Bispecific antibody, antibody-drug conjugate, and application thereof

Abstract

The present disclosure relates to a bispecific antibody targeting B7-H3 and EGFR, an antibody-drug conjugate (ADC) based on the bispecific antibody, a composition containing the bispecific antibody and the ADC, and a pharmaceutical use thereof.

Description

Bispecific antibodies and drug conjugates and their uses Technical Field

[0001] This invention relates to antibodies and antibody-drug conjugates, and more particularly to bispecific antibodies targeting B7-H3 and EGFR, drug conjugates (ADCs) based on said bispecific antibodies, compositions containing said bispecific antibodies and ADCs, and their pharmaceutical uses. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a class of promising innovative targeted therapies. These drugs combine the targeted advantages of monoclonal antibodies with the strong cytotoxic activity of small molecule drugs. Typically, ADCs bind specifically to antigens, are then internalized by cells, and release the small molecule drug under the action of intracellular enzymes to achieve the therapeutic goal (Nasiri, H., et al., Antibody-drug conjugates: promising and efficient tools for targeted cancer therapy. J Cell Physiol. 2018 Sep; 233(9):6441-6457.). Bispecific antibody ADCs are a recently proposed class of ADC drugs using bispecific antibodies / biepitaxy antibodies, and some are currently in clinical development (US20230183358A1, US20230405141A1, US20230416376A1, WO2023083381A1). Compared to traditional ADCs, bispecific ADCs can enhance tumor targeting and reduce the possibility of off-target effects. At the same time, by targeting two different signaling pathways, they can enhance cell killing toxicity and reduce drug resistance (Comer, F., C. Gao, and S. Coats, Bispecific and Biparatopic Antibody Drug M. Damelin, Editor. 2018, Springer International Publishing: Cham. pp. 267-280.).

[0003] Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor with a molecular weight of approximately 170 kDa. It is a transmembrane glycoprotein that spans the cell membrane and is activated by binding to a ligand. It plays an important role in regulating cell proliferation, survival, differentiation, and migration. In various solid tumors, such as lung cancer, breast cancer, kidney cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, and bladder cancer, there is overexpression and / or abnormal mutation activation of EGFR (Rocha-Lima, CM, et al., EGFR targeting of solid tumors. Cancer control: Journal of the Moffitt Cancer Center, 2007.14(3):p.295-304.). EGFR is the most common oncogenic driver gene in non-small cell lung cancer (NSCLC). In Asian NSCLC patients, about 40% of cases are caused by EGFR mutations, and in Caucasian populations, about 15% are caused by EGFR mutations (Arteaga, C.L. and J.A. Engelman, ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics. Cancer Cell, 2014, 25(3): p.282-303.). EGFR is an important tumor target, and there are currently EGFR-TKI small molecule inhibitors (such as afatinib, gefitinib, erlotinib, ametinib, dacomitinib, and osimertinib), monoclonal antibody macromolecules (cetuximab, nimotuzumab, nexituzumab, and panitumumab), and bispecific antibody macromolecules (ervantumab) targeted therapy methods (Seshacharyulu, P., et al., Targeting the EGFR signaling pathway in cancer therapy. Expert Opinion on Therapeutic Targets, 2012.16(1):p.15-31.). However, when TKI small molecule inhibitors or antibody macromolecules are used alone, the treatment effect is often unsatisfactory due to their poor cytotoxicity and tumor penetration.Meanwhile, drug resistance caused by mutations in the target gene and dose adjustments or treatment interruptions due to the expression of EGFR in normal human tissues mean that there is still a high demand in the market for new treatment options targeting this target (Chong, C.R. and PA.Janne, The quest to overcome resistance to EGFR-targeted therapies in cancer. Nature Medicine, 2013, 19(11): p.1389-1400.).

[0004] B7-H3, also known as CD276, is a member of the B7 family of immunomodulatory proteins. B7-H3 is not expressed or is expressed at low levels in normal tissue cells, but it is overexpressed in many solid tumor cells, including prostate cancer, renal cell carcinoma, melanoma, head and neck squamous cell carcinoma, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is closely related to tumor progression, patient survival, and prognosis, and is considered a novel tumor marker and potential therapeutic target (Picarda E. Molecular pathways: targeting B7-H3 (CD276) for human cancer immunotherapy. Clin Cancer Res. 2016; 22:3425-31; Kontos F et al., B7-H3: an attractive target for antibody-based immunotherapy. Clin Cancer Res. 2021 March 01; 27(5):1227–1235). There is evidence that B7-H3 expression may be related to EGFR gene expression status. For example, a search of the Depmap database revealed that EGFR and B7-H3 are co-expressed in approximately 93% of non-small cell lung cancer (NSCLC) cell lines. Currently, there are monoclonal antibodies and ADCs targeting B7-H3 being researched in tumor-related fields, but no approved therapeutic drugs targeting B7-H3 have yet been marketed.

[0005] Despite the rapid development of ADC drugs in recent years, challenges remain in their development and clinical implementation. For example, there is a need to reduce non-specific killing, improve safety, and overcome drug resistance (Junutula, J.R. and HP. Gerber, Next-Generation Antibody-Drug Conjugates (ADCs) for Cancer Therapy. ACS Med Chem Lett, 2016.7(11):p.972-973.). The field still needs to develop new antibody molecules suitable for ADC drug application and ADC-based drugs to suit different tumor treatment scenarios, improve anti-tumor therapeutic effects, reduce toxicity, and expand the therapeutic window.

[0006] Invention Overview

[0007] To address the aforementioned issues, the inventors proposed and designed a bispecific antibody molecule and its drug conjugate (ADC) that simultaneously targets EGFR and B7-H3. Based on the unique properties of the antibodies of this invention, the ADC drugs of this invention not only exhibit significant anti-tumor effects but also have low toxicity and side effects, increasing the maximum tolerated dose (MTD) and demonstrating good safety. Furthermore, the dual-target application is also beneficial in overcoming potential tumor drug resistance in future clinical applications.

[0008] Therefore, in a first aspect, this disclosure provides a multispecific antibody that specifically binds to B7-H3 and EGFR, as well as pharmaceutical compositions and uses thereof. In some embodiments, the multispecific antibody of the present invention comprises a first antigen-binding domain that binds to B7-H3 and a second antigen-binding domain that binds to EGFR. In some embodiments, preferably, the multispecific antibody of the present invention is a bispecific antibody.

[0009] In a second aspect, this disclosure provides an anti-B7-H3 antibody, a pharmaceutical composition thereof, and its use.

[0010] In a third aspect, this disclosure provides a polynucleotide encoding the antibody of the present invention, a vector comprising the same and a host cell, as well as a method for producing the antibody of the present invention.

[0011] In a fourth aspect, this disclosure provides an immunofusion compound and immunoconjugate comprising the antibody of the present invention, pharmaceutical compositions thereof, and uses thereof.

[0012] In a fifth aspect, this disclosure provides an antibody-drug conjugate (ADC) comprising the antibody of the present invention, pharmaceutical compositions thereof, and uses thereof. In some embodiments, the ADC of the present invention comprises an antitumor compound conjugated to the antibody of the present invention, selected from, for example, but not limited to, cytotoxic drugs and cell-inhibiting drugs, particularly topoisomerase I inhibitors. In some embodiments, the topoisomerase I inhibitor is a camptothecin compound. In some embodiments, the topoisomerase I inhibitor is eczemac or a derivative thereof.

[0013] In a sixth aspect, this disclosure provides the use of the antibodies or immunoconjugates or fusions of the present invention, as well as the ADCs of the present invention and their pharmaceutically acceptable salts or solvates, as pharmaceuticals or for the preparation of pharmaceuticals. In some embodiments, the pharmaceuticals are used to treat and prevent cancer. Accordingly, the present invention also provides a method for treating and preventing cancer, comprising administering to a subject in need the antibodies or immunoconjugates or fusions or ADCs of the present invention or their pharmaceutically acceptable salts or solvates.

[0014] In some embodiments, the antibody-drug conjugates of the present invention have one or more of the following advantages:

[0015] (1) Effectively binds to and kills target tumor cells that simultaneously express human EGFR and B7-H3;

[0016] (2) Low non-specific killing activity against non-target cells;

[0017] (3) Obvious bystander effect;

[0018] (4) Minimal toxic side effects;

[0019] (5) Higher maximum tolerated dose (MTD); and

[0020] (6) In vivo circulation stability.

[0021] In other embodiments, the antibody-drug conjugates of the present invention also have one or more of the following advantages:

[0022] (7) Good physicochemical stability;

[0023] (8) Good product uniformity; and

[0024] (9) Good drug-like properties.

[0025] The invention is further illustrated in the following figures and specific embodiments. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily apparent to those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims. Attached Figure Description

[0026] Figure 1: Schematic diagram of the structure of the anti-EGFR / B7-H3 bispecific antibody of the present invention, wherein the anti-B7-H3 half antibody with Hole mutation in the Fc region of immunoglobulin and the anti-EGFR half antibody with Knob mutation in the Fc region of immunoglobulin form a bivalent bispecific antibody through heterodimerization of the two Fc regions.

[0027] Figure 2: Schematic diagram of the structure of the bispecific antibody ADC of the present invention, wherein the antibody Ab unit is connected to the linker-toxin portion via thioether bonds through free thiol groups on the light chain and heavy chain.

[0028] Figure 3: FACS detection of the binding of hybridoma antibody to MDA-MB-231 cells.

[0029] Figure 4: Binding of humanized antibody to B7-H3.

[0030] Figure 5: Binding of humanized antibody to EGFR.

[0031] Figure 6A: SEC-HPLC determination of B035 purity.

[0032] Figure 6B: SEC-HPLC determination of B036 purity.

[0033] Figure 7A: RP-HPLC detection of B035 purity.

[0034] Figure 7B: RP-HPLC detection of B036 purity.

[0035] Figure 8A: The bispecific antibody maintains its affinity for the antigen EGFR.

[0036] Figure 8B: The bispecific antibody maintained its affinity for antigen B7-H3.

[0037] Figure 9A: Internalization of bispecific antibody B036 and parental antibody in H1975 cells.

[0038] Figure 9B: Internalization of bispecific antibody B036 and parental antibody in FaDu cells.

[0039] Figure 9C: Internalization of bispecific antibody B035 and parental antibody in NCI-H226 cells.

[0040] Figure 9D: Internalization of bispecific antibody B035 and parental antibody in NCI-H1650 cells.

[0041] Figure 10: SEC-HPLC determination of the purity of B035-LY22CD.

[0042] Figure 11: RP-HPLC detection of DAR value of B035-LY22CD.

[0043] Figure 12A: Cell viability diagram corresponding to different concentrations of antibody drugs in the FaDu cell model.

[0044] Figure 12B: Cell viability diagram corresponding to different concentrations of antibody drugs in the NCI-H226 cell model.

[0045] Figure 13A: Curve of average tumor volume versus days in the FaDu xenograft model.

[0046] Figure 13B: Curve of relative body weight change versus days in the FaDu xenograft model.

[0047] Figure 14A: Curve of mean tumor volume versus days in the NCI-H1650 xenograft model.

[0048] Figure 14B: Curve of relative body weight change versus days in the NCI-H1650 xenograft model.

[0049] Figure 15A: Curve of mean tumor volume versus days in the NCI-H1975 xenograft model.

[0050] Figure 15B: Curve of relative body weight change versus days in the NCI-H1975 xenograft model.

[0051] Figure 16: Relative tumor volume changes in efficacy ranking of B035-LY22CD3 in different PDX models.

[0052] Figure 17: Relative tumor volume changes in efficacy ranking of B035-LY22CD3 in different PDX models.

[0053] Figure 18A: Blood concentration-time curve of B035-LY22CD3 after administration at 15 mpk in cynomolgus monkeys.

[0054] Figure 18B: Blood concentration-time curve of B036-LY22CD after administration at 15 mpk in cynomolgus monkeys.

[0055] Figure 19: Body weight change curve of B035-LY22CD3 after administration in cynomolgus monkey pretoxicology test.

[0056] Invention Details

[0057] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0058] definition

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0060] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0061] The term “and / or” used to connect two or more options should be understood to mean any one of the options or any combination of two or more of the options.

[0062] As used herein, the terms “comprising” or “including” mean to include the stated 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 specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0063] In this document, the term "B7-H3" refers to B7 homolog 3 (also known as CD276), a member of the B7 family of immunomodulatory proteins. The B7-H3 protein is a transmembrane glycoprotein composed of an extracellular domain, a transmembrane domain, and a short intracellular domain. Human B7-H3 exists in two structurally similar isoforms: 4IgB7-H3, which contains an extracellular region with two identical pairs of IgV-like and IgC-like domains (IgV-IgC-IgV-IgC); and 2IgB7-H3, which contains an extracellular region with a single pair of IgV-like and IgC-like domains (IgV-IgC). In this document, unless otherwise stated, the term "B7-H3" encompasses any isoform of human B7-H3 (including 4Ig and 2Ig), and its variants, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification and conformational variants. An example of B7-H3 is the human B7-H3 protein containing the amino acid sequence UniProtKB-Q5ZPR3. In this document, unless explicitly specified as originating from a non-human species, such as “mouse B7-H3,” “monkey B7-H3,” etc., the term “B7-H3” refers to human B7-H3. Accordingly, in this document, unless otherwise specified, the terms “antigen-binding specificity against B7-H3” and “antigen-binding domain specifically binding to B7-H3” refer to the binding specificity and antigen-binding domain against human B7-H3.

[0064] In this document, the term "B7-H3 positive cell" refers to a cell that is positive for B7-H3 expression on its cell surface. The B7-H3 expression level on the cell surface can be determined by any conventional method known in the art for determining cell surface antigen expression levels, such as FACS detection or immunofluorescence staining. B7-H3 has significantly higher expression levels on a variety of tumor cells than on normal tissues / cells. Preferably, in this document, B7-H3 positive cells are B7-H3 positive tumor cells.

[0065] In this document, the term "EGFR" refers to the epidermal growth factor receptor. Unless otherwise stated, this term encompasses any variant of human EGFR, including sequence variants, particularly naturally occurring variants, allelic variants, and post-translational modification and conformational variants. An example of EGFR is the human EGFR protein containing the amino acid sequence UniProtKB-P00533. Unless explicitly specified as originating from a non-human species, the expression "EGFR" in this document refers to human EGFR. Accordingly, unless otherwise specified, the terms "antigen-binding specificity against EGFR" and "antigen-binding domain specifically binding to EGFR" refer to the binding specificity and antigen-binding domain against human EGFR.

[0066] In this document, the term "EGFR-positive" cell refers to a cell that expresses EGFR on its cell surface. The EGFR expression level on the cell surface can be determined by any conventional method known in the art for determining cell surface antigen expression levels, such as FACS detection or immunofluorescence staining. EGFR is expressed at higher levels on a variety of tumor cells than on normal tissues / cells. Preferably, in this document, EGFR-positive cells are EGFR-positive tumor cells.

[0067] In this document, the terms "first" and "second," when used in conjunction with elements such as Fc regions or antigen-binding domains, are intended to conveniently distinguish two elements belonging to the same category. However, it should be noted that, unless explicitly stated otherwise, the use of these terms is not intended to assign a particular order, orientation, or position to the elements.

[0068] In this document, the term "antigen-binding molecule" refers to a molecule, such as a protein or polypeptide or a molecule derived therefrom, that contains an antigen-binding domain or antigen-binding site capable of binding to a target antigen. In this invention, when the target antigen is B7-H3, an antigen-binding molecule binding B7-H3 is also referred to as a B7-H3-binding molecule; correspondingly, a binding molecule binding dual target antigens B7-H3 and EGFR is also referred to as a B7-H3 / EGFR-binding molecule. Antigen-binding molecules include, for example, antibodies and their antigen-binding fragments, as well as various fusions and conjugates constructed based on antibodies or antigen-binding fragments, such as immunoconjugates, immunofusions, antibody-drug conjugates (ADCs), multi / bispecific antibodies, chimeric antigen receptors (CARs), etc. As will be apparent to those skilled in the art, the antigen-binding site of an antibody typically contains amino acid residues from a "complementarity-determining region" or "CDR". This disclosure covers various antigen-binding molecules containing a B7-H3 antigen-binding site according to this disclosure.

[0069] In this document, the term "antibody" refers to a polypeptide containing one or more domains that bind to epitopes on antigens of interest, wherein the binding domain has a variable region sequence derived from or sequence-identical to that of an immunoglobulin. This term encompasses a wide range of antibody structures, including but not limited to single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human sequence antibodies, full-length antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity. Typically, antibodies form antigen-binding domains on the surface of a VH-VL dimer via three complementarity-determining regions (HCDR1-3) in their heavy chain variable region (VH) and three complementarity-determining regions (LCDR1-3) in their light chain variable region (VL). The six CDRs confer specific binding of the antibody to the antigen. However, in the case of heavy chain antibodies, such as those derived from camelid heavy chain antibodies, the antibody may confer specific binding of the antibody to the antigen via the three complementarity-determining regions (CDR1-3) in a single VH domain (also referred to herein as the VHH domain). In addition to variable regions, antibodies may, in some cases, also contain one or more immunoglobulin constant regions or constant domains. An antibody chain containing the immunoglobulin heavy chain constant region is also referred to herein as a "heavy chain"; correspondingly, an antibody chain containing the immunoglobulin light chain constant region is also referred to herein as a "light chain". It should be understood that, in this disclosure, unless explicitly contrary to context, the term "antibody" encompasses various antibody modifications (e.g., post-translational chemical modifications) that result in non-proteinic portions thereon, such as glycosylated and lipidized forms of antibodies.

[0070] The terms “antibody fragment” and “antigen-binding fragment” used herein are interchangeable and refer to an incomplete portion of an antibody that contains the antibody’s antigen-binding domain and accordingly retains the ability to bind to the antigen to which the antibody is bound. Examples of antibody fragments include, but are not limited to, Fab, scFab, disulfide-linked scFab, Fab', F(ab')2, Fab'-SH, Fv, scFv, disulfide-linked scFv, linear antibodies, diabody, triabody, tetrabody, minibody; single-chain antibodies (e.g., scFv, scFab); single-domain antibodies (sdAb); camelid antibodies (heavy chain antibodies) or fragments thereof (e.g., VHH). Unless otherwise stated herein or explicitly contradicted by the context, the term “antibody” is used herein to mean “antibody or its antigen-binding fragment”.

[0071] In this document, the term "antigen-binding domain" refers to an antibody region or fragment thereof that specifically binds to an antigenic epitope. In some specific embodiments, the antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody, or, when the antibody is a heavy chain antibody, comprises a single heavy chain variable region (VHH). In some embodiments, the antigen-binding domain may also comprise an antibody constant region. Available heavy chain constant regions include any of the following five types: α, δ, ε, γ, or μ. Available light chain constant regions include any of the following two types: κ and λ. Thus, in some aspects, a specific antigen-binding domain may be selected from, including, but not limited to: antibody fragments comprising both the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody, such as Fv, scFv, Fab, scFab, and antibody fragments comprising the heavy chain variable region of a heavy chain antibody, such as VHH. In some embodiments, the B7-H3 antigen-binding domain according to this disclosure comprises, or is substantially composed of, a heavy chain variable domain (VH) and a light chain variable domain (VL) from an anti-B7-H3 antibody. In some embodiments, the EGFR antigen-binding domain according to this disclosure comprises, or is substantially composed of, a heavy chain variable domain (VH) and a light chain variable domain (VL) from an anti-EGFR antibody.

[0072] In this paper, the term "multispecific" refers to the ability of an antibody or antigen-binding molecule to specifically bind to at least two different antigenic epitopes (e.g., different epitopes on different antigens or different epitopes on the same antigen). Correspondingly, "single specific" refers to the ability to bind to only one epitope. "Dual specific" refers to the ability to bind to two different epitopes.

[0073] In this paper, the antibody-related terms "valence" or "valence number" refer to the total number of antigen-binding sites in an antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity. For example, a bivalent antibody means that the antibody molecule contains a total of 2 antigen-binding sites; the antibody molecule can be a "1+1" type bispecific antibody, that is, the antibody has 2 different antigen-binding specificities, where only 1 antigen-binding site exists for one antigen-binding specificity and only 1 antigen-binding site exists for the other antigen-binding specificity. Accordingly, the valence ratio of the two different specific antigen-binding domains in such an antibody is 1:1.

[0074] In this document, the term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody involved in antibody-antigen binding. The VHH domain of a heavy chain antibody, like the variable regions of the heavy and light chains of conventional antibodies, contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs), arranged in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As is well known in the art, in some cases, one or more residues in the variable region of an antibody can be modified; for example, one or more CDR residues and / or one or more framework region residues can be modified, particularly by substitution of conserved residues, while still substantially retaining at least one biological property of the parent antibody (e.g., antigen-binding ability). Furthermore, antibody variable regions can be modified via CDR transplantation to construct antibody variants that mimic the properties of known antibodies. After mutation or engineering, the properties of antibody variants, such as target antigen binding properties or other desired functional properties, such as binding affinity, endocytic activity, pharmacokinetic properties, and / or tumor cell killing activity, can be determined and screened in vitro or in vivo using methods known in the art and described herein to identify functional antibody variants with the desired properties. It should be understood that any such functional variants with variable regions (e.g., VH and / or VL regions) presented herein are within the scope of this invention.

[0075] In this document, the terms "complementarity-determining region" or "CDR region" or "CDR" or "hypervariant region" are used interchangeably to refer to regions within the variable domain of an antibody that are highly variable in sequence and form structurally defined loops ("hypervariant loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. In this document, CDRs of the antibody heavy and light chains are sequentially numbered starting from the N-terminus and are typically referred to as CDR1, CDR2, and CDR3. CDRs located within the variable domain of the antibody heavy chain are also referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. The CDR sequence within a defined variable region (such as a VH / VL domain) can be determined using schemes known in the art, such as, but not limited to, the Kabat, AbM, Contact, and IMGT schemes. The following are exemplary CDR region ranges defined using the Kabat, AbM, IMGT, and Contact schemes.

[0076] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the above-described manner and combinations thereof, but preferably a CDR sequence defined according to the Kabat scheme.

[0077] In this article, "Fv domain" refers to a small antibody fragment that includes both a complete antigen recognition site and a binding site. "Fv" is a dimer (VH-VL dimer) formed by the non-covalent bonding of a VH and a VL.

[0078] In this paper, "scFv domain" refers to a single-chain polypeptide in which two variable regions (usually a VH and a VL) are linked via a linker to form an antigen-binding domain necessary for antigen binding.

[0079] In this paper, the term "Fab domain" refers to an antigen-binding domain similar to that formed in a conventional four-chain IgG antibody by pairing the heavy chain variable region VH and the heavy chain constant region CH1 (VH-CH1) with the complementary light chain variable region VL and the light chain constant region CL (VL-CL).

[0080] In this paper, the term "scFab domain" refers to a Fab domain that is linked into a single polypeptide chain by an artificial linker.

[0081] In this document, the term "VHH domain" refers to the variable heavy chain domain derived from heavy chain antibodies lacking a light chain. Therefore, VHHs differ from the conventional VHs of four-chain immunoglobulins in that they do not require pairing with a light chain variable domain to form an antigen-binding domain. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids may also produce naturally occurring heavy chain antibodies lacking a light chain, and such VHHs are also within the scope of this invention.

