B7-h7 antigen binding molecule and use thereof

Abstract

The present disclosure relates to a B7-H7 antigen binding molecule and the use thereof. Specifically, the present disclosure relates to an antibody specifically binding to B7-H7 or an antigen-binding fragment thereof. The B7-H7 antibody or the antigen-binding fragment thereof of the present disclosure can block the binding of B7-H7 to some receptors, and regulate the activity of immune cells, especially the activity of T cells, thereby enhancing the tumor killing activity thereof. In particular, the B7-H7 antibody of the present disclosure can be used in combination with a PD-1 / PD-L1 axis inhibitor or can be used together with a PD-1 / PD-L1 antibody to construct a multispecific antibody, thereby further enhancing the tumor killing activity of immune cells.

Description

B7-H7 antigen binding molecules and their applications

[0001] The present disclosure claims priority to Chinese patent application No. 202311065627.0, filed on August 23, 2023, entitled “B7-H7 Antigen Binding Molecules and Applications Thereof,” the entire contents of which, including the appendices, are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the field of antibodies, and in particular to a B7-H7 antigen binding molecule and its application. Background Art

[0003] The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

[0004] Human B7-H7 (also known as endogenous retroviral H long terminal repeat-associated protein 2, HHLA2) is a newly discovered member of the B7 family. B7-H7 is aberrantly expressed in solid tumors and exerts either co-stimulatory or co-inhibitory activities that depend on interactions with counter-receptors. B7-H7 represents a co-stimulatory role through interaction with transmembrane and immunoglobulin domain containing 2 (TMIGD2, also known as CD28H), but its interaction with killer cell Ig-like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3) produces a co-inhibitory effect. TMIGD2 is primarily expressed on resting or naive T cells, whereas KIR3DL3 expression occurs on activated T cells. B7-H7 / KIR3DL3 attenuates innate and adaptive anti-tumor immune responses, and activity within this axis is considered a biomarker for poor prognosis in cancer patients. B7-H7 / KIR3DL3 promotes CD8 + T cell exhaustion and macrophage polarity toward a pro-tumor M2 phenotype are induced by B7-H7. B7-H7 represents distinct expression profiles and activities in tumors and the stroma. Tumor expression of B7-H7 may be higher than that of programmed death ligand 1 (PD-L1), and co-expression of B7-H7 and PD-L1 is associated with more severe outcomes. A recommended strategy for patients with B7-H7-high cancers is to use monoclonal antibodies to specifically inhibit the B7-H7 inhibitory receptor KIR3DL3, rather than the B7-H7 ligand. TMIGD2 could serve as a target for the development of agonist bispecific antibodies to mitigate tumor resistance to programmed death-1 (PD-1) / PD-L1 blockade. B7-H7 is also highly expressed in certain tumors. Its expression level is significantly inversely correlated with clinical survival in certain tumor types, such as clear cell renal cell carcinoma, hepatocellular carcinoma, and EGFR-mutant lung adenocarcinoma, with higher B7-H7 expression associated with lower survival. The inventors recognized that B7-H7 may be a potential new target for immuno-oncology therapy.

[0005] Summary of the Invention

[0006] The present disclosure provides a new B7-H7 antibody that regulates the activity of immune cells (such as NK cells and cytotoxic T cells) to enhance their anti-tumor effects, thereby providing a new tumor treatment method.

[0007] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human B7-H7 (also known as endogenous retrovirus-H long terminal repeat-associated protein 2, HHLA2), a multispecific antigen-binding molecule, a nucleic acid fragment, a vector, a host cell, an immune effector cell, a preparation method, a pharmaceutical composition, pharmaceutical uses, and a method for treating tumors or cancer.

[0008] In a first aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to a human B7-H7 protein, wherein the human B7-H7 protein comprises amino acid residues 1 to 341 as shown in SEQ ID NO: 1 or any fragment thereof from amino acid residues 1 to 341 as shown in SEQ ID NO: 1.

[0009] In some specific embodiments, the antibody or antigen-binding fragment thereof binds to one or more amino acids within the amino acid residues of an antigenic fragment selected from:

[0010] (1) amino acid residues 61 to 131 of SEQ ID NO: 1, or amino acid residues on the human B7-H7 protein corresponding to amino acid residues 61 to 131 of SEQ ID NO: 1;

[0011] (2) amino acid residues 138-222 of SEQ ID NO: 1, or amino acid residues on the human B7-H7 protein corresponding to amino acid residues 138-222 of SEQ ID NO: 1; and

[0012] (3) amino acid residues 235-328 of SEQ ID NO: 1, or amino acid residues on human B7-H7 protein corresponding to amino acid residues 235-328 of SEQ ID NO: 1.

[0013] In some specific embodiments, the antibody or antigen-binding fragment thereof binds to one or more amino acids within the amino acid residues of an antigenic fragment selected from:

[0014] (1) amino acid residues 35-44, 45-49, 50-60, 61-67, 68-76, 77-85, 86-94, 95-101, 102-110, 111-116, 117-126, and 127-137 of SEQ ID NO: 1, or the amino acid residues of the human B7-H7 protein corresponding to amino acid residues 35-44, 45-49, 50-60, 61-67, 68-76, 77-85, 86-94, 95-101, 102-110, 111-116, 117-126, and 127-137 of SEQ ID NO: 1;

[0015] (2) amino acid residues 138-143, 144-153, 154-162, 163-167, 168-176, 177-184, 185-193, 194-202, 203-213, 214-221 of SEQ ID NO: 1, or the amino acid residues of the human B7-H7 protein corresponding to amino acid residues 138-143, 144-153, 154-162, 163-167, 168-176, 177-184, 185-193, 194-202, 203-213, 214-221 of SEQ ID NO: 1;

[0016] (3) amino acid residues 228-238, 239-251, 252-262, 263-268, 269-277, 278-287, 288-296, 297-314, 315-335 of SEQ ID NO: 1, or amino acid residues on the human B7-H7 protein corresponding to amino acid residues 228-238, 239-251, 252-262, 263-268, 269-277, 278-287, 288-296, 297-314, 315-335 of SEQ ID NO: 1.

[0017] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human B7-H7, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein:

[0018] (1) the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises LCDR1 of the VL domain set forth in any one of SEQ ID NOs: 22-30, 157, 159, 163, 166, 168, 170, 172, 174, 177, or 178, the LCDR2 comprises LCDR2 of the VL domain set forth in any one of SEQ ID NOs: 22-30, 157, 159, 163, 166, 168, 170, 172, 174, 177, or 178, and the LCDR3 comprises LCDR3 of the VL domain set forth in any one of SEQ ID NOs: 22-30, 157, 159, 163, 166, 168, 170, 172, 174, 177, or 178; and

[0019] (2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the HCDR1 of the VH domain represented by any one of SEQ ID NOs: 13 to 21, 158, 160, 164, 167, 169, 171, 173, 175, 179 or 180, the HCDR2 comprises the HCDR2 of the VH domain represented by any one of SEQ ID NOs: 13 to 21, 158, 160, 164, 167, 169, 171, 173, 175, 179 or 180, and the HCDR3 comprises the HCDR3 of the VH domain represented by any one of SEQ ID NOs: 13 to 21, 158, 160, 164, 167, 169, 171, 173, 175, 179 or 180.

[0020] In some specific embodiments, the LCDR1, LCDR2, and LCDR3 are identified according to the Kabat numbering system and / or the IMGT numbering system.

[0021] In some specific embodiments, the HCDR1, HCDR2, and HCDR3 are identified according to the Kabat numbering system and / or the IMGT numbering system.

[0022] In some specific embodiments, the antibody or antigen-binding fragment thereof comprises the following LCDR1, LCDR2, LCDR3 combination and HCDR1, HCDR2, HCDR3 combination:

[0023] (1) The LCDR1, LCDR2 and LCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination:

[0024] and,

[0025] (2) The HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination:

[0026] In particular, for example, the antibodies or antigen-binding fragments thereof of the present disclosure comprise a combination of light chain CDRs and heavy chain CDRs selected from the group consisting of L1+H1, L2+H2, L3+H3, L4+H4, L5+H5, L6+H6, L7+H7, L8+H8, L9+H9, L10+H10, L11+H11, L12+H12, L13+H13, L14+H14, L15+H15, L16+H16, L17+H17, L18+H18, L19+H19, L3+H20, or L20+H8, as well as a combination of CDRs having 1, 2, 3 or more amino acid insertions, deletions, and / or substitutions compared to the sequence of the light chain and heavy chain CDRs combination.

[0027] In a specific embodiment, the present disclosure provides an antibody or antigen-binding fragment thereof, wherein:

[0028] (1) The light chain variable region sequence comprises a sequence as set forth in SEQ ID NOs: 22-30, 157, 159, 163, 166, 168, 170, 172, 174, 177, or 178, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity to a sequence as set forth in SEQ ID NOs: 22-30, 157, 159, 163, 166, 168, 170, 172, 174, 177, or 178;

[0029] and, (2) a heavy chain variable region sequence comprising a sequence as set forth in SEQ ID NOs: 13-21, 158, 160, 164, 167, 169, 171, 173, 175, 179, 180, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity to a sequence as set forth in SEQ ID NOs: 13-21, 158, 160, 164, 167, 169, 171, 173, 175, 179, 180;

[0030] Preferably, the antibody or antigen-binding fragment thereof has the following combination:

[0031] (1) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 13, respectively;

[0032] (2) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 14, respectively;

[0033] (3) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 24 and SEQ ID NO: 15, respectively;

[0034] (4) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 25 and SEQ ID NO: 16, respectively;

[0035] (5) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 17, respectively;

[0036] (6) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 27 and SEQ ID NO: 18, respectively;

[0037] (7) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 28 and SEQ ID NO: 19, respectively;

[0038] (8) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 29 and SEQ ID NO: 20, respectively;

[0039] (9) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 30 and SEQ ID NO: 21, respectively;

[0040] (10) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 157 and SEQ ID NO: 158, respectively;

[0041] (11) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 159 and SEQ ID NO: 160, respectively;

[0042] (12) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 163 and SEQ ID NO: 164, respectively;

[0043] (13) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 166 and SEQ ID NO: 167, respectively;

[0044] (14) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, respectively;

[0045] (15) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171, respectively;

[0046] (16) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 172 and SEQ ID NO: 173, respectively;

[0047] (17) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 174 and SEQ ID NO: 175, respectively;

[0048] (18) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 177 and SEQ ID NO: 179, respectively;

[0049] (19) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 180, respectively;

[0050] or (20) having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the sequences shown in (1) to (19) above.

[0051] In a specific embodiment, the antibodies or antigen-binding fragments thereof of the present disclosure are chimeric, humanized, or fully human.

[0052] In a specific embodiment, the antibodies or antigen-binding fragments thereof of the present disclosure are capable of binding to human or monkey B7-H7.

[0053] In a specific embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises any constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably comprises a constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or a constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4 with a mutation; further, the antibody or antigen-binding fragment thereof is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug, a cytotoxic agent or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer; more preferably, the cytotoxic agent is selected from methotrexate, anthracycline antibiotics (doxorubicin), taxanes (taxanes), pyrrolobenzodiazepines, (pyrrolobenzodiazepine, PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane.

[0054] In a specific embodiment, the antigen-binding fragment of the present disclosure is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv, nanobody or affibody.

[0055] In a second aspect, the present disclosure further provides a multispecific antigen-binding molecule comprising the aforementioned antibody or antigen-binding fragment thereof and an antigen-binding molecule that binds to an antigen other than B7-H7, or comprising the aforementioned antibody or antigen-binding fragment thereof and an antigen-binding molecule that binds to a B7-H7 epitope different from the B7-H7 epitope bound by the aforementioned antibody or antigen-binding fragment thereof;

[0056] Preferably, the other antigen-binding molecule is an antibody or an antigen-binding fragment thereof;

[0057] Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific;

[0058] Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

[0059] In a third aspect, the present disclosure also provides a chimeric antigen receptor (CAR), which comprises at least a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises any of the aforementioned B7-H7 antibodies or antigen-binding fragments thereof, or the aforementioned multispecific antigen-binding molecules.

[0060] In a fourth aspect, the present disclosure further provides an immune effector cell, which expresses the aforementioned chimeric antigen receptor, or comprises a nucleic acid fragment encoding the aforementioned chimeric antigen receptor; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are preferably selected from cytotoxic T cells (CTLs), regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

[0061] In a fifth aspect, the present disclosure further provides an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule or chimeric antigen receptor.

[0062] In a sixth aspect, the present disclosure further provides a vector comprising the aforementioned nucleic acid fragment.

[0063] In a seventh aspect, the present disclosure further provides a host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

[0064] In an eighth aspect, the present disclosure also provides a method for preparing a product comprising the aforementioned antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule, the method comprising culturing the aforementioned cells, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cells.

[0065] In the ninth aspect, the present disclosure also provides a method for preparing the aforementioned immune effector cells, the method comprising introducing a nucleic acid fragment encoding the aforementioned CAR into the immune effector cells, and optionally, the method further comprising initiating the immune effector cells to express the aforementioned CAR.

[0066] In the tenth aspect, the present disclosure also provides a pharmaceutical composition, which comprises the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, vector or a product prepared according to the aforementioned method; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.

[0067] In some embodiments, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0068] In some embodiments, the anti-tumor agent comprises a PD-1 inhibitor or a PD-L1 inhibitor; in some embodiments, the PD-1 inhibitor comprises pembrolizumab, nivolumab, cemiplimab, camrelizumab, sintilimab, toripalimab, or tislelizumab; in some embodiments, the PD-L1 inhibitor comprises atezolizumab, avelumab, or durvalumab.

[0069] In the eleventh aspect, the present disclosure also provides a method for treating tumors or cancers, comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, vector, product prepared according to the aforementioned method, or pharmaceutical composition; in some embodiments, the tumor or cancer is selected from solid tumors, hematological tumors, or cancers infiltrated with immune cells expressing receptors expressing B7-H7; in some embodiments, the tumor or cancer is selected from melanoma, non-small cell lung cancer, and gastric cancer.

[0070] In some embodiments, the method for treating tumors or cancer further comprises use in combination with a PD-1 inhibitor or a PD-L1 inhibitor; in some embodiments, the PD-1 inhibitor comprises pembrolizumab, nivolumab, cemiplimab, carrelizumab, sintilimab, toripalimab, or tislelizumab; in some embodiments, the PD-L1 inhibitor comprises atezolizumab, avelumab, or durvalumab.

[0071] In the twelfth aspect, the present disclosure also provides the use of the aforementioned antibodies or antigen-binding fragments thereof, multispecific antigen-binding molecules, immune effector cells, nucleic acid fragments, vectors, products prepared according to the aforementioned methods, or pharmaceutical compositions in the preparation of drugs for treating tumors or cancers; in some embodiments, the tumor or cancer is selected from solid tumors, hematological tumors, or cancers infiltrating immune cells expressing receptors expressing B7-H7; in some embodiments, the tumor or cancer is selected from melanoma, non-small cell lung cancer, and gastric cancer.

[0072] In the thirteenth aspect, the present disclosure further provides a kit comprising the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, nucleic acid fragment, vector, product or pharmaceutical composition prepared according to the aforementioned method.

[0073] In a fourteenth aspect, the present disclosure further provides a method for detecting the expression of B7-H7 in a biological sample, the method comprising contacting the biological sample with the aforementioned antibody or antigen-binding fragment thereof under conditions where a complex can be formed between the antibody or antigen-binding fragment thereof and B7-H7; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of B7-H7 in the sample.

[0074] In a fifteenth aspect, the present disclosure further provides use of the aforementioned antibody or antigen-binding fragment thereof in preparing a B7-H7 detection reagent.

[0075] Definition of terms

[0076] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings that are understood by those of ordinary skill in the art.

[0077] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0078] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0079] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the subject term."

[0080] The term "B7-H7" herein, also referred to as human endogenous retrovirus-H long terminal repeat-associated protein 2, HERV-HLTR-associated protein 2, B7y, B7H7, B7-H5, and HHLA2, refers to members of the B7 family. In the present invention, B7-H7 and HHLA2 are synonymous, meaning that a B7-H7 antibody or antigen-binding fragment thereof is equivalent to an HHLA2 antibody or antigen-binding fragment thereof, and a human B7-H7 protein is equivalent to a human HHLA2 protein.

[0081] B7-H7 protein has limited expression in normal human tissues, but is widely expressed in human cancers. B7-H7 protein is a membrane protein with three Ig-like domains (IgV-IgC-IgV), while other members of the B7 family typically have only two Ig domains (IgV-IgC). B7-H7 protein in normal human tissues is expressed in epithelial cells of the kidney, intestine, gall bladder and breast, and in placental trophoblasts. In the immune system, B7-H7 protein is expressed on human mature DC cells, monocytes / macrophages. B7-H7 can regulate human T cell function, including, for example, B7-H7 can inhibit T cell proliferation and cytokine production by interacting with the inhibitory receptor KIR3DL3, or increase T cell production and cytokine production by interacting with the activating receptor TMIGD2. B7-H7 is widely expressed in human cancers, including breast cancer, lung cancer, thyroid cancer, melanoma, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer and esophageal cancer.

[0082] The term "B7-H7" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human B7-H7 cDNA and human B7-H7 protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human B7-H7 variants include variant 1 (NM_007072.3 and NP_009003.1, which represent the longest transcript and encode the longest isoform a), variant 2 (NM_001282556.1 and NP_001269485.1, which represent the use of an alternative promoter and differ in the 5'UTR compared to variant 1), variant 3 (NM_001282557.1 and NP_001269486.1, which represent the use of an alternative promoter and differ in the 5'UTR compared to variant 1), variant 4 (NM_001282558.1 and NP_001282559.1). 1 ) and variant 5 (NM_001282559.1 and NP_001269488.1, encoding isoform c, representing the use of an alternative promoter, differing from variant 1 in the 5'UTR and lacking an alternative in-frame exon in the 3' coding region, resulting in an isoform shorter than isoform a). Nucleic acid and polypeptide sequences of B7-H7 orthologs in organisms other than humans are well known and include, for example, Xenopus tropicalis B7-H7 (NM_001128644.1 and NP_001122116.1). However, B7-H7 is not expressed in mice and rats due to gene loss during evolution. (The foregoing is incorporated herein by reference).

[0083] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an antibody with a specific affinity of about 10 -6 M or lower, 10 -7 M or lower, about 10 -8 M or lower, about 10 -9 The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis (the contents of which are incorporated herein by reference).

[0084] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0085] The term "antibody" is used in the broadest sense herein and refers to a polypeptide or combination of polypeptides that comprises sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain, thereby being able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen-binding activity. The term "antibody" includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portion") or single chains thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed within more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Immunoglobulins differ in their antigenicity due to the amino acid composition and arrangement of their heavy chain constant regions. Accordingly, "immunoglobulins" herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to their heavy chains: μ, δ, γ, α, and ε, respectively. Igs of the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG is divided into IgG1, IgG2, IgG3, and IgG4, and IgA is divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0086] The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0087] The term "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0088] The term "heavy chain antibody" herein refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0089] The term "nanoantibody" in this article refers to heavy chain antibodies that are naturally missing light chains and exist in camels and other organisms. Cloning their variable regions can produce single-domain antibodies consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0090] The terms "VHH domain," "nanobody," and "single-domain antibody" (sdAb) are synonymous and used interchangeably herein. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy-chain variable region, obtained by cloning the variable region of a heavy-chain antibody. This single-domain antibody (sdAb) is the smallest fully functional antigen-binding fragment. Typically, a heavy-chain antibody naturally lacking the light chain and heavy-chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy-chain variable region.

