Anti-B7H4 antibody and bispecific antibody thereof and uses thereof

Abstract

The present invention discloses antibodies targeting B7H4 or variants thereof, as well as bispecific antibodies comprising antibodies targeting B7H4. The B7H4-targeting antibodies comprise a light chain variable region and a heavy chain variable region, and the variants have amino acid mutations in the light chain variable region or heavy chain variable region of the antibody while maintaining the functionality of the antibody. The B7H4-targeting antibodies of the present invention bind to human B7H4 and cynomolgus monkey B7H4 without cross-reactivity with other B7 family members; in vitro and in vivo experiments have demonstrated good antitumor activity, T cell activation activity, and internalization activity, making them suitable for use as ADC drugs. The bispecific antibodies of the present invention have a long half-life, good stability, and hydrophilicity, reducing toxicity while ensuring efficacy.

Description

[Technical Field]

[0001] This application claims priority from Chinese Patent Application No. 202010618159.5, filed June 30, 2020. This application cites the entire text of said Chinese patent application.

[0002] The present invention relates to the biomedical field, in particular to anti-B7H4 antibodies and bispecific antibodies thereof and uses thereof. [Background technology]

[0003] Breast cancer, ovarian cancer, and endometrial cancer are common malignancies in women, with breast cancer being the leading cause of cancer in women. According to 2018 data from the International Agency for Research on Cancer (IARC), the global incidence rate of breast cancer among women was 24.2%. Immunotherapy and targeted therapy are currently hotspots in the treatment of these cancers. For example, hecetin, which targets Her2, has shown good therapeutic effects in Her2-positive breast cancer. For triple-negative breast cancer (TNBC), there are currently few treatment options due to a lack of corresponding targets, and chemotherapy is the primary treatment. In March 2019, the US FDA approved the PD-L1 inhibitor atesolizumab in combination with albumin-bound paclitaxel for the treatment of metastatic triple-negative breast cancer (TNBC), but PD-L1 expression was not high in triple-negative breast cancer.

[0004] Immune checkpoint inhibitors are the most studied form of immunotherapy for breast cancer. Immune checkpoint molecules are often highly expressed in the tumor microenvironment, suppressing T cell activation and inducing T cell exhaustion, allowing tumors to evade immune attack. The B7 family and the TNF family are two major families of costimulatory molecules. The B7 family currently contains 10 molecules: CD80 (B7.1), CD86 (B7.2), B7H1 (PD-L1 / CD274), B7-DC (PD-L2 / CD273), B7H2 (ICOSL), B7H3 (CD276), B7H4 (B7S1 / B7x / Vtcn1), B7H5 (VISTA), B7H6, and B7H7 (HLA2). Several members of the B7 family and their receptors have been proven to function as immune checkpoints, such as PD-L1 / PD1, CTLA4, and VISTA.

[0005] B7H4 is a relatively new member of the B7 family. While its mRNA expression is widespread in living cells, its protein expression is very limited, with low levels only present in certain epithelial cells, such as breast tissue and lobules, fallopian tube epithelium, and endometrium. In contrast, B7H4 is abundantly expressed in various tumor tissues, including breast cancer, particularly triple-negative breast cancer, ovarian cancer, and endometrial cancer. Given its expression spectrum, B7H4 is considered a highly specific tumor-associated antigen. Meanwhile, B7H4 is a novel immune checkpoint molecule. In vitro experiments have demonstrated that B7H4 inhibits T cell proliferation, activation, and cytokine production by interacting with its unidentified T cell surface receptor. Tumor cells overexpress B7H4, and suppressive macrophages expressing B7H4 in the tumor microenvironment inhibit T cell activation, thereby achieving immune evasion. The B7H4 expression spectrum on tumors does not overlap with that of PD-L1. Therapeutic targeting of B7H4 with antibodies and blocking the negative regulatory effects of B7H4 to reactivate the immune system are promising avenues for the treatment of tumors that express B7H4 positively.

[0006] Currently, several pharmaceutical companies are developing monoclonal antibodies, drug conjugates, or bispecific antibodies against B7-H4. Genentech, BMS, Jounce, Jiangsu Hausen, and others are in preclinical development. The most rapidly progressing antibody is FivePrime's anti-B7H4 monoclonal antibody, which is currently in Phase 1 clinical trials and primarily activates T cells by blocking ADCC and immune checkpoints. However, no human-derived anti-B7H4 antibody with high affinity, high selectivity, and high biological activity is currently available in clinical trials. Summary of the Invention

[0007] To address the current lack of high-affinity, high-selectivity, and high-biological activity human-derived anti-B7H4 antibodies, the present invention utilizes the unique Harbour human-derived mouse platform to provide a fully human-derived, high-affinity, high-selectivity, and high-biological activity anti-B7H4 antibody and an anti-B7H4 antibody x CD3 bispecific antibody constructed based on the antibody, for use in the treatment of B7H4-positive tumors.

[0008] A technical aspect of the present invention provides an antibody or variant thereof that targets B7H4, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein: the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 47, 54 and 64, respectively; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 9, 21 and 35, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 48, 54 and 65, respectively; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 11, 22 and 36, respectively; The variants have amino acid mutations in the light chain variable region and / or heavy chain variable region of the antibody, while maintaining the antibody's functionality. In the present invention, the amino acid mutations may be the deletion, substitution, or addition of one or more amino acid residues to the original amino acid sequence, for example, in the CDR. Preferably, the amino acid mutations are amino acid substitutions, and the number of amino acid substitutions is 1-3. Furthermore, the amino acid sequence of the mutants has at least 85% sequence identity with the original amino acid sequence while maintaining or improving the binding of the antibody to the target antigen; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95%, 96%, 97%, or 98% sequence identity; and most preferably at least 99% sequence identity. The antibodies targeting B7H4 are also referred to as anti-B7H4 antibodies in the present invention.

[0009] In a specific embodiment, the antibody or variant thereof has an amino acid substitution at position 3 or 4 of HCDR2 or position 3 of HCDR3 in the heavy chain variable region of the antibody, and / or an amino acid substitution at position 4 of LCDR3 in the light chain variable region of the antibody.

[0010] Preferably, D at position 3 of said HCDR2 is substituted with G or E, and / or G at position 4 is substituted with A, and / or G at position 3 of said HCDR3 is substituted with A; and N at position 4 of said LCDR3 is substituted with S, R or Q. That is, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 48, 55 and 66, respectively; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 11, 22 and 36, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 23 and 35, respectively; or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively; and the heavy chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: or the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 69, respectively; and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 24 and 38, respectively.

[0011] In a specific embodiment, in the above-mentioned antibody or variant thereof, the heavy chain variable region further comprises a heavy chain variable region framework region HFWR, and / or the light chain variable region further comprises a light chain variable region framework region LFWR, in which the HFWR is a heavy chain variable region framework region of a human antibody, and the LFWR is a light chain variable region framework region of a human antibody. Preferably, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 89; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83; or The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91; and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79.

[0012] In the present invention, the amino acid mutation may further involve the deletion, substitution, or addition of one or more amino acid residues in the original amino acid sequence, for example, in the FWR. Preferably, the amino acid mutation is an amino acid substitution, and the number of amino acid substitutions is 1 to 3. The amino acid sequence of the mutant has at least 85% sequence identity with the original amino acid sequence and maintains or improves the binding of the antibody to the target antigen; the at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95%, 96%, 97%, or 98% sequence identity, and most preferably at least 99% sequence identity.

[0013] In a specific embodiment, the antibody or variant thereof further comprises a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region of the antibody is selected from hIgG1, hIgG2, hIgG3, or hIgG4, and the light chain constant region is selected from a κ chain or a λ chain; more preferably, the variant has an amino acid substitution at position 239 and / or position 332 of the Fc of the antibody, preferably S239D and / or I332E.

[0014] In a specific embodiment, among the above-mentioned antibodies or variants thereof, the antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, or a monoclonal or polyclonal antibody prepared from these antibodies.

[0015] In a specific embodiment, among the above antibodies, the antibody comprises the following (1) or (2): (1) a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 95; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 98; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 112; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 98; the light chain comprises the amino acid sequence set forth in SEQ ID NO: 113; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 99; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 114; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 100; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 114; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 101; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 102; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 114; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 106; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 117; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 98; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 115; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 103; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 112; or a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 103; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 115; (2) A heavy chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 132.

[0016] The term "antibody" includes immunoglobulins, which are tetrapeptide chains consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Their antigenicity varies due to differences in the amino acid composition and sequence of the heavy chain constant regions. This leads to five types of immunoglobulins, or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Ig subclasses can be divided into different subclasses based on the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains can be divided into kappa or lambda chains based on the differences in their constant regions. Each of the five types of Ig can have either kappa or lambda chains.

[0017] The antibody light chain variable region described herein may further comprise a light chain constant region, wherein the light chain constant region comprises a human kappa or lambda chain or a variant thereof. The antibody heavy chain variable region described herein may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises a human IgG1, 2, 3, 4 or a variant thereof.

[0018] Within light and heavy chains, the variable and constant regions are connected via a "J" region of about 12 or more amino acids, and heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of the CL domain. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including binding to various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Approximately 110 amino acids near the N-terminus of antibody heavy and light chains are highly variable and comprise the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and comprise the constant region (C region). The variable region contains three highly variable regions (HVRs) and four framework regions (FWRs) with relatively conserved sequences. The three highly variable regions determine the antibody specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FWR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4.

[0019] In order to solve the above technical problem, the second technical aspect of the present invention is: a bispecific antibody targeting B7H4, comprising a protein A functional region and a protein B functional region, wherein the protein A functional region is an antibody targeting B7H4 as described above; wherein the protein B functional region is an antibody that does not target B7H4; preferably, the antibody that does not target B7H4 is an antibody that targets CD3; more preferably, the antibody that targets CD3 comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53, and 63, respectively; the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20, and 34, respectively; or the light chain variable region comprises LCDR1, LCDR2, and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: the heavy chain variable region comprises an HCDR1, an HCDR2 and an HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 10, 20 and 34, respectively; and even more preferably, in the CD3-targeting antibody, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 86; the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 76; or the light chain variable region VL comprises an amino acid sequence set forth in SEQ ID NO: 86; the heavy chain variable region VH comprises an amino acid sequence set forth in SEQ ID NO: 78; or the light chain variable region VL comprises an amino acid sequence set forth in SEQ ID NO: 86; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 84.

[0020] In a specific embodiment, in the bispecific antibody, the protein B functional region comprises a light chain variable region and a heavy chain variable region, and the protein A functional region comprises a light chain variable region and a heavy chain variable region; wherein: In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 64, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 21 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 23 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 24 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 48, 54 and 65, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 11, 22 and 36, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 64, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 21 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 10, 20 and 34, respectively. In the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 23 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 24 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 48, 54 and 65, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 11, 22 and 36, respectively. In the bispecific antibody of the present invention, the protein B functional region comprises a light chain variable region and a heavy chain variable region, and the protein A functional region comprises a light chain variable region and a heavy chain variable region; wherein the protein B functional region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; wherein the protein A functional region comprises the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77; or In said protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; in said protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80; or wherein the protein B functional region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; wherein the protein A functional region comprises the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or In the protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; in the protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79; or wherein the protein B functional region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78. wherein the protein A functional region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77; or In said protein B functional region, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78; in said protein A functional region, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80; or In said protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78; in said protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or In said protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78; in said protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79; or In the protein B functional region, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 84; in the protein A functional region, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 91, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 79.

[0021] In a specific embodiment, among the bispecific antibodies described above, the bispecific antibodies are selected from the following set: (1) The structure is a polypeptide chain-1 of n'-[VL]-CL-c', a polypeptide chain-2 of n'-[VH]-CH1-h-CH2-CH3-c', and a polypeptide chain-3 of n'-{VH-L-VL}-h-CH2-CH3-c' or n'-{VL-L-VH}-h-CH2-CH3-c'; wherein the polypeptide chain-1 and polypeptide chain-2 are protein A functional domains and the polypeptide chain-3 is a protein B functional domain, or the polypeptide chain-1 and polypeptide chain-2 are protein B functional domains and the polypeptide chain-3 is a protein A functional domain; (2) The structures are polypeptide chain-1 of n'-[VL]-CL-c', polypeptide chain-2 of n'-[VH]-CH1-h-CH2-CH3-c', polypeptide chain-3 of n'-{VH}-CH1-h-CH2-CH3-c', and polypeptide chain-4 of n'-{VL}-CL-c'; or the structures are polypeptide chain-1 of n'-[VH]-CH-c', polypeptide chain-2 of n'-[VL]-CL-h-CH2-CH3-c', and polypeptide chain-4 of n'-{VL}-CL-c'. a polypeptide chain-3 of H2-CH3-c' and a polypeptide chain-4 of n'-{VL}-CL-c'; wherein said polypeptide chain-1 and polypeptide chain-2 are a protein A functional domain, and said polypeptide chain-3 and polypeptide chain-4 are a protein B functional domain, or wherein said polypeptide chain-1 and polypeptide chain-2 are a protein B functional domain, and said polypeptide chain-3 and polypeptide chain-4 are a protein A functional domain; (3) The structure is a polypeptide chain-1 of n'-[VH]-CH1-c', a polypeptide chain-2 of n'-{VH}-CH1-L-[VL]-CL-h-CH2-CH3-c' or n'-{VH}-CH1-h-CH2-CH3-L-[VL]-CL-c', a polypeptide chain-3 of n'-{VH}-CH1-h-CH2-CH3-c', and a polypeptide chain-4 of n'-{VL}-CL-c'; wherein said polypeptide chain-1 is an A functional domain, and said polypeptide chain-2 is an A protein functional domain and a B protein functional domain, in the order from n' to c'. and a protein B functional region, or comprises a protein B functional region and a protein A functional region, in order from n' to c', wherein polypeptide chain-1 and polypeptide chain-4 are the B functional region; or polypeptide chain-1 is the B functional region, and polypeptide chain-2 comprises a protein B functional region and a protein A functional region, in order from n' to c', or comprises a protein A functional region and a protein B functional region, in order from n' to c', wherein polypeptide chain-3 and polypeptide chain-4 are the A functional region; (4) The structure is n'-[VH]-CH1-L1-{VL-L2-VH}-h-CH2-CH3-c' or n'-[VH]-CH1-L1-{VH-L2-VL}-h-CH2-CH3-c', polypeptide chain-2 is n'-[VH]-CH1-h-CH2-CH3-c', and polypeptide chain-3 is n'-[VL]-CL-c'; wherein polypeptide chain-1 is a protein A functional domain and a protein B functional domain, and polypeptide chains-2 and 3 are a protein B functional domain, or polypeptide chain-1 is a protein A functional domain and a protein B functional domain, and polypeptide chains-2 and 3 are a protein A functional domain; preferably, polypeptide chain-1 is a B functional domain and an A functional domain, in the order from n' to c', or a A functional domain and a B functional domain, in the order from n' to c'; (5) The structure is polypeptide chain-1 of n'-[VH]-CH1-h-CH2-CH3-L1-{VL-L2-VH}-c' or n'-[VH]-CH1-h-CH2-CH3-L1-{VH-L2-VL}-c', polypeptide chain-2 of n'-[VH]-CH1-h-CH2-CH3-c', and polypeptide chain-3 of n'-[VL]-CL-c'; wherein polypeptide chain-1 is a protein A functional domain and a protein B functional domain, and polypeptide chains-2 and 3 are a protein B functional domain, or wherein polypeptide chain-1 is a protein A functional domain and a protein B functional domain, and polypeptide chains-2 and 3 are a protein A functional domain.