[0082] In this article, the term "immunoglobulin" refers to a protein with a structure that contains naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. In IgG antibody molecules, the VH-CH1 of the heavy chain typically pairs with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG immunoglobulin essentially consists of two Fab molecules linked by immunoglobulin hinge regions and two dimerized Fc regions. Immunoglobulin heavy chains can be classified into one of five types based on the type of their constant domains, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Some of these categories can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can also be classified into one of two types based on the amino acid sequence of their constant domains, called κ and λ.

[0083] In this document, "immunoglobulin constant domain" refers to a constant domain derived from or obtained from the heavy chain of immunoglobulins (e.g., human IgG1 immunoglobulin heavy chain) or the light chain, including heavy chain constant domains CH1, CH2, CH3, and optionally CH4; and light chain constant domain CL. This term includes both native and variant sequence constant domains.

[0084] In this document, the term "type" or "subtype" of an immunoglobulin constant domain refers to the type or subtype determined based on its amino acid sequence. Heavy chain constant domains can be classified into five different types based on their sequence: IgA, IgD, IgE, IgG, and IgM, or further into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Therefore, when referring to the Fc region of the IgG1 isotype in this document, it means that the Fc region can be classified as type IgG based on its amino acid sequence, and can be further classified as subtype IgG1. Similarly, light chain constant domains can be classified into κ and λ light chain CL domains based on their sequence. Those skilled in the art can readily determine the type or subtype of a constant domain by comparing the amino acid sequence of the constant domain with the sequences of the corresponding constant domains of different types or subtypes of native immunoglobulins. In this document, when the antibody or antigen-binding molecule of this disclosure comprises multiple immunoglobulin heavy chain constant domains, it should be understood that these domains can be selected independently of each other according to the intended function or use of the molecule. As an example, for the antibody of this disclosure comprising a CH1 domain as well as CH2 and CH3 domains, all three constant domains may be IgG1 subtypes, such as human IgG1 subtypes, or only the CH2 and CH3 domains may be IgG1 subtypes, such as human IgG1 subtypes.

[0085] In this disclosure, unless otherwise stated, references to the positions of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues) refer to the positions numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0086] In this paper, the term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, for example, an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0087] The term "humanized antibody" refers to an antibody for which a CDR sequence derived from a non-human mammalian species, such as a mouse lineage, is grafted onto a human scaffold sequence. Additional scaffold region modifications can be made within the human scaffold sequence, and / or additional amino acid modifications can be made to the CDR sequence, for example, to facilitate antibody affinity maturation. In some embodiments herein, the humanized antibody of the present invention has a scaffold region sequence "derived" from a specific human lineage sequence. Here, "derived" means that the amino acid sequence of the antibody scaffold region has at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identity with the corresponding scaffold region amino acid sequence encoded by the human lineage immunoglobulin gene, and that the antibody retains antigen-binding activity.

[0088] In this document, "isolated" antibody refers to artificial antibodies, recombinant antibodies, and antibodies that have been at least partially separated from components in the natural environment in which they originated. In some embodiments, the antibodies of the present invention are "isolated" antibodies. In some embodiments, the isolated antibodies are purified to a purity of more than 90%, 95%, or 99%, 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).

[0089] In this document, the terms "binding" or "specific binding" refer to the selective binding of an antigen-binding site to an antigenic epitope, which can be distinguished from unwanted or nonspecific interactions. The binding ability or binding specificity of an antigen-binding site to a specific antigenic epitope can be determined using conventional binding assays known in the art, including but not limited to, detecting antibody-antigen binding by ELISA, detecting antibody binding to cells expressing antigens by FACS, or characterizing the binding affinity constant K using surface plasmon resonance (SPR) or thin-layer interferometry (BLI) techniques. D .

[0090] In this paper, the term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigenic epitope). In this context, "binding affinity" reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair. Binding affinity is typically expressed as the binding dissociation equilibrium constant (K0). D To express it.

[0091] In this paper, the term "affinity" or "binding affinity" refers to the combined strength of interactions between multiple binding sites of a molecule (e.g., an antibody) and the same target. Therefore, a necessary condition for affinity is the multivalent nature of the molecule (e.g., an antibody) to a target.

[0092] The term "K" D "(M) in this paper refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Affinity is related to K..." D The values ​​are inversely correlated; that is, the higher the affinity, the higher the K value. D The smaller the value, the lower the affinity; conversely, the larger the value, the lower the affinity. D The higher the value, the better. Generally, K... D The value depends on the dissociation rate constant (Kd or Kdis or K) between the interacting antibody-antigen pairs. off ,sec -1 ) and binding rate constant (Ka or K) on M -1 x sec -1 ).

[0093] In this document, the term "immune cross-reactivity" refers to an antibody that binds to a specific antigen and also binds to homologs of that antigen from different species. For example, in some embodiments, an antibody specific to human B7-H3 according to the invention may also bind to B7-H3 from other species (e.g., cynomolgus monkey B7-H3). Methods for determining cross-reactivity include the methods described in the examples and standard assays known in the art, such as those using flow cytometry or cell ELISA techniques. Antibodies exhibiting human-monkey species cross-reactivity are advantageous, as this property can facilitate preclinical drug development of the antibody, such as toxicological assays of antigen-binding molecules composed of antibodies.

[0094] In this document, the term "immunoglobulin Fc region" is used interchangeably with "Fc region" and "Fc domain" to define the constant region portion of the immunoglobulin heavy chain that contains the CH3 domain. In some cases, this immunoglobulin portion may also contain one or more other immunoglobulin constant domains or fragments thereof, including hinge regions and / or CH2 domains, but typically excludes variable regions and CH1 domains. When referring to amino acids in the Fc region and constant domains, the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991 is used. When referring to the human IgG1 Fc region, the EU numbering of amino acid residues can also be found in the IMGT Scientific chart (https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html), which is hereby incorporated by reference. According to this numbering, in the heavy chain of human IgG1 immunoglobulin, amino acids 118-215 form the CH1 domain, amino acids 216-230 form the hinge region, amino acids 231-340 form the CH2 domain, and amino acids 341-447 form the CH3 domain. In some cases, the C-terminal lysine (Lys447) of the CH3 domain may or may not be present. Those skilled in the art can readily determine the constant domains contained in an antibody or antigen-binding molecule, their type / subtype, and species origin by sequence alignment with published constant regions of native immunoglobulins.

[0095] In this document, the term "Fc domain" or "Fc region" encompasses both native and variant Fc regions. Specifically, the term "native Fc region" encompasses the Fc region sequences of various naturally occurring immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In this document, the term "variant Fc region" refers to a polypeptide containing a modified Fc region relative to the native Fc region sequence. The modification can be the addition, deletion, or substitution of amino acid residues. Substitution can include both naturally occurring and non-natural amino acids. The purpose of modification can be to alter the physicochemical properties of the Fc region, such as thermal stability and heterodimerization tendency, and / or the binding of the Fc region to its receptor and its effector function, and / or to introduce amino acid residues or modified amino acid residues that can be used for drug molecule conjugation.

[0096] In this paper, when referring to a single mutation, it is described by the position of the amino acid residue where the mutation occurs and the amino acid residues before and after the mutation, denoted as [original amino acid residue] [mutated amino acid residue]. For example, the substitution of threonine at position 366 of the Fc region with tryptophan is denoted as T366W; the substitution of tyrosine at position 407 of the Fc region with valine is denoted as Y407V. When referring to multiple mutations on a polypeptide chain, these mutations are connected by the symbol "-", for example, the mutations T366S, L368A, Y407V, L351Y, and D399R on the same Fc chain can be represented as "T366S-L368A-Y407V-L351Y-D399R".

[0097] In this paper, "IgG conformation" refers to an antibody conformation identical to that of IgG immunoglobulin, essentially consisting of two Fab domains linked to the N-terminus of a dimerized Fc domain via an immunoglobulin hinge region (or, where appropriate, via a peptide linker). Thus, in a typical case, an IgG conformation antibody consists of two heavy chains and two light chains, wherein each heavy chain has VH, CH1, CH2, and CH3 domains from its N- to C-terminus; and each light chain has VL and CL domains from its N- to C-terminus.

[0098] In this document, the terms "endocytosis" and "internalization" are used interchangeably to refer to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or translocated to a suitable intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface. In some embodiments, the antibodies of this disclosure initiate EGFR receptor-mediated endocytosis upon binding to EGFR expressed on the cell surface. The endocytosis rate can be determined, for example, by the methods described in the examples, to characterize the receptor-mediated endocytic activity of the antibody. In some embodiments, the antibodies of this invention having receptor-mediated endocytic activity can be used as a tool for delivering antitumor drugs into cancer cells in the ADCs of this invention.

[0099] In this document, the "percentage of identity (%)" for an amino acid sequence refers to the percentage of positions in the comparison window where a candidate sequence shares the same amino acid residues as the specific amino acid sequence, after comparing the candidate sequence with the specific amino acid sequence shown herein within a comparison window, and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of sequence identity. Unless otherwise specified, the comparison window is the full length of the specific amino acid sequence.

[0100] For polypeptide sequences, “conservative modifications” include substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of a certain amino acid with a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conservedly substituted for each other: 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) (see, for example, Creighton, Proteins (1984)).

[0101] In this document, the term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells are any type of cell system that can be used to produce the antibodies or antigen-binding molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0102] In this document, the term "expression vector" refers to a vector containing recombinant polynucleotides that includes an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including but not limited to, viscera, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0103] In this document, the term "immunofustomy" generally refers to a fusion polypeptide formed by fusing one or more immunoglobulin-related molecules or fragments thereof (e.g., antibodies or fragments thereof) with one or more other molecules of protein nature. The protein-natured molecules may be, for example, peptides, polypeptides, or proteins.

[0104] In this document, the term "immunoconjugate" refers to a chemical modification or derivative formed by covalently linking or conjugating one or more other molecules of non-protein nature (e.g., therapeutic or diagnostic molecules) to one or more immunoglobulin-related molecules or fragments thereof (e.g., antibodies or fragments thereof). Examples of such other molecules include, but are not limited to, markers, pharmaceuticals, and cytotoxic agents, such as: radioisotopes; chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof; fluorescent reporter proteins; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various known antitumor or anticancer agents.

[0105] In this document, the term "amino acid" refers to naturally occurring and synthetic amino acids, amino acid analogs, and their artificially modified forms. Amino acids can be L or D isomers. In this disclosure, common amino acids are represented by single-letter and three-letter abbreviations known in the art. For example, phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn; N), isoleucine (Ile; I), arginine (Arg; R), proline (Pro; P), and glutamine (Gln; Q).

[0106] In this document, the term "peptide linker" refers to a linker that contains or is composed of amino acids. The amino acids used in a peptide linker can be natural amino acids, amino acid analogs, or artificially modified amino acids. In this disclosure, peptide linkers used in peptides (e.g., multispecific antibodies or immunofusions) are composed of natural amino acids to facilitate fusion with the remainder of the peptide via genetic recombination. In this disclosure, peptide linkers used in immunoconjugates (e.g., ADCs according to this disclosure) can be composed of natural amino acids or may contain artificially modified amino acids, provided that they allow the drug load in the ADC to be unloaded from the ADC at the target tissue region. Typically, peptide linkers used in ADCs contain or consist of 2-8 amino acids that can be recognized and cleaved by enzymes in the target tissue region (e.g., tumor tissue).

[0107] In this article, the term "halogen" generally refers to fluorine, chlorine, bromine, and iodine, for example, fluorine and chlorine.

[0108] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms. Specifically, alkyl groups have 1 to 10 carbon atoms, such as 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, as used herein, the term "C1-C6 alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms, examples of which include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, or tert-butyl), pentyl (including n-pentyl, isopentyl, and neopentyl), hexyl (including n-hexyl, 2-methylpentyl, 3-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, and 2-ethylbutyl), etc.

[0109] As used herein, the term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, containing at least one double bond. Specifically, alkenyl groups have 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0110] As used herein, the term "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one triple bond. Specifically, an alkynyl group has 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkynyl" refers to a straight-chain or branched alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-methyl-1-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, etc.

[0111] As used herein, the term "alkylene" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight-chain or branched saturated alkane. Specifically, alkylenes have 1 to 10 carbon atoms, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, as used herein, the term "C1-C6 alkylene" refers to a straight-chain or branched alkylene having 1 to 6 carbon atoms, including, but not limited to, methylene, ethylene, propylene, butylene, etc.

[0112] As used herein, the term "alkenyl" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight-chain or branched unsaturated alkene containing at least one double bond. Specifically, alkenyl groups have 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinylidene, propenide, allylidene, butenide, pentenide, and hexenide.

[0113] As used herein, the term "ynynyl" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight-chain or branched unsaturated alkyne containing at least one triple bond. Specifically, ynynyl groups have 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 ynynyl" refers to a straight-chain or branched ynynyl group having 2 to 6 carbon atoms, such as ethynylene, propynylene, propynylene, butynylene, penynylene, and hexynylene.

[0114] As used herein, the term "cycloalkyl" refers to a monocyclic, fused, bridged, or spirocyclic non-aromatic monovalent hydrocarbon ring structure having a specified number of ring atoms. It can be saturated or unsaturated, for example, containing one or more double bonds. The cycloalkyl group may contain three or more carbon atoms in the ring, for example, 3-18, 3-10, or 3-8 carbon atoms, such as C1. 3-10 cycloalkyl, C 3-8 cycloalkyl, C3-6 cycloalkyl, C 5-6 Cycloalkyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0115] As used herein, the term "heterocyclic" or "heterocyclic group" refers to a 5-20 membered (e.g., 5-14, 5-8, 5-6) aromatic or non-aromatic monocyclic, bicyclic, or polycyclic ring system having 1-4 independent heteroatom ring members selected from N, O, or S. One or more N, C, or S atoms in the heterocycle may be oxidized. Preferably, the heterocycle is a 5-10 membered ring system, and is a monocyclic or fused bicyclic ring. Representative examples include, but are not limited to, pyrrolidine, azaheterocyclic butane, piperidine, morpholine, tetrahydrofuran, tetrahydropyran, benzofuran, benzothiophene, indole, benzopyrazole, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, pyrimidine, pyridine, pyrazine, pyridazine, isothiazine, and isoxazole. It should be understood that this term includes heteroaryl groups as defined herein.

[0116] The term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, for example, 6-12 carbon atoms in the ring moiety. Preferably, the aryl group is (C6-C6) 10 Aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which may optionally be substituted with 1 to 4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, 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, sulfonamide, heterocyclic, etc.

[0117] The term "heteroaryl" refers to a 5-20 membered (e.g., 5-14, 5-8, 5-6) aromatic monocyclic or polycyclic ring system containing 1-4 heteroatoms selected from N, O, or S, which may be substituted or unsubstituted. Preferably, the heteroaryl is a 5-10 membered ring system, which is a monocyclic or fused bicyclic ring. Representative heteroaryl groups include 2- or 3-thienyl, 2- or 3-furanyl, 2- or 3-pyrroleyl, 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-isooxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3- or 4-pyridinyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl.

[0118] Unless otherwise specified, the term “substituted” as used in defining various groups herein means that the corresponding group can be substituted by, for example but not limited to, the following groups: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, halogen, cyano, nitro, azide, carboxyl, hydroxyl, mercapto, amino, mono- or dialkylamino, mono- or dicycloalkylamino, mono- or diarylamino, mono- or diheterocyclicamino, mono- or diheteroarylamino, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-oxy, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-thio, alkyl- or cycloalkyl The groups are alkyl- or heterocyclic- or heteroaryl- or aryl-acyl, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-acylamino, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-acyloxy, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-sulfonyl, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-sulfonyloxy, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-sulfonylamino, or the optional substituted amino-formyl group described above, and each of these groups being further substituted by the remaining optional substituents, wherein the various groups are as defined herein. Examples of substituents include, but are not limited to, one or more groups independently selected from the following: halogen, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, C(O)-amino, OCOCH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO2, phenyl, piperidinyl, piperazine, and pyrimidinyl.

[0119] As used herein, the term “substitution” or “substituted” means that one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a specified atom are replaced by a specified group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and forms a stable compound. Combinations of substituents and variables are only permitted if such combinations form a stable compound.

[0120] In this document, the term "optional" or "optionally" means that the event or situation described below occurs or does not occur, and the description includes instances where the event or situation occurs as well as instances where the event or situation does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0121] In this article, "antibody-drug conjugate (ADC)" refers to a compound obtained by linking an antibody to a (small molecule) drug via a linker.

[0122] In this document, the term "linker" refers to the bifunctional portion in a drug-antibody conjugate that links a drug to an antibody. The linkers of the present invention may have multiple components (e.g., in some embodiments, a linker group responsible for conjugating the antibody; a degradable peptide linker unit; and optionally a spacer group).

[0123] In this article, the portion of the drug compound linked to the antibody via a linker is sometimes referred to as the "payload" or "load".

[0124] 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 that such salt is not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, 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 by the ADC conjugates of the present invention with 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, etc. 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 by reacting with an organic base containing an N group.

[0125] The term "solvent" refers to an association formed by one or more solvent molecules with the ADC conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0126] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0127] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) coupled to the Ab portion as described herein to the Ab portion in an ADC conjugate. In some embodiments described herein, the DAR may be determined by n in Formula I, for example, the DAR may be 1 to 20, such as 2-18, 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, or any integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or a range consisting of any two values ​​between 1 and 10. DAR can also be calculated as the average DAR of the molecular population in the product, which is the overall ratio of the drug fraction (D) coupled to the Ab fraction described herein to the Ab fraction in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC). This DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 20, for example 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, 6-8, or 7-8, for example 1.0-8.0, 2.0-6.0, for example 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4. 6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, and ranges with two of these values ​​as endpoints. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population or mixture of ADC molecules that contains ADC molecules having the same and / or different DAR values.

[0128] The term "drug" as used herein encompasses any substance effective in the prevention or treatment of tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressants).

[0129] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or destruction. Examples of cytotoxic agents that may be mentioned include: camptothecin derivatives, oliguria tinctoria, chlortetracycline, maytansine alkaloids, ricin, ricin A chain, carbetastatin, pyruvic oxytocin, salicylic acid, doxorubicin, doxorubicin, daunomycin, tacrolimus, cisplatin, CC1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, and colchicine.

[0130] The term "small molecule drug" refers to low-molecular-weight organic compounds that can regulate biological processes, particularly altering or preventing pathological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0131] The term "pharmaceutical composition" refers to a composition in which the active ingredient contained herein is present in a biologically effective form and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0132] The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier, or stabilizer that is administered with the active substance.

[0133] In this document, the terms “individual,” “subject,” or “patient” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human.

[0134] The terms “tumor” and “cancer” are used interchangeably herein and refer to a physiological disorder in mammals characterized by unregulated cell growth. The term encompasses both primary and metastatic forms of tumors. It also covers all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and liquid tumors.

[0135] The term "antitumor effect" refers to antitumor biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0136] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0137] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or a particular symptom of a disease or condition. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer. In some implementations, subjects with a family history of cancer are candidates for preventative programs.

[0138] When used herein, the term "effective amount" refers to an amount of a drug (e.g., the antibody-drug conjugate or pharmaceutically acceptable salt or solvate of the present invention, or the antibody or antigen-binding fragment thereof of the present invention, or a combination thereof or a combination of drugs) that, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention.

[0139] When used herein, the term "therapeutic effective amount" refers to an amount that, at the required dose and sustained for the required period of time, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also a amount in which any toxic or harmful effects resulting from the administration of said amount to a subject will outweigh its beneficial therapeutic effects. Compared to untreated individuals, the "therapeutic effective amount" preferably achieves an improvement of at least about 30%, and more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% in measurable parameters (e.g., tumor volume).

[0140] When used in this context, the term "preventive effective dose" refers to the amount at which the desired preventive outcome is effectively achieved, at the required dose and for the required duration. Typically, because the prophylactic dose is administered in subjects before or at an early stage of the disease, the preventive effective dose will be less than the therapeutic effective dose.

[0141] Various aspects of the invention will be further described in the following subsections.

[0142] The antibody of the present invention

[0143] I. Anti-B7-H3 and EGFR antibodies

[0144] Although various monospecific antibody-drug conjugates (ADCs) have been developed for cancer treatment, increasing research indicates that many ADCs are often hampered by factors such as the expression of target tumor antigens in normal healthy tissues, variations in target tumor antigens, and poor internalization activity, making it difficult to meet clinical needs. To address this issue, in in-depth research, the inventors have developed anti-B7-H3 and anti-EGFR antibodies according to the present invention suitable for constructing bispecific antibody-drug conjugates (BADCs). More specifically, the inventors have discovered that although the endocytic activity induced by the B7-H3 antibody itself is weak, by applying the high-affinity B7-H3 binding arm of the present invention, the binding of the lower-affinity EGFR arm to tumor cells can be advantageously enhanced, thereby inducing strong EGFR receptor-mediated endocytic activity and achieving good drug delivery efficacy for the bispecific antibody-drug conjugate. Furthermore, due to the specific tumor tissue distribution characteristics of the B7-H3 antigen, the high affinity of the B7-H3 binding arm in this invention also facilitates greater distribution of the bispecific antibody into the tumor and tumor microenvironment. Simultaneously, by allowing the bispecific antibody to select the EGFR arm with relatively low binding affinity, the binding of the EGFR arm to EGFR expressed in normal tissues is reduced, lowering the risk of on-target detumescent toxicity and reducing antibody internalization and consumption by normal tissues, thus prolonging the antibody's circulating half-life. Based on these findings, this invention establishes an anti-B7-H3 and EGFR antibody suitable for constructing ADCs with high tumor-killing activity and low toxicity.

[0145] In a first aspect, the present invention therefore provides a multispecific antibody that specifically binds to B7-H3 and EGFR. The anti-B7-H3 and EGFR antibody according to the present invention comprises at least one antigen-binding domain specifically binding to B7-H3 and at least one antigen-binding domain specifically binding to EGFR. In some aspects, the antibody of the present invention further comprises an immunoglobulin Fc region. The antibody of the present invention can be in any suitable form, such as the IgG conformation shown in FIG1.

[0146] The components of the multispecific antibody of the present invention are described in detail below. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, the antibody of the present invention (including any form of antibody) may contain any such combination of features.

[0147] Antigen-binding domain

[0148] In some embodiments, the present invention provides a multispecific antibody comprising a B7-H3 binding domain and an EGFR binding domain. In some embodiments, the B7-H3 antigen-binding domain of the antibody according to the present invention has a high binding affinity for B7-H3 (preferably human B7-H3), for example, a binding affinity K0. D Value less than 1x10 -9 M, preferably less than 1x10 -10 M. In some embodiments, the EGFR antigen-binding domain of the antibody according to the invention has a moderate binding affinity for EGFR (preferably human EGFR), for example, a binding affinity K. D The value is greater than approximately 1x10 -10 M. In some embodiments, the binding affinity K of the B7-H3 binding domain to B7-H3. D The value is 5x10 -11 M to 0.5x10 -12 M, wherein the binding affinity of the EGFR binding domain to EGFR is 1 x 10⁻⁶. -7 M to 1x10 -9 M. In some embodiments, the binding affinity K of the B7-H3 binding domain to B7-H3. D The value is 1-5x10 -12 M, wherein the binding affinity of the EGFR binding domain to EGFR is 1-6x10⁻⁶. -8 M. In other embodiments, the binding affinity K of the B7-H3 binding domain to B7-H3. D The value is 1-5x10 -12 M, wherein the binding affinity of the EGFR binding domain to EGFR is 1-6x10⁻⁶. -9 M. In some further embodiments, according to the present invention, the B7-H3 antigen-binding domain of the antibody has a binding dissociation rate constant K with human B7-H3. off Value less than 1x10 -5 s -1 Optionally approximately 1x10 -6 s -1 Up to 1x10 -8 s -1 Preferably approximately 0.5-5x10 -7 s -1 In this disclosure, preferably, the binding affinity K is used. D Value and binding dissociation rate constant K off The values ​​are detected using biofilm thin-layer interferometry (BLI). In some embodiments, the BLI detection is performed at 30°C, for example, as described in Example 5.