[0091] For further description of “heavy chain antibodies” and “single domain antibodies”, “VHH domains” and “nanobodies”, reference is made to the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531; WO 01 / 90190; WO03 / 025020; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 and other prior art mentioned in these applications (the foregoing are incorporated herein by reference).

[0092] The term "multi-specificity" herein refers to the ability of an antibody or its antigen-binding fragment to bind to, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody can bind to. For example, conventional monospecific IgG antibodies have two identical antigen-binding sites (paratopes) and can therefore only bind to the same epitope (rather than binding to different epitopes). In contrast, multispecific antibodies have at least two different types of paratopes / binding sites and can therefore bind to at least two different epitopes. As described herein, "complementarity determining region" refers to the antigen-binding site of an antibody. In addition, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions (the actual number of complementary determining regions / binding sites in a single antibody molecule is referred to as "valence") in a single antibody. For example, a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes. Accordingly, a multispecific antibody comprises at least two (different) complementary determining regions / binding sites. Therefore, the term "multispecific antibody" refers to an antibody having more than one paratope and the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but generally also includes proteins, e.g. antibodies that specifically bind three or more different epitopes, scaffolds, i.e. antibodies with three or more paratopes / binding sites.

[0093] The term "valent" herein refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0094] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0095] The term "chimeric antibody" herein refers to an antibody having variable sequences of an immunoglobulin from one source organism (such as rat, mouse, rabbit or alpaca) and constant regions of an immunoglobulin from a different organism (such as human). Methods for producing chimeric antibodies are known in the art. See, for example, US Pat. No. 5,807,715A; Morrison, 1985, Science 229 (4719): 1202-1207. Transfectomas Provide Novel Chimeric Antibodies; Gillies et al., J Immunol Methods. 1989 Dec 20; 125 (1-2): 191-202; herein incorporated by reference.

[0096] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune response, etc.

[0097] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0098] The term "variable region" herein refers to the region of an antibody heavy or light chain involved in antigen binding. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0099] The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) found in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT), while being considered to be outside the hypervariable region under a different set of criteria (such as KABAT or IMGT). One or more of these positions can also be found in an extended hypervariable region. The present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region can be abbreviated as LCDRs. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3), which form a loop connecting the sheet structure and, in some cases, form a portion of the sheet structure. The CDRs in each chain are held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and contribute to the formation of the antibody antigen-binding site with the CDRs from other antibody chains.

[0100] "CDRs" herein may be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tools such as, but not limited to, the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDRs herein encompass overlaps and subsets of amino acid residues defined using different methods. (The foregoing is incorporated herein by reference.)

[0101] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, and has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" can be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. A "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to that of a native antibody constant region, while the latter only includes "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.

[0102] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0103] The term "Fc region" herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. For example, the human IgG heavy chain Fc region may extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage, removing one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells through expression of a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may be present or absent. Typically, an IgG Fc region comprises the IgG CH2 and IgG CH3 domains, and optionally may further comprise a complete or partial hinge region, but does not comprise a CH1 domain. The "CH2 domain" of a human IgG Fc region typically extends from approximately amino acid residue position 231 to approximately amino acid residue position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues in the Fc region at the C-terminus of the CH2 domain (i.e., from approximately amino acid residue position 341 to approximately amino acid residue position 447 of IgG). The CH3 domain herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" introduced in one chain and a corresponding "hole" introduced in the other chain; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). As described herein, such variant CH3 domains can be used to promote heterodimerization of two different antibody heavy chains.

[0104] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index.

[0105] The term "Fc variant" herein refers to changes in Fc structure or function caused by one or more amino acid substitutions, insertions, or deletions at appropriate sites on the Fc protein. "Inter-Fc variant interactions" refer to interactions between Fc variants engineered to form space-filling effects, electrostatic interactions, hydrogen bonding, hydrophobic interactions, and other interactions. These interactions contribute to the formation of stable heterodimeric proteins. Preferred mutational designs are "knob-into-hole" mutational designs.

[0106] The mutation design technology of Fc variants has been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "knob-into-hole" format proposed by Cater et al. (Protein Engineering vol. 9 no. 7 pp. 617-621, 1996); the use of electrostatic steering by Amgen technicians to form Fc-containing heterodimers (US20100286374 A1); the heterodimers (SEEDbodies) formed by IgG / Ig chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp. 1-8, 2010); the bispecific molecules formed by Genmab's DuoBody (Science, 2007. 317 (5844)) platform technology; and the heterodimers formed by Xencor technicians using a combination of structural calculations and Fc amino acid mutations to combine different modes of action (mAbs 3: 6, 546-557; November / December 2010). 2011); Suzhou Alphamab's charge network-based Fc modification method (CN201110459100.7) to produce a heterodimeric protein form; and other genetic engineering methods based on Fc amino acid changes or functional modification to achieve the formation of heterodimeric functional proteins. The knob / hole structure of the Fc variant fragments described herein refers to mutations in the two Fc fragments, which, after mutation, can combine in a "knob-into-hole" formation. Preferably, the "knob-into-hole" model of Cater et al. is used to perform site-specific mutations in the Fc region, so that the resulting first and second Fc variants can combine in a "knob-into-hole" formation to form a heterodimer. Selecting a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the skill of those skilled in the art. Preferred are Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4, with the Fc region of human antibody IgG1 being more preferred. Randomly select one of the first Fc variant or the second Fc variant to make a knob mutation and the other to make a hole mutation. (The aforementioned contents are incorporated herein by reference).

[0107] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0108] 1) Alanine (A), serine (S), threonine (T);

[0109] 2) Aspartic acid (D), glutamic acid (E);

[0110] 3) Asparagine (N), glutamine (Q);

[0111] 4) Arginine (R), Lysine (K), Histidine (H);

[0112] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0113] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0114] The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences varies as the number of identical positions shared by the sequences changes, taking into account the number of spaces that need to be introduced and the length of each space for optimal comparison of the two sequences.

[0115] Mathematical algorithms can be used to compare sequences between two sequences and calculate percent identity. For example, the Needlema and Wunsch algorithms (available at www.gcg.com) that have been integrated into the GAP program of the GCG software package are used, using a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program (available at www.gcg.com) in the GCG software package is used, using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0116] Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present disclosure can be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences. For example, such searches can be performed using NBLAST and XBLAST programs (version 2.0). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When using BLAST and gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. (the foregoing is incorporated herein by reference).

[0117] The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen, which comprises at least (1) an extracellular antigen binding domain, such as an antibody heavy chain variable region and / or light chain variable region, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR is able to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding domain.

[0118] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as vectors for directly expressing antibodies of the present disclosure in vitro and / or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP2101823B1; the contents of which are incorporated herein by reference).

[0119] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0120] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0121] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0122] The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0123] The term "pharmaceutically acceptable carrier" herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, known to those skilled in the art (see, e.g., Remington Pharmaceutics) (the contents of which are incorporated herein by reference). Except in cases where they are incompatible with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

[0124] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0125] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.

[0126] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0127] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0128] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings that are understood by those of ordinary skill in the art.

[0130] Figure 1 shows the binding activity of anti-B7-H7 chimeric antibodies to human B7-H7-His protein. The anti-B7-H7 chimeric antibodies can effectively bind to human B7-H7-His protein.

[0131] Figure 2 shows the binding activity of anti-B7-H7 chimeric antibodies to CHO-K1-human B7-H7. All chimeric antibodies can effectively bind to CHO-K1-human B7-H7 cells.

[0132] Figure 3 shows the binding activity of anti-B7-H7 chimeric antibodies to Flp-In-CHO-cynomolgus monkey B7-H7. All chimeric antibodies can effectively bind to Flp-In-CHO-cynomolgus monkey B7-H7 cells.

[0133] Figure 4 shows the binding activity of anti-B7-H7 chimeric antibodies to the NUGC-4 human gastric cancer cell line. All chimeric antibodies can effectively bind to the NUGC-4 human gastric cancer cell line.

[0134] Figure 5 shows the binding activity of anti-B7-H7 chimeric antibodies to cynomolgus monkey B7-H7-His protein. Except for Mab06, which binds weakly, other anti-B7-H7 chimeric antibodies can effectively bind to cynomolgus monkey B7-H7-His protein.

[0135] Figure 6 shows the effect of anti-B7-H7 chimeric antibodies in blocking the interaction between KIR3DL3-mFc and CHO-K1-human B7-H7. All chimeric antibodies can significantly block the binding of KIR3DL3-mFc protein to CHO-K1-human B7-H7 cells.

[0136] Figure 7. Shows the effect of anti-B7-H7 chimeric antibodies in blocking the interaction between Bio-hTMIGD2-hFc and CHO-K1-human B7-H7. The experimental results show that compared with the negative control Anti-Fluo-hIgG4, Mab001, Mab004, Mab006 and the control antibody Hz2D7-hIgG4 can completely block the binding of Bio-hTMIGD2-hFc protein to CHO-K1-human B7-H7 cells, while Mab002 and Mab005 show partial blocking effect. Mab003, Mab007, Mab008 and Mab009 cannot block the binding of Bio-hTMIGD2-hFc protein to CHO-K1-human B7-H7 cells.

[0137] Figure 8 shows the biological activity of anti-B7-H7 chimeric antibodies in reverse activation detected by Jurkat-IL-2-human KIR3DL3 reporter gene assay. The experimental results show that compared with the negative control Anti-Fluo-hIgG4, all chimeric antibodies can inhibit the activation of KIR3DL3, thereby enhancing the function of cell activation.

[0138] Figure 9 shows the biological activity of anti-B7-H7 chimeric antibodies in inhibiting activation detected by a Jurkat-NFAT-human TMIGD2 reporter gene assay. The experimental results indicate that, compared with the negative control Anti-Fluo-hIgG4, Mab001, Mab004, and the control antibody Hz2D7-hIgG4 have the biological function of inhibiting TMIGD2 activation, while other chimeric antibodies cannot inhibit TMIGD2 activation.

[0139] Figure 10A shows FACS analysis of B7-H7 expression levels on CHO-K1-OKT3(ScFv)-human B7-H7 cells. The black hollow peaks in the figure indicate B7-H7 expression on CHO-K1-OKT3(ScFv)-human B7-H7 cells, and the gray solid peaks indicate the isotype control corresponding to the detection antibody.

[0140] Figure 10B shows FACS analysis of B7-H7 expression levels on K562 tumor cells overexpressing human B7-H7 (K562-human B7-H7). The black hollow peaks in the figure represent B7-H7 expression on the surface of K562-B7-H7 cells, and the gray solid peaks represent the isotype control corresponding to the detection antibody.

[0141] Figure 11 shows the activation of primary T cells by anti-B7-H7 chimeric antibodies. The results show that all chimeric antibodies stimulated T cell activation and IFN-γ production, with statistically significant differences compared to the negative control (no treatment) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). The percentages in the graph represent the increase in IFN-γ secretion compared to the no treatment group.

[0142] Figure 12A-12B shows FACS detection of KIR3DL3 and TMIGD2 expression on the surface of NK92MI cells. The black hollow peaks in the figure refer to the expression of KIR3DL3 and TMIGD2 on the surface of NK92MI cells, and the gray solid peaks refer to the isotype control antibodies corresponding to the two detection antibodies.

[0143] Figure 13 shows the effect of anti-B7-H7 chimeric antibodies on the cytotoxicity of NK92MI cells to K562-overexpressing human B7-H7 target cells. The horizontal axis represents the concentration of the test antibody, and the vertical axis represents the target cell mortality rate. Compared to the negative control, Anti-Hel-hIgG4, all chimeric antibodies blocked the binding between KIR3DL3 on the surface of NK92MI cells and B7-H7 on the surface of K562-human B7-H7 cells, demonstrating enhanced NK92MI cell cytotoxicity to K562-human B7-H7 target cells, comparable to the positive control, Hz2D7-hIgG4.

[0144] Figure 14 shows the binding activity of anti-B7-H7 humanized antibodies to human B7-H7-His protein. The experimental results show that compared with the negative control Anti-Fluo-hIgG4, the anti-B7-H7 humanized antibodies can effectively bind to the human B7-H7-His protein. Hab03-H6aL1, Hab07-H3L2, Hab08-H2L2a and Hab09-H3L1, Hab09-H4L2 bind relatively weakly, while Hab01-H1L7, Hab02-H2aL1b, Hab04-H1L3, Hab05-H2L1, Hab06-H1L1 and the positive control antibody Hz2D7-hIgG4 bind strongly.

[0145] Figure 15 shows the ELISA method for detecting the binding activity of anti-B7-H7 humanized antibodies to CHO-K1-human B7-H7. The anti-B7-H7 humanized antibodies can effectively bind to CHO-K1-human B7-H7 cells.

[0146] Figure 16 shows the binding activity of anti-B7-H7 humanized antibodies to NUGC-4 human gastric cancer cell lines. Anti-B7-H7 humanized antibodies can effectively bind to NUGC-4 human gastric cancer cell lines.

[0147] Figure 17 shows the binding activity of anti-B7-H7 humanized antibodies to cynomolgus monkey B7-H7-His protein. All anti-B7-H7 humanized antibodies can effectively bind to cynomolgus monkey B7-H7-His protein. Hab06-H1L1 and Hab08-H2L2a bind relatively weakly, while the remaining humanized antibodies have strong specific binding.

[0148] Figure 18 shows the binding activity of anti-B7-H7 humanized antibodies to Flp-In-CHO-cynomolgus monkey B7-H7 cells. The anti-B7-H7 humanized antibodies can effectively bind to Flp-In-CHO-cynomolgus monkey B7-H7 cells.

[0149] Figure 19 shows that anti-B7-H7 humanized antibodies blocked the interaction between KIR3DL3-mFc and CHO-K1-human B7-H7. All humanized antibodies could significantly block the binding of KIR3DL3-mFc protein to CHO-K1-human B7-H7 cells.

[0150] Figure 20 shows that anti-B7-H7 humanized antibodies block the interaction between Bio-hTMIGD2-hFc and CHO-K1-human B7-H7. The experimental results show that compared with the negative control Anti-Fluo-hIgG4, Hab01-H1L7, Hab04-H1L3, Hab06-H1L1 and the control antibody Hz2D7-hIgG4 can completely block the binding of Bio-hTMIGD2-hFc to CHO-K1-human B7-H7, Hab02-H2aL1b, Hab09-H3L1, Hab09-H4L2, and Hab05-H2L1 show partial blocking, and Hab03-H6aL1, Hab07-H3L2 and Hab08-H2L2a cannot block the binding of Bio-hTMIGD2-hFc to CHO-K1-human B7-H7.

[0151] Figure 21 shows the biological activity of anti-B7-H7 humanized antibodies for transactivation detected by the KIR3DL3 IL2 Jurkat reporter gene assay. The experimental results indicate that compared with the negative control Anti-Fluo-hIgG4, all humanized antibodies can inhibit KIR3DL3 activation, thereby enhancing cell activation. The transactivation activities of Hab01-H1L7, Hab04-H1L3, Hab06-H1L1, and the control antibody Hz2D7-hIgG4 are relatively weak, all below 10-fold. The transactivation effects of Hab02-H2aL1b, Hab03-H6aL1, Hab05-H2L1, Hab07-H3L2, Hab08-H2L2a, Hab09-H3L1, and Hab09-H4L2 are relatively strong, all above 10-fold.

[0152] Figure 22 shows the biological activity of anti-B7-H7 humanized antibodies in inhibiting activation using a TMIGD2 NFAT Jurkat reporter gene assay. The experimental results indicate that, compared with the negative control Anti-Fluo-hIgG4, Hab01-H1L7, Hab04-H1L3, and the control antibody Hz2D7-hIgG4 have the biological function of inhibiting TMIGD2 activation, while other humanized antibodies cannot inhibit TMIGD2 activation.

[0153] Figure 23 shows the effect of humanized anti-B7-H7 antibodies on the activation of primary T cells. The results demonstrate that all humanized anti-B7-H7 antibodies stimulated T cell activation and IFN-γ production, with statistically significant differences compared to the negative control (no treatment) (****p<0.0001). The percentages in the graph represent the increase in IFN-γ secretion compared to the no treatment group.

[0154] Figure 24 shows FACS analysis of KIR3DL3 and TMIGD2 expression on primary human NK cells. The black hollow peaks in the figure represent KIR3DL3 / TMIGD2 expression on primary human NK cells, and the gray solid peaks represent isotype control antibodies corresponding to the two detection antibodies.

[0155] Figure 25 shows FACS analysis of B7-H7 expression levels on tumor cells A375 overexpressing human B7-H7 (A375-human B7-H7). The black hollow peaks in the figure represent B7-H7 expression on the surface of A375-B7-H7 cells, and the gray solid peaks represent the isotype control corresponding to the detection antibody.

[0156] Figure 26 shows the effect of humanized anti-B7-H7 antibodies on primary human NK cell activation. The horizontal axis represents the concentration of the test antibody, and the vertical axis represents the percentage of NK cell degranulation (CD107a)-positive cells. Compared with the negative control antibody Anti-Hel-hIgG4, with the exception of Hab04-H1L3 and Hab06-H1L1, which partially inhibited NK cell activation, all other humanized B7-H7 antibodies promoted primary NK cell activation to varying degrees.

[0157] Figure 27 shows the effects of humanized anti-B7-H7 antibodies on primary human NK cell activation. The horizontal axis represents the concentration of the test antibody, and the vertical axis represents the percentage of NK cell populations positive for intracellular IFN-γ. Compared with the negative control antibody, Anti-Hel-hIgG4, with the exception of Hab04-H1L3 and Hab06-H1L1, which partially inhibited NK cell activation, all other humanized B7-H7 antibodies promoted primary NK cell activation to varying degrees.

[0158] Figure 28 shows the effect of humanized anti-B7-H7 antibodies on the cytotoxicity of NK92MI cells to K562-overexpressing human B7-H7 target cells. The horizontal axis represents the concentration of the test antibody, and the vertical axis represents the target cell mortality rate. Compared to the negative control, Anti-Hel-hIgG4, all humanized antibodies blocked the binding of KIR3DL3 on the surface of NK92MI cells to B7-H7 on the surface of K562-human B7-H7 cells, demonstrating enhanced NK92MI cell cytotoxicity to K562-human B7-H7 target cells, comparable to the positive control, Hz2D7-hIgG4.

[0159] Figure 29 shows the efficacy of the combination of anti-B7-H7 antibodies and anti-PD1 / PDL1 antibodies in the Jurkat-IL-2-human KIR3DL3-human PD1 reporter gene assay. The results demonstrate that anti-B7-H7 antibodies synergistically activate the Jurkat-IL-2-human KIR3DL3-human PD1 reporter gene signal with either Keytruda or Tecentriq, significantly outperforming either agent alone.