[0022] Here, n' represents the amino end (also referred to as the N-terminus) of the polypeptide chain, c' represents the carboxyl end (also referred to as the C-terminus) of the polypeptide chain, h represents a hinge region, and L, L1, or L2 represents a connector (or linker). A suitable prior art linker (L) consists of a repeated G4S amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (G4S)4 or (G4S)3 can be used, but variants thereof can also be used, for example, one of the Gs in the G4S can be replaced with a Q, for example, the second or third G can be replaced with a Q. Preferred linker sequences of the present invention are shown in SEQ ID NOs: 133-135. A "-" represents a polypeptide bond connecting different structural regions or is used to separate different structural regions.

[0023] In the present invention, "[]" and "{}" respectively represent different functional regions or structures. For example, {VL-L-VH} and {VH-L-VL} represent scFv structures, and [VH] / {VH} and [VL] / {VL} represent the heavy chain variable region and light chain variable region of a Fab structure, respectively. When VH is the A or B protein functional region, respectively, it can be represented as VH_A (i.e., the heavy chain variable region is the A protein functional region) or VH_B (i.e., the heavy chain variable region is the B protein functional region), respectively; similarly, when VL is the A or B protein functional region, respectively, it can be represented as VL_A (i.e., the light chain variable region is the A protein ribbon) or VL_B (i.e., the light chain variable region is the B protein functional region). In Figure 28, brackets "[]" and "{}" have been removed to facilitate drawing, and therefore VL_B-L-VH_A (wherein the light chain variable region is the protein B functional region, the heavy chain variable region is the protein A functional region, and L is a connector connecting VL_B and VH_A), VH_B-L-VL_A (wherein the heavy chain variable region is the protein B functional region, the light chain variable region is the protein A functional region, and L is a linker connecting VH_B and VL_A; see, for example, polypeptide chain-3 in structure (1)) and the like also represent scFv structures, and a single VL_AB or VH_A (see, for example, polypeptide chain-1 or polypeptide chain-2 in structure (1)) represents the light chain variable region of the protein B functional region and the heavy chain variable region of the protein A functional region in the Fab structure, respectively. In the present invention, polypeptide chain-1, -2, -3, or -4 refers only to the type of polypeptide chain, and when a bispecific antibody is actually composed of polypeptide chains, the number of each polypeptide chain can be 1 or 2. For example, in structures (5) and (6), the number of polypeptide chain-1, -2, or -3 is 1, and the number of polypeptide chain-4 is 2, and in structures (7), (8), (9), and (10), the number of polypeptide chain-1 or -2 is 1, and the number of polypeptide chain-3 is 2. In the present invention, polypeptide chain-1 is also called the first polypeptide chain, polypeptide chain-2 is also called the second polypeptide chain, polypeptide chain-3 is also called the third polypeptide chain, and polypeptide chain-4 is also called the fourth polypeptide chain.

[0024] In a preferred embodiment, the bispecific antibody is selected from the following set: (1) the bispecific antibody comprises three polypeptide chains, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 114; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 120; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 114; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 115; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 122; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 126; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 127; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 110; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 128; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 129; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 110; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 129; (2) the bispecific antibody comprises four polypeptide chains, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 97; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 123; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 120; and a fourth polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 114; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 97; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 123; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 121; a fourth polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 114; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 97; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 123; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 122; a fourth polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 115; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 97; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 124; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 118; and a fourth polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 109; or The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109.

[0025] The protein functional domains of the present invention may be Fab, scFv, or VH in some cases, or F(ab)2 or full-length antibodies in other cases, and are sometimes referred to as antibodies or antigen-binding proteins or binding proteins. In the present invention, a "Fab structure" or "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of a Fab fragment cannot form disulfide bonds with another Fab heavy chain molecule. The "Fc" region contains two heavy chain fragments, the CH2 and CH3 domains that comprise the antibody; the two heavy chain fragments are held together by two or more disulfide bonds and the hydrophobic effect of the CH3 domain. A "Fab' fragment" contains a light chain and a portion of the heavy chain including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains of two Fab' fragments to form a F(ab')2 fragment. The "F(ab')2 fragment" comprises two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single antibody arm, lacking the constant region.

[0026] In the present invention, the scFv (single chain antibody fragment) may be a conventional single chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids, wherein the VL and VH domains are linked by a linker that can be produced as a single polypeptide chain to form a monovalent molecule. Such scFv molecules can have the general structure represented as NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH, or n'-VL-L-VH-c' or n'-VH-L-VL-c'.

[0027] In order to solve the above technical problems, Technical Aspect 3 of the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor is the antibody according to Technical Aspect 1 of the present invention or the bispecific antibody according to Technical Aspect 2 of the present invention.

[0028] In the present application, the antibody or bispecific antibody can be used to prepare a chimeric antigen receptor (CAR) or the like for modifying cells such as T cells or NK cells. The chimeric antigen receptor may be a conventional chimeric antigen receptor in the art, for example, comprising a chimeric antigen receptor of an extracellular antigen-binding domain in the form of an scFv using the antibody.

[0029] Therefore, in order to solve the above technical problem, the fourth technical aspect of the present invention provides a genetically modified cell, which comprises an antibody according to the first technical aspect of the present invention; the cell is preferably a eukaryotic cell, more preferably an isolated human cell, and even more preferably an immune cell, such as a T cell (e.g., in the form of a CAR-T), or an NK cell, such as the NK92 cell line.

[0030] To solve the above technical problems, a fifth technical aspect of the present invention provides an isolated nucleic acid, which encodes the above antibody or the above bispecific antibody, or the chimeric antigen receptor described in the third technical aspect of the present invention.

[0031] The method for preparing the nucleic acid is a conventional method in the art, and preferably includes a step of obtaining a nucleic acid molecule encoding the antibody by gene cloning technology, or a step of obtaining a nucleic acid molecule encoding the antibody by artificial total sequence synthesis.

[0032] Those skilled in the art will recognize that the nucleotide sequence of the amino acid sequence encoding the antibody can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide analog. The polynucleotide analogs of the present invention can be prepared by substituting, deleting, or adding one or more nucleotides encoding the antibody sequence gene within a range that retains antibody activity.

[0033] To solve the above technical problem, the sixth technical aspect of the present invention provides an expression vector, which comprises the above isolated nucleic acid.

[0034] The recombinant expression vector can be obtained by a conventional method in the art, i.e., constructed by ligating the nucleic acid molecule described herein into various expression vectors, which may be various vectors conventional in the art as long as they can accommodate the nucleic acid molecule.

[0035] Preferably, the expression vector comprises a eukaryotic expression vector and / or a prokaryotic expression vector.

[0036] To solve the above technical problem, a seventh technical aspect of the present invention provides a transformant, which comprises the above-mentioned isolated nucleic acid or expression vector. The transformant can be prepared by a conventional method in the art, for example, by transforming the recombinant expression vector into a host cell. The host cell for the transformant may be any of various conventional host cells in the art, as long as it is capable of stably replicating the recombinant expression vector and effectively expressing the nucleic acid carried by it. Preferably, the host cell is a prokaryotic or eukaryotic cell. The prokaryotic cell is preferably an E. coli cell such as TG1 or BL21 (which expresses single-chain or Fab antibodies), and the eukaryotic cell is preferably an HEK293 cell or a CHO cell (which expresses a full-length IgG antibody). The preferred recombinant expression transformant of the present invention can be obtained by transforming the recombinant expression plasmid into a host cell. The transformation method can be a conventional method in the art, preferably a chemical transformation method, a heat stimulation method, or an electrotransformation method.

[0037] In order to solve the above technical problems, technical aspect eight of the present invention provides a method for producing an antibody or bispecific antibody that targets B7H4, wherein the production method comprises the following steps: culturing the transformant described in technical aspect seven of the present invention; and obtaining the antibody or bispecific antibody that targets B7H4 from the culture.

[0038] To solve the above technical problems, the ninth technical aspect of the present invention provides an antibody-drug conjugate, which comprises an antibody moiety and a conjugate moiety, wherein the antibody moiety comprises the antibody described in the first technical aspect of the present invention or the bispecific antibody described in the second technical aspect of the present invention, and the conjugate moiety includes, but is not limited to, a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, and the antibody moiety and the conjugate moiety are conjugated by a chemical bond or a linker.

[0039] To solve the above technical problems, technical aspect 10 of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the antibody according to technical aspect 1 of the present invention or the bispecific antibody according to technical aspect 2 of the present invention, and optionally a pharmaceutically acceptable vector, preferably further comprising a pharmaceutically acceptable vector.

[0040] More preferably, the pharmaceutical composition further comprises another anti-tumor antibody as an active ingredient.

[0041] The pharmaceutically acceptable vector may be a vector commonly used in the art, and this vector may be any suitable physiologically or pharmaceutically acceptable drug additive. The drug additive is a conventional drug additive used in the art, and preferably includes a pharmaceutically acceptable excipient, filler, or diluent. More preferably, the pharmaceutical composition contains 0.01 to 99.99% of the antibody and / or bispecific antibody and 0.01 to 99.99% of the pharmaceutical vector, the percentages being expressed as mass percent of the pharmaceutical composition.

[0042] The pharmaceutical composition of the present invention is preferably administered parenterally, by injection, or orally. Injection preferably includes intravenous, intramuscular, peritoneal, intradermal, or subcutaneous injection. The pharmaceutical composition may be in any of a variety of dosage forms conventional in the art, preferably in solid, semisolid, or liquid form, i.e., aqueous, non-aqueous, or suspension form, more preferably in the form of tablets, capsules, granules, injections, or infusions. More preferably, the pharmaceutical composition is administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the pharmaceutical composition can be administered as an aerosol or coarse spray, i.e., intranasally, or intrathecally, intramedullary, or intraventricularly. More preferably, the pharmaceutical composition can be administered transdermally, transdermally, topically, enterally, intravaginally, sublingually, or rectally. The pharmaceutical composition of the present invention can be prepared in various dosage forms as needed, and a physician can determine the beneficial dosage for a patient based on factors such as the patient's type, age, weight, general disease status, and administration method. The administration method can be, for example, injection or other therapeutic methods.

[0043] The dosage level of the pharmaceutical composition according to the present invention can be adjusted depending on the amount of the composition to achieve the desired diagnostic or therapeutic result. The administration scheme can be a single injection or multiple injections, or can be adjusted accordingly. The selected dose level and scheme can be reasonably adjusted depending on various factors, including the activity and stability (i.e., half-life) of the pharmaceutical composition, formulation, administration route, combination with other drugs or treatments, the disease or disorder to be detected and / or treated, and the health condition and previous medical history of the subject to be treated.

[0044] The therapeutically effective dose of the pharmaceutical composition of the present invention can be estimated initially in cell culture experiments or animal models, such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine the appropriate administration concentration range and route. These can then be used to determine the useful dose and route for administration to humans. In general, the determination and adjustment of the effective amount or dose, and the evaluation of the timing and method of such adjustment, are known to those skilled in the art.

[0045] In combination therapy, the antibodies, bispecific antibodies, and / or additional therapeutic or diagnostic agents can each be used as a single agent for any time period appropriate to effect the desired treatment or diagnosis. Thus, the single agents can be administered substantially simultaneously (i.e., as a single formulation, or within minutes or hours), or sequentially in sequence.

