Anti-b7h3 antibody and use thereof

Abstract

The present application discloses an antibody specifically binding to B7H3 or an antigen-binding fragment thereof, a nucleic acid encoding same, a recombinant vector, a host cell, a preparation method, a pharmaceutical composition, and a use in the treatment of tumor diseases, which can be used for the development of B7H3-targeted therapeutic drugs and the development of detection reagents.

Description

Anti-B7H3 antibodies and their applications

[0001] This disclosure claims priority to Chinese patent application number 202410110647.3, filed with the China Patent Office on January 25, 2024, entitled “Anti-B7H3 Antibodies and Their Applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the field of antibodies, and in particular, to anti-B7H3 antibodies. Background Art

[0003] B7-H3 is a type I transmembrane protein, also known as CD276, and a member of the B7 ligand family. Early studies suggested that B7H3 is a T cell co-stimulatory factor, playing a regulatory role in T cell activation and IFNγ production. Other studies suggest that B7H3 plays an inhibitory role in adaptive immunity, possibly through different receptors, but the specific receptor for B7H3 is currently unclear. The human B7H3 protein is 534 amino acids long and exists in two isoforms: B7H3-4Ig and B7H3-2Ig. The former contains two pairs of immunoglobulin variable region (IgV) and immunoglobulin constant region (IgC)-like domains and is the predominant isoform in humans. The B7H3-2Ig isoform contains only one pair of IgV and IgC domains.

[0004] B7H3 is widely expressed in normal tissues, but expression levels are limited and relatively low, such as in the liver, colon, and prostate. However, it is highly expressed in tumor tissues and is closely associated with cancer progression, survival, and prognosis. Studies have found that B7H3 is highly expressed in a variety of tumor tissues, including small cell lung cancer, prostate cancer, renal cancer, colorectal cancer, and squamous cell lung carcinoma. Within tumor tissues, B7H3 is expressed both in tumor cells and in stromal cells, such as tumor vascular endothelial cells, pericytes, and fibroblasts. This suggests that B7H3 plays an important role in cancer progression, migration, invasion, anti-apoptosis, metabolism, and angiogenesis. Although the physiological functions of B7H3 remain largely undetermined, its high expression levels and high number of positive patients make it an attractive target for cancer therapy. Currently, therapeutic approaches targeting B7H3 are under development, including ADCC-enhancing monoclonal antibodies, CD3 bispecific antibodies, CARTs, and antibody-drug conjugates (ADCs). However, to date, no B7H3-targeting drugs have been approved for marketing. Summary of the Invention

[0005] The present disclosure provides a new tumor treatment method by providing a new antibody or antigen-binding fragment thereof that specifically binds to B7H3.

[0006] In a first aspect of the present disclosure, an antibody or antigen-binding fragment thereof that specifically binds to B7H3 is provided, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0007] (1) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are the HCDR1, HCDR2 and HCDR3 of the VH domain shown in SEQ ID NO. 12; and

[0008] (2) The light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2 and LCDR3 of the VL domain shown in SEQ ID NO.13.

[0009] In some specific embodiments, the HCDR1, HCDR2, and HCDR3 are determined according to the Kabat, Chothia, or IMGT numbering systems, and have an amino acid sequence as shown in SEQ ID NO.14-16, SEQ ID NO.20-22, or SEQ ID NO.26-28, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions, and / or substitutions compared to the amino acid sequences shown in SEQ ID NO.14-16, SEQ ID NO.20-22, or SEQ ID NO.26-28.

[0010] In some specific embodiments, the LCDR1, LCDR2 and LCDR3 are determined according to the Kabat, Chothia or IMGT numbering system, and have an amino acid sequence as shown in SEQ ID NO.17-19, SEQ ID NO.23-25, SEQ ID NO.29-31, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NO.17-19, SEQ ID NO.23-25, SEQ ID NO.29-31.

[0011] In some specific embodiments, the antibody or its antigen binding protein comprises the following LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3:

[0012] (1) According to the Kabat numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 14-19, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 14-19;

[0013] (2) According to the IMGT numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 20-25, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 20-25;

[0014] (3) According to the Chothia numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 26-31 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 26-31.

[0015] In some specific embodiments, the heavy chain variable region sequence comprises the sequence shown in SEQ ID NO.12, or a sequence with 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the sequence shown; the light chain variable region sequence comprises the sequence shown in SEQ ID NO.13, or a sequence with 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to the sequence shown.

[0016] In some specific embodiments, the antibody or antigen-binding fragment thereof is fully human.

[0017] In some specific embodiments, the antibody or antigen-binding fragment thereof can specifically bind to human or monkey B7H3 protein.

[0018] In some specific embodiments, the antibody or antigen-binding fragment thereof may further comprise any constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4 with mutation; further, the antibody or antigen-binding fragment thereof is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic drugs, cytotoxic agents or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from alkaloids, methotrexate, anthracycline antibiotics (doxorubicin), pyrrolobenzodiazepines, (pyrrolobenzodiazepine, PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane.

[0019] In some specific embodiments, the antigen-binding fragment is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv, nanobody or affibody.

