Antibodies specifically binding to CTLA4 and methods of making and uses thereof

Abstract

This invention provides antibodies that specifically bind to CTLA4 or their antigen-binding fragments, as well as their preparation methods and uses. The antibody is a fully human antibody that binds to CTLA4 with high affinity. It can not only specifically kill CTLA4-expressing cells through antibody-dependent cytotoxicity (ADCC), but can also be prepared as a bispecific T-cell conjugate (BiTE) to further enhance the killing effect on CTLA4-expressing cells. It can be used for the prevention or treatment of cancer and has broad clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to antibodies or antigen-binding fragments thereof that can specifically bind to the CTLA4 protein. This invention also relates to methods for preparing and using the antibodies. Background Technology

[0002] Cytotoxic T lymphocyte-associated antigen 4 (CTLA4), also known as CD152, belongs to the immunoglobulin superfamily and is highly homologous to the co-stimulatory molecule CD28, but their functions are opposite. CTLA4 shares ligands B7-1 (CD80) and B7-2 (CD86) with CD28, and CTLA4 has a significantly higher affinity for these ligands than CD28. Upon T cell activation, CTLA4 expression levels are upregulated. By competitively binding to B7 molecules with CD28, it effectively inhibits excessive T cell activation, thereby maintaining immune tolerance. Furthermore, regulatory T cells (Tregs) highly express CTLA4 and can indirectly suppress the immune response of effector T cells by inhibiting the function of antigen-presenting cells (APCs).

[0003] In the tumor microenvironment, overexpression of CTLA4 leads to inhibited activation of T cells infiltrating the tumor microenvironment, significantly suppressing the body's anti-tumor immune response. Simultaneously, the immunosuppressive function of Treg cells is further enhanced, helping tumor cells evade the immune system's surveillance and elimination. Therefore, blocking the CTLA4 signaling pathway can effectively relieve the body's immunosuppressive state and enhance the anti-tumor immune response. Currently, drug development strategies targeting CTLA4 are mainly based on the high affinity binding of anti-CTLA4 antibodies to the CTLA4 molecule, mediating the depletion or functional blockade of Treg cells, thereby enhancing T cell activation levels and the body's immune response to cancer. Specifically, the CTLA4 blocking effect can be achieved through multiple mechanisms: first, it prevents transendocytosis and increases the expression level of CD80 / CD86 on the surface of antigen-presenting cells (APCs), thereby enhancing the activation response of T cells; second, it directly exerts cytotoxic effects on Treg cells; and third, it induces tumor macrophages to exert anti-tumor effects through FcR-IV-mediated antibody-dependent cytotoxicity (ADCC).

[0004] Currently, the first approved monoclonal antibody targeting CTLA4 is ipilimumab, primarily used to treat advanced melanoma and significantly prolonging patient survival. It's important to note that the monotherapy response rate for CTLA4 monoclonal antibodies is approximately 10%-20%. Similar to other immunotherapies, some patients receiving this type of treatment can achieve long-term survival, a phenomenon known as the "long tail" effect. However, CTLA4 monoclonal antibody therapy also brings various side effects, mostly immune-related adverse events (irAEs). These adverse events stem from inflammatory responses triggered by overactivation of the immune system, commonly including colitis, hepatitis, and hypophysitis. Clinically, to improve treatment efficacy, a combination therapy of CTLA4 monoclonal antibodies and PD-1 / PD-L1 inhibitors (such as the "O+Y" regimen) is often used. While this combination significantly improves efficacy, it also leads to a corresponding increase in drug toxicity.

[0005] As a pioneering target in tumor immunotherapy, CTLA4, despite limitations in monotherapy with existing drugs such as limited efficacy and significant toxicity, remains irreplaceable in combination therapy. Therefore, there is an urgent need to develop superior, high-affinity CTLA4-targeting antibodies to further optimize treatment outcomes. Future research targeting CTLA4 will focus on optimizing drug design, reducing therapeutic toxicity, and precisely screening for beneficiaries. Among these, enhancing the binding affinity of antibodies to CTLA4 molecules, optimizing their killing-related mechanisms of action, and reducing their immunogenicity are crucial research directions for optimizing CTLA4-targeted drugs. Summary of the Invention

[0006] Therefore, the purpose of this invention is to address the shortcomings of existing technologies by providing antibodies or antigen-binding fragments that specifically bind to CTLA4, as well as their preparation methods and uses. The CTLA4-specific antibody provided by this invention can bind to CTLA4 with high affinity, and can not only specifically kill CTLA4-expressing cells through antibody-dependent cytotoxicity (ADCC), but can also be prepared into a bispecific T-cell conjugate (BiTE) to further enhance the killing effect on CTLA4-expressing cells. It can be used for the prevention or treatment of cancer and has broad clinical application prospects.

[0007] Furthermore, this invention employs natural human phage display library technology to screen and obtain fully human antibodies capable of specifically binding to CTLA4. The antibody sequences derived from this technology are directly obtained from a natural human antibody library, which can significantly enhance the accuracy of antibody antigen recognition, effectively reduce or even avoid immune rejection reactions, thereby improving the effectiveness and safety of tumor treatment and enhancing the overall anti-tumor effect.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the CTLA4 protein, said antibody or antigen-binding fragment comprising:

[0010] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0011] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 3,

[0012] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 4, and

[0013] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 5;

[0014] and / or

[0015] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions:

[0016] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 8.

[0017] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 9, and

[0018] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 10;

[0019] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0020] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0021] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 13,

[0022] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 14, and

[0023] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 15;

[0024] and / or

[0025] (b) Light chain variable regions containing the following three complementary determinant regions:

[0026] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 18.

[0027] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 19, and

[0028] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 20;

[0029] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0030] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0031] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 23,

[0032] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 24, and

[0033] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 25;

[0034] and / or

[0035] (b) Light chain variable regions containing the following three complementary determinant regions:

[0036] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 28.

[0037] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 29, and

[0038] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 30;

[0039] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0040] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0041] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 33,

[0042] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 34, and

[0043] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 35;

[0044] and / or

[0045] (b) Light chain variable regions containing the following three complementary determinant regions:

[0046] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 38.

[0047] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 39, and

[0048] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 40;

[0049] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0050] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0051] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 43,

[0052] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 44, and

[0053] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 45;

[0054] and / or

[0055] (b) Light chain variable regions containing the following three complementary determinant regions:

[0056] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 48.

[0057] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 49, and

[0058] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 50;

[0059] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0060] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0061] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 53,

[0062] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 54, and

[0063] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 55;

[0064] and / or

[0065] (b) Light chain variable regions containing the following three complementary determinant regions:

[0066] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 58.

[0067] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 59, and

[0068] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 60;

[0069] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0070] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0071] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 63,

[0072] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 64, and

[0073] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 65;

[0074] and / or

[0075] (b) Light chain variable regions containing the following three complementary determinant regions:

[0076] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 68.

[0077] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 69, and

[0078] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 70;

[0079] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0080] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0081] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 73,

[0082] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 74, and

[0083] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 75;

[0084] and / or

[0085] (b) Light chain variable regions containing the following three complementary determinant regions:

[0086] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 78.

[0087] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 79, and

[0088] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 80;

[0089] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0090] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0091] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 83,

[0092] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 84, and

[0093] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 85;

[0094] and / or

[0095] (b) Light chain variable regions containing the following three complementary determinant regions:

[0096] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 88.

[0097] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 89, and

[0098] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 90;

[0099] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0100] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0101] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 93,

[0102] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 94, and

[0103] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 95;

[0104] and / or

[0105] (b) Light chain variable regions containing the following three complementary determinant regions:

[0106] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 98.

[0107] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 99, and

[0108] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 100;

[0109] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0110] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0111] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 103,

[0112] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 104, and

[0113] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 105;

[0114] and / or

[0115] (b) Light chain variable regions containing the following three complementary determinant regions:

[0116] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 108.

[0117] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 109, and

[0118] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 110;

[0119] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0120] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0121] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 113,

[0122] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 114, and

[0123] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 115;

[0124] and / or

[0125] (b) Light chain variable regions containing the following three complementary determinant regions:

[0126] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 118.

[0127] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 119, and

[0128] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 120;

[0129] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0130] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0131] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 123,

[0132] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 124, and

[0133] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 125;

[0134] and / or

[0135] (b) Light chain variable regions containing the following three complementary determinant regions:

[0136] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 128.

[0137] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 129, and

[0138] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 130;

[0139] Alternatively, the antibody or its antigen-binding fragment may comprise:

[0140] (a) Heavy chain variable regions containing the following three complementary determinant regions:

[0141] (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 133,

[0142] (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 134, and

[0143] (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 135;

[0144] and / or

[0145] (b) Light chain variable regions containing the following three complementary determinant regions:

[0146] (iv) VL CDR1, whose amino acid sequence is SEQ ID NO: 138.

[0147] (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 139, and

[0148] (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 140.

[0149] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 3, VH CDR2 as shown in SEQ ID NO: 4, and VHCDR3 as shown in SEQ ID NO: 5; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 8, VL CDR2 as shown in SEQ ID NO: 9, and VL CDR3 as shown in SEQ ID NO: 10.

[0150] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 13, VH CDR2 as shown in SEQ ID NO: 14, and VH CDR3 as shown in SEQ ID NO: 15; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 18, VL CDR2 as shown in SEQ ID NO: 19, and VL CDR3 as shown in SEQ ID NO: 20.

[0151] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 23, VH CDR2 as shown in SEQ ID NO: 24, and VH CDR3 as shown in SEQ ID NO: 25; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 28, VL CDR2 as shown in SEQ ID NO: 29, and VL CDR3 as shown in SEQ ID NO: 30.