[0160] Figure 30 shows the in vivo efficacy of anti-B7-H7 antibodies in a NUGC4 xenograft mouse model reconstituted with human NK cell immunity. Figure 30A shows the tumor growth curve of the NUGC4 subcutaneous xenograft model; Figure 30B shows the weight change curve of the mice after administration. Arrows indicate the time of administration, data points represent the mean tumor volume within the group, and error bars represent the standard error (SEM).

[0161] Figure 31 shows FACS analysis of B7-H7 expression levels on HCC827 tumor cells overexpressing human B7-H7 (HCC827-human B7-H7). The black hollow peaks in the figure represent B7-H7 expression on the surface of HCC827-B7-H7 cells, and the gray solid peaks represent the isotype control corresponding to the detection antibody.

[0162] Figures 32A-32B show the in vivo efficacy of anti-B7-H7 antibodies in an HCC827-B7-H7 xenograft mouse model reconstituted with NK cell immunity; Figure 32A shows the tumor growth curve of the HCC827-B7-H7 subcutaneous xenograft model; Figure 32B shows the weight change curve of mice after administration. Arrows represent the time of administration, data points represent the mean tumor volume within the group, and error bars represent the standard error (SEM).

[0163] Figures 33A-33D show the in vivo efficacy of anti-B7-H7 antibodies in a NUGC4 xenograft tumor model reconstituted with human PBMCs. Figure 33A shows the efficacy curve of the anti-B7-H7 antibody in combination with Keytruda; Figure 33B shows the efficacy curve of the anti-B7-H7 antibody in combination with Tecentriq; Figure 33C shows the body weight change curve of mice after the anti-B7-H7 antibody was combined with Keytruda; Figure 33D shows the body weight change curve of mice after the anti-B7-H7 antibody was combined with Tecentriq. Arrows represent dosing time points, data points represent the mean tumor volume within the group, and error bars represent the standard error (SEM).

[0164] Figures 34A-34D show the in vivo efficacy of anti-B7-H7 antibodies in an A375-B7-H7 xenograft tumor model reconstituted with human PBMCs. Figure 34A shows the efficacy curves of anti-B7-H7 antibodies at different doses; Figure 34B shows the efficacy curves of anti-B7-H7 antibodies in combination with Keytruda; Figure 34C shows the efficacy curves of anti-B7-H7 antibodies in combination with Tecentriq; and Figure 34D shows the body weight changes of mice after dosing. Arrows indicate dosing time points, data points represent the mean tumor volume within each group, and error bars represent the standard error (SEM). DETAILED DESCRIPTION

[0165] The present disclosure is further described below with reference to specific examples, and the advantages and features of the present disclosure will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0166] The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

[0167] In the disclosed embodiments, B7-H7 and HHLA2 are different names for the same target, and they express the same meaning, that is, the B7-H7 antibody or its antigen-binding fragment is equivalent to the HHLA2 antibody or its antigen-binding fragment, and the B7-H7 protein is equivalent to the HHLA2 protein. Beneficial effects:

[0168] The B7-H7 antibodies and related biological products disclosed herein have high binding activity with B7-H7, can strongly block the B7-H7-KIR3DL3 interaction, reduce NK inhibitory signals, and lessen the blockade of the B7-H7-TMIGD2 interaction without affecting B7-H7 activation of NK cells. This, in turn, promotes NK cell activation and enhances NK cell killing of tumor target cells. Furthermore, the antibodies disclosed herein can be used in combination with PD-1 / PD-L1 axis inhibitors to achieve even better tumor suppression.

[0169] The B7-H7 antibody disclosed herein can fill the gap in current clinical tumor treatment, providing a new treatment option for tumor patients with poor response to immune checkpoint inhibitors, better improving prognosis and increasing patient survival rates, and has huge potential clinical needs.

[0170] Example 1. Preparation of immune antigens, detection proteins, control antibodies, and detection antibodies

[0171] 1.1 Preparation of B7-H7 antigen

[0172] Human B7-H7 protein (NCBI sequence number: NP_009003.1) was used as a template for the B7-H7 disclosed herein to design amino acid sequences of the antigens and detection proteins involved in the present disclosure. Optionally, different tags were fused to the B7-H7 protein and cloned into the PTT5 vector (Invitrogen). The proteins were transiently expressed in Expi293F cells or stably expressed and purified in CHO cells to obtain proteins encoding the antigens and detection proteins disclosed herein.

[0173] Fusion protein of the extracellular region of human B7-H7 and His tag: human B7-H7-his (SEQ ID NO: 1), used for detection: Note: The underlined part is the signal peptide, and the italic part is the His tag.

[0174] 1.2 Preparation of KIR3DL3 protein

[0175] Human KIR3DL3 protein (NCBI sequence number: NP_703144) was used as a template for the KIR3DL3 disclosed herein. The amino acid sequences of the antigens and detection proteins disclosed herein were designed based on the extracellular region. A tag was optionally fused to the KIR3DL3 protein and cloned into the PTT5 vector (Invitrogen). Transient expression in Expi293F cells or stable expression and purification in CHO cells were performed to obtain proteins encoding the antigens and detection proteins disclosed herein. Alternatively, KIR3DL3-mFc was biotinylated using the Biotin kit (manufacturer: DOJINDO, catalog number: LK03) and named Bio-KIR3DL3-mFc.

[0176] A fusion protein of the extracellular region of human KIR3DL3 and the Fc segment of mouse IgG2a: human KIR3DL3-mFc (SEQ ID NO: 2), used for detection: Note: The underlined portion is the signal peptide, and the italicized bold portion is mouse IgG2a Fc (mIgG2a Fc: SEQ ID NO: 8).

[0177] 1.3 Preparation of control antibodies and detection antibodies

[0178] Using the control antibody Hz2D7 sequence (patent number WO2020041300A1) and the 26E10 sequence (patent number WO2021067633A2) as templates, they were constructed into the PPT5-hIgG4 (S228P) vector and transiently expressed in Expi293F cells or stably expressed and purified in CHO cells to obtain the positive control antibody Hz2D7-hIgG4. Optionally, the 26E10-hIgG4 antibody was labeled using the AF488 labeling kit (Invitrogen-A20181, Catalog Number: A20181) and named AF488 anti-KIR3DL3 detection antibody. The control antibody sequences are shown in Table 1.

[0179] The light and heavy chain sequences of anti-fluorescein (FITC) were cloned into the pTT5 vector to construct the Anti-Fluo-hIgG4 plasmid as an isotype control antibody. The sequence is shown in Table 1.

[0180] Table 1. Control antibody sequences

[0181] Example 2. Construction of engineered cell lines

[0182] 2.1 Construction of human B7-H7 overexpressing cell line

[0183] The nucleotide sequence encoding the full-length amino acid sequence of human B7-H7 (NCBI: NP_009003.1, SEQ ID NO: 9) was cloned into the pLVX-IRES-Puro vector, and viral particles were prepared in HEK293T cells (purchased from ATCC). CHO-K1 cell line (purchased from ATCC), CHO-K1-OKT3 (ScFv) cell line (purchased from Kangyuan Bochuang), K562 cell line (purchased from ATCC), and A375 cell line (purchased from Beina Biotechnology) were infected with lentivirus, and then selectively cultured in puromycin (purchased from Gibco, catalog number: A1113802) medium for 2 weeks. Monoclonal cells were then plated into 96-well plates and cultured at 37°C, 5% (v / v) CO2. After approximately 2 weeks, some monoclonal wells were selected for expansion, and the amplified clones were screened by flow cytometry to obtain CHO-K1-human B7-H7, CHO-K1-OKT3 (ScFv)-human B7-H7, K562-human B7-H7, and A375-human B7-H7 cell lines overexpressing B7-H7, respectively. Select cell lines with good growth, high fluorescence intensity and monoclonality to continue to expand culture and freeze in liquid nitrogen.

[0184] To generate the HCC827-LUC-GFP cell line, Luc2p and GFP elements were cloned into the pcDNA3.1-Hygro vector (Ubao Bio Product No. VT1979) to construct the pcDNA3.1-Luc2p-IRES-GFP-hygro vector. The pcDNA3.1-Luc2p-IRES-GFP-hygro plasmid was then transfected with Lipofectamine TM HCC827 (Chinese Academy of Sciences, Catalog No. SCSP-538) cells were transiently transfected with 1% FcGFR 3000 (Invitrogen, Catalog No. L3000001) and incubated at 37°C, 5% CO2 for 24 hours. After incubation, the cells were washed with 3 mL of PBS and selectively cultured for 2 weeks in 1640 medium (Gibco, Catalog No. 31870082) containing 10% (w / w) fetal bovine serum and sodium pyruvate (Gibco, Catalog No. 11360070) supplemented with 125 μg / mL hygromycin (Invitrogen, Catalog No. 10687010) to obtain the desired HCC827-LUC-GFP cell line.

[0185] The nucleotide sequence encoding the full-length amino acid sequence of human B7-H7 (NCBI Reference Sequence: NP_009003.1, SEQ ID NO: 9) was cloned into the pLVX-IRES-Puro vector, and viral particles were prepared in HEK293T cells (purchased from ATCC). After lentiviral infection of HCC827-LUC-GFP cells, cells were selectively cultured for two weeks in medium containing 1 μg / mL puromycin (Gibco, Catalog No. A1113802). After that, single clones were plated into 96-well plates and incubated at 37°C in 5% (v / v) CO2. After approximately two weeks, a subset of single clones were selected for expansion, and the expanded clones were screened by flow cytometry. Cell lines with good growth, high fluorescence intensity, and monoclonal activity (HCC827-LUC-GFP-human B7-H7 cells, hereafter referred to as HCC827-human B7-H7 cells) were selected for further expansion and cryopreservation in liquid nitrogen.

[0186] Full-length human B7-H7 (SEQ ID NO: 9, NCBI Reference Sequence: NP_009003.1) was used to construct human B7-H7 overexpressing cell lines: CHO-K1-human B7-H7, CHO-K1-OKT3 (ScFv)-human B7-H7, K562-human B7-H7, A375-human B7-H7, and HCC827-B7-H7: Note: Signal peptide (single underline) + extracellular region + transmembrane region (double underline) + intracellular region (italic part)

[0187] 2.2 Construction of Flp-In-CHO-cynomolgus monkey B7-H7 engineered cell line

[0188] The nucleotide sequence encoding the full-length amino acid sequence of cynomolgus monkey B7-H7 (SEQ ID NO: 10, NCBI Reference Sequence: XP_028700475.1) was cloned into the pcDNA5 vector and a plasmid was prepared. The Flp-In-CHO cell line (purchased from Thermo) was transfected with the plasmid ( 3000 Transfection Kit, purchased from Invitrogen, catalog number: L3000-015), and then selectively cultured for 2 weeks in F12K medium containing 10% (w / w) fetal bovine serum and 600 μg / mL hygromycin (purchased from Invitrogen, catalog number: 10687010) to obtain the desired cell line. The cell line was further expanded and frozen in liquid nitrogen.

[0189] Full-length cynomolgus monkey B7-H7 (SEQ ID NO: 10, NCBI Reference Sequence: XP_028700475.1) was used to construct the cynomolgus monkey B7-H7 overexpression cell line Flp-In-CHO cynomolgus monkey B7-H7: Note: Signal peptide (single underline) + extracellular region + transmembrane region (double underline) + intracellular region (italic part)

[0190] 2.3 Construction of Jurkat-IL-2-human KIR3DL3 cell line

[0191] To generate the parental Jurkat-IL-2 cell line, the NlucP element was first cloned into the pGL4[luc2P / IL2 / Hygro] (Promega Catalog No. CS177203) vector to replace the luc2P element in the original vector and construct the pNL3.2[NlucP / IL-2 / Hygro] vector. The pNL3.2[NlucP / IL-2 / Hygro] vector (containing the IL2 promoter, which drives the transcription of the luciferase reporter gene NanoLuc in response to TCR activation) was then used to transfect the Jurkat cell line (purchased from ATCC) with the plasmid (Lipofectamine LTX Transfection Kit, purchased from Invitrogen, Catalog No. 15338-100). The cells were then cultured in RPMI containing 10% (w / w) fetal bovine serum and 250 μg / mL hygromycin (purchased from Invitrogen, Catalog No. 10687010). The desired Jurkat-IL-2 cell line was obtained after selective culture in 1640 (purchased from Gibco, product number: 31870082) culture medium for 2 weeks.

[0192] The nucleotide sequence encoding the full-length amino acid sequence of human KIR3DL3 (SEQ ID NO: 11, NCBI sequence number: NP_703144) was cloned into the pLVX-IRES-Puro vector, and viral particles were prepared in HEK293T cells (purchased from ATCC). Jurkat-IL-2 cells were infected with lentiviral vectors and selectively cultured for two weeks in RPMI1640 (Gibco, catalog number: 31870082) supplemented with 10% (w / w) fetal bovine serum and 2 μg / mL puromycin (Gibco, catalog number: A1113802). After that, single clones were plated into 96-well plates and incubated at 37°C in 5% (v / v) CO2. After approximately two weeks, a subset of single clones were selected for expansion. Amplified clones were screened by flow cytometry and fluorescence signal intensity. Single clones with good growth, high expression levels, and a wide activation window were selected for further expansion and cryopreservation in liquid nitrogen.

[0193] The full-length amino acid sequence of human KIR3DL3 (SEQ ID NO: 11, NCBI sequence number: NP_703144) was used to construct the Jurkat-IL-2-human KIR3DL3 cell line: Note: Signal peptide (single underline) + extracellular region + transmembrane region (double underline) + intracellular region (italic part)

[0194] 2.4 Construction of Jurkat-NFAT-human TMIGD2 cell line

[0195] To generate the parental Jurkat-NFAT cell line, the pNL[NLucP / NFAT-RE / Hygro] vector (containing NFAT, which drives the transcription of the luciferase reporter gene NanoLuc in response to TCR activation) was used to transfect the Jurkat cell line (purchased from ATCC) with the plasmid (Lipofectamine LTX Transfection Kit, purchased from Invitrogen, catalog number: 15338-100). The desired Jurkat-NFAT cell line was obtained after selective culture for 2 weeks in RPMI1640 (purchased from Gibco, catalog number: 31870082) medium containing 10% (w / w) fetal bovine serum and 125 μg / mL hygromycin (Invitrogen: 10687010).

[0196] The nucleotide sequence encoding the full-length amino acid sequence of human TMIGD2 (SEQ ID NO: 12, NCBI Sequence No. NP_653216.2) was cloned into the pLVX-IRES-Puro vector, and viral particles were prepared in HEK293T cells (purchased from ATCC). Jurkat-NFAT cell lines were infected with lentiviral vectors and selectively cultured for two weeks in RPMI1640 (Gibco, Catalog No. 31870082) supplemented with 10% (w / w) fetal bovine serum (FBS) and 0.125 μg / mL puromycin (Gibco, Catalog No. A1113802). After that, single clones were plated into 96-well plates and incubated at 37°C in 5% (v / v) CO2. After approximately two weeks, a subset of single clones were selected for expansion. Amplified clones were screened by flow cytometry and fluorescence signal intensity. Select monoclonal cell lines with good growth, high expression level and large activation window to continue to expand culture and freeze in liquid nitrogen.

[0197] The full-length amino acid sequence of human TMIGD2 (SEQ ID NO: 12, NCBI sequence number: NP_653216.2) was used to construct the Jurkat-NFAT-human TMIGD2 cell line: Note: Signal peptide (single underline) + extracellular region + transmembrane region (double underline) + intracellular region (italic part)

[0198] 2.5 Construction of CHO-K1-OKT3(ScFv)-human B7-H7-human PDL1 Stable Cell Line

[0199] The nucleotide sequence encoding the full-length amino acid sequence of human PDL1 (NCBI sequence number: NP_054862.1) was cloned into the pLVX-IRES-G418 vector, and viral particles were prepared in HEK293T cells (purchased from ATCC). The CHO-K1-OKT3(ScFv)-human B7-H7 cell line was infected with lentivirus. After selective culture for two weeks in Advanced DMEM / F-12 (Gibco, catalog number: 12634028) supplemented with 10% (w / w) fetal bovine serum (FBS) and 0.8 mg / ml G418 (purchased from Shanghai Peiyuan, catalog number: S150J7), single clones were plated into 96-well plates and incubated at 37°C in 5% (v / v) CO2. After approximately two weeks, a subset of single clones were selected for expansion. Amplified clones were screened by flow cytometry and activation strength. The monoclonal cell line CHO-K1-OKT3 (ScFv)-human B7-H7-human PDL1 with good growth, high expression level and large activation window was selected for further expansion and cryopreservation in liquid nitrogen.

[0200] Human PDL1 full-length amino acid sequence (NCBI sequence number: NP_054862.1): used to construct CHO-K1-OKT3 (ScFv)-human B7-H7-human PDL1 cell line

[0201] Signal peptide (single underline) + extracellular region + transmembrane region (double underline) + intracellular region (italic part)

[0202] 2.6 Construction of Jurkat-IL-2-human KIR3DL3-human PD1 cell line

[0203] The nucleotide sequence encoding the full-length amino acid sequence of human PD-1 (NCBI sequence number: NP_005009.2) was cloned into the pLVX-IRES-G418 vector, and viral particles were prepared in HEK293T cells (purchased from ATCC). The Jurkat-IL-2-human KIR3DL3 cell line was infected with lentivirus and selectively cultured for two weeks in 1640 medium (Gibco, catalog number: 31870082) supplemented with 10% (w / w) fetal bovine serum (FBS) and 0.2 mg / ml G418 (purchased from Shanghai Peiyuan, catalog number: S150J7). After that, single clones were plated into 96-well plates and incubated at 37°C in 5% (v / v) CO2. After approximately two weeks, a subset of single clones were selected for expansion. Amplified clones were screened by flow cytometry and fluorescence signal intensity. The monoclonal cell line Jurkat-IL-2-human KIR3DL3-human PD1 with good growth, high expression level and large activation window was selected to continue to expand the culture and freeze in liquid nitrogen.

[0204] Human PD1 full-length amino acid sequence (NCBI sequence number: NP_005009.2): used to construct Jurkat-IL-2-human KIR3DL3-human PD1 cell line

[0205] Signal peptide (single underline) + extracellular region + transmembrane region (double underline) + intracellular region (italic part)

[0206] Example 3. Generation of anti-human B7-H7 mouse monoclonal antibodies

[0207] Anti-human B7-H7 antibodies are obtained through hybridoma technology. Human B7-H7-hFc fusion protein or the CHO-K1-human B7-H7 cell line overexpressing human B7-H7 protein is used as the immunogen to immunize mice, isolate the spleen cells of the immune mice, and fuse them with mouse myeloma cells by electrofusion. The cells are cultured in HAT selection medium, and the culture supernatant is collected for identification. Clones secreting the target antibody are selected for subcloning, and finally, mouse monoclonal antibodies are obtained through production and purification.

