present invention, the publicly available alignment software BLAST (available from https: / / www.ncbi.nlm.nih.gov / ) can be used to obtain optimal sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using default settings.

[0111] As used herein, the term "aliphatic group" refers to a substituted or unsubstituted linear, branched and / or cyclic, saturated or unsaturated group consisting only of carbon atoms and hydrogen atoms and connected to the rest of the molecule by a single bond, including linear, branched, cyclic or combinations thereof alkyl, alkenyl and alkynyl groups. In some embodiments, the term "aliphatic group" can be used interchangeably with "aliphatic group". In some embodiments, the aliphatic group contains one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 unsaturated carbon-carbon double bonds (-C=C-), carbon-carbon triple bonds (-C≡C-) groups, and / or any combination thereof. Alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl are all defined below. In the present invention, aliphatic group refers to alkyl, alkenyl, alkynyl, cycloalkyl and / or cycloalkenyl, preferably refers to alkyl and / or cycloalkyl. The hydrogen atoms on the aliphatic group may be optionally substituted by any suitable group, such as halogen, hydroxyl, amino, monosubstituted amino, disubstituted amino, alkoxy, heterocyclic group, etc. The term "aliphatic group" refers to a divalent substituted aliphatic group formed by replacing one hydrogen atom on the aliphatic group with a valence bond, and the terms "alkylene", "alkenylene", "alkynylene", "cycloalkylene", "cycloalkenylene" and "cycloalkynylene" have similar meanings.

[0112] As used herein, as an independent group or part of another group, the term "alkyl" means a straight or branched group consisting only of carbon atoms and hydrogen atoms, free of unsaturated bonds, and connected to the rest of the molecule by a single bond. The alkyl group can have, for example, 1 to 18, preferably 1 to 12, and more preferably 1 to 8 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, hexyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl, and decyl, preferably methyl, ethyl, propyl, isopropyl, n-butyl, and more preferably methyl, ethyl, propyl, and isopropyl. The hydrogen on the alkyl group can be optionally substituted by any suitable group, such as halogen, hydroxyl, amino, monosubstituted amino, disubstituted amino, alkoxy, heterocyclic, and the like.

[0113] As used herein, the term "alkenyl" as an independent group or as part of another group refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having, for example, 2 to 18, preferably 2 to 10, more preferably 2 to 8 carbon atoms, and connected to the rest of the molecule by a single bond, including but not limited to ethenyl, propenyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, pent-1,4-dienyl, etc., preferably ethenyl and propenyl. The hydrogen on the alkenyl group may be optionally substituted with any suitable group, such as halogen, hydroxyl, amino, monosubstituted amino, disubstituted amino, alkoxy, heterocyclyl, etc.

[0114] As used herein, as an independent group or as part of another group, the term "alkynyl" means a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing at least one triple bond and optionally one or more double bonds, having, for example, 2 to 18, preferably 2 to 10, more preferably 2 to 8 carbon atoms, and connected to the rest of the molecule by a single bond. Examples of alkynyl groups include, but are not limited to, ethynyl, prop-1-ynyl, pent-1-en-4-ynyl, and the like. The hydrogen on the alkynyl group may be optionally substituted with any suitable group, such as halogen, hydroxy, amino, alkoxy, heterocyclyl, and the like.

[0115] As used herein, as an independent group or as part of another group, the term "cycloalkyl" means a stable, saturated, non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having, for example, 3 to 15, preferably 3 to 10, more preferably 3 to 8 carbon atoms, such as 3 to 6 or 5 to 6 carbon atoms, and connected to the rest of the molecule by a single bond via any suitable carbon atom on the ring. Cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.1.1]heptyl, bicyclo[3.2.1]octyl, and adamantyl, preferably cyclobutyl, cyclopentyl, and cyclohexyl. The hydrogen atoms on the cycloalkyl groups may be optionally replaced with any suitable groups such as halogen, hydroxy, amino, monosubstituted amino, disubstituted amino, alkyl, alkoxy, heterocyclyl, and the like.

[0116] As used herein, as an independent group or as part of other groups, the term "cycloalkenyl" means a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting solely of carbon and hydrogen atoms and containing at least one double bond, which may include a fused ring system or a bridged ring system. It has, for example, 3 to 15, preferably 3 to 10, more preferably 3 to 8 carbon atoms, such as 3 to 6 or 5 to 6 carbon atoms, and is linked to the rest of the molecule via a single bond through any suitable carbon atom on the ring. Examples of cycloalkenyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1,3-cyclohexadiene, 1,4-cyclohexadiene, 1H-indenyl, 2,3-dihydroindanyl, 1,2,3,4-tetrahydro-naphthyl, 5,6,7,8-tetrahydro-naphthyl, 8,9-dihydro-7H-benzocyclohepten-6-yl, 6,7,8,9-tetrahydro-5-hydro-benzocycloheptenyl, 5,6,7,8,9,10-hexahydro-benzocyclooctenyl, fluorenyl, bicyclo[2.2.1]heptenyl, bicyclo[2.2.2]octyl, bicyclo[2.2.2]octenyl, bicyclo[3.2.1]octenyl, octahydro-4,7-methylene-1-hydro-indenyl, and octahydro-2,5-methylene-pentalenyl, and the like. The hydrogen atoms on the cycloalkenyl groups may be optionally replaced with any suitable groups such as halogen, hydroxy, amino, monosubstituted amino, disubstituted amino, alkyl, alkoxy, heterocyclyl, and the like.

[0117] As used herein, as an independent group or part of other groups, the term "cycloalkynyl" means a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon atoms and hydrogen atoms, containing at least one triple bond, which may include a fused ring system or a bridged ring system. It has, for example, 3 to 15, preferably 3 to 10, more preferably 3 to 8 carbon atoms, such as 3 to 6 or 5 to 6 carbon atoms, and is connected to the rest of the molecule by a single bond via any suitable carbon atom on the ring. Examples of cycloalkynyl include, but are not limited to, cyclobutynyl, cyclopentynyl, cyclohexynyl, etc. The hydrogen on the cycloalkynyl group may be optionally substituted by any suitable group, such as halogen, hydroxyl, amino, monosubstituted amino, disubstituted amino, alkyl, alkoxy, heterocyclic, etc.

[0118] As used herein, the term "heteroaliphatic group" refers to a substituted or unsubstituted linear, branched and / or cyclic, saturated or unsaturated hydrocarbon group containing heteroatoms selected from N, O and S, including alkyl, alkenyl and alkynyl groups that are linear, branched, cyclic or a combination thereof. In some embodiments, the term "heteroaliphatic group" can be used interchangeably with "heteroaliphatic group". In some embodiments, the heteroatoms contained in the heteroaliphatic group can form the main chain of the heteroaliphatic group together with the carbon atoms, such as but not limited to -CNC-, -COC-, -COOC, -CSC-, -CSSC and other group structures or any combination thereof. In some embodiments, the heteroatoms contained in the heteroaliphatic group can be substituents attached to carbon atoms, such as but not limited to -C≡N, -C=N-, -CN=, -C=O, -C-OH, -C=S, -C-SH and other substitution structures. In some of the embodiments, the heteroatoms contained in the heteroaliphatic group can be any combination of the group structures listed above. In some embodiments, a heteroaliphatic group contains one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, unsaturated carbon-carbon double bonds (-C=C-), carbon-carbon triple bonds (-C≡C-), -NH-, -NH2-, OH, -OR m 、-O-、-C(O)-、-C(OR n )-、-C(O)O-、-SH、-SR o 、-S-、-C(S)-、-C(SR p )-, -C(S)O-, -P(O)-, and / or any combination thereof, wherein R m 、R n 、R o and R p are each independently substituted or unsubstituted C1-C 14 Aliphatic hydrocarbon groups, such as C1-C 12 、C1-C 10, C1-C8, C1-C6, C1-C4 aliphatic, substituted or unsubstituted C1-C 14 Specific non-limiting examples of aliphatic groups include, but are not limited to, methyl, ethyl, n- and isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, ethylene(vinyl), propenyl, butenyl, pentenyl, 1-methyl-2-buten-1-yl, 5-hexenyl, ethynyl, 1-propynyl, 2-propynyl, and the like.

[0119] As used herein, the term "aryl" or "aromatic ring" refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring system having at least one aromatic ring. Unless otherwise indicated, an aryl group can be 6 to 10 members. In certain embodiments, an aryl group can contain 6 ring carbon atoms. All atoms within a carbocyclic aryl group are carbon atoms. Non-limiting examples of aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, and the like. In the context of the present invention, the terms "aryl" and "aromatic ring" can be used interchangeably.

[0120] As used herein, the term "aliphaticoxy" refers to the group -O-aliphatic, wherein aliphatic has the meaning defined herein.

[0121] As used herein, the term "aliphaticthio" refers to the group -S-aliphatic, wherein aliphatic has the meaning defined herein.

[0122] As used herein, the term "heteroaryl" or "heteroaromatic ring" refers to a monocyclic ring system, or a fused or bridged bicyclic ring system, wherein the ring system contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and at least one ring is an aromatic ring. Unless otherwise specified, a heteroaryl group can be 5 to 10 members. In certain embodiments, a heteroaryl group can be 5-membered or 6-membered. In certain embodiments, a heteroaryl group can contain one, two, or three heteroatoms. In certain embodiments, a heteroaryl group can contain one or two heteroatoms. The limiting examples of heteroaryl groups include benzimidazolyl, benzofuranyl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, quinolyl, quinolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, tetrazolyl, indolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl etc.Heteroaryl groups include at least one heteroatomic ring and at least one aromatic ring with at least one as above.For example, there is at least one heteroatomic ring can be fused to one, two or three carbocyclic rings, for example aryl ring, cyclohexane ring, cyclohexene ring, cyclopentane ring, cyclopentene ring or another monocyclic heterocycle. Non-limiting examples of fused heteroaryl groups include 1,2,3,5,8,8a-hexahydroindolizine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, 2,3-dihydrobenzothiophene, etc. In the context of the present invention, the terms "heteroaryl" and "heteroaromatic ring" are used interchangeably.

[0123] As used herein, the term "heteroatom" refers to nitrogen (N), oxygen (O), and sulfur (S), and can include any oxidized forms of nitrogen and sulfur, and any quaternized forms of basic nitrogen, unless otherwise specified.

[0124] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a cell population is characterized by unregulated cell growth. As used herein, the terms "cancer cell" and "tumor cell" refer to the total number of cells from a tumor, including tumorigenic stem cells (cancer stem cells) and non-tumorigenic cells that make up the majority of the tumor cell population. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include melanoma, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, stomach cancer, head and neck squamous cell carcinoma, and various types of head and neck cancer.

[0125] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive growth or proliferation of cells, whether benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0126] The terms "cancer" and "tumor" are used interchangeably herein and include both solid and hematological tumors.

[0127] As used herein, the term "metastasis" refers to the process by which cancer spreads or metastasizes from its site of origin to other areas of the body as similar cancerous lesions develop in new locations. "Metastatic" cells are cells that have lost their adhesive contacts with neighboring cells and migrated from the primary site of the disease via the blood or lymph to invade neighboring body structures.

[0128] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0129] As used herein, the terms "treat" and "prevent" refer to: 1) curing, slowing, alleviating symptoms and / or stopping the progression of a diagnosed pathological condition or disease; 2) pre-implemented methods to prevent, delay or slow the development of a target pathological condition or disease. Therefore, people in need of treatment include those who already have the disease; those who are susceptible to the disease; and those in need of prevention. A subject is successfully "treated" according to the methods of the present invention if the patient exhibits one or more of the following: a reduction or complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs (these include the spread of cancer cells to soft tissue and bone); inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with a specific cancer; reduction in morbidity and mortality; and improvement in quality of life. In the context of cancer, the term "prevention" refers to the administration of medical treatments such as drugs to a subject, particularly a subject at risk of cancer, before the onset of symptoms or pathological conditions associated with the cancer.

[0130] II. Antibodies

[0131] The present invention provides a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof, which comprises

[0132] a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively,

[0133] The amino acid sequence of HCDR1 is GFTLSGDWIH (SEQ ID NO: 1),

[0134] The amino acid sequence of HCDR2 is EISAAGGYTDYADSVKG (SEQ ID NO: 2),

[0135] The amino acid sequence of HCDR3 is ESRVSFEAAMDY (SEQ ID NO: 3),

[0136] The amino acid sequence of LCDR1 is RASQNIATDVA (SEQ ID NO: 4),

[0137] The amino acid sequence of LCDR2 is SASFLYS (SEQ ID NO: 5),

[0138] The amino acid sequence of LCDR3 is QQSEPEPYT (SEQ ID NO: 6),

[0139] Preferably, HCDR1 is defined using the AbM numbering system; HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined using the Kabat numbering system; and

[0140] heavy chain constant region and light chain constant region, wherein

[0141] The anti-EGFR and HER3 bispecific antibody comprises one or more mutations to cysteine, for example, 1, 2, 3, 4 or more mutations, for example, one or more mutations selected from the following mutations, for example, 1, 2, 3, 4 or more mutations: mutation of position 118 of the heavy chain constant region to cysteine, mutation of position 239 of the heavy chain constant region to cysteine, mutation of position 160 of the light chain constant region to cysteine, and mutation of position 166 of the light chain constant region to cysteine.

[0142] In some embodiments, in the bispecific antibodies of the present invention, the heavy chain variable region (V H )

[0143] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7; or

[0144] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7; or

[0145] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 7, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) does not exceed 10, for example, does not exceed 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids,

[0146] The sequence of SEQ ID NO: 7 is as follows:

[0147] The nucleic acid sequence encoding this amino acid sequence is as follows:

[0148] In some embodiments, in the bispecific antibodies of the present invention, the heavy chain variable region (V H )

[0149] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 8; or

[0150] (ii) comprises or consists of the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 8; or

[0151] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 8, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) does not exceed 10, for example, not more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids.