[0149] Exemplary B7-H3 antigen-binding domain

[0150] In some embodiments of the multispecific antibody according to the invention, the antigen-binding site that specifically binds to B7-H3 is provided by the VH and VL domains from the anti-B7-H3 antibody.

[0151] In some embodiments, the B7-H3 binding domain according to the invention includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) selected from the following heavy chain variable region (VH) and light chain variable region (VL) sequence pairs:

[0152] (a) The VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:2;

[0153] (b) The VH sequence shown in SEQ ID NO:3 and the VL sequence shown in SEQ ID NO:4;

[0154] (c) The VH sequence shown in SEQ ID NO:36 and the VL sequence shown in SEQ ID NO:37; or

[0155] (d) The VH sequence shown in SEQ ID NO:38 and the VL sequence shown in SEQ ID NO:39.

[0156] In some preferred embodiments, the B7-H3 binding domain according to the invention comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) selected from the following heavy chain variable region (VH) and light chain variable region (VL) sequence pairs:

[0157] (a) The VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:2; or

[0158] (b) The VH sequence shown in SEQ ID NO:3 and the VL sequence shown in SEQ ID NO:4.

[0159] In some implementations, the CDR is defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. More preferably, the CDR is defined according to Kabat or AbM, or a combination thereof; more preferably, the CDR is defined according to Kabat. However, it should be understood that the CDR may also be defined in any other manner known in the art.

[0160] In some embodiments, the B7-H3 binding structural domain according to the invention comprises HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3, wherein:

[0161] -HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:8;

[0162] -HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:9;

[0163] -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:10;

[0164] -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:5;

[0165] -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:6 or 34;

[0166] -LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:7.

[0167] In some embodiments, the B7-H3 binding structural domain according to the invention comprises HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3, wherein:

[0168] -HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:43;

[0169] -HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:44;

[0170] -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:45;

[0171] -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:40;

[0172] -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:41;

[0173] -LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:42.

[0174] In some embodiments, the B7-H3 binding structural domain according to the invention comprises HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3, wherein:

[0175] -HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:49;

[0176] -HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:50;

[0177] -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:51;

[0178] -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:46;

[0179] -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:47;

[0180] -LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:48.

[0181] In some preferred embodiments, the B7-H3 binding domain according to the present invention comprises HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3, wherein: HCDR1, HCDR2, and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs:8-10; and LCDR1, LCDR2, and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs:5, 34, and 7.

[0182] In some other preferred embodiments, the B7-H3 binding domain according to the present invention comprises HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, wherein: HCDR1, HCDR2 and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs:8-10; and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs:5-7.

[0183] In some embodiments, the B7-H3 binding domain according to the invention comprises: a variable region sequence of any of the exemplary antibodies of the invention, or a variant thereof, for example, an antibody or fragment thereof having the same CDR sequence as one of the exemplary B7-H3 antibodies and having the same or different framework region sequences, such as a humanized antibody. The retention or improvement of the antigen-binding properties or other functional properties of these antibodies can be evaluated in in vitro or in vivo assays. The exemplary B7-H3 antibodies include 33D6, hz33D6.8, 32F8, and 3H3, whose variable region sequences and Kabat CDR sequences are listed in the sequence listing.

[0184] In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises an amino acid sequence selected from SEQ ID NOs:1, 3, 36 or 38, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof. In other embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VL comprises an amino acid sequence selected from SEQ ID NOs:2, 4, 37 or 39, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed thereof. Preferably, the addition, deletion, and / or substitution of the amino acid does not occur in the CDR region.

[0185] In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO:1, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and (ii) the VL comprises the amino acid sequence of SEQ ID NO:2, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof. Preferably, the addition, deletion, and / or substitution of amino acids does not occur in the CDR region. In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:1 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:2.

[0186] In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO:3, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and (ii) the VL comprises the amino acid sequence of SEQ ID NO:4, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof. Preferably, the addition, deletion, and / or substitution of amino acids does not occur in the CDR region. In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:3 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:4.

[0187] In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO:36, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and (ii) the VL comprises the amino acid sequence of SEQ ID NO:37, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof. Preferably, the addition, deletion, and / or substitution of amino acids does not occur in the CDR region. In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:36 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:37.

[0188] In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO:38, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and (ii) the VL comprises the amino acid sequence of SEQ ID NO:39, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof. Preferably, the addition, deletion, and / or substitution of amino acids does not occur in the CDR region. In some embodiments, the B7-H3 binding domain according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:38 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:39.

[0189] In some embodiments, the B7-H3 antigen-binding domain of the antibody according to the present invention has one or more features selected from the following:

[0190] (a) Binding affinity K with human B7-H3 D Value less than 1x10 -10 M, optionally approximately 5x10 -11 M to 0.5x10 -12 M, preferably approximately 1-5x10 -12 M;

[0191] (b) The rate constant K for the dissociation of B7-H3 with human B7-H3 off Value less than 1x10 -5 s -1 Optionally approximately 1x10 -6 s -1 Up to 1x10 -8 s -1 Preferably approximately 0.5-5x10 -7 s -1 ;and

[0192] (c) It exhibits immune cross-reactivity with monkey B7-H3. Preferably, the K... D Value and K off The values ​​were determined using the BLI assay method.

[0193] In other embodiments, the B7-H3 binding domain according to the invention has weak B7-H3-mediated endocytosis activity.

[0194] Exemplary EGFR combined domain

[0195] In some embodiments of the multispecific antibody according to the invention, the antigen-binding site that specifically binds to EGFR is provided by the VH and VL domains from the anti-EGFR antibody.

[0196] In some embodiments, the binding affinity K of the EGFR binding domain according to the present invention to human EGFR antigen is... D Value greater than 1x10 - 10 M. In some embodiments, the binding affinity K of the EGFR binding domain according to the invention to human EGFR antigen D The value is 1x10 -7 M to 1x10 -9 M. In some further embodiments, the binding affinity K of the EGFR binding domain according to the invention to human EGFR antigen D The value is 1-6x10 -8 M. In some further embodiments, the binding affinity K of the EGFR binding domain according to the invention with human EGFR antigen D The value is 1-6x10 -9 M. Preferably, the K D The values ​​were determined using the BLI assay method.

[0197] In some embodiments, the EGFR binding domain according to the invention has one or more of the following properties: (i) EGFR-mediated endocytosis activity; (ii) no inhibition of EGFR binding to its receptor; and (iii) cynomolgus monkey EGFR cross-reactivity.

[0198] In some embodiments, the EGFR binding domain according to the invention comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) selected from the following heavy chain variable region (VH) and light chain variable region (VL) sequence pairs:

[0199] (a) The VH sequence shown in SEQ ID NO:11 and the VL sequence shown in SEQ ID NO:12;

[0200] (b) The VH sequence shown in SEQ ID NO:13 and the VL sequence shown in SEQ ID NO:14; or

[0201] (c) The VH sequence shown in SEQ ID NO:15 and the VL sequence shown in SEQ ID NO:16.

[0202] In some implementations, the CDR is defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. More preferably, the CDR is defined according to Kabat or AbM, or a combination thereof; more preferably, the CDR is defined according to Kabat. However, it should be understood that the CDR may also be defined in any other manner known in the art.

[0203] In some embodiments, the EGFR binding domain according to the invention comprises HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3, wherein:

[0204] -HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO:20;

[0205] -HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:21 or 35;

[0206] -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:22;

[0207] -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:17;

[0208] -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:18;

[0209] -LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:19.

[0210] In some embodiments, the EGFR binding domain according to the present invention comprises HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3, wherein: HCDR1, HCDR2, and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs:20, 35, and 22; and LCDR1, LCDR2, and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs:17-19.

[0211] In some embodiments, the EGFR binding domain according to the present invention comprises HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3, wherein: HCDR1, HCDR2, and HCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs:20-22; and LCDR1, LCDR2, and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NOs:17-19.

[0212] It is known in the art that although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR directly participate in antigen binding. Therefore, this disclosure also considers the presence of an EGFR-binding domain in any or more of the aforementioned CDRs that has residue substitutions but still retains EGFR-binding activity.

[0213] In some embodiments, the EGFR-binding domain according to the invention comprises: a variable region sequence of any of the exemplary antibodies of the invention or a variant thereof, for example, having the same CDR sequence as one of the exemplary EGFR antibodies and having the same or different framework region sequences, such as humanized variable region sequences. The antigen-binding properties or other functional properties of these EGFR-binding domains can be evaluated in in vitro or in vivo assays.

[0214] In some embodiments, the EGFR-binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises an amino acid sequence selected from SEQ ID NOs:11, 13, or 15, or has at least 85%, 90%, 95%, or 99% identity with respect to said amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or is composed thereof. In other embodiments, the EGFR-binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VL comprises an amino acid sequence selected from SEQ ID NOs:12, 14, or 16, or has at least 85%, 90%, 95%, or 99% identity with respect to said amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or is composed thereof. Preferably, the addition, deletion, and / or substitution of the amino acid does not occur in the CDR region.

[0215] In some embodiments, the EGFR binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO:11, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and (ii) the VL comprises the amino acid sequence of SEQ ID NO:12, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof. Preferably, the addition, deletion, and / or substitution of amino acids does not occur in the CDR region. In some embodiments, the EGFR binding domain according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:11 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:12.

[0216] In some embodiments, the EGFR binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO:13, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and (ii) the VL comprises the amino acid sequence of SEQ ID NO:14, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof. Preferably, the addition, deletion, and / or substitution of amino acids does not occur in the CDR region. In some embodiments, the EGFR binding domain according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:13 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:14.

[0217] In some embodiments, the EGFR binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO:15, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof; and (ii) the VL comprises the amino acid sequence of SEQ ID NO:16, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof. Preferably, the addition, deletion, and / or substitution of amino acids does not occur in the CDR region. In some embodiments, the EGFR binding domain according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:15 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:16.

[0218] In some preferred embodiments, the EGFR binding domain according to the invention comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:15 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:16.

[0219] Immunoglobulin Fc region

[0220] In addition to the aforementioned antigen-binding domain, the multispecific antibody according to the present invention may also include an immunoglobulin Fc region in some embodiments.

[0221] The immunoglobulin Fc region used in the multispecific antibody of the present invention can be an Fc region derived from any immunoglobulin. In some embodiments, the immunoglobulin Fc region comprises at least an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region further comprises a hinge region or a partial hinge region. In some embodiments, the immunoglobulin Fc region comprises, or consists of, an immunoglobulin hinge region or a partial hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region comprises, or consists of, a CH2 domain and a CH3 domain from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from humans.

[0222] The immunoglobulin Fc region can be fused to the C or N terminus of the antigen-binding domain according to the invention. The immunoglobulin Fc can be fused to the antigen-binding domain via a peptide linker or directly to the antigen-binding domain. In embodiments where the immunoglobulin Fc region is fused to the antigen-binding domain at its N-terminus, preferably, the fusion is performed via the immunoglobulin hinge region sequence.

[0223] The immunoglobulin Fc region of the multispecific antibody used in this invention can be the natural Fc region sequence. Alternatively, the Fc region can contain mutations relative to the natural Fc sequence. Mutations include substitutions, insertions, and / or deletions.

[0224] In some embodiments, to facilitate proper antibody assembly, a Knob-into-Hole (KiH) mutation is introduced into the CH3 domain of the Fc region of the antibody of the present invention to promote heterodimerization. With the introduction of the KiH mutation, one Fc chain is designed to contain a large protruding residue (i.e., Knob), while the other Fc chain is designed to contain a complementary pocket (i.e., Hole). Suitable locations for the KiH mutation are known in the art. Exemplary KiH mutations include, but are not limited to, combinations of Knob mutation T366W and Hole mutations T366S, L368A, Y407V; and combinations of Knob mutation T366Y and Hole mutation Y407T.

[0225] Furthermore, it has been found that, based on the introduction of the Knob-into-Hole (KiH) mutation, further introducing a specific CH3 domain interface mutation can make antibodies containing Fc dimers exhibit superior thermostability compared to corresponding antibodies containing only the KiH mutation, thereby further reducing the chain mismatch ratio and increasing the stability of the target antibody product. Such CH3 domain interface mutations applicable to combinations with the KiH mutation are disclosed in the applicant's co-pending applications PCT / CN2023 / 129691 and PCT / CN2024 / 129416, which are hereby incorporated by reference.

[0226] In some embodiments, the antibody of the present invention comprises an Fc dimer having first and second immunoglobulin Fc regions.

[0227] Specifically, the first Fc region and the second Fc region contain the Knob mutation and Hole mutation of the KiH mutation, respectively, in their CH3 domains.

[0228] (a) The Fc region containing the Hole mutation also contains the L351Y mutation; or

[0229] (b) The Fc region containing the Hole mutation also contains the L351Y and D339R mutations, and the Fc region containing the Knob mutation also contains the K409D mutation.

[0230] The amino acid residues are numbered according to the EU index in Kabat.

[0231] In some further embodiments, the first Fc region contains T366W, and the second Fc region contains T366S-L368A-Y407V-L351Y; or the first Fc region contains T366W-K409D, and the second Fc region contains T366S-L368A-Y407V-L351Y-D399R. In further embodiments, the Fc region containing the knob mutation also contains R355Q and Q419E mutations. It has been shown that R355Q and Q419E mutations are beneficial for reducing the isoelectric point (PI) of the Knob chain. Therefore, in some embodiments, the antibody of the present invention comprises an Fc dimer having first and second immunoglobulin Fc regions, wherein the first Fc region comprises T366S-L368A-Y407V-L351Y-D399R and the second Fc region comprises T366W-K409D-R355Q-Q419E; or vice versa.

[0232] In some implementations, the Fc region containing the mutation is an IgG isotype, preferably an IgG1 or IgG4 isotype, especially a human IgG1 or IgG4 isotype.

[0233] In some embodiments, the antibody of the present invention comprises an Fc dimer having first and second immunoglobulin Fc regions, wherein the first and second Fc regions respectively comprise the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:31, or amino acid sequences that are at least 95%, 96%, 98%, or 99% identical thereto. In other embodiments, the antibody of the present invention comprises an Fc dimer having first and second immunoglobulin Fc regions, wherein the first and second Fc regions respectively comprise the amino acid sequences of SEQ ID NO:31 and SEQ ID NO:29, or amino acid sequences that are at least 95%, 96%, 98%, or 99% identical thereto.

[0234] According to the specific application of the antibody or antibody-based molecule of the present invention, the Fc region may also contain other mutations, such as mutations to increase binding to FcRn and / or remove protease sites, and / or introduce amino acid modifications that can be used to couple active molecules. Additionally or alternatively, the Fc region may be mutated to remove or replace amino acids that may undergo post-translational modifications (e.g., glycosylation) to provide improved druggability and developability of the therapeutic antibody.

[0235] Peptide linkers (connectors)

[0236] In the multispecific antibody according to the invention, antibody components (i.e., the antigen-binding domain and optionally immunoglobulin Fc) can be linked using a flexible peptide linker (also referred to as a linker) composed of natural amino acid residues. There are no specific limitations on the linkers that can be used in the antibodies of the invention. The linker sequence is generally flexible. It can consist primarily of amino acids with large side chains that do not limit flexibility, such as glycine, alanine, and serine. Alternatively, it can consist of sequences from the hinge region of immunoglobulins. Depending on the linking location and the components to be linked, those skilled in the art can readily determine the available linker sequence or optimal length. Suitable linker lengths can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid lengths, or longer. In some cases, the linker sequence can be short, for example, less than about 20 or 15 amino acids long, such as 2-15 amino acids or 5-10 amino acids long. Suitable linker sequences include, but are not limited to, (G4S)n (SEQ ID NO:57), where n is an integer equal to or greater than 1; TS(G4S)n (SEQ ID NO:58), where n is an integer equal to or greater than 1; G(G4S)n (SEQ ID NO:59), where n is an integer equal to or greater than 1; (G4)n (SEQ ID NO:60), where n is an integer equal to or greater than 1; and (GRPGS)n (SEQ ID NO:61), where n is an integer equal to or greater than 1.

[0237] antibody configuration

[0238] The anti-B7-H3 and EGFR multispecific antibodies according to the present invention can take any suitable form, such as single-chain or multi-chain, symmetrical or asymmetrical, IgG or non-IgG conformation. The valence (i.e., the total number of antigen-binding domains) of the multispecific antibodies of the present invention can be divalent, trivalent, quadrivalent, or even more valent. In the multispecific antibodies according to the present invention, the ratio of the number or valence of B7-H3 antigen-binding domains to EGFR antigen-binding domains can be adjusted as appropriate (e.g., according to the binding affinity of each domain and the expression of B7-H3 and EGFR antigens on tumor cells), including but not limited to 1:1, 1:2, 2:1, and 2:2. In some cases, preferably, the valence ratio is 1:1. The multispecific antibodies according to the present invention can be bispecific, or they can have binding specificity targeting other antigens in addition to the B7-H3 and EGFR antigen-binding domains, thus exhibiting trispecificity or more specificity. In some embodiments, preferably, the multispecific antibody according to the invention is a bivalent antibody comprising one of the high-affinity B7-H3 binding domains and one of the intermediate-affinity EGFR binding domains.

[0239] In some embodiments, the multispecific antibody according to the present invention comprises:

[0240] -A first structural portion comprising, from the N-terminus to the C-terminus, the following components: a first antigen-binding domain and a first immunoglobulin Fc region; and

[0241] -A second structural portion from the N-terminus to the C-terminus contains the following components: a second antigen-binding domain and a second immunoglobulin Fc region;

[0242] The first and second immunoglobulin Fc regions dimerize to form Fc dimers, wherein the first and second Fc regions are defined as described above.

[0243] The first and second antigen-binding domains bind to different antigens selected from B7-H3 and EGFR, respectively. In some embodiments, the first antigen-binding domain binds to B7-H3 and comprises or is composed of Fab, scFab, or scFv domains. In other embodiments, the second antigen-binding domain binds to EGFR and comprises or is composed of Fab, scFab, scFv, or VHH domains. In some embodiments, the antibody is an IgG conformation antibody comprising two heavy chains and two light chains, optionally wherein the two heavy chains have a tripeptide LSP, SPG, or PGK as the C-terminal amino acid residue. Preferably, the antibody is a bispecific antibody having the structure shown in Figure 1; in some embodiments, the heavy chain containing the Hole mutation and the heavy chain containing the Knob mutation respectively comprise the heavy chain constant regions of SEQ ID NO:28 and SEQ ID NO:30, or the heavy chain constant regions of SEQ ID NO:52 and SEQ ID NO:53, respectively.

[0244] In some embodiments, the multispecific antibody according to the present invention comprises a B7-H3 arm providing at least one B7-H3 antigen-binding domain and an EGFR arm providing at least one EGFR antigen-binding domain. In some embodiments, the multispecific antibody according to the present invention comprises:

[0245] -A first structural portion comprising, from the N-terminus to the C-terminus, the following components: a B7-H3 antigen-binding domain and a first immunoglobulin Fc region; and

[0246] -A second structural portion from the N-terminus to the C-terminus contains the following components: an EGFR antigen-binding domain and a second immunoglobulin Fc region;

[0247] The first and second immunoglobulin Fc regions dimerize to form Fc dimers. In some embodiments, preferably, the B7-H3 antigen-binding domain is a Fab domain that binds B7-H3, and the EGFR antigen-binding domain is a Fab domain that binds EGFR.

[0248] In any of the above embodiments, the B7-H3 antigen-binding domain may be the B7-H3 antigen-binding domain according to any of the foregoing embodiments of the present invention. Preferably, the B7-H3 antigen-binding domain comprises: HCDR1, HCDR2, and HCDR3 respectively comprising or composed of the amino acid sequences shown in SEQ ID NOs:8-10; and LCDR1, LCDR2, and LCDR3 respectively comprising or composed of the amino acid sequences shown in SEQ ID NOs:5-7. In some further embodiments, the B7-H3 antigen-binding domain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:4, or having at least 90%, 95%, or 99% identity with it, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions. Preferably, the B7-H3 antigen-binding domain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:4.

[0249] In any of the above embodiments, the EGFR antigen-binding domain may be an EGFR antigen-binding domain according to any of the foregoing embodiments of the present invention. Preferably, the binding affinity K of the EGFR antigen-binding domain to human EGFR antigen is K. D Value greater than 1x10 -10 M is preferably 1x10 -7 M to 1x10 -9 M, optionally 1-6x10 -8 M or 1-6x10 -9 M. In some embodiments, preferably, the EGFR antigen-binding domain comprises: HCDR1, HCDR2, and HCDR3, respectively comprising or composed of the amino acid sequences shown in SEQ ID NOs:20-22, and LCDR1, LCDR2, and LCDR3, respectively comprising or composed of the amino acid sequences shown in SEQ ID NOs:17-19. More preferably, the EGFR antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein VH and VL comprise or composed of the amino acid sequences shown in SEQ ID NO:13 and SEQ ID NO:14, respectively; or VH and VL comprise or composed of the amino acid sequences shown in SEQ ID NO:15 and SEQ ID NO:16, respectively.

[0250] Exemplary anti-B7-H3 and EGFR antibodies

[0251] In some embodiments, the present invention provides anti-B7-H3 and EGFR antibodies, said antibodies comprising a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain and the first light chain pair and specifically bind to B7-H3; the second heavy chain and the second light chain pair and specifically bind to EGFR, and

[0252] The first heavy chain, the first light chain, the second heavy chain, and the second light chain each contain or consist of the following amino acid sequences:

[0253] (i) The first heavy chain comprises an amino acid sequence of SEQ ID NO: 24 or 54, or has at least 90%, 95% or 99% identity with it, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of it;

[0254] (ii) The first light chain comprises the amino acid sequence of SEQ ID NO: 23, or has at least 90%, 95% or 99% identity with it, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of it;

[0255] (iii) The second heavy chain comprises an amino acid sequence of SEQ ID NO: 26, 27, 55 or 56, or has at least 90%, 95% or 99% identity with it, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of it;

[0256] (iv) The second light chain comprises the amino acid sequence of SEQ ID NO: 25, or has at least 90%, 95% or 99% identity with it, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of it.

[0257] In some preferred embodiments, the present invention provides anti-B7-H3 and EGFR antibodies, wherein the antibodies comprise a first heavy chain, a first light chain, a second heavy chain, and a second light chain, and wherein the first heavy chain, the first light chain, the second heavy chain, and the second light chain each comprise the amino acid sequences of SEQ ID NOs:24,23,26, and 25, or the amino acid sequences of SEQ ID NOs:54,23,56, and 25, or are composed of the latter.