[0152] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 33, VH CDR2 as shown in SEQ ID NO: 34, and VH CDR3 as shown in SEQ ID NO: 35; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 38, VL CDR2 as shown in SEQ ID NO: 39, and VL CDR3 as shown in SEQ ID NO: 40.

[0153] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 43, VH CDR2 as shown in SEQ ID NO: 44, and VH CDR3 as shown in SEQ ID NO: 45; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 48, VL CDR2 as shown in SEQ ID NO: 49, and VL CDR3 as shown in SEQ ID NO: 50.

[0154] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 53, VH CDR2 as shown in SEQ ID NO: 54, and VH CDR3 as shown in SEQ ID NO: 55; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 58, VL CDR2 as shown in SEQ ID NO: 59, and VL CDR3 as shown in SEQ ID NO: 60.

[0155] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 63, VH CDR2 as shown in SEQ ID NO: 64, and VH CDR3 as shown in SEQ ID NO: 65; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 68, VL CDR2 as shown in SEQ ID NO: 69, and VL CDR3 as shown in SEQ ID NO: 70.

[0156] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 73, VH CDR2 as shown in SEQ ID NO: 74, and VH CDR3 as shown in SEQ ID NO: 75; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 78, VL CDR2 as shown in SEQ ID NO: 79, and VL CDR3 as shown in SEQ ID NO: 80.

[0157] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 83, VH CDR2 as shown in SEQ ID NO: 84, and VH CDR3 as shown in SEQ ID NO: 85; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 88, VL CDR2 as shown in SEQ ID NO: 89, and VL CDR3 as shown in SEQ ID NO: 90.

[0158] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 93, VH CDR2 as shown in SEQ ID NO: 94, and VH CDR3 as shown in SEQ ID NO: 95; and the light chain variable region of the antibody or its antigen-binding fragment includes: VLCDR1 as shown in SEQ ID NO: 98, VL CDR2 as shown in SEQ ID NO: 99, and VL CDR3 as shown in SEQ ID NO: 100.

[0159] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 103, VH CDR2 as shown in SEQ ID NO: 104, and VH CDR3 as shown in SEQ ID NO: 105; and the light chain variable region of the antibody or its antigen-binding fragment includes: VL CDR1 as shown in SEQ ID NO: 108, VL CDR2 as shown in SEQ ID NO: 109, and VL CDR3 as shown in SEQ ID NO: 110.

[0160] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 113, VH CDR2 as shown in SEQ ID NO: 114, and VH CDR3 as shown in SEQ ID NO: 115; and the light chain variable region of the antibody or its antigen-binding fragment includes: VL CDR1 as shown in SEQ ID NO: 118, VL CDR2 as shown in SEQ ID NO: 119, and VL CDR3 as shown in SEQ ID NO: 120.

[0161] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 123, VH CDR2 as shown in SEQ ID NO: 124, and VH CDR3 as shown in SEQ ID NO: 125; and the light chain variable region of the antibody or its antigen-binding fragment includes: VL CDR1 as shown in SEQ ID NO: 128, VL CDR2 as shown in SEQ ID NO: 129, and VL CDR3 as shown in SEQ ID NO: 130.

[0162] According to some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 133, VH CDR2 as shown in SEQ ID NO: 134, and VH CDR3 as shown in SEQ ID NO: 135; and the light chain variable region of the antibody or its antigen-binding fragment includes: VL CDR1 as shown in SEQ ID NO: 138, VL CDR2 as shown in SEQ ID NO: 139, and VL CDR3 as shown in SEQ ID NO: 140.

[0163] According to some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises or is any of the amino acid sequences shown in SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122 and 132; and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises or is any of the amino acid sequences shown in SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127 and 137.

[0164] According to some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment comprises or is any one of the nucleotide sequences shown in SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 and 131; the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment comprises or is any one of the nucleotide sequences shown in SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126 and 136.

[0165] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0166] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0167] (i) The amino acid sequence shown in SEQ ID NO: 2;

[0168] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 2; or

[0169] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2;

[0170] And / or,

[0171] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0172] (iv) The amino acid sequence shown in SEQ ID NO: 7;

[0173] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 7; or

[0174] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 7;

[0175] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0176] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0177] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0178] (i) The amino acid sequence shown in SEQ ID NO: 12;

[0179] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 12; or

[0180] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12;

[0181] And / or,

[0182] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0183] (iv) The amino acid sequence shown in SEQ ID NO: 17;

[0184] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 17; or

[0185] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17;

[0186] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0187] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0188] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0189] (i) The amino acid sequence shown in SEQ ID NO: 22;

[0190] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 22; or

[0191] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 22;

[0192] And / or,

[0193] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0194] (iv) The amino acid sequence shown in SEQ ID NO: 27;

[0195] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 27; or

[0196] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 27;

[0197] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0198] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0199] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0200] (i) The amino acid sequence shown in SEQ ID NO: 32;

[0201] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 32; or

[0202] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 32;

[0203] And / or,

[0204] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0205] (iv) The amino acid sequence shown in SEQ ID NO: 37;

[0206] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 37; or

[0207] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 37;

[0208] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0209] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0210] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0211] (i) The amino acid sequence shown in SEQ ID NO: 42;

[0212] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 42; or

[0213] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 42;

[0214] And / or,

[0215] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0216] (iv) The amino acid sequence shown in SEQ ID NO: 47;

[0217] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 47; or

[0218] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 47;

[0219] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0220] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0221] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0222] (i) The amino acid sequence shown in SEQ ID NO: 52;

[0223] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 52; or

[0224] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 52;

[0225] And / or,

[0226] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0227] (iv) The amino acid sequence shown in SEQ ID NO: 57;

[0228] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 57; or

[0229] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 57;

[0230] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0231] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0232] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0233] (i) The amino acid sequence shown in SEQ ID NO: 62;

[0234] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 62; or

[0235] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 62;

[0236] And / or,

[0237] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0238] (iv) The amino acid sequence shown in SEQ ID NO: 67;

[0239] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 67; or

[0240] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 67;

[0241] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0242] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0243] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0244] (i) The amino acid sequence shown in SEQ ID NO: 72;

[0245] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 72; or

[0246] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 72;

[0247] And / or,

[0248] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0249] (iv) The amino acid sequence shown in SEQ ID NO: 77;

[0250] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 77; or

[0251] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 77;

[0252] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0253] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0254] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0255] (i) The amino acid sequence shown in SEQ ID NO: 82;

[0256] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 82; or

[0257] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 82;

[0258] And / or,

[0259] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0260] (iv) The amino acid sequence shown in SEQ ID NO: 87;

[0261] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 87; or

[0262] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 87;

[0263] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0264] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0265] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0266] (i) The amino acid sequence shown in SEQ ID NO: 92;

[0267] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 92; or

[0268] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 92;

[0269] And / or,

[0270] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0271] (iv) The amino acid sequence shown in SEQ ID NO: 97;

[0272] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 97; or

[0273] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 97;

[0274] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0275] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0276] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0277] (i) The amino acid sequence shown in SEQ ID NO: 102;

[0278] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 102; or

[0279] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 102;

[0280] And / or,

[0281] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0282] (iv) The amino acid sequence shown in SEQ ID NO: 107;

[0283] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 107; or

[0284] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 107;

[0285] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0286] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0287] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0288] (i) The amino acid sequence shown in SEQ ID NO: 112;

[0289] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 112; or

[0290] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 112;

[0291] And / or,

[0292] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0293] (iv) The amino acid sequence shown in SEQ ID NO: 117;

[0294] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 117; or

[0295] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 117;

[0296] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0297] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0298] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0299] (i) The amino acid sequence shown in SEQ ID NO: 122;

[0300] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 122; or

[0301] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 122;

[0302] And / or,

[0303] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0304] (iv) The amino acid sequence shown in SEQ ID NO: 127;

[0305] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 127; or

[0306] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 127;

[0307] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0308] According to some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0309] (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0310] (i) The amino acid sequence shown in SEQ ID NO: 132;

[0311] (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 132; or

[0312] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 132;

[0313] And / or,

[0314] (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following:

[0315] (iv) The amino acid sequence shown in SEQ ID NO: 137;

[0316] (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 137; or

[0317] (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 137;

[0318] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0319] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 7.

[0320] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 17.

[0321] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 22, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 27.

[0322] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 37.

[0323] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 42, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 47.

[0324] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises or is the amino acid sequence shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises or is the amino acid sequence shown in SEQ ID NO: 57.

[0325] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 62, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 67.

[0326] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 72, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 77.

[0327] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 82, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 87.

[0328] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 92, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 97.

[0329] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises or is the amino acid sequence shown in SEQ ID NO: 102, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises or is the amino acid sequence shown in SEQ ID NO: 107.

[0330] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises or is the amino acid sequence shown in SEQ ID NO: 112, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises or is the amino acid sequence shown in SEQ ID NO: 117.

[0331] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises or is the amino acid sequence shown in SEQ ID NO: 122, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises or is the amino acid sequence shown in SEQ ID NO: 127.

[0332] According to some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 132, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 137.

[0333] According to some embodiments of the present invention, the antibody or its antigen-binding fragment further comprises:

[0334] (a) The heavy chain constant region of human immunoglobulin or a variant thereof, wherein the amino acid sequence of the variant has one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of the heavy chain constant region of the human immunoglobulin from which it is derived; and

[0335] (b) The light chain constant region of a human immunoglobulin or a variant thereof, wherein the amino acid sequence of the variant has up to 20 conserved substitutions compared to the amino acid sequence of the light chain constant region of the human immunoglobulin from which it is derived.

[0336] Preferably, the heavy chain constant region is the human IgG heavy chain constant region, more preferably the human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region, and even more preferably, it is the human IgG1 or human IgG4 heavy chain constant region.