[0046] For additional guidance on formulations, dosages, methods of administration, and measurable therapeutic outcomes, reference is made to works such as Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey, Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0047] In order to solve the above technical problems, an eleventh technical aspect of the present invention provides use of the antibody according to the first technical aspect of the present invention, the bispecific antibody according to the second technical aspect of the present invention, the chimeric antigen receptor according to the third technical aspect of the present invention, the genetically modified cell according to the fourth technical aspect of the present invention, the antibody-drug conjugate according to the ninth technical aspect of the present invention, or the drug composition according to the tenth technical aspect of the present invention in the production of a drug, a reagent kit, and / or an administration device for treating and / or preventing cancer.

[0048] Preferably, said cancer is a tumor with positive B7H4 expression; said tumor is preferably breast cancer, ovarian cancer and endometrial cancer, said breast cancer is more preferably triple-negative breast cancer.

[0049] To solve the above technical problem, a twelfth aspect of the present invention provides a method for detecting B7H4 in a sample, the method comprising detecting using the above antibody or bispecific antibody. Preferably, the detection method is not for diagnostic purposes.

[0050] The non-diagnostic treatment or testing methods of the present invention include, but are not limited to, laboratory drug screening, preventive medicine research and public health policy formulation, and testing using reagent kits. Those skilled in the art are aware that modern medicine is divided into two parts: preventive medicine and clinical medicine. The "non-diagnostic detection method" of the present invention can be used in preventive medicine to detect samples collected in the environment (including human secretions) and determine whether the environment is contaminated with antigens. In laboratories, experimenters can also use the antibodies or bispecific antibodies of the present invention to detect laboratory reagents to ensure that the antigens used in experiments are not contaminated with antigens other than B7H4, and can further be used to screen new antibodies or small molecule compounds as drug targets.

[0051] In order to solve the above technical problems, a thirteenth technical aspect of the present invention provides a reagent kit, which comprises an antibody according to the first technical aspect of the present invention, a bispecific antibody according to the second technical aspect of the present invention, a chimeric antigen receptor according to the third technical aspect of the present invention, a genetically modified cell according to the fourth technical aspect of the present invention, an antibody-drug conjugate according to the ninth technical aspect of the present invention and / or a drug composition according to the tenth technical aspect of the present invention, and optionally a description.

[0052] To solve the above technical problem, the fourteenth technical aspect of the present invention provides an administration device, which includes (1) an infusion module for administering the drug composition described in the tenth technical aspect of the present invention to a subject in need thereof, and (2) an optional drug efficacy monitoring module.

[0053] To solve the above technical problems, the fifteenth technical aspect of the present invention provides the use of the antibody according to the first technical aspect of the present invention, the bispecific antibody according to the second technical aspect of the present invention, the chimeric antigen receptor according to the third technical aspect of the present invention, the genetically modified cell according to the fourth technical aspect of the present invention, the antibody-drug conjugate according to the ninth technical aspect of the present invention, and / or the drug composition according to the tenth technical aspect of the present invention in the diagnosis, prevention, and / or treatment of tumors. Preferably, the tumor is the one according to the eighth technical aspect of the present invention.

[0054] To solve the above technical problems, technical aspect 16 of the present invention provides a reagent kit, which includes a drug cassette A and a drug cassette B, wherein drug cassette A is an antibody described in technical aspect 1 of the present invention, a bispecific antibody described in technical aspect 2 of the present invention, a chimeric antigen receptor described in technical aspect 3 of the present invention, a genetically modified cell described in technical aspect 4 of the present invention, an antibody-drug conjugate described in technical aspect 9 of the present invention, and / or a drug composition described in technical aspect 10 of the present invention, and drug cassette B is another anti-tumor antibody or a drug composition containing the other anti-tumor antibody. Drug cassette A and drug cassette B can be used simultaneously, or drug cassette A can be used first before drug cassette B, or drug cassette B can be used first before drug cassette A, which can be determined according to the actual needs of a specific application.

[0055] The three-letter and single-letter amino acid codes used in this application are known to those skilled in the art or are described in J. Biol. Chem, 243, p. 3558 (1968). As used herein, the terms "contain" or "comprise" mean that the compositions and methods include the described elements but do not exclude other elements, but also include "consisting of..." depending on the context. Unless otherwise expressly provided, the term "or" is used herein to mean and is used interchangeably with the term "and / or." "About" and "approximately" should generally refer to the degree of tolerance for the measured amount, given the nature or precision of the measurement. Typical tolerances are usually within 10% of the amount, more commonly within 5%, and even within 2% or 1% of the amount. As used herein, EC 50 The term half-maximal effective concentration refers to the concentration that produces 50% of the maximal effect.

[0056] The above-mentioned preferable conditions may be arbitrarily combined in accordance with common knowledge in the art, that is, each preferable example of the present invention can be obtained.

[0057] All of the reagents and raw materials used in the present invention are commercially available.

[0058] The positive and improved effects of the present invention are as follows: 1. The B7H4-targeting monoclonal antibody of the present invention is a naturally occurring fully human antibody that binds to human B7H4 and cynomolgus monkey B7H4 without cross-reacting with other B7 family members. It has a strong ADCC effect due to Fc modification, and in vivo experiments have shown good antitumor activity. Among them, PR003369 has higher T cell activity and internalization activity after affinity maturation, making it more suitable for ADC drug use. 2. The B7H4×CD3 bispecific antibody of the present invention has a bispecific antibody structure of a human Fc fragment, retaining the binding activity of Fc and FcRn, thereby achieving a long half-life. The B7H4 end adopts an ScFv format, simplifying the mismatch between the light and heavy chains while maintaining good stability and hydrophilicity. The activity of the CD3 end has been optimized, and a moderately potent anti-CD3 antibody has been adopted, reducing toxicity while ensuring efficacy. The B7H4 end antibody and CD3 end antibody have good binding activity with cynomolgus monkeys. [Brief explanation of the drawings]

[0059] [Figure 1] The result of the initial antibody binding to human B7H4 on the cell surface; [Figure 2] Results of PR001476 and PR002037 and their PTM mutant / DE mutant antibodies expressing human B7H4 on the surface of supernatant-bound cells; [Figure 3] Results show that the initial antibody binds to cynomolgus monkey B7H4 on the cell surface; [Figure 4] Results of binding of PR001476 and PR002037 and their PTM mutant / DE mutant antibodies to cynomolgus monkey B7H4 on the cell surface; [Figure 5] Results show that the initial antibody binds to mouse B7H4 on the cell surface; [Figure 6] Results of binding of PR001476 and PR002037 and their PTM mutant / DE mutant antibodies to mouse B7H4 on the cell surface; [Figure 7] The initial antibody binds to B7H4 on the surface of tumor cells SK-BR-3; [Figure 8] Results of binding of PR001476, PR002037 and their PTM mutant / DE mutant antibody supernatants to B7H4 on the surface of tumor cell SK-BR-3; [Figure 9] Results of binding of PR001476, PR002037 and their PTM mutant / DE mutant antibodies to B7H4 on the surface of tumor cell SK-BR-3; [Figure 10]Results of PR002418 and PR001476 affinity matured / DE mutant antibody PR003369 binding to B7H4 on the surface of tumor cell MDA-MB-468; [Figure 11] Diagram showing the ADCC killing activity of PR001476 and its PTM mutant / DE mutant antibodies against SK-BR-3 tumor cells; [Figure 12] Diagram showing the ADCC killing activity of PR002037 and its PTM mutant / DE mutant antibodies against SK-BR-3 tumor cells; [Figure 13] Comparison of ADCC killing activity of PR002418, PR002421 and a control antibody from FivePrime against SK-BR-3 tumor cells; [Figure 14] Comparison of ADCC killing activity of PR003369, PR002418, RP002421 and control antibody 2 from FivePrime against MDA-MB-468 and HCC-1954 tumor cells; [Figure 15] Anti-B7H4 antibodies block the immunosuppressive signal of B7H4 and activate T cells; [Figure 16] Internalization results of anti-B7H4 antibody on SK-BR-3 cells; [Figure 17] Internalization-mediated cytotoxic activity of anti-B7H4 antibodies; [Figure 18] Effect of MMAE group cross-linking on the B7H4 binding activity of antibodies on tumor cell surfaces; [Figure 19] The effect of MMAE group crosslinking on the internalization activity of antibodies on tumor cells; [Figure 20] Tumor cell killing efficiency of affinity matured mutant PR003369-ADC and control antibody PR000157-ADC; [Figure 21] Analysis of antibody affinity data with Biacore T200 analysis software 2.0; [Figure 22] If anti-B7H4 antibodies cross-react with other member proteins of the B7 family; [Figure 23]Stability of anti-B7H4 antibodies, PR002418, PR002037, PR003369, and control antibodies in human serum at 37°C for 14 days; [Figure 24] Half-lives of PR002418, PR002421, and control antibody 2 in mice; [Figure 25] In vivo antitumor effect of anti-B7H4 monoclonal antibody molecules in BALB / c nude mouse MDA-MB-468 tumor model; [Figure 26] In vivo antitumor effects of anti-B7H4 monoclonal antibody molecules in the MDA-MB-468 tumor model reconstituting the human PBMC immune system in NSG mice; [Figure 27] Expression of B7H4 in normal tissues (A) and tumors (B) and its IHC score statistics (C); [Figure 28-1] Schematic diagram of the structure of the B7H4×CD3 bispecific molecule; [Figure 28-2] Schematic diagram of the structure of the B7H4×CD3 bispecific molecule; [Figure 28-3] Schematic diagram of the structure of the B7H4×CD3 bispecific molecule; [Figure 29-1] Results of B7H4×CD3 bispecific molecule binding to SK-BR-3 cells (AD) and T cells (EH); [Figure 29-2] Results of binding of B7H4×CD3 bispecific molecules to SK-BR-3 cells (AD) and T cells (EH); [Figure 30-1] B7H4×CD3 bispecific molecules (AL, “1+1” asymmetric structure; MN, “2+1” asymmetric structure) activated T cells and killed target cells; [Figure 30-2] B7H4×CD3 bispecific molecules (AL, “1+1” asymmetric structure; MN, “2+1” asymmetric structure) activated T cells and killed target cells; [Figure 30-3] B7H4×CD3 bispecific molecules (AL, “1+1” asymmetric structure; MN, “2+1” asymmetric structure) activated T cells and killed target cells; [Figure 30-4]B7H4×CD3 bispecific molecules (AL, “1+1” asymmetric structure; MN, “2+1” asymmetric structure) activated T cells and killed target cells; [Figure 31] Tumor suppression rate of B7H4×CD3 bispecific molecules in tumor models reconstituting the human PBMC immune system in NSG mice (A, MDA-MB-468 model; B, HCC-1954 model). DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention will be further described below in the form of examples, but the present invention is not limited to the scope of the examples. Experimental methods for which specific conditions are not specified in the following examples are selected according to conventional methods and conditions or product instructions.

[0061] Example 1. Obtaining anti-B7H4 antibody molecules Antibody molecules that specifically bind to B7H4 can be obtained by immunizing experimental animals with B7H4 recombinant protein or cells overexpressing B7H4. The experimental animals can be mice, rats, rabbits, sheep, camels, etc. The resulting antibody molecules are typically of non-human origin. After obtaining non-human antibodies, these molecules must be humanized using antibody engineering techniques to reduce immunogenicity and enhance drug efficacy. However, the antibody humanization process is technically complex, and humanized molecules often exhibit reduced antigen affinity. Meanwhile, advances in genetic engineering have made it possible to develop genetically engineered mice that contain a human immunoglobulin library and lack the endogenous mouse immune library. The Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying a human immunoglobulin library. The antibodies produced by this transgenic mouse contain fully human sequences, eliminating the need for further humanization, significantly improving the efficiency of therapeutic antibody development.

[0062] 1.1. Mouse immunization Harbour H2L2 mice were immunized multiple times using a soluble recombinant human B7H4-ECD-mFc fusion protein (Sino Biological, #10738-H05H) as the antigen. The antigen protein was mixed with an immunological adjuvant to form an immunogen reagent, which was then injected subcutaneously into the groin or intraperitoneally. Each mouse received a total injection volume of 100 microliters in each immunization cycle. In the first round of immunization, each mouse received an immunogen reagent prepared by mixing 50 micrograms of antigen protein with complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1. In each subsequent round of boosting, each mouse received an immunogen reagent prepared by mixing 25 micrograms of antigen protein with Sigma Adjuvant System adjuvant (Sigma, #S632). The interval between boosting rounds was at least 2 weeks, with six to seven rounds typically. Immunization times were days 0, 14, 28, 42, 56, 70, 84, and 98, and mouse serum antibody titers were measured on days 49 and 77. Five days before H2L2 mouse splenic B cell isolation, the final immunization booster was administered with 25 micrograms of antigen protein per mouse.

[0063] Alternatively, CHO-K1 cells overexpressing human B7H4 (CHO-K1 / huB7H4, HARBOUR BIOMED) were used to transfect a plasmid encoding mouse CD40L, which was then mixed with an immunoadjuvant to form an immunogen reagent, which was then used to immunize mice, with each mouse receiving 5 × 10 6 The immunization process was the same as protein immunization.

[0064] 1.2. Serum titer detection At specific time points, mouse serum was collected, and the antibody binding titer against B7H4 protein in the serum was detected using ELISA, and the antibody binding titer against B7H4-overexpressing cells in the serum was detected using FACS.

[0065] For the ELISA, 100 μL / well of 1 μg / mL hB7H4-ECD-his protein (Sino Biological, #10738-H08H) was coated onto an ELISA plate (Corning, 9018), incubated overnight at 4°C, washed twice, and blocked with 1% BSA in PBST for 2 hours at 37°C. 100 μL / well of gradient-diluted serum was added and incubated for 1 hour at 37°C. After three washes, 100 μL / well of anti-rat-HRP (Sigma, #A5795) diluted 1:5000 was added and incubated for 30 minutes at 37°C. After three washes, 100 μL / well of TMB substrate was added and incubated for approximately 10 minutes. 50 μL / well of 1N HCl was added to stop the color development, and the absorbance was read at 450 nm (Molecular Devices, Plus 384).