[0258] In some other preferred embodiments, the present invention provides anti-B7-H3 and EGFR antibodies, wherein the antibodies comprise a first heavy chain, a first light chain, a second heavy chain, and a second light chain, and wherein the first heavy chain, the first light chain, the second heavy chain, and the second light chain comprise, or consist of, the amino acid sequences of SEQ ID NOs:24,23,27, and 25, or the amino acid sequences of SEQ ID NOs:54,23,57, and 25, respectively.

[0259] Properties of anti-B7-H3 and EGFR antibodies

[0260] In some embodiments, the anti-B7-H3 and EGFR antibodies of the present invention comprise an anti-B7-H3 binding domain derived from an anti-B7-H3 parent antibody and an EGFR binding domain derived from an anti-EGFR parent antibody.

[0261] In some embodiments, the anti-B7-H3 and EGFR antibodies of the present invention have one or more of the following properties:

[0262] (i) with K D It has a high affinity for and specifically binds to the 2Ig and 4Ig subtypes of human B7-H3 with a value less than 0.01 nM;

[0263] (ii) with K D Medium affinity specifically binds to human EGFR with values ​​greater than 1 nM or 10 nM;

[0264] (iii) It specifically binds to tumor cells co-expressing B7-H3 and EGFR;

[0265] (iv) Compared to the corresponding monospecific anti-B7-H3 antibody parent and / or anti-EGFR antibody parent, it has enhanced activity in being internalized by B7-H3 and EGFR double-positive tumors.

[0266] In some further embodiments, the multispecific antibody of the present invention also has one or more of the following properties:

[0267] (v) Specifically binds to extracellular domains I and II of EGFR;

[0268] (vi) It does not inhibit the binding of EGF to EGFR;

[0269] (vii) exhibits immune cross-reactivity with monkey B7-H3 and EGFR.

[0270] In some aspects, the multispecific antibodies of the present invention specifically bind to tumor cells co-expressing B7-H3 and EGFR. The EC50 value and / or maximum binding amount of the antibodies of the present invention to B7-H3 and EGFR-positive tumor cells can be determined by FACS assays (e.g., the assays described in Example 6), and optionally compared with a reference antibody, to reflect the cell-binding activity of the antibodies. In some embodiments, the multispecific antibodies of the present invention have enhanced binding to B7-H3 and EGFR double-positive tumor cells compared to the corresponding monospecific parental B7-H3 antibodies. In other embodiments, the multispecific antibodies of the present invention have enhanced binding to anti-B7-H3 and EGFR double-positive tumor cells compared to the corresponding monospecific parental EGFR antibodies. In some embodiments, the multispecific antibodies of the present invention have enhanced binding to anti-B7-H3 and EGFR double-positive tumor cells compared to a combination of the corresponding monospecific parental B7-H3 antibodies and EGFR antibodies.

[0271] In some aspects, the multispecific antibody of the present invention is capable of being internalized by B7-H3 and EGFR double-positive tumor cells. The internalization rate of the antibody can be determined by FACS assay (e.g., the assay described in Example 6) and optionally compared with a reference antibody to reflect the antibody's internalization activity. In some embodiments, the B7-H3 binding domain and the EGFR binding domain in the multispecific antibody of the present invention synergize with each other in terms of this internalization activity. In some embodiments, the multispecific antibody of the present invention exhibits enhanced internalization activity on B7-H3 and EGFR double-positive tumor cells compared to the corresponding monospecific parental B7-H3 antibody. In other embodiments, the multispecific antibody of the present invention exhibits enhanced internalization activity on B7-H3 and EGFR double-positive tumor cells compared to the corresponding monospecific parental EGFR antibody. In some embodiments, the multispecific antibody of the present invention exhibits enhanced internalization activity on B7-H3 and EGFR double-positive tumor cells compared to a combination of the corresponding monospecific parental B7-H3 antibody and EGFR antibody.

[0272] In some further embodiments, the multispecific antibody of the present invention also has one or more of the following characteristics: (viii) good developability; (ix) good stability; and (x) favorable pharmacokinetic properties.

[0273] In some embodiments, the multispecific antibodies of the present invention achieve a purity of 90% or 95% or higher, for example, 98% or higher, as determined by SEC-HPLC. In other embodiments, the multispecific antibodies of the present invention achieve a purity of 90% or 95% or higher, for example, 98% or higher, as determined by RP-HPLC. The multispecific antibodies of the present invention can be prepared and purified according to the method shown in Example 4, and their purity can be determined.

[0274] II. Anti-B7-H3 antibody

[0275] In a second aspect, the present invention provides an anti-B7-H3 antibody that binds to B7-H3. In some embodiments, the anti-B7-H3 antibody comprises a B7-H3 antigen-binding domain as described in the first aspect of the present invention.

[0276] In some embodiments, the anti-B7-H3 antibody according to the present invention comprises VH and VL domains that specifically bind to B7-H3, wherein the VH and VL domains comprise three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in the heavy chain variable region (VH) and light chain variable region (VL) sequence pairs selected from SEQ ID NOs:1 / 2, SEQ ID NOs:3 / 4, SEQ ID NOs:36 / 37, or SEQ ID NOs:38 / 39.

[0277] In some embodiments, the anti-B7-H3 antibody according to the present invention comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3).

[0278] -HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:8;

[0279] -HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:9;

[0280] -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:10;

[0281] -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:5;

[0282] -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:6 or 34;

[0283] -LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:7;

[0284] In some embodiments, the anti-B7-H3 antibody according to the present invention comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3).

[0285] -HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:43;

[0286] -HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:44;

[0287] -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:45;

[0288] -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:40;

[0289] -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:41;

[0290] -LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:42.

[0291] In some embodiments, the anti-B7-H3 antibody according to the present invention comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3).

[0292] -HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:49;

[0293] -HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:50;

[0294] -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:51;

[0295] -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:46;

[0296] -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:47;

[0297] -LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:48.

[0298] Preferably, HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NOs:8-10 and SEQ ID NOs:5-7, or are composed of.

[0299] In some embodiments, the anti-B7-H3 antibody according to the present invention comprises:

[0300] (i) a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 3, 36 or 38, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of therewith; and / or

[0301] (ii) Light chain variable region (VL), wherein the VL comprises an amino acid sequence selected from SEQ ID NOs:24, 37 or 39, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the VL.

[0302] In some embodiments, the anti-B7-H3 antibody according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the VH and VL respectively comprise the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2, or have at least 90%, 95%, or 99% identity with respect to them, or have one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acid sequences. Preferably, the anti-B7-H3 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:2.

[0303] In some embodiments, the anti-B7-H3 antibody according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the VH and VL respectively comprise the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, or have at least 90%, 95%, or 99% identity with respect to them, or have one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acid sequences. Preferably, the anti-B7-H3 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:4.

[0304] In some embodiments, the anti-B7-H3 antibody according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the VH and VL respectively comprise the amino acid sequences of SEQ ID NO:36 and SEQ ID NO:37, or have at least 90%, 95%, or 99% identity with respect to them, or have one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acid sequences. Preferably, the anti-B7-H3 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:36 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:37.

[0305] In some embodiments, the anti-B7-H3 antibody according to the invention comprises a heavy chain variable region and a light chain variable region, wherein the VH and VL respectively comprise the amino acid sequences of SEQ ID NO:38 and SEQ ID NO:39, or have at least 90%, 95%, or 99% identity with respect to them, or have one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acid sequences. Preferably, the anti-B7-H3 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:38 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:39.

[0306] In some preferred embodiments, the anti-B7-H3 antibody according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein the VH and VL respectively comprise the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, or have at least 90%, 95% or 99% identity with respect to them, or have one or more (preferably 1-10, more preferably 1-5) amino acid sequences with additions, deletions and / or substitutions.

[0307] In some preferred embodiments, the anti-B7-H3 antibody according to the present invention comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:4.

[0308] The anti-B7-H3 antibody according to the present invention can have any suitable antibody structure, including, but not limited to, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), linear antibodies; chimeric antibodies or humanized antibodies. In some embodiments, the anti-B7-H3 antibody according to the present invention comprises an immunoglobulin Fc region linked to its VH or VL domain. In some embodiments, the anti-B7-H3 antibody according to the present invention is a full-length antibody in the IgG configuration.

[0309] Fragments of the anti-B7-H3 antibody according to the present invention include, but are not limited to, Fab, scFab, disulfide-linked scFab, Fab', F(ab')2, Fab'-SH, Fv, scFv, disulfide-linked scFv, linear antibodies, diabody, triabody, tetrabody, minibody; and single-chain antibodies (e.g., scFv, scFab). In some embodiments, the anti-B7-H3 antibody fragment according to the present invention comprises a Fab domain.

[0310] The anti-B7-H3 antibody according to the present invention exhibits good tumor targeting and high tumor cell binding affinity, and possesses cross-reactivity with both human and monkey species. Therefore, the anti-B7-H3 antibody or its antigen-binding domain according to the present invention can serve as a module for constructing bispecific, multispecific, or ADC conjugates or conjugates to provide better applications such as tumor killing, immune modulation, or disease detection.

[0311] III. Polynucleotide, Vector, Host, and Antibody Production Methods

[0312] In a third aspect, this disclosure provides nucleic acids encoding antibodies or fragments thereof according to the first and second aspects of the present invention, vectors containing said nucleic acids, and host cells containing said nucleic acids or said vectors, as well as methods for their preparation. As will be apparent to those skilled in the art, due to codon degeneracy, the amino acid sequence of each antibody or polypeptide chain can be encoded by multiple nucleic acid sequences.

[0313] In some embodiments, this disclosure provides one or more polynucleotides encoding an anti-B7-H3 antibody or an anti-B7-H3 and EGFR antibody or fragments thereof according to the invention. In some embodiments, this disclosure provides one or more vectors comprising the one or more polynucleotides. In some embodiments, the one or more polynucleotides are present in a single vector; in other embodiments, the one or more polynucleotides are present in multiple separate vectors. In some embodiments, the invention also provides host cells comprising the one or more nucleic acids or the one or more vectors. In embodiments involving multispecific antibodies according to the invention, depending on the circumstances, a single or multiple vectors comprising the one or more nucleotides may be introduced into the same host cell to express the desired product in one host cell; or a single or multiple vectors comprising the one or more polynucleotides may be introduced separately into different host cells to express, respectively, intermediates (e.g., haptens) comprising different chains or combinations of different chains of the antibody according to the invention in different host cells, and, under conditions suitable for assembling the antibody according to the invention, the multispecific antibody according to the invention is produced by mixing the intermediates.

[0314] In this disclosure, there are no particular limitations on the vectors that can be used, including cloning vectors and expression vectors. An example of a suitable expression vector is a eukaryotic expression vector, including but not limited to viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In this disclosure, there are no particular limitations on the host cells that can be used. Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as mammalian host cells, insect cells, etc. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7), human embryonic kidney (HEK293 or 293F cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / 0. In some embodiments, mammalian cell lines suitable for suspension culture may be used. In some embodiments, the host cell is CHO or HEK293 cells.

[0315] In some embodiments, this disclosure provides methods for producing the anti-B7-H3 antibody or anti-B7-H3 and EGFR antibody or fragments thereof of the present invention. To produce the antibody of the present invention, the polypeptide chain constituting the antibody of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions. As described above, in the case of recombinant production, the assembly can occur in a host cell for expressing the polypeptide chain; or, if necessary, the assembly of the antibody of the present invention can be performed in vitro after harvesting the expressed intermediate polypeptide chain from the host cell.

[0316] In some embodiments, this disclosure provides a method for producing the antibody of the present invention, the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the antibody; and assembling the polypeptide chain to produce the antibody under conditions suitable for assembling the polypeptide chain into the antibody.

[0317] The antibodies prepared by the method described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. After purification, the purity of the antibody protein of this invention can be determined by any of a variety of well-known analytical methods, such as SEC-HPLC or RP-HPLC.

[0318] The physical / chemical properties and / or biological activity of the antibody molecules provided herein can be identified, screened, or characterized using a variety of assays known in the art.

[0319] IV. Immunoconjugates and immune fusion compounds

[0320] In a fourth aspect, this disclosure provides immunofusions or immunoconjugates produced by fusing or conjugating antibodies according to the first and second aspects of the invention to a heterologous molecule.

[0321] In one embodiment, in the immunofusion, the antibody (or its antigen-binding fragment) of the present invention is linked to a heterologous peptide or polypeptide molecule directly or via an amino acid linker. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that impart another functional activity to the fusion, or tag peptides that facilitate the purification or detection of the immunofusion.

[0322] In one embodiment, in the immunoconjugate, the antibody (or its antigen-binding fragment) of the present invention is conjugated to a therapeutic agent, diagnostic agent, or detectable agent. In the conjugate, linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers, or combinations thereof. Advantageously, the linker is a “cleavable linker” that facilitates the release of the load upon delivery to the target site. For example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used.

[0323] In embodiments where a therapeutic agent is conjugated, the therapeutic agent suitable for the conjugation includes, but is not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), drugs, or radioisotopes.

[0324] In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnostics and detections can be achieved by conjugating antibodies to detectable agents, including but not limited to a variety of enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0325] The antibody-drug conjugate of the present invention

[0326] In a fifth aspect, the present invention provides antibody-drug conjugates (ADCs) comprising the structure of formula (I) below: Ab-(LD) n (I)

[0327] in,

[0328] Ab represents an antibody according to the first and second aspects of the present invention, preferably an antibody according to the first aspect of the present invention;

[0329] L represents the connector;

[0330] D represents a drug, preferably an antitumor compound, such as a cytotoxic drug or a cell-inhibiting drug, especially a topoisomerase I inhibitor; and

[0331] n represents an integer from 1 to 16, for example, n = 1-10, 1-8, 3-8, 4-8, or 6-8.

[0332] The components of the ADC coupling of the present invention and the ADC couplings of the present invention composed therefrom are described in detail below. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, the ADC coupling of the present invention may include any such combination of features.

[0333] Antibody Ab Unit

[0334] In some embodiments, the present invention provides antibody-drug conjugates (ADCs) comprising an antibody according to the first or second aspect of the present invention as the Ab unit of a conjugate of formula I. The antibody of the present invention used as the Ab unit can be of any suitable conformation, including but not limited to, single-chain or multi-chain, having or lacking an immunoglobulin Fc region, symmetrical or asymmetrical, monospecific, bispecific, or multispecific, as long as it has the efficacy of delivering the loaded drug to the target tumor tissue. In some embodiments, preferably, the Ab unit in the ADC of formula (I) according to the present invention is an anti-B7-H3 and EGFR antibody according to the first aspect of the present invention.

[0335] In some embodiments, the Ab unit comprises at least one B7-H3 binding domain according to the invention and at least one EGFR binding domain according to the invention. In some embodiments, the B7-H3 binding domain comprises HCDR1-3 and LCDR1-3 domains selected from the following amino acid sequence groups: (a) SEQ ID NOs:8-10 and SEQ ID NOs:5-7; (b) SEQ ID NOs:8-10 and SEQ ID NOs:5, 34 and 7; (c) SEQ ID NOs:43-45 and SEQ ID NOs:40-42; or (d) SEQ ID NOs:49-50 and SEQ ID NOs:46-48. In some embodiments, the EGFR binding domain comprises HCDR1-3 and LCDR1-3 domains selected from the following amino acid sequence groups: (i) SEQ ID NOs:20-22 and SEQ ID NOs:17-19; and (ii) SEQ ID NOs:20, 35, 22 and SEQ ID NOs:17-19.

[0336] In some embodiments, the Ab unit comprises at least one B7-H3 binding domain according to the invention and at least one EGFR binding domain according to the invention. In some embodiments, the B7-H3 binding domain comprises VH and VL domains selected from the following amino acid sequence pairs: SEQ ID NOs:1 / 2; SEQ ID NOs:3 / 4; SEQ ID NOs:36 / 37; or SEQ ID NOs:38 / 39. In some embodiments, the EGFR binding domain comprises VH and VL domains selected from the following amino acid sequence pairs: SEQ ID NOs:11 / 12; SEQ ID NOs:13 / 14; or SEQ ID NOs:15 / 16.

[0337] In some embodiments, the Ab unit comprises at least one B7-H3 binding domain and at least one EGFR binding domain according to the invention. In some embodiments, the B7-H3 binding domain comprises: HCDR1-3 and LCDR1-3 domains comprising the amino acid sequences shown in SEQ ID NOs:8-10 and SEQ ID NOs:5-7, respectively. In some embodiments, the EGFR binding domain comprises: HCDR1-3 and LCDR1-3 domains comprising the amino acid sequences shown in SEQ ID NOs:20-22 and SEQ ID NOs:17-19, respectively.

[0338] In some embodiments, the Ab unit comprises at least one B7-H3 binding domain according to the invention and at least one EGFR binding domain according to the invention. In some embodiments, the B7-H3 binding domain comprises a heavy chain variable region and a light chain variable region having amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, respectively; and the EGFR binding domain comprises a heavy chain variable region and a light chain variable region having amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0339] In some embodiments, the Ab unit comprises at least one B7-H3 binding domain according to the invention and at least one EGFR binding domain according to the invention. In some embodiments, the B7-H3 binding domain comprises a heavy chain variable region and a light chain variable region having amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, respectively; and the EGFR binding domain comprises a heavy chain variable region and a light chain variable region having amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16, respectively.

[0340] In some embodiments, the Ab unit comprises an antibody according to the invention having an IgG conformation or the conformation shown in FIG1. ​​In some embodiments, the Ab unit comprises a high-affinity B7-H3 binding domain (K). D (Value less than 0.01 nM) and one medium-affinity EGFR binding domain (K D (Value less than 1 nM). In some cases, the heavy chain constant region and light chain constant region contained in the Ab unit are heavy chain and light chain constant regions derived from human immunoglobulins. Preferably, the Ab unit contains a heavy chain constant region of IgG1 or IgG4 isotype, such as the heavy chain constant region of human IgG1 isotype. In some embodiments, the two heavy chains of the Ab unit have heavy chain constant regions of SEQ ID NO:28 and SEQ ID NO:30, respectively, or heavy chain constant regions of SEQ ID NO:52 and SEQ ID NO:53, respectively.

[0341] In some embodiments, the Ab unit comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain and the first light chain pair up and specifically bind B7-H3; the second heavy chain and the second light chain pair up and specifically bind EGFR, and wherein the first heavy chain, the first light chain, the second heavy chain, and the second light chain each comprise or substantially comprise or consist of the following amino acid sequences: (i) amino acid sequences of SEQ ID NOs: 24, 23, 26, and 25; (ii) amino acid sequences of SEQ ID NOs: 54, 23, 56, and 25; (iii) amino acid sequences of SEQ ID NOs: 24, 23, 27, and 25; (iv) amino acid sequences of SEQ ID NOs: 54, 23, 57, and 25; or (v) amino acid sequences having at least 90%, 95%, or 99% identity with (i), (ii), (iii), or (iv).

[0342] In some preferred embodiments, the Ab unit comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs:24,23,26, and 25, respectively.

[0343] In some other preferred embodiments, the Ab unit comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs:54,23,55, and 25, respectively.

[0344] In some other preferred embodiments, the Ab unit comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs:24,23,27, and 25, respectively.

[0345] In some other preferred embodiments, the Ab unit comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs:54,23,56, and 25, respectively.

[0346] To form ADC conjugates, the antibodies according to the present invention can be conjugated to a drug-linker using some natural linker sites thereon. Such natural linker sites include the thiol group of cysteine ​​and the amino group of lysine. Typically, a relatively defined drug-antibody ratio (DAR) can be achieved by using interchain disulfide bridges of the antibody. Therefore, in some embodiments, after reducing the interchain disulfide bonds of the antibody, the drug is conjugated to the antibody of the present invention via thiol chemistry to form an ADC conjugate of formula (I). Alternatively, introducing artificial linker sites into the antibody can also be considered to achieve more site-specific conjugation.

[0347] Drug D Unit

[0348] The drug D unit of the antibody-drug conjugate is also referred to herein as the payload of the ADC drug. There are no particular limitations on the drug D that can be used in the ADC of this invention; it can be any drug or prodrug that is toxic or inhibitory to cells. Those skilled in the art can select appropriate drug molecules as the payload of the ADC based on the desired mechanism of action and cell-killing effect.

[0349] In some embodiments, drug D is a cytotoxic agent. Various cytotoxic agents with different mechanisms suitable as payloads have been reported in the art, including, but not limited to, (1) microtubule inhibitors / disruptors; (2) DNA damaging agents; and (3) topoisomerase inhibitors. Drug D in formula (I) of the present invention can be a pharmaceutical compound selected from these categories.

[0350] In some embodiments, the drug D of the ADC according to the invention is a topoisomerase I inhibitor. A typical example of a topoisomerase I inhibitor is a camptothecin class of drugs, such as, but not limited to, camptothecin (CPT), hydroxycamptothecin, 9-aminocamptothecin, exatecan, topootecan, belutecan, irinotecan, SN-38, and derivatives thereof. See, for example, Vesela Kostova et al., The Chemistry Behind ADCs, Pharmaceuticals 2021, 14, 442. https: / / doi.org / 10.3390 / ph14050442; and WO2019 / 195665, WO2023 / 083381, all of which are incorporated herein by reference.

[0351] In some embodiments, the drug D unit of the ADC of the present invention is Exatecan and its derivatives, for example, Among them, R D Selected from, for example, H, optionally substituted alkyl, optionally substituted alkyl-C(=O)-, optionally substituted alkyl-OC(=O)-, wherein the substituent is, for example, -OH or a substituted or unsubstituted amino group;

[0352] In one implementation scheme, the drug D is:

[0353] In another embodiment, the drug D is:

[0354] In some preferred embodiments, the drug D unit of the ADC of the present invention is an exatecan derivative comprising the structure shown in formula (D-1):

[0355] in:

[0356] R a1 Selected from hydrogen atoms, tritium atoms, and C1-C6 alkyl groups;

[0357] R a2 Selected from key, -C(=O)-CR 1 R 2 -(CR 3 R 4 ) m -O-*,-C(=O)-CR 1 R 2 -(CR 3 R 4 ) m -NH-*,-C(=O)-O-CR 1 R 2 -(CR 3 R 4 ) m -O-*,-C(=O)-O-CR 1 R 2 -(CR 3 R 4 ) m -NH-*,

[0358] Where R 1 and R 2 Each is independently selected from hydrogen atoms, C1-C6 alkyl groups, halogenated C1-C6 alkyl groups, C3-C8 cycloalkyl groups, C3-C8 cycloalkyl groups, C1-C6 alkyl groups, and C6-C6 alkyl groups. 10 aryl C1-C6 alkyl; or R 1 and R 2 It forms a C3-C8 cycloalkyl group with the carbon atom it is attached to;

[0359] R 3 and R 4 Each is independently selected from hydrogen atoms and C1-C6 alkyl groups;

[0360] m is an integer between 0 and 6;

[0361] R a3 Selected from H, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -OR 5 and -SR 5 ;

[0362] R a4 Selected from H, halogen, CN, C1-C6 alkyl, halogenated C1-C6 alkyl and -OR 5 ;and

[0363] R 5Independently selected from H and C1-C4 alkyl groups,

[0364] Where * indicates the connection site with the connector L unit.

[0365] In some implementation schemes, R a2 It is -C(=O)-O-CH2-(CH2) m -O-, where m = an integer from 0 to 6. In some further implementations, R a2 It is -C(=O)-O-(CH2) 4-6 -O-. In some further implementations, R a2 It is -C(=O)-O-(CH2)4-O-.