[0337] Preferably, the light chain constant region is the κ light chain constant region.

[0338] According to some embodiments of the present invention, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, biantibody, and single-domain antibody.

[0339] According to some embodiments of the present invention, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a bispecific antibody, or a multispecific antibody; more preferably, the antibody is a fully human antibody.

[0340] In a second aspect, the present invention provides a bispecific or multispecific molecule comprising an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, wherein the bispecific or multispecific molecule specifically binds to the CTLA4 protein and additionally specifically binds to one or more other targets.

[0341] Preferably, the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity against a target other than the CTLA4 protein, preferably a second antibody.

[0342] Preferably, the bispecific or multispecific molecule further comprises other antibody or antigen-binding fragments that specifically bind to the CTLA4 protein epitope.

[0343] Preferably, the bispecific or multispecific molecule is a bispecific T-cell conjugate.

[0344] More preferably, the nucleotide sequence of the nucleic acid molecule encoding the bispecific T-cell connector is shown in SEQ ID NO: 142, 143, 144 or 145.

[0345] Thirdly, the present invention provides an isolated nucleic acid molecule that encodes an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or a bispecific or multispecific molecule as described in the second aspect of the present invention.

[0346] Fourthly, the present invention provides a carrier comprising the isolated nucleic acid molecules according to the third aspect of the present invention.

[0347] Preferably, the vector is a cloning vector or an expression vector.

[0348] More preferably, the vector is a cloning vector AbVec-hIgKappa, a cloning vector AbVec-hIgG1, or a recombinant viral vector.

[0349] More preferably, the viral backbone of the recombinant viral vector is derived from vaccinia virus strains such as Tian Tan, New York, Copenhagen, Canary, and Ankara, as well as adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, varicella-zoster virus vectors, respiratory syncytial virus, Seneca Forest virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, smallpox virus, molluscum contagiosum virus, sheep pox virus, reovirus, rotavirus, enterovirus, Seneca virus, poliovirus, Coxsackie virus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, capsular virus, alphavirus, eastern equine encephalitis virus, Sindbis virus, rubella virus, coronavirus, flavivirus, hepatitis C virus, Japanese encephalitis virus, and St. Louis virus. Louisiana encephalitis virus, Murray Valley fever virus, yellow fever virus, West Nile virus, Zika virus, dengue virus, Ebola virus, Marburg virus, arena virus, Lassa virus, lymphocytic choriomeningitis virus, Pichend virus, Junin virus, Machupo virus, Hantavirus, Rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, simian virus 5, measles virus, vesicular stomatitis virus, rabies virus, respiratory syncytial virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, simian immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Laureus sarcoma virus, human T-cell leukemia virus type 1, simian foamy virus, hepatitis B virus, hepatitis E virus, human papillomavirus or polyomavirus.

[0350] More preferably, the recombinant viral vector is the recombinant vaccinia virus Tian Tan strain; even more preferably, the recombinant vaccinia virus Tian Tan strain is a bispecific T-cell conjugate vaccinia virus Tian Tan strain; and even more preferably, the recombinant vaccinia virus Tian Tan strain comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NO: 142, 143, 144 or 145.

[0351] Fifthly, the present invention provides a host cell comprising a bispecific or multispecific molecule according to the second aspect of the present invention, an isolated nucleic acid molecule according to the third aspect of the present invention, or a vector according to the fourth aspect of the present invention.

[0352] Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing the antibody or antigen-binding fragment, bispecific antibody, or multispecific antibody; even more preferably, the host cell is a mammalian cell; even more preferably, the host cell is a human, mouse, sheep, horse, dog, or cat cell; most preferably, the host cell is a 293 cell, a 143TK-cell, or a CHO cell.

[0353] In a sixth aspect, the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof according to a first aspect of the present invention, comprising culturing a host cell according to a fifth aspect of the present invention under conditions allowing expression of the antibody or an antigen-binding fragment thereof according to a first aspect of the present invention, and recovering the antibody or an antigen-binding fragment thereof from the cultured host cell culture.

[0354] In a seventh aspect, the present invention provides an immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, and a therapeutic agent attached to said antibody or antigen-binding fragment thereof.

[0355] Preferably, the therapeutic agent is a cytotoxic agent.

[0356] Preferably, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, and any combination thereof.

[0357] Preferably, the immunoconjugate is an antibody-drug conjugate.

[0358] In an eighth aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to a first aspect of the present invention, a bispecific or multispecific molecule according to a second aspect of the present invention, or an immunoconjugate according to a seventh aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0359] Preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents.

[0360] Preferably, the additional pharmaceutically active agent is a drug with antitumor activity; more preferably, the additional pharmaceutically active agent is an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0361] Preferably, the antibody or its antigen-binding fragment, bispecific or multispecific molecule or immunoconjugate is provided as a separate component or as a component of the same composition with the additional pharmaceutically active agent.

[0362] In a ninth aspect, the present invention provides a kit containing an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0363] Preferably, the antibody or its antigen-binding fragment carries a detectable label, radionuclide, fluorescent dye, luminescent substance, or biotin; more preferably, the detectable label is an enzyme, and even more preferably horseradish peroxidase; even more preferably, the luminescent substance is a chemiluminescent substance.

[0364] Preferably, the kit further includes a second antibody that specifically recognizes the antibody or its antigen-binding fragment according to the first aspect of the invention.

[0365] Preferably, the second antibody further includes a detectable label, a radionuclide, a fluorescent dye, a luminescent substance, or biotin; more preferably, the detectable label is an enzyme, and even more preferably horseradish peroxidase; more preferably, the luminescent substance is a chemiluminescent substance.

[0366] In a tenth aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof according to a first aspect of the invention, or a bispecific or multispecific molecule according to a second aspect of the invention, or an immunoconjugate according to a seventh aspect of the invention, or a pharmaceutical composition according to an eighth aspect of the invention, in the preparation of a medicament for the prevention and / or treatment of tumors in a subject.

[0367] Preferably, the drug further comprises additional pharmaceutically active agents.

[0368] Preferably, the additional pharmaceutically active agent is a drug with antitumor activity; more preferably, the additional pharmaceutically active agent is an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus.

[0369] Preferably, the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma (preferably high-grade glioma), blastoma (preferably neuroblastoma), osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer (preferably small cell lung cancer and non-small cell lung cancer), gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, colorectal cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or hematologic malignancies.

[0370] Preferably, the subject is a mammal, more preferably a human.

[0371] The beneficial effects of this invention are as follows:

[0372] 1. Fully human antibody, low immunogenicity, high safety.

[0373] This invention employs natural human phage display library technology to screen and obtain fully human antibodies that specifically bind to CTLA4. The antibody sequences derived from this technology are directly obtained from a natural human antibody library, which can significantly enhance the accuracy of antibody antigen recognition, effectively reduce or even avoid immune rejection reactions, and improve the effectiveness and safety of tumor treatment.

[0374] 2. Significantly superior affinity compared to the control antibody Ipilimumab

[0375] The anti-human CTLA4 antibody provided by this invention has the advantage of high affinity. ELISA detection showed that the affinity of Hu023-3, Hu023-8, Hu023-12, Hu023-13, Hu023-14, Hu023-15, Hu023-19, Hu023-31, Hu023-33, Hu023-34, Hu023-36, Hu023-37, and Hu023-39 was higher than that of the control antibody Ipilimumab; SPR detection showed that the KD values ​​of Hu023-5, Hu023-8, and Hu023-14 for binding to CTLA4 were 1.33E-9 M, 2.03E-10 M, and 1.77E-10 M, respectively, indicating a significant increase in affinity.

[0376] 3. Stronger in vitro cell-killing activity

[0377] Compared to the control antibody ipilimumab, the Hu023-8 and Hu023-14 antibodies of the present invention exhibit significantly higher activity in in vitro ADCC-mediated killing of CTLA4-VERO cells. Furthermore, the BiTE protein prepared based on the Hu023-8, Hu023-14, and Hu023-19 antibodies of the present invention demonstrates significantly superior activity in in vitro T-cell-mediated killing of CTLA4-VERO cells compared to the BiTE protein derived from ipilimumab.

[0378] Furthermore, for the CTLA4-VERO cell line, the BiTE recombinant vaccinia virus prepared from the candidate antibodies Hu023-14, Hu023-19 and Hu023-30 of this invention can all mediate high killing activity of T cells in vitro. Among them, the BiTE recombinant vaccinia virus prepared from the Hu023-14 antibody of this invention also showed significantly higher T cell killing activity in CTLA4-VERO cells in vitro than the control antibody group.

[0379] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0380] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0381] Figure 1 This shows the test results of the supernatant of the ELISA screening clones binding to the antigen CTLA4.

[0382] Figure 2 The images show the expression vectors Cloning vector AbVec-hIgKappa (GenBank: FJ475056.1) and Cloning vector AbVec-hIgG1 (GenBank: FJ475055.1) used in this invention.

[0383] Figure 3 The data are flow cytometry data, showing the affinity analysis of the anti-CTLA4 antibody of the present invention binding to the VERO cell line overexpressing CTLA4.

[0384] Figure 4 shows the affinity results of the candidate antibody of the present invention for binding to CTLA4 detected by ELISA. Among them, Figure 4-A The affinity test results are for Hu023-1 to Hu023-13. Figure 4-B The affinity test results are for Hu023-14 to Hu023-26. Figure 4-C The affinity test results are for Hu023-27 to Hu023-39.

[0385] Figure 5 The results show the affinity of the candidate antibodies Hu023-8 and Hu023-14 of this invention for binding to CTLA4, as determined by flow cytometry.

[0386] Figure 6 The results show the affinity of control antibodies Ipilimumab, Hu023-5, Hu023-8, and Hu023-14 to CTLA4, respectively, using the SPR method.

