Bispecific antibody-drug conjugate targeting B7H3 and EGFR and application thereof

Abstract

The present invention relates to antibody-drug conjugates (ADCs) targeting B7H3 and EGFR and compositions containing said molecules. The invention also relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202311786110.0 and application date of December 22, 2023, and the Chinese patent application with application number 202411857429.2 and application date of December 17, 2024, and claims the priority of the said Chinese patent applications. The entire contents of the said Chinese patent applications are hereby introduced into this application as a reference. Technical Field

[0003] The present invention relates to bispecific antibody-drug conjugates (ADCs) targeting B7H3 and EGFR and compositions containing the same. The present invention also relates to therapeutic and diagnostic uses of these antibodies and antibody fragments. Background of the Invention

[0005] Antibody-drug conjugates (ADCs) are chemotherapy drugs that target tumors. They are generally composed of an antibody and varying numbers of linkers and cytotoxins. The target and cytotoxin-linker are crucial to the efficacy and safety of ADCs. ADCs selectively deliver cytotoxins to tumor tissues, inducing the death of targeted tumor cells through the interaction of the selected cytotoxins with tubulin or DNA in the target cells. Furthermore, the bystander effect further kills surrounding cancer cells or paracancerous tissues, thereby improving the efficacy and therapeutic window of ADC drugs [Chari, et al. Angew. Chem. Int. Ed. 2014: 53(15), 3796–3827]. Mylotarg (gemtuzumab ozogamicin) was the first ADC drug approved by the FDA in 2000. In the past two decades, 14 ADCs have been approved for marketing [Fu Z, et al. Signal Transduct Target Ther. 2022; 7(1):93]. Among them, DS8201 (HER2-DXd) is recognized as the most breakthrough blockbuster drug for the treatment of breast cancer to date. In recent years, the clinical development of other ADC drugs has also shown a rapid growth trend.

[0006] It is known that the receptor tyrosine kinase epidermal growth factor receptor (HER) family, including EGFR (HER1 or ERBB1), HER2 (ERBB2), HER3 (ERBB3) and HER4 (ERBB4), plays an important role in cell proliferation, survival, differentiation and migration. Among the four HER family members, only EGFR induces tumor proliferation through homodimerization, while the homodimerization of others such as HER2, HER3 or HER4 is not oncogenic [Cohen, et al., J.Biol.Chem.1996;271(48):30897–30903]. In tumors with EGFR overexpression or aberrant activation, including lung, head and neck, breast, kidney, gastric, colon, pancreatic, ovarian, prostate, and bladder cancers [Jinfeng Yu et al., Front. Mol. Biosci. 2022; 9. https: / / doi.org / 10.3389 / fmolb.2022.847835], EGFR overexpression or aberrant activation induces and regulates the activation and transduction of multiple EGFR downstream signaling pathways, promoting tumor cell proliferation and migration. It also mediates and promotes tumor angiogenesis through VEGF, playing a crucial role in tumor development, progression, malignancy, and prognosis. EGFR is a well-validated tumor target, and EGFR-targeted tumor therapies (EGFR monoclonal antibodies and small molecules targeting EGFR-TKIs) have been widely used in the treatment of various cancer types, including lung, head and neck, colon, and pancreatic cancers. Although these EGFR-targeted therapies have shown improvements in progression-free survival, overall survival, and quality of life in cancer patients, EGFR-targeted therapies are still limited by toxic side effects and the development of drug resistance mechanisms, such as the most common obvious rash (similar in appearance to acne, usually limited to the face, upper chest, and back) and other side effects including diarrhea, constipation, stomatitis, fatigue, and electrolyte imbalances [Eli Lilly. Package insert (Cetuximab); 2004.Available from: http: / / pi.lilly.com / us / erbitux-uspi.pdf; OSI.Package insert (Erlotinib), 2004, Available from: https: / / www.gene.com / download / pdf / tarceva_prescribing.pdf]; At the same time, due to the mutation status of homologous oncogenic genes such as EGFR, K-Ras, B-Raf, PI3K and PTEN, and their different roles in the EGFR resistance mechanism, these factors also limit the scope of targeted application of EGFR monoclonal antibodies and EGFR-TKI small molecules [De LucaA, et al., Curr Drug Targets. 2010; 11: 851–64; Siena S et al., J Natl Cancer Inst. 2009; 101: 1308–24].

[0007] In order to make full use of EGFR as a tumor target and improve the effectiveness of EGFR drugs, many pharmaceutical companies have designed and developed EGFR-targeted monoclonal antibody ADC drugs, such as Depatuxizumab Mafodotin (ABT-414), AVID100, MRG003, and Serclutamab talirine (Ser-T, formerly ABBV-321), which have entered clinical development. These EGFR-ADCs are in clinical development for different tumor indications, such as MRG003 (Phase I clinical trial - NCT04868344, dose range 0.1-2.5 mg / kg, toxicities include hyponatremia, leukopenia, neutropenia, increased aspartate aminotransferase levels and febrile neutropenia, the results showed manageable safety and promising anti-tumor activity; Phase II clinical trial - NCT05126719, two treatment doses of 2.0 / 2.3 mg / kg for the treatment of recurrent / metastatic nasopharyngeal carcinoma after chemotherapy and immunotherapy, the results showed good anti-tumor activity in patients with advanced r / m NPC, with acceptable tolerability and manageable safety. Based on the higher ORR and potentially better efficacy and well-tolerated safety compared to the 2.0 mg / kg group, 2.3 mg / kg is recommended as the recommended dose for further pivotal studies) [Miaozhen Qiu et al. al.,JAMA Oncol. 2022; 8(7):1042-1046.doi:10.1001 / jamaoncol.2022.0503; F.Han, et al.,Annalsof Oncology. 2023; 2(34).doi.org / 10.1016 / j.annonc.2023.09.2006], low doses showed anti-tumor effects and limited toxicity such as hematopoiesis and elevated blood biochemical levels. In addition to being highly expressed in tumors, EGFR is also expressed in normal tissues and participates in cell growth, proliferation, and differentiation. Although EGFR-ADCs have improved their anti-tumor efficacy and application range compared to EGFR monoclonal antibodies and EGFR-TKI small molecules, EGFR-ADCs themselves also have limited toxicity, which limits the EGFR-ADC dosage and effective safety window.

[0008] B7H3 is a single-pass transmembrane glycoprotein encoded by the CD276 gene and a member of the B7 protein family. B7H3 is highly expressed in a variety of solid tumors, including lung, esophageal, endometrial, prostate, and breast cancers. Although some physiological functions of B7H3 have been reported, the lack of direct evidence for its receptors requires further elucidation of its precise physiological functions and detailed mechanisms of action. Some believe that B7H3 is a T cell co-stimulatory molecule [Chapoval AI et al. Nat Immunol. 2001;2:269–74], while others believe that B7H3 is a co-inhibitory molecule that promotes tumorigenesis by regulating tumor-infiltrating immune cells [Kontos F, et al. Clin Cancer Res. 2021;27:1227–35]. Multiple studies have shown that overexpression of B7H3 is associated with poor prognosis in various cancers [Song J et al., Onco Targets Ther. 2016; 9: 6257-63; Brunner A et al., Gynecol Oncol. 2012; 124: 105–11]. Its expression profile and epidemiological data indicate that B7H3 is an attractive potential anti-tumor target.

[0009] Except for low expression in normal tissues of liver, colon, ovary and prostate, B7H3 is not expressed in other normal tissues and is highly expressed in tumors. DS-7300 (B7H3-DXd) is undergoing safety and tolerability clinical trials in phase I clinical trials (dose range 0.8-16 mg / kg) and has shown good tolerability and acceptable safety in patients with mCRPC [Manish R. et al, Journal of Clinical Oncology. 2022; 6(40). DOI: 10.1200 / JCO.2022.40.6_suppl.087 Journal of Clinical Oncology 40, no. 6_suppl (February 20, 2022) 87-87]. There are various treatment modalities and development strategies for B7H3-targeted anti-tumor therapy, such as enhanced antibody-dependent cellular cytotoxicity (ADCC) antibodies, CD3 bispecific antibodies, chimeric antigen receptor (CAR)-T cells, and antibody-drug conjugates (ADCs). However, to date, no B7H3-targeted therapeutics have been approved. B7H3-ADCs are relatively safe, but less effective than EGFR-ADCs.

[0010] Therefore, there is still an urgent need to develop new antibody molecules and ADC molecules containing the same, in particular, the antibody molecules and ADC molecules have advantages such as high efficacy, high safety (including low side effects), can be used to treat various tumor cancers, can be used to treat drug-resistant tumors and / or have high product homogeneity. SUMMARY OF THE INVENTION

[0012] To further improve the efficacy of B7H3-targeted ADCs and the safety of EGFR-targeted ADCs, the inventors selected a high-affinity B7H3 antibody targeting B7H3 and a low-affinity Zalu antibody targeting EGFR to splice into a bispecific antibody, and then introduced site-directed conjugation technology to prepare a B7H3 / EGFR bispecific ADC to improve the efficacy and safety of ADCs in various tumor cancers.

[0013] The present invention provides an immunoconjugate comprising an antibody targeting B7H3 and EGFR (eg, a bispecific antibody or antigen-binding fragment thereof that specifically binds to B7H3 and EGFR of the present invention) and other payloads.

[0014] In some aspects, the immunoconjugate is an antibody-drug conjugate.

[0015] The present invention provides a bispecific antibody targeting B7H3 and EGFR and a bispecific antibody-drug conjugate (ADC) targeting B7H3 and EGFR, wherein the bispecific antibody has the following advantages:

[0016] (a) specifically binds to one or two antigens with high affinity;

[0017] (b) It is easy to express in cultured cells in vitro, and the chains of the antibody molecules can be correctly coupled or paired;

[0018] (c) having good physical stability, in particular, good long-term thermal stability; and being able to maintain biological activity for a long time;

[0019] (d) After specifically binding to one or two antigens, it exerts biological functions by regulating (e.g., inhibiting or activating) the signal transduction pathways involved in each antigen;

[0020] (e) exerting effector functions;

[0021] (f) Has better anti-tumor activity.

[0022] The antibody-drug conjugate (ADC) has the following advantages:

[0023] (1) Bind to target cells expressing human B7H3 and / or EGFR with high affinity;

[0024] (2) Ability to enter cells through endocytosis and kill target cells; has high endocytosis efficiency;

[0025] (3) There is a significant bystander effect;

[0026] (4) The ADC of the present invention can block EGFR downstream signaling and inhibit cell growth. At the same time, the released Exatecan inhibits topoisomerase I, acts on DNA, and induces target cell death, thus having high anti-tumor efficacy.

[0027] (5) Low toxicity;

[0028] (6) Due to the site-specific conjugation of glycosylation, the Fc function of the antibody is silent, and there is no Fc segment function such as ADCC, ADCP and CDC, thus avoiding the side effects of inducing inflammatory factors; and the site-specific conjugation makes the product more uniform and the efficacy more controllable;

[0029] (7) Good stability;

[0030] (8) Having good drugability;

[0031] (9) It has a broader anti-tumor spectrum and can be applied to the treatment of various cancers including lung cancer, colorectal cancer, pancreatic cancer, breast cancer, oral squamous cell carcinoma, gastric cancer, prostate cancer, melanoma and cervical cancer, thus expanding the scope of ADC indications and applications; at the same time, it can also be combined with IO immune agonists in the later stage to further improve the efficacy or expand clinical indications; and / or

[0032] (10) Effective against drug-resistant cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of proliferation inhibition and endocytosis experiments of B7H3 / EGFR bsAb in NCI-H358 are shown.

[0034] Figure 2 The results of proliferation inhibition and endocytosis experiments of B7H3 / EGFR bsAb in PC9-B7H3 are shown.

[0035] Figure 3 The results of the endocytosis experiment of Hz20G5.26 / Zalu bsAb in various tumor cells are shown.

[0036] Figure 4 The expression levels of EGFR and B7H3 in various tumor cells are shown.

[0037] Figure 5 The pERK signaling blocking effect of Hz20G5.26 / Zalu bsAb in NCI-H358 is shown.

[0038] Figure 6 The results of the endocytosis experiment of Hz20G5.26 / Zalu bsAb in NCI-H358 are shown.

[0039] Figure 7 The results of Hz20G5.26 / Zalu bsAb-Exatecan RP-HPLC analysis are shown.

[0040] Figure 8 The expression of B7H3 and EGFR in different tumor cells is shown.

[0041] Figure 9 The in vitro killing activity of Hz20G5.26 / Zalu bsAb-Exatecan in different tumor types and different tumor cells was shown.

[0042] Figure 10 The in vitro killing activity of Hz20G5.26 / Zalu bsAb-Exatecan in different lung cancer cells is shown.

[0043] Figure 11 The in vitro killing activity of Hz20G5.26 / Zalu bsAb-Exatecan in different colorectal cancer cells was shown.

[0044] Figure 12 The in vitro killing activity of Hz20G5.26 / Zalu bsAb-Exatecan in pancreatic cancer cells was shown.

[0045] Figure 13 The in vitro killing activity of Hz20G5.26 / Zalu bsAb-Exatecan in breast cancer cells was shown.

[0046] Figure 14 The in vitro cytotoxic activity of Hz20G5.26 / Zalu bsAb-Exatecan in oral squamous cell carcinoma cells was shown.

[0047] Figure 15 The in vitro killing activity of Hz20G5.26 / Zalu bsAb-Exatecan in cutaneous melanoma cells was shown.

[0048] Figure 16 The in vitro killing activity of Hz20G5.26 / Zalu bsAb-Exatecan in gastric cancer cells was shown.

[0049] Figure 17 The results showed that Hz20G5.26 / Zalu bsAb-Exatecan has an in vitro killing activity on prostate cancer cells.

[0050] Figure 18 The in vitro activity of Hz20G5.26 / Zalu bsAb-Exatecan in EGFR and B7H3 double-low and double-high expressing tumor cell lines was shown.

[0051] Figure 19 Shown are the results of a bystander effector cell killing assay with Hz20G5.26 / Zalu bsAb-Exatecan.

[0052] Figure 20 The in vivo efficacy of Hz20G5.26 / Zalu bsAb-Exatecan in the H1975 CDX mouse model and the changes in mouse body weight after administration are shown.

[0053] Figure 21 The in vivo efficacy of Hz20G5.26 / Zalu bsAb-Exatecan in the BxPC3 CDX mouse model and the changes in mouse body weight after administration are shown.

[0054] Figure 22 The in vivo efficacy of Hz20G5.26 / Zalu bsAb-Exatecan in the H508 CDX mouse model and the changes in mouse body weight after administration are shown.

[0055] Figure 23 The in vivo efficacy of Hz20G5.26 / Zalu bsAb-Exatecan in the JIMT-1CDX mouse model and the changes in mouse body weight after administration are shown.

[0056] Figure 24 Schematic diagram of the bispecific antibody structure is shown. Detailed Description of the Invention

[0058] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0059] I. Definition

[0060] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0061] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5%, 4%, 3%, 2% or 1% less than the specified numerical value and an upper limit that is 5%, 4%, 3%, 2% or 1% greater than the specified numerical value.

[0062] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0063] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0064] Unless otherwise specified, the terms "B7-H3", "B7H3" and "CD276" are used interchangeably herein. B7-H3 is a type I transmembrane glycoprotein that is a member of the B7 / CD28 superfamily and is similar in sequence to the extracellular domain of PD-L1. B7-H3 has 316 amino acids, including a putative signal peptide consisting of 28 amino acids, an extracellular region consisting of 217 amino acids, a transmembrane region and a cytoplasmic domain consisting of 45 amino acids, with a molecular weight of approximately 45-66 kDa. In humans, due to exon duplication, the extracellular structure of B7-H3 can be an IgV-IgC-like domain (2Ig-B7-H3) or an IgV-IgC-IgV-IgC-like domain (4Ig-B7-H3). The sequence of cynomolgus monkey B7-H3 has approximately 90% homology with its human counterpart. In some embodiments of the invention, B7-H3 is human B7-H3. In some embodiments, B7-H3 is a protein under UniProt database accession number Q5ZPR3.

[0065] As used herein, the term "EGFR" refers to the epidermal growth factor receptor, a tyrosine kinase receptor, a giant transmembrane glycoprotein with a molecular weight of approximately 170 kDa. It is a member of the ErbB receptor family and is the most common carcinogenic driver gene in NSCLC. EGFR activating mutations primarily occur in the EGFR exon 18-21 tyrosine kinase domain. The binding region of EGFR antibodies is primarily located in the EGFR extracellular ligand domain, which prevents the occurrence of drug-resistant mutations. At the same time, EGFR antibodies can inhibit the growth of tumor cells by inhibiting the binding of EGFR to ligands, and can also utilize their own specific ADCC (antibody-dependent cell-mediated cytotoxicity) to kill tumors together with immune cells, thereby exerting an anti-tumor killing effect through multiple mechanisms of action. In some embodiments, EGFR is a protein under Uniprot P00533-1 accession number.

[0066] As used herein, the terms "anti-B7-H3 antibody," "anti-B7-H3," "B7-H3 antibody," or "anti-B7-H3 antibody" refer to antibodies that bind to the B7-H3 protein with sufficient affinity. The antibodies can be used as diagnostic and / or therapeutic agents targeting B7-H3, or used to construct immunoconjugates, such as antibody-drug conjugates.

[0067] As used herein, the terms "anti-EGFR antibody," "anti-EGFR," "EGFR antibody," or "anti-EGFR antibody" refer to antibodies that bind to the EGFR protein with sufficient affinity. The antibodies can be used as diagnostic and / or therapeutic agents targeting EGFR, or used to construct immunoconjugates, such as antibody-drug conjugates.

[0068] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0069] General information concerning the amino acid and nucleotide sequences of 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).

[0070] As used herein, amino acid positions throughout the variable regions of heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and are referred to herein as "Kabat numbers."

[0071] As used herein, when referring to amino acid positions in antibody domains other than the variable region (e.g., constant region, e.g., Fc region), the positions are numbered according to the EU numbering system described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and referred to herein as "EU numbering." 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.

[0072] The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen binding site and encompasses natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, whole antibodies, and antibody fragments.

[0073] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3 (and optionally CH4). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs) interspersed with more conserved regions (framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in antibody-antigen binding but exhibits various effector functions.

[0074] "Half-antibody" or "half-polymer" refers to a monovalent antigen-binding polypeptide. In some embodiments, a half-antibody or half-polymer comprises a VH / VL unit and, optionally, at least a portion of an immunoglobulin constant domain. In some embodiments, a half-antibody or half-polymer comprises an immunoglobulin heavy chain associated with an immunoglobulin light chain, or an antigen-binding fragment thereof. In some embodiments, a half-antibody or half-polymer is monospecific, i.e., binds to a single antigen or epitope. In some specific embodiments, a half-antibody binds to EGFR and does not bind to B7-H3. In some specific embodiments, a half-antibody binds to B7-H3 and does not bind to EGFR. One skilled in the art will readily appreciate that a half-antibody can have an antigen-binding domain composed of a single variable domain (e.g., derived from Camelidae).

[0075] In this article, antibody constant region or antibody constant domain, including CH1, CL and Fc domains and CH2, CH3 and optional CH4 domains constituting the Fc domain, can be selected according to the intended function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG or IgM region, in particular an immunoglobulin constant domain of human IgG, for example, a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1. For another example, the Fab fragment of an antibody can comprise the CH and CL constant regions from IgG1. For another example, the Fc region of an antibody can comprise the CH2 and CH3 domains from IgG1. The immunoglobulin constant region can have a native sequence or a variant sequence.