[0020] In a second aspect of the present disclosure, a multispecific antigen-binding molecule is provided, comprising the antibody or antigen-binding fragment thereof described in the first aspect; preferably, the multispecific antigen-binding molecule further comprises an antibody or antigen-binding fragment thereof that specifically binds to an antigen other than B7H3 or binds to a B7H3 epitope different from the B7H3 epitope bound by the antibody or antigen-binding fragment thereof described in the first aspect;

[0021] Preferably, the antigen other than B7H3 is selected from the following group: (1) tumor-specific antigen (TSA) or tumor-associated antigen (TAA); (2) immune checkpoint; (3) target for recruiting and / or activating immune cells;

[0022] Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific;

[0023] Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

[0024] The third aspect of the present disclosure provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the antibody or antigen-binding fragment thereof according to the first aspect.

[0025] The fourth aspect of the present disclosure provides an immune effector cell, which expresses the chimeric antigen receptor described in the third aspect, or contains a nucleic acid fragment encoding the chimeric antigen receptor; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

[0026] In a fifth aspect of the present disclosure, an isolated nucleic acid fragment is provided, which encodes the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, or the chimeric antigen receptor described in the third aspect.

[0027] In a sixth aspect of the present disclosure, a vector is provided, comprising the nucleic acid fragment.

[0028] The seventh aspect of the present disclosure provides a host cell comprising the vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

[0029] The eighth aspect of the present disclosure provides a method for preparing the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule described in the first aspect, which comprises culturing the host cells, and isolating the antibody or antigen-binding fragment thereof expressed by the host cells, or isolating the multispecific antigen-binding molecule expressed by the host cells.

[0030] The ninth aspect of the present disclosure provides a method for preparing the immune effector cell, which comprises introducing a nucleic acid fragment encoding the CAR described in the third aspect into the immune effector cell, and optionally, further comprises initiating the immune effector cell to express the CAR.

[0031] In a tenth aspect of the present disclosure, a pharmaceutical composition is provided, comprising the antibody or antigen-binding fragment thereof described in the first aspect, or the multispecific antigen-binding molecule described in the second aspect, or the immune effector cell described in the fourth aspect, the nucleic acid fragment described in the fifth aspect, or the vector described in the sixth aspect, or the host cell described in the seventh aspect, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method described in the eighth aspect, or the immune effector cell prepared by the method described in the ninth aspect; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0032] In an eleventh aspect of the present disclosure, there is provided use of the antibody or antigen-binding fragment thereof described in the first aspect, or the multispecific antigen-binding molecule described in the second aspect, or the immune effector cell described in the fourth aspect, or the nucleic acid fragment described in the fifth aspect, or the vector described in the sixth aspect, or the host cell described in the seventh aspect, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method described in the eighth aspect, or the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect, in preparing a medicament for preventing and / or treating tumors; the tumor is selected from a solid tumor, a hematological tumor, or a cancer infiltrating and expressing B7H3;

[0033] Preferably, the tumor is selected from melanoma, prostate cancer, renal cell carcinoma, lung cancer (eg non-small cell lung cancer (NSCLC)) and other solid tumors.

[0034] In a twelfth aspect of the present disclosure, a method for preventing and / or treating tumors is provided, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment thereof of the first aspect, or the multispecific antigen-binding molecule of the second aspect, or the immune effector cell of the fourth aspect, or the nucleic acid fragment of the fifth aspect, or the vector of the sixth aspect, or the host cell of the seventh aspect, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method of the eighth aspect, or the immune effector cell prepared by the method of the ninth aspect, or the pharmaceutical composition of the tenth aspect; wherein the tumor is selected from a solid tumor, a hematological tumor, or a cancer infiltrating and expressing B7H3;

[0035] Preferably, the tumor is selected from melanoma, prostate cancer, renal cell carcinoma, lung cancer (eg non-small cell lung cancer (NSCLC)) and other solid tumors.

[0036] In a thirteenth aspect of the present disclosure, a kit is provided, comprising the antibody or antigen-binding fragment thereof described in the first aspect, or the multispecific antigen-binding molecule described in the second aspect, or the immune effector cell described in the fourth aspect, or the nucleic acid fragment described in the fifth aspect, or the vector described in the sixth aspect, or the host cell described in the seventh aspect, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method described in the eighth aspect, or the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect.

[0037] A fourteenth aspect of the present disclosure provides an in vitro method for detecting B7H3 expression, wherein a sample to be detected is contacted with the antibody or antigen-binding fragment thereof described in the first aspect under conditions where a complex can be formed between the antibody or antigen-binding fragment thereof and B7H3; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of DLL3 in the sample.

[0038] A fifteenth aspect of the present disclosure provides use of the antibody or antigen-binding fragment thereof described in the first aspect in preparing a B7H3 detection reagent.

[0039] Definitions and Explanations of Terms

[0040] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings that are understood by those of ordinary skill in the art.

[0041] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0042] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0043] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0044] The term "B7H3" herein is a member of the B7-CD28 superfamily and is expressed on antigen presenting cells. B7-H3 binds to T cells, but the B7-H3 counter-receptor on the surface of such T cells has not yet been fully characterized. The main human form of B7H3 comprises two extracellular IgV-IgC domains (i.e., IgV-IgC-IgV-IgC) in series. Although initially thought to comprise only two Ig domains (IgV-IgC), a tetra-immunoglobulin extracellular domain variant ("4Ig-B7-H3") has been identified and found to be a more common human form of the protein. The natural murine form (2Ig) and the human 4Ig form show similar functions. The 4Ig-B7-H3 molecule inhibits the lysis of NK cell-mediated cancer cells. B7H3 mRNA expression has been found in heart, kidney, testis, lung, liver, pancreas, prostate, colon, and osteoblasts.