[0387] Figure 7 This demonstrates the results of ADCC mediated by the candidate antibody of the present invention.

[0388] Figure 8 This demonstrates the cytotoxicity results of the BiTE protein prepared using the candidate antibody sequence of the present invention.

[0389] Figure 9 shows the cytotoxicity results of recombinant vaccinia virus prepared using BiTE with the Hu023-14 sequence of the candidate antibody of the present invention; Figure 9-A Image of the pSC65 vector of vaccinia virus; Figure 9-B Results of T cell-mediated killing of CTLA4-VERO cells. Detailed Implementation

[0390] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0391] The term "antigen-binding fragment" in this article refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Antibody fragments can be Fab, F(ab')2, scFv, SMIP, biantibodies, triantibodies, affibody, nanobody, aptamer, or domain antibody.

[0392] The term "bispecific T-cell engager (BiTE)" in this article refers to a class of bispecific antibodies with significant anti-tumor effects, capable of targeting and activating autologous T cells to kill tumor cells. BiTE consists of two single-chain variable fragments (scFvs) linked tandemly by a flexible fusion linker. One scFv recognizes the T cell surface protein CD3ε, while the other recognizes a specific tumor cell surface antigen. This structure and specific protein-binding ability of BiTE allow it to physically bridge T cells to tumor cells, forming a T cell-BiTE-tumor cell complex, inducing immune synapse formation, stimulating T cell activation, and producing tumor-killing cytokines.

[0393] The term “monospecific” in this article refers to having one or more binding sites, where each binding site binds to the same epitope of the same antigen.

[0394] The term "multispecific" in this article refers to having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Therefore, terms such as "bispecific," "trispecific," and "quadrispecific" refer to the number of different epitopes that an antibody / antigen binding molecule can bind to.

[0395] The term “Kabat numbering system” in this article generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0396] The term "humanized antibody" in this article refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to strengthen immune responses.

[0397] The term "fully human antibody" as used herein refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Fully human antibodies as used herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, the term "fully human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0398] The term "conservative amino acid" in this article 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). The following six groups are examples of amino acids considered to be mutually conserved substitutions:

[0399] 1) Alanine (A), Serine (S), Threonine (T);

[0400] 2) Aspartic acid (D), glutamic acid (E);

[0401] 3) Asparagine (N), glutamine (Q);

[0402] 4) Arginine (R), Lysine (K), Histidine (H);

[0403] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0404] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0405] In this invention, "sequence identity" between two polypeptide or polynucleotide sequences typically indicates the percentage of identical amino acids or nucleotides between the sequences. "Sequence identity" indicates the percentage of amino acids or nucleotides substituted with the same amino acid or nucleotide. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST procedure of the NCBI database can be used to determine identity.

[0406] Example 1: Construction and screening of natural human antibody phage display libraries

[0407] CTLA4 antigen: Human recombinant CTLA4 protein was purchased from Suzhou Nearshore Protein Technology Co., Ltd., catalog number: CP33.

[0408] The screening of fully human antibodies against CTLA4 was commissioned to Sanyou Biopharmaceutical (Shanghai) Co., Ltd. Using the company's trillion-capacity fully human phage single-chain antibody (scFv) library, three rounds of solid-phase affinity panning were conducted with recombinant human CTLA4 protein as the target.

[0409] Clones obtained in the third round were selected for initial ELISA screening. Nine plates were used for the initial ELISA screening, yielding a total of 798 single clones. 305 positive clones binding to the human CTLA4 antigen were obtained, and 217 positive clones with an OD value above 0.8 were selected for sequencing analysis. Sequencing analysis yielded 42 unique sequence molecules. From these, 39 molecules were selected for full-length vector construction. The remaining 3 molecules were not used for full-length vector construction because they were predicted to contain sites unfavorable to post-translational modifications or had low sequence diversity.

[0410] The specific implementation method is as follows:

[0411] 1.1 Sequencing and Analysis of Positive Clones

[0412] After initial screening, 217 positive clones capable of binding to the CTLA4 protein were numbered. 2 μL of bacterial culture from each positive clone was transferred to 2 mL of 2×YT medium (17 g peptone, 10 g yeast extract, 5 g sodium chloride, water to 1000 mL) and incubated overnight at 37°C and 220 rpm. Plasmids were then extracted for first-generation sequencing. The sequencing results were used to integrate, align, and remove non-antibody gene sequences from the original AB1 file using SeqMan, generating an integrated antibody gene FASTA file. The DNA sequences were then translated into amino acid sequences using MEGA6, and terminators and non-standard sequences were identified. The FASTA file of the amino acid sequences was then derived, yielding the antibody light and heavy chain sequences of the positive antibody clones. Analysis and alignment confirmed that the sequences were novel antibody sequences, resulting in 42 unique sequence molecules.

[0413] 1.2 Detection of binding between ELISA screening clone supernatant and antigen CTLA4

[0414] 2 μg / mL CTLA4 protein was coated onto an ELISA plate at 4°C for 12 hours. The next day, the coating solution was discarded, the plate was patted dry on sterile absorbent paper, washed three times with 0.05% PBST, and then 350 μL of blocking buffer was added. The plate was incubated at 37°C for 1 hour. Clones selected in the third round of screening were picked and placed in 96-well deep-well plates containing 300 μL of 2-YT medium. The plates were incubated overnight at 37°C. The supernatant contained expressed Fab. The supernatant was serially diluted, the blocking buffer was discarded, the plate was patted dry on sterile absorbent paper, washed three times with 0.05% PBST, and the centrifuged monoclonal supernatant and 2% skim milk powder were added to the corresponding ELISA wells at a 1:1 ratio (100 μL / well). The plates were incubated at 37°C for 1 hour. The supernatant was discarded, and the plate was incubated with 0.1% PBST. Wash 5 times with PBST, then use HRP-labeled goat anti-human Fab as the second antibody (goat anti-human-HRP, Thermo Fisher Scientific, catalog number: 31482, 1:6000 dilution) for detection. A higher signal value (OD450) indicates stronger affinity. Results are as follows: Figure 1 As shown, in the ELISA assay, the Fab values ​​of all 39 antibodies exhibited good affinity activity. The OD450 values ​​of the Fab values ​​of 30 antibodies against the CTLA4 protein were all above 2.0, showing a difference of more than 20-fold from the background, indicating high affinity activity.

[0415] Example 2 Construction, expression, and purification of full-length antibodies

[0416] 2.1 Plasmid preparation

[0417] From the 39 antibody-containing strains obtained through screening, the variable regions of the light and heavy chains of the antibodies were amplified by PCR. These variable regions were then constructed into eukaryotic expression vectors containing the constant regions of the light and heavy chains, namely Cloningvector AbVec-hIgKappa (GenBank: FJ475056.1) or Cloning vector AbVec-hIgG1 (GenBank: FJ475055.1). The plasmid maps are shown below. Figure 2 The complete antibody light and heavy chain genes are composed of these genes.

[0418] The nucleotide sequences of the nucleic acid molecules encoding the heavy chain variable regions of antibodies Hu023-3, Hu023-8, Hu023-12, Hu023-13, Hu023-14, Hu023-15, Hu023-19, Hu023-30, Hu023-31, Hu023-33, Hu023-34, Hu023-36, Hu023-37, and Hu023-39 are shown in SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, and 131, respectively, and their corresponding amino acid sequences are shown in SEQ ID NO: The nucleotide sequences of the nucleic acid molecules encoding the light chain variable regions of antibodies Hu023-3, Hu023-8, Hu023-12, Hu023-13, Hu023-14, Hu023-15, Hu023-19, Hu023-30, Hu023-31, Hu023-33, Hu023-34, Hu023-36, Hu023-37, and Hu023-39 are shown in SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, and 136, respectively, and their corresponding amino acid sequences are shown in SEQ ID NO: As shown in 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127 and 137.

[0419] The constructed vector containing the full-length antibody light and heavy chain genes was transformed into E. coli TOP10 (Video, catalog number DL1010S) and cultured overnight at 37°C. The plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, catalog number D6950-01) to obtain endotoxin-free antibody light and heavy chain plasmids for eukaryotic expression.

[0420] 2.2 Expression of candidate antibodies

[0421] The candidate antibodies Hu023-1 to Hu023-39 and the control antibody ipilimumab (commonly known as drug Y) were expressed using the Expi293 transient expression system (Thermo Fisher Scientific, catalog number A1435101). The specific method is as follows:

[0422] On the day of transfection, the cell density was confirmed to be approximately 7E6 to 1E7 viable cells / mL, with a cell viability >98%. At this point, the cell concentration was adjusted to a final concentration of 6 × 10E6 cells / mL using fresh Expi293 expression medium pre-warmed at 37°C. The target plasmid was diluted with Expi293™ Expression Medium pre-cooled at 4°C (10 μg of plasmid was added to 1 mL of the medium). Simultaneously, FectoPro (Polyplus, catalog number PT-116-001) transfection reagent was diluted with Expi293™ Expression Medium. The two were then mixed in equal volumes and gently pipetted to prepare the Expi293™ Expression Medium / plasmid DNA mixture. The mixture was incubated at room temperature for 15 minutes and then slowly added to the prepared cell suspension while gently shaking. Finally, the mixture was placed in a cell culture shaker and cultured at 37°C and 5% CO2. 18-22 hours after transfection, add FectoPRO booster to the culture medium to a concentration of 0.6 μL / mL, and then place the shake flask in a shaker at 37°C and 5% CO2 for further culture. On day 5 after transfection, add the same volume of Expi293™ Expression Medium, adding it slowly while gently mixing the cell suspension. 7-15 days after transfection, centrifuge the cell culture supernatant expressing the target protein at 4000 g for 10 minutes to remove cell debris, thus obtaining the antibody expression supernatant.