[0076] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residue numbering in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Two Fc regions can achieve dimerization to form a dimeric Fc, and two different Fc heterodimerizations form heterodimeric Fc. In this article, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulin and heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In one embodiment, a human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231, to the carboxyl-terminus of the heavy chain.

[0077] In one embodiment, the human IgG1 Fc region polypeptide (including part of the hinge region) comprises or consists of the following amino acid sequence:

[0078] DKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:47).

[0079] In one embodiment, the Fc region is derived from a human Fc region. In one embodiment, the Fc region comprises all or part of a human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is of the human IgG4 subclass. In one embodiment, the Fc region is of the human IgG1 subclass.

[0080] Herein, a "heterodimeric Fc scaffold" refers to a scaffold comprising two different Fc regions or formed by dimerization of two different Fc regions, which can be connected at its N-terminus or C-terminus to an antigen-binding domain (e.g., an antibody heavy chain and / or light chain variable region or an antibody antigen-binding fragment that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) to form a multispecific antibody such as a bispecific antibody.

[0081] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide that extends from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises or consists of the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 41).

[0082] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen binding fragment. The term "antigen binding fragment" of an antibody is a molecule different from a full-length antibody, which comprises a portion of a full-length antibody, but which can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen binding fragment is derived) for binding to the antigen. Antigen binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb) or nanobody.

[0083] "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable region VH, a heavy chain constant domain CH1, a light chain variable region VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site or antigen binding region. Herein, the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0084] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the variable region of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable region of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The database (IMGT) (www.imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways or in combination. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0085] In one embodiment, the CDRs in an antibody of the invention have boundaries defined by Kabat's rules, or by AbM's rules, or by a combination thereof.

[0086] In one embodiment of the present invention, the HCDR1 of VH in the antigen-binding region that binds to B7H3 is determined by the AbM rule, HCDR2 and HCDR3 are determined by the Kabat rule, and the LCDRs of VL are determined by the Kabat rule.

[0087] In one embodiment of the present invention, the CDRs of VH and VL in the antigen-binding region that binds to EGFR of the present invention are determined by the Kabat rule, respectively.

[0088] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in a wild-type antibody heavy chain, for example, the IgG1 hinge region, such as the sequence from D221 to P230 according to EU numbering. Hinge regions of other IgG subclasses can be identified by aligning the hinge region cysteine ​​residues with those of the IgG1 subclass sequence. In some embodiments, the CH1 region may comprise a portion of the hinge region. In some embodiments, the Fc region may comprise a portion of the hinge region.

[0089] Amino acid mutations are represented by (original amino acid, amino acid position, mutated amino acid). For example, when the mutation site is located in the Fc region, "T366W" means that the T at position 366 of the EU numbering is replaced by W. When referring to a combination of mutations, the combined mutations are connected by "and" or " / ". "R521K / Y1069C" means that the mutations R521K and Y1069C are included at the same time. It should be noted that when describing a mutation, a specific position also covers the corresponding amino acid position in other polypeptide chains. For example, if C220 is mentioned, it covers the 220th amino acid under the EU numbering of the IgG1 heavy chain, as well as the corresponding amino acid in other heavy chains, such as the 131st amino acid in IgG2, IgG3 or IgG4. When describing a mutation, the original amino acid at a specific position may be the described amino acid, or it may be another amino acid at the corresponding position.

[0090] For polypeptide sequences, "conservative changes" include replacements, deletions or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 1) Alanine (A), Glycine (G); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M). In some embodiments, the term "conservative sequence changes" is used to refer to amino acid modifications that do not significantly affect or change the target antigen-binding characteristics of the antibody molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody.

[0091] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule. Where the binding molecule of the present invention relates to a target binding region derived from an antibody, "target" and "antigen" are used interchangeably.

[0092] As used herein, the term "antigen-binding region" refers to any portion of an antibody or antigen-binding fragment thereof, such as a multispecific antibody or bispecific antibody, that binds to a specific target or antigen. The antigen-binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the rest of the multispecific antibody or bispecific antibody and may or may not bind to its antigen / epitope as a separate entity.

[0093] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. A multispecific antibody is an antibody that has binding specificity for at least two different antigens or epitopes. In one embodiment, provided herein is a bispecific antibody that has binding specificity for a first antigen and a second antigen.

[0094] As used herein, the term "bispecific antibody" comprises an antigen-binding domain that specifically binds to two antigens or two epitopes. Unless otherwise indicated, the order of the antigens to which the bispecific antibody binds in the bispecific antibody names listed is arbitrary. That is, in some embodiments, the terms "anti-EGFR / B7H3 bispecific antibody" and "anti-B7H3 / EGFR bispecific antibody" can be used interchangeably. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and optionally at least a portion of a heavy chain constant region and a single light chain variable region and optionally at least a portion of a light chain constant region. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region and does not comprise more than one single heavy chain variable region and does not comprise more than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half antibody binds to a first antigen / epitope and does not bind to a second antigen and the second half antibody binds to the second antigen / epitope and does not bind to the first antigen.

[0095] When referring to a "first antigen-binding region" in a multispecific antibody or bispecific antibody, it refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific antibody or bispecific antibody may contain one or more first antigen-binding regions. For example, a bispecific antibody contains a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0096] When referring to "a target or antigen binding region is derived from an antibody", it means that the binding domain constituting the target / antigen binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, the specific antigen-binding fragment of the antigen binding region, such as Fab, is or is derived from the corresponding fragment, such as Fab, of the antibody, or the heavy chain variable region and / or light chain variable region of the antigen binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen binding region are CDRs of the antibody.

[0097] The term "derived from" means that the fragment in the antigen binding region is substantially identical to the fragment of the antibody from which it is derived, but may have a mutation, such as a substitution, deletion, or addition, at one or more sites. In a specific embodiment, the mutation is not in the CDR of the antibody.

[0098] The multispecific antibodies of the present invention may comprise a linker. As used herein, the term "linker" refers to any molecule that enables the direct connection of the different parts of a multispecific antibody. Examples of linkers for establishing covalent connections between different parts of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol or polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to an amino acid sequence, wherein the sequence connects the amino acid sequences of the various parts of the multispecific antibody together. Preferably, the peptide linker has a length that is sufficient to connect the two entities in a manner that allows them to maintain their conformations relative to each other so as not to interfere with the desired activity. The peptide linker may or may not primarily include the following amino acid residues: Gly, Ser, Ala, or Thr.

[0099] The term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0100] The term "valency" refers to the number of antigen binding sites present in an antibody molecule. "Bivalent," "trivalent," and "tetravalent" antibodies refer to antibodies with two, three, and four antigen binding sites, respectively.

[0101] "knob-into-hole" mutation or "knob-into-knob" mutation or "KIH" mutation is used herein to refer to the introduction of mutations into a first Fc polypeptide and a second Fc polypeptide, respectively, using the "knob-into-knob" technique to form a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and at the interface of the second Fc polypeptide. It is known in the art that the "knob-into-knob" technique can be used to modify the interface between different chains of an antibody molecule to promote the correct association of the chains of the antibody molecule. Typically, the technique involves introducing a "protrusion / knob" at the interface of one chain and a corresponding "hole / knob" at the interface of the other chain to be paired with it, so that the protrusion can be placed in the cavity. A preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to be paired with it. The protrusion can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired. Another optional interface is the CL domain of the light chain and the CH1 domain of the heavy chain of the Fab fragment described above, which promotes correct heterodimerization between the two chains of the Fab fragment by constructing a protrusion-cavity interaction. In some embodiments, the KIH mutation further includes cysteine ​​mutations in two CH3 domains to form non-native disulfide bonds.

[0102] As used herein, the term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assay or surface plasmon resonance (SPR) assays.

[0103] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0104] Carry out the calculating of sequence identity between sequence as follows.For determining the identity percentage of two amino acid sequences or two nucleotide sequences, described sequence is compared for best comparison purpose (for example, can introduce room or can abandon non-homologous sequence for comparison purpose in one or both of first and second amino acid sequences or nucleotide sequences for best comparison).In a preferred embodiment, for comparison purpose, the length of the reference sequence compared is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% reference sequence length.Compare subsequently at the amino acid residue or the nucleotide at corresponding amino acid position or nucleotide position.When the position in the first sequence was occupied by the same amino acid residue or the nucleotide at corresponding position in the second sequence, then described molecule was identical at this position.

[0105] "Immunoconjugate" herein refers to a payload connected to an antibody or its antigen-binding fragment via a linker, such that the antibody or its antigen-binding fragment can be used as a carrier to target and transport the payload to a target site. The term "payload" refers to the active portion of the antibody or antibody fragment of the present invention that is conjugated, and may include any portion for attaching the antibody or antibody fragment. In some embodiments, the payload can be a drug such as a small molecule drug, a radionuclide, DNA, RNA, an enzyme, or a polypeptide. In some embodiments, immunoconjugates include antibody-drug conjugates (ADCs), antibody immunostimulatory drug conjugates (ISACs), antibody oligonucleotide conjugates (AOCs), antibody polypeptide drug conjugates (APCs), antibody nuclide drug conjugates (RDCs), or antibody degradation drug conjugates (ADeCs). Suitable payloads or active moieties for attachment to antibodies include, for example, cytotoxic agents, chemotherapeutic agents, innate immune agonists (e.g., Toll-like receptor agonist (TLR) ISAC drugs SBT6050, SBT6290, BDC-1001; STING agonist ISAC drug XMT-2056, Treg cell regulation ISAC drug ADCT-301, etc.), immunomodulators, therapeutic oligonucleotides (siRNA, PMO, etc.), or radionuclides, etc. In some embodiments, the immunoconjugate of the present invention is an antibody-drug conjugate, i.e., ADC.

[0106] As used herein, "antibody-drug conjugate (ADC)" refers to a compound / molecule obtained by linking an antibody to a drug (eg, a small molecule drug) via a linker.

[0107] The term "linker" refers to a structural fragment that connects a drug (e.g., a small molecule drug) to an antibody portion. It should be understood that the linker has a functional group that can form a bond with a functional group of the antibody or antigen-binding fragment thereof before being attached to the antibody or antigen-binding fragment thereof.

[0108] The term "linker-payload" refers to a compound formed by linking a payload, such as a drug (eg, a small molecule drug), to a linker.

[0109] The term "spacer" refers to a structural segment in a linker that can be used to connect other structural segments in the linker.

[0110] The general term "sugar" used herein refers to monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" used herein refers to a derivative of a monosaccharide, i.e., a monosaccharide comprising a substituent and / or a functional group. Examples of sugar derivatives include amino sugars and sugar acids, such as glucosamine (GlcN), galactosamine (GalN), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc) and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA). Examples of sugar derivatives also include compounds represented herein as E(A)x, where E is a sugar or sugar derivative, and where E includes x functional groups A.

[0111] A core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as being bonded to the GlcNAc of the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. The core-GlcNAc substituent may be present at the native glycosylation site of the antibody, but it may also be introduced at a different site of the antibody. Herein, a core-N-acetylglucosamine substituent is a monosaccharide substituent, or (if the core-GlcNAc substituent is fucosylated) a disaccharide core-(Fucα1-6)GlcNAc substituent - also referred to as GlcNAc(Fuc).

[0112] "Glycosylation modification" refers to the process of modifying the sugar chains of an antibody through glycosylation engineering. The glycosylation of an antibody can be further modified for various purposes to obtain an antibody with a new glycosylation, for example, to remove glycosylation in order to eliminate FcγR affinity and complement binding / effector function, to reduce fucose and sialic acid groups and increase bisecting N-acetylglucosamine, galactose and mannose in order to enhance Fc-mediated ADCC and CDC effects. Methods of glycosylation modification are known in the art, for example, by changing the glycosylation sites of an antibody to increase or decrease the sugar chains on the antibody surface, or by modifying the sugar chains in vitro through chemical or enzymatic methods, or by modifying the glycosylation pathway of the expression system (e.g., composed of enzymes such as glycosidases and glycosyltransferases) to catalyze the glycosylation of the antibody, and the glycosylation of the antibody can also be modified by affecting cell culture conditions. In some embodiments, the "glycosylation modification" of the present invention is performed by modifying the sugar chains by enzymatic methods in vitro. Preferably, the glycosylation modification of the present invention is performed by modifying the sugar chain using a glycosidase (such as an endoglycosidase or a glycosyltransferase).

[0113] The antibodies with engineered glycosylation of the present invention refer to antibodies with an engineered glycosylation pattern compared to an antibody with a native glycosylation pattern. Preferably, the antibodies with engineered glycosylation refer to antibodies obtained after enzymatic modification of the sugar chains of an antibody expressed in an expression system (e.g., a mammalian cell) in vitro (e.g., modification of the sugar chains by a glycosidase (e.g., an endoglycosidase or a glycosyltransferase)). More preferably, the antibodies with engineered glycosylation of the present invention refer to antibodies comprising a core -GlcNAc and a sugar derivative E(A)x attached thereto, wherein the GlcNAc is bonded to the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. If the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucosylated, the fucose is typically attached to C6 of the -GlcNAc substituent via α-1,6. The fucosylated-GlcNAc substituent refers to core-GlcNAc(Fuc), and the fucosylated GlcNAc-E(A)x substituent refers to GlcNAc(Fuc)-E(A)x.

[0114] The term "site-specific conjugation" as used herein refers to conjugation in which a drug / active substance is specifically linked to a specific site of an antibody via a linker.

[0115] The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon group. The alkyl group preferably contains 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0116] The term "alkylene" refers to an alkyl group as defined above, but it is divalent, i.e., has two single bonds connected to two other groups. Non-limiting examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)-, or -CH2CH(-CH3)-.

[0117] The term "halogen" or "halo" refers to fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).

[0118] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen groups as defined herein. Halogenated alkyl groups may preferably be monohalogenated alkyl, dihalogenated alkyl, or polyhalogenated alkyl (including perhalogenated alkyl). Monohalogenated alkyl groups may contain one iodine, bromine, chlorine, or fluorine in the alkyl group. Dihalogenated alkyl and polyhalogenated alkyl groups may contain two or more identical halogen atoms or a combination of different halo groups in the alkyl group. Preferably, polyhalogenated alkyl groups contain up to 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhalogenated alkyl groups refer to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0119] The term "aryl" refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 20, for example 6 to 12, carbon atoms in the ring portion. Preferably, aryl is (C6-C 10 ) aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which may be optionally substituted with 1 to 4 substituents such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, heterocyclyl, and the like.

[0120] The term "arylene" as used herein refers to a divalent aromatic group as defined above, including but not limited to phenylene, such as 1,4-phenylene, 1,3-phenylene or 1,2-phenylene.

[0121] The term "cycloalkyl" is a cyclic alkyl group, that is to say, a monovalent, saturated or unsaturated hydrocarbon group with a ring structure. Cycloalkyl includes fully saturated or partially saturated hydrocarbon groups (containing 1 or 2 double bonds) with a ring structure. Cycloalkyl groups can comprise 3 or more, for example 3-18, 3-10 or 3-8 carbon atoms in the ring, and usually according to the present invention, comprise 3 to 6 atoms. The example of cycloalkyl groups includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0122] As used herein, the term "heteroaryl" refers to a 5-20 membered (e.g., 5-14 membered, 5-8 membered, 5-6 membered) monocyclic, bicyclic, or fused polycyclic ring system containing 1-8 heteroatoms selected from N, O, or S. Preferably, the heteroaryl is a 5-10 membered ring system. Representative heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, 2-, 4-, or 5-pyrimidinyl.

[0123] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, when a group or structure is "optionally substituted," the group or structure may be substituted or unsubstituted.

[0124] In this document, when not specifically stated and not contradictory to the context, when substituted by a "substituent", it may be substituted by 1 or more (e.g. 1, 2, 3 or 4) substituents, which may be selected from, for example, hydrogen, halogen, C1-C 12 Alkyl O-, -NO2, -CN, -S(O)2R2, C1-C 12 Alkyl, halogenated C1-C 12 Alkyl, cycloalkyl, hydroxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, thiol, alkyl-S-, aryl-S-, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonylamino, and heterocyclyl.

[0125] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention and is not biologically or otherwise undesirable. The ADC conjugates of the present invention may exist as pharmaceutically acceptable salts thereof, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable, non-toxic acid addition salt refers to a salt formed between the ADC conjugates of the present invention and an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed with organic bases containing an N group.

[0126] The term "solvate" refers to an association formed between one or more solvent molecules and the ADC conjugate of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0127] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0128] The term "drug:antibody ratio," "DAR," or similar terms refers to the ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in an antibody-drug conjugate molecule. In some embodiments described herein, the DAR can be determined by r and p in Formula (I), or by r, x, and y in Formula (III), for example, the DAR can be 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the total ratio of the drug portion (D) coupled to the Ab portion described herein to the Ab portion in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), and this DAR is referred to herein as the average DAR. The average DAR can be an integer or a decimal. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 16, e.g., 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 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. 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, or any 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.

[0129] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0130] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0131] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0132] The term "drug" refers to an organic compound capable of modulating biological processes, particularly altering or preventing pathological processes.

[0133] The term "prodrug" refers to a chemically modified active or inactive compound that, after administration to a subject, becomes an active drug through physiological actions in the body (e.g., hydrolysis, metabolism, etc.). The techniques for making and using prodrugs are well known to those skilled in the art.

[0134] An "angiogenesis inhibitor" refers to a compound that blocks or interferes to some extent with the development of blood vessels. An angiogenesis inhibitor can be, for example, a small molecule or an antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis.

[0135] The term "small molecule drug" refers to low molecular weight organic compounds that can modulate biological processes, particularly alter or prevent pathological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD, more preferably less than 500 kD. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules. "Anti-tumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773, camptothecin compounds such as Exitecan (topoisomerase I inhibitor, Exatecan), Dxd (a new topoisomerase I inhibitor, Exatecan derivative), auristatin compounds such as monomethyl auristatin E (MMAE) or maytansine compounds such as small molecule microtubule inhibitor DM1, taxanes such as paclitaxel or docetaxel, anthracyclines, epothilones, mitomycins, combretastatins, vinca alkaloids, calicheamicins, duocarmycins, tubulysins, amatoxins, bleomycins, MEK inhibitors, KSP inhibitors, etc. It should be understood that the anti-tumor compounds may be substituted by isotopes including but not limited to deuterium, tritium, etc. For example, when deuterium is substituted, replacing a carbon-hydrogen bond with a carbon-deuterium bond can directly affect the absorption, distribution, metabolism, and excretion properties of certain drugs, thereby improving the drug's efficacy, safety, and tolerability. Therefore, the "anti-tumor compound" in this application can include compounds substituted with deuterium or tritium.

[0136] "Deuterium substituted" means that hydrogen in a molecule is replaced by deuterium, for example, one or more hydrogens, for example 1-10 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) hydrogens are replaced by deuterium. Tritiated has the same or similar interpretation.

[0137] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or regulates (e.g., activates) an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants or immune agonists. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0138] The term "effective amount" refers to an amount or dosage of an ADC molecule, composition, or combination of the present invention that, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. Depending on the desired effect, both "therapeutically effective amount" and "prophylactically effective amount" may be included.