[0208] 3.1 Animal immunization

[0209] Female SJL / J or BALB / c mice, 6-8 weeks old (purchased from Shanghai Slake Laboratory Animal Co., Ltd.), were used in the experiments. The animals were housed in an SPF-grade animal facility (Heyuan Biotechnology (Shanghai) Co., Ltd.) for one week to acclimate to the laboratory environment. The mice were maintained on a 12 / 12 hr light / dark cycle, at 20-25°C and 40-60% humidity. The acclimatized mice were immunized according to the following protocol.

[0210] The immune antigen is human B7-H7 protein (extracellular segment Ile23-Asn344) with hFc tag (Acro, catalog number: B77-H5257). Gold Adjuvant (Sigma catalog number: T2684) and Thermo Alum (Thermo, Cat. No. 77161) adjuvant cross immunization. Antigen and adjuvant ( Gold Adjuvant) ratio was 1:1, antigen and adjuvant (Thermo The ratio of emulsified antigen to emulsified antigen was 2:1, 50 μg / animal / time for the initial immunization and 25 μg / animal / time for the booster immunization. Emulsified antigen was administered on days 1, 8, 15, 22, 29, 36, 43, and 50. On day 1, 50 μg / animal of emulsified antigen was injected intraperitoneally (IP). On day 8, 25 μg / animal was injected subcutaneously at multiple sites (SC, typically at four sites on the back and groin). Intraperitoneal and subcutaneous injections were performed alternate weeks apart.

[0211] The immune antigen is the cell line CHO-K1-human B7-H7 that overexpresses human B7-H7 protein. Immunization plan: prepare cell suspension with normal saline, and the cell number is 5×10 6 On the first day, emulsified blank adjuvant ( Gold Adjuvant (50 μL / mouse) was injected intraperitoneally 15 minutes later. Cell suspension was injected intraperitoneally on days 8, 15, 22, 29, 36, 43, and 50.

[0212] Blood was collected on days 20, 27, 34, and 48, and the antibody titer of the mouse serum was determined by ELISA. After the 6th to 8th immunization, spleen and lymph node cells from mice with high antibody titers in the serum were selected for fusion. Three days before fusion, a surge immunization was performed with an intraperitoneal (IP) injection of 50 μg / mouse of an antigen solution prepared in physiological saline.

[0213] 3.2 Cell fusion

[0214] Spleen and lymph node cells were fused with myeloma SP2 / 0 cells ( CRL-1581) were fused to obtain hybridoma cells. The fused hybridoma cells were 5×10 5 The cells were resuspended at a density of 1:1 / mL in complete medium containing 20% ​​FBS (Excell, catalog number: FND500), 1× HT (Sigma, catalog number: H0262-10VL, and 1× NEAA in DMEM (Gibco, catalog number: 10569044), and 100 μL / well was plated in a 96-well flat-bottom plate. After incubation at 37°C, 5% CO2 for 6-7 days, the supernatant was removed, and 200 μl of DMEM containing HT complete medium containing 10% FBS, 1× HT (Sigma, catalog number: H0137-10VL), and 1× NEAA was added to each well. The cells were cultured overnight at 37°C, 5% CO2, and then assayed by ELISA.

[0215] 3.3 Screening of hybridoma cells

[0216] 7 to 10 days after fusion, the hybridoma cell supernatant was collected and tested for binding activity to human B7-H7-his (produced in-house, SEQ ID NO: 1) by ELISA, and positive clones were selected. The next day, the supernatant of the positive clone was tested for binding activity to CHO-K1-human B7-H7 (constructed in-house, see Section 2.1 of Example 2), binding activity to Flp-In-CHO-cynomolgus monkey B7-H7 (constructed in-house, see Section 2.2 of Example 2), and the effect of blocking the binding of KIR3DL3-mFc (SEQ ID NO: 2, optionally further labeled with Biotin) and TMIGD2-hFc (ACRO, Catalog No.: CDH-H5251, optionally further labeled with Biotin) to CHO-K1-human B7-H7 (constructed in-house, see Section 2.1 of Example 2), and the target clones were selected for subcloning.

[0217] After 7-10 days of subclonal cell culture, the target hybridoma monoclonal cells were selected by ELISA screening and expanded to 24-well culture. After 2-3 days, the culture supernatant was tested for binding activity to human B7-H7-his (SEQ ID NO: 1), CHO-K1-human B7-H7, and Flp-In-CHO-cynomolgus monkey B7-H7, as well as the effect of blocking the binding of KIR3DL3-mFc, TMIGD2-hFc and CHO-K1-human B7-H7. The target clones were selected for production and purified using protein L to obtain 189 strains of mouse monoclonal antibodies.

[0218] 3.4 Identification of mouse monoclonal antibodies

[0219] A total of 189 anti-B7-H7 monoclonal antibodies were obtained through hybridoma technology. After identification through binding experiments, blocking experiments, reporter gene experiments, etc., 69 candidate antibodies were selected and further identified and screened using the following experiments.

[0220] (a) Binding activity assay of anti-B7-H7 mouse monoclonal antibody to human B7-H7-His protein

[0221] Human B7-H7-His (extracellular fragment Ile23-Asn344) (ACRO, catalog number: B77-H52H5) was diluted to 1 μg / mL with PBS pH 7.4 and added to an ELISA plate (corning, catalog number: 9018) at 50 μL / well. The plate was incubated at 4°C overnight. The coating solution was discarded and 3% BSA-PBS was added at 250 μL / well. The plate was incubated at 37°C for 2-4 hours. The plate was washed three times with 0.05% Tween20-PBS on a plate washer. Purified mouse monoclonal antibody (diluted to 15 nM with 1% BSA and serially diluted to 8 concentrations in 5-fold steps) was added at 50 μL / well. The plate was incubated at 37°C for 1.5-2 hours. The plate was washed three times with 0.05% Tween20-PBS on a plate washer. HRP enzyme-labeled Goat anti-HRP antibody was diluted 1:5000 with 1% BSA-PBS. Anti-Mouse antibody (Jackson, catalog number: 115-035-003) was added to the ELISA plate at 50 μL / well and incubated at 37°C for 1 hour. The plate was washed three times with 0.05% Tween20-PBS on a plate washer. TMB colorimetric solution was added at 50 μL / well and incubated at room temperature for 5-10 minutes. 1 M HCL was added at 50 μL / well to terminate the reaction, and the OD450 nm was read on a microplate reader.

[0222] (b) ELISA assay for the binding activity of anti-B7-H7 mouse monoclonal antibody to CHO-K1-human B7-H7 and Flp-In-CHO-cynomolgus monkey B7-H7

[0223] The cells were collected and the concentration was adjusted to 5×10 5 / mL, added to a 96-well cell culture plate (Corning, Catalog No.: 3599), 100 μL / well, incubated at 37°C 5% CO2 overnight, discarded the culture supernatant, added cell fixative (Biyuntian, Catalog No.: P0098), 50 μL / well, fixed at room temperature for 1 hour, washed once with 0.05% Tween20-PBS on a plate washer, added 5% skim milk powder (Sangon Biotechnology, Catalog No.: A600669-0250)-PBS, 250 μL / well, incubated at 37°C for 2-4 hours, washed once with 0.05% The cells were washed three times with Tween20-PBS, and purified mouse monoclonal antibody diluent was added and diluted with 1% BSA to 15 nM. The cells were diluted 5-fold to 8 concentration points, 50 μL / well, and incubated at 37°C for 1.5 to 2 hours. The cells were washed three times with 0.05% Tween20-PBS on a plate washer, and HRP enzyme-labeled Goat anti-mouse antibody was diluted with 1% BSA-PBS at a dilution ratio of 1:5000, added to the cell plate, 50 μL / well, and incubated at 37°C for 1 hour. The cells were washed three times with 0.05% Tween20-PBS on a plate washer, and TMB color development solution was added, 50 μL / well, and incubated at 37°C for 10 minutes. 1M HCL was added, 50 μL / well, to terminate the reaction, and the OD450nm was read with a microplate reader.

[0224] (c) FACS assay to detect the binding activity of anti-B7-H7 mouse monoclonal antibody to NUGC-4 human gastric cancer cell line

[0225] NUGC-4 human gastric cancer cell line was purchased from Nanjing Kebai (Cat. No. CBP60493). The cells were collected, washed once with PBS, and resuspended in 1% BSA-PBS to 2×10 5 / 50μL, 1% BSA-PBS diluted the antibody to 100nM, 4-fold continuous gradient dilution to 8 concentration points, 50μL cells were mixed with 50μL antibody dilution solution, incubated at 4℃ for 60 minutes, washed twice with PBS, and added Alexa Resuspend the cells in 100 μL / well of 647 fluorescein-labeled Goat Anti-Mouse secondary antibody (1:800) (Jackson, catalog number: 115-605-003), incubate at 4°C for 40 minutes, wash twice with PBS, resuspend in 1% BSA-PBS, 100 μL / well, and analyze the cell samples on a flow cytometer (BD, CantoⅡ).

[0226] (d) FACS analysis of the blocking effect of anti-B7-H7 mouse monoclonal antibody on the interaction between Bio-KIR3DL3-mFc and CHO-K1-human B7-H7.

[0227] CHO-K1-human B7-H7 cells were collected, washed once with PBS, and resuspended to 2×105 The antibody was diluted to 1200 nM in 1% BSA-PBS in 40 μL / well, and 8 concentration points were serially diluted 4-fold. Bio-KIR3DL3-mFc (SEQ ID NO: 2, Biotinylated, produced in-house) was diluted to 1.5 μg / mL in 1% BSA-PBS. Then 40 μL of cells were mixed with 40 μL of antibody dilution and 40 μL of Bio-KIR3DL3-mFc dilution, incubated at 4°C for 60 minutes, washed twice with PBS, and APC-labeled streptavidin (working dilution 1:1700, Biolegend, cat. no.: 405243) was added and resuspended in 100 μL / well. The cells were incubated at 4°C for 40 minutes, washed twice with PBS, and resuspended in 100 μL / well of 1% BSA-PBS. The cell samples were analyzed on a flow cytometer (BD, CantoⅡ).

[0228] (e) FACS analysis of the blocking effect of anti-B7-H7 mouse monoclonal antibody on the interaction between hTMIGD2-hFc and CHO-K1-human B7-H7

[0229] CHO-K1-human B7-H7 cells were collected, washed once with PBS, and resuspended to 2×10 5 / 40μL, 1% BSA-PBS diluted the antibody to 1200nM, 4-fold serial dilution to 8 concentration points, 1% BSA-PBS diluted hTMIGD2-hFc (ACRO, product number: CDH-H5251) to 3μg / mL, then 40μL cells were mixed with 40μL antibody dilution and 40μL hTMIGD2-hFc dilution, incubated at 4℃ for 60 minutes, washed twice with PBS, and added Alexa Fluor 500 μL PBS. Resuspend the cells in 100 μL / well of 647 fluorescein-labeled Goat anti-human secondary antibody (1:800) (Jackson, catalog number: 109-605-098), incubate at 4°C for 40 minutes, wash twice with PBS, resuspend in 1% BSA-PBS, 100 μL / well, and analyze the cell samples on a flow cytometer (BD, CantoⅡ).

[0230] (f) Jurkat-IL-2-human KIR3DL3 cell reporter gene assay to detect the biological activity of anti-B7-H7 mouse monoclonal antibody in reverse activation

[0231] One day before the experiment, CHO-K1-OKT3 (ScFv)-human B7-H7 cells (prepared in-house, see Section 2.1 of Example 2) were collected and the cell density was adjusted to 2×10 5 / mL, and then the cell suspension was transferred to a 96-well white-bottom microplate (Corning, Cat. No. 3610), 2×10 4 / 100 μL / well, place the microplate with cells in a 37°C, 5% CO2 incubator and culture overnight.

[0232] On the day of the experiment, the antibody was diluted to 1200 nM with culture medium and diluted 4-fold continuously to 8 concentration points. After removing the cell culture supernatant plated the day before, the antibody dilution solution was transferred to the microplate at 50 μL / well. The microplate with the sample was placed in a 37°C 5% CO2 incubator and incubated for 1 hour.

[0233] During the incubation, Jurkat-IL-2-human KIR3DL3 cells (prepared in-house, see Section 2.3 of Example 2) were collected and the cell density was adjusted to 1×10 cells using cell culture medium containing 1 μg / mL anti-human CD28 antibody (Thermo, Cat. No. 16-0289-85). 6 / mL, and at the end of the sample incubation time, Jurkat-IL-2-human KIR3DL3 cell suspension was added to the microplate, 5×10 4 / 50μL / well, then place the microplate in a 37℃ 5% CO2 incubator and continue incubating for 5 hours. After the incubation, remove the microplate and add Nano- Luciferase Reporter Gene Assay System Reagent (Promega, Cat. No. N1130) was added at 50 μL / well and shaken at 450 rpm for 10 minutes. Luminescence was then read on a microplate reader. A stronger luminescence signal indicates a stronger transactivation effect of the antibody, and vice versa. A four-parameter effect curve was fitted using the luminescence signal and the corresponding antibody concentration. The transactivation activity of the antibody was compared by comparing the IC50 value and the fold increase in activation relative to the negative control.

[0234] (g) Jurkat-NFAT-human TIMGD2 cell reporter gene assay to detect the biological activity of anti-B7-H7 monoclonal antibody in inhibiting activation

[0235] One day before the experiment, CHO-K1-OKT3(ScFv)-human B7-H7 cells were collected and the cell density was adjusted to 2×10 5 / mL, and then the cell suspension was transferred to a 96-well white-bottom microplate (Corning, Cat. No. 3610), 2×10 4 / 100 μL / well, place the microplate with cells in a 37°C 5% CO2 incubator and culture overnight.

[0236] On the day of the experiment, the antibody was diluted to 1200 nM with culture medium and diluted 4-fold continuously to 8 concentration points. After removing the cell culture supernatant plated the day before, the antibody dilution solution was transferred to the microplate at 50 μL / well. The microplate with the sample was placed in a 37°C 5% CO2 incubator and incubated for 1 hour.

[0237] During the incubation, Jurkat-NFAT-human TIMGD2 cells (prepared in-house, refer to Section 2.4 of Example 2) were collected and the cell density was adjusted to 1×10 6 / mL, and at the end of the sample incubation time, Jurkat-NFAT-human TIMGD2 cell suspension was added to the microplate, 5×10 4 / 50μL / well, then place the microplate in a 37℃ 5% CO2 incubator and continue incubation for 5 hours. After the incubation is completed, remove the microplate and add Luciferase reporter gene assay system reagent, 50 μL / well, shake at 450 rpm for 10 minutes, and then read the luminescence signal on a microplate reader. Weaker luminescence signals indicate stronger inhibitory effects of the antibody, and vice versa. A four-parameter effect curve was fitted based on the luminescence signal and the corresponding antibody concentration. The inhibitory activity of the antibody was analyzed by comparing the IC50 value and inhibition rate.

[0238] Example 4. Identification of anti-human B7-H7 chimeric antibodies

[0239] After the identification test of 69 murine monoclonal antibodies in Example 3, the better antibodies were selected and the hIgG4(S228P)Fc (SEQ ID NO: 7) human-mouse chimeric antibody was constructed. 9 chimeric antibodies were further identified and confirmed, namely Mab001, Mab002, Mab003, Mab004, Mab005, Mab006, Mab007, Mab008, and Mab009.

[0240] The sequences were determined by conventional methods. Table 2 shows the VH / VL sequences of the chimeric antibodies, Table 3 shows the results of KABAT analysis of the chimeric antibodies, and Table 4 shows the results of IMGT analysis of the chimeric antibodies.

[0241] Table 2 B7-H7 chimeric antibody VH / VL sequences

[0242] Table 3 Kabat analysis results of B7-H7 chimeric antibody

[0243] Table 4 IMGT analysis results of B7-H7 chimeric antibody

[0244] 4.1 Detection of Binding Activity of Anti-B7-H7 Chimeric Antibodies to Human B7-H7-His Protein

[0245] For specific methods, refer to step (a) in section 3.4 of Example 3. A gradient dilution of chimeric antibody was added to a protein-coated 96-well plate. After incubation, HRP-labeled Goat anti-human antibody was added. The color was developed with TMB, and the OD450nm was read on a microplate reader. As shown in Table 5 and Figure 1, the results demonstrate that the anti-B7-H7 chimeric antibody effectively binds to human B7-H7-His protein compared to the negative control Anti-Fluo-hIgG4 (i.e., an anti-FITC antibody, used as an isotype control antibody), and its binding activity is comparable to that of the positive control antibody Hz2D7-hIgG4.

[0246] 4.2 ELISA assay for binding activity of anti-B7-H7 chimeric antibodies to CHO-K1-human B7-H7 and Flp-In-CHO-cynomolgus monkey B7-H7

[0247] For specific methods, refer to step (b) of Section 3.4 of Example 3. After cell fixation, a gradient of diluted chimeric antibodies was added. After incubation, HRP-labeled Goat anti-human antibody was added, and color was developed using TMB. The OD450nm was read on a microplate reader. As shown in Table 5 and Figures 2 and 3, the results demonstrate that all chimeric antibodies effectively bind to CHO-K1-human B7-H7 and Flp-In-CHO-cynomolgus monkey B7-H7, compared to the negative control Anti-Fluo-hIgG4, and have comparable binding activity to the positive control antibody Hz2D7-hIgG4.

[0248] 4.3 FACS detection of the binding activity of anti-B7-H7 chimeric antibody to NUGC-4 human gastric cancer cell line

[0249] For specific methods, refer to step (c) of section 3.4 of Example 3. The collected cells were mixed with equal volumes of chimeric antibodies, and then Alexa Fluor was added after incubation. Cell samples were analyzed on a flow cytometer (BD, Canto II) using a 647 fluorescein-conjugated goat anti-human secondary antibody (1:800) (Jackson, Cat. No. 109-605-088). As shown in Table 5 and Figure 4, all chimeric antibodies effectively bound to the NUGC-4 human gastric cancer cell line compared to the negative control, Anti-Fluo-hIgG4.

[0250] 4.4 Binding activity assay of anti-B7-H7 chimeric antibody to cynomolgus monkey B7-H7-His protein

[0251] Cynomolgus monkey B7-H7-His (extracellular segment Ile21-Asn345 and His tag fusion protein) (ACRO, Catalog No.: B77-C52H3) was diluted to 2 μg / mL with PBS pH 7.4 and added to an ELISA plate (Corning, Catalog No.: 9018) at 50 μL / well. The plate was incubated at 4°C overnight. The coating solution was discarded and 3% BSA-PBS was added at 250 μL / well. The plate was incubated at 37°C for 2-4 hours. The plate was washed three times with 0.05% Tween20-PBS on a plate washer. The chimeric antibody (diluted to 15 nM with 1% BSA and serially diluted to 8 concentrations in 5-fold steps) was added at 50 μL / well. The plate was incubated at 37°C for 1.5-2 hours. The plate was washed three times with 0.05% Tween20-PBS on a plate washer. The HRP enzyme-labeled Goat anti-antibody was diluted 1:5000 with 1% BSA-PBS. Human antibodies were added to an ELISA plate at 50 μL / well and incubated at 37°C for 1 hour. The plates were washed three times with 0.05% Tween 20-PBS on a plate washer. TMB colorimetric solution (50 μL / well) was added and incubated at room temperature for 5-10 minutes. The reaction was terminated by adding 50 μL / well of 1 M HCl and the OD450 nm was read on a microplate reader. As shown in Table 5 and Figure 5, the results demonstrate that both anti-B7-H7 chimeric antibodies exhibited binding activity to cynomolgus monkey B7-H7-His protein, compared to the negative control, Anti-Fluo-hIgG4.