[0423] 2.3 Purification of candidate antibodies

[0424] The antibody in the expression supernatant was purified by affinity using a Protein G agarose column (GE, catalog number 28903134). The specific method is as follows:

[0425] Centrifuge at 200 g for 20 seconds to remove the protective buffer (20% ethanol) from the Protein G agarose column. Add 600 µL of PBS buffer and centrifuge at 200 g for 20 seconds to wash away any remaining protective buffer. Add 600 µL of PBS buffer, mix well, and add the mixed Protein G agarose to the expression supernatant. Incubate at 4°C in a four-dimensional shaker for 6 hours. After incubation, centrifuge at 1000 g for 5 minutes at 4°C to remove the expression supernatant. Transfer the Protein G agarose to the column, add 600 µL of PBS buffer to wash the Protein G agarose, and centrifuge at 200 g for 20 seconds to remove the supernatant. Repeat this washing process three times. Elute the target protein with 400 µL of 100 mM glycine (pH 2.9), centrifuge at 200 g for 20 seconds, and then neutralize the elution buffer with 10 µL of 1M Tris-HCl (pH 9.0). Repeat this process once more. The neutralized eluent was ultrafiltered through a 30 kD ultrafiltration concentrator (Millipore, catalog number UFC901096), and the resulting protein was replaced in PBS buffer at a dilution factor >1000.

[0426] 2.4 Antibody Concentration Determination

[0427] The relative molecular weight and purity of 39 candidate antibodies Hu023-01~39 and the control antibody Ipilimumab were determined by SDS-PAGE. The concentration of the purified antibody protein was determined using a validated micro-volume spectrophotometer (Thermo Fisher NanoDrop One C). The A280 value was divided by the theoretical extinction coefficient of the antibody and used as the antibody concentration value for subsequent studies. After passing quality control, the antibody was aliquoted and stored at -80℃.

[0428] Example 3: Test for specific binding of candidate antibodies to CTLA4-expressing target cells

[0429] The affinity of candidate antibodies for specific binding to CTLA4-expressing target cells was determined by flow cytometry. The VERO cell line overexpressing CTLA4 protein (CTLA4-VERO) was used as the target cell line for flow cytometry analysis using the fluorescently labeled antibody PE anti-human IgG Fc Antibody (BioLegend, catalog number 410708). The specific method is as follows:

[0430] CTLA4-VERO cells were cultured in DMEM medium (10% FBS, 1% penicillin / streptomycin) in cell culture flasks. After culturing at 37°C and 5% CO2 for 48 hours, the supernatant was discarded, and the cells were collected by digestion with 0.25% trypsin and then centrifuged at 800g for 3 minutes.

[0431] Discard the supernatant and resuspend the cells in 2% FBS wash buffer (1E6 / mL). Each group contains 1E5 cells. Add the candidate antibodies Hu023-1~39 of this invention to each group and incubate at room temperature for 15 minutes. Centrifuge at 800g for 3 minutes, discard the supernatant, and wash the cells with 200µL of 2% FBS wash buffer. Centrifuge again and discard the supernatant. For the control group, resuspend the cells in 200µL of 2% FBS wash buffer. For the candidate antibody group, add a 200-fold diluted fluorescently labeled secondary antibody PE anti-human IgG Fc Antibody (BioLegend, catalog number 410708) to a final volume of 50µL and continue incubating at room temperature in the dark for 15 minutes. Centrifuge at 800g for 3 minutes, discard the supernatant, and resuspend the cells in 200µL of 2% FBS wash buffer. Analyze the cells along with the control group. Higher signal values ​​indicate stronger affinity.

[0432] The results of the candidate antibody's specific binding to CTLA4-expressing target cells are as follows: Figure 3 All 39 candidate antibodies were able to bind to target cells. Among them, Hu023-1, Hu023-3, Hu023-5, Hu023-8, Hu023-9, Hu023-11, Hu023-14, Hu023-16, Hu023-19, Hu023-20, Hu023-22, Hu023-24, Hu023-25, Hu023-26, Hu023-27, and Hu023-29 showed higher affinity for target cells than the control antibody.

[0433] Example 4: ELISA was used to determine the affinity of the candidate antibody for binding to CTLA4.

[0434] 2 μg / mL CTLA4 protein was coated onto an ELISA plate and coated at 4°C for 12 hours. The next day, the coating solution in the ELISA plate was discarded, the plate was patted dry on sterile absorbent paper, washed three times with 0.05% PBST, and then 350 μL of blocking buffer was added. After incubation at 37°C for 1 hour, the blocking buffer was discarded. The candidate antibodies Hu023-1~39 of this invention were diluted with 0.05% PBST to eight concentration gradients: 3, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, and 0.00015 μg / mL. Then, using a 12-channel pipette, the antibody dilution was mixed with 2% skim milk powder at a 1:1 ratio and added to the corresponding ELISA wells, 100 μL per well. The mixture was incubated at 37°C for 1 hour. The supernatant was discarded, and the sample was washed 5 times with 0.1% PBST. Then, HRP-labeled goat anti-human Fab was used as the second antibody (goat anti-human-HRP, ThermoFisher, 31482, 1:6000 dilution) for detection. The results were read using an ELISA microplate reader (BioTek, model 800TS) at a wavelength of 450 nm.

[0435] like Figure 4-A , Figure 4-B and Figure 4-C As shown, Hu023-3, Hu023-8, Hu023-12, Hu023-13, Hu023-14, Hu023-15, Hu023-19, Hu023-31, Hu023-33, Hu023-34, Hu023-36, Hu023-37, and Hu023-39 exhibited higher affinity than the control antibody. Table 1 provides the nucleotide and amino acid sequence information of these antibodies, with the CDR region information for each antibody determined using the Kabat numbering system.

[0436] Table 1. Antibody Information

[0437]

[0438]

[0439] Example 5: Flow cytometry was used to determine the affinity of the candidate antibody for specific binding to CTLA4-expressing target cells.

[0440] 5.1 Cell Plating: Transfer CTLA4-VERO cells from the culture flask to centrifuge tubes, centrifuge to remove the supernatant, resuspend in culture medium, count the cells, and adjust the cell density to 1E6 cells / mL. Take a 96-well round-bottom plate and add 100 μL of cells to each well using a pipette. Centrifuge at 300 g / min for 5 min. Discard the supernatant.

[0441] 5.2 Addition of protein antibodies: Hu023-8 and Hu023-14 antibodies were diluted to eight concentration gradients using FACS buffer containing 2% FBS: 150.000, 37.500, 9.375, 2.3438, 0.5859, 0.1465, 0.0366, and 0.0092 nM. Using a 100 μL 12-line pipette, 100 μL of the antibody dilution was added to each well of a 96-well cell culture plate. After mixing, the plates were incubated at 4°C for 1 h.

[0442] 5.3 Addition of Secondary Antibody: Dilute the secondary antibody PE anti-human IgG Fc Antibody (BioLegend, catalog number 410708) 1:200 with FACS buffer containing 2% FBS. Centrifuge the cell culture plate to remove the supernatant, and use a 100 μL 12-channel pipette to add 100 μL of the secondary antibody dilution to each well of the cell culture plate. Incubate the cell culture plate at 4°C for 30 min. Centrifuge to remove the supernatant, wash the plate twice with FACS buffer, and resuspend the cells in 120 μL of FACS buffer in each well.

[0443] 5.4 Data Acquisition: Turn on the flow cytometer and, after the instrument has finished cleaning, perform FACS binding detection.

[0444] like Figure 5 As shown, ECG of the control antibody Ipilimumab 50 The values ​​were 2.8 times and 3.2 times that of the Hu023-8 and Hu023-14 antibodies, respectively, indicating that the Hu023-8 and Hu023-14 antibodies had higher affinity for CTLA4-expressing target cells than the control antibody. Furthermore, the results of SPR method analysis on the affinity of the control antibody Ipilimumab, Hu023-5, Hu023-8, and Hu023-14 antibodies for CTLA4 showed ( Figure 6 The KD values ​​of the control antibody Ipilimumab were approximately 12 times, 79 times, and 91 times that of Hu023-5, Hu023-8, and Hu023-14, respectively, indicating that the candidate antibodies Hu023-5, Hu023-8, and Hu023-14 of the present invention have higher affinity for CTLA4. The above two experimental data further illustrate that the candidate antibodies Hu023-8 and Hu023-14 not only have significantly better binding affinity to CTLA4 than the control antibody Ipilimumab, but also exhibit stable and stronger binding ability at both the cellular and molecular levels, resulting in more outstanding technical effects.

[0445] Example 6: In vitro killing activity assay mediated by candidate antibodies Hu023-8 and Hu023-14

[0446] This embodiment validates the in vitro killing activity of candidate antibodies Hu023-8 and Hu023-14 in CTLA4-VERO cells.

[0447] Principle: The Cell Counting Kit-8 (CCK-8 kit) is a rapid, highly sensitive colorimetric assay kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt), widely used for cell proliferation and cytotoxicity. Its detection principle is as follows: In the presence of the electron coupling agent 1-Methoxy PMS, WST-8 can be reduced by certain dehydrogenases in mitochondria to generate an orange-yellow, water-soluble formazan. The intensity of the formazan color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at 450 nm using a microplate reader, indirectly reflecting the number of viable cells.