[0366] In some implementation schemes, R a2 It is -C(=O)-O-CH2-(CH2) m -O-,R a1 For H; R a3 It is a C1-C6 alkyl group; and R a4 It is a halogen. In some further embodiments, R a3 It is methyl. In some further embodiments, R a4 For F. In some preferred embodiments, R a2 It is -C(=O)-O-(CH2) 4-6 -O-;R a1 For H; R a3 Methyl; R a4 It is F.

[0367] In some embodiments, the drug unit D has the structure shown in formula (D-1a) or formula (D-1b):

[0368] Where R a1 R a2 R a3 and R a4 As defined above, * indicates the connection site with the L unit of the connector.

[0369] In some preferred embodiments, the drug unit D has the structure shown in formula (D-2):

[0370] Preferably, D has the structure shown in formula (D-2a) or formula (D-2b):

[0371] In some other preferred embodiments, the drug unit D has the structure shown in formula (D-3):

[0372] Preferably, D has the structure shown in formula (D-3a) or formula (D-3b), especially the structure shown in formula (D-3a):

[0373] The camptothecin compounds applicable to this disclosure can be prepared and their activity determined by referring to the embodiments of this disclosure and the methods disclosed in patent applications WO2024 / 175069, WO2023 / 083381, Vesela Kostova et al., The Chemistry Behind ADCs, Pharmaceuticals 2021, 14, 442. https: / / doi.org / 10.3390 / ph14050442; and WO2019 / 195665. These patent applications and documents are hereby incorporated herein by reference in their entirety.

[0374] Connector L unit

[0375] The linker L suitable for use in this invention can be any linker capable of conjugating the antibody of this invention to a drug. Suitably, the addition of the linker should ensure adequate stability of the ADC of this invention in the circulatory system, while providing rapid and efficient release of the active form of the drug at the target site (e.g., tumor cells or the tumor environment).

[0376] In some embodiments, the linker L in formula (I) of the present invention is a degradable linker. Typically, such a degradable linker consists of a coupling portion, a degradable portion, and optionally a spacer portion. The coupling portion is responsible for the connection between the antibody and the linker-drug and can be selected according to the desired antibody-drug conjugation chemistry. The degradable portion will contain a peptide or peptide analog that can be recognized by an enzyme in the event of an enzyme-based release mechanism, such as oligopeptides or oligopeptide analogs that can be cleaved by proteolytic enzymes, such as Val-Ala, Val-Cit, Phe-Lys, Gly-Phe-Leu-Gly (SEQ ID NO:62), Ala-Leu-Ala-Leu (SEQ ID NO:63), Gly-Gly-Phe-Gly (SEQ ID NO:64), cyclobutyl-Ala, cyclobutyl-Cit, etc. In this document, such enzyme-degradable linkers based on peptides or peptide analogs are also referred to as "enzyme-cleavable peptide linkers". In some cases, spacer groups are introduced between the degradable portion of the linker and the drug D, as needed, to promote the release (especially, traceless release) of the remaining portion of the self-coupled drug active molecule. Examples include spontaneously eliminable p-aminobenzylcarbamate (PABA) or aminomethylene (-NHCH2-) spacer groups in acidic media. Furthermore, in cases where the drug is highly hydrophobic, the addition of units such as PEG may be considered (but not required) to improve the properties of the ADC, such as reducing precipitation and aggregation. Linkers applicable to this invention include, but are not limited to, those disclosed in WO2024 / 175069, PCT / CN2023 / 107489, and WO2018 / 025168 (these documents are hereby incorporated by reference).

[0377] In some embodiments, the connector L in formula (I) of the present invention has the structure of formula (II): -ZYM- (II),

[0378] in

[0379] Z is the linker base connected to Ab.

[0380] Y is an enzyme-cleavable peptide linker containing amino acids.

[0381] M is absent, or it is a spacer group used to link with drug D.

[0382] According to the present invention, the linker L may, in some aspects, comprise a Z linker group of a heterocyclic (e.g., maleimide) or a heterocyclic (e.g., pyrimidine and pyridothiazole) class; and, in some cases, preferably comprise a heterocyclic linker group to provide conjugates with higher stability in blood circulation. For this purpose, the linker comprising a leaving group-substituted heterocyclic ring can be subjected to a nucleophilic substitution reaction with a free thiol group in an antibody molecule to obtain an antibody-drug conjugate.

[0383] Z unit

[0384] In some implementations, Z in equation (II), in the direction from the Ab element side to the Y element side, has the following structure:

[0385] -Z1-Z2-Z3-Z4-,

[0386] in,

[0387] Z1 is a 5-10 membered heterocyclic group, preferably containing 1-3 heteroatoms selected from N, S, and O; more preferably, Z1 is selected from

[0388] Z2 is selected from key, -C(=O)-, *-C1-C 10 Alkylene -C(=O)-, *-C3-C 10 Ethyne-C(=O)-, *-C3-C 10 alkenyl-C(=O)- and *-heteroaryl-C1-C 10 Alkylene-C(=O)-,

[0389] Z3 does not exist or is *-R 6 -(CH2-CH2-O-) x -R 7 - where x is an integer from 0 to 5, preferably x = 1 or 2;

[0390] Z4 does not exist or is *-R 8 -(CH2-CH2-O-) y -R 9 - where y is an integer from 0 to 12, preferably y = 6, 7, 8, 9 or 10;

[0391] R 6 and R 8 Each is independently selected from key, C 1-4 Alkylene, -NH-, -heteroaryl-, *-NH-C 1-4 alkylene-heteroaryl-, wherein the heteroaryl group is a 5- or 6-membered nitrogen-containing heteroaryl group, preferably a triazolyl group; R 7 and R 9 Independently selected from -C(=O)-, *-C(=O)-NH-, C 1-4 Alkylene, *-C 1-4 Alkylene-C(=O)-, *-C 1-4 Alkylene -C(=O)-NH-, *-NH-C(=O)-(CH2OCH2)-C(=O)-, *-C 1-4 Alkylene-NH-C(=O)-(CH2OCH2)-C(=O)-,

[0392] Preferably R 6 and R 8 Each is independently selected from -NH-, and R 7 and R 9 Independently selected from -C(=O)-, *-C 1-4 Alkylene-C(=O)-, *-C 1-4 Alkylene-NH-C(=O)-(CH2OCH2)-C(=O)-,

[0393] The heteroaryl group is a 5- or 6-membered nitrogen-containing heteroaryl group, preferably a triazole group.

[0394] Where * indicates the connection site facing the Ab unit side.

[0395] In some embodiments, Z1 is a 5-10 membered heteroaryl group. In some embodiments, Z1 is a 5-10 membered heteroaryl group comprising 1-3 heteroatoms selected from N, S, and O. In some embodiments, Z1 is a heteroaryl group selected from the following...

[0396] The asterisk on the left indicates the connection site with the Ab unit; the wavy line on the right indicates the connection site with Z2. In a preferred embodiment, Z1 is a pyrimidine group, preferably... More In a preferred embodiment, Z1 is a pyridinothiazolyl group, preferably... More

[0397] In some implementations, Z1 is Z3 does not exist. Furthermore, in some implementations, Z2 is selected from: bond, -C (=O)-, *-C3-C. 10 Ethyne-C(=O)-, *-C3-C 10 alkenyl-C(=O)- and *-heteroaryl-C1-C 10 Alkylene -C(=O)-; in some cases, Z2 is preferably -C(=O)-. *-(C≡C)-C 1-8 Alkylene -C(=O)- or *-(CH=CH)-C 1-8 Alkylene-C(=O)-, and more preferably, Z2 is *-(C≡C)-C. 1-8 Alkylene-C(=O)-, where * indicates the connection site with Z1. Furthermore, in some embodiments, Z4 is absent; or Z4 is *-R. 8 -(CH2-CH2-O-) y -R 9 -, where y is an integer from 0 to 10, and R8 It is -NH-, and R 9 Selected from *-C 1-4 Alkylene-C(=O)- or *-C 1-4 Alkylene -NH-C(=O)-(CH2OCH2)-C(=O)-.

[0398] In some implementations, Z1 is Z2 is -C (=O)-. Furthermore, in some implementations, Z3 is *-R. 6 -(CH2-CH2-O-) x -R 7 - where x = 0-5 or preferably x = 1 or 2; and Z4 does not exist or is *-R 8 -(CH2-CH2-O-) y -R 9 -, where y is an integer from 0 to 12, and R 6 and R 8 They are independently -NH-, and R 7 and R 9 Selected independently from *-C 1-4 Alkylene-C(=O)- or *-C 1-4 Alkylene -NH-C(=O)-(CH2OCH2)-C(=O)-.

[0399] In some implementations, Z in equation (II) has the following order from the Ab element side to the Y element side: -Z1-Z2-Z3-Z4-, where:

[0400] -Z1-Z2-Z3-Selected from

[0401] in:

[0402] n1 is an integer from 1 to 8, for example, 1, 2, 3, 4, 5 or 6;

[0403] n2 is an integer from 0 to 5, for example, 1 or 2;

[0404] n3 is an integer from 1 to 4, for example, 1 or 2;

[0405] Z4 does not exist or is Where n4 is an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0406] Where * indicates the connection site facing the Ab unit side. This indicates the connection point facing the Y-unit side.

[0407] In some implementations, Z in equation (II) has a structure selected from the following:

[0408] Where n1 is an integer from 1 to 6, n2 is an integer of 1 or 2, and n4 is an integer from 1 to 10. The asterisk on the left indicates the binding site with antibody Ab, and the wavy line on the right indicates the binding site with Y unit.

[0409] In some embodiments, preferably, Z has a structure selected from the following:

[0410] The asterisk on the left indicates the binding site with antibody Ab, and the wavy line on the right indicates the binding site with Y unit.

[0411] Y unit

[0412] In some embodiments, the Y unit in formula (II) is an enzyme-cleavable peptide linker containing 1-8 amino acids. In some embodiments, each amino acid in the Y unit is independently selected from natural or non-natural amino acids, such as alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, substituted lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, β-alanine, citrulline, cyclobutane-1,1-dicarboxamide-citrulline (cBu-Cit), and derivatives thereof. In some aspects, after the ADC is internalized into tumor cells, the amide bond in the Y unit is recognized and degraded by enzymes in the lysosomes of the tumor cells, releasing drug fraction D. In other aspects, the amide bond in the Y unit can be recognized and degraded by enzymes in the tumor environment, releasing drug fraction D.

[0413] In some embodiments, the Y unit comprises a peptide or peptide analog selected from the following: *-Gly-, *-Val-, *-Gly-Phe-Gly-, *-Phe-Gly-, *-Gly-Val-Cit-, *-Val-Cit-, *-Gly-Val-Arg-, *-Val-Arg-, *-Gly-Val-Ala-, *-Val-Ala-, *-Gly-Phe-, *-Phe-Gly-, *-Gly-Gly-Gly-, *-Gly-Gly-, *-Gly-Val-Gly-, *-Gly-Ala-Gly-, *- Gly-Phe-Cit-, *-Gly-Phe-Val-, *-Gly-Phe-Ala-, *-Gly-Phe-Lys-, *-Phe-Lys-, *-Gly-Val-, *-Gly-Cit-, *-Gly-Ala-, *-Gly-Gly- Lys-, *-Gly-Lys-, *-Ala-Ala-Ala-, *-Gln-Val-Ala-, *-Gln-Val-Cit-, *-Asp-Val-Ala-, *-Asp-Val-Cit-, *-Lys-Gly-Val-Ala- (SEQ ID NO: 65), *-Lys-Gly-Val-Cit- (SEQ ID NO: 66), *-Lys-Gly-Gly-Val-Ala- (SEQ ID NO: 67), *-Lys-Gly-Gly-Val-Cit- (SEQ ID NO: 68), *-Gly-Gly-Phe-Gly- (SEQ ID NO:64), *-Lys-Gln-Val-Cit-(SEQ ID NO:69), *-Lys-Gln-Val-Ala-(SEQ ID NO:70), *-Lys-Glu-Val-Cit-(SEQ ID NO:71), *-Lys-Glu-Val-Ala-(SEQ ID NO:72), *-Lys-Asp-Val-Cit-(SEQ ID NO:72) NO:73),*-Lys-Asp-Val-Ala-(SEQ ID NO:74), *-Glu-Val-Cit-, *-Glu-Val-Ala-, *-Lys-Val-Ala-, *-Lys-Val-Cit-, *-Val-Lys-Gly-, *-Val-Lys-, where * indicates the connection site with Z.

[0414] In some embodiments, the Y unit comprises a peptide or peptide analog selected from the following: *-Val-Ala-, *-Val-Cit-, *-Glu-Val-Cit-, *-Glu-Gly-Cit-, *-Gly-Phe-Gly-, *-Gly-Val-Cit-, *-Gly-Val-Ala-, *-Gly-Phe-Gly-, *-Phe-Gly-, *-Gly-Phe-Lys-, *-Phe-Lys-, *-Gln-Val-Ala-, *-Gln-Val-Cit-, *-Asp-Val-Ala-, *-Lys-Gly-Val-Ala-(SEQ ID NO:65), *-Lys-Gly-Val-Cit-(SEQ ID NO:66), *-Lys-Gly-Gly-Val-Ala-(SEQ ID NO:65). NO:67), *-Lys-Gly-Gly-Val-Cit-(SEQ ID NO:68), *-Gly-Gly-Phe-Gly-(SEQ ID NO:64), *-Lys-Gln-Val-Cit-(SEQ ID NO:69), *-Lys-Gln-Val-Ala-(SEQ ID NO:70), *-Lys-Glu-Val-Cit-(SEQ ID NO:71), *-Lys-Glu-Val-ALa-(SEQ ID NO:72), *-Lys-Asp-Val-Cit-(SEQ ID NO:73), *-Lys-Asp-Val-Ala-(SEQ ID NO:74), *-Glu-Val-Ala-, *-Lys-Val-Ala-, *-Lys-Val-Cit-, *-Val-Lys-, *-Asp-Val-Cit-, *-Ala-Ala-Ala-, *-cyclobutyl-Ala, *-cyclobutyl-Cit, or *-cBu-Cit-, where * indicates the connection site with Z.

[0415] In other embodiments, the Y unit comprises a peptide or peptide analog selected from the following: *-Val-Ala, *-Val-Cit, *-cBu-Cit-, *-Glu-Val-Cit-, *-Glu-Gly-Cit-, *-Gly-Val-Ala-, *-Gly-Gly-Phe-Gly (SEQ ID NO:64), where * indicates a linking site with Z.

[0416] In some embodiments, the Y unit comprises 1-5 amino acids. In some embodiments, the Y unit comprises a dipeptide, tripeptide, or tetrapeptide linker.

[0417] In some embodiments, the Y unit is preferably *-Val-Cit- or *-Val-Ala-, and more preferably, Y has the following structure:

[0418] The asterisk on the left indicates a connection to unit Z, and the wavy line on the right indicates a connection to unit M in equation (II) (if M exists) or a direct connection to drug D.

[0419] M unit

[0420] The ADC conjugate of the present invention may or may not have a spacer group (M) between the releasable peptide unit (Y) and the drug (D).

[0421] In some embodiments, there is no M spacer group between the Y unit and the drug unit D. In some embodiments, the Y unit is linked to a free hydroxyl or amino group on the drug unit D via an amide bond or an ester bond.

[0422] In some embodiments, an M spacer group is present between the Y unit and the drug unit D. The spacer group may be a functional group that facilitates the attachment of the peptide unit (Y) to the drug unit D, or may provide additional structural components to further facilitate the release of the drug D from the remainder of the conjugate (e.g., a self-dissolving group, such as a p-aminobenzyl (PAB) component). In some embodiments, the M spacer group is an amino-C1-C3 alkylene group, for example, -NH-CH2-, or amino-phenyl-C1-C3 alkylene group-OC(=O)-. In some embodiments, the M spacer group has a structure selected from:

[0423] Where R 10 Selected from: H and C1-C6 alkyl groups; n5 is 1, 2, 3, or 4; n6 is 1, 2, 3, 4, 5, or 6, wherein the asterisk on the left indicates the connection site with the Y unit, and the wavy line on the right indicates the connection site with the drug unit D. In some preferred embodiments, the M spacer group is:

[0424] Exemplary connector L

[0425] In some implementations, the connector L has the following structure (L-1):

[0426] Or it can have the following structure (L-2):

[0427] Where Y is the enzyme-cleavable peptide Y unit as defined above, where n1 is an integer from 1 to 6, n2 is an integer of 1 or 2, and n4 is an integer from 0 to 10.

[0428] In some embodiments, the Y unit comprises 1-5 amino acids. In some embodiments, the Y unit comprises a dipeptide, tripeptide, tetrapeptide, or pentapeptide linker.

[0429] In some embodiments, the Y unit comprises a peptide or peptide analog selected from the following: *-Val-Ala-, *-Val-Cit-, *-Glu-Val-Cit-, *-Glu-Gly-Cit-, *-Gly-Phe-Gly-, *-Gly-Val-Cit-, *-Gly-Val-Ala-, *-Gly-Phe-Gly-, *-Phe-Gly-, *-Gly-Phe-Lys-, *-Phe-Lys-, *-Gln-Val-Ala-, *-Gln-Val-Cit-, *-Asp-Val-Ala-, *-Lys-Gly-Val-Ala-(SEQ ID NO:65), *-Lys-Gly-Val-Cit-(SEQ ID NO:66), *-Lys-Gly-Gly-Val-Ala-(SEQ ID NO:65). NO:67), *-Lys-Gly-Gly-Val-Cit-(SEQ ID NO:68), *-Gly-Gly-Phe-Gly-(SEQ ID NO:64), *-Lys-Gln-Val-Cit-(SEQ ID NO:69), *-Lys-Gln-Val-Ala-(SEQ ID NO:70), *-Lys-Glu-Val-Cit-(SEQ ID NO:71), *-Lys-Glu-Val-ALa-(SEQ ID NO:72), *-Lys-Asp-Val-Cit-(SEQ ID NO:73), *-Lys-Asp-Val-Ala-(SEQ ID NO:74), *-Glu-Val-Ala-, *-Lys-Val-Ala-, *-Lys-Val-Cit-, *-Val-Lys-, *-Asp-Val-Cit-, *-Ala-Ala-Ala-, *-cyclobutyl-Ala, *-cyclobutyl-Cit, or *-cBu-Cit-, where * indicates the connection site with Z.

[0430] In other embodiments, the Y unit comprises a peptide or peptide analog selected from the following: *-Val-Ala, *-Val-Cit, *-cBu-Cit-, *-Glu-Val-Cit-, *-Glu-Gly-Cit-, *-Gly-Val-Ala-, *-Gly-Gly-Phe-Gly (SEQ ID NO:64), where * indicates a linking site with Z.

[0431] In other embodiments, the Y unit comprises a peptide or peptide analog selected from the following: *-Val-Ala, *-Val-Cit, *-cBu-Cit-, *-Glu-Val-Cit-, *-Gly-Gly-Phe-Gly (SEQ ID NO:64), where * indicates a linking site with Z.

[0432] In some implementations, Y is preferably *-Val-Cit.

[0433] In other embodiments, Y is preferably *-Val-Ala, and preferably has the following structure:

[0434] In some further embodiments, n1 is an integer from 1 to 4, n2 is 1, and n4 is an integer from 1 to 10. Preferably, n4 is an integer of 6, 7, 8, 9, or 10, and more preferably, n4 is 8.

[0435] In some embodiments, the connector L has a structure of formula (L-1), where Y is *-Val-Cit, and n1 is an integer from 1 to 4, n4 is an integer from 1 to 10, and preferably, n4 is an integer of 6, 7, 8, 9 or 10, more preferably, n4 is 8.

[0436] In some implementations, the connector L has a structure of formula (L-2), where Y is *-Val-Cit, and n2 is 1, n4 is an integer from 1 to 10, and preferably, n4 is an integer of 6, 7, 8, 9 or 10, more preferably, n4 is 8.

[0437] In some implementations, the connector L has a structure of formula (L-1), where Y is *-Val-Ala, and n1 is an integer from 1 to 4, n4 is an integer from 1 to 10, and preferably, n4 is an integer of 6, 7, 8, 9 or 10, more preferably, n4 is 8.

[0438] In some embodiments, the connector L has a structure of formula (L-2), where Y is *-Val-Ala, and n2 is 1, n4 is an integer from 1 to 10, and preferably, n4 is an integer of 6, 7, 8, 9 or 10, more preferably, n4 is 8.

[0439] In some preferred embodiments, the connector L has the following structure:

[0440] The asterisk on the left indicates the binding site with the antibody Ab unit, and the wavy line on the right indicates the binding site with the drug D unit.

[0441] In some other preferred embodiments, the connector L has the following structure:

[0442] The asterisk on the left indicates the binding site with the antibody Ab unit, and the wavy line on the right indicates the binding site with the drug D unit.

[0443] In the above embodiments, in some embodiments, the connector L is connected to a pharmaceutical unit D according to formula (D-1), or formula (D-1a), or formula (D-1b) according to the present invention. In some embodiments, the connector L is connected to a pharmaceutical unit D according to formula (D-2) according to the present invention. In some embodiments, the connector L is connected to a pharmaceutical unit D according to formula (D-2a) or formula (D-2b) according to the present invention. In some embodiments, the connector L is connected to a pharmaceutical unit D according to formula (D-3) according to the present invention. In some embodiments, the connector L is connected to a pharmaceutical unit D according to formula (D-3a) or formula (D-3b) according to the present invention. The connector LD unit according to this disclosure can be prepared according to the method described in WO2024 / 175069 or a similar method.

[0444] In some embodiments, the LD unit of Formula I of the present invention is linked to the antibody by forming a thioether bond with the thiol group of the free cysteine ​​residue of the light chain and / or heavy chain of the antibody.

[0445] Exemplary ADC Coupler

[0446] In some embodiments, the present invention provides antibody-drug conjugates (ADCs) or pharmaceutically acceptable salts or solvates thereof, wherein the ADC has a structure selected from formulas (I-1) and (I-2):

[0447] Where Ab and n are defined as above,

[0448] Preferably, the Ab comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences of SEQ ID NOs:24,23,26, and 25, respectively; or a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences of SEQ ID NOs:54,23,55, and 25, respectively; or the Ab comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences of SEQ ID NOs:24,23,27, and 25, respectively; or a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences of SEQ ID NOs:54,23,56, and 25, respectively.

[0449] ADC preparation and average DAR

[0450] The generation of the antibody-drug conjugate according to the present invention can be accomplished by any technique known to those skilled in the art. In some aspects, the conjugation of the drug-linker to the antibody is accomplished by reacting with the amino acid residues of the antibody. In some embodiments, a heteroaryl linker L with a leaving group is used to conjugate the drug D to the cysteine ​​residues of the antibody to prepare the conjugate of formula I of the present invention. In some embodiments, the interchain disulfide bonds of the antibody can be disrupted and free thiol groups exposed for conjugation with the heteroaryl linker-drug by controlling the conditions of treating the antibody with a reducing agent such as tris(2-hydroxyethyl)phosphine (TCEP).