[0139] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the ADC molecule or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0140] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0141] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0142] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0143] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0144] An "isolated" antibody or other molecule is one that has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0145] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0146] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer can be in the early, middle or late stages or be a metastatic cancer. Cancers suitable for treatment by the molecules of the present invention are, for example, selected from epithelial cancers or cancers of the digestive tract, such as gastric cancer or pancreatic cancer or intestinal cancer or colon cancer or colorectal cancer, head and neck squamous cell carcinoma, gastric adenocarcinoma, breast cancer, oral squamous cell carcinoma, prostate cancer, melanoma, cervical cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), esophageal cancer, kidney cancer, bladder cancer, ovarian cancer and head and neck cancer, including metastatic forms of those cancers.

[0147] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Tumor" encompasses solid tumors and hematological tumors, as well as metastatic lesions. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0148] "Tumor immune escape" refers to the process by which a tumor evades immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when this evasion is compromised, allowing the tumor to be recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0149] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0150] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0151] The term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an ADC molecule of the present invention, and (ii) an additional therapeutic agent) are administered to a patient as separate entities simultaneously, without specific time restrictions, or sequentially at equal or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient. In some embodiments, the ADC molecule of the present invention and the additional therapeutic agent used in the pharmaceutical combination are administered at levels no greater than when they are administered alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of the components can produce an effect greater than that achieved by using any one component alone in the treatment of a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0152] The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. Such administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in a plurality of or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, such administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

[0153] As used herein, "treat," ...

[0154] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0155] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0156] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0157] Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples, or frozen tumor samples.

[0158] II. Antibody-Drug Conjugates

[0159] The present invention provides an antibody-drug conjugate having formula (I) or a pharmaceutically acceptable salt or solvate thereof:

[0160] Ab-(L-(D) r ) p (I),

[0161] in:

[0162] Ab is a bispecific antibody or a fragment thereof that specifically binds to B7H3 and EGFR (e.g., human B7H3 and human EGFR);

[0163] L is a linker;

[0164] D is a drug, preferably an anti-tumor compound; and

[0165] p is an integer selected from 1 to 20, for example an integer selected from 1-9, 2-8, 3-7, 4-6 or 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0166] r is an integer selected from 1-5, such as 1, 2, 3, 4 or 5, preferably 1 or 2.

[0167] In some embodiments, the antibody Ab in formula (I) of the present invention is an antibody as defined below, such as a bispecific antibody.

[0168] In some embodiments, L and D in formula (I) of the present invention are as defined below.

[0169] II-1. Bispecific Antibodies

[0170] In one aspect, the present invention provides a multispecific antibody that specifically binds to EGFR and B7H3.

[0171] In some embodiments, the multispecific antibodies of the invention comprise a first binding specificity for EGFR and a second binding specificity for B7H3, and optionally additional binding specificities.

[0172] In some embodiments, the multispecific antibody is a bispecific antibody.

[0173] Therefore, the present invention relates to a bispecific antibody that specifically binds to EGFR and B7H3.

[0174] Therefore, one aspect of the present invention relates to a multispecific antibody comprising

[0175] A first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to EGFR, and / or the second antigen-binding region specifically binds to B7H3, and optionally other antigen-binding regions.

[0176] Therefore, one aspect of the present invention relates to a bispecific antibody comprising

[0177] a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to EGFR, and / or the second antigen-binding region specifically binds to B7H3.

[0178] The bispecific antibodies of the present invention can be prepared using bispecific antibody forms or technologies known in the art. Specific exemplary bispecific forms that can be used in the context of the present invention are described, for example, in Labrijn, et al., Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18 (8): 1-24. In one embodiment, the bispecific antibody format includes IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8: 130). The most common IgG-like antibody type comprises two Fab regions and two Fc regions, and the heavy chain and light chain of each Fab can be from a separate monoclonal antibody. In some embodiments, the bispecific antibody of the present invention is an IgG-like bispecific antibody, which comprises a Fab fragment specifically binding to EGFR as one antigen-binding region and a Fab fragment specifically binding to B7H3 as another antigen-binding region.

[0179] The following describes in detail the components of the multispecific antibodies, such as bispecific antibodies, of the present invention. Those skilled in the art will appreciate that, unless the context clearly indicates otherwise, any combination of any technical features of these components falls within the scope of the present invention. Furthermore, those skilled in the art will appreciate that, unless the context clearly indicates otherwise, the antibodies of the present invention (including any form of antibodies) may include any such combination.

[0180] II-1-1 Antigen binding region specifically binding to EGFR

[0181] In some embodiments, the first antigen-binding region of the anti-EGFR / B7H3 bispecific antibody suitable for use in the present invention may comprise an anti-EGFR antibody or an antigen-binding fragment thereof (e.g., the EGFR antibody disclosed in WO02100348A2 (incorporated in its entirety) such as Zalutumumab monoclonal antibody) or consist thereof, as long as it can specifically bind to EGFR, including but not limited to, for example, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), heavy chain antibodies or linear antibodies that specifically bind to EGFR.

[0182] In some embodiments, the antigen binding region that specifically binds to EGFR is derived from an antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody.

[0183] In some embodiments, the antigen binding region that specifically binds to EGFR comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as zalutumumab monoclonal antibody.

[0184] In some embodiments, the antigen binding region that specifically binds to EGFR comprises one, two, or three heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as zalutumumab monoclonal antibody.

[0185] In some embodiments, the antigen binding region that specifically binds to EGFR comprises one, two, or three light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as zalutumumab monoclonal antibody.

[0186] In some embodiments, the antigen binding region that specifically binds to EGFR comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to EGFR, such as the EGFR antibody disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody.

[0187] In some embodiments, the antigen binding region that specifically binds to EGFR comprises the heavy chain variable region and / or light chain variable region of a known antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody.

[0188] In some embodiments, the antigen binding region that specifically binds to EGFR comprises a known antibody that specifically binds to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, for example, Fab of the zalutumumab monoclonal antibody.

[0189] In some embodiments, the antigen binding region that specifically binds to EGFR comprises three complementary determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen binding region that specifically binds to EGFR comprises three complementary determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen binding region that specifically binds to EGFR comprises three complementary determining regions (HCDRs) from the heavy chain variable region and three complementary determining regions (LCDRs) from the light chain variable region.

[0190] In some aspects, the antigen binding region that specifically binds to EGFR comprises a heavy chain variable region (VH). In some aspects, the antigen binding region that specifically binds to EGFR comprises a light chain variable region (VL). In some aspects, the antigen binding region that specifically binds to EGFR comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0191] The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH and / or VL comprised in the antigen-binding region that specifically binds to EGFR of the present invention are as defined herein, respectively.

[0192] In some embodiments, the heavy chain variable region of the antigen binding region that specifically binds to EGFR of the present invention is

[0193] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1; or

[0194] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 1; or

[0195] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 1, preferably, the amino acid changes do not occur in the CDR regions.

[0196] In some embodiments, the light chain variable region of the antigen binding region that specifically binds to EGFR of the present invention is

[0197] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2; or

[0198] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 2; or

[0199] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 2, preferably, the amino acid changes do not occur in the CDR regions.

[0200] In some embodiments, the three complementary determining regions (HCDRs) of the antigen binding region that specifically binds to EGFR of the present invention are from the heavy chain variable region, HCDR1, HCDR2 and HCDR3 are selected from

[0201] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1, or

[0202] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence in (i),

[0203] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0204] For example, the HCDR1-3 are determined by the Kabat scheme respectively.

[0205] In some embodiments, the three complementary determining regions (LCDRs) of the antigen binding region that specifically binds to EGFR of the present invention are from the light chain variable region, LCDR1, LCDR2 and LCDR3 are selected from

[0206] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2, or

[0207] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence in (i),

[0208] wherein the LCDR can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0209] For example, the LCDRs 1 to 3 are each determined by the Kabat protocol.

[0210] In some embodiments, the antigen-binding region that specifically binds to EGFR of the present invention comprises three complementarity-determining regions (HCDRs) contained in a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 and three complementarity-determining regions (LCDRs) contained in a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2;

[0211] wherein the HCDR and LCDR can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0212] For example, the HCDR1-3 are determined by the Kabat scheme, and the LCDR1-3 are determined by the Kabat scheme, respectively.

[0213] In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 9, or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 9.

[0214] In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10, or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10.

[0215] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11, or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11.

[0216] In some embodiments, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, or consists of the amino acid sequence, or the LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 12.

[0217] In some embodiments, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, or consists of the amino acid sequence, or the LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 13.

[0218] In some embodiments, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 14, or consists of the amino acid sequence, or the LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 14.

[0219] In some specific embodiments of the present invention, the antigen binding region that specifically binds to EGFR of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as described above.

[0220] In some specific embodiments of the present invention, the antigen binding region that specifically binds to EGFR of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.

[0221] In some specific embodiments of the present invention, the antigen binding region that specifically binds to EGFR of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein

[0222] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 9;

[0223] HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10;

[0224] HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11;

[0225] LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12;

[0226] LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13; and

[0227] LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0228] In some specific embodiments of the present invention, the antigen binding region that specifically binds to EGFR of the present invention comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11; LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13 and LCDR3 as shown in SEQ ID NO:14.

[0229] In some specific embodiments of the present invention, the antigen binding region that specifically binds to EGFR of the present invention comprises VH and VL, wherein

[0230] The VH comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0231] In some specific embodiments of the present invention, the antigen binding region that specifically binds to EGFR of the present invention comprises VH and VL, wherein the VH and VL respectively comprise or consist of the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 2.

[0232] In some specific embodiments of the present invention, the antigen binding region that specifically binds to EGFR of the present invention is an anti-EGFR Fab.

[0233] II-1-2 Antigen binding region specifically binding to B7H3

[0234] In some embodiments, the second antigen-binding region of the anti-EGFR / B7H3 bispecific antibody suitable for use in the present invention may comprise an anti-B7H3 antibody or an antigen-binding fragment thereof (e.g., the B7-H3 antibody described in PCT / CN2021 / 140449 (incorporated in its entirety) such as the monoclonal antibody numbered Hz20G5) or consist thereof, as long as it can specifically bind to B7H3, including but not limited to, for example, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), heavy chain antibodies or linear antibodies that specifically bind to EGFR.

[0235] In some embodiments, the antigen binding region that specifically binds to B7H3 is derived from an antibody that specifically binds to B7H3, such as the B7H3 antibody described in PCT / CN2021 / 140449, such as the monoclonal antibody numbered Hz20G5 (such as Hz20G5.26 mentioned in the Examples of the present invention).

[0236] In some embodiments, the antigen binding region that specifically binds to B7H3 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to B7H3, such as the B7H3 antibody described in PCT / CN2021 / 140449, such as the monoclonal antibody numbered Hz20G5.

[0237] In some embodiments, the antigen binding region that specifically binds to B7H3 comprises one, two, or three heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to B7H3, such as the B7H3 antibody described in PCT / CN2021 / 140449, for example, the monoclonal antibody numbered Hz20G5.

[0238] In some embodiments, the antigen binding region that specifically binds to B7H3 comprises one, two, or three light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to B7H3, such as the B7H3 antibody described in PCT / CN2021 / 140449, for example, the monoclonal antibody numbered Hz20G5.

[0239] In some embodiments, the antigen binding region that specifically binds to B7H3 comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to B7-H3, such as the B7H3 antibody described in PCT / CN2021 / 140449, for example, the monoclonal antibody numbered Hz20G5.

[0240] In some embodiments, the antigen binding region that specifically binds to B7H3 comprises the heavy chain variable region and / or light chain variable region of a known antibody that specifically binds to B7-H3, such as the B7H3 antibody described in PCT / CN2021 / 140449, such as the monoclonal antibody numbered Hz20G5.

[0241] In some embodiments, the antigen binding region that specifically binds to B7H3 comprises a Fab of a known antibody that specifically binds to B7-H3, such as the B7H3 antibody described in PCT / CN2021 / 140449, such as the monoclonal antibody numbered Hz20G5.

[0242] In some embodiments, the antigen binding region that specifically binds to B7H3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen binding region that specifically binds to B7H3 comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen binding region that specifically binds to B7H3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0243] In some aspects, the antigen binding region that specifically binds to B7H3 comprises a heavy chain variable region (VH). In some aspects, the antigen binding region that specifically binds to B7H3 comprises a light chain variable region (VL). In some aspects, the antigen binding region that specifically binds to B7H3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0244] The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH and / or VL comprised in the antigen-binding region that specifically binds to B7-H3 of the present invention are as defined herein, respectively.

[0245] In some embodiments, the heavy chain variable region of the antigen binding region that specifically binds to B7H3 of the present invention is

[0246] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3, 5 or 7; or

[0247] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 3, 5 or 7; or

[0248] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3, 5 or 7, or consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions.

[0249] In some embodiments, the light chain variable region of the antigen binding region that specifically binds to B7H3 of the present invention is

[0250] (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4, 6 or 8; or

[0251] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4, 6 or 8; or

[0252] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4, 6 or 8, or consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions.

[0253] In some embodiments, the three complementarity determining regions (HCDRs) of the antigen binding region that specifically binds to B7H3 of the present invention are from the heavy chain variable region, HCDR1, HCDR2 and HCDR3 are selected from

[0254] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 3, 5 or 7, or

[0255] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of any one of (i),

[0256] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0257] For example, the HCDR1 is determined using the Abm protocol, and the HCDR2 and HCDR3 are determined using the Kabat protocol, respectively.

[0258] In some embodiments, the three complementarity determining regions (LCDRs) of the antigen binding region that specifically binds to B7H3 of the present invention are from the light chain variable region, LCDR1, LCDR2 and LCDR3 are selected from

[0259] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 4, 6 or 8, or

[0260] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions relative to the sequence of any one of (i),

[0261] wherein the LCDR can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0262] For example, the LCDRs 1 to 3 are each determined by the Kabat protocol.

[0263] In some embodiments, the antigen-binding region that specifically binds to B7H3 of the present invention comprises three complementarity determining regions (HCDRs) contained in a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, 5, or 7, and three complementarity determining regions (LCDRs) contained in a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, 6, or 8;

[0264] wherein the HCDR and LCDR can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0265] For example, the HCDR1 is determined using the Abm protocol, the HCDR2 and HCDR3 are determined using the Kabat protocol, and the LCDR1-3 are determined using the Kabat protocol.

[0266] In some embodiments, the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, 21 or 27, or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 15, 21 or 27.

[0267] In some embodiments, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16, 22 or 28, or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16, 22 or 28.

[0268] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17, 23 or 29, or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 17, 23 or 29.

[0269] In some embodiments, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, 24 or 30, or the LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 18, 24 or 30.

[0270] In some embodiments, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, 25 or 31, or the LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 19, 25 or 31.

[0271] In some embodiments, the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20, 26 or 32, or the LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 20, 26 or 32.

[0272] In some specific embodiments of the present invention, the antigen binding region that specifically binds to B7H3 of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 as described above.

[0273] In some specific embodiments of the present invention, the antigen binding region that specifically binds to B7-H3 of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as described above.

[0274] In some specific embodiments of the present invention, the antigen binding region that specifically binds to B7H3 of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein

[0275] (i) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17,

[0276] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, and LCDR2 comprises

[0277] The amino acid sequence shown in SEQ ID NO: 19 or consists of the said sequence, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20; or

[0278] (ii) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23,

[0279] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, and LCDR2 comprises

[0280] The amino acid sequence shown in SEQ ID NO: 25 or consists of the said sequence, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; or

[0281] (iii) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 29,

[0282] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 30, and LCDR2 comprises

[0283] The amino acid sequence shown in SEQ ID NO: 31 or consists of the said sequence, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 32 or consists of the said sequence.

[0284] In some specific embodiments of the present invention, the antigen binding region that specifically binds to B7H3 comprises

[0285] (i) HCDR1 as set forth in SEQ ID NO: 15, HCDR2 as set forth in SEQ ID NO: 16, HCDR3 as set forth in SEQ ID NO: 17; LCDR1 as set forth in SEQ ID NO: 18, LCDR2 as set forth in SEQ ID NO: 19, and LCDR3 as set forth in SEQ ID NO: 20;

[0286] (ii) HCDR1 as set forth in SEQ ID NO:21, HCDR2 as set forth in SEQ ID NO:22, HCDR3 as set forth in SEQ ID NO:23; LCDR1 as set forth in SEQ ID NO:24, LCDR2 as set forth in SEQ ID NO:25, and LCDR3 as set forth in SEQ ID NO:26; or

[0287] (iii) HCDR1 as shown in SEQ ID NO: 27, HCDR2 as shown in SEQ ID NO: 28, HCDR3 as shown in SEQ ID NO: 29; LCDR1 as shown in SEQ ID NO: 30, LCDR2 as shown in SEQ ID NO: 31 and LCDR3 as shown in SEQ ID NO: 32.

[0288] In some specific embodiments of the present invention, the antigen binding region that specifically binds to B7H3 of the present invention comprises VH and VL, wherein

[0289] (i) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:3, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4;

[0290] (ii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6; or

[0291] (iii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:8.

[0292] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7H3 of the present invention comprises VH and VL, wherein the VH and VL respectively comprise or consist of the amino acid sequences shown below:

[0293] SEQ ID NO: 3 and SEQ ID NO: 4;

[0294] SEQ ID NO:5 and SEQ ID NO:6; or

[0295] SEQ ID NO:7 and SEQ ID NO:8.

[0296] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7H3 of the present invention is Fab.

[0297] II-1-3 Fab fragment

[0298] In some embodiments, the first antigen-binding region and / or the second antigen-binding region of the present invention is a Fab fragment. A Fab fragment suitable for use as an antigen-binding region of a multispecific antibody, such as a bispecific antibody, described herein is composed of two polypeptide chains comprising the antibody VH, CH1, VL, and CL domains, wherein the VH is paired with the VL and the CH1 is paired with the CL to form an antigen-binding region.

[0299] In some embodiments, in Fab, one chain comprises VH and CH1 (i.e., VH-CH1) or consists of VH and CH1 from N-terminus to C-terminus, and the other chain comprises VL and CL (i.e., VL-CL) or consists of VL and CL from N-terminus to C-terminus. In some embodiments, in the multispecific antibodies or bispecific antibodies of the present invention, Fab can be connected to the N-terminus of the Fc domain of the antibody through the C-terminus of the chain comprising VH. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and can be connected to the antibody Fc domain through the C-terminus of the CH1 of the VH-CH1 chain. In some embodiments, the connection is a direct connection or a connection through a linker. Herein, the Fab chain comprising VH-CH1 is also referred to as the Fab heavy chain, and the Fab chain comprising VL-CL is also referred to as the Fab light chain.

[0300] In some embodiments, the CH1 is a CH1 from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 from IgG1.

[0301] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 41;

[0302] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 41; or

[0303] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 41.

[0304] In some embodiments, the CL is a kappa light chain constant region or a lambda light chain constant region. In some embodiments, the CL is a kappa light chain constant region. In some embodiments, the CL

[0305] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 54;

[0306] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 54; or

[0307] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 54, or consists of said amino acid sequence.

[0308] In some embodiments, the first antigen-binding region is a Fab that specifically binds to EGFR, wherein the Fab fragment is from an anti-EGFR antibody and comprises the heavy chain variable region VH and the light chain variable region VL of the anti-EGFR antibody. In some embodiments, the Fab that specifically binds to EGFR as the first antigen-binding region comprises the 6 CDRs of the antigen-binding region that specifically binds to EGFR as described herein. In some embodiments, the Fab that specifically binds to EGFR as the first antigen-binding region comprises the VH or VL of the antigen-binding region that specifically binds to EGFR as described herein, or comprises the VH and VL of the antigen-binding region that specifically binds to EGFR as described herein.