[0045] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an antibody with a specific affinity of about 10 -6 M or lower, 10 -7 M or lower, about 10 -8 M or lower, about 10 -9The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0046] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0047] The term "antibody" herein is used in the broadest sense and refers to a polypeptide or polypeptide combination comprising sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer, IP, Beste, G. & Skerra, A. (2003). Proteins, 53, 121-129.; Roque, ACA, Lowe, CR & Taipa, MA Antibodies and genetically engineered related molecules: production and purification. Biotechnol. Prog. 20, 639-654 (2004); the contents of which are incorporated herein in their entirety. Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0048] The term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Differences in the amino acid composition and order of arrangement of the constant region of immunoglobulins' heavy chains result in varying antigenicity. Consequently, "immunoglobulins" can be classified into five classes, or isotypes, herein: IgM, IgD, IgG, IgA, and IgE, corresponding to their corresponding heavy chains: μ, δ, γ, α, and ε. Within the same class, Ig can be further divided into subclasses based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

[0049] The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0050] The term "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0051] The term "heavy chain antibody" herein refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0052] As used herein, the term "nanoantibody" refers to a naturally occurring heavy chain antibody lacking a light chain that exists in camels. Cloning its variable region can yield a single-domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0053] The terms "nanobody" and "single-domain antibody" (sdAb) are used interchangeably and have the same meaning. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy-chain variable region by cloning the variable region of a heavy-chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy-chain antibody naturally lacking the light chain and heavy-chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy-chain variable region.

[0054] For further description of “heavy chain antibodies” and “nanobodies”, see: Hamers-Casterman et al., Nature. 1993; 363; 446-8; the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001); and the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531; WO 01 / 90190; WO 03 / 025020; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 and other prior art mentioned in these applications. (The above contents are incorporated herein in their entirety).

[0055] The term "multi-specificity" herein refers to the ability of an antibody or its antigen-binding fragment to bind to, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody can bind to. For example, conventional monospecific IgG antibodies have two identical antigen-binding sites (paratopes) and can therefore only bind to the same epitope (rather than binding to different epitopes). In contrast, multispecific antibodies have at least two different types of paratopes / binding sites and can therefore bind to at least two different epitopes. As described herein, "complementarity determining region" refers to the antigen-binding site of an antibody. In addition, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions (the actual number of complementary determining regions / binding sites in a single antibody molecule is referred to as "valence") in a single antibody. For example, a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes. Accordingly, a multispecific antibody comprises at least two (different) complementary determining regions / binding sites. Therefore, the term "multispecific antibody" refers to an antibody having more than one paratope and the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but generally also includes proteins, e.g. antibodies that specifically bind three or more different epitopes, scaffolds, i.e. antibodies with three or more paratopes / binding sites.

[0056] The term "valent" herein refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0057]

[0014] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody.

[0058] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0059] The term "chimeric antibody" herein refers to an antibody having variable sequences of an immunoglobulin from one source organism (such as rat, mouse, rabbit or alpaca) and constant regions of an immunoglobulin from a different organism (such as human). Methods for producing chimeric antibodies are known in the art. See, for example, US Pat. No. 5,807,715A; Morrison, 1985, Science 229 (4719): 1202-1207. Transfectomas Provide Novel Chimeric Antibodies; Gillies et al., J Immunol Methods. 1989 Dec 20; 125 (1-2): 191-202; the entire contents are incorporated herein.

[0060] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune response, etc.

[0061] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted onto human framework sequences.

[0062] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007); the contents of which are incorporated herein in their entirety. A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0063] The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) found in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are referred to as framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered as hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of standards (such as IMGT, Chothia or KABAT), while being considered to be outside the hypervariable region under different sets of standards (such as KABAT, Chothia or IMGT). One or more of these positions can also be found in an extended hypervariable region. The present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region can be abbreviated as LCDRs. The variable domains of native heavy and light chains each contain four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3), which form loops connecting the sheet structure and, in some cases, form part of the sheet structure. The CDRs in each chain are held together tightly by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen-binding site of the antibody with the CDRs from other antibody chains (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; the contents of which are incorporated herein in their entirety).

[0064] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways. (The foregoing is incorporated herein in its entirety).

[0065] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0066] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0067] The term "IMGT numbering system" herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003 (incorporated herein in its entirety).

[0068] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, and has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" can be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. A "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to that of a native antibody constant region, while the latter only includes "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.

[0069] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0070] The term "Fc region" herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. For example, the human IgG heavy chain Fc region may extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage, removing one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells through expression of a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may be present or absent. Typically, an IgG Fc region comprises an IgG CH2 and IgG CH3 domains, and optionally, may further comprise a complete or partial hinge region, but does not comprise a CH1 domain. The "CH2 domain" of a human IgG Fc region typically extends from approximately amino acid residue position 231 to approximately amino acid residue position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues at the C-terminus of the CH2 domain in the Fc region (i.e., from approximately amino acid residue position 341 to approximately amino acid residue position 447 of IgG). The CH3 domain herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" introduced into one chain and a corresponding "hole" introduced into the other chain; see U.S. Patent No. 5,821,333, the contents of which are incorporated herein in their entirety). As described herein, such variant CH3 domains can be used to promote heterodimerization of two different antibody heavy chains.