[0451] In some aspects of this disclosure, the drug loading of the conjugate is represented by the average DAR. In any of the foregoing embodiments of the ADC according to this disclosure, in some aspects, the ADC according to the invention has an average DAR value of about 1 to 8 or a range consisting of any two values ​​between 1 and 8, for example, about 1, 2, 3, 4, 5, 6, 7, 8, about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 2 to 8, 2 to 6, 2 to 4, 3 to 8, 4 to 6, 4 to 8, 6 to 8, or 6 to 8. In some embodiments, the ADC according to the invention has an average DAR of 2-10 or 4-8. In some embodiments, the ADC according to the invention has an average DAR of 4-8. In some embodiments, the ADC according to the invention has an average DAR of about 6-8. In some embodiments, the ADC according to the invention has an average DAR of about 7-8. In some embodiments, the ADC according to the invention has an average DAR of about 8.

[0452] In some preferred embodiments, the present invention provides antibody-drug conjugates (ADCs) or pharmaceutically acceptable salts or solvates thereof, wherein the ADC has the structure of formula (I-1) as defined above, and wherein the Ab consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs: 24, 23, 26, and 25, respectively. Preferably, the ADC has an average DAR of about 6-8, more preferably about 7-8, for example, about 8.

[0453] In other preferred embodiments, the present invention provides antibody-drug conjugates (ADCs) or pharmaceutically acceptable salts or solvates thereof, wherein the ADC has the structure of formula (I-1) as defined above, and wherein the Ab consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs: 54, 23, 55, and 25, respectively. Preferably, the ADC has an average DAR of about 6-8, more preferably about 7-8, for example about 8.

[0454] In some further preferred embodiments, the present invention provides an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof, wherein the ADC has the structure of formula (I-2) as defined above, and wherein the Ab comprises, or is composed of, a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs: 24, 23, 26, and 25, respectively. Preferably, the ADC has an average DAR of about 6-8, more preferably about 7-8, for example, about 8.

[0455] In some further preferred embodiments, the present invention provides an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof, wherein the ADC has the structure of formula (I-2) as defined above, and wherein the Ab consists of a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs: 54, 23, 55, and 25, respectively. Preferably, the ADC has an average DAR of about 6-8, more preferably about 7-8, for example about 8.

[0456] The compositions and combinations of the present invention

[0457] In some aspects, this disclosure provides compositions comprising antibodies according to the first and second aspects of the invention, immunoconjugates or fusions according to the fourth aspect of the invention, and ADCs according to the fifth aspect of the invention.

[0458] In some embodiments, the compositions according to this disclosure are pharmaceutical compositions. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Available pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. In some embodiments, the pharmaceutical composition further comprises a second active agent, such as a second antitumor drug.

[0459] The pharmaceutical compositions of the present invention can be formulated into preparations according to methods well known to those skilled in the art. Furthermore, pharmaceutically acceptable carriers or media, such as sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, solvents, preservatives, binders, etc., can be appropriately combined with the antibodies or ADCs of the present invention to formulate unit dose forms suitable for drug administration. For example, a pharmaceutically acceptable carrier can be used to prepare a sterile solution or suspension for non-gastrointestinal administration in the form of an injection. The amount of the active ingredient in the formulation will be set to produce an effective amount within a predetermined range after administration to an individual.

[0460] The pharmaceutical compositions of the present invention are suitable for various routes of administration, including but not limited to intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). Accordingly, the pharmaceutical compositions of the present invention can be formulated into dosage forms for the intended route of administration, such as injectable, nasal, pulmonary, or transdermal dosage forms. For injectable dosage forms, the route of administration may include, but is not limited to, systemic or local administration such as intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or intratumoral injection. Furthermore, the specific method of administration of the pharmaceutical compositions of the present invention can be appropriately selected according to the patient's age and symptoms.

[0461] In other embodiments, detection or diagnostic kits comprising antibodies, immunoconjugates, fusions, or ADCs disclosed herein are also within the scope of this invention. The kit may include one or more other elements, such as: instructions for use; other reagents, such as markers or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject.

[0462] In some embodiments, this disclosure also provides combination products comprising the compositions of the present invention with other active agents. In some aspects, the combination products are pharmaceutical combinations. The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination product" means that the product allows two or more active ingredients (e.g., (i) the ADC molecule or antibody of the present invention, and (ii) other therapeutic agents) to be administered to a patient simultaneously or sequentially, without specific time restrictions or at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of the two or more active ingredients. The term "fixed combination product" means that the product allows two or more active ingredients to be administered to a patient simultaneously in the form of a single entity.

[0463] The treatment methods and uses of the present invention

[0464] In some aspects, this disclosure also provides antibodies according to the first and second aspects of the invention, immunoconjugates or fusions according to the fourth aspect of the invention, and ADCs according to the fifth aspect of the invention for use as pharmaceuticals or for the treatment and prevention of cancer, and methods thereof.

[0465] B7-H3 is overexpressed on the surface of cancer cells from various sources. In some embodiments, this disclosure provides the use of the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salts or solvates, and the antibody of the present invention or its antigen-binding fragments, for the prevention and / or treatment of B7-H3-positive tumors in a subject. In some embodiments, preferably, the tumor is a B7-H3 and EGFR double-positive tumor. In said applications, the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salts or solvates, or the antibody or antigen-binding fragment of the present invention, may be administered to the subject as the sole active agent, or may be administered to the subject in combination with other therapies or therapeutic agents. The other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells to eliminate the tumor by binding to and / or blocking these molecules; and drugs that activate the subject's immune system to spontaneously eliminate the tumor.

[0466] In some embodiments, this disclosure provides a method for treating or preventing cancer, comprising administering to an individual in need an antibody according to the first or second aspect of the invention, an immune conjugate or fusion according to the fourth aspect of the invention, or an ADC according to the fifth aspect of the invention.

[0467] The antibody or its binding fragment according to the invention and the ADC or its pharmaceutically acceptable salt or solvate according to the invention, and other therapeutic agents optionally used in combination therewith, can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0468] In some embodiments, the cancer treated according to the method of the present invention is a B7-H3 and EGFR double-positive solid tumor or hematologic malignancy, selected from, for example, bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, glioma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastrointestinal cancer, skin cancer, squamous cell carcinoma, and adenocarcinoma. In some embodiments, the cancer is skin cancer (e.g., squamous cell carcinoma of the skin), head and neck cancer, pharyngeal cancer (e.g., squamous cell carcinoma of the pharynx), esophageal cancer (e.g., squamous cell carcinoma of the esophagus), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung).

[0469] In some embodiments, the method according to the invention is used to treat tumors with a high percentage of B7-H3 positive cells, for example, tumors with at least 25%, 50%, 75%, or 100% B7-H3 positive cells.

[0470] In some embodiments, the tumors treated according to the method of the present invention have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% B7-H3 and EGFR double-positive cells. The expression levels of B7-H3 and EGFR on the tumor biopsy can be assessed by immunohistochemistry.

[0471] Tumors suitable for treatment by the method of the present invention can be early, intermediate or late stage or metastatic cancer, or tumors that have previously received treatment (e.g. anti-EGFR therapy) and have experienced immune escape.

[0472] In some embodiments, the application of the method of the present invention to said cancer results in tumor growth inhibition. In some embodiments, the application of the method of the present invention to said cancer induces tumor regression.

[0473] In some embodiments, the subject is receiving or has received other treatments, such as chemotherapy and / or radiotherapy, or other immunotherapies, prior to receiving treatment using the method of the present invention.

[0474] In any embodiment of the methods and applications of the present invention described above, the compositions, multispecific antibodies, or immunoconjugates / fusions, or combination products of the present invention may be applied instead of the antibodies or antigen-binding portions thereof, or instead of the ADCs or pharmaceutically acceptable salts or solvates thereof. Alternatively, in these methods, in addition to applying the antibodies or antigen-binding portions thereof, or the ADCs or pharmaceutically acceptable salts or solvates thereof, the compositions, multispecific antibodies, or immunoconjugates / fusions, or combination products of the present invention may be further applied.

[0475] In some embodiments, the present invention also provides the use of the ADC of the present invention or its pharmaceutically acceptable salts or solvates in the preparation of medicaments for the aforementioned therapeutic and preventive methods. In other embodiments, the present invention also provides the use of the antibodies or antigen-binding fragments, compositions, immunoconjugates / fusions, and multispecific antibodies of the present invention in the preparation of medicaments for the aforementioned therapeutic and preventive methods.

[0476] Any or all of the features described above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention; however, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation.

[0477] Example 1: Isolation and Identification of Anti-B7-H3 Antibody 33D6

[0478] Anti-B7-H3 antibody molecules were obtained through hybridoma screening. (Kearney JF, Radbruch A, Liesegang B, Rajewsky K (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 123:1546–1550)

[0479] Immunization and serum titer testing

[0480] Six-week-old SPF-grade female Balb / C healthy mice (purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd.) were randomly divided into two groups and immunized every two weeks. The immunoantigen was recombinant hB7-H3 (Acro, B7B-H52E7). The first immunization used Freund's complete adjuvant (Sigma, F5881), and subsequent immunizations used Freund's incomplete adjuvant (Sigma, F5506), for a total of four immunizations.

[0481] Hybridoma fusion screening and cloning

[0482] Spleens from qualified Balb / C mice were used to prepare cell suspensions. Spleen cells were then fused with SP2 / 0 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, TCM42) using an electrofusion method. After fusion, cells were seeded at 20,000 cells per well into 96-well plates and screened using HAT medium. Seven days later, the plate supernatant was collected for ELISA and FACS analysis to screen for positive clones. Positive hybridoma clones were expanded for antibody production, purification, and sequencing of antibody expression genes.

[0483] ELISA detection of the binding ability of mouse antibodies to proteins

[0484] Dilute mouse B7-H3 (Acro, B73-M52H4), monkey B7-H3 (Acro, B73-C52Ha), human B7-H3-2Ig (Acro, B73-H52E2), and human B7-H3-4Ig (Acro, B7B-H52E7) protein to a concentration of 1 μg / mL, add to a 96-well ELISA plate, and incubate overnight at 4°C. Discard the protein, and block with 300 μL of 3% skim milk powder (dissolved in PBST, with 0.5‰ Tween 20 added to PBS) for 2 h. Wash three times with 300 μL of PBST, dilute the antibody to be tested to 20 μg / mL, add 100 μL to each well of the blocked ELISA plate, and incubate at room temperature for 1 h. Discard the supernatant, wash three times with 300 μL of PBST, add 100 μL of HRP-labeled goat anti-mouse secondary antibody (Jackson, catalog number 115-035-071, 1:10000 dilution), and incubate at room temperature for 1 h. Discard the antibody, wash three times with 300 μL of PBST. Add 100 μL of TMB chromogenic buffer and develop at room temperature in the dark for 10 min. Stop the reaction by adding 2M sulfuric acid, and take readings within 30 min.

[0485] FACS detection of the binding ability of murine antibodies to cells

[0486] Add 5x10 to each well of the 96-well V-shaped microplate 5 MDA-MB-231 (ATCC, CRM-HTB-26) human breast cancer cells endogenously expressing B7-H3 were centrifuged at 1500 rpm for 1 min, and the supernatant was discarded. Antibody was serially diluted with PBS, and 50 μL / well was added to each well of a microplate. After incubation on ice for 30 min, 150 μL of PBS was added to each well, and the plates were centrifuged at 1500 rpm for 1 min. The plates were washed twice. 50 μL of APC-labeled goat anti-mouse IgG (Jackson, catalog number 115-605-164, diluted 1:800 in PBS) was added to each well, and the plates were incubated on ice for 30 min. Finally, the plates were washed twice with PBS, and 150 μL of PBS was added to each well to resuspend the cells. Detection was performed using CytoFLEX (Beckman).

[0487] BLI method for detecting the affinity between murine antibodies and antigens

[0488] The BLI detection method was performed according to the instrument's instruction manual. A Fortebio Octet Red 96 instrument was used for detection, and the equilibrium dissociation constant (KL) was calculated. DOne AMC (Fortebio, Cat 18-5088) sensor was immersed in a standard buffer solution (1×PBS, pH 7.4, containing 0.1% BSA and 0.02% Tween-20). After equilibration in the standard buffer solution, 5 μg / mL of the target antibody was immobilized, then bound to 50 nM antigen hB7-H3-2Ig (Acro, B73-H52E2) and dissociated in the standard buffer solution. Instrument operation steps: Baseline 1 (60s), Loading (~15s), Baseline 2 (60s), Association (60s), and Dissociation (120s), at a rotation speed of 1000 rpm and a temperature of 30℃.

[0489] Table 1A and Figure 3 show exemplary screening results for murine antibodies. Table 1A shows the ELISA absorbance values ​​of the tested antibodies for binding to different antigens. As the results indicate, the three hybridoma monoclonal antibodies 32F8, 33D6, and 3H3 all showed significant binding to both splice isoforms of human B7-H3, 2IgB7-H3 and 4IgB7-H3; and exhibited species-binding properties, binding to monkey B7-H3 but not to murine B7-H3 antigen. Figure 3 shows that, by FACS detection, hybridoma monoclonal antibodies 32F8, 33D6, and 3H3 showed significant binding to MDA-MB-231 cells endogenously expressing B7-H3.

[0490] The affinity of murine antibodies for antigens was assessed using bio-layer interferometry (BLI). The murine antibody 33D6 showed superior antigen-binding affinity compared to two other murine antibodies, 32F8 and 3H3 (Table 1B). The study also found that 33D6 exhibited better drug-like properties. Therefore, the murine antibody 33D6, which can simultaneously bind to both human and monkey B7-H3 antigens and showed binding activity in human B7-H3-expressing cells, was selected for subsequent molecular development.

[0491] Table 1A shows the binding of hybridoma monoclonal antibodies to different B7-H3 proteins.

[0492] Table 1B shows the affinity of mouse antibodies for B7-H3 as determined by BLI.

[0493] Example 2: Humanization of anti-B7-H3 antibody 33D6

[0494] Human-centered design

[0495] The murine antibody 33D6 sequence was analyzed and compared with the human germline gene of IMGT. The framework region sequence of IGKV1-NL1*01 was determined to be the humanized framework sequence of the light chain, and the framework region sequence of IGHV3-23*01 was determined to be the humanized framework sequence of the heavy chain. Through CDR transplantation (CDRs were determined using the Kabat coding method), the CDRs of the heavy and light chains were respectively placed in parallel with the selected humanized framework sequences. Simultaneously, reversion mutations were designed at key sites in the framework regions to obtain the humanized antibody hz33D6.8. The murine and humanized 33D6 antibody sequences are shown in Table 2. The affinity between the humanized antibody and the antigen was detected using ELISA and thin-layer chromatography (BLI).

[0496] Table 2 Sequences of the murine and humanized heavy and light chain variable regions of anti-B7-H3 antibody 33D6

[0497] Expression and purification of chimeric and humanized antibodies

[0498] Genes encoding the amino acid sequences of the heavy and light chain variable regions of murine and humanized antibodies were synthesized and constructed into expression vectors. After plasmid synthesis, ExpiCHO cells (Invitrogen, A14635) were transfected. After several days of culture, the cell culture supernatant was collected by centrifugation. The supernatant was purified using a protein A chromatography column. The expressed chimeric antibody ch33D6 shares the same human IgG1 heavy chain constant region and human Kappa light chain constant region as the humanized antibody hz33D6.8.

[0499] ELISA method for detecting the binding ability of chimeric and humanized antibodies to antigens.

[0500] The ELISA detection method is briefly described below. Dilute hB7-H3-2Ig (Acro, B73-H52E2) protein to a concentration of 1 μg / mL, add 100 μL / well to a 96-well microplate, and incubate overnight at 4°C. Discard the protein, and block with 300 μL of 3% bovine serum albumin (dissolved in PBST) for 2 h. Wash three times with 300 μL of PBST, serially dilute the antibody to be tested, and add 100 μL to each well of the blocked microplate, incubating at room temperature for 1 h. Discard the supernatant, wash three times with 300 μL of PBST, add 100 μL of HRP-labeled goat anti-human secondary antibody (Bethyl, A80-304P, 1:5000 dilution), and incubate at room temperature for 1 h. Discard the antibody, and wash three times with 300 μL of PBST. Add 100 μL of TMB colorimetric solution and develop the color at room temperature in the dark for 10 min. Then, add 2 M sulfuric acid to terminate the reaction and complete the reading within 30 min.

[0501] BLI method for detecting the affinity of chimeric and humanized antibodies for antigens

[0502] The BLI detection method was performed according to the instrument's instruction manual. A Fortebio Octet Red 96 instrument was used for detection, and the equilibrium dissociation constant (KL) was calculated. D One AHC (Fortebio, Cat 18-5060) sensor was immersed in standard buffer (PBS, pH 7.4, containing 0.1% BSA and 0.02% Tween-20). After equilibration in the standard buffer, 5 μg / mL of the target antibody was immobilized, then bound to 50 nM antigen hB7-H3-2Ig (Acro, B73-H52E2) and dissociated in the standard buffer. Instrument operation steps: Baseline 1 (60s), Loading (~15s), Baseline 2 (60s), Association (60s), and Dissociation (120s), at a speed of 1000 rpm and a temperature of 30℃.

[0503] The ELISA results are shown in Figure 4, and the BLI results are shown in Table 3. The humanized antibody hz33D6.8 and the chimeric antibody ch33D6 showed comparable binding ability to the B7-H3 protein, with no significant loss of affinity.

[0504] Table 3. Affinity of humanized antibodies to B7-H3 as determined by BLI.

[0505] Example 3: Humanization of anti-EGFR antibody A001

[0506] The A001 antibody is derived from patent US20090004192A1, clone number 1260, and can specifically bind to the extracellular domains I and II of EGFR.

[0507] Human-centered design

[0508] Using standard methods, the mouse antibody A001 sequence was analyzed and compared with the human germline gene of IMGT. This confirmed that the framework region sequence of IGKV1-39*-01 was the humanized framework sequence for the light chain, and the framework region sequence of IGHV4-59*01 was the humanized framework sequence for the heavy chain. Through CDR transplantation (CDRs were determined using the Kabat coding method), the CDRs for the heavy and light chains were placed into the selected humanized framework sequences, respectively. Simultaneously, reversion mutations were designed at key sites in the framework regions, thereby obtaining the humanized antibodies hzA001.8 and hzA001.12. The corresponding amino acid sequences of the light and heavy chains are shown in Table 4.

[0509] Table 4. Sequences of anti-EGFR antibody A001 and its humanized heavy chain and light chain variable regions.

[0510] Expression and purification of chimeric and humanized antibodies

[0511] Genes encoding the amino acid sequences of the heavy and light chain variable regions of A001 and its humanized antibody were synthesized and constructed into an expression vector. After plasmid synthesis, ExpiCHO cells (Invitrogen, A14635) were transfected. After several days of culture, the cell culture supernatant was collected by centrifugation. The supernatant was purified using a protein A chromatography column. The expressed chimeric antibody chA001 shares the same human IgG1 heavy chain constant region and human Kappa light chain constant region as the humanized antibodies hzA001.12 and hzA001.8.

[0512] ELISA method for detecting the binding ability of chimeric and humanized antibodies to antigens.

[0513] The ELISA detection method is as described in Example 2, and the antigen to be detected is EGFR (Acro, EGR-H5222).

[0514] BLI method for detecting the affinity of chimeric and humanized antibodies

[0515] The BLI detection method is described in Example 2, and the antigen to be detected is EGFR (Acro, EGR-H5222).

[0516] The ELISA results are shown in Figure 5. An antibody, hzA001.8, with an affinity close to chA001, and a humanized antibody, hzA001.12, with an affinity several times weaker, were obtained. The BLI results are shown in Table 5. The affinity K of hzA001.8 is... D The value is 5.3 nM, and the affinity K is 1.12 hzA001.12. D The value is 23.3nM.

[0517] Table 5. Affinity of humanized antibodies to EGFR detected by BLI.

[0518] Example 4: Preparation of Bispecific Antibody

[0519] Half antibody preparation

[0520] Humanized antibodies hzA001.8 and hzA001.12 have the same light chain variable region sequence. This light chain variable region sequence was fused with the human Kappa light chain constant region sequence to form the light chain sequence of a full-length anti-EGFR antibody (SEQ ID No:25). The heavy chain variable region sequences of humanized antibodies hzA001.8 and hzA001.12 were fused with the human IgG1 type heavy chain constant region sequence (with Knob mutation T366W-K409D-R355Q-Q419E) to form the heavy chain sequence of a full-length anti-EGFR antibody (SEQ ID No:56; SEQ ID No:55). The light chain variable region sequence of the humanized antibody hz33D6.8 was fused with the human Kappa light chain constant region sequence to form the light chain sequence of the anti-B7-H3 antibody (SEQ ID No: 23); the heavy chain variable region sequence of the humanized antibody hz33D6.8 was fused with the human IgG1 type heavy chain constant region sequence (with the Hole mutation T366S-L368A-Y407V-L351Y-D399R) to form the full-length heavy chain sequence of the anti-B7-H3 antibody (SEQ ID No: 54). The synthesized antibody heavy chain and light chain genes were inserted into the vector pcDNA3.4 (Invitrogen) to obtain plasmids for expressing anti-EGFR haptens or anti-B7-H3 haptens.

[0521] Plasmid transfection was performed according to the instructions of the ExpiCHO expression system kit (Gibco, A29133). The light and heavy chain plasmids of the hapten were mixed with ExpiFectamine CHO transfection reagent in OptiPRO SFM medium (Gibco, 12309019). After incubation, the mixture was added to ExpiCHO cells (Invitrogen, A14635), and the supernatant was harvested after 8 days. The supernatant was captured using a Protein A Diamond chromatography column (Borglon, AA0273) to obtain either an anti-EGFR hapten or an anti-B7-H3 hapten.

[0522] Bispecific antibody preparation

[0523] Bispecific antibody assembly was performed in vitro. The obtained half-antibodies were mixed at a 1:1 molar ratio, and an appropriate amount of 2-MEA reducing agent (2-mercaptoethylamine, Sigma) was added. The mixture was reacted at 25°C for 4 hours, and the reducing agent was removed by ultrafiltration to terminate the reaction. The in vitro assembly efficiency was detected by SEC-HPLC and RP-HPLC. The samples were incubated overnight at 4°C.

[0524] Fine purification was performed using a Capto S ImpAct cation exchange column (Cytiva, catalog number 17371751). The buffer consisted of 20 mM sodium phosphate (pH 6.0) and 20 mM sodium phosphate with 1 M sodium chloride (pH 6.0), with an elution gradient of 0-30% (10 column volumes). The resulting protein solution was analyzed by SEC-HPLC and RP-HPLC.

[0525] SEC-HPLC analysis of purity

[0526] The purified product was analyzed for purity using a Theromo Vanquish Core high-performance liquid chromatograph via SEC-HPLC. The column used was a Waters BioResolve SEC mAb (5 μm, 7.8 × 300 mm). Isocratic elution was performed using PBS as the mobile phase at a flow rate of 0.5 mL / min, with a detection wavelength of 280 nm and a column temperature of 25 °C.

[0527] The SEC-HPLC analysis results of the in vitro assembled and purified bispecific antibodies B035 (half-antibody combination: hzA001.12-K26+hz33D6.8-H18) and B036 (half-antibody combination: hzA001.8-K26+hz33D6.8-H18) are shown in Table 6. The heterodimer content of both the assembled and purified B035 and B036 bispecific antibodies was greater than 98%. The SEC-HPLC chromatogram of B035 is shown in Figure 6A, and the SEC-HPLC chromatogram of B036 is shown in Figure 6B. The main component in the chromatograms is the target bispecific antibody heterodimer, and the proportion of other impurities is less than 2%.