[0309] In some embodiments, the second antigen-binding region is a Fab that specifically binds to B7H3, wherein the Fab fragment is from an anti-B7H3 antibody and comprises the heavy chain variable region VH and light chain variable region VL of the anti-B7H3 antibody. In some embodiments, the Fab that specifically binds to B7H3 as the second antigen-binding region comprises the six CDRs of the antigen-binding region that specifically binds to B7-H3 as described herein. In some embodiments, the Fab that specifically binds to B7-H3 as the second antigen-binding region comprises the VH or VL of the antigen-binding region that specifically binds to B7H3 as described herein, or comprises the VH and VL of the antigen-binding region that specifically binds to B7H3 as described herein.

[0310] II-1-4Fc region

[0311] In some embodiments, the bispecific antibodies of the present invention further comprise an Fc region, wherein the Fc regions comprised may be the same or different.

[0312] In some embodiments, an antibody molecule of the invention comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

[0313] In some embodiments, the first and second Fc regions are different and are capable of dimerizing to form a heterodimeric Fc scaffold.

[0314] In this article, Fc district refers to the C-terminal region of the immunoglobulin heavy chain containing the constant region of at least a portion, and can include native sequence Fc district and variant Fc district.Native sequence F district covers naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc district (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520) of its allotype.In some embodiments, Fc district of the present invention comprises antibody CH2 and CH3.In some embodiments, antibody Fc district can also carry IgG hinge region or part IgG hinge region at N end, such as, IgG1 hinge region or part IgG1 hinge region, such as according to EU numbering, the sequence of D221 to P230.In the hinge region, can contain sudden change.

[0315] Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0316] In some embodiments, the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 46 or 47, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0317] As will be appreciated by those skilled in the art, to promote the formation of heterodimers of the multispecific antibodies of the present invention, the Fc regions comprised by the multispecific antibodies of the present invention may include mutations that facilitate heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of both Fc regions.

[0318] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 region of a first Fc region and the CH3 region of a second Fc region are engineered in a complementary manner such that each CH3 region (or a heavy chain comprising it) is no longer able to homodimerize with itself but is forced to heterodimerize with the other complementary engineered CH3 region (such that the CH3 regions of the first and second Fc regions heterodimerize and homodimers are not formed between the two first CH3 regions or the two second CH3 regions).

[0319] Preferably, based on the Knob-into-hole technique, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region. This technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In some embodiments, the Knob mutation and Hole mutation may further comprise a cysteine ​​mutation, thereby introducing a disulfide bond. The disulfide bond can be used to stabilize the final antibody product or antibody ADC.

[0320] In a specific embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (numbering according to the EU index).

[0321] In some embodiments, the Fc region may further comprise cysteine ​​residue replacements, thereby obtaining non-native disulfide bonds. In some embodiments, the serine residue at position 354 is replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C) in one Fc region (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue), while in another Fc region, the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C) (numbering according to the EU index). Thus, in a further embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine ​​residue); whereas in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index), optionally the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C) (numbering according to the EU index).

[0322] In a specific embodiment, one Fc region comprises the amino acid substitution T366W and the other Fc region comprises the amino acid substitutions T366S, L368A, and Y407V (numbering is according to the EU index).

[0323] In a specific embodiment, one Fc region comprises amino acid substitutions S354C and T366W, and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering is according to the EU index).

[0324] Corresponding mutations can also be introduced into the first and second Fc regions based on the Innobody technology. This technology is described, for example, in PCT / CN2021 / 143141, which is incorporated herein in its entirety.

[0325] In a specific embodiment,

[0326] The first CH3 region comprises an S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region comprises a K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region comprises an S364R / K mutation, and the second CH3 region comprises a K370S / T / A / V mutation. In some embodiments, the first CH3 region comprises an S364R mutation, and the second CH3 region comprises a K370S mutation.

[0327] In some embodiments, the first CH3 region comprises S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region comprises K370S / T / A / V (preferably K370S) mutations and K409D / E (preferably K409D) mutations. In some embodiments, the first CH3 region comprises S364R / K+D399K / R, and the second CH3 region comprises K370S / T / A / V+Y349T / S / A / V. In some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T. In some embodiments, the first CH3 region further comprises E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further comprises K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0328] In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T + K409D. In some embodiments, the first CH3 region further comprises E357N. In some embodiments, the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region further comprises E357N, and the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region and the second CH3 region further comprise T350V, or both comprise T350V.

[0329] Thus, in some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N+T350V, and the second CH3 region comprises K370S+Y349T+K409D+Q347D+T350V.

[0330] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E), and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D+T411R / K, and the second CH3 region comprises D399K / R+K370T / S / A / V. In some embodiments, the CH3 region comprises K409E+T411R, and the second CH3 region comprises D399K+K370T.

[0331] In some specific embodiments, the first and second CH3 regions have the following mutation combinations:

[0332] First CH3 region Second CH3 region S364R,D399K,E357N,T350V K370S,Y349T,Q347D,K409D,T350V K409E,T411R D399K,K370T S364R,D399K,E357N K370S,Y349T,Q347D,K409D S364R,D399K K370S,Y349T,Q347D,K409D S364R,D399K K370S,Y349T,K409D

[0333] In one embodiment, the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S, and K409D mutations.

[0334] Thus, in a specific embodiment, the multispecific antibody of the present invention comprises two Fc regions that heterodimerize, wherein

[0335] a) one Fc region polypeptide comprises the mutation T366W, and the other Fc region polypeptide comprises

[0336] T366S, L368A and Y407V, or

[0337] b) one Fc-region polypeptide comprises the mutations S354C and T366W and the other Fc-region polypeptide comprises the mutations Y349C, T366S, L368A and Y407V, or

[0338] c) one Fc-region polypeptide comprises the mutations S364R and D399K, and the other Fc-region polypeptide comprises the mutations Y349T, K370S and K409D.

[0339] Thus, in a specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or 50, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 52 or 53.

[0340] Thus, in a specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49 or 50, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52 or 53.

[0341] Thus, in a specific embodiment, the multispecific antibody of the invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 49 or 50 and comprises the mutations Y349T, K370S and K409D, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 52 or 53 and comprises the mutations S364R and D399K.

[0342] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0343] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0344] In a preferred embodiment, the Fc region has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector functions, for example, comprising mutations that achieve the above functions. As will be appreciated by those skilled in the art, depending on the intended use of the binding molecules of the present invention, such as antibody molecules, the binding molecules of the present invention, such as antibody molecules, may also comprise modifications in the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises mutations that reduce binding to Fcγ receptors. For example, in some embodiments, the Fc region used in the present invention has mutations that reduce binding to Fcγ receptors, such as L234A / L235A mutations. In another preferred embodiment, the Fc fragment may have mutations that result in increased serum half-life, such as mutations that improve binding of the Fc fragment to FcRn.

[0345] II-1-5 Exemplary bispecific antibody molecules

[0346] In some embodiments, the anti-B7H3 / EGFR bispecific antibodies of the present invention have one or more of the following properties:

[0347] (1) The bispecific antibodies of the present invention can, on the one hand, block the binding of EGFR ligands to EGFR, inhibit biological signal transmission, and block the corresponding biological activity of tumors; on the other hand, they can stimulate the endocytosis of EGFR and ultimately be degraded by intracellular lysosomes and the like;

[0348] (2) The bispecific antibody of the present invention uses the EGFR antibody parent sequence with low affinity for EGFR, which greatly reduces the toxic side effects of EGFR monoclonal antibodies on normal epithelial tissues such as skin;

[0349] (3) The bispecific antibody of the present invention uses the antibody parent sequence of B7H3 with high affinity on the basis of low affinity for EGFR, which greatly improves the EGFR signal blocking activity and enhances the pharmacodynamic biological activity and pharmacodynamic safety window of the bispecific antibody of the present invention;

[0350] (4) The bispecific antibodies of the present invention have high pharmacodynamic biological activity and safety;

[0351] (5) The bispecific antibodies of the present invention have excellent tumor killing and inhibitory effects; and / or

[0352] (6) The ADC constructed with the bispecific antibody of the present invention has reduced toxic side effects and is highly effective and safe.

[0353] In some embodiments, in the antibody molecules of the present invention, the heavy chain variable region of the antigen binding region that specifically binds to EGFR as described herein is connected to the heavy chain constant region CH, for example, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecules of the present invention, the light chain variable region of the antigen binding region that specifically binds to EGFR as described herein is connected to the light chain constant region CL, for example, wherein the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecules of the present invention, the heavy chain variable region of the antigen binding region that specifically binds to EGFR as described herein is connected to the heavy chain constant region CH and its light chain variable region is connected to the light chain constant region CL.

[0354] In some embodiments, in the antibody molecules of the present invention, the heavy chain variable region of the antigen binding region that specifically binds to B7H3 described herein is connected to the heavy chain constant region CH, for example, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecules of the present invention, the light chain variable region of the antigen binding region that specifically binds to B7H3 described herein is connected to the light chain constant region CL, for example, wherein the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecules of the present invention, the heavy chain variable region of the antigen binding region that specifically binds to B7-H3 described herein is connected to the heavy chain constant region CH and its light chain variable region is connected to the light chain constant region CL.

[0355] In some embodiments, the heavy chain constant region comprises a CH1 and Fc region described herein, connected with or without a hinge region.

[0356] In some embodiments, the heavy chain constant region is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4.

[0357] In some embodiments, the CH1 comprises the amino acid sequence shown in SEQ ID NO:41 or 42, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:41 or 42, or consists of the amino acid sequence.

[0358] In some embodiments, the hinge region comprises or consists of the amino acid sequence shown in SEQ ID NO:43, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:43.

[0359] In some embodiments, the CH1 comprises a portion of the hinge region. In some embodiments, the Fc region comprises a portion of the hinge region. Thus, when the CH1 and Fc regions are linked to form a heavy chain constant region, if the CH1 and Fc regions comprise a portion of the hinge region, the heavy chain constant region after the two regions are linked will comprise the hinge region, and thus there is no need to add a hinge region.

[0360] In some embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region, such as a human kappa or human lambda light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 54.

[0361] In some preferred embodiments, the present invention provides a bispecific antibody molecule comprising a Fab fragment that specifically binds to EGFR as described herein, a Fab fragment that specifically binds to B7H3 as described herein, and an Fc dimer as described herein, wherein the Fab fragment that specifically binds to EGFR forms a half antibody that specifically binds to EGFR with an Fc, and the Fab fragment that specifically binds to B7-H3 forms a half antibody that specifically binds to B7H3 with an Fc.

[0362] In some embodiments, the bispecific antibody is an IgG-like antibody having Figure 24 Configuration shown.

[0363] In a specific embodiment, the first Fc region and the second Fc region comprise Innobody mutations.

[0364] In some embodiments, the bispecific antibody comprises heavy chain 1 and light chain 1, and heavy chain 2 and light chain 2, wherein heavy chain 1 and light chain 1 constitute a first half antibody, and heavy chain 2 and light chain 2 constitute a second half antibody; wherein heavy chain 1 comprises a heavy chain variable region and a first heavy chain constant region of a first antigen binding region; light chain 1 comprises a light chain variable region and a first light chain constant region of a first antigen binding region; and heavy chain 2 comprises a heavy chain variable region and a second heavy chain constant region of a second antigen binding region; and light chain 2 comprises a light chain variable region and a second light chain constant region of a second antigen binding region.

[0365] In a specific embodiment, the bispecific antibody of the present invention specifically binds to EGFR and B7H3, and comprises or consists of:

[0366] Heavy chain 1: comprises or consists of the following from N-terminus to C-terminus: a heavy chain variable region of a Fab that specifically binds to EGFR - a heavy chain constant region CH1 - a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region);

[0367] Light chain 1: From N-terminus to C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to EGFR - a light chain constant region;

[0368] Heavy chain 2: comprises or consists of, from N-terminus to C-terminus: a heavy chain variable region of a Fab that specifically binds to B7H3 - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region);

[0369] Light chain 2: From N-terminus to C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to B7-H3 - a light chain constant region,

[0370] Preferably, each domain is directly linked;

[0371] Optionally, the first Fc region comprises mutations K370S, Y349T and K409D, and the second Fc region comprises S364R and D399K. The respective domains of the bispecific antibodies of the present invention are as described herein.

[0372] In some embodiments, in the bispecific antibody, heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO:33, or 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:33.

[0373] In some embodiments, in the bispecific antibody, light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO:34, or 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:34.

[0374] In some embodiments, in the bispecific antibody, heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 33, or 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: 33, and light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or 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: 34.

[0375] In some embodiments, in the bispecific antibody, heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, 37 or 39, or 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 shown in SEQ ID NO: 35, 37 or 39.

[0376] In some embodiments, in the bispecific antibody, light chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 36, 38 or 40, or 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: 36, 38 or 40.

[0377] In some embodiments, in the bispecific antibody,

[0378] (1) heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or 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: 35; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 36, or 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: 36;

[0379] (2) heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 37, or 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: 37; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 38, or 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: 38;

[0380] (3) Heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:39, or 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:39; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or 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:40.

[0381] In some embodiments, in the bispecific antibody,

[0382] Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:33, or 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:33, and light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or 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:34; and heavy chain 2 and light chain 2 each comprise, or comprise, or consist of, the amino acid sequence of SEQ ID NO:

[0383] i) SEQ ID NO:35 and SEQ ID NO:36;

[0384] ii) SEQ ID NO:37 and SEQ ID NO:38;

[0385] iii) SEQ ID NO: 39 and SEQ ID NO: 40.

[0386] In some embodiments, in the bispecific antibody,

[0387] (i) the heavy chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 34,

[0388] Heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 36; or,

[0389] (ii) the heavy chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 34,

[0390] Heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 37, and light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 38; or,

[0391] (iii) the heavy chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 34,

[0392] Heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 39, and light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 40.

[0393] In some embodiments, the bispecific antibody comprises or consists of heavy chain 1, heavy chain 2, light chain 1, and light chain 2 described herein.

[0394] II-2 Modified glycosylated antibodies

[0395] The antibody Ab in formula (I) of the present invention may be an antibody with modified glycosylation. In some embodiments, the antibody is an antibody obtained after enzymatic modification of the sugar chain in vitro (e.g., modification of the sugar chain by a glycosidase (e.g., endoglycosidase or glycosyltransferase)). In some embodiments, the antibody with modified glycosylation refers to an antibody in which the sugar chain at the antibody glycosylation site is modified from a non-uniform structure N sugar chain to a single structure N sugar chain with a reactive group (e.g., any reactive group that can react with a linker moiety, such as an azide group, a ketone group, and an alkyne group). In a preferred embodiment, the N-glycosylation site of the antibody is a conserved N-glycosylation site on the antibody Fc domain, such as Asn297.

[0396] Methods for engineering antibody glycosylation suitable for the present invention are described, for example, in PCT / NL2013 / 050744, PCT / EP2016 / 059194 or PCT / EP2017 / 052792, which are incorporated herein in their entirety.

[0397] In a preferred embodiment, the antibody of the invention having engineered glycosylation is a GlcNAc-E(A) x An antibody comprising a substituent, wherein GlcNAc is N-acetylglucosamine, wherein E(A)x is a sugar derivative comprising x functional groups A, wherein A is independently selected from azide, keto, and alkynyl, and x is 1, 2, 3, or 4; wherein the GlcNAc-E(A)x substituent is bonded to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent, wherein the N-acetylglucosamine is optionally fucosylated. In the case where the N-acetylglucosamine is fucosylated, it is bonded to fucose (Fuc) at C6 via a glycosidic bond.

[0398] In one embodiment, the antibody of the invention with engineered glycosylation is an antibody of formula (II), wherein Ab represents an antibody, GlcNAc represents N-acetylglucosamine, Fuc represents fucose, b represents 0 or 1, and wherein E(A)x represents a sugar derivative comprising x functional groups A, wherein A represents an azide group, a keto group, and an alkyne group, and x represents 1, 2, 3, or 4. In a preferred embodiment, y represents 1 to 10, more preferably, y represents 1, 2, 3, 4, 5, 6, 7, or 8, even more preferably, y represents 1, 2, 3, or 4, and most preferably, y represents 1 or 2.

[0399]

[0400] The sugar derivative E(A)x in the GlcNAc-E(A)x substituent in the antibody with engineered glycosylation can be, for example, bonded to C4 of the GlcNAc via a β(1,4)- or β(1,3)-glycosidic bond or bonded to C3 of the GlcNAc via an α(1,4)- or α(1,3)-glycosidic bond, preferably bonded to C4 of the GlcNAc via a β(1,4)-glycosidic bond. The N-acetylglucosamine of the GlcNAc-E(A)x substituent is bonded to the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom in the side chain of asparagine of the antibody (GlcNAcβ1-Asn). The GlcNAc on the GlcNAc-E(A)x substituent is optionally fucosylated. Accordingly, the GlcNAc-E in the antibody-drug conjugate, when present, can also be linked as described above.

[0401] In a preferred embodiment, the functional group A is an azido group. When A is an azido group, preferably, A is bonded to C2, C3, C4, or C6. As described above, one or more azide substituents in E(A)x may be bonded to C2, C3, C4, or C6 of the sugar or sugar derivative E, replacing the hydroxyl (OH) at the position. It should be understood that the bonding position of the functional group A corresponds to the position where the sugar containing A is attached to the linker.

[0402] In a preferred embodiment, the sugar derivative E(A)x is derived from a sugar or a sugar derivative E. In a preferred embodiment, E is a sugar or a sugar derivative selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), preferably Gal, GlcNAc, glucose and GalNAc, most preferably GalNAc.

[0403] In another preferred embodiment, the E(A)x is GalNAc-N3, preferably, E(A)x is 6-azido-6-deoxy-2-acetamidogalactose.

[0404] It should be understood that, unless otherwise stated, the description and illustration of sugars in Formula (II) (including but not limited to the glycosidic linkage of GlcNAc-E(A)x) can be used to explain or define the corresponding sugars in the conjugate of Formula (III), such as the sugar linkage therein. Those skilled in the art will understand that, although in some embodiments, a reactive group (e.g., functional group A) is attached to the sugar chain of the antibody during antibody glycosylation modification, and thus an "antibody with modified glycosylation" is defined as an antibody comprising such a reactive group, when the antibody-drug conjugate (Formula (I) or Formula (III)) is formed, the reactive group can react with the linker portion to form a new group with the linker portion, and thus, in the antibody-drug conjugate, the new group is considered to be part of the linker.

[0405] When the modified antibody is a bispecific antibody of the present invention, the antibody preferably comprises two or more, more preferably two, GlcNAc-E(A)x substituents, wherein the GlcNAc-E(A)x substituents are optionally fucosylated. However, if the modified antibody is an antibody fragment, such as a Fab or Fc fragment, the antibody may have only one GlcNAc-E(A)x substituent, which is optionally fucosylated. The GlcNAc-E(A)x substituent may be located at any position in the antibody, provided that the substituent does not interfere with the binding of the antigen to the antigen-binding site of the antibody. In one embodiment, the GlcNAc-E(A)x substituent is located in the Fc domain of the antibody, more preferably, in the CH2 domain.

[0406] As described above, the antibodies of the present invention with engineered glycosylation comprise more than one GlcNAc-E(A)x substituent, such as two GlcNAc-E(A)x substituents.