[0071] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; the contents of which are incorporated herein in their entirety.

[0072] The term "Fc variant" herein refers to changes in Fc structure or function caused by one or more amino acid substitutions, insertions, or deletions at appropriate sites on the Fc protein. "Inter-Fc variant interactions" refer to interactions between Fc variants engineered to form space-filling effects, electrostatic interactions, hydrogen bonding, hydrophobic interactions, and other interactions. These interactions contribute to the formation of stable heterodimeric proteins. Preferred mutational designs are "knob-into-hole" mutational designs.

[0073] The mutation design technology of Fc variants has been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "knob-into-hole" format proposed by Cater et al. (Protein Engineering vol. 9 no. 7 pp. 617-621, 1996); the use of electrostatic steering by Amgen technicians to form Fc-containing heterodimers (US20100286374 A1); the heterodimers (SEEDbodies) formed by IgG / Ig chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp. 1-8, 2010); the bispecific molecules formed by Genmab's DuoBody (Science, 2007. 317 (5844)) platform technology; and the heterodimers formed by Xencor technicians using a combination of structural calculations and Fc amino acid mutations to combine different modes of action (mAbs 3: 6, 546-557; November / December 2010). 2011); Suzhou Alphamab's charge network-based Fc modification method (CN201110459100.7) to produce a heterodimeric protein form; and other genetic engineering methods based on Fc amino acid changes or functional modification to achieve the formation of heterodimeric functional proteins. The knob / hole structure of the Fc variant fragments described herein refers to mutations in the two Fc fragments, which, after mutation, can combine in a "knob-into-hole" formation. Preferably, the "knob-into-hole" model of Cater et al. is used to perform site-specific mutations in the Fc region, so that the resulting first and second Fc variants can combine in a "knob-into-hole" formation to form a heterodimer. Selecting a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the skill of those skilled in the art. Preferred are Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4, with the Fc region of human antibody IgG1 being more preferred. Randomly select one of the first Fc variant or the second Fc variant to make a knob mutation and the other to make a hole mutation. (The above contents are incorporated herein in their entirety).

[0074] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0075] 1) Alanine (A), serine (S), threonine (T);

[0076] 2) Aspartic acid (D), glutamic acid (E);

[0077] 3) Asparagine (N), glutamine (Q);

[0078] 4) Arginine (R), Lysine (K), Histidine (H);

[0079] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0080] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0081] The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences varies as the number of identical positions shared by the sequences changes, taking into account the number of spaces that need to be introduced and the length of each space for optimal comparison of the two sequences.

[0082] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and one that should be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4. (The aforementioned contents are incorporated herein in their entirety.)

[0083] Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present disclosure can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov (incorporated herein in their entirety).

[0084] The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen, which comprises at least (1) an extracellular antigen binding domain, such as an antibody heavy chain variable region and / or light chain variable region, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR is able to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding domain.

[0085] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as vectors for directly expressing antibodies of the present disclosure in vitro and / or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP2101823B1; the foregoing is incorporated herein in its entirety).

[0086] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0087] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0088] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0089] The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0090] The term "pharmaceutically acceptable carrier" herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed. Mack Printing Company, 1990, pp. 1289-1329; the contents of which are incorporated herein in their entirety). Except in the case of incompatibility with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

[0091] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0092] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.

[0093] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0094] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIG1A is an ELISA assay for the binding activity of SCR-10437 to human B7H3(4Ig)-his protein;

[0096] FIG1B is an ELISA test showing the binding activity of SCR-10437 to human B7H3(2Ig)-his protein;

[0097] FIG1C is an ELISA test of the binding activity of SCR-10437 to monkey B7H3-his protein;

[0098] Figures 2A-2D show the expression levels of B7H3 in tumor cells A375, A549, 786-O, and NCI-H1975;

[0099] FIG3A is a FACS assay showing the binding reaction between SCR-10437 and the human endogenous tumor cell line A375;

[0100] FIG3B is a FACS assay showing the binding reaction between SCR-10437 and the human endogenous tumor cell line A549;

[0101] FIG4A is a FACS assay showing the endocytic activity of B7H3 antibodies in the tumor cell line 786-O;

[0102] FIG4B is a FACS assay showing the endocytic activity of B7H3 antibodies on the tumor cell line NCI-H1975. DETAILED DESCRIPTION

[0103] The present disclosure is further described below with reference to specific examples, and the advantages and features of the present disclosure will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0104] The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

[0105] Example 1. Preparation and purification of recombinant protein and control antibody

[0106] 1.1 Design and expression of recombinant proteins

[0107] Construction of B7H3-4Ig recombinant protein: Using human B7H3-4Ig protein (UniProt No.: Q5ZPR3-1) as a template sequence, a tagged fusion protein was designed and cloned into the pTT5 vector (Ubi Biotech, VT2202) to construct the B7H3-4Ig plasmid. Similarly, construction of B7H3-2Ig recombinant protein: Using human B7H3-2Ig protein (UniProt No.: Q5ZPR3-2) as a template sequence, a tagged fusion protein was designed and cloned into the pTT5 vector to construct the B7H3-2Ig plasmid. The antigens and detection proteins disclosed herein were transiently expressed in Expi 293F cells (Gibco, A14527). The preparation method for cynomolgus monkey B7H3 recombinant protein is similar to that for human recombinant protein. The cynomolgus monkey B7H3 sequence is from UniProt No.: A0A2K5U2B3. The specific sequence information of the recombinant protein is shown in Table 1.