[0066] For FACS, gradient-diluted mouse serum was incubated with HEK293-B7H4 cells at 4°C for 1 hour, washed twice, and then incubated with the secondary antibody anti-rat IgG (H+L) (Life Technologies, A11006) at 4°C for 1 hour. After washing twice, the resuspended cells were analyzed using a flow cytometer (BD, Flibur). HEK293 cells served as a background control.

[0067] 1.3. Screening for anti-B7H4 antibodies using hybridoma technology Mice with high serum titers were selected and subjected to primary and terminal immunization. The mice were sacrificed, and spleen cells and SP2 / 0 myeloma cells (ATCC, CRL-1581) were collected and electrofusion was performed. The cell ratio was 4:1, and the electrofusion parameters were V1:50V, t1:15s, V2:600V, t2:20 μs, t3:0.5s, n:1, t4:7s, V+ / -:+, fade:on. The cells were resuspended in DMEM culture medium containing 20% FBS and HT, and 1 × 10 5The cells were plated at 100 μL per well. After 24 hours, 100 μL per well of DMEM containing 20% FBS and 2xHT was added and the incubation continued. The supernatants were then collected and the antibody titers were measured. Generally, 9-15 days after fusion, supernatants from protein-immunized mice were subjected to initial sieving using an Acumen filter to detect binding to CHO-K1 / huB7H4 cells; supernatants from cell-immunized mice were subjected to initial sieving using a Mirrorball (SPT Labtech, mirrorball® fluorescence cytometer) to detect binding to HEK-293 / huB7H4 cells. Positive clones were then further confirmed using ELISA and FACS to detect their binding to CHO-K1 cell lines overexpressing human B7H4 (CHO-K1 / huB7H4), cynomolgus monkey B7H4 (CHO-K1 / cynoB7H4), and mouse B7H4 (CHO-K1 / mB7H4). Positive wells were further subcloned by finite dilution and further screened by ELISA and FACS. Clones showing good binding to human and monkey B7H4 were selected and sequenced.

[0068] 1.4. Screening of anti-B7H4 antibodies using in vitro B cell clonal technology The spleens of the mice were removed, polished, and filtered through a 200-mesh filter. The single-cell suspension was sorted using a mouse memory B cell sorting reagent kit (Miltenyi, #130-095-838). The sorted cells were subjected to immunofluorescence staining.

[0069] B200-positive (BioLegend, #103227), IgM-negative (BioLegend, #406506), and B7H4-specific positive cells (BioLegend, #405207) were sorted on a flow cytometer S3e. The sorted cells were cultured at a density of 5 cells per well in a 96-well cell culture plate, and pre-irradiated EL4 cells were layered on the plate.

[0070] After 14 days of culture, culture supernatants were collected and subjected to ELISA detection. Cells from wells showing binding activity to B7H4 protein were removed and subjected to RT-PCR using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (#634892) and I-5® 2x High-Fidelity Master Mix (#I5HM-5000). The light and heavy chains obtained by amplification were combined into scFv by overlap PCR and expressed in E. coli. The expressed supernatants were subjected to ELISA detection, and positive clones were sequenced.

[0071] 1.5. Sequence analysis and sequence optimization of anti-B7H4 antibodies The nucleotide sequences encoding the variable domains of the antibody molecules and the corresponding amino acid sequences were obtained using conventional sequencing techniques. Three monoclonal sequences were obtained. In this example, the variable domain sequences of the anti-B7H4 monoclonal antibody molecules obtained from immunized Harbour H2L2 mice were human-derived antibody sequences, and their germline genetic analysis and post-translational modification (PTM) analysis are shown in Table 1-1.

[0072] After translational synthesis in cells, proteins or polypeptide amino acid chains may undergo chemical modifications called post-translational modifications (PTMs). For antibodies, some PTM sites are highly conserved. For example, the conservative amino acid asparagine Asn at position 297 (EU numbering) in the constant domain of human IgG1 antibodies is typically glycosylated to form glycans, which are crucial for antibody structure and associated effector functions. However, when PTMs are present in the variable domain of an antibody, particularly in the antigen-binding region such as the CDR, the presence of these PTMs can significantly affect antigen binding and can also alter the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, and oxidation can all increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding potential PTMs is crucial for therapeutic antibody development. With increasing experience, it has been discovered that some PTMs are highly correlated with the amino acid sequence composition, particularly the "pattern" of adjacent amino acid composition, allowing us to predict potential PTMs from the primary amino acid sequence of a protein. For example, the sequence pattern NxS / T (asparagine at position 1, any amino acid except proline at position 2, and serine or threonine at position 3) predicts an N-linked glycosylation site. The amino acid sequence pattern that causes PTM may be derived from a germline gene sequence, for example, the human germline gene fragment IGHV3-33 naturally has the glycosylation pattern NST in the FR3 region; it may also be derived from somatic hypermutation.

[0073] PTM amino acid sequence patterns can be disrupted by amino acid mutation to reduce or eliminate the formation of specific PTMs. Different mutation design methods exist depending on the antibody sequence and PTM sequence pattern. One method is to replace "hot spot" amino acids, such as N or S in an N-S pattern, with physicochemically similar amino acids (e.g., mutating N to Q). If the PTM sequence pattern is derived from somatic hypermutation and does not exist in the germline gene sequence, another method is to replace the sequence pattern with the corresponding germline gene sequence. In practice, multiple mutation design methods can be employed for the same PTM sequence pattern.

[0074] Table 1-2 shows the new antibody molecule sequences obtained by amino acid mutation of the sequences of antibodies PR001476 and PR002037.

[0075] [Table 1-1]

[0076] [Table 1-2]

[0077] 1.6. PR001476 Antibody Affinity Maturation The molecule, PR001476, is engineered by site-directed mutagenesis to increase its binding affinity to B7H4. This affinity maturation process is divided into two rounds.

[0078] In the first round, we performed point-by-point scanning of the heavy and light chain CDR3s (defined as Chothia CDRs) of the PR001476 molecule to construct single-point saturation mutation libraries at multiple amino acid positions. We then screened the saturation mutation libraries and selected positive molecules with signals greater than twice those of the wild type for sequencing. These positive molecules were further characterized and selected several mutation hotspots based on their binding ability to human B7H4.

[0079] In the second round, the hotspots identified in the first round of saturation mutations were randomly combined to create a library containing all possible mutation combinations. The combinatorial library was then screened. For positive molecules, several mutants were selected by sequencing and their ability to bind to human hB7H4. The screened mutants are represented by their corresponding clone numbers, e.g., PR001476-R1-25B3, PR001476-R1-26D7, etc.

[0080] The mutants were constructed in a mammalian expression vector for protein expression and purification. The mutants were then tested for their B7H4 binding ability using FACS and a Fortebio Octet. PR003369 in Tables 1-2 is a preferred mutant derived from PR001476.

[0081] 1.7. Preparation of recombinant antibodies and physicochemical characterization analysis 1.7.1. Antibody Expression and Purification This example introduces a general method for producing antibodies using mammalian host cells (e.g., human embryonic kidney cells (HEK 293) or Chinese hamster ovary cells (CHO) and their derivatives) and techniques such as instantaneous transfection, expression, and affinity capture isolation. This method applies to target antibodies containing an Fc region; target antibodies can be composed of one or more protein polypeptide chains and can be derived from one or more expression plasmids.

[0082] The amino acid sequence of the antibody polypeptide chain is converted to a nucleotide sequence using codon optimization; the encoded nucleotide sequence is synthesized and cloned into an expression vector compatible with the host cell. Plasmids encoding the antibody polypeptide chains are co-transfected into mammalian host cells at a specific ratio, and conventional recombinant protein expression and purification techniques are used to obtain recombinant antibodies with correct folding and polypeptide chain assembly. Specifically, FreeStyle® 293-F cells (Thermo, #R79007) were expanded in FreeStyle® F17 Expression Medium (Thermo, #A1383504). Before the start of instantaneous transfection, the cell concentration was adjusted to 6–8 × 10 5 The cells were cultured at 37°C in an 8% CO2 shaker for 24 hours, with the cell concentration adjusted to 1.2 x 10 6The cell density is measured at 1000kJ / mL. A 30 mL sample was prepared from the cultured cells. Plasmids encoding antibody polypeptide chains were mixed at a fixed ratio. A total of 30 μg of plasmid (1 μg:1 mL) was dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo, #31985088) and sterilized by filtration through a 0.22 μm filter. Another 1.5 mL of Opti-MEM was dissolved in 120 μL of 1 mg / mL PEI (Polysciences, #23966-2) and allowed to stand for 5 minutes. The PEI was slowly added to the plasmids, and the mixture was incubated at room temperature for 10 minutes. The plasmid-PEI mixture was then slowly added dropwise while rocking the culture flask. The cells were then cultured for 5 days at 37°C in an 8% CO2 shaker. Cell viability was monitored after 5 days. The culture was collected and centrifuged at 3300 x g for 10 minutes, after which the supernatant was removed. The supernatant was then centrifuged at high speed to remove impurities. Equilibrate a gravity column (Bio-Rad, #7311550) containing MabSelect® (GE Healthcare, #71-5020-91) with PBS pH 7.4 buffer and wash with 2-5 column volumes. The supernatant sample is passed through the column, washed with 5-10 column volumes of PBS buffer, and the target protein is eluted with 0.1 M glycine, pH 3.5, then adjusted to neutral with Tris-HCl, pH 8.0. Finally, the purified recombinant antibody solution is concentrated and exchanged into PBS buffer or other buffers containing other components using an ultrafiltration tube (Millipore, #UFC 901024). Finally, the concentration is measured using a NanoDrop (Thermo, NanoDrop® One), aliquoted, and stored in reserve.

[0083] 1.7.2. Analysis of Protein Purity and Multimerization by SEC-HPLC In this example, analytical size exclusion chromatography (SEC) was used to analyze the purity and multimeric form of protein samples. An analytical chromatography column, TSKgel G3000 SWxl (Tosoh Bioscience, #08541, 5 μm, 7.8 mm × 30 cm), was connected to a high-pressure liquid chromatography (HPLC) system (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μg) was filtered through a 0.22 μm filter membrane and injected into the system. The HPLC program was set to run the sample through the column in PBS buffer at a flow rate of 1.0 mL / min for a maximum of 25 minutes. The HPLC generated an analytical report reporting the retention times of different molecular size components within the sample.

[0084] 1.7.3. Analysis of Protein Purity and Hydrophobicity by HIC-HPLC Analytical hydrophobic interaction chromatography (HIC) was used to analyze the purity and hydrophobicity of protein samples. An analytical column, TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6 mm x 3.5 cm), was connected to a high-pressure liquid chromatography (HPLC) column (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. The setup consisted of a linear gradient from 100% mobile phase A (20 mM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0) within 16 minutes at a flow rate of 0.7 mL / min, a protein sample concentration of 1 mg / mL, an injection volume of 20 μL, and a detection wavelength of 280 nm. After collection, the chromatogram was integrated using ChemStation software, relevant data were calculated, and an analytical report was generated, reporting the retention times of components of different molecular sizes within the sample.

[0085] 1.7.4. Measuring the thermal stability of protein molecules by DSF Differential scanning fluorimetry (DSF) is a common high-throughput method for measuring protein thermal stability. Using a real-time fluorescent quantitative PCR system, changes in the fluorescence intensity of dyes bound to unfolded protein molecules are monitored to reflect the process of protein denaturation and thus the thermal stability of the protein molecules. In this example, the DSF method was used to measure the thermal denaturation temperature (Tm) of protein molecules. 10 μg of protein was added to a 96-well PCR plate (Thermo, #AB-0700 / W), followed by 2 μl of 100x diluted SYPRO™ dye (Invitrogen, #2008138), followed by buffer to a final volume of 40 μl per well. The PCR plate was sealed and placed in a real-time fluorescent quantitative PCR system (Bio-Rad CFX 96 PCR System). After 5 minutes at 25°C, the temperature was gradually increased from 25°C to 95°C at a 0.2°C / 0.2 minute gradient, and then reduced to 25°C at the end of the experiment. Data analysis was performed using FRET scan mode and Bio-Rad CFX Maestro software to calculate the Tm of the samples.

[0086] 1.8. Generation of anti-B7H4 fully human recombinant antibodies The anti-B7H4 fully human IgG antibodies and the optimized antibodies obtained in 1.3 to 1.6 were produced and analyzed using the method described in 1.7.1. Tables 1 to 3 and Tables 1 to 4 show the results of small-volume and large-volume instantaneous expression and purification, respectively. Additionally, anti-B7H4 antibody sequences were obtained from previous literature (Tables 1 to 5) and used as controls in subsequent experiments.

[0087] [Table 1-3]

[0088] [Table 1-4]

[0089] [Table 1-5]

[0090] 1.9. Anti-B7H4 antibody sequences and numbers In the present invention, the amino acid sequences of the CDRs described above are all shown according to the Chothia definition rules. However, those skilled in the art are aware of various methods in the art for defining antibody CDRs, such as the Kabat definition rules based on sequence variability (see Kabat et al., Immunological Protein Sequences, 5th Edition, National Institutes of Health, Bethesda, MD (1991)) and the Chothia definition rules based on the location of structural ring regions (see J Mol Biol 273:927-481997). In embodiments of the present invention, a combined definition rule, which includes the Kabat and Chothia definitions, can also be used to determine the amino acid residues in the variable domain sequence. Here, the combined definition rule combines the scope of the Kabat and Chothia definitions and has a broader scope based on this. Details are shown in Tables 1-6. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or a region thereof (e.g., a variable region) encompass the complementarity determining region defined by any of the above-mentioned known embodiments described by the present invention. The scope of protection in the present invention is the sequence shown based on the Chothia definition rules, but corresponding amino acid sequences according to other CDR definition rules should also be included in the scope of protection of the present invention.