[0152] In some embodiments, in the bispecific antibodies of the present invention, the light chain variable region (V L )

[0153] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; or

[0154] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9; or

[0155] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 9, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) does not exceed 10, for example, does not exceed 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids,

[0156] The amino acid sequence of SEQ ID NO: 9 is as follows:

[0157] The nucleic acid sequence encoding this amino acid sequence is as follows:

[0158] In some embodiments, in the bispecific antibodies of the present invention, the light chain variable region (V L )

[0159] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 10; or

[0160] (ii) comprises or consists of the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 10; or

[0161] (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 10, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) does not exceed 10, for example, not more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids.

[0162] In some embodiments, the bispecific antibodies of the present invention include anti-EGFR and HER3 bispecific antibodies with cysteine ​​mutations at relatively hidden sites in the constant region, thereby providing improved stability and / or hydrophilicity to the ADC containing the cysteine ​​mutations. As defined herein, "cysteine ​​mutation" refers to the substitution of a non-cysteine ​​amino acid in a protein with a cysteine ​​amino acid.

[0163] In some embodiments, in the bispecific antibody of the present invention, the anti-EGFR and HER3 bispecific antibody comprises any two mutations selected from the following mutations: mutation of position 118 of the heavy chain constant region to cysteine, mutation of position 239 of the heavy chain constant region to cysteine, mutation of position 160 of the light chain constant region to cysteine, and mutation of position 166 of the light chain constant region to cysteine.

[0164] In some embodiments, in the bispecific antibodies of the present invention, the light chain constant region is a λ light chain constant region or a κ light chain constant region.

[0165] In some embodiments, the bispecific antibody of the present invention further comprises a heavy chain constant region wherein positions 234 and 235 are mutated to alanine (LALA mutation).

[0166] In some embodiments, in the bispecific antibodies of the present invention, the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4.

[0167] In some embodiments, in the bispecific antibodies of the present invention, the light chain constant region is a human λ light chain constant region or a human κ light chain constant region.

[0168] In some embodiments, in the bispecific antibodies of the present invention, amino acid residue positions are numbered according to the EU numbering system, for example, amino acid residue positions in the constant region are numbered according to the EU numbering system.

[0169] In some embodiments, in the bispecific antibodies of the present invention, the constant region mutations are each independently relative to the parent heavy chain constant region or the parent light chain constant region. The parent heavy chain constant region or the parent light chain constant region can be a native heavy chain constant region or light chain constant region, or a variant heavy chain constant region or light chain constant region, as long as the amino acid at the position to be mutated is different from the amino acid to be mutated. For example, in some embodiments, the parent heavy chain constant region is relative to the heavy chain constant region or light chain constant region of a native human IgG1 or native human IgG4 immunoglobulin.

[0170] In some embodiments, in the bispecific antibodies of the invention, the mutations are each independently compared to the heavy chain constant region or the light chain constant region of a native human IgG1 immunoglobulin.

[0171] In some embodiments, the amino acid sequence of the native human IgG1 heavy chain constant region is shown in SEQ ID NO: 35. In some embodiments, the amino acid sequence of the native human lambda light chain constant region is shown in SEQ ID NO: 36. In some embodiments, the amino acid sequence of the native human kappa light chain constant region is shown in SEQ ID NO: 17.

[0172] In some embodiments, the bispecific antibodies of the present invention comprise any one of the following mutation combinations compared to the natural human IgG1 light chain constant region and / or heavy chain constant region: (1) a mutation of position 160 of the λ light chain constant region to cysteine ​​and a mutation of position 166 of the λ light chain constant region to cysteine; (2) a mutation of position 118 of the heavy chain constant region to cysteine ​​and a mutation of position 239 of the heavy chain constant region to cysteine; (3) a mutation of position 160 of the λ light chain constant region to cysteine ​​and a mutation of position 118 of the heavy chain constant region to cysteine; (4) a mutation of position 166 of the λ light chain constant region to cysteine ​​and a mutation of position 118 of the heavy chain constant region to cysteine; (5) a mutation of position 160 of the λ light chain constant region to cysteine ​​and a mutation of position 239 of the heavy chain constant region to cysteine; and (6) a mutation of position 166 of the λ light chain constant region to cysteine ​​and a mutation of position 239 of the heavy chain constant region to cysteine.

[0173] In some embodiments, the bispecific antibodies of the present invention comprise any one of the following combinations of cysteine ​​mutations compared to the natural human IgG1 light chain constant region and / or heavy chain constant region: (1) position 160 of the λ light chain constant region is mutated to cysteine ​​and position 166 of the λ light chain constant region is mutated to cysteine; (2) position 118 of the heavy chain constant region is mutated to cysteine ​​and position 239 of the heavy chain constant region is mutated to cysteine; (3) position 160 of the λ light chain constant region is mutated to cysteine ​​and position 239 of the heavy chain constant region is mutated to cysteine; constant region is mutated to cysteine; (4) position 166 of the lambda light chain constant region is mutated to cysteine ​​and position 118 of the heavy chain constant region is mutated to cysteine; (5) position 160 of the lambda light chain constant region is mutated to cysteine ​​and position 239 of the heavy chain constant region is mutated to cysteine; and (6) position 166 of the lambda light chain constant region is mutated to cysteine ​​and position 239 of the heavy chain constant region is mutated to cysteine, and the bispecific antibody optionally further comprises a LALA mutation in the heavy chain constant region.

[0174] In some embodiments, the bispecific antibodies of the invention comprise the following heavy chain constant region and light chain constant region combinations compared to native human IgG1 light chain constant region and / or heavy chain constant region:

[0175] (i) a lambda light chain constant region in which positions 160 and 166 are mutated to cysteines; and a heavy chain constant region without the cysteine ​​mutations;

[0176] (ii) a kappa light chain constant region without a cysteine ​​mutation and a heavy chain constant region with cysteine ​​mutations at positions 118 and 239;

[0177] (iii) a lambda light chain constant region in which position 160 is mutated to cysteine; and a heavy chain constant region in which position 118 is mutated to cysteine;

[0178] (iv) a lambda light chain constant region with a mutation at position 166 to cysteine; and a heavy chain constant region with a mutation at position 118 to cysteine;

[0179] (v) a lambda light chain constant region in which position 160 is mutated to cysteine; and a heavy chain constant region in which position 239 is mutated to cysteine;

[0180] (vi) a lambda light chain constant region in which position 166 is mutated to cysteine; and a heavy chain constant region in which position 239 is mutated to cysteine;

[0181] Optionally, the heavy chain constant region further comprises a LALA mutation.

[0182] In some embodiments, in the bispecific antibodies of the present invention, the heavy chain constant region

[0183] (i) comprising or consisting of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 11, 15, 19 and 23; or

[0184] (ii) comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 11, 15, 19 and 23; or

[0185] (iii) an amino acid sequence comprising or consisting of one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of any one of SEQ ID NOs: 11, 15, 19 and 23, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) is no more than 10, for example no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids. In some embodiments, in the bispecific antibodies of the invention, the heavy chain constant region comprising the LALA mutation comprises or consists of an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 11, or the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain constant region comprising the LALA mutation and mutations at positions 118 and 239 to cysteine ​​comprises, or consists of, an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 15, or the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain constant region comprising the LALA mutation and mutation at position 118 to cysteine ​​comprises, or consists of, an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 19, or the amino acid sequence of SEQ ID NO: 19. In some embodiments, the heavy chain constant region comprising a LALA mutation and a mutation at position 239 to cysteine ​​comprises, or consists of, an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 23, or the amino acid sequence of SEQ ID NO: 23.

[0186] In some embodiments, in the bispecific antibodies of the present invention, the light chain constant region

[0187] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21 and 25; or

[0188] (ii) comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 13, 17, 21 and 25; or

[0189] (iii) an amino acid sequence comprising or consisting of one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21 and 25, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) is no more than 10, for example, no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids.

[0190] In some embodiments, the lambda light chain constant region comprising a cysteine ​​mutation at position 160 and 166 comprises, or consists of, an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13, or the amino acid sequence of SEQ ID NO: 13. In some embodiments, the lambda light chain constant region comprising a cysteine ​​mutation at position 160 comprises, or consists of, an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, or the amino acid sequence of SEQ ID NO: 21. In some embodiments, in the bispecific antibodies of the invention, the lambda light chain constant region comprising a mutation to cysteine ​​at position 160 comprises, or consists of, an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 25, or the amino acid sequence of SEQ ID NO: 25. In some embodiments, in the bispecific antibodies of the invention, the kappa light chain constant region comprises, or consists of, an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 17, or the amino acid sequence of SEQ ID NO: 21.

[0191] In some embodiments, the bispecific antibodies of the present invention comprise two identical heavy chain constant regions or two different heavy chain constant regions, for example, a cysteine ​​mutation is included in one heavy chain constant region but not in the other heavy chain constant region. In some embodiments, the bispecific antibodies of the present invention comprise two identical light chain constant regions or two different light chain constant regions, for example, a cysteine ​​mutation is included in one light chain constant region but not in the other light chain constant region. In some embodiments, the bispecific antibodies of the present invention comprise two identical heavy chain constant regions and two identical light chain constant regions, for example, the same cysteine ​​mutation is included in the two heavy chain constant regions and / or the same cysteine ​​mutation is included in the two light chain constant regions.

[0192] In some embodiments, in the bispecific antibodies of the present invention,

[0193] heavy chain

[0194] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 12, 16, 20 and 24; or

[0195] (ii) comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 12, 16, 20 and 24; or

[0196] (iii) an amino acid sequence comprising or consisting of one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of any one of SEQ ID NOs: 12, 16, 20 and 24, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) is no more than 10, for example, no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids; and

[0197] light chain

[0198] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 14, 18, 22 and 26; or

[0199] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 14, 18, 22 and 26; or

[0200] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to an amino acid sequence selected from any one of SEQ ID NOs: 14, 18, 22 and 26, wherein the number of amino acid changes (i.e., substitutions, insertions or deletions) is no more than 10, for example, no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1, and the amino acid substitutions are, for example, conservative substitutions of amino acids.

[0201] In some embodiments, the bispecific antibodies of the invention are selected from the following groups of antibodies:

[0202] (I)Group

[0203] heavy chain

[0204] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12; or

[0205] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12; or

[0206] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 12; and

[0207] light chain

[0208] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14; or

[0209] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 14; or

[0210] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 14.

[0211] Group (II)

[0212] heavy chain

[0213] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16; or

[0214] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 16; or

[0215] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 16; and

[0216] light chain

[0217] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; or

[0218] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 18; or

[0219] (iii) comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 18, or consisting of said amino acid sequence.

[0220] Group (III)

[0221] heavy chain

[0222] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; or

[0223] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 20; or

[0224] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 20; and

[0225] light chain

[0226] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; or

[0227] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 22; or

[0228] (iii) comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 22, or consisting of said amino acid sequence.

[0229] Group (IV)

[0230] heavy chain

[0231] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; or

[0232] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 20; or

[0233] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 20; and

[0234] light chain

[0235] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26; or

[0236] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 26; or

[0237] (iii) comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 26, or consisting of said amino acid sequence.

[0238] Group (V)

[0239] heavy chain

[0240] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24; or

[0241] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 24; or

[0242] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 24; and

[0243] The light chain

[0244] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; or

[0245] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 22; or

[0246] (iii) comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 22, or consisting of said amino acid sequence.

[0247] and

[0248] Group (VI)

[0249] heavy chain

[0250] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24; or

[0251] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 24; or

[0252] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 24; and

[0253] light chain

[0254] (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26; or

[0255] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 26; or

[0256] (iii) comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 26, or consisting of said amino acid sequence.

[0257] In some embodiments, the bispecific antibodies of the present invention are selected from the following antibodies:

[0258] (1) Duligo-LC1, the heavy chain is represented by the amino acid sequence of SEQ ID NO: 12, and the light chain is represented by the amino acid sequence of SEQ ID NO: 14;

[0259] (2) DU-v-01, the heavy chain of which is represented by the amino acid sequence of SEQ ID NO: 16, and the light chain of which is represented by the amino acid sequence of SEQ ID NO: 18;

[0260] (3) DU-v-02, the heavy chain of which is represented by the amino acid sequence of SEQ ID NO: 20, and the light chain of which is represented by the amino acid sequence of SEQ ID NO: 22;

[0261] (4) DU-v-03, the heavy chain of which is represented by the amino acid sequence of SEQ ID NO: 20, and the light chain of which is represented by the amino acid sequence of SEQ ID NO: 26;

[0262] (5) DU-v-04, the heavy chain of which is represented by the amino acid sequence of SEQ ID NO: 24, and the light chain of which is represented by the amino acid sequence of SEQ ID NO: 22; and

[0263] (6) DU-v-05, the heavy chain is represented by the amino acid sequence of SEQ ID NO: 24, and the light chain is represented by the amino acid sequence of SEQ ID NO: 26.

[0264] III. Immunoconjugates

[0265] In one aspect, the present invention provides an immunoconjugate comprising the anti-EGFR and HER3 bispecific antibody or its antigen-binding fragment described in the present invention. In some embodiments, the immunoconjugate comprises the anti-EGFR and HER3 bispecific antibody or its antigen-binding fragment described in the present invention and one or more other substances conjugated thereto, such as a label, therapeutic agent or diagnostic agent. In the immunoconjugate, a linker can be used to covalently link the different parts of the conjugate. For example, a linker is used to covalently link the antibody or its antigen-binding fragment to the one or more other substances. Suitable linkers for the conjugate include chemical linkers or peptide linkers, and include cleavable linkers and non-cleavable linkers.

[0266] In some embodiments, the therapeutic agent includes but is not limited to a cytotoxic compound.