[0108] Recombinant protein purification using a nickel column: Cell expression supernatant samples were centrifuged at high speed to remove impurities. The nickel column was equilibrated with 20mM PBS + 500mM NaCl solution and rinsed for 2-5 column volumes. The culture supernatant was loaded onto a Ni affinity chromatography column (purchased from GE Healthcare) while monitoring the UV absorbance (A280nm) using a UV detector. The column was rinsed with equilibration solution until the A280 reading dropped to baseline. The column was then eluted with a gradient of equilibration solution containing 10mM, 20mM, 40mM, 90mM, 250mM, and 500mM imidazole. The eluted peaks were collected and the target protein was identified based on SDS-PAGE gel images. The collected eluted product containing the target protein was concentrated and further purified using a Superdex200 gel chromatography (GE) with PBS as the mobile phase to remove aggregates and impurity peaks. The target product elution peak was collected. The resulting protein was confirmed to be positive by electrophoresis, peptide mapping, and LC-MS and then aliquoted for use. The proteins purified by this protocol include human B7H3 4Ig-His, human B7H3 2Ig-His and monkey B7H3-His.

[0109] Table 1 Sequences of human and monkey B7H3-His recombinant proteins

[0110] 1.2 Design and expression of control antibodies

[0111] The control antibodies used in this disclosure are all from published patents. The DS7300 antibody is derived from the published patent CN103687945B, the MGC018 antibody is derived from the published patent WO2017180813A, and the enoblituzumab antibody is derived from the published patent WO2011109400A2. Unless otherwise specified, the DS7300, enoblituzumab, and MGC018 control antibodies were all recombinantly expressed using the human IgG1+κ subtype.

[0112] The control antibody was expressed and purified as follows: the antibody sequence was synthesized and cloned into the expression vector pTT5, then transiently transfected into Expi293F cells (purchased from Gibco, A14527). After 7 days of incubation at 37°C on a shaker, the cell supernatant was collected for Protein A antibody purification. The Protein A affinity column was washed with 0.1M NaOH for 3-5 column volumes, followed by 3-5 column volumes of pure water. The column was equilibrated with 1× PBS (pH 7.4) as the equilibration buffer for 3-5 column volumes. The cell supernatant was loaded and bound at a low flow rate, controlling the flow rate to achieve a retention time of approximately 1 minute or longer. After binding, the column was washed with 1× PBS (pH 7.4) for 3-5 column volumes until the UV absorbance returned to baseline. The sample was eluted using 50mM citric acid / sodium citrate (pH 3.0-3.5) buffer, and the elution peak was collected according to ultraviolet detection. The eluted product was quickly adjusted to pH 5-6 using 1M Tris-HCl (pH 8.0) and temporarily stored. The eluted product can be subjected to solution replacement using methods well known to those skilled in the art, such as ultrafiltration concentration and solution replacement to the desired buffer system using an ultrafiltration tube, or replaced with the desired buffer system using molecular exclusion such as G-25 desalting, or using a high-resolution molecular exclusion column such as Superdex 200 to remove polymer components in the eluted product to improve sample purity. The obtained control antibodies were named DS7300-hIgG1, MGC018-hIgG1, and Enoblituzumab-hIgG1. The specific sequence information of the antibodies is shown in Table 2.

[0113] Table 2 Control antibody sequence list

[0114] Example 2: Preparation of antibodies against B7H3

[0115] 2.1 Animal immunization

[0116] The monoclonal antibodies disclosed herein are produced by immunizing mice. The mice used in the experiment were female mice aged 6 to 8 weeks. The immunogen was the human B7H3 4Ig-his protein prepared in Example 1. During the initial immunization of the protein, the immunogen was emulsified with TiterMax (purchased from Sigma, T2684-1M) and injected subcutaneously (SC) and intraperitoneally (IP) with 0.1 mL, i.e., each mouse was injected with 50 μg of the immunogen; during the booster immunization, the immunogen was injected subcutaneously and intraperitoneally with Imject Alum (purchased from Thermo) with 0.1 mL, i.e., each mouse was injected with 25 μg of the immunogen. The immunization frequency was once a week, and blood was collected on days 3, 19, 47, and 61. The binding to the human B7H3 protein (His tag, coating concentration 2 μg / mL) was detected by ELISA, and the presence and antibody titer of antibodies recognizing human B7H3 in the serum of the immunized animals were tested. Based on the serum titer, mice with high antibody titer in serum and titer approaching a plateau were selected for booster immunization.

[0117] 2.2 Antibody Screening and Sequencing

[0118] After the booster immunization, antigen-specific B cells were directly isolated from the immunized mice without fusion with myeloma cells. Plasma cells secreting antigen-specific monoclonal antibodies were screened using an Optofluidic System (Berkeley Lights Inc.). The antibody light and heavy chain variable region sequences were directly obtained from antigen-specific B cells by reverse transcription and PCR sequencing. The resulting humanized anti-B7H3 antibody, designated SCR10437, was screened using conventional methods. The amino acid sequences of its heavy chain variable region (HCVR) and light chain variable region (LCVR) are shown below:

[0119] SCR10437 HCVR (SEQ ID NO.12):

[0120] SCR10437 LCVR (SEQ ID NO.13):

[0121] The CDR regions of the B7H3 monoclonal antibody were analyzed. The CDR regions were identified and annotated using the Kabat numbering system, the Chothia numbering system, and the IMGT numbering system. The specific results are shown in Table 3.