[0252] Table 5 Binding activity of anti-B7-H7 chimeric antibodies

[0253] 4.5 FACS detection of anti-B7-H7 chimeric antibody blocking the interaction between KIR3DL3-mFc and CHO-K1-human B7-H7

[0254] For specific experimental methods, refer to step (d) of section 3.4 of Example 3. The collected cells were mixed with equal volumes of chimeric antibody and KIR3DL3-mFc (SEQ ID NO: 2, produced in-house) dilution, and Alexa Fluor was added. Cell samples were analyzed on a flow cytometer (BD, Canto II) using 647 fluorescein-conjugated goat anti-mouse secondary antibody (1:800) (Jackson, Cat. No. 115-006-071). As shown in Table 6 and Figure 6, all chimeric antibodies significantly blocked the binding of KIR3DL3-mFc protein to CHO-K1-human B7-H7 cells compared to the negative control Anti-Fluo-hIgG4.

[0255] 4.6 FACS detection of anti-B7-H7 chimeric antibody blocking the interaction between Bio-hTMIGD2-hFc and CHO-K1-human B7-H7

[0256] For specific experimental methods, refer to step (e) of section 3.4 of Example 3. The collected cells were mixed with equal volumes of chimeric antibody and Bio-hTMIGD2-hFc diluent, and APC-labeled streptavidin (working dilution 1:1700, Biolegend, cat. no. 405243) was added. The cell samples were analyzed on a flow cytometer (BD, Canto II).

[0257] As shown in Table 6 and Figure 7, the results showed that compared with the negative control Anti-Fluo-hIgG4, Mab001, Mab004, Mab006 and the control antibody Hz2D7-hIgG4 could completely block the binding of Bio-hTMIGD2-hFc protein to CHO-K1-human B7-H7 cells, Mab002 (maximum inhibition rate 25.3%) and Mab005 (maximum inhibition rate 93.1%) showed partial blocking, and Mab003, Mab007, Mab008 and Mab009 could not block the binding of Bio-hTMIGD2-hFc protein to CHO-K1-human B7-H7 cells.

[0258] 4.7 Jurkat-IL-2-human KIR3DL3 cell reporter gene assay to detect the biological activity of anti-B7-H7 chimeric antibody transactivation

[0259] For the specific experimental method, refer to step (f) of section 3.4 of Example 3. After the incubation of the antibody and cell system is completed, a luciferase reporter gene detection reagent is added and the luminescent signal is read on a microplate reader. The stronger the luminescent signal, the stronger the inverse activation effect of the antibody, and vice versa. The luminescent signal and the corresponding antibody concentration are subjected to a four-parameter fitting effect curve, and the inverse activation activity of the antibody is compared by comparing the IC50 and the multiple of activation enhancement relative to the negative control.

[0260] As shown in Table 6 and Figure 8, the results indicate that all chimeric antibodies can inhibit the activation of KIR3DL3 compared with the negative control Anti-Fluo-hIgG4, thereby enhancing the function of cell activation.

[0261] 4.8 Jurkat-NFAT-human TMIGD2 cell reporter gene assay to detect the biological activity of anti-B7-H7 chimeric antibodies in inhibiting activation

[0262] Specific experimental method refers to step (g) of section 3.4 in Example 3. After the system of antibody and cell is incubated, luciferase reporter gene detection reagent is added and the luminescent signal is read on a microplate reader. The weaker the luminescent signal, the stronger the inhibitory effect of the antibody, and vice versa. The inhibitory activity of the antibody is analyzed by comparing the IC50 and the inhibition rate using the luminescent signal and the corresponding antibody concentration through a four-parameter fitting effect curve.

[0263] As shown in Table 6 and Figure 9, the results showed that compared with the negative control Anti-Fluo-hIgG4, Mab001, Mab004 and the positive control antibody Hz2D7-hIgG4 had the biological function of inhibiting TMIGD2 activation, while the other chimeric antibodies could not inhibit TMIGD2 activation.

[0264] Table 6 Blocking activity of anti-B7-H7 chimeric antibodies against KIR3DL3

[0265] 4.9 Primary T cell activation assay to detect the biological activity of anti-B7-H7 chimeric antibodies

[0266] Human peripheral blood mononuclear cells (PBMCs) were resuscitated (purchased from Shanghai Saili Biotechnology Co., Ltd.) and cultured in complete medium (RPMI 1640-Glutamax medium + 10% FBS + 1% P / S + (1×) 2-β-mercaptoethanol). After overnight static culture, T cells were sorted using a sorting kit (Stemcell, Cat. No. 17951) and resuspended in complete medium to adjust the density to 0.5×10 6 / mL. CHO-K1-OKT3(ScFv)-human B7-H7 cells were collected as presenting cells and treated with 10μg / mL Mitomycin C (Selleckchem, Cat. No. S8146) at 37°C in the dark for 1 hour. After the reaction, the cells were terminated with complete medium and washed once, and the cell concentration was adjusted to 0.04×10 6 / mL, and anti-human CD28 antibody was added to the above cell-presenting cell suspension to a concentration of 4μg / mL (4× concentration). Anti-B7-H7 antibody and control antibody were diluted to 40μg / mL (4× concentration). To a 96-well U-bottom plate (Thermo, Cat. No. 163320), 50μL of antibody, 100μL of T cell suspension, and 50μL of Mitomycin C-treated CHO-K1-OKT3(ScFv)-human B7-H7 (cell suspension containing 4μg / mL of anti-human CD28 antibody) were added sequentially and gently aerated with a dispenser. The final drug concentration in this system was 10μg / mL, with 50,000 T cells per well and 2,000 CHO-K1-OKT3(ScFv)-human B7-H7 cells per well. The final anti-human CD28 antibody concentration was 1μg / mL. The positive control in this system was the Hz2D7-hIgG4 antibody, and the negative controls were the Anti-Hel-hIgG4 antibody and the no-treatment group (i.e., no drug addition). The cells were incubated at 37°C with 5% CO2 for 5 days. Anti-B7-H7-PE-Cy7 antibody (Invitrogen, Catalog No. 25-6537-42) was used to analyze B7-H7 expression on CHO-K1-OKT3(ScFv)-human B7-H7 cells by flow cytometry (Figure 10A). Figure 10A shows that CHO-K1-OKT3(ScFv)-human B7-H7 cells express high levels of human B7-H7. After incubation, the culture plates were centrifuged at 350g, and the supernatant was assayed for human IFN-γ levels using an ELISA kit (Dakoway, Catalog No. 1110003). As shown in Figure 11, after treatment with the anti-B7-H7 test antibodies, IFN-γ secretion in the cell supernatant was significantly higher than that in the negative control (no treatment group), with significant differences (****P<0.0001, analyzed using one-way anova analysis). All antibodies were superior to the positive control (Hz2D7-hIgG4). The percentages in each bar graph represent the percentage increase in IFN-γ secretion compared to the no treatment group.

[0267] 4.10 Anti-B7-H7 chimeric antibody promotes NK92MI cells to kill K562-human B7-H7 target cells

[0268] FACS was used to detect the expression of B7-H7 on the surface of K562-human B7-H7 target cells. First, K562-human B7-H7 cells were counted using a cell counter. Two flow cytometry tubes were taken and 1×10 5K562 human B7-H7 cells were washed twice with PBS, the supernatant discarded, and 100 μL of diluted live / dead stain, Zombie Violet (diluted 500-fold in PBS, Biolegend, Cat. No. 423114), was added to each tube, mixed, and incubated at room temperature for 15 minutes. The cells were then washed twice with staining buffer, and the supernatant discarded. Next, 100 μL of diluted PE / Cy7 Mouse anti-B7-H7 antibody staining solution was added to one tube, and an equivalent concentration of isotype control antibody, PE / Cy7 Mouse IgG1κ staining solution (Thermo, Cat. No. 25-4714-80), was added to the other tube. The cells were mixed, and the cells were incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with staining buffer, centrifuged, and 300 μL / tube of staining buffer was added and mixed. Flow cytometry (Thermo, Attune NxT) was used to analyze the PE / Cy7 signal of the Zombie Violet negative cell population. Figure 10B shows that K562-human B7-H7 cells have a high expression level of B7-H7 on their surface.

[0269] The expression of KIR3DL3 and TMIGD2 on NK92MI cells was detected by FACS. First, NK92MI cells were counted using a cell counter, and two flow cytometry tubes were taken, and 1×10 5NK92MI cells were washed twice with PBS, the supernatant discarded, and 100 μL of diluted live / dead stain, Zombie Violet (Biolegend, Catalog No. 77477), was added to each tube, mixed thoroughly, and incubated at room temperature for 15 minutes. The cells were then washed twice with staining buffer, the supernatant discarded, and 50 μL of diluted FcX blocker (diluted 50-fold with staining buffer, Biolegend, Catalog No. 422302) was added to each tube, mixed thoroughly, and incubated at 4°C for 15 minutes. Then, dye solution was added to each tube. 50 μL of 2× antibody dye solution (staining buffer + AF488-anti-KIR3DL3 detection antibody + PE Mouse anti-human TMIGD2 detection antibody (R&D, catalog number: FAB83162P-100)) was added to one tube, and 50 μL of isotype control antibody dye solution of the same mass concentration (staining buffer + AF488-labeled Anti-Hel-hIgG4 detection antibody (labeled using AF488 labeled antibody kit) + PE Mouse IgG2b detection antibody (Biolegend, catalog number 400314)) was added to the other tube. After mixing, the cells were incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with dyeing buffer, centrifuged, and 300 μL / tube of dyeing buffer was added and mixed. Zombie cells were detected and analyzed using a flow cytometer (Thermo, Attune NxT). Figures 12A and 12B show that NK92MI cells only express KIR3DL3 and barely express TMIGD2.

[0270] FACS was used to assess the mortality of target cells in a K562-B7-H7 (in-house constructed, see Section 2.1 of Example 2) target cell model using NK92MI cells, and the agonistic effect of the test antibodies on NK92MI cell cytotoxicity was evaluated. The cytotoxicity assay was performed as follows: NK92MI cells (Nanjing Kebai, Catalog No. CBP61113) and K562-human B7-H7 cells were collected in the logarithmic growth phase and counted using a cell counter (Beckman Coulter, Vi-Cell). The experimental system was cultured in NK92MI complete medium (Nanjing Kebai, Catalog No. CBP61113M). First, an appropriate number of K562-human B7-H7 cells were removed, washed twice with 2 mL of PBS, and centrifuged at 350 g for 5 minutes. After centrifugation and discarding the supernatant, pre-prepared CellTrace Violet (Invitrogen, Catalog No. C34557A) working solution was added to a final concentration of 5 μM. The target cell suspension containing CellTrace Violet dye was mixed by blowing thoroughly and placed in a 37°C incubator for 10 minutes, with gentle shaking once. After the reaction, 5 volumes of complete medium were added to the labeled cell suspension to terminate the reaction. After standing at room temperature for 5 minutes, the suspension was centrifuged at 350 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete medium and counted. The density was adjusted to 0.2 × 10 based on the count results. 6 During the target cell labeling period, anti-B7-H7 chimeric antibodies were prepared, and the test antibody was diluted to 80 μg / mL (4× final concentration) as the initial concentration working solution, and then 5-fold gradient dilution was performed for a total of 7 concentrations, so that the working solution concentrations of the test antibody (4× final concentration) were 80 μg / mL, 16 μg / mL, 3.2 μg / mL, 0.64 μg / mL, 0.128 μg / mL, 0.0256 μg / mL and 0.00512 μg / mL, respectively, for use. Next, NK92MI cells were collected and the density was adjusted to 1.2×10 6Cells / mL were added to a 96-well U-bottom plate (Corning, Cat. No. 3799). CellTrace Violet-labeled K562-human B7-H7 target cells (100 μL / well, 20,000 cells / well) and antibody working solution (50 μL / well, 4× final concentration) were added sequentially. After incubation for 15 minutes, NK92MI cells (50 μL / well, 60,000 cells / well) were added and mixed by pipetting. The final volume was 200 μL / well. A separate well containing target cells (100 μL / well K562-human B7-H7 + 100 μL / well experimental culture medium) was set up as a control group for spontaneous target cell death. The plate was placed in a 37°C, 5% CO2 incubator and the drug and cells were incubated for 4 hours. After the incubation, the 96-well plate was removed and centrifuged at 350 g for 5 minutes. 100 μL / well of the cell supernatant was aspirated with a pipette, and 20 μL / well of diluted PI stain (Propidium Iodide, diluted 20-fold with PBS, Invitrogen, Cat. No. P3566) was added to the plate, mixed, and incubated at room temperature for 5 minutes. The percentage of PI-positive cell populations among CellTrace Violet-positive cells was analyzed using a flow cytometer (BD, Canto II). The higher the percentage of PI-positive cells, the stronger the killing effect on NK92MI cells. As shown in Table 7 and Figure 13, compared with the negative control Anti-Hel-hIgG4, all tested antibodies blocked the interaction between KIR3DL3 on the surface of NK92MI cells and B7-H7 on the surface of K562-human B7-H7, demonstrating an effect of promoting NK92MI cell killing of K562-human B7-H7 target cells, which was comparable to the positive control Hz2D7-hIgG4.

[0271] Table 7 The promoting effect of anti-B7-H7 chimeric antibodies on NK92MI cells killing K562-human B7-H7 target cells Note: “NA” means no data.

[0272] Example 5. Humanization of anti-human B7-H7 monoclonal antibody

[0273] By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes, we selected heavy and light chain variable region germline genes with high homology to murine antibodies as templates. The CDRs of the murine antibodies were transplanted into the corresponding humanized templates, resulting in a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The sequences of the humanized light and heavy chain templates are shown in Table 8. As needed, key amino acids in the framework sequence were backmutated to their corresponding amino acids in the murine antibody to maintain the original affinity, resulting in humanized anti-B7-H7 monoclonal antibodies. The amino acid residues of the antibody CDRs are typically assigned and annotated using the Kabat numbering system. The constant region sequence was hIgG4 S228P (SEQ ID NO: 7).

[0274] Table 8 Humanized light chain and heavy chain template sequence list

[0275] 5.1 Humanization of Mab001

[0276] The humanized light chain templates for the murine antibody Mab001 are IGKV3-11*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV4-28*02 and IGHJ6*01. The CDRs of the murine antibody Mab001 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequence of the humanized Mab001 antibody were backmutated to their murine counterparts to maintain the original affinity. Specific backmutation designs are shown in Table 9.

[0277] Table 9 Back mutation design of humanized antibody Mab001 Note: Graft(IGKV3-11*01) represents the grafting of murine antibody CDRs into the human germline IGKV3-11*01 FR region sequence; Y53F indicates the Y to F mutation at position 53 of Graft, and so on. The backmutated amino acids are numbered in natural order.

[0278] The specific sequence of the variable region of Hab01-H1L7 humanized antibody is as follows:

[0279] The amino acid sequence of Hab01.VL7 is shown in SEQ ID NO: 157:

[0280] The amino acid sequence of Hab01.VH1 is shown in SEQ ID NO: 158:

[0281] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibody VH and VL are shown in Table 10.

[0282] Table 10 Kabat analysis results of Hab01 humanized antibody VH and VL sequences

[0283] 5.2 Humanization of Mab002

[0284] The humanized light chain templates for the murine antibody Mab002 are IGKV2-40*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV7-4-1*02 and IGHJ6*01. The CDRs of the murine antibody Mab002 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized Mab002 antibody were backmutated to their murine counterparts to maintain the original affinity. Antibodies contain sites susceptible to chemical modification, and point mutations were performed at these sites to eliminate this risk. Detailed backmutation designs are shown in Table 11.

[0285] Table 11 Humanized antibody backmutation design of Mab002 Note: Graft(IGKV2-40*01) represents the grafting of murine antibody CDRs into the human germline IGKV2-40*01 FR region sequence; Q50K indicates the Q to K mutation at position 50 of Graft, and so on. The backmutated amino acids are numbered in natural order.

[0286] The specific sequence of the variable region of Hab02-H2aL1b humanized antibody is as follows:

[0287] The amino acid sequence of Hab02.VL1b is shown in SEQ ID NO: 159:

[0288] The amino acid sequence of Hab02.VH2a is shown in SEQ ID NO: 160:

[0289] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibodies VH and VL are shown in Table 12.

[0290] Table 12 Kabat analysis results of Hab02 humanized antibody VH and VL sequences

[0291] 5.3 Humanization of Mab003

[0292] The humanized light chain templates for the murine antibody Mab003 are IGKV3-20*02 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-69-2*01 and IGHJ6*01. The CDRs of the murine antibody Mab003 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized Mab003 antibody were backmutated to their murine counterparts to maintain the original affinity. Antibodies contain sites susceptible to chemical modification, and point mutagenesis was performed at these sites to eliminate this risk. Detailed backmutation designs are shown in Table 13.

[0293] Table 13 Humanized antibody backmutation design of Mab003 Note: Graft(IGKV3-20*02) represents the grafting of murine antibody CDRs into the human germline IGKV3-20*02 FR region sequence; Y35F indicates the Y to F mutation at position 35 of Graft, and so on. The backmutated amino acids are numbered in natural order.

[0294] The specific sequence of the variable region of Hab03-H6aL1 humanized antibody is as follows:

[0295] The amino acid sequence of Hab03.VL1 is shown in SEQ ID NO: 163:

[0296] The amino acid sequence of Hab03.VH6a is shown in SEQ ID NO: 164:

[0297] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibody VH and VL are shown in Table 14.

[0298] Table 14 Kabat analysis results of Hab03 humanized antibody VH and VL sequences

[0299] 5.4 Humanization of Mab004

[0300] The humanized light chain templates for the murine antibody Mab004 are IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV3-7*01 and IGHJ6*01. The CDRs of the murine antibody Mab004 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized Mab004 antibody were backmutated to their murine counterparts to maintain the original affinity. Specific backmutation designs are shown in Table 15.

[0301] Table 15 Humanized antibody backmutation design of Mab004 Note: Graft(IGKV1-12*01) represents the grafting of murine antibody CDRs into the human germline IGKV1-12*01 FR region sequence; A43S indicates the A to S mutation at position 43 of Graft, and so on. The backmutated amino acids are numbered in natural order.

[0302] The specific sequence of the variable region of Hab04-H1L3 humanized antibody is as follows:

[0303] The amino acid sequence of Hab04.VL3 is shown in SEQ ID NO: 166:

[0304] The amino acid sequence of Hab04.VH1 is shown in SEQ ID NO: 167:

[0305] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibody VH and VL are shown in Table 16.