[0091] [Table 1-6]

[0092] Here, Laa-Lbb can refer to the amino acid sequence from position aa (Chothia coding convention) to position bb (Chothia coding convention) from the N-terminus of the antibody light chain, and Haa-Hbb can refer to the amino acid sequence from position aa (Chothia coding convention) to position bb (Chothia coding convention) from the N-terminus of the antibody heavy chain. For example, L24-L34 can refer to the amino acid sequence from positions 24 to 34 of the antibody light chain from the N-terminus according to the Chothia coding convention, and H26-H35 can refer to the amino acid sequence from positions 26 to 35 of the antibody heavy chain from the N-terminus according to the Chothia coding convention. Those skilled in the art should recognize that insertion sites can exist at several positions when encoding CDRs with Chothia (see http: / / bioinf.org.uk / abs / ).

[0093] Tables 1 to 7 show the sequence numbers of the CDRs, variable regions, and light and heavy chains corresponding to the sequences of the anti-B7H4 antibodies of the present invention and control antibody molecules. PR003366 is a single-chain variable region (scFv) homodimer molecule (scFv-Fc structure) constructed using the variable region sequence of PR002410.

[0094] [Table 1-7]

[0095] Tables 1 to 8 show the SEQ ID NOs of the framework regions and Fv corresponding to the sequences of the anti-B7H4 antibodies of the present invention and control antibody molecules.

[0096] [Table 1-8]

[0097] Tables 1 to 9 show the CDR sequences corresponding to the sequences of the anti-B7H4 antibodies of the present invention and control antibody molecules.

[0098] [Table 1-9]

[0099] Example 2. FACS detection of the ability of anti-B7H4 antibodies to bind to B7H4 This example was conducted to investigate the in vitro binding activity of anti-human B7H4 H2L2 monoclonal antibodies to human, cynomolgus monkey, and mouse B7H4. Antibody binding experiments were performed using CHOK1 cell lines overexpressing human B7H4 (CHOK1 / hu B7H4, HARBOUR BIOMED), cynomolgus monkey B7H4 (CHOK1 / cyno B7H4, HARBOUR BIOMED), mouse B7H4 (CHOK1 / m B7H4, HARBOUR BIOMED), and the SK-BR-3 (ATCC® HTB-30) cell line, which highly expresses human B7H4. Briefly, CHOK1 / hu B7H4 cells, CHOK1 / cyno B7H4 cells, CHOK1 / m B7H4 cells, or SK-BR-3 cells were digested and resuspended in PBS containing 2% BSA at a cell density of 1 × 10 6 The antibody was adjusted to 100 cells / mL. 100 μL of cells / well were seeded into a 96-well V-bottom plate (Corning, #3894), and 100 μL / well of the antibody to be measured, diluted 3-fold at twice the final concentration, was added. The cells were placed at 4°C and incubated for 2 hours, protected from light. Then, the cells were washed twice with 100 μL / well of pre-chilled 2% BSA in PBS, centrifuged at 500 x g for 5 minutes at 4°C, and the supernatant was discarded. The cells were then incubated with 100 μL / well of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, 1:500 dilution) for 1 hour at 4°C, protected from light. The cells were washed twice with 100 μL / well of pre-chilled 2% BSA in PBS, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-chilled 2% BSA in PBS, and the fluorescence signal was read using an ACEA Novocyte3000 flow cytometer.

[0100] The binding of the antibody to human B7H4, cynomolgus monkey B7H4, and mouse B7H4 on the cell surface, as well as to B7H4 on the surface of tumor cells SK-BR-3, is summarized below (Tables 2-1, 2-2, and 2-3). The affinity-matured variant PR003369 showed significantly improved binding to tumor cells compared to PR002418 (Tables 2-3).

[0101] [Table 2-1]

[0102] [Table 2-2]

[0103] [Table 2-3]

[0104] The binding results of the initial antibodies to human B7H4 on the cell surface are shown in Figure 1. The results show that PR001476 and PR002037 have relatively good human B7H4 binding activity, while PR002038 has relatively poor binding activity. The binding results of the supernatants expressing PR001476, PR002037, and their PTM mutant / DE mutant antibodies to human B7H4 on the cell surface are shown in Figure 2. The results show that the PTM mutant / DE mutations of PR002037 and PR001476 did not significantly affect the binding activity of the antibodies to human B7H4. The binding results of the initial antibodies to cell surface cynomolgus monkey B7H4 are shown in Figure 3, and the results showed that PR002037 and PR001476 had relatively good cross-binding activity to cynomolgus monkey B7H4, while PR002038 had relatively poor cross-binding activity. The binding results of PR001476, PR002037, and their PTM mutant / DE mutant antibodies to cell surface cynomolgus monkey B7H4 are shown in Figure 4, and the results showed that the PTM mutant / DE mutations of PR002037 and PR001476 did not significantly affect the cross-binding activity of the antibodies to cynomolgus monkey B7H4. The binding results of the initial antibodies to cell surface mouse B7H4 are shown in Figure 5, and the results showed that PR002037 had relatively good mouse B7H4 cross-binding activity, PR001476 had relatively weak mouse B7H4 cross-binding activity, and PR002038 had no mouse B7H4 cross-binding activity.The binding results of PR001476, PR002037, and their PTM mutant / DE mutant antibodies to cell surface mouse B7H4 are shown in Figure 6, and the results showed that PR002037 and PR001476 PTM mutant / DE mutant antibodies maintained similar mouse B7H4 cross-binding activity compared to their parent antibodies. The binding results of the initial antibodies to B7H4 on the surface of SK-BR-3 tumor cells are shown in Figure 7, and the results showed that PR002037 and PR001476 had relatively good binding activity to B7H4 on the surface of SK-BR-3 tumor cells, while PR002038 had relatively poor binding activity. The binding results of PR001476, PR002037, and their PTM mutant / DE mutant antibody supernatants to B7H4 on the surface of SK-BR-3 tumor cells are shown in Figure 8, and the results showed that the PTM mutant / DE mutation of PR002037 and PR001476 did not significantly affect the binding activity of the antibodies to B7H4 on the surface of SK-BR-3 tumor cells.The binding results of PR001476, PR002037, and their PTM mutant / DE mutant antibodies to B7H4 on the surface of SK-BR-3 tumor cells are shown in Figure 9. The results showed that the PTM mutant / DE mutant antibodies PR002037 and PR001476 maintained their binding activity to B7H4 on the surface of SK-BR-3 tumor cells. Among them, the PTM mutant / DE mutant antibody PR001476 had stronger binding activity (lower EC50) than control antibody 1. The binding results of PR002418 and the affinity-matured / DE mutant antibody PR003369 of PR001476 to B7H4 on the surface of MDA-MB-468 tumor cells are shown in Figure 10. Compared to the PTM mutant PR002418, the affinity-matured mutant PR003369 exhibited significantly improved binding to B7H4 on the surface of MDA-MB-468 tumor cells.

[0105] Example 3. Detection of ADCC activity This example investigated the in vitro ADCC activity of anti-human B7H4 H2L2 monoclonal antibody (HBsAg) in mediating NK cell killing of target cells. In this experiment, human PBMCs were used as effector cells, and the B7H4-highly expressing cell lines SK-BR-3 and MDA-MB-468, and the B7H4-moderately expressing cell line HCC-1954 were used as target cells. The electrical conductivity of the target cells was measured using an ACEA RTCA instrument to reflect the killing efficiency. A 96-well e-plate was first equilibrated with 50 μl of complete medium. SK-BR-3, MDA-MB-468, or HCC-1954 cells were digested and resuspended in RPM1640 complete medium containing 10% fetal bovine serum. 4 × 10 cells were then cultured. 5 / ml and place 50 μl / well on an e-plate 96-well plate, i.e., 2 × 10 4 The cells were plated at 2 × 10 cells / well and cultured overnight at 37°C. 550 μl of fresh culture medium containing PBMCs was added to each well, followed by 50 μl of 4x gradient diluted antibodies, with the highest final antibody concentration being 100 nM. Each antibody was diluted to a total of eight concentrations, with two replicates. The electrical conductivity of the target cells was measured in real time, and the target cell killing efficiency was calculated using the data from the 24-hour time point: target cell killing efficiency = (1 - sample / blank control) × 100%.

[0106] The ADCC killing activity of PR001476 and its PTM mutant / DE mutant antibody against SK-BR-3 tumor cells is shown in Figure 11, and the DE mutation at the Fc terminus (PR002418) clearly enhances the ADCC killing activity of the antibody against SK-BR-3 tumor cells. The ADCC killing activity of PR002037 and its PTM mutant / DE mutant antibody against SK-BR-3 tumor cells is shown in Figure 12, and the DE mutation at the Fc terminus (PR002421) clearly enhances the ADCC killing activity of the antibody against SK-BR-3 tumor cells. A comparison of the ADCC killing activity of PR002418, PR002421, and the control antibody from FivePrime against SK-BR-3 tumor cells is shown in Figure 13 (where A is data from donor 1 and B is data from donor 2), and the results showed that PR002418 had similar ADCC killing activity against SK-BR-3 tumor cells compared to the control antibody from FivePrime, while PR002421 had slightly weaker killing activity. The ADCC killing activity of PR003369, PR002418, RP002421, and control antibody 2 from FivePrime against MDA-MB-468 and HCC-1954 tumor cells is compared in Figure 14 (where A represents MDA-MB-468 data and B represents HCC-1954 data). The results showed that PR003369, PR002418, and RP002421 all had ADCC killing activity against MDA-MB-468 and HCC-1954 tumor cells, and the killing activity was positively correlated with B7H4 expression, i.e., the killing activity was stronger on MDA-MB-468 cells, which highly express B7H4, and weaker on HCC-1954 cells, which moderately express B7H4. The affinity-matured mutant PR003369 showed further improved ADCC killing activity compared to PR002418 and PR002421.

[0107] Example 4. Detection of T cell activating activity To determine whether anti-B7H4 antibody activates T cells by blocking T cell immune checkpoints, this experiment used HEK293T cells overexpressing full-length B7H4 and the scFv form of the anti-human CD3 antibody OKT3 as artificial antigen-presenting cells (HEK293T / OS8 / hB7H4, KYinno). T cells were isolated using a human T cell isolation kit (Miltenyi, #130-096-535) according to the manufacturer's instructions. The artificial antigen-presenting cells and T cells were then co-cultured to measure the effect of anti-B7H4 antibody on T cell activation. Specifically, HEK293T-OS8-hB7H4 cells were cultured at 1 x 10 4 Human primary T cells were isolated and plated at a density of 2 x 10 / well and cultured overnight. 5 The cells were added to HEK293T / OS8 / hB7H4 cells at a density of 100 μl / well. 100 μL / well of 5-fold gradient diluted antibodies (2x final concentration) were then added at 100 μL / well, with the highest final antibody concentration at 10 nM. Each antibody was diluted at six concentrations, with two replicates. After 3 days of incubation, supernatants were collected and IFN-γ levels were measured by ELISA. Results showed that PR003369, PR002418, PR002421, and the control antibody all promoted T cell activation. Among these, the affinity-matured mutant PR003369 had stronger T cell activation activity than the PTM mutant PR002418 and the control antibody 2. Its mechanism of action may be blocking the interaction between B7H4 and its unknown receptor on T cells. The results showing that anti-B7H4 antibody blocked the immunosuppressive signal of B7H4 and activated T cells are shown in FIG. 15 (where A is donor 1 and B is donor 2).

[0108] Example 5. Antibody internalization experiments Antibody internalization was detected using the Zenon pHrodo iFL IgG Labeling Reagents Kit (Invitrogen, #Z25611). This reagent is a fluorescent dye-conjugated secondary antibody that is non-fluorescent at neutral pH and binds to the primary antibody, which is internalized into lysosomal bodies along with the antibody. After internalization, it automatically emits bright fluorescence in an acidic pH environment, allowing detection by FACS. The specific method is as follows: SK-BR-3 cells were harvested, centrifuged, and the supernatant discarded. The cells were then resuspended in medium to a cell concentration of 3 x 10. 6 The cell suspension was added to a 96-well plate, 50 μl per well, and then incubated overnight at 37°C in an incubator. A 4x dilution of the target antibody was prepared, with the highest concentration being 40 nM (4x), followed by 3-fold dilutions for a total of eight dilutions. A 4x Zenon solution was prepared, and 25 μl of the target antibody was mixed with 25 μl of the 4x Zenon labeling solution and left at room temperature for 5 minutes. 50 μl of the labeled antibody was then added to the 96-well plate containing the cells and incubated at 37°C for 24 hours. The cells were digested, and fluorescence readings were measured on a flow cytometer. Compared to the control antibody RP000014 and other antibodies, PR003369 exhibited the highest internalization activity. Figure 16 shows the internalization results of the anti-B7H4 antibody on SK-BR-3 cells.