[0122] Table 3 Antibody CDR sequences and numbers

[0123] 2.3 Construction, expression and purification of fully human antibodies

[0124] Based on the sequencing results of the variable region genes of the SCR10437 antibody obtained in Section 2.2 above, primers were designed and PCR was used to construct the VH / VL gene segments of each antibody. The obtained VH / VL gene segments were homologously recombined with the expression vector pTT5 to construct a full-length expression vector plasmid for a fully human antibody. The SCR10437 antibody was expressed in the form of human IgG1 (the sequences of the heavy chain constant region and the light chain constant region are shown in Table 2). After the plasmid preparation was completed, it was transfected into Expi293F cells and cultured on a shaker at 37°C for 7 days. The supernatant was collected, centrifuged, and the antibody was purified according to the purification method described in Section 1.2 of Example 1. The resulting antibody was named SCR10437-hIgG1.

[0125] Example 3: Antibody affinity determination

[0126] The binding strength of antibodies to antigens was determined using a Protein A capture assay using a BIAcore 8K instrument. Protein A was first immobilized onto a CM4 chip (GE, BR-1005-34) using the amino coupling method. Following the instructions for the Amine Coupling Kit (GE, BR100633), the chip was activated for approximately 600 seconds using a mixture of NHS and EDC using HBS-EP+, pH 7.4, as the mobile phase. Protein A was then diluted to 50 μg / mL with 10 mM sodium acetate, pH 4.5, and injected for 600 seconds. Finally, any remaining active sites were blocked with ethanolamine. Then, the affinity between the antibody and the antigen was determined by a multi-cycle kinetic method. In each cycle, the antibody to be tested was first captured using a Protein A chip, and then a single concentration of antigen protein was injected, and the binding and dissociation processes of the antibody and antigen protein were recorded. Finally, the chip was regenerated with Glycine pH 1.5. The mobile phase was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20), the flow rate was 30μL / min, the regeneration time was 30s, and the detection temperature was 25°C. Finally, according to the 1:1 binding model, the data were analyzed and the antibody-antigen binding kinetic parameters were fitted, including the association rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax.

[0127] The association rate (Ka), dissociation rate (Kd) and binding affinity (KD) of SCR10437-hIgG1 antibody to human and monkey B7H3 protein are shown in Table 4.

[0128] Table 4 Affinity of antibodies and B7H3 protein detected by SPR (biacore)

[0129] Example 4: Identification of Antibody Binding Activity

[0130] 4.1 ELISA detection of binding of fully human antibodies to human and monkey B7H3 proteins

[0131] Human B7H3 (4Ig) -his protein was diluted with PBS to a final concentration of 1 μg / mL, then 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed twice with PBST and blocked with blocking solution [PBS + 2% (w / w) BSA] for 2 hours at room temperature. The blocking solution was discarded, and 50 μl of the antibody starting at 50 nM and serially diluted 4-fold, as well as positive and negative control antibodies, were added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. HRP (horseradish peroxidase)-conjugated secondary antibody (purchased from Merck, Cat. No.: AP113P) was added, incubated at 37°C for 1 hour, and the plate was washed five times with PBST. TMB substrate (50 μl) was added to each well, incubated at room temperature for 10 minutes, and then stop solution (1.0 M HCl) was added to each well. OD was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer) 450nm The binding activity of the antibodies to human B7H3(4Ig)-his protein is shown in Figure 1A. The results showed that the negative control anti-FITC-hIgG1 (derived from the literature J Biol Chem. 1990 Jan 5; 265(1): 133-8) did not bind to B7H3, while the SCR10437-hIgG1 antibody effectively bound to human B7H3(4Ig)-his protein, with a binding ability comparable to that of the positive control antibody DS7300-hIgG1.

[0132] Human B7H3(2Ig)-his protein was diluted with PBS to a final concentration of 2 μg / mL, and 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The binding activity of the B7H3 antibody to human B7H3(2Ig)-his protein was analyzed using the same ELISA assay described above. The results showed that the SCR10437-hIgG1 antibody effectively bound to human B7H3(2Ig)-his protein, with binding activity comparable to that of the positive control antibody DS7300-hIgG1 (Figure 1B).

[0133] Monkey cynoB7H3-his protein was diluted with PBS to a final concentration of 2 μg / mL, and 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The binding activity of the B7H3 antibody to the cynoB7H3-his protein was analyzed using the same assay described above. The results showed that the SCR10437-hIgG1 antibody effectively bound to the cynoB7H3-his protein, with binding activity comparable to that of the positive control antibody DS7300-hIgG1 (Figure 1C).