[0267] In some embodiments, the label or diagnostic agent includes, but is not limited to, enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances such as radiolabeled tracer compounds and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission tomography techniques.

[0268] In some embodiments, the immunoconjugate is an antibody-drug conjugate.

[0269] In one aspect, the present invention provides a bispecific antibody-drug conjugate having formula (I), a stereoisomer or a pharmaceutically acceptable salt or solvate thereof: Ab-(LD) n (Ⅰ)

[0270] in,

[0271] Ab is the anti-EGFR and HER3 bispecific antibody or its antigen-binding fragment according to the present invention,

[0272] L is a linker,

[0273] D is a biologically active compound moiety, such as a cytotoxic compound,

[0274] n represents the number of connections, i.e., the number of -LD groups connected to Ab, and n is a natural number selected from 1-15, for example, a natural number in the range of 1-14, 2-13, 3-12, 4-11, 5-10, 6-9, 7-8, 1-6, 1-5, 1-4, 2-4, 3-4, for example, a natural number in a numerical range with any two values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 as endpoints, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0275] As used herein, the term "immunoconjugate" generally refers to a molecule formed by conjugating one or more immunoglobulin-related molecules or fragments thereof (e.g., antibodies or fragments thereof herein) to one or more other molecules. In some cases, the other molecules may be proteinaceous molecules, such as peptides, polypeptides, or proteins. In some cases, the other molecules may also be non-proteinaceous molecules. In some embodiments, the other molecules are, for example, markers, therapeutic agents, or diagnostic agents. In some cases, the one or more other molecules may be the same or different from each other. For example, the other molecules may be target binding elements and / or effector elements, such as chemotherapeutic agents, cytotoxic compounds, radioactive elements, probes, or signaling molecules.

[0276] As used herein, the term "antibody-drug conjugate" refers to a targeted biologic drug obtained by connecting a bioactive compound fragment (drug molecule such as a cytotoxic compound) to an antibody or its antigen-binding fragment portion through a connecting unit. The connecting unit, also known as a linker, can break in a specific environment (such as an intracellular low pH environment) or under a specific action (such as the action of a lysosomal protease), thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment. ADC can utilize the high affinity and specificity of the target antibody to recognize tumor cell surface antigens, and deliver bioactive compounds, such as small molecule cytotoxic drugs, to tumor cells through strong intracellular internalization, thereby achieving accurate and efficient killing of tumor cells. It should be understood that in antibody-drug conjugates, when referring to a bioactive compound such as a cytotoxic compound, it means the bioactive compound portion / fragment (such as a cytotoxic compound portion / fragment) connected to the rest of the antibody-drug conjugate.

[0277] As used herein, the term "linker" refers to a fragment that connects a biologically active compound fragment (drug molecule) to an antibody or antigen-binding fragment thereof.

[0278] As used herein, the term "biologically active compound fragment" refers to a portion of an antibody-drug conjugate (ADC) known in the art that, after cleavage / degradation / enzymatic cleavage of the linker between tumor tissues or within tumor cells, can form a biologically active drug, such as a small molecule cytotoxic drug. This portion may also be referred to as the "biologically active compound portion."

[0279] As used herein, the term "DAR (drug to antibody ratio)," also known as "drug to antibody ratio," refers to the number of drug moieties conjugated to the antibody in an antibody-drug conjugate molecule. The DAR can vary and will be limited by the number of available sites on the antibody. It should be understood that for the antibody-drug conjugate of Formula (I), the DAR is a natural number greater than 0, i.e., a positive integer. In some embodiments, the antibody-drug conjugates of the present invention have a DAR of 1-15, such as 1-14, 2-13, 3-12, 4-11, 5-10, 6-9, 7-8, 1-6, 1-5, 1-4, 2-4, or 3-4, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or an integer range of any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. During the conjugation process, a heterogeneous mixture of different antibody-drug conjugate (ADC) molecules (i.e., ADC molecules with different DARs) is typically produced. Therefore, the term "antibody-drug conjugate (ADC)" also refers to a mixture of such ADC molecules with different DARs. The term "average DAR" refers to the average value of the DAR of a population of ADC molecules in such a mixture, also referred to herein as the "average number of attachments". As is well known in the art, DAR and drug loading distribution can be determined, for example, by using hydrophobic interaction chromatography (HIC) or reversed phase high-performance liquid chromatography (RP-HPLC), wherein HIC is particularly suitable for determining the average DAR. In some embodiments, the average DAR (average number of attachments) of the antibody-drug conjugates of the present invention is a value in the range of 1-15, such as 1-14, 2-13, 3-12, 4-11, 5-10, 6-9, 7-8, 1-6, 1-5, 1-4, 2-4, or 3-4, such as a value in a range of values ​​having any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 as endpoints. It should be understood that for average DAR, the "value" mentioned includes integers and decimals. A value between 1 and 15 refers to any value between 1 and 15, including integers and decimals, and includes endpoints.In some embodiments, the average DAR (average number of connections) is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, or a value selected from a range having two of these values ​​as endpoints. It should be understood that when referring to an average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules comprising ADC molecules with the same and / or different DARs.

[0280] "Reacted cysteine" or "free cysteine" refers to a cysteine ​​residue that has a thiol functional group (-SH) (sulfhydryl group) and is not paired with or otherwise part of an intra- or intermolecular disulfide bridge.

[0281] In some embodiments, in the bispecific antibody-drug conjugate of formula (I) of the present invention, L is a linker that can link the antibody portion (Ab) and the biologically active compound portion together.

[0282] In some embodiments, in the bispecific antibody-drug conjugate of formula (I) of the present invention, Ab comprises cysteine. In some embodiments, in the bispecific antibody-drug conjugate of formula (I) of the present invention, the Ab is connected to the linker via its cysteine, for example, the linker is coupled to the Ab via the sulfhydryl group of cysteine. In some embodiments, the Ab comprises a cysteine ​​mutation, whereby the linker is coupled to the antibody via the sulfhydryl group of the mutated cysteine. In some embodiments, the Ab comprises one or more non-cysteine ​​mutations to cysteine ​​on its heavy or light chain, whereby the linker is coupled to the cysteine, for example, site-specific coupling. Anti-EGFR and HER3 antibodies or antigen-binding fragments thereof comprising the cysteine ​​mutation suitable for ADC of the present invention are described in detail herein, wherein the cysteine ​​obtained after the mutation is coupled to the linker, for example, site-specific coupling.

[0283] In some embodiments, the Ab is conjugated to a linker via a cysteine ​​obtained by mutation as follows:

[0284] (i) cysteine ​​residues obtained by mutation of cysteine ​​residues at positions 160 and 166 in one or both Lambda light chain constant regions;

[0285] (ii) cysteine ​​residues obtained by mutation of cysteine ​​residues at positions 118 and 239 in one or both heavy chain constant regions;

[0286] (iii) a cysteine ​​obtained by mutation at position 160 in one or both Lambda light chain constant regions and a cysteine ​​obtained by mutation at position 118 in one or both heavy chain constant regions;

[0287] (iv) a cysteine ​​obtained by mutation at cysteine ​​position 166 in one or both Lambda light chain constant regions and a cysteine ​​obtained by mutation at cysteine ​​position 118 in one or both heavy chain constant regions;

[0288] (v) a cysteine ​​obtained by mutation of cysteine ​​at position 160 in one or both Lambda light chain constant regions and a cysteine ​​obtained by mutation of cysteine ​​at position 239 in one or both heavy chain constant regions;

[0289] (vi) a cysteine ​​obtained by mutation of cysteine ​​at position 166 in one or both Lambda light chain constant regions and a cysteine ​​obtained by mutation of cysteine ​​at position 239 in one or both heavy chain constant regions.

[0290] In some specific embodiments, the Ab with cysteine ​​mutations comprises two heavy chains and two light chains, and the cysteine ​​obtained by the following mutations is coupled to the linker:

[0291] (i) Cysteine ​​gained by mutation of cysteine ​​at positions 160 and 166 in the constant regions of the two Lambda light chains;

[0292] (ii) cysteine ​​residues obtained by mutation of cysteine ​​residues at positions 118 and 239 in the constant regions of both heavy chains;

[0293] (iii) cysteine ​​obtained by mutation of cysteine ​​at position 160 in the two Lambda light chain constant regions and cysteine ​​obtained by mutation of cysteine ​​at position 118 in the two heavy chain constant regions;

[0294] (iv) cysteine ​​obtained by mutation of cysteine ​​at position 166 in both Lambda light chain constant regions and cysteine ​​obtained by mutation of cysteine ​​at position 118 in both heavy chain constant regions;

[0295] (v) cysteine ​​obtained by mutation of cysteine ​​at position 160 in both Lambda light chain constant regions and cysteine ​​obtained by mutation of cysteine ​​at position 239 in both heavy chain constant regions;

[0296] (vi) Cysteine ​​obtained by mutation of cysteine ​​at position 166 in the two Lambda light chain constant regions and cysteine ​​obtained by mutation of cysteine ​​at position 239 in the two heavy chain constant regions.

[0297] Any cysteine ​​residue obtained after any cysteine ​​mutation of any antibody or antigen-binding fragment thereof mentioned herein can be used for coupling with a linker, such as site-directed coupling, to obtain the ADC molecule of the present invention.

[0298] In some embodiments, the linker L has a structure represented by formula (II): Q-L'(II),

[0299] Q represents a linker coupled to Ab via a thioether bond (-S-);

[0300] L' represents a linker moiety that connects Q to the cytotoxic compound D and has the following structure:

[0301] Wherein, L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, optionally including at least one amino acid residue with a side chain carboxylic acid, and "-C(=O)-" represents the carbonyl group of the amino acid residue at the C-terminus of the polypeptide residue;

[0302] L2 does not exist or is a hydrophilic group connected to the carbonyl group after the reaction of the side chain carboxylic acid of the amino acid residue of the polypeptide residue L1, and L2 is -NHR L2 , R L2 is selected from C optionally substituted by 1 to 6 hydroxyl groups 1-6 alkyl;

[0303] represents the N-terminus of the polypeptide residue to which the linking moiety Q is covalently linked.

[0304] In some embodiments, the linking moiety Q has the following structure:

[0305] Among them, Q a It is the functional group coupled to Ab;

[0306] A is selected from optionally substituted aliphatic or optionally substituted heteroaliphatic, wherein the aliphatic and heteroaliphatic groups are optionally independently selected from halogen, -CN, -OR Qa1 、-SR Qa1 、-N(R Qa1 )2, wherein each R Qa1 independently selected from hydrogen, C 1-6 Alkyl, C 1-6 In some embodiments, A is selected from optionally substituted C 1-10 In some embodiments, A is selected from an optionally substituted C containing 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. 1-18 Aliphatic group.

[0307] Preferably, A is optionally substituted C 1-10 Alkylene, C 1-8 Alkylene, C 1-6 Alkylene, C 3-6 Alkylene, or C 4- 6. Alkylene groups, for example, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene.

[0308] In some embodiments, the functional group Q a The functional group Q is absent or selected from maleimide, iodoacetamide, bromoacetamide, pyrimidine, pyrimidyl sulfide, vinylpyrimidine, vinyltriazine, vinylpyridine, disulfide, pyridyl disulfide, haloacetamide, α-haloacetyl, active ester such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, acid anhydride, acid chloride, sulfonyl chloride, isocyanate and isothiocyanate. Preferably, the functional group Q a Selected from maleimide, such as the following structure or pyrimidine, such as the following structure:

[0309] "*" indicates the site of covalent attachment to A;

[0310] Indicates the site of covalent attachment to antibody Ab.

[0311] In some embodiments, the linking moiety Q has the following structure:

[0312] In some embodiments, the linking moiety L1 has the following structure: NH -AA 1 AA 2 AA 3 …AA p-C(＝O) ,

[0313] Among them AA 1 AA 2 AA 3 、......AA p Each of which is independently an optionally substituted amino acid residue, optionally AA 1 AA 2 AA 3 、......AA p At least one of them is an amino acid residue with a side chain carboxylic acid, such as Glu or Asp;

[0314] p is an integer from 3 to 8, for example, from 3 to 5;

[0315] "NH-" indicates the N-terminus of a polypeptide residue;

[0316] "-C(=O)" indicates the C-terminus of a polypeptide residue.

[0317] In some embodiments, AA 1 AA 2 AA 3 、......AA p Each of the amino acid residues is independently an optionally substituted amino acid residue selected from the group consisting of Glu, Asp, Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, Gly, and N-alkyl amino acids, and AA 1 AA 2 AA 3 、......AA p At least one of them is Glu or Asp.

[0318] In some embodiments, the linking moiety L1 is NH -Glu-Val-Ala- C(＝O) .

[0319] In some embodiments, L2 is selected from the structures shown below:

[0320] In some embodiments, L2 has the following structure:

[0321] In some embodiments, the linking moiety L' has the following structure:

[0322] In some embodiments, linker L has the following structure:

[0323] In some embodiments, the linker L is attached to the cysteine ​​side chain of the antibody via a maleimide linker, such as via an -S- linkage.

[0324] In some embodiments, the linker L can be composed of one or more linker building blocks. Exemplary linker building blocks include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), hexanoyl-glycine-glycine-phenylalanine-glycine (GGFG), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"), ethyleneoxy -CH2CHO- as one or more repeating units ("EO" or "PEO").

[0325] Exemplary linkers L include, but are not limited to, maleimido-caproyl-valinealaine (mc-va) linker, maleimidobutanoic acid-valine-citrulline (mb-vc) linker, or maleimido-caproyl-glycine-glycine-phenylalanine-glycine linker (mc-GGFG).

[0326] In some embodiments, in the bispecific antibody-drug conjugate of formula (I) of the present invention, D is a biologically active compound, for example, an active compound that exerts a biological effect on tumors (also known as an anti-tumor compound). The anti-tumor compound can be a cytotoxic compound.