[0407] In a preferred embodiment, the GlcNAc-E(A)x substituent is present at a native N-glycosylation site (e.g., a native conserved N-glycosylation site) of the antibody, such as a glycosylation site in the Fc region (more preferably, a CH2 domain). In another preferred embodiment, the antibody is an IgG antibody and the GlcNAc-E(A)x substituent is present at a native N-glycosylation site (a native conserved N-glycosylation site) of the IgG antibody. In another preferred embodiment, the native site is the Asn297-glycosylation site of the IgG antibody. The Asn297-glycosylation site is present in the Fc region of the heavy chain of the IgG antibody. In a preferred embodiment, the GlcNAc-E(A)x substituent is present at the Asn297-glycosylation site of both heavy chains of the antibody.

[0408] II-3. Linkers and Drugs

[0409] In some embodiments, L in formula (I) of the present invention is a linker. Any linker known in the art can be used to connect to the anti-human B7H3 and EGFR antibodies of the present invention. Preferably, the linker can achieve site-specific coupling of the ADC.

[0410] In some embodiments, the linker suitable for use in the present invention can be any linker that can achieve conjugation of an antibody to a drug. In some embodiments, the linker can be a linker used in a technology that can achieve site-specific conjugation.

[0411] In a preferred embodiment, the linker of the present invention is a linker connected to an antibody oligosaccharide. As defined herein, a "linker connected to an antibody oligosaccharide" refers to any linker that is connected to a reactive group on a sugar chain at an antibody glycosylation site to couple the antibody to a drug. The sugar chain on the antibody glycosylation site is generally an N-sugar chain, and is usually modified to transform the non-uniform structure N-sugar chain into a single structure N-sugar chain with a reactive group, and then the reactive group carried by the sugar chain is connected to the "linker" to achieve site-specific coupling of the drug to the antibody to obtain an antibody-drug conjugate. In a preferred embodiment, the N-glycosylation site of the antibody is the antibody Fc domain, preferably a conserved N-glycosylation site on the CH2 domain, such as Asn297. Therefore, in one embodiment, the "linker attached to an antibody oligosaccharide" of the present invention is particularly a linker obtained by reacting with a reactive group on an N-sugar chain at a conserved N-glycosylation site (e.g., Asn297) on the Fc domain of an antibody to achieve site-specific coupling, such as the linker described in PCT / NL2013 / 050744 or the linker of PCT / EP2021 / 075401, which are incorporated herein by reference in their entirety. In one embodiment, the reactive group of the present invention is an azido group, a keto group, or an alkynyl group. In one embodiment, the linker of the present invention is a linker comprising an alkynyl group. In one embodiment, the reactive group of the present invention is an azido group, a keto group, or an alkynyl group, preferably an azido group, and the linker of the present invention is a linker comprising an alkynyl group. When the present invention refers to such linkers, since a new group is formed after the reactive group reacts with the group of the linker, the group formed after the reaction of the reactive group in the antibody-drug conjugate can also be defined as part of the "linker", such as shown in formula (III) of the present invention.

[0412] Linkers suitable for the present invention also include, for example, cathepsin-degradable linkers, such as Val-Cit linkers (e.g., vc-PAB), cBu-Cit linkers, and CX linkers; non-cleavable linkers such as SMCC linkers or MD linkers; acid-sensitive linkers, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, and ultraviolet-sensitive linkers such as PC4AP (Antibody–drug conjugates: Recent advances in linker chemistry, Su, Z., Xiao, D., Xie, F., Liu, L., Wang, Y., Fan, S., … Li, S. (2021). Antibody–drug conjugates: Recent advances in linker chemistry. Acta Pharmaceutica Sinica B.). Linkers suitable for use in the present invention may also be combinations of one or more linkers. For example, cathepsin-degradable linkers can be combined with other types of linkers to form new linkers. Therefore, the term "linker" as used herein encompasses a single type of linker or a combination of different types of linkers, as long as they are capable of conjugating the antibodies of the present invention to the drug. Therefore, in one embodiment, a linker suitable for use in the present invention is MC-VC-PAB, vc-PAB, SMCC, or MC-GGFG.

[0413] D in formula (I) of the present invention can be any antitumor compound, without particular limitation, as long as it has an antitumor effect and has a substituent or partial structure that can be attached to the linker structure. For example, the antitumor compound can be a pharmaceutically active compound that has an effect on tumors. For antitumor compounds, a portion or all of the linker can preferably be cleaved within tumor cells, releasing the antitumor compound portion, thereby exhibiting an antitumor effect. When the linker is cleaved from the drug-attached portion, the antitumor compound is released in its unmodified structure, allowing it to exert its original antitumor effect.

[0414] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent or a chemotherapeutic agent, such as a camptothecin compound exitecan (topoisomerase I inhibitor Exatecan), DXd (a novel topoisomerase I inhibitor Exatecan derivative), an auristatin compound such as monomethyl auristatin E (MMAE), a maytansine compound such as a small molecule microtubule inhibitor DM1, a taxane such as paclitaxel or docetaxel, an anthracycline, an epothilone, a mitomycin, a combretastatin, a vinca alkaloid, a calicheamicin, a multicarboxycin, a tubulysin, a phalloidin, a bleomycin, a MEK inhibitor, a KSP inhibitor, and the like.

[0415] II-4. Antibody-drug conjugates linked via carbohydrates

[0416] The present invention relates to an antibody-drug conjugate of formula (III) or a pharmaceutically acceptable salt or solvate thereof

[0417]

[0418] Ab is a bispecific antibody or fragment thereof of the present invention that specifically binds to B7H3 and EGFR (eg, human B7H3 and EGFR), as defined herein,

[0419] L1 is a linker;

[0420] E is a sugar or a sugar derivative; for example a sugar or a sugar derivative as defined above;

[0421] And GlcNAc is N-acetylglucosamine, Fuc is fucose;

[0422] D is as defined above in formula I;

[0423] r is 1-5, such as 1, 2, 3, 4 or 5, preferably 1 or 2;

[0424] b is 0 or 1, for example, 0;

[0425] x is 1, 2, 3 or 4; preferably 1 or 2;

[0426] y is an integer selected from 1 to 20, such as an integer selected from 1 to 10, more preferably, y is 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably, y is 1, 2, 3 or 4.

[0427] In some embodiments, x is 1 or 2, more preferably 1, and / or y is 1 or 2, more preferably 2.

[0428] In some embodiments, in formula (III), E is selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), preferably Gal, GlcNAc, glucose and GalNAc, most preferably GalNAc, such as 6-deoxy-2-acetamidogalactose, which is linked to L1 through C2, C3, C4 and / or C6, preferably through the C atom at position C6.

[0429] In some embodiments, in formula (III), the monosaccharide or sugar derivative E can be bonded to C4 of the GlcNAc through a β(1,4)- or β(1,3)-glycosidic bond or bonded to C3 of the GlcNAc through an α(1,4)- or α(1,3)-glycosidic bond, preferably bonded to C4 of the GlcNAc through a β(1,4)-glycosidic bond, and the GlcNAc attached to the Ab is bonded to the antibody through C1, preferably bonded to the amide nitrogen atom in the side chain of asparagine of the antibody through an N-glycosidic bond (GlcNAcβ1-Asn), and the GlcNAc is optionally fucosylated.

[0430] In a preferred embodiment, the GlcNAc connected to Ab is present on the native N-glycosylation site (e.g., native conserved N-glycosylation site) of the antibody, such as the glycosylation site in the Fc region. In another preferred embodiment, the antibody is IgG and the GlcNAc is present on the native N-glycosylation site (e.g., native conserved N-glycosylation site) of IgG, such as the glycosylation site in the Fc region. In another preferred embodiment, the native site is the Asn297-glycosylation site of IgG. The Asn297-glycosylation site is present in the Fc region of the heavy chain of the IgG antibody. In a preferred embodiment, the GlcNAc group is present on the Asn297-glycosylation site of both heavy chains of the antibody.

[0431] In some embodiments, in formula (III), L1 is the following structure

[0432]

[0433] Q is -NHC(O)CH2- or -CH2-;

[0434] Z is

[0435]

[0436] R1 is independently selected from hydrogen, halogen, -OR2, -NO2, -CN, -S(O)2R2, C1-C 12 Alkyl-, C6-C 20 Aryl-, 5-20 membered heteroaryl-, C1-C 12 Alkyl C6-C 20 Aryl-, C1-C 12 Alkyl 5-20 membered heteroaryl-, C6-C 20 Aryl C1-C 12 Alkyl- and 5-20 membered heteroaryl C1-C 12 Alkyl-, and wherein the alkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, arylalkyl and heteroarylalkyl are optionally substituted, wherein two substituents R1 can be linked together to form a fused, bridged or spiro C3-C 18 Cycloalkyl or fused or spiro aromatic or heteroaromatic substituents, and wherein R2 is independently selected from hydrogen, C1-C 24 Alkyl-, C6-C 20 Aryl-, 5-20 membered heteroaryl-, C1-C 12 Alkyl C6-C 20 Aryl-, C1-C 12 Alkyl 5-20 membered heteroaryl-, C6-C 20 Aryl C1-C 12 Alkyl- and 5-20 membered heteroaryl C1-C 12 alkyl-;

[0437] R3 and R4 are each independently selected from hydrogen, halogen, C1-C 24 Alkyl-, C6-C 20 Aryl-, 5-20 membered heteroaryl-, C1-C 12 Alkyl C6-C 20 Aryl-, C1-C 12 Alkyl 5-20 membered heteroaryl-, C6-C 20 Aryl C1-C 12 Alkyl- and 5-20 membered heteroaryl C1-C 12 alkyl-;

[0438] Y is -O-, -S-, -NR2- or absent;

[0439] X1 is -O-, -S- or -NR2-;

[0440] G1 is -C(O)-, or direct key;

[0441] G2 is

[0442] R7 are each independently selected from hydrogen and C1-C 12 alkyl;

[0443] R8 is hydrogen, C1-C 12 Alkyl or

[0444] R9 is

[0445] L2 and L3 are each independently C1-C 12 an alkylene group in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced by a heteroatom selected from O, N, and S, with the proviso that said O, N, and / or S are not directly bonded to each other;

[0446] L4 is each independently a cleavable spacer;

[0447] n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0448] n2 are each independently 0, 1 or 2;

[0449] n3 are each independently 0, 1, 2, 3, 4 or 5;

[0450] c is 0, 1, 2, 3, or 4;

[0451] m is 0 or 1.

[0452] It should be understood that, unless otherwise specified and not contradictory according to the context, for the ADC of the present invention, the left-side bond of the divalent group shown herein is connected to Ab or a group near the end of Ab, and the right-side bond of the divalent group is connected to D or a group near the end of D. For example, when G1 is The carbonyl group is connected to the Y group, N is connected to L2; the Z group The bond on the triazole is connected to the sugar E on the left side of Z, and the bond on the cyclopropyl is connected to the CH2 on the right side of Z.

[0453] It should be understood that when m is 0, it means that Q is absent and the relevant nitrogen atom of the triazole is directly linked to the E moiety via a covalent single bond.

[0454] In some embodiments, Z is The symbols are as defined above.

[0455] In some embodiments, Z is The symbols are as defined above.

[0456] In some embodiments, n2 is 1.

[0457] In some embodiments, R3 and R4 are H.

[0458] In some embodiments, Z is

[0459] In some embodiments, Z is

[0460] In some embodiments, for

[0461] In some embodiments, m is 0.

[0462] In some embodiments, L2 is n4 is 0, 1, 2 or 3, or C1-C 12 Alkylene, preferably

[0463] In some embodiments, each L3 is independently n4 is 0, 1, 2 or 3, or C1-C 12 Alkylene, preferably

[0464] In some embodiments, each L4 is independently selected from

[0465] wherein R5 and R6 are each independently selected from hydrogen, C1-C 12 Alkyl or -(CH2)3NHC(O)NH2;

[0466] or

[0467] -CO-L5-L6-,

[0468] wherein L5 is a peptide residue consisting of 2, 3, 4 or 5 amino acids;

[0469] L6 is selected from the group consisting of: absent, -NH-CH2-,

[0470] where R a1 Each independently selected from C 1-6 Alkyl-, C 1-6 Alkoxy-, halogenated C 1-6 alkyl-, halogen, nitro, and cyano; and

[0471] t is 0, 1, 2, 3, or 4.

[0472] In some embodiments, the amino acid is selected from, for example, glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine, and proline.

[0473] In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine.

[0474] In some embodiments, L5 is selected from the group consisting of: -Val-Ala-, -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, -Ala-Ala-, -Ala-Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val-, -Cit-Ala-, -Cit-Asn-, - Cit-Asp-, -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, -Ile-Val-, -Leu- Cit-, -Lys-Cit-, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val-Asp-, -Cit-Val-, -Val-Glu-, -Val-Lys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, -Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit-Ala-Glu-, -Val-Lys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-Gly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Ly s-Gly-, -Val-Lys-Leu-, -Ala-Ala-Ala-, -Asn-Ala-Ala-, -Gly-Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -G ly-Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly-, and -Val-Lys-Gly-Gly.

[0475] In some embodiments, L6 is selected from -NH-CH2-,

[0476] In some embodiments, L6 is selected from

[0477] In some embodiments, each L4 is independently -CO-L5-L6-, wherein each symbol (L5, L6) is as defined above.

[0478] In some embodiments, each L4 is independently

[0479] wherein R5 and R6 are each independently selected from hydrogen, C1-C 12 Alkyl or -(CH2)3NHC(O)NH2; preferably, R5 is isopropyl and R6 is methyl;

[0480] Ar is selected from arylene, preferably phenylene, more preferably

[0481] In some embodiments, each L4 is independently

[0482] In some embodiments, in formula (III), -L1- is the following structure

[0483]

[0484] Among them, Q, R 1、 R 2、 R3, R4, c, m, n1, Y, L2, L3 and L4 are as defined above.

[0485] In some embodiments, in formula (III), -L1- is the following structure

[0486]

[0487] In some embodiments, D is represented by formula (D-1a), formula (D-1b), formula (D-1c), or formula (D-1d):

[0488]

[0489]

[0490] Each R 10 Independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 12 and-SR 12 ; R 11 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 12 ; or R 10 and R 11 Together they form -O(CH2) n5 O- or -O(CF2) n5 O-, where n5 is 1 or 2;

[0491] Each R 12 Independently selected from H or C1-C4 alkyl;

[0492] Among them, each R 13 independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0493] Each Q1 is independently -O- or -S-;

[0494] L a1 Yes-(C1-C 10 alkylene)C(O)-;

[0495] Each L b1 Independently is -(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-C(O)N(R 14 )-or-(C1-C 10 Alkylene)-N(R 14 )C(O)-, where R 14 It is H or C1-C6 alkyl.

[0496] It should be understood that, consistent with the above description, the divalent groups shown herein, such as L a1 and L b1 (For example -(C1-C 10 The left end of the alkylene) C(O)-) is covalently linked to Q1. The wavy lines on the structures / fragments herein have commonly understood meanings and indicate the attachment position of the structure / fragment.

[0497] In some embodiments, L a1 It is -(C1-C4 alkylene)C(O)-.

[0498] In some embodiments, L a1 It is -CH2-C(O)-.

[0499] In some embodiments, L b1 It is -(C1-C6 alkylene)-.

[0500] In some embodiments, L b1 It is -CH2-CH2-CH2-CH2- or -CH2-CH2-CH2-.

[0501] In some embodiments, Q1 is -O-.

[0502] In some embodiments, D is a structure represented by formula (D-1a):

[0503]

[0504] R 10 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 12 and-SR 12 ; R 11 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 12 ; or R 10 and R 11 Together they form -O(CH2) n5 O- or -O(CF2) n5 O-, where n5 is 1 or 2;

[0505] R 12 Selected from H or C1-C4 alkyl.

[0506] In some embodiments, R 10 is C1-C6 alkyl or C1-C4 alkoxy, preferably methyl or methoxy, and R 11 is halogen, preferably F;

[0507] or R 10 and R 11 Together they form -OCH2O-.

[0508] In some embodiments, R 10 and R 11 Together they form -OCH2O-.

[0509] In some embodiments, R 10 is a C1-C6 alkyl group, preferably a methyl group, and R 11 is halogen, preferably F.

[0510] In some embodiments, each R 13 Independently selected from H and C1-C6 alkyl, preferably H.

[0511] In some embodiments, D is represented by formula (D-2a), (D-2b), (D-2c), or (D-2d):

[0512]

[0513]

[0514] Among them, each symbol (R 10 、R 11 、R 13 ,Q1,L a1 and L b1 ) as defined above.

[0515] In some embodiments, D is a structure represented by formula (D-2a):

[0516]

[0517] where R 10 and R 11 As defined above.

[0518] In some embodiments, D is a structure represented by formula (D-3a):

[0519]

[0520] In some embodiments, the antibody-drug conjugate has an average DAR of 1-10, such as 2-8, 2-6, or 3-5. In some embodiments, the antibody-drug conjugate of the present invention is selected from:

[0521]

[0522] wherein Ab is a bispecific antibody of the present invention, preferably Hz20G5.26 / Zalu bsAb;

[0523] y is 1 or 2, preferably 2.

[0524] Preferably, the antibody-drug conjugate has an average DAR of, for example, 2-6, 3-5, or 3-4.

[0525] In another aspect, the present invention provides an antibody with engineered glycosylation represented by formula (II).

[0526] III. Preparation of ADC molecules of the present invention

[0527] In one aspect, the present invention provides a method for preparing an antibody having modified glycosylation, the method comprising

[0528] (1) Preparation of glycosylated antibodies: culturing a host cell containing a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium) to obtain an antibody containing a core N-acetylglucosamine substituent (core-GlcNAc substituent), wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated;

[0529] (2) preparing a trimmed antibody: deglycosylation of the antibody prepared in step (1) in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated;

[0530] (3) contacting the trimmed antibody obtained in (2) with a compound of formula E(A)xP in the presence of a suitable catalyst to obtain an antibody comprising a GlcNAc-E(A)x substituent, wherein the GlcNAc-E(A)x substituent is bonded to the antibody via the C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent; wherein the catalyst is a glycosyltransferase, wherein P is selected from uridine diphosphate (UDP), guanosine diphosphate (GDP) and cytidine diphosphate (CDP).

[0531] To perform step (1), nucleic acid encoding an antibody (e.g., an antibody described above, e.g., any one polypeptide chain and / or multiple polypeptide chains) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

[0532] The endoglycosidase in step (2) can be selected according to the properties of the glycosylated antibody, for example, selected from Endo S, Endo S2, Endo A, Endo F, Endo M, Endo D and Endo H enzymes and / or combinations thereof, and further for example, Endo S, Endo S2, Endo S49, Endo F or a combination thereof. In a preferred embodiment, the endoglycosidase is the endoglycosidase described in PCT / EP2017 / 052792, most preferably Endo SH in PCT / EP2017 / 052792.

[0533] The glycosyltransferase in step (3) is preferably a glycosyltransferase or a glycosyltransferase derived from β-(1,4)-N-acetylgalactosamine transferase, more preferably any β-(1,4)-GalNAcT enzyme described in PCT / EP2016 / 059194. In some embodiments, the β-(1,4)-GalNAcT enzyme is or is derived from an invertebrate β-(1,4)-GalNAcT enzyme. The β-(1,4)-GalNAcT enzyme can be or can be derived from any invertebrate β-(1,4)-GalNAcT enzyme known to those skilled in the art. Preferably, the β-(1,4)-GalNAcT enzyme is or is derived from a β-(1,4)-GalNAcT enzyme from the phylum Nematoda, preferably from the class Chromadorea or Secernentea, or from the phylum Arthropoda, preferably from the class Insecta. More preferably, the β-(1,4)-GalNAcT enzyme is or is derived from Caenorhabditis elegans, Ascaris suum, Trichoplusia ni, Drosophila melanogaster, saprophytic fruit nematodes, Caeno rhabditis briggsae, Wuchereria elegans, Loa filariae, B. bispezii, Coptotermes floridans, Camponotus floridans, Crassostrea gigas, and Platyrrhineia rubripes. Preferably, the glycosyltransferase suitable for step (3) is the Trichoplusia ni β-(1,4)-GalNAcT enzyme designated TnGalNAcT disclosed in PCT / EP2016 / 059194 (eg His-TnGalNacT).