[0109] Antibody internalization was detected using a-HFc-CL-MMAF reagent (Moradec, #AH-102-AF). This reagent is a toxic group-containing MMAF secondary antibody that binds to the primary antibody and is internalized along with the antibody. Afterwards, it releases the toxic group inside the cell, killing the target cell. The mechanism is similar to the action of ADC. The specific method is as follows: SK-BR-3 cells were harvested, centrifuged, and the supernatant discarded. The cells were then resuspended in medium to a cell concentration of 1 x 10. 5The cell suspension was added to a 96-well plate, 50 μl per well, and then incubated overnight at 37°C in an incubator. A 4x MMAF solution (4 μg / mL) was prepared. The highest concentration was 40 nM (4x), followed by 5-fold dilutions for a total of eight dilutions. A 4x MMAF solution (4 μg / mL) was also prepared. 25 μl of the 4x MMAF solution and 25 μl of the 4x MMAF solution were added to each well of the 96-well plate containing cells, and the plate was incubated at 37°C for 72 hours. 100 μl of CTG solution was added to the experimental wells, and the CTG luminescence signal was read using a microplate reader. The results in Figure 17 demonstrate that PR003369 has the highest internalization-mediated cytotoxicity activity compared to the control antibody RP000014 and other antibodies.

[0110] Example 6. Antibody Drug Conjugates (ADCs) In this example, the toxic group MMAE was crosslinked to an anti-B7H4 antibody (PR003369 antibody or control antibody 1) using ADC conjugation technology to form an ADC. The purity parameters of the product are as follows, and the HPLC measurement method is the same as in 1.7.2.

[0111] [Table 3]

[0112] To investigate whether crosslinking MMAE groups affects the antibody's binding activity to the B7H4 target, a cellular antibody binding experiment was performed using the MDA-MB-468 cell line, which highly expresses human B7H4, using the same method as in Example 2. The results in Figure 18 show that crosslinking MMAE groups does not substantially affect the antibody's binding activity to B7H4 on the tumor cell surface.

[0113] To examine whether MMAE group crosslinking affects the internalization activity of the antibody, antibody internalization was detected using the Zenon pHrodo iFL IgG Labeling Reagents kit (Invitrogen, #Z25611). The experimental method was the same as in Example 5. The results in Figure 19 show that MMAE group crosslinking does not substantially affect the internalization activity of the antibody on tumor cells.

[0114] This example was conducted to investigate the cell-killing activity of antibody-MMAE-group-crosslinked ADCs. MDA-MB-468 cells, a cell line highly expressing B7H4, were used as target cells. The electrical conductivity of the target cells was measured using an ACEA RTCA instrument to reflect the killing efficiency. A 96-well e-plate was first equilibrated with 50 μl of complete medium. MDA-MB-468 cells were digested and resuspended in RPM1640 complete medium containing 10% fetal bovine serum. 1 × 10 cells were then cultured. 5 / mL and place 50 μl / well on an e-plate 96-well plate, i.e., 5 × 10 3 Plated at 100 μl per well and incubated overnight at 37°C. The next day, 100 μl of 2x gradient diluted antibody was added per well, with the highest final antibody concentration at 50 nM. Each antibody was diluted to a total of eight concentrations, with two replicates. The electrical conductivity of the target cells was measured in real time, and the target cell killing efficiency was typically calculated using the data from the 96-hour time point: target cell killing efficiency = (1 - sample / blank control) × 100%. The results in Figure 20 demonstrate that the affinity-matured variant PR003369-ADC has a higher tumor cell killing efficiency than the control antibody 1-ADC.

[0115] Example 7. Affinity measurement of anti-B7H4 antibodies to human B7H4 recombinant protein 7.1. Affinity measurement by SPR method 10x HBS-EP+ (GE Healthcare, #BR-1006-69) was diluted 10-fold and used as the experimental buffer. The flow rate was set to 10 μL / min. Protein A was conjugated to four channels of a chip CM5 (GE Healthcare, #BR-1005-30): 1) 50 mM NHS and 200 mM EDC were freshly mixed at a 1:1 volume ratio and injected into all four channels for an 800-second injection time; 2) Protein A was diluted to 20 μg / mL with sodium acetate, pH 4.5 (GE Healthcare, #BR-1003-50), and injected into each channel for 800 seconds; and 3) 1 M ethanolamine, pH 8.5, was injected for 800 seconds to cap any remaining active carboxyl groups on the chip surface. After capping, the chip was equilibrated in the 1x HBS-EP+ buffer equilibrator for 2 hours. The final conjugated amount of protein A was approximately 2,000 RU.

[0116] The Biacore T200 was configured in multi-cycle kinetic mode, with each cycle including antibody capture, analyte binding, and chip regeneration. Antibodies PR002418, PR002421, control antibody 1, and control antibody 2 were each diluted to 1 μg / mL and injected into channels 2, 3, and 4 for 30 seconds at a flow rate of 10 μl / min. Each antibody was captured using pre-conjugated Protein A, resulting in a capture yield of approximately 160 RU. Human B7-H4 (Sino biological, #10738-H08H) was injected sequentially into four channels at a concentration gradient of 0 nM, 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM (with one highest concentration of 100 nM added to control antibody 1) at a flow rate of 30 μl / min. The dissociation time was set to 200 s for PR002418, PR002421, and control antibody 2, and 500 s for control antibody 1. The injection time was 180 s for all antibodies. Finally, 10 mM glycine-HCl pH 1.5 (GE Healthcare, #BR-1003-54) was injected for 30 s at the same flow rate to regenerate the chip.

[0117] The experimental results were analyzed using Biacore T200 analysis software 2.0. One channel was subtracted as the reference channel, and a 1:1 kinetic fitting model was selected for the analysis model. The results are shown in Table 7-1 and Figure 21A-D. The results showed that PR002421 had the highest protein affinity.

[0118] [Table 4-1]

[0119] 7.2. Measurement of affinity by BLI 10x kinetics buffer (ForteBio, #18-1105) was diluted to 1x and used for affinity testing and dilution of antigens and antibodies. Binding kinetics between antigens and antibodies was analyzed using biofilm interference (BLI) technology with an Octet Red 96e (ForteBio) molecular interaction analyzer.

[0120] When measuring the affinity of an antigen and an antibody, the sensor rotation speed was 1,000 rpm. The column-mounted AHC sensor (Fortebio, #18-5060) was equilibrated in the test buffer for 10 minutes, after which the B7-H4 antibody was captured by the AHC sensor at a capture height of 0.7 nm. The AHC sensor was equilibrated in the buffer for 120 seconds, then bound to a 2-fold gradient of human B7-H4 (concentrations of 50-3.125 nM and 0 nM) for 180 seconds and then dissociated for 300 seconds. Finally, the AHC sensor was regenerated by immersing it in 10 mM glycine-HCl, pH 1.5, and the bound protein was eluted.

[0121] Data analysis was performed using Octet Data Analysis software (Fortebio, version 11.0). 0 nM was used as the reference well, and reference subtraction was subtracted. Data fitting was performed using the "1:1 Global fitting" method to calculate the kinetic parameters of the binding between the antigen and the antigen-binding protein. on (1 / Ms) value, k disThe (1 / s) and KD(M) values were obtained (see Table 7-2), and the affinity-matured variant PR003369 showed significantly improved protein affinity compared to PR002418.

[0122] [Table 4-2]

[0123] Example 8. Measurement of epitope competition for binding of anti-B7H4 antibodies to B7H4 using the BLI method Epitope competition experiments were performed using the ForteBio Octet Red96e platform with B7-H4 antibodies PR002418, PR002421, control antibody 1, and control antibody 2. The experimental buffer was the same as described above. In the first step, 100% antibody signal was obtained: B7-H4 (Acro Biosystems, #B74-H82E2-200 μg) was captured using an SA sensor (ForteBio, #18-5019) at a capture height of 0.25 nm. After equilibrating in buffer for 120 s, the sensor was immersed in each antibody diluted to 100 nM. After 240 s, the final signal upon antibody binding to B7-H4 was recorded as 100% antibody signal. In the second step, epitope competition experiments were performed: B7-H4 was captured using an SA sensor at a capture height of 0.25 nm. The sensor was immersed in the first antibody (concentration 100 nM) for 240 seconds, and then the SA sensor was immersed in a mixture of the first and second antibodies (final concentrations of both antibodies were 100 nM). The difference in signal after the sensor was immersed in the antibody mixture for 240 seconds was recorded as the signal of the second antibody. The inhibition rate was calculated using the following formula: Suppression rate (%)=(AB) / A*100 A: 100% signal of one antibody (obtained from the first step), B: this antibody as the signal of the second antibody (obtained from the second step).

[0124] If the obtained inhibition rate exceeds 85%, it means that the epitopes of the two types of antibodies completely overlap, and if the inhibition rate is less than 85%, it means that the epitopes bound by the two types of antibodies do not completely overlap.

[0125] The results in Table 8-1 show that PR002418 and PR002421 bind to different epitopes of B7-H4, both of which are different from the epitopes of Control Antibody 1 and Control Antibody 2. Here, PR002418 binds to one unique epitope (first epitope), PR002421 binds to a different epitope (second epitope), and Control Antibody 1 and Control Antibody 2 bind to the same epitope (third epitope).

[0126] [Table 5]

[0127] Example 9. Cross-reactivity with other members of the B7 family B7 family proteins (see Table 9-1 for details) were diluted to 1 μg / mL in PBS and added to a 96-well plate (Corning, #9018), 100 μl per well, and incubated overnight at 4°C. After discarding the liquid, the plate was washed three times with PBST buffer (pH 7.4, containing 0.05% Tween-20). 250 μl of 2% BSA blocking solution was added and incubated at 37°C for 1 hour. The blocking solution was discarded, and the plate was washed three times with PBST buffer (pH 7.4, containing 0.05% Tween-20). The target antigen-binding protein was diluted to two concentrations, 10 nM and 1 nM, and 100 μl of each was added to each well. The plate was incubated at 37°C for 1 hour. The same antibody was used as a control. After washing three times with PBST buffer (pH 7.4, containing 0.05% Tween-20), a 5000-fold diluted goat anti-human F(ab') gallium HRP secondary antibody (Jackson ImmunoResearch, 109-035-097) was added and incubated at 37°C for 1 hour, protected from light. After washing three times with PBST buffer (pH 7.4, containing 0.05% Tween-20), 100 μl / well of TMB (Biopanda, #TMB-S-003) was added and the plate was incubated at room temperature for approximately 30 minutes, protected from light. The reaction was stopped by adding 50 μl / well of stop solution (BBI Life Sciences, #E661006-0200) to each well, and the optical density at 450 nm (OD450) was measured using a microplate reader (PerkinElmer, #Enspire). Figure 22 demonstrates that the antibodies of the present invention do not cross-react with other B7 family members.

[0128] [Table 6]

[0129] Example 10. Serum stability analysis Thirty microliters of antibody was diluted in 270 μl of normal human serum (serum concentration 90%), and five aliquots of antibody were incubated at 37°C for 0, 1, 4, 7, and 14 days, respectively, removed, snap-frozen in liquid nitrogen, and then stored at -80°C. A flow method was used to detect antibody binding to B7H4 on SK-BR-3 cells.

[0130] SK-BR-3 or CHOK1 / h B7H4 cells were digested and resuspended in PBS containing 2% BSA at a cell density of 1 × 10 6 The antibody was adjusted to 100 cells / mL. After seeding 100 μL cells / well into a 96-well V-bottom plate (Corning, #3894), 100 μL / well of the antibody to be measured, diluted 3-fold at twice the final concentration, was added. The cells were placed at 4°C and incubated for 2 hours, protected from light. Then, 100 μL / well of pre-chilled 2% BSA in PBS was added and the cells were washed twice. The cells were centrifuged at 500 x g for 5 minutes at 4°C and the supernatant was discarded. The cells were then incubated with 100 μL / well of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, 1:500 dilution) for 60 minutes at 4°C, protected from light. The cells were washed twice with 100 μL / well of pre-chilled 2% BSA in PBS, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-chilled 2% BSA in PBS, and the fluorescence signal was read using an ACEA NovoCyte 3000 flow cytometer. The results in Figure 23 demonstrate that the anti-B7H4 antibodies PR002418, PR002037, PR003369, and control antibodies 1 and 2 have good stability in human serum at 37°C for 14 days.

[0131] Example 11. Pharmacokinetics in C57BL / 6 mice Six female Nu / Nu mice weighing 18-22 grams were selected and administered the drug at a dose of 20 mg / kg via tail vein injection. Whole blood samples were collected from one group of three mice before administration, 15 minutes, 24 hours (day 1), days 4, and 10 after administration, and from another group of three mice before administration, 5 hours, days 2, 7, and 14 after administration. The whole blood was allowed to clot for 30 minutes, then centrifuged at 2000 rpm for 5 minutes at 4°C. The separated serum samples were frozen and stored at -80°C until analysis. In this example, ELISA was used to quantitatively measure drug concentrations in mouse serum. The ELISA Fc-terminal full detection method involved capturing human Fc-containing fusion proteins in mouse serum with a goat anti-human Fc polyclonal antibody coated on a 96-well plate, followed by detection with an HRP-conjugated goat anti-human Fc secondary antibody. Phoenix WinNonlin software version 8.2 was used to select a non-compartmental model (NCA) to analyze the blood concentration data and evaluate their pharmacokinetics.

[0132] Table 11-1 shows the pharmacokinetic parameters of PR002418, PR002421, and control antibody 2 (PR002962). The results in Figure 24 show that, using the entire Fc tail detection method and calculated from the data over the previous 14 days, the half-life in mice of PR002418 was approximately 6.56 days, the half-life of PR002421 was approximately 6.64 days, and the half-life of control antibody 2 was approximately 5.90 days. These results indicate that the half-lives of PR002418 and PR002421 in mice are slightly longer than those of control antibody 2.