[0327] In some embodiments, the cytotoxic compound D has the structure shown in formula (III): Z-D'(III)

[0328] Z is absent or selected from -NH- or -NH-R z1 -YR z2 -, where R z1 and R z2 Each independently absent or selected from optionally substituted C 1-8 aliphatic, optionally substituted C 0-8 aliphatic-arylene, optionally substituted C 0-8 aliphatic-carbonyl (-C(O)-), optionally substituted C 0-8 Aliphatic-oxycarbonyl (-C(O)O- or -OC(O)-), optionally substituted C 0- 8 aliphatic-carbonyl-imino, optionally substituted arylene-carbonyl (-C(O)-), optionally substituted arylene-oxycarbonyl (-C(O)O- or -OC(O)-), optionally substituted arylene-carbonyl-imino, wherein optionally substituted refers to optionally independently selected from C 1-8 Aliphatic, halogen, -CN, -OR z3 、-SR z3 、-N(R z3 )2, wherein each R z3 independently selected from hydrogen, C 1-6 Aliphatic, C 1-6 Halogenated aliphatic, Y is absent or selected from -O- or -S-;

[0329] D' is a camptothecin compound, or an analog or derivative thereof.

[0330] In some embodiments, Z is -NH-CH2-O-C3-C4alkylene-.

[0331] In some embodiments, Z is -NH-CH2-O-C3H6-, -NH-CH2-O-C4H8-.

[0332] In some embodiments, Z is -NH-CH2-O-CH2-CH2-CH2- or -NH-CH2-O-CH2-CH2-CH2-CH2-.

[0333] In some embodiments, D' is a camptothecin compound having the structure:

[0334] where R D1 、R D2 and R D3 Each is independently selected from H, hydroxy, cyano, halogen, optionally substituted C 1-6 aliphatic, optionally substituted C 1-6 Aliphatic oxy, optionally substituted C 1-6Aliphatic thio, optionally substituted C 1-6 A group consisting of halogenated aliphatic groups, or R D1 and R D2 Together with the carbon atoms to which it is attached, it forms a 5-8 membered cyclic or heterocyclic group, wherein optionally substituted refers to being optionally independently selected from C 1-8 Aliphatic, halogen, -CN, -OR D’ 、-SR D’ 、-S(O)R D’ and -S(O)2R D’ , and -N(R D’ )2, wherein each R D’ independently selected from hydrogen, C 1-6 Aliphatic, C 1-6 Halogenated aliphatic, -SR D” 、-S(O)R D” and -S(O)2R D” , each R D” independently selected from hydrogen, C 1-6 Aliphatic, C 1-6 Halogenated aliphatic groups.

[0335] In some embodiments, the cytotoxic compound D is selected from the following compounds:

[0336] In some embodiments, D The chiral carbon atoms marked with * in the structural fragments are in S configuration.

[0337] In some embodiments, the bispecific antibody-drug conjugates of the present invention have the following structure:

[0338] Wherein, Ab and n are as defined in the present invention. In the present invention, the -LD portion of the bispecific antibody-drug conjugate is referred to as NT3, i.e., the structural portion shown in square brackets.

[0339] In some embodiments, the bispecific antibody-drug conjugates of the present invention have the following structure:

[0340] wherein Ab and n are as defined herein. In the present invention, the -LD portion of the bispecific antibody-drug conjugate is mc-GGFG-DXd (which may also be simplified to DXd when describing the name of the antibody-drug conjugate), i.e., the structural portion shown in square brackets.

[0341] In some embodiments, the antibody-drug conjugate has an average DAR of 1-10, 2-8, 3-5, 3.0-4.0, 3.5-4.5, 6-10, 7-9, or 7.5-8.5.

[0342] The bispecific antibody-drug conjugates provided herein are described with reference to general formulae and specific compounds. In addition, the bispecific antibody-drug conjugates of the present invention may exist in a variety of different forms or derivatives, all of which are within the scope of the present invention. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and active metabolites.

[0343] As used herein, unless otherwise indicated, the term "pharmaceutically acceptable salt" includes salts that maintain the biological effectiveness of the free acid / base form of the specific compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts can include salts formed with inorganic bases or acids and organic bases or acids. In the case where the bispecific antibody-drug conjugates of the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically acceptable salts. Therefore, the bispecific antibody-drug conjugates of the present invention containing acidic groups (e.g., carboxyl groups) can exist in salt form and can be used according to the present invention, for example, alkali metal salts, alkaline earth metal salts, aluminum salts or ammonium salts. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts or salts with ammonia or organic amines (e.g., ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids). For example, these salts are readily obtained by reacting a compound having an acidic group with a suitable base (e.g., lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide). Other alkaline salts of the bispecific antibody-drug conjugates of the present invention include, but are not limited to, copper (I), copper (II), iron (II), iron (III), manganese (II), and zinc salts. The bispecific antibody-drug conjugates of the present invention contain one or more basic groups, such as groups that can be protonated, and can exist in the form of salts and can be used according to the present invention in the form of addition salts thereof with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, malonic acid, maleic acid, malic acid, pamoic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, as well as others known to those skilled in the art. The salts formed are, in particular, hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate (mesylate), toluenesulfonate, carbonate, bicarbonate, formate, acetate, sulfoacetate, triflate, oxalate, malonate, maleate, succinate, tartrate, malate, pamoate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate and glutamate. In addition, the stoichiometry of the salts formed by the bispecific antibody-drug conjugates of the present invention can be an integer multiple or a non-integer multiple of 1.

[0344] The bispecific antibody-drug conjugates of the present invention containing basic nitrogen groups can be synthesized using reagents such as C 1-4Quaternization of alkyl halides, for example, methyl, ethyl, isopropyl and tert-butyl chloride, bromine and iodine; di-C 1-4 Alkyl sulfates, such as dimethyl sulfate, diethyl sulfate, and dipentyl sulfate; C 10-18 Alkyl halides, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aryl C 1-4 Alkyl halides, such as benzyl chloride and phenethyl bromide.

[0345] If the bispecific antibody-drug conjugates of the present invention contain both acidic and basic groups in the molecule, the present invention also includes internal salts or betaines (zwitterions) in addition to the above-mentioned salt forms. The corresponding salts can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the bispecific antibody-drug conjugates of the present invention, which are not directly suitable for pharmaceutical use due to low physiological compatibility, but can be used, for example, as intermediates in chemical reactions or for the preparation of pharmaceutically acceptable salts. For a review of more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, 2002).

[0346] The bispecific antibody-drug conjugate of formula (I) and its pharmaceutically acceptable salts can exist in unsolvated and solvated forms. As used herein, the term "solvate" refers to a molecular complex comprising a bispecific antibody-drug conjugate of formula (I), its stereoisomers or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable solvent molecules. For example, the term "hydrate" is used when the solvent is water.

[0347] The bispecific antibody-drug conjugate of formula (I) may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomeric forms of the bispecific antibody-drug conjugate of formula (I). The asymmetric centers present in the bispecific antibody-drug conjugate of formula (I) may have (R) or (S) configurations independently of one another. When the bond of a chiral carbon is described as a straight line in the structural formula of the present invention, or when the compound name is described without the (R) or (S) chiral name of the chiral carbon, it should be understood that the (R) and (S) configurations of each such chiral carbon and therefore each enantiomer or diastereomer and mixtures thereof are included in the formula or name. The production of a specific stereoisomer or mixture thereof can be identified in the example of obtaining such stereoisomers or mixtures, but this in no way limits all stereoisomers and mixtures thereof to being included within the scope of the present invention. When the bond of a chiral carbon is depicted as a triangular solid line or a dashed line in the structural formula of the present invention, or when the name of a compound is depicted with a (R) or (S) chiral designation of a chiral carbon, it should be understood that the compound represented by the structural formula or name has a definite stereo configuration at the chiral carbon position and will be distinguished from other stereoisomers, enantiomers, diastereomers, or mixtures thereof.

[0348] The present invention includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, such as mixtures of enantiomers and / or diastereomers in all ratios. Therefore, enantiomers are enantiomerically pure forms (as left-handed and right-handed enantiomers), racemic forms, and mixtures of two enantiomers in all ratios of the subject matter of the present invention. In the case of cis / trans isomers, the present invention includes mixtures of all ratios of cis-form and trans-form and these forms. If necessary, single stereoisomers can be prepared by conventional methods (such as by chromatography or crystallization, by using stereochemically uniform synthetic starting materials or by stereoselective synthesis). Optionally, derivatization can be carried out before stereoisomer separation. The separation of stereoisomer mixtures can be carried out in an intermediate step during the synthesis of the bispecific antibody-drug conjugate of formula (I), or can be carried out on the final racemic product. Absolute stereochemistry can be determined by X-ray crystallography of crystalline products or crystalline intermediates which have been derivatized, if necessary, with reagents containing stereocenters of known configuration. Alternatively, absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopy.

[0349] Unless otherwise indicated, structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number, but with an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Such compounds are termed "isotopic variants." The present invention is intended to include all pharmaceutically acceptable isotopic variants of the bispecific antibody-drug conjugates of Formula (I). Examples of isotopes suitable for inclusion in the bispecific antibody-drug conjugates of the present invention include, but are not limited to, isotopes of hydrogen, such as 2 H (i.e., D, deuterium) and 3 H (i.e. tritium); carbon, e.g. 11 C. 13 C and 14 C; chlorine, e.g. 36 Cl; fluorine, e.g. 18 F; iodine, e.g. 123 I and 125 I; nitrogen, e.g. 13 N and 15 N; oxygen, e.g. 15 O. 17 O and 18 O; phosphorus, e.g. 32 P; and sulfur, e.g. 35 Certain isotopic variants of the bispecific antibody-drug conjugates of formula (I), such as those incorporating radioactive isotopes, are useful in drug and / or substrate tissue distribution studies. In particular, those having isotopic variations that are only observed upon substitution with heavier isotopes, such as deuterium ( 2 H or D) replaces hydrogen) in the depicted structure of different compounds can provide certain therapeutic advantages, for example, due to greater metabolic stability, increased in vivo half-life or reduced dosage requirements, and thus can be used in some specific situations. Isotopic variants of the bispecific antibody-drug conjugates of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and by using appropriate isotopically labeled reagents instead of the non-labeled reagents previously used for synthesis.

[0350] When describing the structures of compounds herein, the undesignated hydrogen atoms in the shown formula are D (i.e., 2 H), it is generally understood that the hydrogen at this position is a hydrogen isotope " 1 hydrogen( 1 H)" or in a form with natural isotopic abundance in its natural state. The structures shown indicate that the hydrogen atoms are D (i.e., 2 H, deuterium), it should be understood that the hydrogen at this position is a hydrogen isotope " 2 hydrogen( 2H, D, deuterium)" or in a form in which deuterium is present at a greater isotopic abundance than natural deuterium (e.g., a deuterium abundance of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or 100%).

[0351] Pharmaceutically acceptable solvates according to the present invention may include those wherein the solvent of crystallization may be isotopically substituted, for example D2O, d6-acetone, d6-DMSO.

[0352] III. Use and Administration

[0353] The bispecific antibody-drug conjugates of the present invention (bispecific antibody-drug conjugates of Formula (I), stereoisomers thereof) or pharmaceutically acceptable salts, solvates thereof, including mixtures thereof in all ratios, can be used as drugs. They have been found to exhibit pharmacological activity that targets EGFR and / or HER3 and thereby kills cancer cells that overexpress EGFR and / or HER3. Through this activity, the bispecific antibody-drug conjugates of the present invention can be used to treat conditions or diseases associated with EGFR and / or HER3 activity, such as EGFR and / or HER3-positive tumors.

[0354] Therefore, the bispecific antibody-drug conjugates of the present invention that are capable of targeting EGFR and / or HER3 are particularly suitable for treating diseases and conditions associated with EGFR and / or HER3 activity, such as cancers and tumors, including but not limited to the following: lymphoma, blastoma, sarcoma, leukemia, melanoma, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, gastric cancer, head and neck squamous cell carcinoma, and various types of head and neck cancer, etc., in particular, breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer and bladder cancer, etc.

[0355] The bispecific antibody-drug conjugates of the present invention can be administered in an amount effective to treat the diseases or conditions described herein. The bispecific antibody-drug conjugates of the present invention can be administered as the antibody-drug conjugate itself, or alternatively, as a pharmaceutically acceptable salt. For the purpose of administration and dosing, the bispecific antibody-drug conjugates of the present invention (the bispecific antibody-drug conjugates of Formula (I), their stereoisomers) or their pharmaceutically acceptable salts, solvates will be referred to simply as the bispecific antibody-drug conjugates of the present invention.

[0356] The bispecific antibody-drug conjugates of the present invention are administered by any suitable route in the form of a pharmaceutical composition suitable for such route, and at a dose effective for the intended treatment. The bispecific antibody-drug conjugates of the present invention can be administered intravenously, subcutaneously, intranasally, orally, rectally, vaginally, parenterally, or topically, etc.

[0357] As used herein, the term "administer" refers to absorbing, ingesting, injecting, inhaling, implanting or otherwise introducing a bispecific antibody-drug conjugate of the present invention or its pharmaceutical composition. The term "treating" refers to reversing, alleviating, delaying the onset of a "pathological condition" (e.g., a disease, disorder or condition, or one or more signs or symptoms thereof) as described herein or inhibiting its progression. In certain embodiments, treatment may be administered after one or more signs or symptoms of the disease or condition have developed or have been observed. In other embodiments, treatment may be performed in the absence of signs or symptoms of the disease or condition. For example, susceptible individuals may be treated before the onset of symptoms (e.g., according to a history of symptoms and / or according to genetic or other susceptibility factors). Treatment may also be continued after the symptoms subside, for example, to delay or prevent recurrence. As used herein, the terms "disease," "disorder," "condition," and "pathological condition" are used interchangeably.