[0534] Another aspect of the present invention provides a method for preparing an ADC using the antibody of the present invention. "ADC" in the present invention is defined as an antibody coupled to a biologically and / or pharmaceutically active active substance (D) via a linker (L). The method comprises coupling the antibody of the present invention to one or more active substances D via one or more linkers (L) as defined herein. Preferably, the linker-active substance is site-specifically coupled to the antibody.

[0535] In some embodiments, a method for preparing an ADC of the present invention is provided, the method comprising glycosylated antibody of formula (II)

[0536]

[0537] The meanings of the variables or symbols are as defined above;

[0538] reacting with a linker-drug compound containing an alkynyl group and one or more (e.g., 1, 2, 3, or 4) drug molecules,

[0539] Generation of Antibody-Drug Conjugates of Formula (III)

[0540]

[0541] The meanings of the variables and symbols are as defined above.

[0542] In some embodiments, the linker-drug compound has the following structure:

[0543]

[0544] Where Z1 is N3- or

[0545] Each variable in each linker-drug compound (including the variable in Z1) is as defined above.

[0546] IV. Pharmaceutical Compositions

[0547] In some embodiments, the present invention provides a composition comprising any ADC molecule described herein or a pharmaceutically acceptable salt thereof, preferably a pharmaceutical composition or pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an ADC molecule of the present invention and a combination of one or more other therapeutic agents.

[0548] The present invention also includes compositions (including pharmaceutical compositions) comprising the ADC molecules of the present invention or pharmaceutically acceptable salts thereof. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0549] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0550] For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0551] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0552] A medicament comprising the ADC described herein can be prepared by mixing the ADC molecule of the invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0553] The pharmaceutical composition or preparation of the present invention may also include more than one active ingredient, which is required for the specific indication being treated, preferably having those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists). The active ingredient is suitably present in combination in an amount effective for the purpose.

[0554] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the ADC molecules, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0555] VII. Pharmaceutical Combinations and Kits

[0556] In some embodiments, the present invention also provides a drug combination or drug combination product comprising an ADC molecule of the present invention and one or more other therapeutic agents (e.g., chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists), etc.).

[0557] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0558] In some embodiments, the kit of parts of the present invention comprises, in the same package:

[0559] - a first container containing a pharmaceutical composition comprising the ADC molecule of the present invention;

[0560] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0561] VIII. Use and Methods

[0562] In one aspect, the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the ADC molecule, pharmaceutical composition, pharmaceutical combination or kit of the present invention.

[0563] In some embodiments, the disease is a B7H3 and / or EGFR-related disease. In some embodiments, the disease is associated with abnormal expression or abnormal activity of B7H3 and / or EGFR.

[0564] In some embodiments, the disease is a tumor, such as cancer.

[0565] In some embodiments, the tumor, e.g., cancer, is B7H3 positive, e.g., it comprises tumor cells that express B7H3. In some embodiments, the patient's tumor comprises tumor cells that express B7H3. In some embodiments, the patient's tumor cells express B7H3, e.g., moderately express B7H3, preferably highly express B7H3. In some embodiments, the tumor (e.g., cancer) patient's tumor tissue has (e.g., elevated levels, e.g., nucleic acid or protein levels or activity of) B7H3, e.g., compared to the level of B7-H3 in the same tissue of a healthy individual or in healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the patient's tumor cells have (e.g., elevated levels, e.g., nucleic acid or protein levels or activity of) B7H3, e.g., compared to the level of B7H3 in the same cell of a healthy individual or in healthy cells adjacent to the patient's tumor cells.

[0566] In some embodiments, the tumor such as cancer is EGFR positive, for example, it comprises the tumor cell expressing EGFR. In some embodiments, the patient's tumor comprises the tumor cell expressing EGFR. In some embodiments, the patient's tumor cell expresses EGFR, for example, medium expression EGFR, preferably high expression EGFR. In some embodiments, there is (for example, elevated levels, for example, nucleic acid or protein levels or activity) EGFR in the tumor tissue of the tumor (for example cancer) patient, for example, compared with the EGFR level in the same tissue of a healthy individual or the healthy tissue adjacent to the patient's tumor tissue. In some embodiments, there is (for example, elevated levels, for example, nucleic acid or protein levels or activity) EGFR in the patient's tumor cell, for example, compared with the EGFR level in the same cell of a healthy individual or the healthy cell adjacent to the patient's tumor cell.

[0567] In some embodiments, the tumor, e.g., cancer, is B7H3 positive and EGFR positive, e.g., it comprises tumor cells expressing B7H3 and EGFR. In some embodiments, the patient's tumor comprises tumor cells expressing B7H3 and EGFR. In some embodiments, the patient's tumor cells express B7H3 and EGFR, e.g., moderate or high expression of B7H3 and EGFR. In some embodiments, the tumor (e.g., cancer) patient's tumor tissue has (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) B7H3 and EGFR, e.g., compared to the levels of B7H3 and EGFR in the same tissue of a healthy individual or in healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the patient's tumor cells have (e.g., elevated levels, e.g., nucleic acid or protein levels or activity) B7H3 and EGFR, e.g., compared to the levels of B7H3 and EGFR in the same cell of a healthy individual or in healthy cells adjacent to the patient's tumor cells.

[0568] In some embodiments, the tumor, such as cancer, includes solid tumors and blood tumors and metastatic lesions. In some embodiments, examples of solid tumors include malignant tumors. Cancer can be in the early, middle or late stages or be metastatic cancer.

[0569] In a specific embodiment, the ADC molecule of the present invention is capable of killing tumor cells and / or inhibiting tumor cell proliferation, such as tumor cells expressing B7H3 and / or EGFR, such as epithelial tumor cells, digestive tract tumor cells, such as gastric cancer cells or pancreatic cancer cells or intestinal cancer cells or colon cancer cells or colorectal cancer cells, head and neck squamous cell carcinoma cells, gastric adenocarcinoma cells, breast cancer cells, oral squamous cell carcinoma cells, prostate cancer cells, melanoma cells, cervical cancer cells, lung cancer (such as small cell lung cancer and non-small cell lung cancer) cells, head and neck cancer cells.

[0570] In some embodiments, the tumor is an immune evasion tumor.

[0571] In some embodiments, the tumor is a cancer, such as an epithelial cancer or a digestive tract cancer, such as selected from gastric cancer, pancreatic cancer, intestinal cancer, colon cancer, colorectal cancer, head and neck squamous cell carcinoma, gastric adenocarcinoma, breast cancer, oral squamous cell carcinoma, prostate cancer, melanoma, cervical cancer, lung cancer (such as small cell lung cancer and non-small cell lung cancer) and head and neck cancer.

[0572] In some embodiments, the tumor is a TKI small molecule resistant tumor.

[0573] In some embodiments, the tumor is a mutant tumor, e.g., having Figure 11 and Figure 12A tumor with the mutation described in the foregoing, such as a tumor with EGFR and / or KRAS mutations, wherein the mutation can be the following mutation EGFR L858R / T790M 、KRAS G12C , EGFR(L858R / T790M / C797S) and / or Exon20ins S768 _ D770dup .

[0574] In some embodiments, the tumor cells of the tumor have one or more of the following characteristics compared to normal cells of the same tissue or normal cells of adjacent normal tissue from the same subject, or compared to normal cells of the same tissue from a healthy subject:

[0575] (i) overexpressing wild-type EGFR (e.g., wild-type EGFR with elevated nucleic acid or protein levels) and / or expressing mutated EGFR compared to normal cells in adjacent tissues or normal cells in the same tissue in healthy subjects, such as EGFR comprising a mutation listed in Passaro A, et al. Nat Cancer. 2021, preferably, the mutated EGFR comprises one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion (Del19), Exon20ins (e.g., S768_D770dup), preferably, the mutated EGFR comprises R521K / Y1069C, R521K, L858R / T790M / C797S, Del19 / T790M / C797S or S768_D770dup;

[0576] (ii) overexpressing wild-type KRAS (e.g., having elevated nucleic acid or protein levels of wild-type KRAS) compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects, or expressing a mutant KRAS, preferably, the mutant KRAS comprises a G12 or G13 mutation, such as G12D or G12C;

[0577] (iii) having elevated nucleic acid or protein levels of B7H3 compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects;

[0578] (iv) The tumor cells are resistant to tyrosine kinase inhibitors, such as first-generation (erlotinib) and third-generation (osimertinib), for example, resistant to osimertinib.

[0579] The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., cancer) or is at risk of suffering from a disease described herein. In certain embodiments, the subject receives or has received other treatments, e.g., chemotherapy and / or radiotherapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0580] In other aspects, the present invention provides the use of the above-mentioned ADC molecules or pharmaceutical compositions or pharmaceutical combinations or kits in the production or preparation of drugs for the purposes described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0581] In other aspects, the present invention provides the above-mentioned ADC molecules or pharmaceutical compositions or pharmaceutical combinations or kits for use in therapy, such as for the uses described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0582] In some embodiments, the ADC molecule or pharmaceutical composition or pharmaceutical combination or kit of the invention delays the onset of a disorder and / or symptoms associated with a disorder.

[0583] In some embodiments, the ADC molecules or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, such as for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0584] In some embodiments, treatment modalities include surgery (eg, tumor resection); radiation therapy, localized or focused radiation, and the like.

[0585] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist).

[0586] Exemplary additional antibodies include antibodies that specifically bind to immune checkpoints.

[0587] In some embodiments, the small molecule drug is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (such as AMG510 (Sotorasib) or GFH925), a KRAS G12D (such as MRTX1133), or a KRAS G12S inhibitor.

[0588] Immunomodulators include immune checkpoint inhibitors and co-stimulatory molecule activators.

[0589] In further embodiments, the ADC molecules of the invention are used in combination with a small molecule inhibitor of KRAS, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133), or a KRAS G12S inhibitor.

[0590] In some embodiments, the ADC molecules of the invention can be administered in combination with a therapy comprising adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR).

[0591] In some embodiments, the ADC molecules of the present invention can be administered in combination with an anti-tumor agent.

[0592] In some embodiments, the ADC molecules of the present invention can be administered in combination with cytokines. Cytokines can be administered as fusion molecules with antibody molecules of the present invention, or as separate compositions. In some embodiments, the ADC molecules of the present invention are administered in combination with one, two, three or more cytokines (e.g., as fusion molecules or as separate compositions).

[0593] In some embodiments, the ADC molecules of the present invention can be combined with conventional cancer therapies in the art, including but not limited to: (i) radiation therapy; (ii) chemotherapy, or the use of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or agents that specifically affect the deregulation of cancer cell proteins; (iv) immunotherapy, or enhancing the host immune response (e.g., vaccines); (v) hormone therapy, or blocking hormones (e.g., when the tumor is hormone-sensitive), (vi) angiogenesis inhibitors, or blocking blood vessel formation and growth, and (vii) palliative care.

[0594] In some embodiments, the ADC molecules of the present invention can be combined with conventional methods to enhance host immune function.

[0595] The various combination therapies described above can be further combined for treatment.

[0596] Such combination therapies encompass combined administration (where two or more therapeutic agents are contained in the same formulation or in separate formulations), and separate administration, in which case administration of the ADC molecules of the invention can occur before, concurrently with, and / or after administration of the other therapy, e.g., treatment modality and / or therapeutic agent. The ADC molecules and / or other therapies, e.g., therapeutic agents or treatment modalities can be administered during active disease or during remission or less active disease. The ADC molecules can be administered before, concurrently with, after, or during remission of the disease.

[0597] Combination therapies of the present invention encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of the ADC molecules of the invention can occur prior to, concurrently with, and / or after administration of the other therapeutic and / or active agents.

[0598] The route of administration of the pharmaceutical composition is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscularly, intraocularly, intraarterially, intraportally or intralesionally; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implant device.

[0599] The composition can also be administered topically via an implant membrane, sponge, or another suitable material onto which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implant device is used, the device can be implanted in any suitable tissue or organ and the desired molecule can be delivered via diffusion, a timed-release bolus, or continuous administration.

[0600] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0601] The following information is related to some of the cell lines used in the experiments:

[0602] NCI-H1975:ATCC,CRL-5908;

[0603] NCI-H358:Cobioer,CBP60136;

[0604] H508:CoBioer,CBP60795;

[0605] SW620:CoBioer,CBP60036;

[0606] HT55:CoBioer,CBP60012;

[0607] BxPC3:CoBioer,CBP60542;

[0608] PE / PJ-41:CoBioer,CBP60606;

[0609] SW601:CoBioer,CBP60507;

[0610] LNCaP:CoBioer,CBP60346;

[0611] PC-9: Shanghai Yubo Biotechnology, YB-H3210;

[0612] NCI-H1703:ATCC,CRL-5889;

[0613] NCI-H2030:ATCC,CRL-5914.

[0614] H322-EGFR20ins(Exon20ins S768 _ D770dup ): Construction and packaging of Lentvirus+EGFR S768 _ D770dup , using Lentvirus+EGFR S768 _ D770dup Virus infection NCI-H322 (Cobioer, CBP60134), through pressure screening, obtained H322-EGFR S768 _ D770dup Stable cell lines.

[0615] MDA-MB-453 OX : Lentvirus+EGFR and Lentvirus+B7-H3(4Ig) were constructed and packaged respectively, MAD-MB-453 (Cobioer, CBP60386) were infected with Lentvirus+EGFR and Lentvirus+B7-H3(4Ig) viruses, and the MDA-MB-453+EGFR+B7H3 stable cell line was obtained through pressure screening, wherein B7H3 is a protein under the UniProt database accession number Q5ZPR3, and EGFR is a protein under the UniProt database accession number P00533-1.

[0616] Example 1 Design, Preparation and Effect Verification of B7-H3 / EGFR bsAb Bispecific Antibody

[0617] Example 1.1B7-H3 / EGFRbsAb Design

[0618] The anti-B7-H3 / EGFR bispecific antibody molecule of the present invention is an IgG1 antibody format assembled from an anti-B7H3 antibody parent and an anti-EGFR antibody parent through the Innobody technology platform of Innovent Biologics (Suzhou) Co., Ltd. (Application No.: PCT / CN2021 / 143141 (WO2022143912A1), Invention Name: Protein Containing Heterodimer Antibody Fc and Preparation Method Thereof). This bispecific antibody format contains four polypeptide chains and can bind to two antigens, antigen A is B7H3 and antigen B is EGFR.

[0619] Among them, the parent antibodies used to construct the bispecific antibody are the anti-EGFR monoclonal antibody Zalutumumab (hereinafter referred to as Zalu, Publication No.: WO02100348A2, Invention Name: Human monoclonal antibodies to Epidermal Growth Factor Receptor (EGFR)) and the anti-B7-H3 monoclonal antibody, which are derived from the Innovent Biological hybridoma technology screening platform and humanized to obtain the antigen binding region sequences of the anti-B7H3 monoclonal antibodies Hz20G5.26, Hz19A2.25 and Hz5C2.9 (Application No.: PCT / CN2021 / 140449, Invention Name: Anti-B7-H3 Antibodies and Their Uses); At the same time, the Innobody technology is used to mutate the Fc region to improve the formation of specific antibody heterodimers (Application No.:

[0620] PCT / CN2021 / 143141, Invention Name: Protein Containing Heterodimeric Antibody Fc and Preparation Method Thereof). Three bispecific antibodies that simultaneously bind to B7H3 and EGFR were screened and obtained, numbered as Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb, and Hz20G5.26 / Zalu bsAb bispecific antibody molecules, respectively.

[0621] The sequence information is as follows:

[0622] Table 1. B7H3 / EGFR bsAb molecular sequence details

[0623]

[0624] Example 1.2 Preparation of B7H3 / EGFR bsAb bispecific antibody

[0625] The heavy chain sequence and light chain sequence of EGFR antibody, and the heavy chain sequence and light chain sequence of B7H3 antibody were inserted into the vector pcDNA3.1 (Invitrogen, V790-20) to obtain the anti-EGFR end heavy chain plasmid, light chain plasmid, and anti-B7H3 end heavy chain plasmid, light chain plasmid, respectively.

[0626] The heavy chain plasmid and light chain plasmid of the EGFR parent antibody and the heavy chain plasmid and light chain plasmid of the B7H3 parent antibody were transiently transfected into ExpiCHO (Invitrogen, A29133) cells, respectively. After 7 days, the cell fermentation broth was harvested, filtered and clarified, and captured using Hitrap Mabselect Sure chromatography columns (GE Healthcare, 11-0034-95) to obtain the EGFR antibody parent and B7H3 antibody parent.

[0627] After the concentration was detected by the A280 method, the antibody parents were mixed in a molar ratio of 1:1, an appropriate amount of reducing agent GSH was added, the reaction was allowed to react overnight at room temperature, the reducing agent was removed by ultrafiltration, and the reaction was terminated. Fine purification was performed using a MonoS cation exchange chromatography column (GE Healthcare, 17-5168-01). The B7-H3 antibody parent solution was 20mM sodium phosphate buffer (pH 6.6), and the EGFR antibody parent solution was 20mM sodium phosphate buffer (pH 6.6) containing 1M sodium chloride. The elution gradient was 0-50% (30 column volumes). The eluted protein solution was ultrafiltered and exchanged with PBS (Gibco, 70011-044), and the purity was tested by SEC-HPLC.

[0628] Example 1.3B7-H3 / EGFRbsAb bispecific antibody screening and mechanism

[0629] Our design of a B7-H3 / EGFR bsAb-ADC aims to enhance the efficacy and safety of bsAb-ADCs. While retaining the enhanced blocking effect of B7-H3 on EGFR downstream signaling, we sought to identify molecules with strong tumor cell internalization effects on B7-H3 / EGFR bsAbs. Through proliferation inhibition experiments that block EGFR signaling, Fab-ZAP endocytosis assays, and affinity KD testing, we ultimately identified the Hz20G5.26 / Zalu bsAb as the preferred bispecific antibody sequence, demonstrating its strong EGFR signaling blockade and tumor cell endocytosis-promoting activity. We also validated the tumor cell endocytosis-promoting activity of the Hz20G5.26 / Zalu bsAb in various tumor cells. Furthermore, we designed in vitro activity experiments to evaluate and explain the mechanistic principles underlying the suitability of the Hz20G5.26 / Zalu bsAb for the preparation of bsAb-ADCs.

[0630] Experimental methods

[0631] 1. Proliferation Inhibition Experiment

[0632] (1) 1000 cells / 100 μl were plated in 96-well white-bottom plates (NUNC, 136101) for 2D cell culture.

[0633] (2) Add the antibody molecules prepared in advance (maximum concentration 10 nM, 4-fold dilution) to the corresponding cell wells, mix well, and culture at 37°C with 5% CO2 for 4 days.