[0133] [Table 7]

[0134] Example 12. Antitumor efficacy of anti-B7H4 antibodies 12.1. BALB / c nude mouse MDA-MB-468 tumor model On the day of cell inoculation, 1 × 10 cells were added to each BALB / c nude mouse. 7MDA-MB-468 tumor cells were inoculated subcutaneously. The cells were resuspended in a 1:1 mixture of PBS and Matrigel (0.1 mL / mouse) and inoculated subcutaneously. The average tumor volume in mice was 135 mm 3 The 25 mice were divided into 5 groups, and the administration cycle was twice a week, for a total of 12 doses, administered intraperitoneally. After the start of administration, body weight and tumor volume were measured twice a week, and tumor volume was calculated as tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experiment was terminated on day 39 after administration, and all mice were euthanized.

[0135] The in vivo antitumor effect of BALB / c nude mouse MDA-MB-468 tumor model is shown in Figure 25. Specifically, the average tumor volume of the vehicle control group mice on day 39 after administration was 1054 mm. 3 The mean tumor volume in the PR002418 (5 mg / kg) treatment group on day 39 after administration was 606 mm 3 The mean tumor volume in the PR002418 (15 mg / kg) treatment group on day 39 was 532 mm , which was significantly different from the vehicle control group (p value 0.015). 3 The mean tumor volume in the PR002421 (5 mg / kg) treatment group on day 39 was 665 mm. 3 There was no significant difference compared to the vehicle control group (p value 0.07), and the tumor inhibition rate TGI (%) was 36.86%. The mean tumor volume on day 39 after administration of the test drug PR002421 (15 mg / kg) treatment group was 335 mm 3 There was a significant difference (p value 0.018) compared to the vehicle control group, and the tumor inhibition rate TGI (%) was 68.23%.

[0136] 12.2. MDA-MB-468 tumor model reconstituting the human PBMC immune system in NSG mice On the day of cell inoculation, each NCG mouse was given 5 × 10 6MDA-MB-468 tumor cells were inoculated subcutaneously. The cells were resuspended in a 1:1 mixture of PBS and Matrigel (0.1 mL / mouse) and inoculated subcutaneously. The average tumor volume in mice was 126 mm 3 Once grouped, the 30 mice were divided into 5 groups, each containing 5 × 10 6 Human PBMCs were inoculated intravenously, and the cells were resuspended in 200 μl of PBS. Administration began the following day, with the administration cycle being twice a week, for a total of eight doses, administered intraperitoneally. After the start of administration, body weight and tumor volume were measured twice a week, and tumor volume was calculated as tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Experimental observation was terminated on the 36th day after administration, after which all mice were euthanized.

[0137] The in vivo antitumor effect of the MDA-MB-468 tumor model in which the human PBMC immune system was reconstituted in NSG mice was shown in Figure 26. Specifically, the mean tumor volume of the vehicle control group mice on day 36 after administration was 942 mm. 3 The mean tumor volume on day 36 after administration of the test drug PR002418 (15 mg / kg) was 585 mm 3 There was no significant difference compared to the vehicle control group (p value 0.073), and the tumor inhibition rate (TGI) (%) was 37.91%. The mean tumor volume on day 36 after administration of the test drug PR002421 (15 mg / kg) treatment group was 670 mm 3 There was no significant difference compared to the vehicle control group (p value 0.200), and the tumor inhibition rate TGI (%) was 28.87%. The mean tumor volume on day 36 after administration of the test drug PR003369 (15 mg / kg) treatment group was 354 mm 3 The mean tumor volume in the test drug control antibody 2 (15 mg / kg) treatment group on day 36 after administration was 533 mm 3 There was a significant difference (p value 0.028) compared to the vehicle control group, and the tumor inhibition rate TGI (%) was 43.41%.

[0138] Example 13. Immunohistochemical staining (IHC) Pathological tissue chips were purchased from Guilin Fanpu Biotech, Inc., including BRC1021 breast cancer tissue chip, EMC1021 endometrial cancer tissue chip, OVC1021 ovarian cancer tissue chip, and MNO1021 normal tissue chip. Paraffin sections were 4 μm thick and included positive control tissues. Dewaxing and washing with water; antigen retrieval: pH 6 (citric acid), heated at 125°C for 5 minutes, left for 10 minutes, then cooled at room temperature for 30 minutes; after washing with water, washed with 0.3% hydrogen peroxide for 5 minutes, then washed three times with TBST for 5 minutes, and blocked in an incubation box at room temperature for 20 minutes using Dako blocking solution directly; after shaking off the blocking solution, primary antibody was applied, and antibody diluent was directly used from Dako, and incubated in an incubation box at room temperature for 60 minutes, and the control was replaced with Rabbit IgG; washed three times with TBST, 5 minutes each time; secondary antibody, Anti-Rabbit (EnVision+System-HRPLabelled Polymer), incubated in an incubation box at room temperature for 30 minutes, and washed three times with TBST, 5 minutes each time; DAB color development, distilled water 0.85 50 μL of each reagent was added in the order of 1 mL, 2 mL, 3 mL, 4 mL, 50 μL of each reagent, and the mixture was incubated at room temperature in an incubation box for 5 minutes. The mixture was then washed with distilled water and restained with hematoxylin. The mixture was then observed under a microscope, sealed, and read.

[0139] The results in Figure 27 show that B7H4 was weakly expressed in normal tissues, including the adrenal gland, renal cortex, bladder, mammary gland, fallopian tube, esophagus, ureter, and endometrium, but not in other tissues (Figure 27, A). Conversely, it was highly expressed in breast cancer, ovarian cancer, and endometrial cancer (Figure 27, B). For example, in ovarian cancer, 67.65% of the 102 samples had an IHC score of 2-4, while in endometrial cancer, 62.24% of the 98 samples had an IHC score of 2-4 (Figure 27, C).

[0140] Example 14. Structure and design of B7H4xCD3 bispecific antibodies The selected anti-B7H4 and anti-CD3 antibodies are used to prepare a bispecific antibody. This B7H4xCD3 bispecific antibody can simultaneously bind to two targets: one end recognizes B7H4 specifically expressed on the surface of tumor cells, and the other end binds to the CD3 molecule on T cells. After binding to the surface of tumor cells, the B7H4xCD3 bispecific antibody molecule can recruit and activate T cells near the tumor cells and kill them.

[0141] In Figure 28, A and B are "1 + 1" Fab-FC-scFv asymmetric structure molecules. C and D are B7H4 x CD3 bispecific antibody molecules with "1 + 1" Fab-FC-crossFab asymmetric structure molecules. For the "1 + 1" asymmetric structure molecules, structures (1) and (2) relate to three protein chains, including the heavy and light chains of the corresponding anti-B7H4 antibody and the scFv polypeptide chain of the anti-CD3 antibody, respectively (see Figure 28, A and B). For the "1 + 1" asymmetric structure molecules, structures (3) and (4) relate to four protein chains, including the heavy and light chains of the corresponding anti-B7H4 antibody and the heavy and light chains of the anti-CD3 antibody, respectively (see Figure 28, C and D).

[0142] E and F, G and H, I and J are B7H4xCD3 bispecific antibody molecules with a "2+1" asymmetric structure. For the "2+1" asymmetric structure molecules, structures (5) and (6) relate to four protein chains, including the heavy and light chains of the corresponding anti-B7H4 antibody and the heavy and light chains of the anti-CD3 antibody, respectively (see Figure 28, E and F). For the "2+1" asymmetric structure molecules, structures (7), (8), and (9), (10) relate to three protein chains, including the heavy and light chains of the corresponding anti-B7H4 antibody and the polypeptide chains of the scFvs of the anti-B7H4 and CD3 antibodies, respectively (see Figure 28, G, H, I and J).

[0143] To minimize by-product formation from mismatched heavy chains (e.g., two mismatched heavy chains of an anti-CD3 antibody), a heterodimeric Fc region with a "knob-hole" mutation and modified disulfide bonds was used, as described in WO2009080251 and WO2009080252. The B7H4xCD3 bispecific antibody has an IgG1 Fc and carries L234A, L235A, and P329G (according to EU index numbering) mutations in the CH3 of the Fc. Each bispecific antibody was produced by simultaneously cotransfecting three or four different mammalian expression vectors, each encoding: 1) the heavy chain of the corresponding anti-B7H4 antibody carrying "hole" mutations in the Fc region and L234A, L235A, and P329G mutations in the CH3 of the Fc to generate a heterodimeric antibody; 2) The heavy chain of the corresponding anti-CD3 antibody, which carries a "knob" mutation in the Fc region to produce a heterodimeric antibody, and the CH3 of the Fc region carries L234A, L235A, and P329G mutations. 3) The light chain of the corresponding anti-CD3 antibody. 4) The light chain of the corresponding anti-B7H4 antibody. The "knob" mutation in the human IgG1 Fc region consists of T366W, and the "hole" mutations consist of T366S, L368A, and Y407V. Furthermore, the "knob" Fc region contains S354C and the "hole" Y349C, which can form a pair of disulfide bonds to increase stability and heterodimeric antibody yield.

[0144] Tables 14-1, 14-2, and 14-3 show the B7H4xCD3 bispecific antibody molecules constructed according to this embodiment, and the structure numbers in the tables correspond to Figure 28. Table 14-4 shows the sequences of the connecting peptides. Table 14-5 shows the SEQ ID NOs corresponding to the CD3 monoclonal antibody molecules. The B7H4 monoclonal antibody molecules are derived from Tables 1 to 7. Table 14-6 shows the SEQ ID NOs corresponding to the B7H4xCD3 bispecific antibody molecules. Table 14-7 shows the SEQ ID NOs of the CDR sequences corresponding to the first and second antigen-binding domains of the bispecific antibody molecules.

[0145] [Table 8-1]

[0146] [Table 8-2]

[0147] [Table 8-3]

[0148] [Table 8-4]

[0149] [Table 8-5]

[0150] [Table 8-6]

[0151] [Table 8-7]

[0152] [Table 8-8]

[0153] Example 15. FACS detection of the ability of B7H4xCD3 bispecific antibodies to bind to B7H4 SK-BR-3 cells were digested. T cells were isolated using a human T cell isolation reagent kit (Miltenyi, #130-096-535) according to the manufacturer's instructions. They were resuspended in PBS containing 2% BSA. The cell density was adjusted to 1 x 10 6The antibody was adjusted to 100 cells / mL. After seeding 100 μL cells / well into a 96-well V-bottom plate (Corning, #3894), 100 μL / well of the antibody to be measured, diluted 3-fold at twice the final concentration, was added. The cells were placed at 4°C and incubated for 2 hours, protected from light. The cells were then washed twice with 100 μL / well of pre-chilled 2% BSA in PBS, centrifuged at 500 x g for 5 minutes at 4°C, and the supernatant was discarded. The cells were then incubated with 100 μL / well of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-098, 1:500 dilution) for 1 hour at 4°C, protected from light. The cells were washed twice with 100 μL / well of pre-chilled 2% BSA in PBS, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-chilled 2% BSA in PBS, and the fluorescence signal was read using an ACEA Novocyte3000 flow cytometer.

[0154] Figure 29A shows the binding activity of bispecific antibody molecules with a "1+1" asymmetric structure (B7H4×CD3) to SK-BR-3 cells. The results demonstrate that the bispecific antibody molecules have relatively good binding activity to B7H4 on the surface of SK-BR-3 tumor cells. Figure 29A shows the binding activity of bispecific antibody molecules with an anti-B7H4 end in a Fab structure and an anti-CD3 end in an ScFv polypeptide chain. Among these, bispecific antibody molecules PR002849, PR002850, and PR002851, whose anti-B7H4 end is derived from the ScFv polypeptide chain of PR001476 and its PTM mutant antibody, have stronger binding activity than bispecific antibody molecule PR002852, whose anti-B7H4 end is derived from the ScFv polypeptide chain of PR002037 PTM mutant antibody. Figure 29B shows the binding activity of bispecific antibody molecules with an anti-B7H4 end in a Fab structure and an anti-CD3 end in a cross-Fab structure. Among these, bispecific antibody molecules PR002855, PR002856, and PR002857, whose anti-B7H4 end is derived from PR001476 and its PTM mutant antibody, have stronger binding activity than bispecific antibody molecule PR002858, whose anti-B7H4 end is derived from PR002037 PTM mutant antibody. Figure 29C shows the binding activity of bispecific antibody molecules with an anti-B7H4 end in an ScFv polypeptide chain and an anti-CD3 end in a Fab structure. Because PR003733 and PR003899 share the same ScFv polypeptide chain at the anti-B7H4 end, their binding activity is identical. The binding results of the "2 + 1" asymmetric B7H4 x CD3 bispecific antibody molecule to SK-BR-3 cells are shown in Figure 29D. The results showed that the bivalent anti-B7H4 end also exhibited high binding activity. The results of binding of the B7H4xCD3 bispecific antibody molecule with a "1 + 1" asymmetric structure to human T cells are shown in Figure 29, E and G. The results of binding of the B7H4xCD3 bispecific antibody molecule with a "2 + 1" asymmetric structure to human T cells are shown in Figure 29, H. The results revealed that both the anti-CD3 end in the Fab structure or the ScFv polypeptide chain were able to bind to the T cell surface, and that the bispecific antibody molecule PR003899 with a weak anti-CD3 end showed no binding at the FACS level.