[0358] Those skilled in the art can determine the dosage level to be administered by routine experimentation. The dosage regimen of the bispecific antibody-drug conjugates of the present invention and / or compositions comprising the same is based on a variety of factors, including the type, age, weight, sex, and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the specific antibody-drug conjugate used. Therefore, the dosage regimen can vary widely.

[0359] In some embodiments, the bispecific antibody-drug conjugates of the present invention can be used in combination with one or more other therapeutic agents. In some embodiments, non-limiting examples of such other therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, immunomodulators, and targeted degradation agents. These therapeutic agents can be administered before, after, or simultaneously with the administration of the bispecific antibody-drug conjugates of the present invention, or can be administered sequentially in any order.

[0360] IV pharmaceutical composition

[0361] In some aspects, the present invention relates to a pharmaceutical composition comprising a bispecific antibody-drug conjugate of Formula (I) as provided herein, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0362] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier or excipient that can be used to prepare a pharmaceutical composition, which is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes carriers or excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used herein, a pharmaceutically acceptable carrier or excipient includes one or more such carriers or excipients. The specific carrier or excipient used will depend on the manner and purpose of using the bispecific antibody-drug conjugate of the present invention. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, for example, Ansel, Howard C et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R. et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. One or more of buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavorings, flavorings, diluents, and other known additives may also be included to improve the therapeutic performance of the drug (i.e., the bispecific antibody-drug conjugate or pharmaceutical composition provided herein) or to facilitate the production of a pharmaceutical product (i.e., a drug).

[0363] The compositions of the present invention can be formulated into a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, suppositories, etc. The form depends on the intended mode of administration and therapeutic application.

[0364] The pharmaceutical compositions of the present invention can be prepared by any well-known pharmaceutical techniques (e.g., effective formulations and administration procedures). The above considerations regarding effective formulations and administration procedures are well known in the art and are described in standard textbooks. For example, the formulation of drug products is discussed in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients, 3rd ed., American Pharmaceutical Association, Washington, 1999.

[0365] In yet another aspect, the present invention relates to a kit for treating diseases and conditions associated with HER3 activity, such as cancer, comprising a bispecific antibody-drug conjugate of Formula (I) as provided herein, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a bispecific antibody-drug conjugate of Formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein, optionally a container and optionally a package insert or label indicating treatment.

[0366] V Treatment Methods

[0367] In another aspect, the present invention relates to a method for treating diseases and conditions associated with EGFR and / or HER3 activity, such as cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody-drug conjugate of Formula (I) as provided herein, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention.

[0368] As used herein, the term "subject in need thereof" refers to a subject having a disease associated with EGFR and / or HER3 activity, such as cancer, or a subject having an increased risk of developing a disease or condition associated with EGFR and / or HER3 activity relative to the population as a whole. In certain embodiments, the subject is a warm-blooded animal. In certain embodiments, the warm-blooded animal is a mammal. In certain embodiments, the warm-blooded animal is a human.

[0369] As used herein, the term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody drug conjugate or pharmaceutical composition are outweighed by the therapeutically beneficial effects. Relative to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100%.

[0370] As used herein, the term "diseases and conditions associated with EGFR and / or HER3 activity" refers to any pathophysiological situation in which the inhibition of EGFR and / or HER3 would be beneficial. In certain embodiments, the disease and condition associated with EGFR and / or HER3 activity is cancer. In some embodiments, the disease and condition associated with EGFR and / or HER3 activity is an EGFR and / or HER3 positive tumor, such as an EGFR and / or HER3 overexpressing tumor. In some embodiments, There is EGFR and / or HER3 expression, especially EGFR and / or HER3 overexpression, in the subject (particularly an adult subject). In some embodiments, there is (e.g., elevated levels of, for example, nucleic acid or protein levels or activity) EGFR and / or HER3 (e.g., compared to healthy subjects) in the subject. In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) has (e.g., elevated levels of, e.g., greater than 10%, greater than 20%, greater than 50%, greater than 80%, greater than 90%, greater than 1-fold, greater than 2-fold, greater than 3-fold, greater than 5-fold, greater than 10-fold, greater than 50-fold, greater than 100-fold, or a range between these values ​​(e.g., 10%-2-fold), such as nucleic acid or protein levels or activity) EGFR and / or HER3 (e.g., compared to a biological sample from a healthy subject (e.g., a corresponding tissue or cell in a healthy subject), or compared to an adjacent healthy tissue or cell of the subject). In certain embodiments, the diseases and conditions associated with EGFR and / or HER3 activity are cancers or tumors selected from the group consisting of lymphoma, blastoma, sarcoma, leukemia, melanoma, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, gastric cancer, head and neck squamous cell carcinoma, and various types of head and neck cancer, in particular, breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, ovarian cancer, melanoma, prostate cancer and bladder cancer, etc.

[0371] In some embodiments, the treatment methods of the present invention further comprise co-administration with one or more other therapeutic agents. Non-limiting examples of such other therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, immunomodulators, and targeted degradation agents. These therapeutic agents can be administered before, after, or simultaneously with the administration of the bispecific antibody-drug conjugates of the present invention, or sequentially in any order.

[0372] In some embodiments, the method further comprises administering to the patient one or more treatment modalities selected from the group consisting of radiation therapy, cell therapy, gene therapy, RNA therapy, and surgery.

[0373] In another aspect, the present invention relates to a bispecific antibody-drug conjugate of formula (I) as provided herein, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention for use in treating diseases and conditions associated with EGFR and / or HER3 activity, such as cancer or tumors. In some embodiments, the diseases and conditions associated with EGFR and / or HER3 activity are as defined herein.

[0374] In another aspect, the present invention relates to the use of a bispecific antibody-drug conjugate of formula (I) as provided herein, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present invention in the preparation of a medicament for treating diseases and conditions associated with EGFR and / or HER3 activity, such as cancer or tumors. In some embodiments, the diseases and conditions associated with EGFR and / or HER3 activity are as defined herein.

[0375] Example

[0376] The technical solutions of the present invention will be further described in detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased on the market. Those skilled in the art will appreciate that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present invention.

[0377] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

[0378] Example 1: Preparation of Antibody Molecules of Duligotuzumab, Duligo-LC1, Patritumab, SIB001, IgG1LALA, and Cetuximab

[0379] Expi293F cells (purchased from Gibco, A14527) were used for transient transfection to prepare the recombinant antibodies Duligotuzumab (heavy chain sequence: SEQ ID NO: 29; light chain sequence: SEQ ID NO: 30), Duligo-LC1 (an antibody with cysteine ​​mutations based on Duligotuzumab), Patritumab (heavy chain sequence: SEQ ID NO: 33; light chain sequence: SEQ ID NO: 34), SIB001 (heavy chain sequence: SEQ ID NO: 27; light chain sequence: SEQ ID NO: 28), IgG1LALA (heavy chain sequence: SEQ ID NO: 31; light chain sequence: SEQ ID NO: 32) and Cetuximab (heavy chain sequence: SEQ ID NO: 38; light chain sequence: SEQ ID NO: 39).

[0380] Subculture Expi293 cells according to the required transfection volume and adjust the cell density to 3 × 10 on the day of transfection. 6 Cells / ml. Take 1 / 10 the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, including the light and heavy chain plasmid pCDNA3.1 at a 1:1 ratio, mix thoroughly, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix thoroughly, incubate at room temperature for 20 minutes, then gently pour the mixture into the Expi293F cell suspension, shaking while pouring. Incubate the cells on a shaker at 8% CO2, 36.5°C, and 120 rpm. After 16 to 18 h of culture, 2% (volume ratio) of 200 g / L Feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution with a final concentration of 5 g / L, and VPA (Merck, Cat# P4543-100G) with a final concentration of 2.2 mM were added to the cell suspension, gently mixed, and cultured at 8% CO2, 36.5°C, and 120 rpm for another 7 days.

[0381] Cell supernatant was collected for antibody purification. Affinity capture was performed using a HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column. Prior to purification, the column was treated with 0.1M NaOH for 2 hours. The tubing and column were then rinsed with 10-20 column volumes of distilled water. The column was equilibrated with 5 column volumes of 1× PBS (Gibco). The filtered cell feed was passed through the column, and the column was then rinsed with 10 column volumes of 1× PBS to remove nonspecifically bound proteins. The column was then rinsed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5). The eluate was collected, adjusted to pH 6.0 with 2M Tris, and sterilized by filtration. Antibody coupling was performed after SEC purity testing (>98%) was completed.

[0382] In the following examples, antibody-drug conjugates (NT3-ADC) comprising NT3 (NT3, i.e., MB3 in Example 4 of WO2021173773A1) and antibody-drug conjugates (DXd-ADC) comprising Dxd were synthesized, as shown below. It should be understood that only one drug molecule portion is specifically shown in the following structural diagram for example, but these antibody-drug conjugates may have a DAR greater than 1, such as a natural number in the range of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 2-4, or 3-4. The measured DAR, i.e., the average DAR, of the antibody-drug conjugates prepared in the examples is provided in the following section.

[0383] The schematic diagram of the NT3-ADC structure is as follows:

[0384] Specifically, examples of the antibody portion include Duligo-LC1, DU-v-01, DU-v-02, DU-v-03, DU-v-04, DU-v-05, and IgG1LALA, the amino acid sequences of which are detailed in the sequence listing. Furthermore, the antibody portion may include a monoclonal antibody, a bispecific antibody, a polyclonal antibody, or an antigen-binding fragment. The schematic diagram herein uses a Y-shaped antibody as an example.

[0385] The schematic diagram of DXd-ADC structure is as follows

[0386] Specifically, examples of the antibody portion include SIB001, Patritumab, Duligotuzumab (also referred to as Duligo), and IgG1LALA, whose amino acid sequences are detailed in the sequence listing. Furthermore, the antibody portion may be a monoclonal antibody, a bispecific antibody, a polyclonal antibody, or an antigen-binding fragment. The schematic diagram herein uses a Y-shaped antibody as an example.

[0387] Example 2: Synthesis and Characterization of Duligo-LC1-NT3

[0388] FIG1 shows a schematic diagram of the molecular structure of the bispecific antibody-drug conjugate Duligo-LC1-NT3 of the present invention.

[0389] Duligo-LC1 (36.8 mg) was dissolved in 20 mM histidine (8.6 mL, pH 5.5), and 10 mM TCEP solution (513 μL, 20 equivalents) was added. The mixture was mixed thoroughly and reacted at 37°C for 3 hours. RP-HPLC confirmed the reduction of all interchain disulfide bonds. The reaction solution was transferred to a dialysis cassette (Thermo Scientific, Slide-A-Lyzer 20K MWCO) and dialyzed against 20 mM histidine (800 mL, pH 6.5). The buffer was replaced with fresh buffer every 2 hours and then 4 hours, followed by overnight dialysis to remove small sulfhydryl-containing impurities and TCEP. After dialysis, 35 mg (95%) of antibody was obtained.

[0390] To the dialyzed antibody solution (2.55 mg / mL), 10 mM dhAA (dehydroascorbic acid, 975 μL, 40 equivalents) was added, mixed thoroughly, and incubated at 37°C for 3 hours. RP-HPLC confirmed the disappearance of the free light chain signal. DMSO (164 μL) and NT3 in DMSO (277 μL, 10 mg / mL) were then added, mixed thoroughly, and incubated at 25°C for 2 hours. Following the reaction, the product was purified using a desalting column to remove small molecule impurities, and the solution was replaced with 20 mM histidine, pH 5.5 buffer to yield the target ADC product, Duligo-LC1-NT3 (32 mg, 90%).

[0391] Duligo-LC1-NT3 (50 μg) was diluted to 1 mg / mL with H2O. Dithiothreitol (DTT) solution (1 M, 1.0 μL) was added and the pH was adjusted to 7.5. After incubation at 37°C for 30 minutes, RP-HPLC analysis was performed as follows.

[0392] The RP-HPLC determination results are shown in FIG2 , and the average DAR value was calculated to be 3.64.

[0393] Duligo-LC1-NT3 (50 μg) was diluted to 3 mg / mL with H2O and analyzed by SEC as follows.

[0394] The SEC measurement results are shown in Figure 3. The high molecular weight component and monomer ADC accounted for 0.59% and 99.41%, respectively, and no low molecular weight fragments were observed.

[0395] Reagents and chromatographic columns

[0396] equipment

[0397] Example 3: Preparation of different coupling site mutants, synthesis and characterization of corresponding site-specific coupling ADCs, and selection of coupling sites

[0398] With reference to Example 1, based on Duligotuzumab, the heavy chain constant region or the light chain constant region was mutated to prepare five antibody molecules, DU-v-01, DU-v-02, DU-v-03, DU-v-04 and DU-v-05 (see the sequence list for the full-length sequence), each with a different cysteine ​​(Cysteine) mutation site. With reference to Example 2, five ADC molecules, DU-v-01-NT3, DU-v-02-NT3, DU-v-03-NT3, DU-v-04-NT3 and DU-v-05-NT3, were synthesized and characterized. The sites of each mutant, protein expression yield and one-step purification efficiency, average DAR value of ADC coupling and SEC purity after ADC coupling are summarized in the following table.

[0399] The Duligo-LC1 mutant exhibited excellent properties in terms of antibody expression yield, one-step purification efficiency, DAR value of the ADC molecule after coupling, and SEC purity (aggregation). Compared with other mutation combinations, the LLC160-LLC166 mutation site combination demonstrated the best coupling efficacy.

[0400] Example 4: Synthesis and Characterization of SIB001-DXd

[0401] The schematic diagram of the molecular structure of SIB001-DXd is shown in Figure 4.