[0134] 4.2 Flow cytometry (FACS) detection of B7H3 antibody binding to tumor cells

[0135] Endogenous tumor cells A375 (Nanjing Kebai, CBP60329, B7H3 expression levels are shown in Figure 2A) were expanded in T-175 culture flasks to the logarithmic growth phase. The culture medium was aspirated and the cells were washed twice with PBS buffer. The cells were trypsinized and then digested with complete culture medium. The cells were pipetted to a single cell suspension. After cell counting, the cells were centrifuged and the cell pellet was resuspended in FACS buffer (PBS + 10% fetal bovine serum) to 2×10 6 For every milliliter of cells, 100 μl was added to each well of a 96-well FACS reaction plate, centrifuged, the supernatant was discarded, 50 μl of the antibody sample to be tested (200 nM as the starting concentration, 4-fold serial dilution) was added to each well, mixed with the cells, and incubated at 4°C for 1 hour. Washed three times by centrifugation with PBS buffer, 50 μl of Alexa Fluor® was added to each well. Incubate with 647AffiniPure Goat Anti-Human IgG, Fcγ fragment-specific secondary antibody (purchased from Jackson, Cat. No. 109-605-098) at 4°C for 1 hour. Wash cells three times with PBS buffer by centrifugation, resuspend in 100 μl of PBS, and analyze using FACS (FACS Canto™, purchased from BD Biosciences). Data were analyzed using FlowJo software to obtain the mean fluorescence intensity (MFI) of the cells. GraphPad Prism 8 software was then used for data fitting and EC50 calculation. As shown in Figure 3A and Table 5, SCR10437-hIgG1 antibody specifically binds to A375 cells, with stronger binding than DS7300-hIgG1 and Enoblituzumab-hIgG1.

[0136] Table 5 Binding of B7H3 antibody to A375 cells

[0137] The same method was used to detect the binding of SCR10437-hIgG1 antibody to the lung cancer cell line A549 (Nanjing Kebai, CBP60084, B7H3 expression level is shown in Figure 2B), which has moderate B7H3 expression. The results are shown in Figure 3B and Table 6. SCR10437-hIgG1 has good binding activity with A549, and its binding ability is stronger than DS7300-hIgG1 and MGC018-hIgG1.

[0138] Table 6 Binding of B7H3 antibody to A549 cells

[0139] Example 5: Antibody endocytosis activity detection

[0140] To evaluate the endocytic activity of the anti-B7H3 antibody SCR10437, 786-O (Nanjing Kebai, CBP60442, B7H3 expression levels are shown in Figure 2C) and NCI-H1975 (Nanjing Kebai, CBP60121, B7H3 expression levels are shown in Figure 2D) cells were cultured to 80% confluence. The cells were harvested and stained with the anti-B7H3 antibody to be tested (100 nM) and the secondary antibody, Aleax Flour488-conjugated anti-human IgG, Fcγ fragment-specific antibody (2.2 μg / ml, Jackson, 109-545-098) at 4°C. The cells were washed and incubated at 37°C for 0 h (pre-incubation) and 4 h. Cells were washed and pre-incubated or incubated with an anti-Alexa 488 antibody (Invitrogen, A11094) at a concentration of 10 μg / ml to quench cell surface fluorescence, or unquenched to detect the entire signal, and analyzed by flow cytometry. The internalization rate (%) was calculated using the following formula: [1-(Na-Qa) / (Na-NaxQi / Ni)]x100, and the internalization amount (MFI value) was calculated using the following formula: Qa-Qi; Na: mean fluorescence intensity (MFI) of samples at each incubation time under unquenched conditions, Qa: MFI of samples at each incubation time under quenched conditions, Ni: MFI of samples pre-incubated under unquenched conditions, Qi: MFI of samples pre-incubated after quenching. The internalization amount represents the total amount of antibody internalized into the cell within 4 hours. The results are shown in Figures 4A and 4B. All antibodies were able to be internalized, and SCR10437-hIgG1 was internalized better than the positive control antibody.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to B7H3, wherein, The antibody or its antigen-binding fragment comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), wherein, (1) the heavy-chain variable region comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are the HCDR1, HCDR2 and HCDR3 of the VH domain shown in SEQ ID NO.12; and (2) the light-chain variable region comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2 and LCDR3 of the VL domain shown in SEQ ID NO.

13.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The HCDR1, HCDR2, and HCDR3 are determined according to the Kabat, Chothia, or IMGT numbering system and have the amino acid sequences shown in SEQ ID NOs. 14-16, 20-22, 26-28 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions, and / or substitutions as compared to the amino acid sequences shown in SEQ ID NOs. 14-16, 20-22, 26-28.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The LCDR1, LCDR2, and LCDR3 are determined according to the Kabat, Chothia, or IMGT numbering system and have the amino acid sequences shown in SEQ ID NOs. 17-19, 23-25, 29-31 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions, and / or substitutions as compared to the amino acid sequences shown in SEQ ID NOs. 17-19, 23-25, 29-31.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The antibody or its antigen binding comprises the following LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3: (1) According to the Kabat numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 14-19 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions, and / or substitutions as compared to the amino acid sequences shown in SEQ ID NOs. 14-19; (2) According to the IMGT numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 20-25 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions, and / or substitutions as compared to the amino acid sequences shown in SEQ ID NOs. 20-25; (3) According to the Chothia numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 26-31 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions, and / or substitutions as compared to the amino acid sequences shown in SEQ ID NOs. 26-31.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein, The heavy chain variable region sequence comprises the sequence shown in SEQ ID NO.12, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the shown sequence; the light chain variable region sequence comprises the sequence shown in SEQ ID NO.13, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the shown sequence.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein, The antibody or its antigen-binding fragment is fully human.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein, The antibody or its antigen-binding fragment can specifically bind to human or monkey B7H3 protein.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein, The antibody or its antigen-binding fragment may further comprise a constant region sequence of any one of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it comprises a constant region sequence of human or murine antibodies IgG1, IgG2, IgG3 or IgG4, or a constant region sequence of a human or murine antibody IgG1, IgG2, IgG3 or IgG4 with mutations; further, the antibody or its antigen-binding fragment is further conjugated with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic drugs, cytotoxic agents or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from alkaloids, methotrexate, doxorubicin, pyrrolobenzodiazepine (PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is taxanes. (pyrrolobenzodiazepine, PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is taxanes.