[0155] Example 16. T cell killing experiments In this experiment, human primary T cells were used as effector cells, and the target cells were the B7H4-highly expressing cell lines SK-BR-3 or MDA-MB-468, the B7H4-expressing cell line HCC-1954, or the B7H4-negative cell line MDA-MB-231. The electrical conductivity of the target cells was measured using an ACEA RTCA instrument to reflect the killing efficiency. A 96-well e-plate was first equilibrated with 50 μl of complete medium. The target cells were digested and resuspended in RPM1640 complete medium containing 10% fetal bovine serum, and 4 × 10 5 / mL and place 50 μl / well on an e-plate 96-well plate, i.e., 2 × 10 4 The cells were plated at 2 × 10 cells / well and cultured overnight at 37°C. The next day, primary T cells were isolated using the Miltenyi T cell isolation reagent kit (Miltenyi, #130-096-535) according to the manufacturer's instructions. 5 50 μl of fresh culture medium containing T cells was added to each well, followed by 50 μl of 4x gradient diluted antibodies, with the highest final antibody concentration at 10 nM. Each antibody was tested at eight concentrations, with two replicates. The target cell conductivity was measured in real time, and the target cell killing efficiency was typically calculated using the 24-hour data as follows: target cell killing efficiency = (1 - sample / blank control) × 100%. The supernatant was collected after 24 hours, and IFN-γ levels were detected using an ELISA. For ELISA detection, refer to the instructions for the IFN gamma Human Uncoated ELISA Kit (Thermo, #88-7316-77).

[0156] However, the B7H4×CD3 bispecific antibody molecule with a "1+1" asymmetric structure activated T cells and killed the target cell SK-BR-3, as shown in Figure 30, A and B. Figure 30, A and B, show the killing activity and cytokine IFN-γ production of bispecific antibody molecules in which the anti-B7H4 end is an Fab structure and the anti-CD3 end is an ScFv polypeptide chain. Among these, the bispecific antibody molecule PR002852, in which the anti-B7H4 end is derived from the ScFv polypeptide chain of the PR002037PTM mutant antibody, has the strongest killing activity and cytokine IFN-γ production. Figure 30C and D show the killing activity and cytokine IFN-γ production of bispecific antibody molecules with an anti-B7H4 end in a Fab structure and an anti-CD3 end in a Cross Fab structure. Among them, the bispecific antibody molecule PR002852, whose anti-B7H4 end is derived from the PR002037PTM mutant antibody, has the strongest killing activity and cytokine IFN-γ production. Figure 30E and F show the killing activity and cytokine IFN-γ production of bispecific antibody molecules with an anti-B7H4 end in an ScFv polypeptide chain and an anti-CD3 end in a Fab structure. The bispecific antibody molecule PR003733, which has a strong CD3 end, had higher killing activity and cytokine IFN-γ production than the bispecific antibody molecule PR003899, which has a weak CD3 end. Figure 30 (GL) shows the results of comparing the killing activity and cytokine secretion of PR003733 and PR003899 on other tumor cell lines, MDA-MB-468, HCC-1954, and MDA-MB-231. Results showed that PR003733 outperformed PR003899 in killing MDA-MB-468 and HCC-1954 cells in vitro and in producing the cytokine IFN-γ. MDA-MB-231 cells were used as a negative control, but neither antibody had any effect on them. The "2+1" asymmetric B7H4×CD3 bispecific antibody molecule activated T cells and killed the target cell line SK-BR-3. See Figure 30 (MN).Both bispecific antibody molecules with a "2+1" asymmetric structure can kill SK-BR-3 cells and produce the cytokine IFN-γ, and the bispecific antibody molecules PR003001 and PR003008, in which the anti-CD3 terminus is a cross-Fab structure, have stronger killing activity and IFN-γ production than the bispecific antibody molecules PR002987 and PR002994, in which the anti-CD3 terminus is an ScFv polypeptide chain.

[0157] Example 17. Tumor model reconstituting the human PBMC immune system in NSG mice On the day of cell inoculation, each NCG mouse was given 5 × 10 6 MDA-MB-468 tumor cells were inoculated subcutaneously. The cells were resuspended in a 1:1 mixture of PBS and Matrigel (0.1 mL / mouse) and inoculated subcutaneously. The average tumor volume in mice was 126 mm 3 Once grouped, the 18 mice were divided into three groups, each containing 5 × 10 6 Human PBMCs were inoculated intravenously, and the cells were resuspended in 200 μl of PBS. Administration began the following day, with the administration cycle being once a week, for a total of three doses. After the start of administration, body weight and tumor volume were measured twice a week, and tumor volume was calculated as tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experimental observation was terminated on the 36th day after administration, after which all mice were euthanized.

[0158] The mean tumor volume of vehicle control mice at day 36 after treatment was 942 mm 3 The mean tumor volume in the PR003733 (2 mg / kg) treatment group on day 36 after administration was 590 mm 3 There was a significant difference (p value 0.048) compared with the vehicle control group, and the tumor inhibition rate TGI (%) was 37.31%. In the test drug PR003899 (2 mg / kg) treatment group, the mean tumor volume was 0 mm 36 days after administration. 3 The tumor completely disappeared, which was significantly different from the vehicle control group (p value 0.0001), and the tumor inhibition rate TGI (%) was 100% (see Figure 31, A).

[0159] For the HCC-1954 model, on the day of cell inoculation, each NCG mouse received 5 × 10 6 HCC-1954 tumor cells were resuspended in a 1:1 mixture of PBS and Matrigel (0.1 mL / mouse) and inoculated subcutaneously. The average tumor volume in mice was 102 mm 3 Once grouped, the 15 mice were divided into three groups, each containing 3 × 10 6 Human PBMCs were inoculated intravenously, and the cells were resuspended in 200 μl of PBS. Administration began the following day, with the administration cycle being once a week, for a total of two intravenous administrations. After the start of administration, body weight and tumor volume were measured twice a week, and tumor volume was calculated as tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 Experimental observations were terminated on the 16th day after administration, after which all mice were euthanized.

[0160] The mean tumor volume of vehicle control mice at day 16 after treatment was 622 mm 3 The mean tumor volume on day 16 after administration of the test drug PR003733 (0.5 mg / kg) treatment group was 450 mm 3 There was no significant difference (p value 0.1) compared to the vehicle control group, and the tumor inhibition rate (TGI) (%) was 27.64%. The mean tumor volume on day 16 after administration of the test drug PR003899 (0.5 mg / kg) treatment group was 322 mm 3 There was a significant difference (p value 0.0028) compared with the vehicle control group, and the tumor inhibition rate TGI (%) was 48.27% (see Figure 31, B).

[0161] Efficacy studies in two tumor models revealed that PR003899 was more effective than PR003733.

Claims

1. An antibody targeting B7H4, The antibody comprises a light chain variable region and a heavy chain variable region, (1) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 47, 54, and 64, respectively; and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 9, 21, and 35, respectively; or (2) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 48, 54, and 65, respectively; and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 11, 22, and 36, respectively; or (3) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 47, 54, and 67, respectively; and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 9, 23, and 35, respectively; or (4) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 47, 54, and 67, respectively; and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 9, 24, and 35, respectively; or (5) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 47, 54, and 69, respectively; and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 9, 24, and 38, respectively; antibody.

2. the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 88; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 93; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83; or The antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 91; and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

79.

3. The antibody of claim 1 or 2, wherein the antibody further comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region of the antibody is selected from hIgG1, hIgG2, hIgG3, or hIgG4, and the light chain constant region is selected from a κ chain or a λ chain.

4. The antibody of claim 3, wherein the heavy chain constant region of the antibody is in the form of hIgG1, and the antibody has amino acid substitutions of S239D and / or I332E in the Fc of the antibody.

5. The antibody may be a full-length antibody, Fab, Fab', F(ab') 2 5. The antibody of any one of claims 1-4, which is an Fv, Fv, or scFv.

6. (1) a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 95; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 98; the light chain comprises the amino acid sequence set forth in SEQ ID NO: 112; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 99; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 114; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 100; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 114; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 101; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 102; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 114; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 106; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 117; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 98; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 115; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 103; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 112; or the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 103; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 115; or (2) The antibody of claim 1 or 2, comprising a polypeptide chain comprising an scFv and an Fc region, wherein the polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:

132.

7. A bispecific antibody targeting B7H4, comprising a protein A functional domain and a protein B functional domain, wherein the protein A functional domain is an antibody targeting B7H4 described in any one of claims 1 to 6; and the protein B functional domain is an antibody that does not target B7H4.

8. The bispecific antibody targeting B7H4 of claim 7, wherein the antibody that does not target B7H4 is an antibody that targets CD3.

9. 9. The bispecific antibody targeting B7H4 of claim 8, wherein the antibody targeting CD3 comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 46, 53, and 63, respectively; the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 8, 20, and 34, respectively; or the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 46, 53, and 63, respectively; and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 10, 20, and 34, respectively.

10. 10. The B7H4-targeting bispecific antibody of claim 8 or 9, wherein the CD3-targeting antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; or wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 86; the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO: 78; or wherein the light chain variable region VL comprises the amino acid sequence set forth in SEQ ID NO: 86; and the heavy chain variable region VH comprises the amino acid sequence set forth in SEQ ID NO:

84.

11. wherein the protein B functional region comprises a light chain variable region and a heavy chain variable region, and the protein A functional region comprises a light chain variable region and a heavy chain variable region; In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 64, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 21 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 23 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 24 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 48, 54 and 65, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 11, 22 and 36, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 64, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 21 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 23 and 35, respectively; or In the B protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 46, 53 and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 10, 20 and 34, respectively; in the A protein functional region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 47, 54 and 67, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 9, 24 and 35, respectively; or 11. The bispecific antibody according to claim 7 , wherein in the protein B functional region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 46, 53, and 63, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 10, 20, and 34, respectively; and in the protein A functional region, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 48, 54, and 65, respectively, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are set forth in SEQ ID NOs: 11, 22, and 36, respectively.

12. wherein the protein B functional region comprises a light chain variable region and a heavy chain variable region, and the protein A functional region comprises a light chain variable region and a heavy chain variable region; wherein the protein B functional region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; wherein the protein A functional region comprises the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77; or In the protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; in the protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80; or In said protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; in said protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or In the protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76; in the protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79; or wherein the protein B functional region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78; wherein the protein A functional region comprises the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 87 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 77; or In the protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78; in the protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80; or In the protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78; in the protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 90 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 81; or In said protein B functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 78; in said protein A functional domain, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 79; or 12. The bispecific antibody of claim 11 , wherein, in said protein B functional domain, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86 and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 84; and in said protein A functional domain, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 91 and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

79.

13. (1) the bispecific antibody comprises three polypeptide chains, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; and the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 119; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 114; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 120; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 119; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 114; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 121; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 119; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 115; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 122; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 119; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 126; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 118; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 109; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 127; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 118; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 109; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 110; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 128; and a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 129; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 110; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 129; (2) the bispecific antibody comprises four polypeptide chains, wherein the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 123; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 97; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 123; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 120; and a fourth polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 114; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 97; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 123; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 121; a fourth polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 114; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 97; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 123; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 122; a fourth polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 115; or a first polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 97; a second polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 124; a third polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 118; and a fourth polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO: 109; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 97; the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 109; 13. The bispecific antibody of claim 12, selected from the set:

14. A chimeric antigen receptor comprising the antibody according to any one of claims 1 to 6 or the bispecific antibody according to any one of claims 7 to 13.

15. 1. A genetically modified cell comprising: A cell comprising the chimeric antigen receptor of claim 14.

16. 16. The genetically modified cell of claim 15, which is a T cell or an NK cell.

17. 15. An isolated nucleic acid encoding the antibody according to any one of claims 1 to 6, the bispecific antibody according to any one of claims 7 to 13, or the chimeric antigen receptor according to claim 14.

18. 18. An expression vector comprising the isolated nucleic acid of claim 17.

19. A transformant, A transformant comprising the expression vector according to claim 18; the transformant is obtained by transforming the recombinant expression vector into a host cell.

20. 1. A method for producing an antibody or bispecific antibody that targets B7H4, comprising: A method for producing the antibody or bispecific antibody that targets B7H4, comprising the steps of culturing the transformant described in claim 19 and obtaining the antibody or bispecific antibody that targets B7H4 from the culture.

21. 14. An antibody-drug conjugate comprising an antibody portion and a conjugate portion, wherein the antibody portion comprises the antibody of any one of claims 1 to 6 or the bispecific antibody of any one of claims 7 to 13, and wherein the conjugate portion comprises, but is not limited to, a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, and wherein the antibody portion and the conjugate portion are conjugated by a chemical bond or a linker.

22. 14. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 6 or the bispecific antibody according to any one of claims 7 to 13, and optionally a pharmaceutically acceptable vector.

23. Use of the antibody according to any one of claims 1 to 6, the bispecific antibody according to any one of claims 7 to 13, the chimeric antigen receptor according to claim 14, the genetically modified cell according to claim 15 or 16, the antibody-drug conjugate according to claim 21, or the drug composition according to claim 22 in the manufacture of a drug, a reagent kit, and / or an administration device for treating and / or preventing cancer.

24. 24. The use of claim 23, wherein the cancer is a tumor with positive B7H4 expression.

25. 25. The use of claim 24, wherein the cancer is selected from breast cancer, ovarian cancer and endometrial cancer.

26. 26. The use of claim 25, wherein the breast cancer is triple-negative breast cancer.

27. 1. A method for detecting B7H4 in a sample, comprising: A method comprising performing detection using the antibody according to any one of claims 1 to 6 or the bispecific antibody according to any one of claims 7 to 13, wherein the detection method is not for diagnostic purposes.

Images