[0448] The specific steps are as follows: CTLA4-VERO cells were seeded in 96-well plates (1E4 / well) and cultured at 37°C and 5% CO2 for 24 hours. After removing the culture medium, the candidate antibodies Hu023-8 and Hu023-14 of this invention, as well as the control antibody ipilimumab, were added at a dose of 2 μg / mL, 200 μL per well. Activated NK cells were then added to each well, with the ratio of NK cells to CTLA4-VERO cells (E:T) being 1:1, 2:1, and 4:1. A blank control group and a non-specific killing group (NK cells only) were also set up. After further culturing at 37°C and 5% CO2 for 24 hours, the supernatant was removed, and 100 μL of CCK8 reaction solution (Novizan, catalog number A311-02) was added to each well. After incubation at 37°C for 2 hours, the readings were taken using an ELISA microplate reader (BioTek, model 800TS) at a wavelength of 450 nm. The specific killing data were calculated using the values ​​of the blank group without NK cells as a control, and the calculation formula is as follows:

[0449] Cytotoxicity (%) = [(BA) / (BC)] × 100%

[0450] A: Absorbance values ​​of the experimental group (containing culture medium, target cells, antibody, CCK-8 reagent, and NK cells).

[0451] B: Absorbance of the control group (containing culture medium, target cells, antibody, and CCK-8 reagent, but without NK cells).

[0452] C: Absorbance value of the blank group (absorbance value of the cell-free group containing culture medium and CCK-8 reagent).

[0453] The results are as follows Figure 7As shown, for the CTLA4-VERO cell line, the candidate fully human antibodies Hu023-8 and Hu023-14 of this invention can mediate high NK cell killing activity in vitro. Compared with the control antibody Ipilimumab, Hu023-8 and Hu023-14 antibodies have similar killing activity at E:T ratios of 1:1 and 2:1; however, at an E:T ratio of 4:1, the ADCC-mediated killing activity of Hu023-8 and Hu023-14 antibodies is significantly higher than that of the control antibody, with p values ​​<0.0001 and <0.01, respectively. This indicates that the anti-CTLA4 antibodies Hu023-8 and Hu023-14 of this invention have the activity of mediating ADCC killing tumors in vitro.

[0454] Example 7: BiTE-mediated synthesis prepared from candidate antibodies Hu023-8, Hu023-14, Hu023-19, and Hu023-30 In vitro killing activity experiment

[0455] This embodiment validated the in vitro killing activity of BiTE protein mediated by candidate antibodies Hu023-8, Hu023-14, Hu023-19, and Hu023-30 against CTLA4-VERO cells. BiTE DNA sequences of ipilimumab, Hu023-8, Hu023-14, Hu023-19, and Hu023-30 were constructed, as shown in SEQ ID NO. 141, SEQ ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, and SEQ ID NO. 145, respectively, and were loaded into Cloningvector AbVec-hIgG1 (GenBank: FJ475055.1).

[0456] 7.1 Cell Preparation: CTLA4-VERO target cells were seeded at a density of 1E4 cells / well in 96-well clear lamellae and incubated overnight at 37°C until adherence. 24 hours after seeding, 50 μL of supernatant from different BiTEs prepared with Hu023-8, Hu023-14, Hu023-19, and Hu023-30 antibodies and the control antibody ipilimumab were added, along with 50 μL of activated T cells (E:T ratio 1:1). Controls were set up with supernatant and T cells alone, no T cells, and no cells. Cells were incubated overnight at 37°C for cell killing.

[0457] 7.2 Cell killing test: Cell killing test was performed after about 18 hours (cell killing can be observed under a microscope). The supernatant in the 96-well plate was discarded, and the plate was gently tapped on absorbent paper. The prepared solution containing 10% CCK-8 (e.g., 10 μL CCK-8 solution + 90 μL culture medium) was added to the culture plate at 100 μL / well. The culture plate was incubated in the dark for 2 hours and the absorbance at 450 nm was measured (BioTek, model 800TS).

[0458] The results are as follows Figure 8 As shown, for the CTLA4-VERO cell line, the BiTE proteins prepared by Hu023-8, Hu023-14, Hu023-19 and Hu023-30 of this invention can all mediate high killing activity of T cells in vitro. Moreover, compared with BiTE prepared by the control antibody Ipilimumab, the killing effect is enhanced. Among them, when using T cells derived from donor-1, the killing effect of Hu023-8 and Hu023-19 on CTLA4-VERO cells is significantly enhanced, with p values ​​of 0.04 and 0.02, respectively. This indicates that the anti-CTLA4 antibody of this invention has a strong activity in mediating T cell killing of CTLA4-VERO cells in vitro.

[0459] Example 8: Construction of a vaccinia virus vector carrying the BiTE target gene (a candidate antibody)

[0460] 8.1 Construction of pSC65 vector carrying BiTE target gene (candidate antibody)

[0461] BiTE DNA sequences containing antibodies against Ipilimumab, Hu023-8, Hu023-14, Hu023-19, and Hu023-30 (as shown in SEQ ID NO. 141, SEQ ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, and SEQ ID NO. 145, respectively) were cloned into a shuttle plasmid containing the vaccinia virus TK gene locus. The plasmid construction map is shown below. Figure 9-A As shown.

[0462] 8.2 Construction of recombinant vaccinia virus

[0463] 8.2.1 Cell preparation: Seed 143TK- cells into 6-well plates, approximately 1E6 cells per well. Incubate for about 24 hours. When the cells adhere to the plates and cover the entire bottom surface, proceed to the next step.

[0464] 8.2.2 Vaccine virus incubation: Wild-type vaccinia virus strain Tiantan was used to infect 143TK- cells with MOI = 0.0042 (MOI = virus PFU / cell number, PFU is plaque forming unit, virus titer). After incubation at 37℃ for 1 h, the cells were removed, the supernatant was aspirated, and the cells were washed once with 1 mL PBS. Then, 1 mL of complete culture medium was added.

[0465] 8.2.3 Plasmid transfection: Transfect 143TK- cells with the above shuttle plasmid. Incubate at 37℃ for approximately 48 h, the exact time depending on the cytopathic effect.

[0466] 8.2.4 Collecting viruses:

[0467] Prepare 2×DMEM maintenance medium (containing 2% PS and 4% FBS) for virus plaque formation, add 2% preheated low-melting-point agarose, and then add X-gal (final concentration 200 μg / mL). Aspirate the supernatant from the 6-well plate and add 1 mL of the plaque formation mixture to each well. Carefully place the plate in a 4°C freezer to promote solidification. Once the low-melting-point agarose has solidified, transfer it to a 37°C incubator and invert it until clear blue spots appear.

[0468] Before picking the blue spots (recombinant vaccinia virus), 143TK- cells were seeded in 6-well plates, approximately 1E6 cells per well, and cultured for about 24 hours. Once the cells adhered and covered the entire bottom surface, the blue spots were picked and transferred to 500 μL of complete culture medium to obtain the virus solution. The virus solution was subjected to a freeze-thaw cycle at -80°C at least three times to release as much virus as possible. The virus solution was then repeatedly pipetted until the blue spots were completely dispersed. The complete culture medium for the 143TK- cells was replaced with maintenance medium, and then the virus solution containing the blue spots was added. The cells were incubated at 37°C for 3-4 hours. A selection pressure of 50 μg / mL BrdU was added, and the cells were incubated at 37°C for approximately 48 hours. Plating was then performed based on the formation of the viral plaques. This purification process needs to be repeated at least 5 times.

[0469] Then, a small-scale amplification of recombinant vaccinia virus was performed. 143TK- cells were seeded into six-well plates, with 1E6 cells per well, filling approximately 100% of the plate's bottom area. Before inoculation, the culture medium in each well was replaced with 2 mL of maintenance medium. The purified virus solution containing blue spots was repeatedly pipetted until the spots dispersed. Approximately 100 μL of virus solution was added to each well. The mixture was incubated at 37°C for approximately 48 h, and samples were collected based on the formation of viral spots.

[0470] Sample collection: Carefully aspirate 1 mL of the culture medium supernatant from the well. Use another 1 mL of culture medium to thoroughly blow the cells down and collect them in an EP tube. This can be used for subsequent genome extraction and as a seed for amplification.

[0471] 8.2.5 Amplification and Purification of Recombinant Vaccine Virus

[0472] VERO cell plating: 10 cm dishes, approximately 5E6 cells per dish, ensuring 100% cell density before vaccinia virus inoculation the next day. Before virus inoculation, replace the complete culture medium with 8 mL of maintenance medium (DMEM + 2% FBS + 1% PS). Inoculate the cells in the maintenance medium with the virus at an inoculation volume of approximately 0.02 MOI. Continue incubation at 37°C, 5% CO2 for approximately 48 h, and collect samples based on viral plaque formation.

[0473] Collect recombinant vaccinia virus: Discard 8 mL of culture medium in the dish, take 2 mL of maintenance culture medium, blow off the remaining cells, and collect them in a 15 mL centrifuge tube.

[0474] After freezing for 24 h, the collected virus solution was repeatedly frozen and thawed twice. It was then subjected to density gradient centrifugation with 36% sucrose solution at 16000 g and 4℃ for 90 min. The supernatant was carefully discarded, and the virus precipitate in the centrifuge tube was dissolved with PBS buffer. The tubes were then aliquoted and stored at -80℃ for virus titer determination.

[0475] 8.2.6 Titer determination of recombinant vaccinia virus

[0476] Preparation of 143TK cells: Seed 143TK cells into 24-well plates, approximately 2E5 cells per well, ensuring the cell density reaches 100% of the bottom area of ​​the plate. Dilute the virus using maintenance medium, starting with a 1:100 dilution and performing 10-fold serial dilutions to a final volume of 1100 μL. Discard the complete medium in the 24-well plate and add 500 μL of the diluted virus solution to each well, performing two replicates. Incubate at 37°C with 5% CO2 for approximately 48 hours, adjusting the plaque formation time according to the progress of plaque formation.