[0306] Table 16 Kabat analysis results of Hab04 humanized antibody VH and VL sequences

[0307] 5.5 Humanization of Mab005

[0308] The humanized light chain templates for the murine antibody Mab005 are IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ1*01. The CDRs of the murine antibody Mab005 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized Mab005 antibody were backmutated to their murine counterparts to maintain the original affinity. Specific backmutation designs are shown in Table 17.

[0309] Table 17 Humanized antibody backmutation design of Mab005 Note: Graft(IGKV1-39*01) represents the grafting of murine antibody CDRs into the human germline IGKV1-39*01 FR region sequence; I48V indicates the mutation of position 48 of Graft from I to V, and so on. The backmutated amino acids are numbered in natural order.

[0310] The specific sequence of the variable region of Hab05-H2L1 humanized antibody is as follows:

[0311] The amino acid sequence of Hab05.VL1 is shown in SEQ ID NO: 168:

[0312] The amino acid sequence of Hab05.VH2 is shown in SEQ ID NO: 169:

[0313] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibody VH and VL are shown in Table 18.

[0314] Table 18 Kabat analysis results of Hab05 humanized antibody VH and VL sequences

[0315] 5.6 Humanization of Mab006

[0316] The humanized light chain templates for the murine antibody Mab006 are IGKV6-21*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDRs of the murine antibody Mab006 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized Mab006 antibody were backmutated to their murine counterparts to maintain the original affinity. Specific backmutation designs are shown in Table 19.

[0317] Table 19 Humanized antibody backmutation design of Mab006 Note: Graft(IGKV6-21*01) represents the grafting of murine antibody CDRs into the human germline IGKV6-21*01 FR region sequence; K48Y indicates the mutation of K to Y at position 48 of Graft, and so on. The backmutated amino acids are numbered in natural order.

[0318] The specific sequence of the variable region of Hab06-H1L1 humanized antibody is as follows:

[0319] The amino acid sequence of Hab06.VL1 is shown in SEQ ID NO: 170:

[0320] The amino acid sequence of Hab06.VH1 is shown in SEQ ID NO: 171:

[0321] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibodies VH and VL are shown in Table 20.

[0322] Table 20 Kabat analysis results of Hab06 humanized antibody VH and VL sequences

[0323] 5.7 Humanization of Mab007

[0324] The humanized light chain templates for the murine antibody Mab007 are IGKV3-11*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDRs of the murine antibody Mab007 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized Mab007 antibody were backmutated to their murine counterparts to maintain the original affinity. Specific backmutation designs are shown in Table 21.

[0325] Table 21 Humanized antibody backmutation design of Mab007 Note: Graft(IGKV4-1*01) represents the grafting of murine antibody CDRs into the human germline IGKV4-1*01 FR region sequence; Y53I indicates the Y at position 53 of Graft was mutated to an I, and so on. The backmutated amino acids are numbered in natural order.

[0326] The specific sequence of the variable region of Hab07-H3L2 humanized antibody is as follows:

[0327] The amino acid sequence of Hab07.VL2 is shown in SEQ ID NO: 172:

[0328] The amino acid sequence of Hab07.VH3 is shown in SEQ ID NO: 173:

[0329] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibodies VH and VL are shown in Table 22.

[0330] Table 22 Kabat analysis results of Hab07 humanized antibody VH and VL sequences

[0331] 5.8 Humanization of Mab008

[0332] The humanized light chain templates for the murine antibody Mab008 are IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01. The CDRs of the murine antibody Mab008 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized Mab008 antibody were backmutated to their murine counterparts to maintain the original affinity. Antibodies contain sites susceptible to chemical modification, and point mutations were performed at these sites to eliminate this risk. Detailed backmutation designs are shown in Table 23.

[0333] Table 23 Humanized antibody backmutation design of Mab008 Note: Graft(IGKV4-1*01) represents the grafting of murine antibody CDRs into the human germline IGKV4-1*01 FR region sequence; P49S indicates the mutation of P at position 49 of Graft to S, and so on. The backmutated amino acids are numbered in natural order.

[0334] The specific sequence of the variable region of Hab08-H2L2a humanized antibody is as follows:

[0335] The amino acid sequence of Hab08.VL2a is shown in SEQ ID NO: 174:

[0336] The amino acid sequence of Hab08.VH2 is shown in SEQ ID NO: 175:

[0337] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibody VH and VL are shown in Table 24.

[0338] Table 24 Kabat analysis results of Hab08 humanized antibody VH and VL sequences

[0339] 5.9 Humanization of Mab009

[0340] The humanized light chain templates for the murine antibody Mab009 are IGKV2-29*02 / IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-69-2*01 and IGHJ6*01. The CDRs of the murine antibody Mab009 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized Mab009 antibody were backmutated to their murine counterparts to maintain the original affinity. Antibodies contain sites susceptible to chemical modification, and point mutagenesis was performed at these sites to eliminate this risk. Detailed backmutation designs are shown in Table 25.

[0341] Table 25 Humanized antibody backmutation design of Mab009 Note: Graft(IGKV2-29*02) represents the grafting of murine antibody CDRs into the human germline IGKV2-29*02 FR region sequence; I48F indicates the mutation of position 48 of Graft from I to F, and so on. The backmutated amino acids are numbered in natural order.

[0342] Hab09-H3L1 comprises a heavy chain variable region VH3 and a light chain variable region VL1, and Hab09-H4L2 comprises a heavy chain variable region VH4 and a light chain variable region VL2. The specific sequences of the humanized antibody variable regions are as follows:

[0343] The amino acid sequence of Hab09.VL1 is shown in SEQ ID NO: 177:

[0344] The amino acid sequence of Hab09.VL2 is shown in SEQ ID NO: 178:

[0345] The amino acid sequence of Hab09.VH3 is shown in SEQ ID NO: 179:

[0346] The amino acid sequence of Hab09.VH4 is shown in SEQ ID NO: 180:

[0347] The present disclosure selects different light chain and heavy chain sequences from the back mutation designs of the light chain and heavy chain variable regions of the above-mentioned Hab09 humanized antibody for cross-combination, and ultimately obtains a variety of Hab09 humanized antibodies.

[0348] According to the Kabat numbering system, the results of sequence analysis of the above humanized antibodies VH and VL are shown in Table 26.

[0349] Table 26 Kabat analysis results of Hab09 humanized antibody VH and VL sequences

[0350] Example 6. Identification of anti-B7-H7 humanized antibodies

[0351] 6.1 Preliminary Screening of Humanized Anti-B7-H7 Antibodies

[0352] The affinity of the above-mentioned anti-B7-H7 humanized antibodies to human B7-H7-His protein and the binding activity to NUGC-4 human gastric cancer cell line were determined by the Biacore method, and 10 anti-B7-H7 humanized antibodies were confirmed, namely Hab01-H1L7, Hab02-H2aL1b, Hab03-H6aL1, Hab04-H1L3, Hab05-H2L1, Hab06-H1L1, Hab07-H3L2, Hab08-H2L2a and Hab09-H3L1, Hab09-H4L2.

[0353] 6.2 BIAcore Detection of Affinity of Humanized B7-H7 Antibody to Human and Cynomolgus Monkey B7-H7 Protein

[0354] The specific binding of 10 humanized anti-B7-H7 antibodies to human and cynomolgus macaque B7-H7 proteins was assessed using a BIAcore 8K assay. Antibodies were captured using a Protein A chip (purchased from Cytiva, 29-1275-56). Human B7-H7-His (ACRO) and cynomolgus macaque B7-H7-His (ACRO) were diluted in equal gradients, and multi-cycle kinetics was used to measure antibody-antigen affinity. In each cycle, the antibodies were captured and then injected with gradients of human and cynomolgus macaque B7-H7 proteins, and the binding and dissociation processes between the antigen and antibody were recorded. After each cycle, the Protein A chip was regenerated with Glycine pH 1.5 (purchased from Cytiva, BR-1003-54) to remove the protein from the chip. Finally, the data were analyzed using a 1:1 binding model to fit the antibody-antigen binding kinetic parameters, including the association rate constant ka, dissociation rate constant kd, equilibrium dissociation constant KD, and maximum binding signal Rmax. The results in Table 27 indicate that all tested anti-B7-H7 antibodies specifically bind to human or cynomolgus monkey B7-H7 proteins with high affinity.

[0355] Table 27 Affinity of anti-B7-H7 humanized antibodies Note: “ / ” represents no data.

[0356] 6.3 Detection of Binding Activity of Anti-B7-H7 Humanized Antibody to Human B7-H7-His Protein

[0357] The specific experimental method is as described in step (a) of section 3.4 of Example 3. A gradient dilution of chimeric antibody was added to a protein-coated 96-well plate. After incubation, HRP-labeled goat anti-human antibody was added, and color was developed with TMB. The OD450 nm was read on a microplate reader.

[0358] As shown in Table 28 and Figure 14, the results indicate that the anti-B7-H7 humanized antibodies can effectively bind to human B7-H7-His compared to the negative control Anti-Fluo-hIgG4.

[0359] 6.4 ELISA assay for binding activity of anti-B7-H7 humanized antibodies to CHO-K1-human B7-H7 cells

[0360] For specific experimental procedures, refer to step (b) in section 3.4 of Example 3. After cell fixation, a gradient of diluted chimeric antibodies was added. After incubation, HRP-labeled Goat anti-human antibodies were added, and color was developed using TMB. The OD450nm was read on a microplate reader. As shown in Table 28 and Figure 15, the results demonstrate that the anti-B7-H7 humanized antibodies effectively bind to CHO-K1-human B7-H7 cells, compared to the negative control, Anti-Fluo-hIgG4.

[0361] 6.5 FACS detection of the binding activity of anti-B7-H7 humanized antibody to NUGC-4 human gastric cancer cell line

[0362] For specific methods, refer to step (c) of section 3.4 of Example 3. The collected cells were mixed with equal volumes of chimeric antibodies, and then Alexa Fluor was added after incubation. Cell samples were analyzed on a flow cytometer (BD, Canto II) using a 647 fluorescein-labeled goat anti-human secondary antibody (1:800). As shown in Table 28 and Figure 16, the anti-B7-H7 humanized antibody effectively bound to the NUGC-4 human gastric cancer cell line, compared to the negative control, Anti-Fluo-hIgG4.

[0363] 6.6 Binding activity assay of humanized anti-B7-H7 antibody to cynomolgus monkey B7-H7-His protein

[0364] For specific experimental methods, refer to Example 4.4. Cynomolgus monkey B7-H7-His protein was coated in a 96-well plate, and serially diluted humanized antibody was added. Then, HRP-labeled goat anti-human antibody was added. TMB colorimetric solution (50 μL / well) was used for incubation at room temperature for 5-10 minutes. The reaction was terminated by adding 1 M HCl (50 μL / well), and the OD at 450 nm was read using a microplate reader.

[0365] As shown in Table 28 and Figure 17, the results indicate that the anti-B7-H7 humanized antibodies can effectively cleave cynomolgus monkey B7-H7-His protein compared to the negative control Anti-Fluo-hIgG4.

[0366] 6.7 FACS detection of the binding activity of anti-B7-H7 humanized antibody to Flp-In-CHO-cynomolgus monkey B7-H7 cells

[0367] The cells were collected, washed once with PBS, and resuspended to 2 × 10 5 / 50μL, 1% BSA-PBS diluted the antibody to 100nM, 4-fold continuous gradient dilution to 8 concentration points, 50μL cells were mixed with 50μL antibody dilution solution, incubated at 4℃ for 60 minutes, washed twice with PBS, and added Alexa Cells were resuspended in 100 μL / well of 647 fluorescein-conjugated goat anti-human secondary antibody (1:800), incubated at 4°C for 40 minutes, washed twice with PBS, and resuspended in 100 μL / well of 1% BSA-PBS. Cell samples were analyzed on a flow cytometer (BD, Canto II). As shown in Table 28 and Figure 18, the results demonstrate that the anti-B7-H7 humanized antibody effectively binds to Flp-In-CHO-cynomolgus monkey B7-H7 cells, compared to the negative control, Anti-Fluo-hIgG4.

[0368] Table 28 Binding activity of anti-B7-H7 humanized antibodies

[0369] 6.8 FACS detection of anti-B7-H7 humanized antibody blocking the interaction between KIR3DL3-mFc and CHO-K1-human B7-H7

[0370] For specific experimental methods, refer to step (d) of section 3.4 of Example 3. The collected cells were mixed with equal volumes of humanized antibody and KIR3DL3-mFc (SEQ ID NO: 2, produced in-house) dilution, and Alexa Fluor was added. Cell samples were analyzed on a flow cytometer (BD, Canto II) using 647 fluorescein-conjugated goat anti-mouse secondary antibody (1:800). As shown in Table 29 and Figure 19, all humanized antibodies significantly blocked the binding of KIR3DL3-mFc protein to CHO-K1-human B7-H7 cells compared to the negative control, Anti-Fluo-hIgG4.

[0371] 6.9 FACS detection of anti-B7-H7 humanized antibody blocking the interaction between Bio-hTMIGD2-hFc and CHO-K1-human B7-H7

[0372] For specific experimental methods, refer to step (e) of section 3.4 of Example 3. The collected cells were mixed with equal volumes of humanized antibody and Bio-hTMIGD2-hFc diluent, and APC-labeled streptavidin was added (working dilution 1:1700). The cell samples were analyzed on a flow cytometer (BD, Canto II).

[0373] As shown in Table 29 and Figure 20, the results showed that compared with the negative control Anti-Fluo-hIgG4, Hab01-H1L7, Hab04-H1L3, Hab06-H1L1 and the control antibody Hz2D7-hIgG4 could completely block the binding of Bio-hTMIGD2-hFc to CHO-K1-human B7-H7, Hab02-H2aL1b, Hab09-H3L1, Hab09-H4L2, and Hab05-H2L1 showed partial blocking, and Hab03-H6aL1, Hab07-H3L2 and Hab08-H2L2a could not block the binding of Bio-hTMIGD2-hFc to CHO-K1-human B7-H7.

[0374] 6.10 Jurkat-IL-2-human KIR3DL3 cell reporter gene assay to detect the transactivation biological activity of humanized anti-B7-H7 antibodies

[0375] For the specific experimental method, refer to step (f) of section 3.4 of Example 3. After the incubation of the antibody and cell system is completed, a luciferase reporter gene detection reagent is added and the luminescent signal is read on a microplate reader. The stronger the luminescent signal, the stronger the inverse activation effect of the antibody, and vice versa. The luminescent signal and the corresponding antibody concentration are subjected to a four-parameter fitting effect curve, and the inverse activation activity of the antibody is compared by comparing the IC50 and the multiple of activation enhancement relative to the negative control.

[0376] As shown in Table 29 and Figure 21, the results indicate that all humanized antibodies can inhibit the activation of KIR3DL3 compared to the negative control Anti-Fluo-hIgG4, thereby enhancing the function of cell activation.

[0377] 6.11 Jurkat-NFAT-human TMIGD2 cell reporter gene assay to detect the biological activity of anti-B7-H7 humanized antibodies in inhibiting activation

[0378] For specific experimental methods, refer to step (g) of section 3.4 of Example 3. After the antibody and cell system is incubated, add the luciferase reporter gene detection reagent and read the luminescence signal on the microplate reader. Luciferase reporter gene assay system reagent, 50 μL / well, shake at 450 rpm for 10 minutes, and then read the luminescence signal on a microplate reader. Weaker luminescence signals indicate stronger inhibitory effects of the antibody, and vice versa. A four-parameter effect curve was fitted based on the luminescence signal and the corresponding antibody concentration. The inhibitory activity of the antibody was analyzed by comparing the IC50 value and inhibition rate.

[0379] As shown in Table 29 and Figure 22, the results indicate that, compared with the negative control Anti-Fluo-hIgG4, Hab01-H1L7, Hab04-H1L3 and the control antibody Hz2D7-hIgG4 have the biological function of inhibiting TMIGD2 activation, while other humanized antibodies cannot inhibit TMIGD2 activation.

[0380] Table 29 Effects of anti-B7-H7 humanized antibodies blocking KIR3DL3

[0381] 6.12 Primary T Cell Activation Assay to Detect the Biological Activity of Anti-B7-H7 Humanized Antibodies in Promoting T Cell Activation

[0382] For specific experimental methods, please refer to Section 4.9 of Example 4. The positive control in this system is the Hz2D7-hIgG4 antibody, and the negative control is the Anti-Hel-hIgG4 antibody and the No treatment group (i.e., no drug addition group). The level of human IFN-γ in the supernatant was detected using an ELISA kit (Dakoway, Cat. No.: 1110003). As shown in Figure 23, after the anti-B7-H7 test antibody was applied, the secretion of IFN-γ in the cell supernatant was significantly higher than that of the negative control No treatment group, and there was a significant difference (****P<0.0001, analyzed by One-way Anova Analysis). All antibodies were superior to the positive control Hz2D7-hIgG4 group. The percentage on each bar graph is the percentage of IFN-γ secretion increased compared to the No treatment group.

[0383] 6.13 Promoting Effect of Humanized Anti-B7-H7 Antibody on Primary NK Cell Activation

[0384] FACS was used to detect the expression of KIR3DL3 and TMIGD2 on primary human NK cells. First, NK cells were counted using a cell counter. Two flow cytometry tubes were taken and 1×10 5NK cells were washed twice with PBS, the supernatant discarded, and 100 μL of diluted Zombie Violet (500-fold diluted with PBS) was added to each tube, mixed thoroughly, and incubated at room temperature for 15 minutes. Subsequently, the cells were washed twice with staining buffer, the supernatant discarded, and 50 μL of diluted FcX blocker (Biolegend, Cat. No. 422302) was added to each tube, mixed thoroughly, and incubated at 4°C for 15 minutes. Then, dye solution was added to each tube. 50 μL of 2× antibody dye solution (staining buffer + AF488 anti-KIR3DL3 detection antibody + PE mouse anti-human TMIGD2 detection antibody) was added to one tube, and 50 μL of isotype control antibody dye solution of equal mass concentration (staining buffer + AF488-labeled anti-Hel-hIgG4 detection antibody + PE mouse IgG2b detection antibody) was added to the other tube. After mixing, the cells were incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with dyeing buffer, centrifuged, and 300 μL / tube of dyeing buffer was added and mixed. Flow cytometry (Thermo, Attune NxT) was used to analyze the AF488 and PE signals of the Zombie Violet-negative cell population. Figure 24 shows that KIR3DL3 and TMIGD2 receptors are co-expressed on the surface of NK cells.