[0402] SIB001 (30.0 mg) was dissolved in 20 mM histidine (7.5 mL, pH 6.5). 10 mM TCEP solution (228 μL, 15 equivalents) was added, mixed thoroughly, and reacted at 37°C for 2 hours. RP-HPLC confirmed the reduction of all interchain disulfide bonds. DMSO (545 μL) and a DMSO solution of mc-GGFG-DXd (205 μL, 10 mg / mL) were then added, mixed thoroughly, and reacted at 25°C for 1 hour. Following the reaction, the product was purified using a desalting column to remove small molecule impurities. The solution was then exchanged with 20 mM histidine, pH 6.5 buffer to yield the target ADC product, SIB001-DXd (28 mg, 91%).

[0403] Among them, mc-GGFG-DXd has the following structure:

[0404] SIB001-DXd (50 μg) was diluted to 1 mg / mL with H2O and analyzed by RP-HPLC as follows.

[0405] The RP-HPLC determination results are shown in FIG5 , and the average DAR value was calculated to be 7.51.

[0406] SIB001-DXd (50 μg) was diluted to 3 mg / mL with H2O and analyzed by SEC as follows.

[0407] The SEC measurement results are shown in Figure 6. The high molecular weight component and monomer ADC accounted for 2.24% and 97.76%, respectively, and no low molecular weight fragments were observed.

[0408] Example 5: Synthesis and Characterization of Patritumab-DXd

[0409] The molecular structure of Patritumab-DXd is shown in Figure 7.

[0410] Patritumab (35.0 mg) was dissolved in 20 mM histidine (7.8 mL, pH 6.5), and 10 mM TCEP solution (477 μL, 20 equivalents) was added. The mixture was mixed thoroughly and reacted at 37°C for 2 hours. RP-HPLC confirmed the reduction of all interchain disulfide bonds. DMSO (457 μL) and a DMSO solution of mc-GGFG-DXd (321 μL, 10 mg / mL) were then added, mixed thoroughly, and reacted at 25°C for 1 hour. Following the reaction, the product was purified using a desalting column to remove small molecule impurities. The solution was then exchanged with 20 mM histidine, pH 6.5 buffer to yield the target ADC product, Patritumab-DXd (29 mg, 83%).

[0411] Patritumab-DXd (50 μg) was diluted with H2O to 1 mg / mL and analyzed by RP-HPLC as follows.

[0412] The RP-HPLC determination results are shown in FIG8 , and the average DAR value was calculated to be 7.75.

[0413] Patritumab-DXd (50 μg) was diluted to 3 mg / mL with H2O and analyzed by SEC as follows.

[0414] The SEC measurement results are shown in Figure 9. The high molecular weight component and monomer ADC accounted for 0.22% and 99.78%, respectively, and no low molecular weight fragments were observed.

[0415] Example 6: Synthesis and Characterization of Duligo-DXd

[0416] The molecular structure of Duligo-DXd is shown in Figure 10.

[0417] Duligotuzumab (20.0 mg) was dissolved in 20 mM histidine (6.7 mL, pH 6.5), and 10 mM TCEP solution (276 μL, 20 equivalents) was added. The mixture was mixed thoroughly and reacted at 37°C for 2 hours. RP-HPLC confirmed the reduction of all interchain disulfide bonds. DMSO (481 μL) and a DMSO solution of mc-GGFG-DXd (186 μL, 10 mg / mL) were then added, mixed thoroughly, and reacted at 25°C for 1 hour. After the reaction, the product was purified using a desalting column to remove small molecule impurities, and the solution was replaced with 20 mM histidine, pH 6.5 buffer to yield the target ADC product, Duligo-DXd (17.1 mg, 85%).

[0418] Duligo-DXd (50 μg) was diluted to 1 mg / mL with H2O and analyzed by RP-HPLC as follows.

[0419] The RP-HPLC determination results are shown in FIG11 , and the average DAR value was calculated to be 7.70.

[0420] Duligo-DXd (50 μg) was diluted to 3 mg / mL with H2O and analyzed by SEC as follows.

[0421] The SEC measurement results are shown in Figure 12. The high molecular weight component and monomer ADC accounted for 0.80% and 99.20%, respectively, and no low molecular weight fragments were observed.

[0422] Example 7: Synthesis and Characterization of IgG1LALA-NT3 (DAR=8)

[0423] The molecular structure of IgG1LALA-NT3 (DAR=8) is shown in FIG13 .

[0424] IgG1LALA (20.0 mg) was dissolved in 20 mM histidine (5.0 mL, pH 6.5), and 10 mM TCEP solution (271 μL, 20 equivalents) was added. The mixture was mixed thoroughly and reacted at 37°C for 2 hours. RP-HPLC confirmed the reduction of all interchain disulfide bonds. DMSO (300 μL) and NT3 DMSO solution (200 μL, 10 mg / mL) were then added, mixed thoroughly, and reacted at 25°C for 1 hour. After the reaction, small molecule impurities were removed using a desalting column, and the solution was exchanged with 20 mM histidine, pH 6.5 buffer to obtain the target ADC product, IgG1LALA-NT3 (DAR = 8) (18.2 mg, 91%).

[0425] IgG1LALA-NT3 (DAR=8) (50 μg) was diluted to 1 mg / mL with H 2 O and analyzed by RP-HPLC as follows.

[0426] The RP-HPLC determination results are shown in FIG14 , and the average DAR value was calculated to be 7.75.

[0427] IgG1LALA-NT3 (DAR=8) (50 μg) was diluted to 3 mg / mL with H 2 O and analyzed by SEC as follows.

[0428] The SEC measurement results are shown in Figure 15. The high molecular weight component and monomer ADC accounted for 1.09% and 98.91%, respectively, and no low molecular weight fragments were observed.

[0429] Example 8: Synthesis and Characterization of IgG1LALA-NT3 (DAR=4)

[0430] The molecular structure of IgG1LALA-NT3 (DAR=4) is shown in FIG16 .

[0431] IgG1LALA (1.0 mg) was dissolved in 20 mM histidine (0.25 mL, pH 6.5). 5 mM TCEP solution (4.06 μL, 3 equivalents) was added, mixed thoroughly, and incubated at 37°C for 2 hours. DMSO (18.8 μL) and NT3 DMSO solution (6.2 μL, 10 mg / mL) were then added, mixed thoroughly, and incubated at 25°C for 1 hour. After the reaction, the mixture was purified using a desalting column to remove small molecule impurities. A 50 μg sample was diluted to 1 mg / mL in H2O and analyzed by RP-HPLC as follows to determine the average DAR.

[0432] The above steps were repeated to determine the DARs of the ADCs prepared at TCEP equivalents of 4.0 and 5.0. A linear relationship between DAR and TCEP equivalents was fitted, confirming that the theoretical DAR was 4.0 at a TCEP equivalent of 2.5. IgG1LALA (40.0 mg) was dissolved in 20 mM histidine (10.0 mL, pH 6.5), and 5 mM TCEP solution (135 μL, 2.5 equivalents) was added. The mixture was mixed thoroughly and reacted at 37°C for 2 hours. After cooling to room temperature, DMSO (754 μL) and NT3 solution in DMSO (246 μL, 10 mg / mL) were added, mixed thoroughly, and reacted at 25°C for 1 hour. After completion of the reaction, the product was purified using a desalting column to remove small molecule impurities, and the solution was replaced with 20 mM histidine, pH 6.5 buffer to obtain the target ADC product, IgG1LALA-NT3 (DAR = 4) (35.2 mg, 88%).

[0433] The results of RP-HPLC determination according to the above method are shown in FIG17 , and the average DAR value was calculated to be 3.89.

[0434] IgG1LALA-NT3 (DAR=4) (50 μg) was diluted to 3 mg / mL with H 2 O and analyzed by SEC as follows.

[0435] The SEC measurement results are shown in FIG18 . The monomeric ADC accounted for 100%, and no high molecular weight components or low molecular weight fragments were observed.

[0436] Example 9: Synthesis and Characterization of IgG1LALA-DXd

[0437] The molecular structure of IgG1LALA-DXd is shown in Figure 19 .

[0438] IgG1LALA (15.0 mg) was dissolved in 20 mM histidine (5.0 mL, pH 6.5), and 10 mM TCEP solution (196 μL, 20 equivalents) was added. The mixture was mixed thoroughly and reacted at 37°C for 2 hours. RP-HPLC confirmed that all interchain disulfide bonds were reduced. DMSO (378 μL) and DXd in DMSO (122 μL, 10 mg / mL) were then added, mixed thoroughly, and reacted at 25°C for 1 hour. After the reaction, the product was purified using a desalting column to remove small molecule impurities. The solution was then exchanged with 20 mM histidine, pH 6.5 buffer to obtain the target ADC product, IgG1LALA-DXd (DAR = 8) (13.4 mg, 89%).

[0439] IgG1LALA-DXd (DAR=8) (50 μg) was diluted to 1 mg / mL with H2O and analyzed by RP-HPLC according to the following method.

[0440] The RP-HPLC determination results are shown in FIG20 , and the calculated DAR value is 7.97.

[0441] IgG1LALA-DXd (DAR=8) (50 μg) was diluted to 3 mg / mL with H2O and analyzed by SEC as follows.

[0442] The SEC measurement results are shown in Figure 21. The high molecular weight component and monomer ADC accounted for 0.20% and 99.80%, respectively, and no low molecular weight fragments were observed.

[0443] Example 10: Stability test of Duligo-LC1-NT3 in mouse and monkey plasma

[0444] Duligo-LC1-NT3 (100 μg) was diluted to 100 μL in mouse or monkey plasma (five samples were prepared for each type of plasma). The cells were then incubated at 37°C for 0, 1, 3, 7, and 14 days. ADC samples were purified by affinity chromatography and analyzed by RP-HPLC and LC-MS to determine the DAR trend over time. The results, shown in Figures 22 and 23, demonstrate that Duligo-LC1-NT3 was very stable in both plasmas, with no significant drug shedding observed.

[0445] RP-HPLC analysis

[0446] LC-MS analysis

[0447] Example 11: Detection of EGFR and HER3 expression in different tumor cell lines

[0448] Detection method:

[0449] After 2-3 passages, the cells were revived and the culture medium was discarded. The cells were rinsed with 10 mL of PBS (Cat. No. 10010-023, Manufacturer: Gibco) and then digested with 4 mL of trypsin (Cat. No. 25200-072, Manufacturer: Gibco). After the culture was terminated, the cells were centrifuged at 400 x g for 3 min and the supernatant was discarded. 2% FBS (Cat. No. 10091-148, Manufacturer: Gibco) was added to PBS to prepare FACS Buffer. The cells were resuspended and counted with FACS Buffer and the cell density was adjusted to 4x10 6 / mL, 50uL per well was added to a round-bottom 96-well plate and centrifuged at 400xg for 5min;

[0450] Antibodies PE anti-human EGFR (catalog number: 352904, manufacturer: Bio Legend) and PE anti-human erbB3 / HER-3 (catalog number: 324706, manufacturer: Bio Legend) were diluted 1:100 with FACS Buffer, and cells were resuspended in 50uL per well of two wells. At the same time, two wells were resuspended in FACS Buffer as a control and incubated at 4°C in the dark for 1 hour; 150uL FACS buffer was added to each well, and the cells were centrifuged at 400xg for 5 minutes; each well was resuspended in 200uL FACS buffer and centrifuged at 400xg for 5 minutes, and finally, the cells were resuspended in 100uL FACS Buffer per well for flow cytometry detection. For each sample, 20,000 data acquisition points were collected in the PE channel.

[0451] Use software to analyze and export the MFI value of the PE channel and then take the average. Divide the value of each antibody-stained well by the value of the control well to obtain the corresponding value, and finally draw a graph.

[0452] EGFR&HER3 expression detection results in different tumor cell lines:

[0453] By measuring the expression levels of EGFR and HER3 on a series of cell lines, it was verified that EGFR and HER3 have diverse expression profiles in different tumor cell lines. The results are shown in Figure 24.

[0454] Example 12: Fab-ZAP endocytosis-killing experiment of antibody molecules

[0455] Detection method:

[0456] After 2-3 passages, the revived AsPC1 cells were first passaged and the culture medium was discarded. The cells were rinsed with 10 mL of PBS and then digested with 4 mL of trypsin. After the culture was terminated, the cells were centrifuged at 400 × g for 3 minutes, the supernatant was discarded, and the culture medium was resuspended and counted. Adjust the cell density to 3E4 / mL and add 50uL per well to a flat-bottom 96-well plate. Dilute the test antibody to 160nM (maximum concentration 40nM) and Fab-ZAP to 640nM (maximum concentration 160nM, Fab-ZAP:antibody = 4:1) in complete medium. Then, take an equal volume of antibody and Fab-ZAP and mix them at a ratio of 1:1. Use this as the initial concentration and serially dilute 10 wells at a ratio of 1 / 4. Incubate at room temperature for 30 minutes. Add 50uL per well to the cells, mix well, and incubate at 37°C for 5 days. Remove the 96-well plate, equilibrate the plate to room temperature, add 100uL of CTG detection solution to each well, shake at 300rpm for 20 minutes, pipette 100uL into a white-bottom 96-well plate, and measure the luminescence value using a multi-function microplate reader.

[0457] Fab-ZAP endocytosis assay results:

[0458] Fab-ZAP is a chemical conjugate of monoclonal antibody and ribosome-inactivating protein saporin. It recognizes the Fc end of the target antibody through its F(ab) fragment. After coupling with the antibody, the antibody guides the ZAP complex to the target cell, and then binds to and internalizes the target cell. After the ZAP complex enters the cytoplasm, it releases saporin through enzymatic hydrolysis, affecting the normal function of the ribosome and thus killing the cell. Finally, the antibody internalization rate is calculated by detecting the cell-killing efficiency of the antibody complex.