[0634] (3) Proliferation inhibition experiment After culturing for 4 days, the prepared Cell-Titer reagent (Promega, G7572) was added to the corresponding cell wells and kept in the dark at room temperature for 30 minutes before detection using a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0635] 2. Fab-ZAP endocytosis assay

[0636] (1) 1000 cells / 100 μl were plated in a 96-well white-bottom plate (NUNC, 136101) and the cells were cultured overnight.

[0637] (2) The pre-diluted antibody molecules (maximum concentration 10 nM, 4-fold dilution or maximum concentration 12.5 nM, 4-fold dilution) were mixed evenly with Fab-ZAP (ATSBIO, IT-51) at a molar ratio of 1:4 and reacted at room temperature for 0.5 hours.

[0638] (3) Add the Ab-ZAP from (2) above to the corresponding cell wells, mix well, and culture at 37°C with 5% CO2 for 4 days.

[0639] (4) After 4 days of cell culture and killing, the prepared Cell-Titer reagent (Promega, G7572) was added to the corresponding cell wells, kept in the dark at room temperature for 30 minutes, and detected by a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0640] 3.K D Detection

[0641] The affinity (K) of the bispecific antibody of the present invention for binding to B7H3 and EGFR was determined by biofilm thin layer interferometry (BLI). D ).

[0642] Half an hour before the start of the experiment, depending on the number of samples, an appropriate number of AHC sensors (18-5060, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). The antibodies prepared above, human B7H3 (B73-H52E2, Acro Biosystem), and human EGFR (EGR-H5222, Acro Biosystem) were diluted to 100 nM. SD buffer, antibody solution, human B7H3, and human EGFR were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a Fortebio Octet Red96e, with the sensor positions selected based on the sample positions. The instrument parameters were set as follows: Run steps: 120 s baseline equilibration, 100 s addition of immobilized antibody, 120 s baseline equilibration, 100 s antigen binding, and 120 s dissociation. The rotation speed was 1000 rpm and the temperature was 30°C. After the experiment was completed, the KD values ​​were analyzed using ForteBio Octet analysis software.

[0643] 4. Cisbio pERK (Cisbio, 64AERPEG) activity assay

[0644] (1) 25,000 cells / 100 μl were plated in a 96-well white-bottom plate (Nest, 701001) and the cells were cultured for 6 hours.

[0645] (2) Add the pre-diluted antibody molecules (maximum concentration 300 nM, 3.5-fold dilution) to the corresponding cell wells, mix well, and culture at 37°C with 5% CO2 for 16 hours.

[0646] (3) Remove the culture medium, add 50 μl lysis buffer, and shake for 0.5 h.

[0647] (4) Mix the lysate evenly and transfer 16 μl to a HTRF 96-well cell culture plate.

[0648] (5) Add 4 μl of the pre-prepared mixture of Eu Cryptate antibody and d2 antibody to (4) and place it at room temperature in the dark for 4 hours.

[0649] (6) After 4 hours, the cells were detected using a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0650] Experimental results:

[0651] 1. B7H3 / EGFR bsAb sequence screening

[0652] from Figure 1 and Figure 2 The results showed that the endocytosis of Hz20G5.26 / Zalu bsAb and Hz5C2.9 / Zalu bsAb was stronger than that of Hz19A2.25 / Zalu bsAb in different tumor cells. The endocytosis of Hz20G5.26 / Zalu bsAb and Hz5C2.9 / Zalu bsAb was not significantly different. Figure 1 and Figure 2 The results of the proliferation inhibition experiment by blocking EGFR signaling showed that among the three B7-H3 / EGFR bsAb candidate molecules, Hz20G5.26 / Zalu bsAb had a stronger proliferation inhibition effect than Hz5C2.9 / Zalu bsAb and Hz19A2.25 / Zalu bsAb in blocking EGFR signaling. At the same time, the affinity detection data in Table 1 showed that among the three B7-H3 / EGFR bsAb candidate molecules, the Hz20G5.26 antibody parent had the highest affinity for B7H3.

[0653] Therefore, based on the proliferation inhibition and tumor cell endocytosis activity induced by blocking EGFR signaling and the affinity test results, we preferred the Hz20G5.26 / Zalu bsAb sequence as the preferred target sequence for B7H3 / EGFR bsAb-ADC.

[0654] 2. Hz20G5.26 / Zalu bsAb as a B7H3 / EGFR bsAb-ADC mechanism of action

[0655] The primary mechanism of action of ADCs is that tumor cells utilize the robust endocytosis of targeted antibodies to transport the toxin payload into target tumor cells, exerting their anti-tumor effects. We conjugated the Hz20G5.26 / Zalu bsAb to the ZAP toxin (Fab-ZAP human (Ats-Bio, IT-51)). We rapidly validated the potential of the Hz20G5.26 / Zalu bsAb as a B7H3 / EGFR bsAb-ADC in multiple tumor cell lines, following the product datasheet. Figure 3 and Figure 4 The results showed that Hz20G5.26 / Zalu bsAb-ADC had a good anti-tumor killing effect in a variety of tumor cells and was positively correlated with the expression levels of EGFR and B7H3; at the same time, Figure 3 and Figure 4 The results showed that EGFR endocytosis was stronger than B7H3 endocytosis.

[0656] Our goal in designing the Hz20G5.26 / Zalu bsAb-ADC is to improve the efficacy and safety of the B7-H3 / EGFR bsAb-ADC while retaining the enhanced activity of the B7H3 antibody parent in blocking EGFR signaling. Figure 5 The results showed that Hz20G5.26 / Zalu bsAb significantly enhanced the effect of blocking EGFR downstream signaling. Figure 6 The results showed that the endocytosis of Hz20G5.26 / Zalu bsAb was significantly stronger than that of the parental mAb. Figure 3 and Figure 4 As shown, Zalu mAb has stronger endocytosis than Hz20G5.26 mAb. By splicing the high-affinity Hz20G5.26 antibody parent with the low-affinity Zalu antibody parent, we can not only reduce the toxic side effects of EGFR mAb-ADC, but also enhance the efficacy of B7H3 mAb-ADC, thus comprehensively improving the efficacy and safety of B7H3 / Zalu bsAb-ADC.

[0657] Table 1. B7H3 / EGFR bsAb affinity detection

[0658]

[0659] Example 2 Preparation of Hz20G5.26 / Zalu bsAb-Exatecan

[0660] We used glycosylation site-specific conjugation technology (see, for example, PCT / EP2017 / 052792 and

[0661] PCT / CN2022 / 139637), Hz20G5.26 / Zalu bsAb was glycosylated and site-specifically coupled to prepare Hz20G5.26 / Zalu bsAb-Exatecan (DAR=4).

[0662]

[0663] The preparation process is as follows:

[0664]

[0665] The Hz20G5.26 / Zalu bsAb (20.5 mg) prepared as described above was dissolved in 25 mM Tris (pH 8.0) to a final concentration of 10 mg / mL. Endo-SH (enzyme to antibody mass ratio of 0.1% w / w), transglycosylase His-TnGalNAcT (final concentration of 0.25 mg / mL), glycosyl donor 6-N3-GalNAc-UDP (glycosyl donor to antibody molar ratio of 20:1) (see PCT / EP2017 / 052792) were added in sequence, and 1 M MnCl2 solution was added to adjust the final concentration to 10 mM Mn. 2+ The reaction solution was mixed evenly and reacted at 30°C for 20 hours.

[0666] The above reaction product (glycosylated antibody Hz20G5.26 / Zalu bsAb-(GlcNAc(Fuc) 0,1 The antibody (-6-N3-GalNAc) was dissolved in a 50 mM PB / DMF (3:1, v / v) mixture (pH 7.0). SYNtecan E (commercially available from synaffix) in DMF was slowly added at a molar ratio of SYNtecan E to antibody of 4:1 to 6:1. After mixing, the mixture was reacted at 25°C for 20 hours. Protein A affinity chromatography was used to purify the antibody and remove small molecule impurities. The buffer was then replaced with 20 mM histidine (pH 6.5) to obtain the target product, Hz20G5.26 / Zalu bsAb-Exatecan. The measured molecular weight was 150,730, consistent with the theoretical molecular weight.

[0667] The obtained Hz20G5.26 / Zalu bsAb-Exatecan was subjected to RP-HPLC analysis. The results were as follows: Figure 7 As shown, the actual DAR was measured to be 3.28.

[0668] Example 3 In vitro efficacy experiment of Hz20G5.26 / Zalu bsAb-Exatecan

[0669] B7H3 and EGFR are highly expressed in many tumors. We conducted in vitro efficacy validation of Hz20G5.26 / Zalu bsAb-Exatecan in different tumor types and different tumor cells.

[0670] Example 3.1 Detection of EGFR and B7H3 Expression in Tumor Cells

[0671] Cells were resuspended in FACS buffer and plated at 200,000 cells in a 96-well plate (Corning, CLS3799-50EA). 10 μg / ml EGFR antibody (Cetuximab), 10 μg / ml B7-H3 antibody (Hz20G5.26mAb), and 10 μg / ml IgG were added to the plate and incubated at 4°C for 1 hour. The plates were then washed twice with PBS, and the prepared APC-anti-human Fc antibody (Biolegend, 410712) was added to the corresponding plate and incubated at 4°C for 30 minutes. The cells were then washed twice with PBS and analyzed by FACS (BD, Celesta).

[0672] from Figure 8 The experimental results found that B7H3 and EGFR were significantly expressed in different types of tumors and different tumor cells, indicating that Hz20G5.26 / Zalu bsAb-Exatecan can be used to treat a variety of different types of tumors.

[0673] Example 3. In vitro pharmacodynamic activity of 2Hz20G5.26 / Zalu bsAb-Exatecan in various tumor cells

[0674] 2D cell killing assay

[0675] (1) Cells were plated at 1000 cells / 100 μl in the middle 60 wells of a 96-well white-bottom plate (NUNC, 136101) and cultured overnight.

[0676] (2) Add the pre-diluted drug (400 nM, 4-fold dilution) to the corresponding cell well plate, and kill and culture the cells for 6 days.

[0677] (3) Cell killing experiment: The cells were cultured for 6 days. The prepared Cell-Titer reagent (Promega, G7572) was added to the corresponding cell wells and kept in the dark at room temperature for 30 minutes before detection using a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0678] 3D cell killing assay

[0679] The following information is related to some of the cell lines used in the experiments:

[0680] Calu-6: ATCC, HTB-56; JIMT-1: CoBioer, CBP60378; AsPC-1: CoBioer, CBP60546;

[0681] A375:ATCC,CRL-1619TM.

[0682] NCI-H1975 LTC : Construction and packaging of Lentvirus+EGFR L858R / T790M / C797S , use Lentvirus+

[0683] EGFR L858R / T790M / C797S Virus infection NCI-H1975 (ATCC, CRL-5908), through pressure screening, obtained H1975-EGFR L858R / T790M / C797S Stable cell lines.

[0684] (1) Cells were plated at 1500 cells / 100 μL in the middle 60 wells of a 96-well low-adhesion plate (Corning, CLS7007-24EA).

[0685] (2) Add the pre-diluted drug (400 nM, 4-fold dilution) to the corresponding cell well plate, and kill and culture the cells for 6 days.

[0686] (3) After 6 days of continuous culture for cell killing experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the corresponding cell wells and kept at room temperature in the dark for 30 minutes.

[0687] (4) Transfer the Cell-Titer and cell mixture into a 96-well white-bottom plate (NUNC, 136101) and detect using a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0688] Experimental results

[0689] Hz20G5.26 / Zalu bsAb and Hz20G5.26 / Zalu bsAb-Exatecan were used to kill tumor cells in different types of tumors and different tumor cells. Figure 9 The experimental results showed that Hz20G5.26 / Zalu bsAb-Exatecan has both proliferation inhibitory activity by blocking EGFR signaling and ADC-induced target tumor cell death.

[0690] like Figure 9 and Figure 10 Hz20G5.26 / Zalu bsAb-Exatecan not only has anti-tumor activity in different types of lung cancer, but also has anti-tumor activity against EGFR mutations such as H1975 (NSCLC, EGFR L858R / T790M ), EGFR downstream signal mutations such as H358 (NSCLC, EGFR WT ,KRAS G12C) caused by EGFR-TKI small molecule drug-resistant cell lines also have in vitro anti-tumor effects, especially against some rare EGFR mutation tumor cell lines such as H1975-EGFR (L858R / T790M / C797S) and H322-EGFR20ins (Exon20ins S768 _ D770dup ) also has good anti-tumor efficacy in vitro.

[0691] The experimental results showed that Hz20G5.26 / Zalu bsAb-Exatecan can be used not only for B7H3 and EGFR-expressing tumor cell lines, but also for TKI small molecule-resistant tumor cell lines.

[0692] from Figures 9-17 Analysis of in vitro efficacy results showed that Hz20G5.26 / Zalu bsAb-Exatecan can be used not only to treat various types of lung cancer, but also colorectal cancer, pancreatic cancer, breast cancer, oral squamous cell carcinoma, gastric cancer, prostate cancer, melanoma and cervical cancer.

[0693] Hz20G5.26 / Zalu bsAb-Exatecan itself has proliferation inhibitory activity by blocking EGFR signaling and ADC-induced target cell death. In EGFR-insensitive tumor cells, the overall tumor cell killing results only reflect the ADC-mediated cell killing effect; in EGFR-sensitive cells, the overall killing results reflect not only ADC-induced target cell death activity but also proliferation inhibitory activity by blocking EGFR signaling. In both mechanisms of action, ADC-induced target tumor cell death plays a dominant role.

[0694] Example 4 Hz20G5.26 / Zalu bsAb-Exatecan Bystander Effect

[0695] Hz20G5.26 / Zalu bsAb-Exatecan exhibits EGFR signal blocking activity and ADC-induced target cell death activity in EGFR-sensitive tumor cells. To further verify whether Hz20G5.26 / Zalu bsAb-Exatecan has an ADC bystander effect, we constructed a stable transgenic cell line MDA-MB-453 (MDA-MB-453) with dual high expression of EGFR and B7H3 based on the MDA-MB-453 (EGFR and B7H3 dual low expression) tumor cell line. OX .

[0696] MDA-MB-453 and MDA-MB-453 OX The killing experiment method is the same as the 2D cell killing experiment in Example 3.1. Figure 18 The experimental results showed that MDA-MB-453WT was insensitive to Hz20G5.26 / Zalu bsAb and Hz20G5.26 / Zalu bsAb-Exatecan, but sensitive to Exatecan; OX The cells were insensitive to the Hz20G5.26 / Zalu bsAb but sensitive to Hz20G5.26 / Zalu bsAb-Exatecan and Exatecan. We used 10nM Hz20G5.26 / Zalu bsAb-Exatecan, 10nM Hz20G5.26 / Zalu bsAb, and 10nM Exatecan for the bystander effect assay, while 10nM IgG1-Exatecan and IgG1 were used as negative controls.

[0697] Experimental methods for the bystander effect

[0698] 70,000 cells / MDA-MB-453 WT and 90,000 cells / MDA-MB-453 OX Six-well plates were plated and cultured overnight. Pre-diluted drugs or controls were added to the corresponding cell wells, with a final concentration of 10 nM for both drugs and controls. Mix thoroughly and incubate at 37°C with 5% CO2 for 5 days. On day 5, cells were digested and the total cell count was performed. MDA-MB-453 cells were labeled by FACS (BD, Celesta) based on B7H3 expression. OX and MDA-MB-453 WT Percentage relationship.

[0699] Absolute cell number calculation method:

[0700] MDA-MB-453 OX =N*FACS(B7H3 + -MDA-MB-453 OX )*100

[0701] MDA-MB-453=N*FACS(B7H3 - -MDA-MB-453)*100

[0702] Experimental results

[0703] from Figure 19 Experimental results showed that Hz20G5.26 / Zalu bsAb-Exatecan has a strong bystander effect and can further enhance anti-tumor activity.

[0704] Example 5 Hz20G5.26 / Zalu bsAb-Exatecan in vivo efficacy experiment

[0705] In vitro activity verified that Hz20G5.26 / Zalu bsAb-Exatecan has proliferation inhibitory activity by blocking EGFR signaling, ADC-induced target tumor cell death activity, and ADC bystander effect.

[0706] We designed NCI-H508 (CRC), JIMT-1 (BCAR), BxPC3 (PAAD), and NCI-H1975 (NSCLC) CDX mouse tumor models to validate the in vivo pharmacodynamic activity of Hz20G5.26 / Zalu bsAb-Exatecan.

[0707] Example 5.1 In vivo efficacy study of 20G5.26 / Zalu bsAb-Exatecan in the NCI-H1975 (NSCLC) CDX mouse tumor model

[0708] Experimental methods

[0709] NCI-H1975 cells were subcultured and resuspended in equal proportions of PBS and Matrigel Matix. 8E6 / 200ul were inoculated into the right abdomen of CB17 / SCID mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.). On the 7th day, the mice were grouped and administered via the tail vein. The administration cycle was once a week for a total of two doses. Tumor size and body weight were measured twice a week.

[0710] The calculation formula of tumor growth inhibition rate (TGI%) is as follows: TGI% = 100%*(tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - initial tumor volume of control group).

[0711] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L * W 2 / 2

[0712] Experimental results

[0713] On day 36 of NCI-H1975 CDX modeling, the mouse tumor growth inhibition rate was calculated to evaluate the in vivo efficacy of Hz20G5.26 / ZalubsAb-Exatecan.

[0714] like Figure 20The experimental results showed that after administration twice a week, 1 mg / kg and 3 mg / kg Hz20G5.26 / ZalubsAb-Exatecan had significant in vivo anti-tumor efficacy, and the anti-tumor efficacy of 3 mg / kg was better than that of 1 mg / kg.

[0715] On day 36, the tumor growth inhibition rate of mice was calculated. At a dose of 1 mg / kg, the average tumor size was 112 mm. 3 The tumor growth inhibition rate was 104% at a dose of 3 mg / kg, and the average tumor size was 31 mm. 3 , the tumor growth inhibition rate was 109%.

[0716] Analysis of body weight changes in mice in the 1 mg / kg and 3 mg / kg groups showed that Hz20G5.26 / Zalu bsAb-Exatecan was safe and effective.

[0717] Example 5. In vivo efficacy study of 2Hz20G5.26 / Zalu bsAb-Exatecan in the BxPC3 (PAAD) CDX mouse tumor model

[0718] Experimental methods

[0719] BxPC3 cells were subcultured and resuspended in equal proportions of PBS and Matrigel Matix. 3E6 / 200ul were inoculated into the right abdomen of CB17 / SCID mice. The mice were grouped and administered via the tail vein on day 8. The administration was done once. Tumor size and body weight were measured twice a week for the first 46 days and once a week thereafter.

[0720] The calculation formula of tumor growth inhibition rate (TGI%) is as follows: TGI% = 100%*(tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - initial tumor volume of control group).

[0721] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L * W 2 / 2

[0722] Experimental results

[0723] On day 53 of BxPC3 CDX modeling, the mouse tumor growth inhibition rate was calculated to evaluate the in vivo efficacy of Hz20G5.26 / ZalubsAb-Exatecan.

[0724] like Figure 21As shown, after a single dose, Hz20G5.26 / Zalu bsAb showed an anti-tumor effect by blocking EGFR signaling and significantly inhibited the growth of BxPC3 tumors. However, the tumor growth inhibition effect of Hz20G5.26 / Zalu bsAb-Exatecan was far superior to that of Hz20G5.26 / Zalu bsAb at the same dose to block EGFR signaling, indicating that Hz20G5.26 / Zalu bsAb-Exatecan, which has multiple tumor killing mechanisms, has a better anti-tumor effect than Hz20G5.26 / Zalu bsAb.