[0385] FACS was used to detect the expression of B7-H7 on the surface of A375-human B7-H7 target cells. First, the target cells A375-B7-H7 (constructed in-house) were digested with trypsin. After the digestion was terminated by the culture medium, the cells were counted using a cell counter. Two flow cytometry tubes were taken, and 1×10 5 A375-human B7-H7 cells were washed twice with PBS, the supernatant discarded, and 100 μL of diluted Zombie Violet stain (500-fold diluted in PBS) was added to each tube, mixed, and incubated at room temperature for 15 minutes. The cells were washed twice with staining buffer, and the supernatant discarded. Next, staining solutions were added to each tube: 100 μL of diluted PE / Cy7 Mouse anti-B7-H7 detection antibody stain was added to one tube, and 100 μL of an isotype control antibody PE / Cy7 Mouse IgG1κ stain of equal concentration was added to the other tube. The cells were mixed, and incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with staining buffer, centrifuged, and 300 μL of staining buffer was added to each tube, mixed, and the Zombie Violet-negative cell population was analyzed using a flow cytometer (Thermo, Attune NxT). Figure 25 shows that A375-B7-H7 cells have high levels of B7-H7 expression on their surface.

[0386] After co-incubation of primary human natural killer (NK) cells and A375-human B7-H7 target cells (constructed in-house, see Section 2.1 of Example 2), FACS was used to detect the expression levels of CD107a on the NK cell surface and intracellular IFN-γ to indicate the effect of the test antibody on the NK cell activation function.

[0387] Human peripheral blood mononuclear cells (PBMCs, purchased from Stemexpress) were resuscitated one day in advance, NK cells were isolated (Stemcell, Catalog No. 17955), and stimulated overnight with 200 IU / mL of rh-IL-2 (R&D, Catalog No. 202-IL). The next day, the antibody was diluted to a maximum concentration of 20 μg / mL (4× the final concentration) in assay medium (RPMI1640-Glutamax medium + 10% FBS + 1× P / S + 1× β-mercaptoethanol). A four-fold serial dilution was then performed in assay medium, resulting in six working concentrations: 20 μg / mL, 5 μg / mL, 1.25 μg / mL, 0.3125 μg / mL, 0.078 μg / mL, and 0.02 μg / mL. The diluted antibody was added to a 96-well ultra-low-binding U-bottom plate (Costar, Catalog No. 7007) at a volume of 50 μL per well. Next, the target cells A375-B7-H7 were digested with trypsin. After the digestion was terminated, the cells were counted using a cell counter (Beckman Coulter, Vi-CELL). Based on the count results, an appropriate number of target cells were removed and centrifuged at 300 g for 5 minutes. The supernatant was discarded and the cells were resuspended in assay medium to a concentration of 0.6 × 10 6 50 μL / well of target cell suspension was added to the drug-coated 96-well U-bottom plate, mixed well, and incubated at room temperature for 15 minutes. During the incubation period, NK cells were collected and counted, and resuspended in assay medium to a density of 0.3×10 6 Cells were plated at a density of 100 cells / mL. A protein transport inhibitor (Invitrogen, Catalog No. 00498093) and an APC mouse anti-human CD107a detection antibody (Biolegend, Catalog No. 328620) were added to the cell suspension. After incubation, 100 μL / well of the NK cell suspension was added to the plate, resulting in each well containing 30,000 NK cells, 30,000 target cells, and a gradient of test antibody concentrations. Wells containing only NK cells served as resting controls, while wells containing both NK cells and target cells served as no-drug controls. Each well was mixed thoroughly and incubated in a 37°C incubator for 18 hours.

[0388] FACS staining was used to detect changes in CD107a expression and intracellular IFN-γ on NK cells: cells were transferred to a 96-well V-bottom plate (Thermo, Catalog No. 277143) in parallel, washed twice with PBS, and the supernatant discarded. 100 μL / well of diluted Live / Dead Fixable Near-IR stain (1000-fold diluted in PBS, Invitrogen, Catalog No. L34976) was added to each well and incubated at room temperature in the dark for 15-20 minutes. Cells were washed twice with staining buffer, centrifuged, and the supernatant discarded. 100 μL of diluted PE mouse anti-human CD56 detection antibody (Biolegend, Catalog No. 318306) was added to each well, mixed thoroughly, and incubated at 4°C for 30 minutes. Wash the cells twice with staining buffer, centrifuge, and discard the supernatant. Fix and permeabilize the cells using the Fixation / Permeabilization Kit (BD, Cat. No. 554714) for intracellular staining. First, add 100 μL of 1× Fixation / Permeabilization Solution to each well and incubate at 4°C for 20 minutes. Then, wash the cells twice with 1× Perm buffer, centrifuge, and discard the supernatant. Add 100 μL of diluted PE anti-human IFN-γ detection antibody (Biolegend, Cat. No. 506507) to each well, mix, and incubate at 4°C for 30 minutes. Wash the cells twice with 1× Perm buffer, centrifuge, and discard the supernatant. Then, add 150 μL of staining buffer to each well, resuspend, and analyze on an immunoassay (BD, Canto II).

[0389] The proportion of CD107a-positive cells and the proportion of intracellular IFN-γ-positive cells in CD56-positive cells were analyzed. The higher the proportion of CD107a-positive cells or the proportion of intracellular IFN-γ-positive cells, the higher the degree of NK cell activation. Figures 26 and 27 show that the negative control Anti-Hel-hIgG4 has no effect on CD107a on the surface of NK cells and intracellular IFN-γ. Hab04-H1L3 and Hab06-H1L1 antibodies showed partial inhibition of NK cell activation function. Other tested antibodies Hab01-H1L7, Hab02-H2aL1b, Hab03-H6aL1, Hab07-H3L2, Hab08-H2L2a, Hab09-H3L1, and Hab09-H4L2 can promote NK cell activation function to varying degrees.

[0390] 6.14 Anti-B7-H7 Humanized Antibody Promotes Killing of K562-Human B7-H7 Target Cells by NK92MI Cells

[0391] For specific experimental methods, refer to Section 4.10 of Example 4. K562-human B7-H7 cells were labeled with CellTrace Violet. A blank control group and a humanized antibody group were set up. PI stain was added and the control and drug-treated groups were detected by flow cytometry (BD, Canto II). The proportion of PI-positive cells among CellTrace Violet-positive cells was analyzed. A higher proportion of PI-positive cells indicates a stronger killing effect on NK92MI cells.

[0392] As shown in Figure 28 and Table 30, the tested antibodies Hab01-H1L7, Hab02-H2aL1b, Hab03-H6aL1, Hab04-H1L3, Hab05-H2L1, Hab06-H1L1, Hab07-H3L2, Hab08-H2L2a, Hab09-H3L1, and Hab09-H4L2 can all effectively block the binding of KIR3DL3 to B7-H7 on cells, and show the effect of promoting NK92MI cells to kill K562-human B7-H7 target cells, which is equivalent to the positive control Hz2D7-hIgG4.

[0393] Table 30: Effect of anti-B7-H7 humanized antibodies on the killing of K562-human B7-H7 target cells by NK92MI cells Note: “ / ” represents no data.

[0394] Example 7. Jurkat-IL-2-human KIR3DL-human PD1 cell reporter gene assay to detect the combined activity of anti-B7-H7 antibody and anti-PD1 / PDL1 antibody

[0395] One day before the experiment, CHO-K1-OKT3 (ScFv)-human HHLA-human PDL1 cells were collected and the cell density was adjusted to 2 × 10 5 / mL, and then the cell suspension was transferred to a 96-well white-bottom microplate (Corning, Cat. No. 3610), 2×10 4 / 100 μL / well, place the microplate with cells in a 37°C 5% CO2 incubator and culture overnight.

[0396] On the day of the experiment, the antibody was diluted to 1200 nM with culture medium and diluted 4-fold continuously to 8 concentration points. After removing the cell culture supernatant plated the day before, the antibody dilution solution was transferred to the microplate at 50 μL / well. The microplate with the sample was placed in a 37°C 5% CO2 incubator and incubated for 1 hour.

[0397] During the incubation, Jurkat-IL-2-human KIR3DL3-human PD1 cells were collected and the cell density was adjusted to 1×10 cells using cell culture medium containing 1 μg / mL anti-human CD28 antibody (Thermo, catalog number: 16-0289-85). 6 / mL, and at the end of the sample incubation time, Jurkat-IL-2-human KIR3DL3-human PD1 cell suspension was added to the microplate, 5×10 4 / 50μL / well, then place the microplate in a 37℃ 5% CO2 incubator and continue incubating for 5 hours. After the incubation, remove the microplate and add Nano- Luciferase reporter gene assay system reagent (Promega, catalog number: N1130), 50 μL / well, shaken at 450 rpm for 10 minutes, and then the luminescence signal was read on a microplate reader.

[0398] As shown in Figure 29, the anti-B7-H7 antibody synergistically activated the signaling of Jurkat-IL-2-human KIR3DL3-human PD1 reporter cells with either Keytruda or Tecentriq, significantly outperforming either agent alone. Furthermore, the synergistic effect of Hab02-H2aL1b with the anti-PD1 / PDL1 antibody was significantly superior to that of the control antibody Hz2D7-hIgG4 with the anti-PD1 / PDL1 antibody.

[0399] Example 8. Evaluation of the efficacy of anti-B7-H7 in a NUGC4 xenograft tumor mouse model reconstituted with human NK cell immunity

[0400] Human gastric cancer cell NUGC4 cells 1×10 7 2×10 human NK cells 7 After mixing, 0.2 mL of the mixture was inoculated subcutaneously on the right side of the back of female 5-6 week old B-NDG-IL15 mice (strain: B-NDG-IL15; Biocytogen Jiangsu Gene Biotechnology Co., Ltd.). 3 When the level of human CD56 reconstitution in the peripheral blood of mice exceeded 12%, 48 mice were randomly divided into 6 groups according to the tumor volume, namely: PBS control group (10 mL / kg, 8 mice), Hz2D7 group (20 mg / kg, 8 mice), Hab03-H6aL1 group (20 mg / kg, 8 mice), Hab02-H2aL1b group (20 mg / kg, 8 mice), Hab06-H1L1 group (20 mg / kg, 8 mice) and Hab04-H1L3 group (20 mg / kg, 8 mice). All groups were administered by intraperitoneal injection twice a week, and the experiment was terminated on the 14th day after administration. During the observation period, the body weight and tumor volume of the mice were measured twice a week, and the measured values ​​were recorded to calculate the tumor volume (long diameter × short diameter).2 / 2) and growth inhibition rate (TGI TV (%) = (1-(Tn-T0) / (Vn-V0) x 100%). Tn is the average tumor volume of the treatment group at each measurement, T0 is the average tumor volume of the treatment group at the time of grouping, Vn is the average tumor volume of the control group at each measurement, and V0 is the average tumor volume of the control group at the time of grouping.

[0401] Efficacy results: On the 14th day after group administration, the average tumor volume of mice in the PBS control group reached 418.18 mm 3 The average tumor volume of mice in the Hz2D7 control group reached 361.41 mm 3 There was no significant difference between the two groups. The average tumor volume of mice in the Hab03-H6aL1 group reached 326.72 mm 3 The average tumor volume of mice in the Hab02-H2aL1b group reached 269.26 mm. 3 The tumor volume of mice in the Hab06-H1L1 group was significantly lower than that in the PBS control group (P<0.0001), with a TGI of 46.58%. The average tumor volume in the Hab06-H1L1 group reached 231.15 mm 3 The tumor volume of mice in the Hab04-H1L3 group was significantly lower than that in the PBS control group (P<0.0001), with a TGI of 57.77%. The average tumor volume in the Hab04-H1L3 group reached 262.97 mm 3 , which was significantly lower than that of the PBS control group (P<0.0001), with a TGI of 47.47%. Among the four candidate molecules, Hab06-H1L1 showed the best efficacy, as detailed in Figure 30A and Table 31.

[0402] Table 31. Antitumor effect of humanized B7-H7 molecules on NUGC4 xenograft tumor model reconstituted with human NK cell immunity Note: a. Mean ± standard deviation (SD). b. Comparisons among the five groups in this experiment were performed using two-way ANOVA followed by multiple comparisons analysis. P values ​​for each group compared with the G1 PBS control group are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

[0403] Body weight results: 14 days after administration of the humanized B7-H7 molecule, the mice showed a steady upward trend in body weight. The mice were in good condition, with no mortality or other abnormalities, demonstrating that the mice tolerated the 20 mg / kg dose of humanized B7-H7. Detailed changes in body weight for each group are shown in Figure 30B and Table 32.

[0404] Table 32. Effects of humanized B7-H7 molecules on the body weight of mice in the NUGC4 xenograft tumor model reconstituted with human NK cell immunity Note: a. Mean ± standard deviation (SD).

[0405] The results showed that all tested humanized anti-B7-H7 molecules inhibited the growth of NUGC4 subcutaneous xenografts reconstituted with human NK cell immunity to a certain extent, with Hab06-H1L1 demonstrating the greatest tumor suppression. Furthermore, no abnormalities such as weight loss or mortality were observed in the mice during dosing, demonstrating that the humanized B7-H7 molecule is well tolerated in this model.

[0406] Example 9. Evaluation of the efficacy of anti-B7-H7 antibodies in a HCC827-B7-H7 xenograft mouse model reconstituted with human NK cell immunity

[0407] FACS was used to detect the expression of B7-H7 on the surface of HCC827-human B7-H7 target cells. First, the target cells HCC827-human B7-H7 were digested with trypsin. After the digestion was terminated by the culture medium, the cells were counted using a cell counter. Two flow cytometry tubes were taken and 1×10 5 HCC827 human B7-H7 cells were washed twice with PBS, the supernatant discarded, and 100 μL of the diluted live / dead stain Zombie Violet (500-fold diluted in PBS) was added to each tube, mixed, and incubated at room temperature for 15 minutes. The cells were washed twice with staining buffer, and the supernatant discarded. Next, staining solutions were added to each tube: 100 μL of the diluted PE / Cy7 mouse anti-B7-H7 detection antibody was added to one tube, and 100 μL of the isotype control antibody PE / Cy7 mouse IgG1κ was added to the other tube at the same concentration. The cells were mixed, and incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with staining buffer, centrifuged, and 300 μL of staining buffer was added / tube, mixed. The PE / Cy7 signal in the Zombie Violet-negative cell population was analyzed using a flow cytometer (Thermo, Attune NxT). FIG31 shows that HCC827-human B7-H7 cells have a high expression level of B7-H7 on their surface.

[0408] Human lung cancer HCC827-B7-H7 overexpression modified cells 1×10 7 1×10 human NK cells 7 After mixing, 0.2 mL of the mixture was inoculated subcutaneously on the right side of the back of female 5-6 week old B-NDG-IL15 mice (strain: B-NDG-IL15; Biocytogen Jiangsu Gene Biotechnology Co., Ltd.). 3, and when the level of human CD56 reconstitution in the peripheral blood of mice exceeded 20%, 16 mice were randomly divided into 2 groups according to the tumor volume, namely: PBS control group (10 mL / kg, 8 mice) and Hab02-H2aL1b group (20 mg / kg, 8 mice). All groups were administered by intraperitoneal injection twice a week, and the experiment was terminated on the 14th day after administration. During the observation period, the body weight and tumor volume of mice were measured twice a week, and the measured values ​​were recorded. The tumor volume (long diameter × short diameter) was calculated. 2 / 2) and growth inhibition rate (TGI TV (%)=(1-(Tn-T0) / (Vn-V0)×100%).

[0409] Efficacy results: On the 14th day after group administration, the average tumor volume of mice in the PBS control group reached 299.27 mm 3 The average tumor volume of mice in the Hab02-H2aL1b group reached 181.42 mm 3 , which was significantly lower than that of the PBS control group (P<0.0001), and the TGI was 58.88%, indicating that Hab02-H2aL1b had a good anti-tumor effect in this model. For detailed data, please see Figure 32A and Table 33.

[0410] Table 33. Anti-tumor effect of anti-B7-H7 antibodies on HCC827-B7-H7 xenograft tumor model reconstituted with human NK cell immunity Note: a. Mean ± standard deviation (SD). b. Comparisons between the two groups in this experiment were analyzed using two-way ANOVA. P values ​​for each group compared with the G1 PBS control group are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

[0411] Body weight results: 14 days after administration of the anti-B7-H7 antibody Hab02-H2aL1b, the mice maintained stable weight and were in good condition, with no mortality or other abnormalities, demonstrating that the mice tolerated the 20 mg / kg dose of the anti-B7-H7 antibody Hab02-H2aL1b. Detailed changes in body weight for each group are shown in Figure 32B and Table 34.

[0412] Table 34. Effects of anti-B7-H7 antibodies on body weight in HCC827-B7-H7 xenograft tumor model mice reconstituted with human NK cell immunity Note: a. Mean ± standard deviation (SD).

[0413] The results showed that the anti-B7-H7 antibody Hab02-H2aL1b significantly inhibited the growth of HCC827-B7-H7 subcutaneous xenografts reconstituted with human NK cell immunity. Furthermore, no abnormalities such as weight loss or mortality were observed in the mice during the dosing period, indicating that the anti-B7-H7 antibody Hab02-H2aL1b was tolerated in this model.

[0414] Example 10. Evaluation of the combined efficacy of anti-B7-H7 and anti-PD1 / PDL1 antibodies in a NUGC4 xenograft mouse model reconstituted with human PBMC immunity

[0415] Human gastric cancer cell NUGC4 cells were cultured at 1×10 7 The cells were inoculated subcutaneously on the right side of the back of female 5-6-week-old NPG mice (strain: NPG; Beijing Weitongda Biotechnology Co., Ltd.) at a concentration of 5×10 6 The concentration of 0.2 mL was injected into the tail vein of mice, and the tumor volume reached 82.9 mm. 3 When the number of human CD45 cells in the peripheral blood of mice reached more than 0.5%, 80 mice were randomly divided into 10 groups according to the tumor volume, namely: PBS control group (10 mL / kg, 8 mice), isotype anti-fluorescence control group (20 mg / kg, 8 mice), Hab03-H6aL1 group (20 mg / kg, 8 mice), Hab02-H2aL1b group (20 mg / kg, 8 mice), Keytruda (10 mg / kg, 8 mice), Tecentriq (10 mg / kg, 8 mice), and combination groups Hab02-H2aL1b 20 mpk + Keytruda 10 mpk (8 mice), Hab02-H2aL1b 20 mpk + Tecentriq 10 mpk (8 mice), Hab03-H6aL1 20 mpk + Keytruda 10 mpk (8 mice), Hab03-H6aL1 20mpk+Tecentriq 10mpk (8 mice). All groups were administered by intraperitoneal injection twice a week, and the experiment was terminated on the 17th day after administration. During the observation period, the body weight and tumor volume of mice were measured twice a week, and the measured values ​​were recorded. The tumor volume (long diameter × short diameter) was calculated. 2 / 2) and growth inhibition rate (TGI TV (%)=(1-(Tn-T0) / (Vn-V0)×100%).

[0416] Efficacy results: On the 17th day after group administration, the average tumor volume of mice in the PBS control group reached 903.26 mm 3The average tumor volume of mice in the Anti-fluo control group reached 778.78 mm 3 The average tumor volume of mice in the Keytruda control group reached 556.76 mm 3 , which was significantly smaller than that of the PBS control group (P=0.003), with a TGI of 33.49% and an average tumor volume of 564.11 mm in the Tecentriq control group. 3 , which was significantly smaller than that of the PBS control group (P=0.005), with a TGI of 31.47% and an average tumor volume of 808.08 mm in the Hab03-H6aL1 group. 3 , with little efficacy, and the average tumor volume of mice in the Hab02-H2aL1b group reached 548.01 mm 3 , which was significantly lower than that of the PBS control group (P=0.0007), and the TGI was 33.81%, which was comparable to the efficacy of Keytruda or Tecentriq.