[0459] Duligotuzumab and Cetuximab were more strongly internalized than Patritumab in human pancreatic cancer cells AsPC1, which have high expression of both EGFR and HER3 (MFI-fold (EGFR) = 186, MFI-fold (HER3) = 12.4), and the IC50 was reduced by approximately 80 times (IC50 of Duligotuzumab is 0.028 nM, IC50 of Cetuximab is 0.021 nM, and IC50 of Patritumab is 2.127 nM), as shown in Figure 25.

[0460] Duligotuzumab and Cetuximab were more strongly internalized than Patritumab in human gastric cancer cells NUGC4, where both EGFR and HER3 were moderately expressed (MFI-fold (EGFR) = 14.7, MFI-fold (HER3) = 44.9), as shown in Figure 26 ; Duligotuzumab and Patritumab were more strongly internalized than Cetuximab in Chinese hamster ovary CHO-HER3 cells, where EGFR was not expressed and HER3 was overexpressed, as shown in Figure 27 .

[0461] Example 13: In vitro cell killing assay of antibody drug conjugates Duligo-DXd and SIB001-DXd

[0462] Detection method:

[0463] After 2-3 passages, the revived MCF7 cells were first decanted for culture, rinsed once with 10 mL of PBS, and then digested with 4 mL of trypsin. After the culture was terminated, the cells were centrifuged at 400 x g for 3 minutes, the supernatant discarded, and the cells were resuspended in culture and counted. The cell density was adjusted to 3E4 / mL, and 50 μL was added to each well of a round-bottom, low-adhesion 96-well plate. The antibody to be tested was serially diluted and added to the corresponding cells at 50 μL per well. After incubation at 37°C for 5 days, the 96-well plate was removed and equilibrated to room temperature. 100 μL of CTG detection solution was added to each well and shaken at 300 rpm for 20 minutes. 100 μL of the solution was then transferred to a white-bottom 96-well plate and luminescence was measured using a multi-function microplate reader.

[0464] In vitro cell killing assay results:

[0465] Duligo-DXd has a significantly stronger killing effect than SIB001-DXd on human breast cancer cells MCF7 with low EGFR expression and high HER3 expression (MFI-fold (EGFR) = 3.3, MFI-fold (HER3) = 19.1), with the IC50 reduced by approximately 250 times (IC50 of Duligo-DXd is 0.049 nM, IC50 of SIB001-DXd is 13.23 nM), as shown in Figure 28.

[0466] Duligo-DXd also has a significantly stronger killing effect than SIB001-DXd on human breast cancer cells such as MDA-MB-453 cells, as shown in Figure 29; Duligo-DXd and SIB001-DXd have similar in vitro killing effects on human colorectal cancer cells GP2D, human lung cancer cells H1568, and human colorectal cancer cells SW480 overexpressing HER3, as shown in Figures 30-32.

[0467] Example 14: In vitro cell killing assay of antibody drug conjugates Duligo-LC1-NT3 and SIB001-DXd

[0468] Detection method:

[0469] After thawing and passaged 2-3 times, remove the culture medium from the revived MDA-MB-453 cells, rinse once with 10 mL of PBS, and then digest the cells with 4 mL of trypsin. After the culture is terminated, centrifuge at 400 x g for 3 minutes, discard the supernatant, and resuspend the cells in the culture medium for counting. Adjust the cell density to 3E4 / mL and add 50 μL per well of a round-bottom, low-adhesion 96-well plate. Add 50 μL of the test antibody to each well of the corresponding cells after serial dilutions. Incubate at 37°C for 5 days, then remove the 96-well plate, equilibrate to room temperature, add 100 μL of CTG detection solution to each well, shake at 300 rpm for 20 minutes, aspirate 100 μL of the solution into a white-bottom 96-well plate, and measure luminescence using a multi-function microplate reader.

[0470] In vitro cell killing assay results:

[0471] Duligo-LC1-NT3 had a significant killing effect on human breast cancer MDA-MB-453 cells with low EGFR expression and high HER3 expression (MFI-fold (EGFR) = 2.2, MFI-fold (HER3) = 55.2), compared with SIB001-DXd, with the IC50 reduced by approximately 250 times (IC50 of Duligo-LC1-NT3 is 0.037 nM, IC50 of SIB001-DXd is 9.36 nM). The results are shown in Figure 33.

[0472] Duligo-LC1-NT3 (DAR=4) and SIB001-DXd (DAR=8) exhibited similar in vitro cytotoxic activity on lung cancer, colorectal cancer, pancreatic cancer, and some HER3-overexpressing cell lines. The results are shown in Figures 34-39.

[0473] Example 15: ELISA detection of the affinity of Duligo-LC1-NT3 and SIB001-DXd to human EGFR and human HER3

[0474] Affinity detection method:

[0475] Coat a 96-well ELISA plate one day in advance, dilute the EGFR or HER3 antigen to 0.25 μg / mL using the coating solution, add 100 μL to each well, seal the plate with a sealing film, and incubate at 4°C overnight; pour out the pre-coating solution in the 96-well ELISA plate, pat dry on absorbent paper, then add 230 μL of washing solution to each well, pat dry the washing solution, and repeat washing 3 times; add 200 μL of blocking solution to each well with a gun, seal the plate with a sealing film, block at 37°C for 2 hours, and then wash the plate; dilute the sample to be tested 12 wells at the highest concentration of 100 nM, 1:4, and add 100 μL to each well after dilution. Incubate in the dark for 2 hours at room temperature and wash the plate; add 100 μL of Goat anti-Human IgG diluted 1:80,000 to each well Fc-HRP, incubate at room temperature in the dark for 1 hour, wash the plate; add 100 μL of TMB substrate to each well of the 96-well ELISA plate, develop color at room temperature in the dark for 2-5 minutes, then add 50 μL of ELISA stop solution to each well, read the OD within 30 minutes. 450nm and OD 620nm The value of .

[0476] Affinity experiment results:

[0477] Duligo-LC1-NT3 has a stronger affinity for EGFR than SIB001-DXd, with Kd reduced by approximately 2.5 times (Kd(Duligo-LC1-NT3)=0.03517nM, Kd(SIB001-DXd)=0.08833nM), as shown in Figure 40; Duligo-LC1-NT3 has a stronger affinity for HER3 (Kd(Duligo-LC1-NT3)=0.02574nM), and SIB001-DXd binds very weakly to HER3, as shown in Figure 41.

[0478] Example 16: Therapeutic Effects of EGFR / HER3-ADCs Antibodies in the H508 Model

[0479] In this experiment, H508 cells were inoculated into BALB / c NUDE mice to determine the anti-tumor effect of the EGFR / HER3-ADCs antibody of the present invention.

[0480] BALB / c NUDE mice:

[0481] Female BALB / c NUDE mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SPF grade). The quality inspection unit is Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the certificate number is No. 110011230103807467. Mice were acclimated for 3 days after arrival before the study began.

[0482] cell:

[0483] H508 cells were purchased from Nanjing Kebai (CAT#: CBP60795) and routine subculture was performed in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 25×10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of BALB / c nude mice to establish an H508 tumor-bearing mouse model.

[0484] Dosage:

[0485] 37 days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice with tumor volume of about 120-140 mm were selected. 3 The mice were divided into groups according to the average tumor volume (6 mice per group). The dosage and method of administration are shown in Table 1. h-IgG (purchased from EQUITECH-BIO, SLH56-0001) was used as a negative control and administered on the 37th and 44th days after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 55th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0486] Table 1. Experimental design

[0487] The tumor inhibition rates for the Duligo-LC1-NT3 group were 147%, and for the SIB001-DXd group were 155%. The tumor inhibition effect of the SIB001-DXd group was similar to that of the Duligo-LC1-NT3 group, and both were stronger than that of the h-IgG monotherapy group. The efficacy results are shown in Figure 42. We also monitored the body weights of the mice, and as shown in Figure 43, there were no significant differences in their body weights. Therefore, the EGFR / HER3-ADCs of the present invention have a significant inhibitory effect on tumors.

[0488] Table 2. Tumor inhibition rate on day 55

[0489] Example 17: Therapeutic Effect of EGFR-HER3 ADC in NUGC-4 Model

[0490] In this experiment, BALB / c-NUDE mice were inoculated with NUGC-4 cells to determine the anti-tumor effect of EGFR-HER3 ADC.

[0491] BALB / c-NUDE mice:

[0492] Female BALB / c-NUDE mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SPF grade) and quality-inspected by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a certificate number of 110011230103807533. Mice were acclimated for 3 days upon arrival before the study began.

[0493] cell:

[0494] NUGC-4 cells were purchased from NIBIOHN (CAT#: JCRB0834) and routine subculture was performed strictly according to the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 20×10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of BALB / c-NUDE mice to establish a NUGC-4 tumor-bearing mouse model.

[0495] Dosage:

[0496] Thirteen days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of approximately 200 mm were selected. 3The mice were divided into groups according to the average tumor volume (7 mice per group). The dosage and method of administration are shown in Table 3. h-IgG (purchased from EQUITECH-BIO, SLH56-0001) was used as a negative control and administered on the 13th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 32nd day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0497] Table 3. Experimental design

[0498] The tumor inhibition rates are shown in Figure 44 and Table 4. On day 32 post-inoculation, the tumor inhibition rates for Patritumab-DXd, SIB001-DXd, Duligo-LC1-NT3, and Duligo-DXd were 51% / 70% / 95% / 75%, respectively. Duligo-LC1-NT3 was statistically more potent than both SIB001-DXd and Duligo-DXd. Mouse body weights were also measured, and as shown in Figure 45, no significant differences were observed. Therefore, the EGFR / HER3 ADC demonstrated a significant inhibitory effect against NUGC-4 tumors.

[0499] Table 4. Tumor inhibition rate on day 32

[0500] Example 18: Therapeutic Effect of EGFR-HER3 ADC in SW620 Model

[0501] In this experiment, BALB / c-NUDE mice were inoculated with SW620 cells to determine the anti-tumor effect of EGFR-HER3 ADC.

[0502] BALB / c-NUDE mice:

[0503] Female BALB / c-NUDE mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SPF grade). The quality inspection unit was Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and the certificate number is 20230614Abzz0619000190. Mice were acclimated for 3 days after arrival before the study began.

[0504] cell:

[0505] SW620 cells were obtained from Nanjing Kebai Biotechnology Co., Ltd. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 10 × 10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal area of ​​BALB / c-NUDE mice to establish the SW620 tumor-bearing mouse model.

[0506] Dosage:

[0507] Fourteen days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of approximately 200 mm were selected. 3 The mice were divided into groups according to the average tumor volume (7 mice per group). The dosage and method of administration are shown in Table 5. h-IgG (purchased from EQUITECH-BIO, SLH56-0001) was used as a negative control and administered on days 14, 21, and 28 after inoculation. The tumor volume and body weight of the mice were monitored twice a week. Body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on day 42 after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0508] Table 5. Experimental design

[0509] The tumor inhibition rates are shown in Figure 46 and Table 6. On day 42 post-inoculation, the tumor inhibition rates of IgG1LALA-NT3 (DAR = 4) / SIB001-DXd / Duligo-LC1-NT3 were 45% / 39% / 100%, respectively. Duligo-LC1-NT3 was more potent than SIB001-DXd. Mouse body weights were also measured, and as shown in Figure 47, no significant differences were observed. Therefore, the EGFR-HER3 ADC demonstrated a significant inhibitory effect against SW620 tumors.

[0510] Table 6. Tumor inhibition rate on day 42

[0511] Example 19: Therapeutic Effect of EGFR-HER3 ADC in ASPC1 Model

[0512] In this experiment, ASPC1 cells were inoculated into BALB / c-NUDE mice to determine the anti-tumor effect of EGFR-HER3 ADC.

[0513] BALB / c-NUDE mice:

[0514] Female BALB / c-NUDE mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with an SPF grade and a quality inspection unit, Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with a certificate number of 20230531Abzz0619000787. The mice were purchased from Weitonglihua and acclimated for 3 days after arrival before the study began.

[0515] cell:

[0516] ASPC1 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. and routine subcultured in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 25×10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of BALB / c-NUDE mice to establish an ASPC1 tumor-bearing mouse model.

[0517] Dosage:

[0518] Seven days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of approximately 200 mm were selected. 3 The mice were divided into groups according to the average tumor volume (7 mice per group). The dosage and method of administration are shown in Table 7. h-IgG (purchased from EQUITECH-BIO, SLH56-0001) was used as a negative control and administered on the 7th, 14th, and 21st days after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 28th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0519] Table 7. Experimental design

[0520] The tumor inhibition results are shown in Figure 48 and Table 8. On day 28 after inoculation, the tumor inhibition rates of SIB001-DXd and Duligo-LC1-NT3 were 67% and 75%, respectively. Duligo-LC1-NT3 was slightly more potent than SIB001-DXd. We also measured mouse body weights, and as shown in Figure 49, no significant differences were observed. Therefore, the EGFR-HER3 ADC demonstrated a significant inhibitory effect on ASPC1 tumors.

[0521] Table 8. Tumor inhibition rate on day 28

[0522] Example 20: Therapeutic Effect of EGFR-HER3 ADC in the NCI-H1568 Model

[0523] In this experiment, BALB / c-NUDE mice were inoculated with NCI-H1568 cells to determine the anti-tumor effect of EGFR-HER3 ADC.

[0524] BALB / c-NUDE mice:

[0525] Female BALB / c-NUDE mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SPF grade) and quality-inspected by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a certificate number of 110011231105795417. Mice were acclimated for 3 days upon arrival before the study began.

[0526] cell:

[0527] NCI-H1568 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. and routinely subcultured according to the manufacturer's instructions for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in sterile PBS at a cell density of 25 × 10⁶ cells / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right abdominal region of BALB / c-NUDE mice to establish the NCI-H1568 tumor-bearing mouse model.