[0528] RP-HPLC analysis of purity

[0529] RP-HPLC can sensitively detect the assembly efficiency of heterodimer molecules of bispecific antibodies. The RP-HPLC instrument was an Agilent 1200 high-performance liquid chromatograph; the column was an Agilent Zorbax 300SB-C18 (5μm, 4.6×250mm). Gradient elution was performed using 0.1% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile-isopropanol-trifluoroacetic acid (80:20:0.1) as mobile phase B (0-20 min 30% B-50% B). The detection wavelength was 280 nm, the flow rate was 0.5 mL / min, and the column temperature was 70℃.

[0530] The RP-HPLC analysis results are shown in Table 6. The proportion of heterodimers of the assembled and purified B035 and B036 bispecific antibodies was greater than 98%. The RP-HPLC chromatogram of B035 is shown in Figure 7A, and the RP-HPLC chromatogram of B036 is shown in Figure 7B.

[0531] Table 6. Purity data of bispecific antibodies (SEC and RP-HPLC)

[0532] Example 5: Analysis of the antigen-binding characteristics of bispecific antibodies

[0533] ELISA assay for bispecific antibody-antigen binding activity

[0534] The binding activity of purified B035 and B036 antibodies from Example 4 to the antigen was determined by ELISA. The ELISA detection method was the same as in Example 2. The antigens were recombinant human EGFR protein (ACRO Biosystems, EGR-H5222) and B7-H3 protein (ACRO Biosystems, B73-H52E2). The maximum starting concentration of the penicillin antibodies B035 and B036 was 2 μg / mL, and they were serially diluted.

[0535] The ELISA results for binding to human EGFR are shown in Figure 8A, and the ELISA results for binding to human B7-H3 protein are shown in Figure 8B. Bispecific antibody B036 showed strong EGFR binding ability, with an EC50 value of 15.65 ng / mL. Bispecific antibody B035 showed weak EGFR binding ability, with an EC50 value of 205.2 ng / mL. Bispecific antibodies B036 and B035 showed comparable binding abilities to B7-H3, with EC50 values ​​ranging from 1.5 to 2.2 ng / mL.

[0536] BLI method for determining the affinity of bispecific antibodies

[0537] The affinity of the purified bispecific antibody from Example 4 for EGFR and B7-H3 was determined using the BLI method. The BLI assay method was the same as in Example 2. The BLI test results are shown in Table 7. The affinity of the bispecific antibody B035 for EGFR was 4.29 x 10⁻⁶. -8 M; The affinity of the bispecific antibody B036 for EGFR is 4.74 x 10⁻⁶. -9 M, B035 has approximately 10 times weaker affinity for EGFR than B036. The bispecific antibody B036 has an affinity for B7-H3 of 2.75 x 10⁻⁶. -12 M to 2.92 x 10 -12 Between M.

[0538] Table 7. BLI assay for the affinity of the penicillin antibody for EGFR and B7-H3.

[0539] Example 6: Monitoring Bispecific Antibody Internalization Using Flow Cytometry

[0540] Human pharyngeal squamous cell carcinoma cells FaDu (TCHu132, China Academy of Sciences Cell Bank), human lung adenocarcinoma cells NCI-H1975 (SCSP-597, China Academy of Sciences Cell Bank), human lung squamous cell carcinoma cells NCI-H226 (SCSP-5073, China Academy of Sciences Cell Bank), and human non-small cell lung cancer cells NCI-H1650 (SCSP-592, China Academy of Sciences Cell Bank) expressing endogenous EGFR and B7-H3 were used as experimental models to evaluate the in vitro internalization effect of antibodies. Cell suspensions were prepared using serum-free MEM (Gibco, 11095-080) or 1640 (Gibco, 22400-089) fresh cell culture media, with a cell density of 2 × 10⁶ cells / mL. 6 Cells / mL. A mixture of 2 μg / mL antibody and 4 μg / mL Alexa Fluor 647-affiniPure Fab Fragment Goat Anti-Human IgG (Jackson, 109-607-008) fluorescent antibody was prepared using serum-free medium. 200 μL of cells were mixed with 200 μL of the antibody mixture and incubated on ice for 30 minutes. After incubation, 100 μL was added to each well of a cell culture plate. Binding wells were incubated on ice, and endocytosis wells were incubated at 37°C for 1 hour and 2 hours in a 5% CO2 incubator, respectively. After washing, 200 μL of papain (final concentration 0.5 mg / mL) was added to each endocytosis well, and the cells were incubated for 15 minutes at room temperature. After incubation, the cells were washed with FACS buffer (PBS + 2% FBS) and resuspended. Cells were then analyzed using a Beckman flow cytometer.

[0541] Experimental results showed that in tumor cell lines H1975 and FaDu, which co-express EGFR and B7-H3 targets, the endocytosis capacity of the bispecific antibody B036 of this invention was stronger than that of the corresponding monospecific parental antibodies hzA001.8 and hz33D6.8. Furthermore, bispecific antibody B036 exhibited stronger endocytosis capacity than the combination of the two monoclonal antibodies (Figures 9A and 9B). In tumor cell lines NCI-H226 and NCI-H1650, which co-express EGFR and B7-H3 targets, the endocytosis capacity of the bispecific antibody B035 of this invention was stronger than that of the corresponding monospecific parental antibodies hzA001.12 and hz33D6.8. Furthermore, bispecific antibody B035 exhibited stronger endocytosis capacity than the combination of the two monoclonal antibodies (Figures 9C and 9D).

[0542] Example 7: Preparation and Analysis of Antibody-Drug Conjugates

[0543] Example 7.1 Synthesis of drug-linker intermediate compounds

[0544] The synthesis of the drug-linker compounds and the preparation of the ADC used in this embodiment were performed according to the procedures described in the applicant's co-pending application WO2024 / 175069, which is hereby incorporated herein by reference in its entirety. Specifically, the intermediate compounds LY22CD and LY22CD3 were synthesized according to the descriptions in Examples 28 and 30 of WO2024 / 175069:

[0545] Example 7.2 ADC Fabrication and Characterization

[0546] Preparation of antibody-drug conjugate B035-LY22CD

[0547] At 37°C, a solution of tris(2-carboxyethyl)phosphine (TCEP) (40 mM, 23.3 μL, 931 nmol) was added to a PBS buffer solution (pH = 7.4, 0.01 M PBS buffer solution) containing bispecific antibody B035 (10.0 mg / mL, 2 mL, 133 nmol). The solution was placed in a water bath and reacted at 37°C for 2 hours, after which the reaction was stopped. The reaction solution was then cooled to room temperature in a water bath.

[0548] Compound LY22CD (2.85 mg, 1995 nmol) was dissolved in 125 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and reacted at 25 °C for 30 minutes. The reaction solution was then analyzed using a Slide-A-Lyzer dialyzer. TM The antibody-drug conjugate B035-LY22CD was purified by dialysis using a dialysis kit (10K MWCO) to obtain histidine buffer (20mM histidine-hydrochloride buffer, pH=5.5), and stored at 4℃. It can be stored at -80℃ for long-term preservation.

[0549] SEC-HPLC analysis

[0550] The monomer yield of B035-LY22CD was determined to be 98.9% using the SEC-HPLC method. The SEC-HPLC instrument was a Theromo Vanquish Core high-performance liquid chromatograph, and the chromatographic column was a Waters BioResolve SEC mAb (2.5 μm, 7.8 × 300 mm). Isopropanol (95:5) was used as the mobile phase for isocratic elution for 30 minutes at a flow rate of 0.5 mL / min. The detection wavelength was 280 nm, and the column temperature was 25 °C. The results are shown in Figure 10.

[0551] DAR value measurement

[0552] The drug loading average (DAR) of B035-LY22CD was calculated by RP-HPLC. The RP-HPLC instrument was an Agilent 1200 high-performance liquid chromatograph; the chromatographic column was a Thermo MAbPac RP (4μm, 3×100mm); gradient elution was performed using 0.1% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile-isopropanol-trifluoroacetic acid (80:20:0.1) as mobile phase B (0-28 min 30% B-50% B), with a flow rate of 0.4 mL / min, a detection wavelength of 280 nm, and a column temperature of 70℃. The results are shown in Figure 11. The calculated DAR value of B035-LY22CD was 7.86.

[0553] Preparation and analysis of other ADCs

[0554] Following a similar method, antibody-drug conjugates B035-LY22CD3 and B036-LY22CD were prepared by conjugating bispecific antibodies with the small molecule LY22CD3, and the SEC purity and drug loading average (DAR) of the conjugate products were detected. The results are shown in Table 8 below.

[0555] Table 8. Average (DAR) data for the purity and drug loading of antibody-drug conjugates (SECs).

[0556] Following a similar method, conjugates of parental monospecific antibodies hzA001.8 and hz33D6.8 with compound LY22CD were prepared and used as controls in subsequent examples.

[0557] Example 8: Determination of the in vitro cell activity of antibody-drug conjugates

[0558] The in vitro activity of antibody-drug conjugates (ADCs) was measured using human pharyngeal squamous cell carcinoma FaDu (TCHu132, Chinese Academy of Sciences Cell Bank) and human non-small cell lung squamous cell carcinoma NCI-H226 (SCSP-5073, Chinese Academy of Sciences Cell Bank). Cells were cultured at 1000 cells / 100 μL in 96-well plates. Pre-diluted antibody-drug conjugates (maximum concentration 400 nM, 4-fold dilution, 9 concentrations) were added to the corresponding wells, mixed, and incubated at 37°C with 5% CO2 for 6 days. After co-incubating the cells with the antibody-drug conjugates for 6 days, the pre-diluted ADCs were added to the wells. Luminescent Cell reagent (Promega, G7571) was added to the cell wells and incubated at room temperature in the dark for 3 minutes. The plate was then read using a microplate reader (Tecan, Spark), and the half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism software.

[0559] In human pharyngeal squamous cell carcinoma cells (FaDu), the bispecific antibody ADC B036-LY22CD showed a significant killing effect on cells, with a maximum killing rate of 97.9% and an IC50 of 5.25 nM. Its cell-killing effect was not only superior to the corresponding monoclonal antibody ADCs hzA001.8-LY22CD and hz33D6.8-LY22CD, but also superior to the combination of the corresponding monoclonal antibody ADCs hzA001.8-LY22CD and hz33D6.8-LY22CD (Figure 12A).

[0560] In non-small cell lung squamous cell carcinoma NCI-H226, the bispecific antibody ADC B036-LY22CD showed a certain degree of cell killing effect, with a maximum killing rate of approximately 90% and an IC50 of approximately 90 nM. Its cell-killing effect was not only superior to the corresponding monoclonal antibody ADCs hzA001.8-LY22CD and hz33D6.8-LY22CD, but also superior to the combination of the corresponding monoclonal antibody ADCs hzA001.8-LY22CD and hz33D6.8-LY22CD (Figure 12B).

[0561] Example 9: In vivo FaDu cell xenograft experiment in mice

[0562] CB17 SCID mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. FaDu cells were routinely passaged for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in an equal ratio of RPMI-1640 and Matrigel Matix (Corning) to prepare a cell concentration of 5 x 10⁻⁶ cells / mL. 7 Cells / mL suspension. 0.1 mL of cell suspension was subcutaneously injected into the right dorsal region of CB17 SCID mice to establish a FaDu tumor-bearing mouse model. When the tumor volume reached 250-300 mm²... 3 Animals were divided into groups and administered the corresponding ADC drugs via tail vein injection as a single dose. The groups included a mouse treatment group receiving hzA001.8-LY22CD, hz33D6.8-LY22CD, hzA001.8-LY22CD+hz33D6.8-LY22CD, B035-LY22CD, and B036-LY22CD at a dose of 1.5 mg / kg, a mouse treatment group receiving B036-LY22CD at a dose of 5 mg / kg, and a control group receiving PBS injection.

[0563] Tumor volume and body weight in mice were monitored twice weekly. The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = L * W 2 / 2. Body weight was measured using an electronic balance. For the entire group with an average tumor volume exceeding 2000 mm²... 3Mice that have lost more than 20% of their body weight are euthanized.

[0564] The tumor growth inhibition rate (%TGI) was calculated as follows: TGI% = (1 - Ti / Ci) × 100; where Ti and Ci are the average tumor volumes of the treatment group and the control group, respectively, at a certain time point. The inhibition rates of each tested ADC drug on day 20 after administration are shown in Table 9.

[0565] Figure 13A shows that within 60 days after administration, each tested ADC drug significantly inhibited tumor growth in FaDu tumors. At a dose of 1.5 mg / kg, the bispecific antibody ADCs B035-LY22CD and B036-LY22CD showed superior tumor-suppressive effects compared to their corresponding monoclonal antibody ADCs hzA001.8-LY22CD and hz33D6.8-LY22CD, and were also superior to the combination of monoclonal antibody ADCs (hzA001.8-LY22CD + hz33D6.8-LY22CD combo). Moreover, the tumor-suppressive activity of the bispecific antibody ADCs increased with increasing concentration.

[0566] Meanwhile, the results of monitoring the mouse body weight (Figure 13B) showed that there was no significant decrease in mouse body weight within 60 days after administration.

[0567] Table 9. Tumor suppression effect of FaDu cell xenograft experiments *Complete release rate: Complete tumor regression, volume 0.

[0568] Example 10: In vivo NCI-H1650 cell xenograft experiment in mice

[0569] A subcutaneous xenograft model of CB17 SCID mice was established according to the method in Example 9, using the human tumor cell line (NCI-H1650). When the tumor volume reached 150-250 mm... 3 The drugs were administered via a single tail vein injection, with the test substances B036-LY22CD, hzA001.8-LY22CD, hz33D6.8-LY22CD, hzA001.8-LY22CD+hz33D6.8-LY22CD, and B035-LY22CD all administered at a dose of 3 mg / kg.

[0570] Tumor volume and body weight in mice were monitored twice weekly. The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = L * W 2 / 2. Body weight was measured using an electronic balance. For the entire group with an average tumor volume exceeding 2000 mm²... 3 Mice that have lost more than 20% of their body weight are euthanized.

[0571] The tumor growth inhibition rate (%TGI) was calculated as follows: TGI% = (1 - Ti / Ci) × 100; where Ti and Ci are the average tumor volumes of the treatment group and the control group, respectively, at a certain time point. The inhibition rates of each tested ADC drug on day 25 after administration are shown in Table 10.

[0572] Figure 14A shows that within 48 days after administration, each tested ADC drug significantly inhibited tumor growth in NCI-H1650 tumors. At a dose of 3 mg / kg, the bispecific antibody ADCs B035-LY22CD and B036-LY22CD showed better tumor-suppressive effects than the monoclonal antibody ADCs hzA001.8-LY22CD and hz33D6.8-LY22CD, and were also superior to the combination of monoclonal antibody ADCs (hzA001.8-LY22CD + hz33D6.8-LY22CD combo).

[0573] Meanwhile, the results of monitoring the mouse body weight (Figure 14B) showed that there was no significant decrease in mouse body weight within 48 days after administration.

[0574] Table 10. Tumor suppression effect of NCI-H1650 cell xenografts

[0575] *Complete release rate: Complete tumor regression, volume 0.

[0576] Example 11: In vivo NCI-H1975 cell xenograft experiment in mice

[0577] A subcutaneous xenograft model of CB17 SCID mice using the human tumor cell line (NCI-H1975) was established according to the method in Example 9. When the tumor volume reached 150-300 mm... 3 At that time, the drug was administered by a single tail vein injection, and the dosage of the test substances B035-LY22CD3, B043-BYBL, B043-LY22CD and B035-LY22CD was 10 mg / kg. The preparation method of the control antibody B043 used in this example comes from the patent document CN106659779B (B043 corresponds to the anti-EGFR and HER3 bispecific antibody named "SI-1X6.4" in the document), and the preparation method of the bispecific antibody ADC B043-BYBL comes from the patent application WO2023083381A1 (B043-BYBL corresponds to "ADC-6" in the document, and the drug form of the ADC BL-B01D1 is in clinical trials, see Lancet Oncology, vol 25, NO.7, DOI:10.1016 / S1470-2045(24)00159-1).

[0578] Tumor volume and body weight in mice were monitored twice weekly. The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = L * W 2 / 2. Body weight was measured using an electronic balance. For the entire group with an average tumor volume exceeding 2000 mm²... 3 Mice that have lost more than 20% of their body weight are euthanized.

[0579] The tumor growth inhibition rate (%TGI) was calculated as follows: TGI% = (1 - Ti / Ci) × 100; where Ti and Ci are the average tumor volumes of the treatment group and the control group, respectively, at a certain time point. The inhibition rates of each tested ADC drug on day 24 after administration are shown in Table 11.

[0580] Figure 15A shows that within 40 days after administration, each tested ADC drug significantly inhibited tumor growth in NCI-H1975 tumors. At a dose of 10 mg / kg, the bispecific anti-tumor ADCs B035-LY22CD and B035-LY22CD3 showed similar tumor-inhibiting effects, and were significantly superior to B043-BYBL and B043-LY22CD.

[0581] Meanwhile, the results of monitoring the mouse body weight (Figure 15B) showed that there was no significant decrease in mouse body weight within 40 days after administration.

[0582] Table 11 Tumor suppression effect of NCI-H1975 cell xenografts *Complete release rate: Complete tumor regression, volume 0.

[0583] Example 12 In vivo pharmacodynamics trial in a tumor xenograft (PDX) model

[0584] Mouse PDX experiments were conducted at Crown Bioscience (Beijing) Co., Ltd. Tumor tissue was collected from tumor-bearing mice in a lung cancer xenograft model and cut into tumor blocks with a diameter of 2-3 mm. These blocks were then subcutaneously injected into the right anterior scapula of BALB / c nude mice. Before administration, all animals were weighed, and tumor volume was measured using calipers. Tumors were allowed to grow to an appropriate initial tumor volume range (170-200 mm²). 3 Dosage was administered to patients in random groups based on tumor size.

[0585] The LU0894 model was administered the drug on day 0 (grouping day), and the other eight lung cancer models were administered the drug on day 1 (grouping day), according to the experimental design. All animals in all groups received a single intravenous dose of 10 mg / kg. Following tumor cell inoculation, tumor growth and the effect of treatment on normal animal behavior were routinely monitored, including animal activity, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. After the start of administration, mouse body weight and tumor size were measured twice weekly.

[0586] Figure 16 shows the relative tumor volume changes in the efficacy ranking of B035-LY22CD3 in different PDX models after drug administration. The results show that, based on measurements taken from 14 to 28 days post-administration, the bispecific antibody ADC B035-LY22CD3 induced tumor growth inhibition or regression in the tested PDX models. Partial remission (PR) was achieved in the LU0357, LU0377, LU2512, LU1204, and LU0894 PDX models; stable disease (SD) was achieved in the LU11554, LU6437, LU0350, and LU1219 PDX models, with an overall disease control rate of 100%.

[0587] Example 13: In vivo pharmacodynamics trial in a tumor xenograft (PDX) model

[0588] In vivo pharmacodynamic studies were conducted in mouse PDX models at Crown Bioscience (Beijing) Co., Ltd., Shanghai Lidi Biotechnology Co., Ltd., and WuXi AppTec Co., Ltd. The experimental models included EGFR wild-type or mutant non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), breast cancer, head and neck squamous cell carcinoma (HNSCC), and small cell lung cancer (SCLC). Tumor tissue was collected from xenograft tumor-bearing mice, cut into tumor blocks approximately 3 mm in diameter, and subcutaneously injected into the right back of immunodeficient mice. Before administration, the weight of all animals was measured, and the tumor volume was measured using calipers. The tumors were allowed to grow to an appropriate initial tumor volume range (140-200 mm) before administration. 3 Dosage was administered to patients in random groups based on tumor size, with the day of grouping defined as day 0.

[0589] All models were administered the drug according to the experimental design on day 0 or day 1, with all animals in all groups receiving a single intravenous dose of 10 mg / kg. Following tumor cell inoculation, tumor growth and the effect of treatment on normal animal behavior were routinely monitored, including animal activity, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. After the start of administration, mouse body weight and tumor size were measured twice weekly.

[0590] Figure 17 shows the relative tumor volume changes in the efficacy ranking of B035-LY22CD3 in different PDX models. The results showed that, based on measurements taken 14 to 28 days post-drug administration, the bispecific antibody ADC B035-LY22CD3 induced tumor growth inhibition or regression in the tested PDX models. In 26 PDX models, 46% achieved complete remission (CR), 35% achieved partial remission (PR), and 19% achieved stable disease (SD), resulting in a disease control rate (DCR) of 100% (Table 12).

[0591] Table 12: Summary of the efficacy of B035-LY22CD3 in different PDX models

[0592] Example 14 Pharmacokinetics of Bispecific Antibody ADC in Cynomolgus Monkeys

[0593] Toxicological and pharmacokinetic experiments were conducted on cynomolgus monkeys at Jiangsu Dingtai Biotechnology Co., Ltd. Cynomolgus monkeys aged 3–5 years were randomly assigned to two groups and administered a single intravenous bolus injection of 15 mg / kg B035-LY22CD3 and B036-LY22CD, respectively. Blood samples were collected before administration (0 min) and at 1 h, 3 h, 8 h, 24 h, 72 h, 168 h, 240 h, and 336 h after administration, approximately 0.25 mL at each time point. These samples were transferred to pre-chilled EDTA-K2 tubes and placed on wet ice until centrifugation. After centrifugation, plasma samples were collected and rapidly frozen on dry ice until ELISA analysis.

[0594] Total antibody ELISA

[0595] Coat a 96-well microplate with EGFR (ACRO Biosystems, EGR-H5222) and B7-H3 (ACRO Biosystems, B73-H52E2) and incubate overnight at 4°C. Discard the coating solution, add 300 μL of 3% BSA in PBST solution, and incubate at room temperature for 2 h to block the microplates. Wash three times with PBST, and add 100 μL / well of serum samples from different time points to a gradient. Set up a standard sample gradient (ADC standard starting concentration 50 ng / mL, 2-fold serial dilution), and incubate at 37°C for 1 h. Wash three times with PBST, add 100 μL of HRP-labeled goat anti-human IgG (Southern Biotech, 2049-05, 1:5000 dilution), incubate at 37°C for 0.5 h, and wash three times with PBST. Add 100 μL of TMB and develop color for 10 min. Add 100 μL of 2M sulfuric acid to stop the color development. Use a multi-functional microplate reader (Tecan, Spark) to read the OD450 wavelength absorbance value.

[0596] ADC ELISA

[0597] Coat 96-well microplates with EGFR (ACRO Biosystems, EGR-H5222) and B7-H3 (ACRO Biosystems, B73-H52E2) and incubate overnight at 4°C. Discard the coating solution, add 300 μL of 3% BSA in PBST solution, and block the wells at room temperature for 2 h. Wash three times with PBST. Dilute serum samples from different time points to a gradient and add 100 μL / well to the wells. Set up a standard sample gradient (ADC standard starting concentration 50 ng / mL, 2-fold serial dilution), and incubate at 37°C for 1 h. Wash three times with PBST, add 100 μL of anti-payload antibody (self-made, 19E9F1H7) to a final concentration of 1 μg / mL, incubate at 37°C for 1 h, and wash three times with PBST. Add 100 μL of HRP-labeled goat anti-mouse IgG (Jackson ImmunoResearch, 115-035-071, 1:5000 dilution), incubate at 37°C for 0.5 h, and wash three times with PBST. Add 100 μL of TMB for 10 min of color development, and add 100 μL of 2M sulfuric acid to stop the color development. Read the absorbance at OD450 using a multi-functional microplate reader (Tecan, Spark).