9. The antibody or its antigen-binding fragment according to any one of claims 1 to 8, wherein the antigen-binding fragment is selected from one or more of F(ab’)2, Fab’, Fab, Fv, scFv, nanobody or affibody.

10. A multispecific antigen-binding molecule, which comprises the antibody or its antigen-binding fragment according to any one of claims 1 to 9; preferably, the multispecific antigen-binding molecule further comprises an antibody or its antigen-binding fragment that specifically binds to an antigen other than B7H3 or binds to a B7H3 epitope different from the B7H3 epitope bound by the antibody or its antigen-binding fragment according to any one of claims 1 to 9; Preferably, the antigen other than B7H3 is selected from the following group: (1) tumor-specific antigen (TSA) or tumor-associated antigen (TAA); (2) immune checkpoint; (3) target for recruiting and / or activating immune cells; Preferably, the multispecific antigen-binding molecule can be bispecific, trispecific or tetravalent; Preferably, the multispecific antigen-binding molecule can be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

11. A chimeric antigen receptor (CAR), which at least comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen-binding domain comprises the antibody or its antigen-binding fragment according to any one of claims 1 to 9.

12. An immune effector cell, which expresses the chimeric antigen receptor according to claim 11, or comprises a nucleic acid fragment encoding the chimeric antigen receptor according to claim 11; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

13. An isolated nucleic acid fragment, which encodes the antibody or its antigen-binding fragment according to any one of claims 1 to 9, or the multispecific antigen-binding molecule according to claim 10, or the chimeric antigen receptor according to claim 11.

14. A vector that comprises the nucleic acid fragment recited in claim 13.

15. A host cell that comprises the vector recited in claim 14; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as a bacterium (Escherichia coli), a fungus (yeast), an insect cell, or a mammalian cell (CHO cell line or 293T cell line).

16. A method for preparing the antibody or antigen-binding fragment thereof recited in any one of claims 1-9, or the multispecific antigen-binding molecule recited in claim 10, which comprises culturing the host cell recited in claim 15, and isolating the antibody or antigen-binding fragment thereof expressed by the host cell, or isolating the multispecific antigen-binding molecule expressed by the host cell.

17. A method for preparing the immune effector cell recited in claim 12, which comprises introducing the nucleic acid fragment encoding the CAR recited in claim 11 into the immune effector cell; optionally, the method further comprises activating the immune effector cell to express the CAR recited in claim 11.

18. A pharmaceutical composition that comprises the antibody or antigen-binding fragment thereof recited in any one of claims 1-9, or the multispecific antigen-binding molecule recited in claim 10, or the immune effector cell recited in claim 12, or the nucleic acid fragment recited in claim 13, or the vector recited in claim 14, or the host cell recited in claim 15, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule obtained by the method recited in claim 16, or the immune effector cell obtained by the method recited in claim 17; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant.

19. Use of the antibody or antigen-binding fragment thereof recited in any one of claims 1-9, or the multispecific antigen-binding molecule recited in claim 10, or the immune effector cell recited in claim 12, or the nucleic acid fragment recited in claim 13, or the vector recited in claim 14, or the host cell of claim 15, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule obtained by the method recited in claim 16, or the immune effector cell obtained by the method recited in claim 17; or the pharmaceutical composition recited in claim 18 in the preparation of a medicament for preventing and / or treating tumors; the tumors are selected from solid tumors, hematological tumors, or cancers infiltrating and expressing B7H3; Preferably, the tumors are selected from melanoma, prostate cancer, renal cell carcinoma, lung cancer (such as non-small cell lung cancer (NSCLC)), and other solid tumors.

20. A method for preventing and / or treating tumors, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the multispecific antigen-binding molecule according to claim 10, or the immune effector cell according to claim 12, or the nucleic acid fragment according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule obtained by the method according to claim 16, or the immune effector cell obtained by the method according to claim 17, or the pharmaceutical composition according to claim 18; wherein the tumors are selected from solid tumors, hematological tumors or cancers infiltrating and expressing B7H3; Preferably, the tumors are selected from melanoma, prostate cancer, renal cell carcinoma, lung cancer (such as non-small cell lung cancer (NSCLC)) and other solid tumors.

21. A kit, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the multispecific antigen-binding molecule according to claim 10, or the immune effector cell according to claim 12, or the nucleic acid fragment according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule obtained by the method according to claim 16, or the immune effector cell obtained by the method according to claim 17, or the pharmaceutical composition according to claim 18.

22. A method for in vitro detecting B7H3 expression, wherein, Under the conditions capable of forming a complex between the antibody or antigen-binding fragment thereof according to any one of claims 1-9 and B7H3, contacting a sample to be detected with the antibody or antigen-binding fragment thereof according to any one of claims 1-9; preferably, the method further comprises detecting the formation of the complex to indicate the presence or expression level of DLL3 in the sample.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a B7H3 detection reagent.

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