[0528] Dosage:

[0529] Eleven days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of approximately 200 mm were selected. 3 The mice were divided into groups according to the average tumor volume (7 mice per group). The dosage and method of administration are shown in Table 9. h-IgG (purchased from EQUITECH-BIO, SLH56-0001) was used as a negative control and administered on the 11th, 18th, and 27th days after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 30th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0530] Table 9. Experimental design

[0531] The tumor inhibition rates are shown in Figure 50 and Table 10. On day 30 after inoculation, the tumor inhibition rates for SIB001-DXd and Duligo-LC1-NT3 were 61% and 81%, respectively. Duligo-LC1-NT3 demonstrated greater efficacy than SIB001-DXd. We also measured mouse body weights, and as shown in Figure 51, the treated group showed a slight decrease in body weight. Therefore, the EGFR-HER3 ADC demonstrated significant inhibitory activity against NCI-H1568 tumors.

[0532] Table 10. Tumor inhibition rate on day 30

[0533] The amino acid sequences of the antibody molecules involved in this article and the nucleic acid sequences encoding the antibody molecules are as follows.

Claims

1. A bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof, comprising A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; A heavy chain constant region and a light chain constant region, wherein the anti-EGFR and HER3 bispecific antibody comprises one or more mutations to cysteine, for example, one or more mutations selected from the following mutations: a mutation of position 118 of the heavy chain constant region to cysteine, a mutation of position 239 of the heavy chain constant region to cysteine, a mutation of position 160 of the light chain constant region to cysteine, and a mutation of position 166 of the light chain constant region to cysteine; wherein, Amino acid residue position numbering is according to the EU numbering system.

2. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7; or (iii) comprises or consists of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 7, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions.

3. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The light chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 9; or (iii) comprises or consists of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 9, optionally wherein the amino acid substitutions, insertions or deletions do not occur in the CDR regions.

4. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The bispecific antibody against EGFR and HER3 comprises any two mutations selected from the following mutations: mutation of position 118 of the heavy chain constant region to cysteine, mutation of position 239 of the heavy chain constant region to cysteine, mutation of position 160 of the light chain constant region to cysteine, and mutation of position 166 of the light chain constant region to cysteine.

5. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein: The light chain constant region is a lambda light chain constant region or a kappa light chain constant region.

6. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein: The anti-EGFR and HER3 bispecific antibody further comprises a heavy chain constant region where positions 234 and 235 are mutated to alanine.

7. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein: The heavy chain constant region is that of human IgG1.

8. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein: The light chain constant region is a human lambda light chain constant region or a human kappa light chain constant region.

9. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein: The mutations are each independently compared to the heavy chain constant region or the light chain constant region of a native human IgG1 immunoglobulin.

10. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein: The anti-EGFR and HER3 bispecific antibody comprises any one of the following cysteine ​​mutation combinations: (1) position 160 of the λ light chain constant region is mutated to cysteine ​​and position 166 of the λ light chain constant region is mutated to cysteine; (2) position 118 of the heavy chain constant region is mutated to cysteine ​​and position 239 of the heavy chain constant region is mutated to cysteine; (3) position 160 of the λ light chain constant region is mutated to cysteine ​​and position 118 of the heavy chain constant region is mutated to cysteine; (4) position 166 of the λ light chain constant region is mutated to cysteine ​​and position 118 of the heavy chain constant region is mutated to cysteine; (5) position 160 of the λ light chain constant region is mutated to cysteine ​​and position 239 of the heavy chain constant region is mutated to cysteine; and (6) position 166 of the λ light chain constant region is mutated to cysteine ​​and position 239 of the heavy chain constant region is mutated to cysteine, and the bispecific antibody optionally further comprises a LALA mutation in the heavy chain constant region.

11. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein: Heavy chain constant region of the anti-EGFR and HER3 bispecific antibody (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 11, 15, 19 and 23; or (ii) comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 11, 15, 19 and 23; or (iii) comprises an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of any one of SEQ ID NOs: 11, 15, 19 and 23, or consists of said amino acid sequence.

12. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein: Light chain constant region of the anti-EGFR and HER3 bispecific antibody (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21 and 25; or (ii) comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 13, 17, 21 and 25; or (iii) comprises an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21 and 25, or consists of the amino acid sequence.

13. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein: The anti-EGFR and HER3 bispecific antibody comprises a heavy chain and a light chain, wherein The heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 12, 16, 20 and 24; or (ii) comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 12, 16, 20 and 24; or (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of any one of SEQ ID NOs: 12, 16, 20 and 24; and The light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 14, 18, 22 and 26; or (ii) comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 14, 18, 22 and 26; or (iii) comprises an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of any one of SEQ ID NOs: 14, 18, 22 and 26, or consists of said amino acid sequence.

14. The anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein: The anti-EGFR and HER3 bispecific antibody comprises a heavy chain and a light chain, wherein (I) the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 12; or (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 12; and The light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 14; or (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 14; (II) the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 16; or (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 16; and The light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 18; or (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 18; (III) the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 20; or (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 20; and The light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 22; or (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 22; (IV) the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 20; or (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 20; and The light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 26; or (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 26; (V) the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 24; or (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 24; and The light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 22; or (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 22; or (VI) the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 24; or (iii) comprising or consisting of an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 24; and The light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 26; or (iii) comprises an amino acid sequence having one or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 26, or consists of said amino acid sequence.

15. An immunoconjugate comprising the anti-EGFR and HER3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

16. A bispecific antibody-drug conjugate having formula (I), a stereoisomer or a pharmaceutically acceptable salt or solvate thereof: Ab-(LD) n (Ⅰ) in, Ab is a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof according to any one of claims 1 to 14, L is a linker, D is a cytotoxic compound, n represents the number of connections, and n is a natural number selected from 1-15; Preferably, the L is coupled to a mutated cysteine ​​in the Ab.

17. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to claim 16, wherein: The linker L has a structure shown in formula (II): Q-L' (II), Q represents a linker moiety coupled to Ab via a thioether bond (-S-); L' represents a linking moiety that connects Q to the cytotoxic compound D and has the following structure: Wherein, L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, optionally including at least one amino acid residue with a side chain carboxylic acid, and "-C(=O)-" represents the carbonyl group of the amino acid residue at the C-terminal end of the polypeptide residue; L2 does not exist or is a hydrophilic group connected to the carbonyl group after the reaction of the side chain carboxylic acid of the amino acid residue of the polypeptide residue L1, and L2 is -NHR L2 , R L2 is selected from C optionally substituted by 1 to 6 hydroxyl groups 1-6 alkyl; represents the N-terminus of the polypeptide residue to which the linking moiety Q is covalently linked.

18. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to claim 17, wherein: The connecting part Q has the following structure: Among them, Q a It is the functional group coupled to Ab; A is selected from optionally substituted aliphatic or optionally substituted heteroaliphatic, wherein the aliphatic and heteroaliphatic groups are optionally independently selected from halogen, -CN, -OR Qa1 、-SR Qa1 、-N(R Qa1 )2, wherein each R Qa1 independently selected from hydrogen, C 1-6 Alkyl, C 1-6 haloalkyl; and represents the site for covalent attachment to the linking moiety L.

19. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to claim 18, wherein: Functional Group Q a It is absent or selected from maleimide, iodoacetamide, bromoacetamide, pyrimidine, pyrimidinyl sulfide, vinyl pyrimidine, vinyl triazine, vinyl pyridine, disulfide, pyridyl disulfide, haloacetamide, α-haloacetyl, active ester, preferably maleimide, the following structure: "*" indicates the site of covalent attachment to A; Indicates the site of covalent attachment to antibody Ab.

20. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 17 to 19, wherein: The connecting part Q has the following structure:

21. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 17 to 20, wherein: The connection part L1 has the following structure: NH -AA 1 AA 2 AA 3 …AA p_C(＝O) , Among them, AA 1 AA 2 AA 3 ,......AA p Each of which is independently an optionally substituted amino acid residue, optionally AA 1 AA 2 AA 3 ,......AA p At least one of them is an amino acid residue with a side chain carboxylic acid, such as Glu or Asp; p is an integer from 3 to 8, for example, from 3 to 5; "NH-" indicates the N-terminus of a polypeptide residue; "-C(=O)" indicates the C-terminus of a polypeptide residue.

22. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to claim 21, wherein: AA 1 AA 2 AA 3 ,......AA p Each of the amino acids is independently an optionally substituted amino acid residue selected from the group consisting of Glu, Asp, Pro, Nva, Leu, Ile, Met, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Arg, Phe, Lys, Val, Ala, Cit, Gly and N-alkyl amino acids, and AA 1 AA 2 AA 3 ,......AA p At least one of them is Glu or Asp.

23. The bispecific antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to claim 21 or 22, wherein: The connection part L1 is NH -Glu-Val-Al a- C(＝O) .

24. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 17 to 23, wherein: L2 has the following structure:

25. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 17 to 24, wherein: The connecting part L' has the following structure:

26. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 17 to 25, wherein: The linker L has the following structure:

27. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 26, wherein: The linker L is connected to Ab via the side chain of a cysteine ​​residue.

28. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 27, wherein: The cytotoxic compound D has a structure shown in formula (III): Z-D' (III) Z is absent or selected from -NH- or -NH-R z1 -YR z2 -, where R z1 and R z2 Each independently absent or selected from optionally substituted C 1-8 aliphatic, optionally substituted C 0-8 aliphatic-arylene, optionally substituted C 0-8 aliphatic-carbonyl (-C(O)-), optionally substituted C 0-8 aliphatic-oxycarbonyl (-C(O)O- or -OC(O)-), optionally substituted C 0- 8 aliphatic alkylene-carbonyl-imino, optionally substituted arylene-carbonyl (-C(O)-), optionally substituted arylene-oxycarbonyl (-C(O)O- or -OC(O)-), optionally substituted arylene-carbonyl-imino, wherein optionally substituted refers to optionally independently selected from C 1-8 Aliphatic, halogen, -CN, -OR z3 、-SR z3 、-N(R z3 )2, wherein each R z3 independently selected from hydrogen, C 1-6 Aliphatic, C 1-6 Halogenated aliphatic, Y is absent or selected from -O- or -S-; D' is a camptothecin compound, or an analog or derivative thereof.

29. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to claim 28, wherein: D' is a camptothecin compound having the following structure, or an analog or derivative thereof: Where R D1 , R D2 and R D3 Each independently selected from H, hydroxyl, cyano, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, and C 1-6 A group consisting of a haloalkyl group, or R D1 and R D2 Together with the carbon atoms to which it is attached, it forms a 5-8 membered cyclic or heterocyclic group, wherein optionally substituted refers to being optionally independently selected from C 1-8 Aliphatic, halogen, -CN, -OR D’ 、-SR D’ 、-S(O)R D’ and -S(O)2R D’ , and -N(R D’ )2, wherein each R D’ independently selected from hydrogen, C 1-6 Aliphatic, C 1-6 Halogenated aliphatic, -SR D” 、-S(O)R D” and -S(O)2R D” , each R D” independently selected from hydrogen, C 1-6 Aliphatic, C 1-6 Halogenated aliphatic.

30. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 29, wherein: The cytotoxic compound D is selected from the following compounds:

31. The bispecific antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to claim 16, wherein the antibody-drug conjugate has the following structure: wherein Ab and n are as defined in claim 16.

32. The bispecific antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 31, having an average DAR of 1-10, such as 3-5 or 7-9.

33. A pharmaceutical composition comprising the bispecific antibody-drug conjugate according to any one of claims 16 to 32, its stereoisomer or pharmaceutically acceptable salt or solvate, and a pharmaceutically acceptable carrier or excipient.

34. Use of the bispecific antibody-drug conjugate, its stereoisomer or pharmaceutically acceptable salt or solvate according to any one of claims 16 to 32 in the preparation of a medicament for treating or preventing a disease or condition associated with EGFR and / or HER3 activity.

35. The use according to claim 34, wherein the disease or disorder associated with EGFR and / or HER3 activity is an EGFR and / or HER3 positive tumor.

36. The method of claim 35, wherein the EGFR and / or HER3 positive tumor is selected from the group consisting of lymphoma, blastoma, sarcoma, leukemia, melanoma, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, gastric cancer, head and neck squamous cell carcinoma, and various types of head and neck cancer.

37. The bispecific antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 16 to 32 for use in treating or preventing a disease or disorder associated with EGFR and / or HER3 activity.

38. The bispecific antibody-drug conjugate for treating or preventing a disease or condition associated with EGFR and / or HER3 activity, a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof according to claim 37, wherein the disease or condition associated with EGFR and / or HER3 activity is an EGFR and / or HER3 positive tumor.

39. The bispecific antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof for treating or preventing a disease or condition associated with EGFR and / or HER3 activity according to claim 38, wherein the EGFR and / or HER3 positive tumor is selected from the group consisting of lymphoma, blastoma, sarcoma, leukemia, melanoma, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, gastric cancer, head and neck squamous cell carcinoma, and various types of head and neck cancer.

40. A method for treating or preventing a disease or condition associated with EGFR and / or HER3 activity, the method comprising administering to a subject an effective amount of a bispecific antibody-drug conjugate, a stereoisomer, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 16-32.

41. The method of claim 40, wherein the disease or condition associated with EGFR and / or HER3 activity is an EGFR and / or HER3 positive tumor.

42. The method of claim 41, wherein the EGFR and / or HER3 positive tumor is selected from the group consisting of lymphoma, blastoma, sarcoma, leukemia, melanoma, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, gastric cancer, head and neck squamous cell carcinoma, and various types of head and neck cancer.

43. The method according to any one of claims 40-42, wherein the method further comprises co-administering one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, immunomodulators and targeted degradation agents.

44. The method of claim 43, wherein the antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof and one or more therapeutic agents are administered simultaneously or sequentially in any order.

45. The method of claims 40-44, wherein the method further comprises administering to the patient one or more treatment modalities selected from the group consisting of radiation therapy, cell therapy, gene therapy, RNA therapy, and surgery.

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