[0598] The results (Figures 18A and 18B) showed that after intravenous injection of the sample (15 mg / kg) into cynomolgus monkeys, the plasma concentrations of total antibody and ADC reached their peak at approximately 1 hour, followed by a gradual decrease in drug concentration. Calculation of the terminal half-life (T1 / 2) revealed that B035-LY22CD3 exhibited higher drug exposure and a longer half-life in cynomolgus monkeys (Table 13). Compared to B036-LY22CD, B035-LY22CD3 showed better PK stability.

[0599] Table 13. Half-life results of B035-LY22CD3 and B036-LY22CD in PK experiments

[0600] Example 15: Pretoxicology test of B035-LY22CD3 primates

[0601] Non-GLP toxicology studies in primates were conducted at WuXi AppTec Co., Ltd. in Suzhou. Six cynomolgus monkeys were randomly divided into three groups (n=2 per group, half male and half female), receiving low-dose (10 mg / kg), medium-dose (30 mg / kg), and high-dose (60 mg / kg) B035-LY22CD3, respectively. Administered to each group on days 1, 22, and 43, for a total of three administrations.

[0602] During the trial, all cynomolgus monkeys in the treatment groups tolerated the drug well, with normal weight and food intake, and no gastrointestinal side effects. The weight change curves of the cynomolgus monkeys are shown in Figure 19. The results indicate that B035-LY22CD3 has excellent safety. The highest non-severely toxic dose (HNSTD) of B035-LY22CD3 is defined as ≥30 mg / kg.

[0603] Sequence List Description

Claims

1. An antibody that specifically binds to B7-H3 and EGFR, comprising a first antigen-binding domain specifically binding to B7-H3 and a second antigen-binding domain specifically binding to EGFR, wherein the B7-H3 antigen-binding domain comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) selected from the following heavy chain variable region (VH) and light chain variable region (VL) sequence pairs: (a) The VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:2; (b) The VH sequence shown in SEQ ID NO:3 and the VL sequence shown in SEQ ID NO:4; (c) The VH sequence shown in SEQ ID NO:36 and the VL sequence shown in SEQ ID NO:37; or (d) The VH sequence shown in SEQ ID NO:38 and the VL sequence shown in SEQ ID NO:

39.

2. The antibody according to claim 1, wherein the B7-H3 antigen-binding domain comprises an amino acid sequence selected from the following combinations: (a) SEQ ID NOs: 8, 9, 10, 5, 6 and 7; (b) SEQ ID NOs: 8, 9, 10, 5, 34 and 7; (c) SEQ ID NOs: 43, 44, 45, 40, 41 and 42; or (d) SEQ ID NOs: 49, 50, 51, 46, 47 and 48; Preferably, where: -HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; -HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:9; -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:10; -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:6 or 34; -LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; More preferably, HCDR1, HCDR2 and HCDR3 respectively contain or are composed of the amino acid sequences shown in SEQ ID NOs:8-10; and LCDR1, LCDR2 and LCDR3 respectively contain or are composed of the amino acid sequences shown in SEQ ID NOs:5-7.

3. The antibody according to claim 1 or 2, wherein, The B7-H3 antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (i) The VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 3, 36 or 38, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of such amino acids; and / or (ii) The VL comprises an amino acid sequence selected from SEQ ID NOs:2, 4, 37 or 39, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the VL. Preferably, the VH and VL respectively comprise the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, or have at least 90%, 95% or 99% identity with them, or have one or more (preferably 1-10, more preferably 1-5) amino acid sequences with additions, deletions and / or substitutions. More preferably, the B7-H3 antigen-binding domain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

4.

4. The antibody of any one of claims 1-3, wherein the B7-H3 antigen-binding domain has one or more features selected from the following: (a) Binding affinity K with human B7-H3 D Value less than 1x10 -10 M, optionally approximately 5x10 -11 M to 0.5x10 -12 M, preferably approximately 1-5x10 -12 M; (b) The rate constant K for the dissociation of B7-H3 with human B7-H3 off Value less than 1x10 -5 s -1 Optionally approximately 1x10 -6 s -1 Up to 1x10 -8 s -1 Preferably approximately 0.5-5x10 -7 s -1 ;and (c) It has immune cross-reactivity with monkey B7-H3.

5. The antibody of any one of claims 1-4, wherein the antibody has one or more features selected from: (a) The binding affinity K between the EGFR antigen-binding domain and the human EGFR antigen. D Value greater than 1x10 -10 M, preferably 1x10 -7 M to 1x10 -9 M, optionally 1-6x10 -8 M or 1-6x10 -9 M; (b) The ratio of the valence of the B7-H3 antigen-binding domain to the EGFR antigen-binding domain in the antibody is 1:1; (c) The antibody is a bispecific antibody comprising an antigen-binding domain that specifically binds to B7-H3 and an antigen-binding domain that specifically binds to EGFR; and (d) The antibody is a bivalent bispecific antibody.

6. The antibody according to any one of claims 1-5, wherein the antibody comprises an Fc dimer having first and second immunoglobulin Fc regions, and optionally wherein: (i) The first Fc region and the second Fc region are IgG isotypes. (ii) The first Fc region and the second Fc region are IgG1 or IgG4 isotypes, especially human IgG1 or IgG4 isotypes; (iii) The first Fc region and the second Fc region contain amino acid mutations that promote the formation of the Fc dimer. (iv) The first Fc region contains T366S-L368A-Y407V-L351Y-D399R, and the second Fc region contains T366W-K409D-R355Q-Q419E, and / or (v) The first and second Fc regions respectively contain the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:31, or amino acid sequences that are at least 95%, 96%, 98% or 99% identical to them.

7. The antibody of any one of claims 1-6, wherein the antibody comprises -A first structural portion comprising, from the N-terminus to the C-terminus, the following components: a first antigen-binding domain and a first immunoglobulin Fc region; and -A second structural portion from the N-terminus to the C-terminus contains the following components: a second antigen-binding domain and a second immunoglobulin Fc region; The Fc regions of the first and second immunoglobulins dimerize to form Fc dimers. The first and second antigen-binding domains bind to different antigens selected from B7-H3 and EGFR, respectively. Preferably, the first antigen-binding domain binds to B7-H3 and comprises or is composed of Fab, scFab, or scFv domains; and / or the second antigen-binding domain binds to EGFR and comprises or is composed of Fab, scFab, scFv, or VHH domains. More preferably, the antibody is a bispecific antibody having the structure shown in FIG1, wherein the heavy chain optionally containing the Hole mutation and the heavy chain containing the Knob mutation respectively contain the heavy chain constant regions of SEQ ID NO:28 and SEQ ID NO:30, or respectively contain the heavy chain constant regions of SEQ ID NO:52 and SEQ ID NO:

53.

8. The antibody of any one of claims 1-7, wherein the antibody comprises an EGFR antigen-binding domain, and the EGFR antigen-binding domain comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) selected from the following heavy chain variable region (VH) and light chain variable region (VL) sequence pairs: (a) The VH sequence shown in SEQ ID NO:11 and the VL sequence shown in SEQ ID NO:12; (b) The VH sequence shown in SEQ ID NO:13 and the VL sequence shown in SEQ ID NO:14; or (c) The VH sequence shown in SEQ ID NO:15 and the VL sequence shown in SEQ ID NO:16; Preferably, wherein: -HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO:20; -HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:21 or 35; -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:22; -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:17; -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:18; -LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19; More preferably, HCDR1, HCDR2 and HCDR3 respectively contain or are composed of the amino acid sequences shown in SEQ ID NOs:20-22, and LCDR1, LCDR2 and LCDR3 respectively contain or are composed of the amino acid sequences shown in SEQ ID NOs:17-19. More preferably, the EGFR antigen-binding domain comprises: (i) a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 11, 13 or 15, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of therewith; and / or (ii) Light chain variable region (VL), wherein the VL comprises an amino acid sequence selected from SEQ ID NOs:12, 14 or 16, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the thereof; More preferably, the EGFR antigen-binding domain comprises a heavy chain variable region and a light chain variable region, and the VH and VL respectively comprise or consist of the following amino acid sequences: (i) The amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12, or amino acid sequences having at least 90%, 95% or 99% identity with them, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions. (ii) The amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14, or amino acid sequences having at least 90%, 95%, or 99% identity with them, or having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids; or (iii) The amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16, or amino acid sequences having at least 90%, 95% or 99% identity with them, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions. Most preferably, the EGFR antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the VH and VL respectively comprise or consist of the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14; or the VH and VL respectively comprise or consist of the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:

16.

9. The antibody of any one of claims 1-8, wherein the antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain and the first light chain pair and specifically bind to B7-H3; the second heavy chain and the second light chain pair and specifically bind to EGFR, and The first heavy chain, the first light chain, the second heavy chain, and the second light chain each contain or consist of the following amino acid sequences: (i) The first heavy chain comprises an amino acid sequence of SEQ ID NO: 24 or 54, or has at least 90%, 95% or 99% identity with it, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of it; (ii) The first light chain comprises the amino acid sequence of SEQ ID NO: 23, or has at least 90%, 95% or 99% identity with it, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of it; (iii) The second heavy chain comprises an amino acid sequence of SEQ ID NO: 26, 27, 55 or 56, or has at least 90%, 95% or 99% identity with it, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of it; (iv) The second light chain comprises the amino acid sequence of SEQ ID NO: 25, or has at least 90%, 95% or 99% identity with it, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of it.

10. The antibody of any one of claims 1-9, wherein the antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, and wherein... - The first heavy chain, the first light chain, the second heavy chain, and the second light chain respectively contain, or are composed of, the amino acid sequences of SEQ ID NOs:24,23,26, and25; - The first heavy chain, the first light chain, the second heavy chain, and the second light chain respectively contain, or are composed of, the amino acid sequences of SEQ ID NOs:54,23,55, and25; - The first heavy chain, the first light chain, the second heavy chain, and the second light chain respectively contain, or are composed of, the amino acid sequences of SEQ ID NOs:24,23,27, and 25; or - The first heavy chain, the first light chain, the second heavy chain, and the second light chain respectively contain, or are composed of, the amino acid sequences of SEQ ID NOs:54,23,56, and25.

11. An anti-B7-H3 antibody comprising three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained in a heavy chain variable region (VH) and light chain variable region (VL) sequence pair selected from SEQ ID NOs:1 / 2, SEQ ID NOs:3 / 4, SEQ ID NOs:36 / 37, or SEQ ID NOs:38 / 39. Optionally, the anti-B7-H3 antibody comprises an amino acid sequence combination selected from the following: (a) SEQ ID NOs: 8, 9, 10, 5, 6 and 7; (b) SEQ ID NOs: 8, 9, 10, 5, 34 and 7; (c) SEQ ID NOs: 43, 44, 45, 40, 41 and 42; or (d) SEQ ID NOs: 49, 50, 51, 46, 47 and 48; Preferably, wherein: -HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; -HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:9; -HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:10; -LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; -LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:6 or 34; -LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; More preferably, HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs:8-10 and SEQ ID NOs:5-7, or are composed of, Optionally, the B7-H3 antibody comprises: (i) a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence selected from SEQ ID NOs: 1, 3, 36 or 38, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of therewith; and / or (ii) Light chain variable region (VL), wherein the VL comprises an amino acid sequence selected from SEQ ID NOs:2, 4, 37 or 39, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed of the above; Preferably, the B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, and the VH and VL respectively comprise the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, or have at least 90%, 95% or 99% identity with them, or have one or more (preferably 1-10, more preferably 1-5) amino acid sequences with additions, deletions and / or substitutions. Most preferably, the B7-H3 antibody comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

4.

12. A polynucleotide encoding an antibody as claimed in any one of claims 1-11.

13. A vector, preferably an expression vector, comprising the polynucleotide of claim 12.

14. A host cell comprising the polynucleotide of claim 12 or the vector of claim 13, wherein the host cell is optionally a mammalian cell.

15. A method for producing the antibody according to any one of claims 1-11, the method comprising: Culture host cells containing the polypeptide chain encoding the antibody under conditions suitable for expressing the polypeptide chain; The polypeptide chain is assembled to produce the antibody under conditions suitable for assembly into the antibody.

16. An immunoconjugate or immunofusion comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-11.

17. Antibody-drug conjugates (ADCs) containing the structure shown in formula (I): Ab-(LD) n (I) Or its pharmaceutically acceptable salts or solvates, in: Ab is the antibody according to any one of claims 1-11; L is the connector; D represents a drug, preferably an anti-tumor compound; and n is an integer selected from 1 to 16, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or a range of any two values ​​between 1 and 10.

18. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 17, wherein the antitumor compound is a cytotoxic agent, such as a camptothecin compound, preferably eczemab, Dxd or a derivative thereof.

19. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-18, wherein D is a camptothecin compound containing the structure shown in formula (D-1): in: R a1 Selected from hydrogen atoms, tritium atoms, and C1-C6 alkyl groups; R a2 selected from a bond, -C(=O)-CR 1 R 2 -(CR 3 R 4 ) m -O-*, -C(=O)-CR 1 R 2 -(CR 3 R 4 ) m -NH-*, -C(=O)-O-CR 1 R 2 -(CR 3 R 4 ) m -O- *,-C(=O)-O-CR 1 R 2 -(CR 3 R 4 ) m -NH-*, Where R 1 and R 2 Each is independently selected from hydrogen atoms, C1-C6 alkyl groups, halogenated C1-C6 alkyl groups, C3-C8 cycloalkyl groups, C3-C8 cycloalkyl groups, C1-C6 alkyl groups, and C6-C6 alkyl groups. 10 aryl C1-C6 alkyl; or R 1 and R 2 It forms a C3-C8 cycloalkyl group with the carbon atom it is attached to; R 3 and R 4 Each is independently selected from hydrogen atoms and C1-C6 alkyl groups; m is an integer between 0 and 6; R a3 Selected from H, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -OR 5 and -SR 5 ; R a4 Selected from H, halogen, CN, C1-C6 alkyl, halogenated C1-C6 alkyl and -OR 5 ;and R 5 Independently selected from H and C1-C4 alkyl groups, Where * indicates the connection site with the connector L unit.

20. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt or solvate thereof, wherein... R a1 For H; R a2 It is -C(=O)-O-CH2-(CH2) m -O-, where m = an integer of 0, 1, 2, 3, 4, 5 or 6, preferably m = 3-5; preferably R a2 It is -C(=O)-O-(CH2)4-O-; R a3 It is a C1-C6 alkyl group, preferably methyl; R a4 It is a halogen, preferably F.

21. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-20, wherein D has the structure shown in formula (D-1a) or formula (D-1b): Where R a1 R a2 R a3 and R a4 As defined in claim 19 or 20.

22. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-21, wherein D has the structure shown in formula (D-2): Preferably, D has the structure shown in formula (D-2a) or formula (D-2b):

23. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-22, wherein D has the structure shown in formula (D-3): Preferably, D has the structure shown in formula (D-3a) or formula (D-3b), especially the structure shown in formula (D-3a):

24. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-23, wherein, L is a peptide linker containing the structure of formula (II): -ZYM-, (II) in Z is the linker base connected to Ab. Y is an enzyme-cleavable peptide linker containing amino acids. M is absent, or it is a spacer group used to link with drug D.

25. The antibody-drug conjugate of claim 24 or a pharmaceutically acceptable salt or solvate thereof, wherein, The Y unit contains a peptide or peptide analog selected from the following: *-Val-Ala-, *-Val-Cit-, *-Glu-Val-Cit-, *-Glu-Gly-Cit-, *-Gly-Phe-Gly-, *-Gly-Val-Cit-, *-Gly-Val-Ala-, *-Gly-Phe-Gly-, *-Phe-Gly-, *-Gly-Phe-Lys-, *-Phe-Lys-, *-Gln-Val-Ala-, *-Gln-Val-Cit-, *-Asp-Val-Ala-, *-Lys-Gly-Val-Ala-, *-Lys-Gly-Val-Cit-, *-Lys-Gly-Gly-Val-Ala- a-, *-Lys-Gly-Gly-Val-Cit-, *-Gly-Gly-Phe-Gly-, *-Lys-Gln-Val-Cit-, *-Lys-Gln-Val-Ala-, *-Lys-Glu-Val-Cit-, *-Lys-Glu-Val-ALa-, *-Lys-Asp-Val-Cit-, *-Lys-Asp-Val-Ala-, *-Glu-Val-Ala-, *-Lys-Val-Ala-, *-Lys-Val-Ala-, *-Lys-Val-Cit-, -Val-Lys-, *-Asp-Val-Cit-, *-Ala-Ala-Ala-, or *-cBu-Cit-, where * indicates the connection site with Z; Preferably, Y is -Val-Cit- or -Val-Ala-, and more preferably, Y has the following structure: The asterisk on the left indicates a connection to the Z-base, and the wavy line on the right indicates a connection to M (if M exists) or a direct connection to drug D.

26. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 24 or 25, wherein, Following the direction from the Ab element side to the Y element side, Z has the following structure: -Z1-Z2-Z3-Z4-, in, Z1 is a 5-10 membered heterocyclic group, preferably containing 1-3 heteroatoms selected from N, S, and O; more preferably, Z1 is selected from Z2 is selected from key, -C(=O)-, *-C1-C 10 Alkylene -C(=O)-, *-C3-C 10 Ethyne-C(=O)-, *-C3-C 10 alkenyl-C(=O)- and *-heteroaryl-C1-C 10 Alkylene-C(=O)-, Z3 does not exist or is *-R 6 -(CH2-CH2-O-) x -R 7 - where x is an integer from 0 to 5, preferably x = 1 or 2; Z4 does not exist or is *-R 8 -(CH2-CH2-O-) y -R 9 - where y is an integer from 0 to 12, preferably y = 6, 7, 8, 9 or 10; R 6 and R 8 Each is independently selected from key, C 1-4 Alkylene, -NH-, -heteroaryl-, *-NH-C 1-4 alkylene-heteroaryl-, wherein the heteroaryl group is a 5- or 6-membered nitrogen-containing heteroaryl group, preferably a triazolyl group; R 7 and R 9 Independently selected from -C(=O)-, *-C(=O)-NH-, C 1-4 Alkylene, *-C 1-4 Alkylene-C(=O)-, *-C 1-4 Alkylene -C(=O)-NH-, *-NH-C(=O)-(CH2OCH2)-C(=O)-, *-C 1-4 Alkylene-NH-C(=O)-(CH2OCH2)-C(=O)-, Preferably R 6 and R 8 Each is independently selected from -NH-, and R 7 and R 9 Independently selected from -C(=O)-, *-C 1-4 Alkylene-C(=O)-, *-C 1-4 Alkylene-NH-C(=O)-(CH2OCH2)-C(=O)-, The heteroaryl group is a 5- or 6-membered nitrogen-containing heteroaryl group, preferably a triazole group. Where * indicates the connection site facing the Ab unit side.

27. The antibody-drug conjugate of claim 26 or a pharmaceutically acceptable salt or solvate thereof, in, -Z1-Z2-Z3-Selected from in: n1 is an integer from 1 to 8, for example, 1, 2, 3, 4, 5 or 6; n2 is an integer from 0 to 5, for example, 1 or 2; n3 is an integer from 1 to 4, for example, 1 or 2; Z4 does not exist or is Where n4 is an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Where * indicates the connection site facing the Ab unit side. This indicates the connection point facing the Y-unit side.

28. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 24-27, wherein Z has a structure selected from: Where n1 is an integer from 1 to 6, n2 is an integer of 1 or 2, and n4 is an integer from 1 to 10. Preferably, Z has a structure selected from the following: The asterisk on the left indicates the binding site with antibody Ab, and the wavy line on the right indicates the binding site with Y unit.

29. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 24-28, wherein M is absent or is an amino-C1-C3 alkylene group, for example, -NH-CH2-, or amino-phenyl-C1-C3 alkylene group-OC(=O)-. Preferably, M is selected from: Where R 10 Selected from: H and C1-C6 alkyl groups; n5 is 1, 2, 3, or 4; n6 is 1, 2, 3, 4, 5, or 6. The preferred option for M is: The asterisk on the left indicates the connection site with unit Y, and the wavy line on the right indicates the connection site with unit D of the drug.

30. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 24-29, wherein: The L-connector has the following structure: Or, the L-connector may have a structure: Wherein, Y is as defined in claim 25, preferably Y is -Val-Cit- or Val-Ala, Where n1 is an integer from 1 to 6, n2 is an integer of 1 or 2, and n4 is an integer from 1 to 10. Preferably, the L-connector has a structure selected from the following: The asterisk on the left indicates the binding site with the antibody Ab unit, and the wavy line on the right indicates the binding site with the drug D unit.

31. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-30, wherein the LD unit of formula I is linked to the antibody by forming a thioether bond with the thiol group of cysteine ​​in the light chain and / or heavy chain of the Ab.

32. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-31, wherein, The ADC comprises a structure selected from the following formulas (I-1) and (I-2): Wherein, Ab and n are as defined in claim 17, Preferably, the Ab comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences of SEQ ID NOs:24,23,26, and 25, respectively; or comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences of SEQ ID NOs:24,23,27, and 25, respectively, or The Ab comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences of SEQ ID NOs:54,23,55, and 25, respectively; or comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences of SEQ ID NOs:54,23,56, and 25, respectively.

33. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-32, wherein, The ADC has an average DAR of 2-10, 2-6, 4-8, 6-8, or 7-8.

34. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 17-33, wherein, The ADC comprises the structure of the following formula (I-2): The Ab comprises, or consists of, a first heavy chain, a first light chain, a second heavy chain, and a second light chain having amino acid sequences having SEQ ID NOs: 54, 23, 55, and 25, respectively, and The ADC has an average DAR of approximately 6-8, preferably approximately 8.

35. A pharmaceutical composition comprising an antibody according to any one of claims 1-11, or an immunoconjugate or immunofusion of claim 16, or an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 17-34, and a pharmaceutically acceptable carrier.

36. The antibody of any one of claims 1-11, or the immunoconjugate or immunofusion of claim 16, or the antibody-drug conjugate or pharmaceutically acceptable salt or solvate of any one of claims 17-34, used as a drug or for the preparation of a drug, preferably said drug for the treatment and / or prevention of cancer in an individual.

37. A method of treating or preventing cancer, comprising administering to an individual in need an antibody according to any one of claims 1-11, or an immunoconjugate or immunofusion of claim 16, or an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 17-34.

38. The use of claim 36 or the method of claim 37, wherein the cancer is a B7-H3 or EGFR-positive solid tumor or hematologic malignancy, selected from, for example, bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, glioma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastrointestinal cancer, skin cancer, squamous cell carcinoma, and adenocarcinoma. Optionally, the cancer is skin cancer (such as squamous cell carcinoma of the skin), head and neck cancer, pharyngeal cancer (such as squamous cell carcinoma of the pharynx), esophageal cancer (such as squamous cell carcinoma of the esophagus), intestinal cancer (such as colon cancer, rectal cancer, colorectal cancer), or lung cancer (such as non-small cell lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung).

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