[0725] The tumor growth inhibition rate of mice was calculated on day 53. At a dose of 1 mg / kg Hz20G5.26 / Zalu bsAb-Exatecan, the average tumor size was 468 mm. 3 , tumor growth inhibition rate was 79% at 3mg / kg Hz20G5.26 / Zalu bsAb dose, and the average tumor size was 1050mm 3 The tumor growth inhibition rate was 44% at a dose of 3 mg / kg Hz20G5.26 / Zalu bsAb-Exatecan, and the average tumor size was 410 mm 3 The tumor growth inhibition rate was 83% at a dose of 10 mg / kg Hz20G5.26 / ZalubsAb-Exatecan, and the average tumor size was 84 mm. 3 , the tumor growth inhibition rate was 103%.

[0726] Analysis of body weight changes in the BxPC3 CDX mouse model showed that high-dose Hz20G5.26 / Zalu bsAb-Exatecan had good safety.

[0727] Example 5. In vivo efficacy study of 3Hz20G5.26 / Zalu bsAb-Exatecan in the H508 (CRC) CDX mouse tumor model

[0728] Experimental methods

[0729] H508 cells were subcultured, resuspended in PBS and Matrigel Matix in equal proportions, and 6E6 / 200ul were inoculated into the right abdomen of NOG mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.). On the 7th day, the cells were grouped and administered by tail vein. The cells were administered twice a week for a total of 8 low-dose administrations. The tumor size and body weight were measured twice a week.

[0730] The calculation formula of tumor growth inhibition rate (TGI%) is as follows: TGI% = 100%*(tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - initial tumor volume of control group).

[0731] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L * W 2 / 2

[0732] Experimental results

[0733] Cetuximab is an EGFR monoclonal antibody used for first-line treatment of colorectal cancer. In the H508 tumor model, we designed a low-dose, multiple-dose approach to compare the efficacy of Hz20G5.26 / Zalu bsAb and Hz20G5.26 / ZalubsAb-Exatecan with cetuximab.

[0734] from Figure 22 The results showed that the anti-tumor activity of low-dose Cetuximab was stronger than that of Hz20G5.26 / Zalu bsAb, but the anti-tumor activity of Hz20G5.26 / Zalu bsAb-Exatecan was far superior to that of Cetuximab.

[0735] The tumor growth inhibition rate of mice was calculated on day 53 of H508 modeling. The average tumor size was 486 mm at a dose of 1 mg / kg Cetuximab. 3 The tumor growth inhibition rate was 64% at a 1mg / kg Hz20G5.26 / Zalu bsAb dose, and the average tumor size was 687mm 3 The tumor growth inhibition rate was 40% at a 1mg / kg Hz20G5.26 / Zalu bsAb-Exatecan dose, and the average tumor size was 11mm. 3 , the tumor growth inhibition rate was 121%, and the tumors were completely eliminated in 3 out of 6 mice.

[0736] Analysis of body weight changes in the mouse model showed that Hz20G5.26 / Zalu bsAb-Exatecan was safe and effective after multiple low-dose administration.

[0737] Example 5.4 In vivo efficacy study of 20G5.26 / Zalu bsAb-Exatecan in the JIMT-1 (BCAR) CDX mouse tumor model

[0738] Experimental methods

[0739] JIMT-1 cells were subcultured and resuspended in PBS and Matrigel Matix in equal proportions. 8E6 / 200ul were inoculated into the right abdomen of CB17 / SCID mice. The mice were grouped and administered via the tail vein on day 8. The administration was once, and the tumor size and body weight were measured twice a week.

[0740] The calculation formula of tumor growth inhibition (TGI%) is as follows: TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-initial tumor volume of control group).

[0741] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L * W 2 / 2

[0742] Experimental results

[0743] from Figure 23 The results showed that Hz20G5.26 / Zalu bsAb-Exatecan had anti-tumor activity in the JIMT-1CDX mouse tumor model at different doses.

[0744] On day 28, the tumor growth inhibition rate of mice was calculated. At a dose of 1 mg / kg, the average tumor size was 583 mm. 3 The tumor growth inhibition rate was 59% at a dose of 3 mg / kg, and the average tumor size was 31 mm. 3 The tumor growth inhibition rate was 107% at a dose of 10 mg / kg, and the average tumor size was 21 mm. 3 , the tumor growth inhibition rate was 108%, and the tumor was completely eliminated in one of the five mice.

[0745] Analysis of body weight changes in mouse models showed that high-dose Hz20G5.26 / Zalu bsAb-Exatecan is safe.

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

Claims

1. An antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt or solvate thereof: Ab-(L-(D) r ) p (I), in: Ab is a bispecific antibody or a fragment thereof that specifically binds to B7H3 and EGFR (e.g., human B7H3 and human EGFR); L is a linker; D is a drug, preferably an anti-tumor compound; and p is an integer selected from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r is an integer selected from 1-5, and is preferably 1 or 2.

2. The antibody-drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, Wherein Ab in formula (I) is a bispecific antibody that specifically binds to B7H3 and EGFR, which comprises a first antigen binding region that specifically binds to EGFR and a second antigen binding region that specifically binds to B7H3, wherein the second antigen binding region that specifically binds to B7H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein (i) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20; or, (ii) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:21, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:22, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:23, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:24, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:25, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:26; or, (iii) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:30, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:31, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

32.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 2, wherein the second antigen-binding region that specifically binds to B7H3 comprises VH and VL, wherein (i) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:3, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4; (ii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5, and the VL comprises, or consists of the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6; or (iii) the VH comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:8, or consists of said amino acid sequence.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, wherein the second antigen-binding region that specifically binds to B7H3 comprises VH and VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below: SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; or SEQ ID NO:7 and SEQ ID NO:

8.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4, wherein the first antigen binding region that specifically binds to EGFR comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:9; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:10; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:11; and the LCDR1 of the first antigen binding region comprises or consists of the amino acid sequence of SEQ ID NO:12; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:13; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

14.

6. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 5, wherein the first antigen-binding region that specifically binds to EGFR comprises VH and VL, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1, and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:

2.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 6, wherein the first antigen-binding region that specifically binds to EGFR comprises VH and VL, wherein VH and VL comprise or consist of the amino acid sequences shown below, respectively: SEQ ID NO: 1 and SEQ ID NO:

2.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 7, wherein the bispecific antibody comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

9. The antibody-drug conjugate of claim 8 or a pharmaceutically acceptable salt or solvate thereof, wherein the first Fc region and the second Fc region are respectively human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprising or consisting of an amino acid sequence of SEQ ID NO: 46 or 47 or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 8 or 9, wherein mutations that promote heterodimerization of the first Fc region and the second Fc region of the bispecific antibody are introduced into the first Fc region and the second Fc region.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 10, wherein the mutation in the Fc region is introduced based on the Innobody technology.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of claim 11, wherein the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 of the other Fc region comprises Y349T, K370S and K409D mutations.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 11, wherein a) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or 50, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 52 or 53; b) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:49 or 50, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:52 or 53.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 10, wherein the mutation in the Fc region is introduced based on the Knob-into-Hole technology, wherein corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region, respectively.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 8 to 14, wherein the first antigen-binding region of the bispecific antibody is a Fab fragment, and / or the second antigen-binding region is a Fab.

16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 15, wherein the Fab as the first antigen-binding region or the second antigen-binding region comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 16, wherein the CH1 (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:41; or (ii) comprises the amino acid sequence of SEQ ID NO: 41 or consists of the amino acid sequence.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 17, wherein the Fab as the first antigen-binding region or the second antigen-binding region comprises a light chain constant region, wherein the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region.

19. The antibody-drug conjugate of claim 18 or a pharmaceutically acceptable salt or solvate thereof, wherein the Kappa light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:54; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:

54.

20. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof of any one of claims 15 to 19, wherein the first antigen-binding region that specifically binds to EGFR comprises a first Fab, and the second antigen-binding region that specifically binds to B7-H3 comprises a second Fab, wherein the first Fab is connected at the C-terminus of its CH1 to the N-terminus of the first Fc region (via or without a linker, such as a hinge region), and the second Fab is connected at the C-terminus of its CH1 to the N-terminus of the second Fc region (via or without a linker, such as a hinge region).

21. The antibody-drug conjugate of claim 20 or a pharmaceutically acceptable salt or solvate thereof, wherein the bispecific antibody comprises a first Fab as a first antigen-binding region that specifically binds to EGFR and a second Fab as a second antigen-binding region that specifically binds to B7H3, wherein the bispecific antibody comprises or consists of: Heavy chain 1: From N-terminus to C-terminus, it comprises or consists of: A heavy chain variable region of a Fab that specifically binds to EGFR, a heavy chain constant region CH1, and a first Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the first Fc region via or without a linker (e.g., a hinge region); Light chain 1: from N-terminus to C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to EGFR - a light chain constant region; Heavy chain 2: comprising or consisting of the following from N-terminus to C-terminus: a heavy chain variable region of a Fab that specifically binds to B7-H3 - a heavy chain constant region CH1 - a second Fc region, wherein the heavy chain constant region CH1 is connected at its C-terminus to the N-terminus of the second Fc region via or without a linker (e.g., a hinge region); Light chain 2: from N-terminus to C-terminus, it comprises or consists of: a light chain variable region of a Fab that specifically binds to B7H3 - a light chain constant region, Preferably, each domain is directly linked; Optionally, the first Fc region comprises mutations K370S, Y349T and K409D, and the second Fc region comprises S364R and D399K.

22. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 21, wherein Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:33, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:33, and light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:34; and heavy chain 2 and light chain 2 each comprise, or comprise, or consist of, the amino acid sequence of SEQ ID NO: i) SEQ ID NO:35 and SEQ ID NO:36; ii) SEQ ID NO:37 and SEQ ID NO:38; iii) SEQ ID NO:39 and SEQ ID NO:

40.

23. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 22, wherein the linker is MC-VC-PAB, vc-PAB, SMCC or MC-GGFG.

24. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 23, wherein the linker is a linker capable of achieving site-specific coupling, preferably a linker linked to an antibody oligosaccharide, preferably, the oligosaccharide linker is a linker obtained by reacting with a reactive group (e.g., an azide group) on an N-sugar chain at a conserved N-glycosylation site (e.g., Asn297) on an antibody Fc domain for specific site coupling.

25. The antibody-drug conjugate of any one of claims 1 to 24, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-tumor compound is a cytotoxic agent or a chemotherapeutic agent, such as a camptothecin compound, an auristatin compound, a maytansine compound, a taxane, an anthracycline, a vinca alkaloid, a MEK inhibitor, or a KSP inhibitor.

26. The antibody-drug conjugate of claim 25 or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-tumor compound is selected from Exatecan, DXd, MMAE or DM1, paclitaxel, docetaxel, epothilone, mitomycin, combretastatin, calicheamicin, duocarmycin, tubulysin, amatoxin and bleomycin.

27. An antibody-drug conjugate represented by formula (III) or a pharmaceutically acceptable salt or solvate thereof in Ab is as defined in any one of claims 1 to 22, L1 is a linker; E is a sugar or a sugar derivative, preferably selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid); And GlcNAc is N-acetylglucosamine, Fuc is fucose; D and r are as defined in formula I according to any one of claims 1 to 26; b is 0 or 1; x is 1, 2, 3, or 4; y is an integer selected from 1-10.

28. The antibody-drug conjugate according to claim 27 or a pharmaceutically acceptable salt or solvate thereof, where x is 1 or 2; and y is 1, 2, 3, or 4; Preferably x is 1 and y is 2.

29. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 27, wherein E is GalNAc.

30. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 27 to 29, wherein E is 6-deoxy-2-acetamidogalactose and is linked to L1 via the C atom at position C6.

31. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 27 to 29, wherein the GlcNAc linked to Ab is present at the Asn297-glycosylation site of both heavy chains of the antibody.

32. The antibody-drug conjugate of any one of claims 27 to 31 or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is the following structure Q is -NHC(O)CH2- or -CH2-; Z is R1 is independently selected from hydrogen, halogen, -OR2, -NO2, -CN, -S(O)2R2, C1-C 12 Alkyl, C6-C 20 Aryl, 5-20 membered heteroaryl, C1-C 12 Alkyl C6-C 20 Aryl, C1-C 12 Alkyl 5-20 membered heteroaryl, C6-C 20 Aryl C1-C 12 Alkyl and 5-20 membered heteroaryl C1-C 12 wherein the alkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, arylalkyl and heteroarylalkyl are optionally substituted, wherein two substituents R1 may be linked together to form a fused, bridged or spiro C3-C 18 cycloalkyl or fused or spiro aromatic or heteroaromatic substituents, and wherein R2 is independently selected from hydrogen, C1-C 24 Alkyl, C6-C 20 Aryl, 5-20 membered heteroaryl, C1-C 12 Alkyl C6-C 20 Aryl, C1-C 12 Alkyl 5-20 membered heteroaryl, C6-C 20 Aryl C1-C 12 Alkyl and 5-20 membered heteroaryl C1-C 12 alkyl; R3 and R4 are each independently selected from hydrogen, halogen, C1-C 24 Alkyl, C6-C 20 Aryl, 5-20 membered heteroaryl, C1-C 12 Alkyl C6-C 20 Aryl, C1-C 12 Alkyl 5-20 membered heteroaryl, C6-C 20 Aryl C1-C 12 Alkyl and 5-20 membered heteroaryl C1-C 12 alkyl; Y is -O-, -S-, -NR2- or absent; X1 is -O-, -S- or -NR2-; G1 is -C(O)-, or direct key; G2 is R7 are each independently selected from hydrogen and C1-C 12 alkyl; R8 is hydrogen, C1-C 12 Alkyl or R9 is L2 and L3 are each independently C1-C 12 Alkylene, wherein one or more (e.g. 1, 2, 3 or 4) carbon atoms are optionally replaced by heteroatoms selected from O, N and S, provided that said O, N and / or S are not directly connected to each other; L4 is each independently a cleavable spacer; n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n2 is each independently 0, 1 or 2; n3 is each independently 0, 1, 2, 3, 4 or 5; c is 0, 1, 2, 3, or 4; m is 0 or 1.

33. The antibody-drug conjugate of claim 32, or a pharmaceutically acceptable salt or solvate thereof, wherein Z is 34. The antibody-drug conjugate of claim 33 or a pharmaceutically acceptable salt or solvate thereof, wherein L4 is each independently wherein R5 and R6 are each independently selected from hydrogen, C1-C 12 Alkyl or -(CH2)3NHC(O)NH2; and Ar is selected from arylene; or L4 is each independently -CO-L5-L6-, wherein L5 is a peptide residue consisting of 2, 3, 4 or 5 amino acids selected from the group consisting of glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine and proline; L6 is selected from the group consisting of absent, -NH-CH2-, Where R a1 Each independently selected from C 1-6 Alkyl-, C 1-6 Alkoxy-, C 1-6 haloalkyl-, halogen, nitro and cyano; and t is 0, 1, 2, 3, or 4.

35. The antibody-drug conjugate of claim 34 or a pharmaceutically acceptable salt or solvate thereof, Where L4 is -CO-L5-L6-, L5 is selected from -Val-Ala-, -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, -Ala-Ala-, -Ala-Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val-, -Cit-Ala-, -Cit-Asn-, -Cit-Asp- , -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, -Ile-Val-, -Leu-Cit-, -L ys-Cit-, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val-Asp-, -Cit-Val-, -Val-G lu-, -Val-Lys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, -Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit- Ala-Glu-, -Val-Lys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-Gly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Lys-Gl y-, -Val-Lys-Leu-, -Ala-Ala-Ala-, -Asn-Ala-Ala-, -Gly-Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -Gly -Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly- and -Val-Lys-Gly-Gly; and L6 is selected from -NH-CH2-, 36. The antibody-drug conjugate according to any one of claims 32 to 35, or a pharmaceutically acceptable salt or solvate thereof, Where L2 is n4 is 0, 1, 2 or 3; or C1-C 12 Alkylene, preferably L3 n4 is 0, 1, 2 or 3; or C1-C 12 Alkylene, preferably 37. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 32 to 36, wherein L4 is independently 38. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 32 to 37, wherein -L1- is the following structure Among them, Q, R 1、 R 2、 R3, R4, c, m, n1, Y, L2, L3 and L4 are as defined in any one of claims 32-37.

39. The antibody-drug conjugate of claim 32 or a pharmaceutically acceptable salt or solvate thereof, wherein -L1- is a structure 40. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 39, wherein D is a structure represented by formula (D-1a), formula (D-1b), formula (D-1c) or formula (D-1d): Each R 10 independently selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 12 and-SR 12 ; R 11 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 12 ; or R 10 and R 11 Together they form -O(CH2) n5 O- or -O(CF2) n5 O-, where n5 is 1 or 2; Each R 12 Independently selected from H or C1-C4 alkyl; Each R 13 independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl; Each Q1 is independently -O- or -S-; L a1 Yes - (C1-C 10 Alkylene)C(O)-; Each L b1 Independently is -(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-C(O)N(R 14 )-or-(C1-C 10 Alkylene)-N(R 14 )C(O)-, where R 14 is H or C1-C6 alkyl; Preferably, D is a structure represented by formula (D-2a), (D-2b), (D-2c) or (D-2d): R 10 is C1-C6 alkyl or C1-C4 alkoxy, preferably methyl or methoxy, and R 11 is halogen, preferably F; or R 10 and R 11 Together they form -OCH2O-; R 13 is H; Each Q1 is independently -O- or -S-; L a1 is -(C1-C4alkylene)C(O)-; and L b1 is -(C1-C6 alkylene)-; More preferably, D is a structure represented by formula (D-3a):

41. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 40, having an average DAR of 1-10, such as 3-5.

42. The antibody-drug conjugate of claim 27 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is selected from: The Ab is a bispecific antibody that specifically binds to EGFR and B7H3, comprising or consisting of heavy chain 1, light chain 1, heavy chain 2 and light chain 2: wherein the heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33, and the light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34, Heavy chain 2 comprises the amino acid sequence set forth in SEQ ID NO:35, and light chain 2 comprises the amino acid sequence set forth in SEQ ID NO:36; wherein y is 1 or 2, preferably 2; Preferably, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof has an average DAR of 2-6 or 3-4.

43. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 42 or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optional pharmaceutical excipients.

44. A drug combination comprising the antibody-drug conjugate of any one of claims 1 to 42, or a pharmaceutically acceptable salt or solvate thereof, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.

45. A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the antibody-drug conjugate of any one of claims 1 to 42 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 43, or the pharmaceutical combination of claim 44.

46. ​​The method of claim 45, wherein the tumor is a cancer, preferably, the cancer has an elevated level (e.g., nucleic acid or protein level) of B7H3 and / or EGFR, preferably, the subject's tumor cells moderately express or highly express B7H3 and / or EGFR, and the cancer is, for example, an epithelial cancer or a digestive tract cancer, such as gastric cancer, pancreatic cancer, intestinal cancer, colon cancer, colorectal cancer, head and neck squamous cell carcinoma, gastric adenocarcinoma, breast cancer, oral squamous cell carcinoma, prostate cancer, melanoma, cervical cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), esophageal cancer, kidney cancer, bladder cancer, ovarian cancer, and head and neck cancer.

47. The method of any one of claims 45-46, wherein the method further comprises administering to the patient one or more therapies, such as treatment modalities and / or other therapeutic agents, preferably, the treatment modalities comprise radiation therapy or surgery, or the therapeutic agents comprise chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

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