[0417] Further analysis of the combination of anti-B7-H7 antibody and anti-PD-1 antibody Keytruda showed that the average tumor volume of mice in the Hab03-H6aL1 and Keytruda combination group reached 632.12 mm 3 , TGI was 21.58%, which showed no superior efficacy compared with single drug. The average tumor volume of mice in the Hab02-H2aL1b and Keytruda combination group reached 414.42 mm 3 , TGI was 53.74%, and the efficacy of the combination was significantly better than that of the two single drugs; at the same time, the combination of anti-B7-H7 antibody and anti-PDL1 antibody Tecentriq was analyzed. The average tumor volume of mice in the Hab03-H6aL1 and Tecentriq combination group reached 513.47 mm 3 , TGI was 38.8%, and the average tumor volume of mice in the Hab02-H2aL1b and Tecentriq combination group reached 478.29 mm 3 , TGI was 43.87%, indicating that the combined efficacy was superior to the efficacy of the two drugs alone. Detailed data are shown in Figures 33A and 33B and Table 35.

[0418] Table 35. Anti-tumor effect of anti-B7-H7 antibodies on NUGC4 xenograft tumor model reconstituted with human PBMC immunity Note: a. Mean ± standard deviation (SD). b. Comparisons among the 10 groups in this experiment were performed using two-way ANOVA followed by multiple comparisons analysis. P values ​​for each group compared with the G1 PBS control group are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

[0419] Body weight results: On day 17 after group dosing, mouse body weight continued to increase. One mouse in the Hab03-H6aL1 group died, while the other mice were in good condition with no other abnormalities. This indicates that mice tolerated the anti-B7-H7 antibody at a dose of 20 mg / kg and also tolerated the combination with the PD1 / PDL1 antibody. Detailed changes in mouse body weight for each group are shown in Figures 33C and 33D and Table 36.

[0420] Table 36. Effects of anti-B7-H7 antibodies on body weight of mice in the NUGC4 xenograft tumor model reconstituted with human PBMCs Note: a. Mean ± standard deviation (SD).

[0421] The final results showed that anti-B7-H7 antibodies had a certain degree of inhibitory effect on the growth of NUGC4 subcutaneous transplanted tumors reconstituted by human PBMC immunity. Among them, Hab02-H2aL1b had the best tumor inhibition effect and had a good synergistic effect with anti-PD1 / PDL1 antibodies.

[0422] Example 11. Evaluation of the efficacy of anti-B7-H7 antibodies in a human PBMC-immune reconstituted A375-B7-H7 xenograft mouse model

[0423] Human melanoma cells A375-B7-H7 were transformed (refer to section 2.1 of Example 2 for the transformation method) at a rate of 5×10 6 A375-B7-H7 cells were inoculated at a concentration of 5×10 cells / 0.1 mL on the right back of female 5-6 week old NPG mice (strain: NPG; Beijing Weitongda Biotechnology Co., Ltd.). 6 The concentration of 0.2 mL was injected into the tail vein of mice, and the mean tumor volume reached 89.09 mm 3When the number of human CD45 cells in the peripheral blood of mice reached 0.5% or more, 64 mice were randomly divided into 8 groups according to tumor volume: PBS control group (10 mL / kg, 8 mice), Hab02-H2aL1b group (5 mg / kg, 8 mice), Hab02-H2aL1b group (10 mg / kg, 8 mice), Hab02-H2aL1b group (20 mg / kg, 8 mice), Tecentriq (10 mg / kg, 8 mice), Keytruda (10 mg / kg, 8 mice), and combination groups: Hab02-H2aL1b 10 mpk + Keytruda 10 mpk, and Hab02-H2aL1b 10 mpk + Tecentriq 10 mpk. All groups were administered by intraperitoneal injection twice weekly, and the experiment ended on day 16 after dosing. During the observation period, the body weight and tumor volume of mice were measured twice a week, and the measured values ​​were recorded. The tumor volume (long diameter × short diameter) was calculated. 2 / 2) and growth inhibition rate (TGI TV (%)=(1-(Tn-T0) / (Vn-V0)×100%).

[0424] Efficacy results: On the 17th day after group administration, the average tumor volume of mice in the PBS control group reached 2552.95 mm 3 The average tumor volume of mice in the Hab02-H2aL1b 5mg / kg group reached 1566.32mm 3 The average tumor volume of mice in the Hab02-H2aL1b 10mg / kg group reached 1420.32mm. 3 The average tumor volume of mice in the Hab02-H2aL1b 20mg / kg group reached 1083.42mm. 3 , significantly lower than the PBS control group (P<0.0001), with a TGI of 59.56%, and one mouse's tumor disappeared. All three doses of Hab02-H2aL1b demonstrated significant efficacy in a dose-dependent manner. Detailed data are shown in Figures 34A, 34B, and 34C and Table 37.

[0425] Combination efficacy results with anti-PD1 / PDL1 antibodies: The average tumor volume of mice in the Tecentriq group reached 943.20 mm 3 The tumor volume in the Keytruda group was significantly lower than that in the PBS control group (P<0.001), with a TGI of 65.24%, and the tumor in one mouse disappeared. The average tumor volume in the Keytruda group reached 1685.92 mm 3The mean tumor volume in the Hab02-H2aL1b 10 mpk and Tecentriq 10 mpk combination group reached 1517.98 mm 3 The mean tumor volume in the Hab02-H2aL1b 10 mpk plus Keytruda 10 mpk combination group reached 631.44 mm 3 , which was significantly lower than that of the PBS control group (P<0.001), with a TGI of 77.84%, and better than the efficacy of keytruda alone and Hab02-H2aL1b 10mpk alone. At the same time, the tumor volume of two mice was smaller than the initial tumor volume when they were grouped, indicating that Hab02-H2aL1b and keytruda have a good synergistic effect.

[0426] Table 37. Anti-tumor effect of anti-B7-H7 antibodies on A375-B7-H7 xenograft tumor model reconstituted with human PBMC immune system Note: a. Mean ± standard deviation (SD). b. Comparisons among the eight groups in this experiment were performed using two-way ANOVA followed by multiple comparisons analysis. P values ​​for each group compared with the G1 PBS control group are shown in the table (ns: not significant, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001).

[0427] Body weight results: After 17 days of administration of the anti-B7-H7 antibody Hab02-H2aL1b and its combination with an anti-PD1 / PDL1 antibody, the mice showed a steady upward trend in body weight. The mice were in good condition, with no mortality or other abnormalities, demonstrating that the anti-B7-H7 antibody Hab02-H2aL1b was well tolerated at a dose of 20 mg / kg. Detailed changes in body weight for each group are shown in Figure 34D and Table 38.

[0428] Table 38. Effect of anti-B7-H7 antibodies on body weight of mice bearing NUGC4 xenograft tumor model Note: a. Mean ± standard deviation (SD).

[0429] The results demonstrated that the anti-B7-H7 antibody Hab02-H2aL1b significantly inhibited the growth of A375-B7-H7 subcutaneous xenografts reconstituted with human PBMCs in a dose-dependent manner. The anti-B7-H7 antibody Hab02-H2aL1b exhibited a strong synergistic effect with Keytruda. Furthermore, no abnormalities such as weight loss or mortality were observed in mice during the dosing period, indicating that mice in this model tolerated the anti-B7-H7 antibody Hab02-H2aL1b at a dose of 20 mg / kg, and also tolerated it in combination with a PD-1 / PD-L1 antibody.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human B7-H7 protein, wherein: The human B7-H7 protein comprises amino acid residues 1 to 341 as shown in SEQ ID NO: 1 or any fragment of amino acid residues 1 to 341 as shown in SEQ ID NO:

1.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof binds to one or more amino acids within the amino acid residues of an antigen fragment selected from: (1) amino acid residues 61 to 131 of SEQ ID NO: 1, or amino acid residues on human B7-H7 protein corresponding to amino acid residues 61 to 131 of SEQ ID NO: 1; (2) amino acid residues 138-222 of SEQ ID NO: 1, or amino acid residues on human B7-H7 protein corresponding to amino acid residues 138-222 of SEQ ID NO: 1; and (3) amino acid residues 235-328 of SEQ ID NO: 1, or amino acid residues on human B7-H7 protein corresponding to amino acid residues 235-328 of SEQ ID NO:

1.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof binds to one or more amino acids within the amino acid residues of an antigen fragment selected from: (1) amino acid residues 35-44, 45-49, 50-60, 61-67, 68-76, 77-85, 86-94, 95-101, 102-110, 111-116, 117-126, 127-137 of SEQ ID NO: 1, or the amino acid residues on the human B7-H7 protein corresponding to amino acid residues 35-44, 45-49, 50-60, 61-67, 68-76, 77-85, 86-94, 95-101, 102-110, 111-116, 117-126, 127-137 of SEQ ID NO: 1; (2) amino acid residues 138-143, 144-153, 154-162, 163-167, 168-176, 177-184, 185-193, 194-202, 203-213, 214-221 of SEQ ID NO: 1, or the amino acid residues on the human B7-H7 protein corresponding to amino acid residues 138-143, 144-153, 154-162, 163-167, 168-176, 177-184, 185-193, 194-202, 203-213, 214-221 of SEQ ID NO: 1; (3) amino acid residues 228-238, 239-251, 252-262, 263-268, 269-277, 278-287, 288-296, 297-314, 315-335 of SEQ ID NO: 1, or amino acid residues on human B7-H7 protein corresponding to amino acid residues 228-238, 239-251, 252-262, 263-268, 269-277, 278-287, 288-296, 297-314, 315-335 of SEQ ID NO:

1.

4. An antibody or antigen-binding fragment thereof that specifically binds to human B7-H7, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein: (1) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises LCDR1 of the VL domain as shown in any one of SEQ ID NOs: 22 to 30, 157, 159, 163, 166, 168, 170, 172, 174, 177 or 178, the LCDR2 comprises LCDR2 of the VL domain as shown in any one of SEQ ID NOs: 22 to 30, 157, 159, 163, 166, 168, 170, 172, 174, 177 or 178, and the LCDR3 comprises LCDR3 of the VL domain as shown in any one of SEQ ID NOs: 22 to 30, 157, 159, 163, 166, 168, 170, 172, 174, 177 or 178; and (2) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the HCDR1 of the VH domain as shown in any one of SEQ ID NOs: 13 to 21, 158, 160, 164, 167, 169, 171, 173, 175, 179 or 180, the HCDR2 comprises the HCDR2 of the VH domain as shown in any one of SEQ ID NOs: 13 to 21, 158, 160, 164, 167, 169, 171, 173, 175, 179 or 180, and the HCDR3 comprises the HCDR2 of the VH domain as shown in any one of SEQ ID NOs: HCDR3 of the VH domain represented by any one of sequences 13-21, 158, 160, 164, 167, 169, 171, 173, 175, 179 or 180.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The LCDR1, LCDR2 and LCDR3 are identified according to the Kabat numbering system and / or the IMGT numbering system.

6. The antibody or antigen-binding fragment thereof according to claim 4, wherein The HCDR1, HCDR2 and HCDR3 are determined according to the Kabat numbering system and / or the IMGT numbering system.

7. The antibody or antigen-binding fragment thereof according to claim 5 or 6, wherein The antibody or antigen-binding fragment thereof comprises the following LCDR1, LCDR2, LCDR3 combination and HCDR1, HCDR2, HCDR3 combination: (1) The LCDR1, LCDR2 and LCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination: and, (2) The heavy chain CDRs combination comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are determined according to the Kabat numbering system and the IMGT numbering system; the HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination: In particular, the antibody or its antigen-binding fragment comprises a combination of light chain CDRs and heavy chain CDRs selected from the following: L1+H1, L2+H2, L3+H3, L4+H4, L5+H5, L6+H6, L7+H7, L8+H8, L9+H9, L10+H10, L11+H11, L12+H12, L13+H13, L14+H14, L15+H15, L16+H16, L17+H17, L18+H18, L19+H19, L3+H20 or L20+H8, as well as a combination of CDRs having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence of the light chain and heavy chain CDRs combination.

8. An antibody or antigen-binding fragment thereof according to claim 7, wherein: (1) a light chain variable region sequence comprising a sequence as shown in SEQ ID NO: 22-30, 157, 159, 163, 166, 168, 170, 172, 174, 177 or 178, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence as shown in SEQ ID NO: 22-30, 157, 159, 163, 166, 168, 170, 172, 174, 177 or 178; and, (2) the heavy chain variable region sequence comprises a sequence as shown in SEQ ID NO: 13-21, 158, 160, 164, 167, 169, 171, 173, 175, 179 or 180, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to a sequence as shown in SEQ ID NO: 13-21, 158, 160, 164, 167, 169, 171, 173, 175, 179 or 180; Preferably, the antibody or antigen-binding fragment thereof has the following combination: (1) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 13, respectively; (2) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 14, respectively; (3) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 24 and SEQ ID NO: 15, respectively; (4) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 25 and SEQ ID NO: 16, respectively; (5) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 17, respectively; (6) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 27 and SEQ ID NO: 18, respectively; (7) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 28 and SEQ ID NO: 19, respectively; (8) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 29 and SEQ ID NO: 20, respectively; (9) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 30 and SEQ ID NO: 21, respectively; (10) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 157 and SEQ ID NO: 158, respectively. List; (11) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 159 and SEQ ID NO: 160, respectively; (12) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 163 and SEQ ID NO: 164, respectively; (13) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 166 and SEQ ID NO: 167, respectively; (14) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, respectively; (15) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171, respectively; (16) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 172 and SEQ ID NO: 173, respectively; (17) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 174 and SEQ ID NO: 175, respectively; (18) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 177 and SEQ ID NO: 179, respectively; (19) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 180, respectively; Or (20) has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the sequences shown in (1) to (19) above.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein Is chimeric, humanized or fully human.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein The antibody or antigen-binding fragment thereof can bind to human or monkey B7-H7.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein It may also contain any constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it contains the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4 with mutation; further, the antibody or antigen-binding fragment thereof is also coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioactive isotopes, chemotherapeutic drugs, cytotoxic agents or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from anthracyclines (doxorubicin), taxanes (taxanes), pyrrolobenzodiazepines, (pyrrolobenzodiazepine, PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane.

12. The antigen-binding fragment according to claim 11 is selected from one or more of F(ab')2, Fab', Fab, Fv, scFv, nanobody or affibody.

13. A multispecific antigen-binding molecule, wherein: The multispecific antigen-binding molecule comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and an antigen-binding molecule that binds to an antigen other than B7-H7, or comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and an antigen-binding molecule that binds to a B7-H7 epitope different from the B7-H7 epitope bound by the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12; Preferably, the other antigen-binding molecule is an antibody or an antigen-binding fragment thereof; Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific; Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

14. A chimeric antigen receptor (CAR), wherein: The chimeric antigen receptor comprises at least a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the multispecific antigen-binding molecule according to claim 13.

15. An immune effector cell, wherein: The immune effector cell expresses the chimeric antigen receptor of claim 14, or comprises a nucleic acid fragment encoding the chimeric antigen receptor of claim 14; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are preferably selected from cytotoxic T cells (CTLs), regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

16. An isolated nucleic acid fragment, wherein: The nucleic acid fragment encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the multispecific antigen-binding molecule according to claim 13, or the chimeric antigen receptor according to claim 14.

17. A recombinant vector, wherein: The vector comprises the isolated nucleic acid fragment of claim 16.

18. A host cell, wherein The host cell comprises the recombinant vector of claim 17; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

19. A method for preparing a product comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the multispecific antigen-binding molecule according to claim 13, the method comprising the steps of culturing the host cell according to claim 18, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell.

20. A method for preparing the immune effector cell of claim 15, the method comprising the step of introducing a nucleic acid fragment encoding the CAR of claim 14 into the immune effector cell, and optionally, the method further comprises the step of starting the immune effector cell to express the CAR of claim 14.

21. A pharmaceutical composition, wherein: The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to claims 1 to 12, the multispecific antigen-binding molecule according to claim 13, the immune effector cell according to claim 15, the nucleic acid fragment according to claim 16, the recombinant vector according to claim 17, or a product prepared according to the method of claim 19; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.

22. The pharmaceutical composition according to claim 21, wherein The pharmaceutical composition may further comprise an additional anti-tumor agent.

23. The pharmaceutical composition according to claim 22, wherein The anti-tumor agent comprises a PD-1 inhibitor or a PD-L1 inhibitor; preferably, the PD-1 inhibitor comprises pembrolizumab, nivolumab, cemiplimab, carrelizumab, sintilimab, toripalimab or tislelizumab, and the PD-L1 inhibitor comprises atezolizumab, avelumab or durvalumab.

24. A method for treating a tumor or cancer, wherein: The method comprises administering to a subject an effective amount of the antibody or antigen-binding fragment thereof of claims 1 to 12, the multispecific antigen-binding molecule of claim 13, the immune effector cell of claim 15, the nucleic acid fragment of claim 16, the recombinant vector of claim 17, the product prepared by the method of claim 19, or the pharmaceutical composition of any one of claims 21 to 23; preferably, the tumor or cancer is selected from a solid tumor, a hematological tumor, or a cancer infiltrating immune cells expressing a receptor of B7-H7.

25. The method according to claim 24, wherein: It also includes combination with a PD-1 inhibitor or a PD-L1 inhibitor; preferably, the PD-1 inhibitor includes pembrolizumab, nivolumab, cemiplimab, carrelizumab, sintilimab, toripalimab or tislelizumab, and the PD-L1 inhibitor includes atezolizumab, avelumab or durvalumab.

26. Use of the antibody or antigen-binding fragment thereof of claims 1 to 12, the multispecific antigen-binding molecule of claim 13, the immune effector cell of claim 15, the nucleic acid fragment of claim 16, the recombinant vector of claim 17, the product prepared according to the method of claim 19, or the pharmaceutical composition of any one of claims 21 to 23 in the preparation of a drug for treating tumors or cancer; preferably, the tumor or cancer is selected from a solid tumor, a hematological tumor, or a cancer infiltrating immune cells expressing a receptor of B7-H7.

27. A kit comprising the antibody or antigen-binding fragment thereof according to claims 1 to 12, the multispecific antigen-binding molecule according to claim 13, the immune effector cell according to claim 15, the nucleic acid fragment according to claim 16, the recombinant vector according to claim 17, the product prepared according to the method according to claim 19, or the pharmaceutical composition according to any one of claims 21 to 23.

28. A method for detecting the expression of B7-H7 in a biological sample, the method comprising contacting the biological sample with the antibody or antigen-binding fragment thereof according to claims 1 to 12 under reaction conditions in which a complex can be formed between the antibody or antigen-binding fragment thereof and B7-H7; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of B7-H7 in the sample.

29. Use of the antibody or antigen-binding fragment thereof according to claims 1 to 12 in the preparation of a B7-H7 detection reagent.

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