[0477] Plastering method: Prepare 8 mL of plastering medium containing 2×DMEM medium + 4% FBS + 2% PS and 8 mL of low-melting-point agarose melted in a boiling water bath and placed in a 37℃ water bath. Mix the two, then add X-gal to the mixture to a final concentration of 200 μg / mL. Remove the supernatant from the 24-well plate, and immediately add the above mixture to each well (500 μL). Carefully place the plate in a 4℃ refrigerator to promote solidification. After the low-melting-point agarose has solidified, transfer it to a 37℃ incubator and invert it until clear blue spots appear. Virus plaque counting: First, observe whether the number of virus plaques decreases tenfold. Then, count the number of single-digit blue spots in the two replicates of the seed virus. The sum of the blue spot values ​​in the two wells, multiplied by the reciprocal of the dilution of that well, is the virus titer per mL.

[0478] Example 9: In vitro killing activity assay of recombinant vaccinia virus carrying the BiTE target gene (candidate antibody)

[0479] This invention validates the in vitro killing activity of BiTE-based recombinant vaccinia virus-mediated activated T cells carrying candidate antibodies Hu023-8, Hu023-14, Hu023-19, and Hu023-30 against CTLA4-VERO cells.

[0480] 9.1 Cell Preparation: CTLA4-VERO target cells were seeded at a density of 1E4 cells / well in 96-well clear slabs and incubated overnight at 37°C until adherence. 24 hours after seeding, vaccinia virus solution prepared from Hu023-8, Hu023-14, Hu023-19, Hu023-30, and the control antibody ipilimumab was added to each well at a volume of 250 PFU (100 μL). 50 μL of activated T cells were then added. Two donors were used, with an E:T ratio of 1:1 and 2:1. Controls were set up with either supernatant and T cells alone, or without T cells and cells. Cells were incubated overnight at 37°C for cell killing.

[0481] 9.2 Cell killing test: Cell killing test was performed after about 22 hours (the cell killing situation can be observed under a microscope). The supernatant in the 96-well plate was discarded, and the plate was gently tapped on absorbent paper. The prepared solution containing 10% CCK-8 (e.g., 10 μL CCK-8 solution + 90 μL culture medium) was added to the culture plate at 100 μL / well. The culture plate was incubated in the dark for 2 hours and the absorbance at 450 nm was measured (BioTek, model 800TS).

[0482] The results are as follows Figure 9-B As shown, for the CTLA4-VERO cell line, the vaccinia virus prepared by the candidate antibodies Hu023-14, Hu023-19 and Hu023-30 of this invention can all mediate high killing activity of T cells in vitro. Compared with BiTE prepared by the control antibody Ipilimumab, the killing mediated by the recombinant vaccinia virus prepared by Hu023-14 is significantly improved. Among them, when using T cells derived from donor-1, at E:T ratios of 1:1 and 2:1, Hu023-14 killed CTLA4-VERO cell lines significantly more than the control antibody Ipilimumab, with p values ​​of 0.0028 and 0.0093, respectively. When using T cells derived from donor-2, at an E:T ratio of 2:1, Hu023-14 killed CTLA4-VERO cell lines significantly more than the control antibody Ipilimumab, with a p value of 0.0296. This invention demonstrates that the recombinant vaccinia virus of BiTE carrying the candidate antibody Hu023-14 has potent cytotoxic activity against CTLA4-VERO cells.

[0483] SEQ ID NO. 1:

[0484] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGCGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGCACCTCTCCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATATGTTTCAGCCATTACTCCTAGTGGGGATAGAACATATTATGCAGACTCTGTGAAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAATTGGGCAGCCTGAGAACTGAGGACATGGCAGTTTATTACTGTGCGAGACGGGGTGATGGTTATTACTACGACTACTGGGGCCAAGGAACCCCGGTCACCGTCTCATCA

[0485] SEQ ID NO. 2:

[0486] EVQLLESGGGLVQPGGSLRLSCVASGFTFSTSPMHWVRQAPGKGLEYVSAITPSGDRTYYADSVKGRFTISRDNSKNTLYLQLGSLRTEDMAVYYCARRGDGYYYDYWGQGTPVTVSS

[0487] SEQ ID NO. 3:

[0488] TSPMH

[0489] SEQ ID NO. 4:

[0490] AITPSGDRTYYADSVKG

[0491] SEQ ID NO. 5:

[0492] RGDGYYYDY

[0493] SEQ ID NO. 6:

[0494] AACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGAGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCGTCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAGAGTTACAAAACCCCTCTGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0495] SEQ ID NO. 7:

[0496] NIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTVSSLQPEDFATYYCQQSYKTPLTFGQGTKVEIK

[0497] SEQ ID NO. 8:

[0498] RASQSISSYLN

[0499] SEQ ID NO. 9:

[0500] AASSLQS

[0501] SEQ ID NO. 10:

[0502] QQSYKTPLT

[0503] SEQ ID NO. 11:

[0504] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGCGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGCACCTCTCCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATATGTTTCAGCCATTACTCCTAGTGGGGATAGAACATATTATGCAGACTCTGTGAAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAATTGGGCAGCCTGAGAACTGAGGACATGGCAGTTTATTACTGTGCGAGACGGGGTGATGGTTATTACTACGACTACTGGGGCCAAGGAACAACGGTCACCGTCTCATCA

[0505] SEQ ID NO. 12:

[0506] EVQLVESGGGLVQPGGSLRLSCVASGFTFSTSPMHWVRQAPGKGLEYVSAITPSGDRTYYADSVKGRFTISRDNSKNTLYLQLGSLRTEDMAVYYCARRGDGYYYDYWGQGTTVTVSS

[0507] SEQ ID NO. 13:

[0508] TSPMH

[0509] SEQ ID NO. 14:

[0510] AITPSGDRTYYADSVKG

[0511] SEQ ID NO. 15:

[0512] RGDGYYYDY

[0513] SEQ ID NO. 16:

[0514] GCCATCAGGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0515] SEQ ID NO. 17:

[0516] AIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK

[0517] SEQ ID NO. 18:

[0518] RASQSISSYLN

[0519] SEQ ID NO. 19:

[0520] AASSLQS

[0521] SEQ ID NO. 20:

[0522] QQSYSTPLT

[0523] SEQ ID NO. 21:

[0524] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGTACGACCCGGCGCTTTTGATATCTGGGGCCAGGGAACAACGGTCACCGTCTCATCA

[0525] SEQ ID NO. 22:

[0526] EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVRPGAFDIWGQGTTVTVSS

[0527] SEQ ID NO. 23:

[0528] SYAMH

[0529] SEQ ID NO. 24:

[0530] VISYDGSNKYYVDSVKG

[0531] SEQ ID NO. 25:

[0532] GVRPGAFDI

[0533] SEQ ID NO. 26:

[0534] AACATCCAGTTGACCCAGTCTCCATCTTCCCTGTCTGCTTATGTAGGAGAGAGAGTCACTATCACCTGTCGGGCGAGTCAGGATGTTGGCAACTGGTTAGCCTGGTATCAGCACAAACCAGGGAAAGTCCCTAAACTCCTGATCCATTCTGCATCCAATTTGCAAAGTGGGGTCCCGTCACGGTTCAGCGGCAGTGGATTCGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCCGACGATTTTGCAACTTACTATTGCCAACAGGCTAGCAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0535] SEQ ID NO. 27:

[0536] NIQLTQSPSSLSAYVGERVTITCRASQDVGNWLAWYQHKPGKVPKLLIHSASNLQSGVPSRFSGSGFGTDFTLTISSLQPDDFATYYCQQASSFPLTFGGGTKVEIK

[0537] SEQ ID NO. 28:

[0538] RASQDVGNWLA

[0539] SEQ ID NO. 29:

[0540] SASNLQS

[0541] SEQ ID NO. 30:

[0542] QQASSFPLT

[0543] SEQ ID NO. 31:

[0544] AGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGACGCAGACTTGGGGAACTTGACTACTGGGGCCAGGGGACAATGGTCACCGTCTCATCA

[0545] SEQ ID NO. 32:

[0546] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRRLGELDYWGQGTMVTVSS

[0547] SEQ ID NO. 33:

[0548] SYAMH

[0549] SEQ ID NO. 34:

[0550] VISYDGSNKYYADSVKG

[0551] SEQ ID NO. 35:

[0552] GRRLGELDY

[0553] SEQ ID NO. 36:

[0554] GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0555] SEQ ID NO. 37:

[0556] DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK

[0557] SEQ ID NO. 38:

[0558] RASQGISSWLA

[0559] SEQ ID NO. 39:

[0560] AASSLQS

[0561] SEQ ID NO. 40:

[0562] QQANSFPLT

[0563] SEQ ID NO. 41:

[0564] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGAGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACATCCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAAAGGTCCCGTCGGGGAGTCAGTCCTCTACTACTACTACGGTATGGACGTCTGGGGCCAAGGAACCCCGGTCACCGTCTCATCA

[0565] SEQ ID NO. 42:

[0566] EVQLLESGGGLVQPGESLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNILYLQMNSLRAEDTAVYYCAKGPVGESVLYYYYGMDVWGQGTPVTVSS

[0567] SEQ ID NO. 43:

[0568] SYAMS

[0569] SEQ ID NO. 44:

[0570] AISGSGGSTYYADSVKG

[0571] SEQ ID NO. 45:

[0572] GPVGESVLYYYYGMDV

[0573] SEQ ID NO. 46:

[0574] GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACCTGTCGGGCGAGTCAGGGTATTAGCAGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTGACAGTTTCCCTCTCACTTTCGGCCCAGGGACCAAAGTGGATATCAA

[0575] SEQ ID NO. 47:

[0576] DIQLTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQKPGKAPNLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADSFPLTFGPGTKVDIK

[0577] SEQ ID NO. 48:

[0578] RASQGISRWLA

[0579] SEQ ID NO. 49:

[0580] AASSLQS

[0581] SEQ ID NO. 50:

[0582] QQADSFPLT

[0583] SEQ ID NO. 51:

[0584] CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGCGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGCGCAGCGTCTGGATTCAACTTCAGGAATTCTGGCATGCACTGGGTCCGCCAGGCTCCCGGCAAGGGTCTGGAATGGGTGGCAGTTATTTGGCATGACGGCAGTCAAAAACACTATGCACCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACTCCACGAACACACTGTCCCTGCAAATGAACAGCCTGCGAGTCGAGGACACGGCTGTATATTTTTGCGCGCGAAGTGGTGTCTACAACGAGAAGAATCACTGGGGCCAAGGAACCACGGTCACCGTCTCATCA

[0585] SEQ ID NO. 52:

[0586] QVQLLESGGGAVRPGRSLRLSCAASGFNFRNSGMHWVRQAPGKGLEWVAVIWHDGSQKHYAPSVKGRFTISRDDSTNTLSLQMNSLRVEDTAVYFCARSGVYNEKNHWGQGTTVTVSS

[0587] SEQ ID NO. 53:

[0588] NSGMH

[0589] SEQ ID NO. 54:

[0590] VIWHDGSQKHYAPSVKG

[0591] SEQ ID NO. 55:

[0592] SGVYNEKNH

[0593] SEQ ID NO. 56:

[0594] AACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

[0595] SEQ ID NO. 57:

[0596] NIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK

[0597] SEQ ID NO. 58:

[0598] RASQSISSYLN

[0599] SEQ ID NO. 59:

[0600] AASSLQS

[0601] SEQ ID NO. 60:

[0602] QQSYSTPFT

[0603] SEQ ID NO. 61:

[0604] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATATGTTTCAGCTATTAGTAGTAATGGGGGTAGCACATACTACGCAGACTCAGTGAAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAGCAGTCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGTGAAAGCCTACAGGCGAGAAAAAGAAAAGTATTATGATTACGTTTGGGGGAGTTATCGTTCCCCCAACTGGTTCGACCCCTGGGGCCAAGGAACCCTGGTCACCGTCTCATCA

[0605] SEQ ID NO. 62:

[0606] EVQLLESGGGLVQPGGSLRLSCSASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGSTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCVKAYRREKEKYYDYVWGSYRSPNWFDPWGQGTLVTVSS

[0607] SEQ ID NO. 63:

[0608] SYAMH

[0609] SEQ ID NO. 64:

[0610] AISSNGGSTYYADSVKG

[0611] SEQ ID NO. 65:

[0612] AYRREKEKYYDYVWGSYRSPNWFDP

[0613] SEQ ID NO. 66:

[0614] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTTTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCGGGGCATCGCCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAGGTTCCTAGCCTCCTGATCTATGGTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGCCTGCAGCCTGAAGATGTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0615] SEQ ID NO. 67:

[0616] DIQLTQSPSSLSASLGDRVTITCRASRGIANYLAWYQQKPGKVPSLLIYGASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQSYSTPWTFGQGTKVEIK

[0617] SEQ ID NO. 68:

[0618] RASRGIANYLA

[0619] SEQ ID NO. 69:

[0620] GASTLQS

[0621] SEQ ID NO. 70:

[0622] QQSYSTPWT

[0623] SEQ ID NO. 71:

[0624] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGGGGGAGGCAGCTAGGGGCTTTTGATATCTGGGGCCAGGGAACAATGGTCACCGTCTCATCA

[0625] SEQ ID NO. 72:

[0626] EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGRQLGAFDIWGQGTMVTVSS

[0627] SEQ ID NO. 73:

[0628] SYAMH

[0629] SEQ ID NO. 74:

[0630] VISYDGSNKYYADSVKG

[0631] SEQ ID NO. 75:

[0632] GRQLGAFDI

[0633] SEQ ID NO. 76:

[0634] AACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTAGAGACCAAG

[0635] SEQ ID NO. 77:

[0636] NIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVETK

[0637] SEQ ID NO. 78:

[0638] RASQGISSWLA

[0639] SEQ ID NO. 79:

[0640] AASSLQS

[0641] SEQ ID NO. 80:

[0642] QQANSFPLT

[0643] SEQ ID NO. 81:

[0644] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGTATTAGGTCGGGGGCTTTTGATATCTGGGGCCAGGGGACCCTGGTCACCGTCTCATCA

[0645] SEQ ID NO. 82:

[0646] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIRSGAFDIWGQGTLVTVSS

[0647] SEQ ID NO. 83:

[0648] SYAMH

[0649] SEQ ID NO. 84:

[0650] VISYDGSNKYYADSVKG

[0651] SEQ ID NO. 85:

[0652] GIRSGAFDI

[0653] SEQ ID NO. 86:

[0654] GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTTGGAGACAGAGTCTCCATCACTTGCCGGGCGAGTCAGGGAATTAGTAAGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAGCGCCCCTAAGCTCCTGATCTCTGCTACATCCAACTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACACATTTCACTCTCACCATCAACAGCCTACAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAAGTCAAA

[0655] SEQ ID NO. 87:

[0656] DIQLTQSPSSVSASVGDRVSITCRASQGISKWLAWYQQKPGSAPKLLISATSNLQSGVPSRFSGSGSGTHFTLTINSLQPEDFATYYCQQANSFPLTFGGGTKVEVK

[0657] SEQ ID NO. 88:

[0658] RASQGISKWLA

[0659] SEQ ID NO. 89:

[0660] ATSNLQS

[0661] SEQ ID NO. 90:

[0662] QQANSFPLT

[0663] SEQ ID NO. 91:

[0664] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCATCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGTCAGGCCGGGGGGTATGGACGTCTGGGGCCAAGGGACAACGGTCACCGTCTCATCA

[0665] SEQ ID NO. 92:

[0666] EVQLVESGGGLVKPGGSLRLSCASSGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVRPGGMDVWGQGTTVTVSS

[0667] SEQ ID NO. 93:

[0668] SYAMH

[0669] SEQ ID NO. 94:

[0670] VISYDGSNKYYADSVKG

[0671] SEQ ID NO. 95:

[0672] GVRPGGMDV

[0673] SEQ ID NO. 96:

[0674] GACATCCAGATGACCCAGTCTCCATCTTCTGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACTATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0675] SEQ ID NO. 97:

[0676] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIK

[0677] SEQ ID NO. 98:

[0678] RASQGISSWLA

[0679] SEQ ID NO. 99:

[0680] AASSLQS

[0681] SEQ ID NO. 100:

[0682] QQANSFPIT

[0683] SEQ ID NO. 101:

[0684] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATTCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGATGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGCGTAGGTTCTACTACTACTACGGTATGGACGTCTGGGGCCAGGGGACAACGGTCACCGTCTCATCA

[0685] SEQ ID NO. 102:

[0686] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCARGGRRFYYYYGMDVWGQGTTVTVSS

[0687] SEQ ID NO. 103:

[0688] SYAIH

[0689] SEQ ID NO. 104:

[0690] VISYDGSNKYYADSVKG

[0691] SEQ ID NO. 105:

[0692] GGRRFYYYYGMDV

[0693] SEQ ID NO. 106:

[0694] AACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGTCAAGTCAGACCATTAACACTTATTTGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAGGCTCCTCGTCTATGCGGCATCCACTTTACAAAGTGGGGTCCCTCCAAGGTTCACTGGCAGTGAATCTGGGACAGATTTCAGTCTCACCATCAGCAGTCTGCAACCTGAAGATCTTGCAACTTATTACTGTCAACTTGGACGGACGTTCGGCCACGGGACCAAGGTGGATATCAAA

[0695] SEQ ID NO. 107:

[0696] NIQLTQSPSSLSASVGDRVTITCRSSQTINTYLNWYQQKPGKAPRLLVYAASTLQSGVPPRFTGSESGTDFSLTISSLQPEDLATYYCQLGRTFGHGTKVDIK

[0697] SEQ ID NO. 108:

[0698] RSSQTINTYLN

[0699] SEQ ID NO. 109:

[0700] AASTLQS

[0701] SEQ ID NO. 110:

[0702] QLGRT

[0703] SEQ ID NO. 111:

[0704] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCTTGCGGGGCCCGCCTGCCTGGGGCCAGGGAACAACGGTCACCGTCTCATCA

[0705] SEQ ID NO. 112:

[0706] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGLRGPPAWGQGTTVTVSS

[0707] SEQ ID NO. 113:

[0708] SYGMH

[0709] SEQ ID NO. 114:

[0710] VISYDGSNKYYADSVKG

[0711] SEQ ID NO. 115:

[0712] GLRGPPA

[0713] SEQ ID NO. 116:

[0714] GACATCCAGATGACCCAGTCTCCATACTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGGTCCTGATCTATGGTGCATCCAGCTTGCAAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTTCTGTCACCAGACTTACGGTACCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

[0715] SEQ ID NO. 117:

[0716] DIQMTQSPYSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKVLIYGASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCHQTYGTPFTFGPGTKVDIK

[0717] SEQ ID NO. 118:

[0718] RASQSISSYLN

[0719] SEQ ID NO. 119:

[0720] GASSLQD

[0721] SEQ ID NO. 120:

[0722] HQTYGTPFT

[0723] SEQ ID NO. 121:

[0724] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGAATTAGGGGGGGGGCTTTTGATATCTGGGGCCAAGGGACCCCGGTCACCGTCTCATCA

[0725] SEQ ID NO. 122:

[0726] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIRGGAFDIWGQGTPVTVSS

[0727] SEQ ID NO. 123:

[0728] SYAMH

[0729] SEQ ID NO. 124:

[0730] VISYDGSNKYYADSVKG

[0731] SEQ ID NO. 125:

[0732] GIRGGAFDI

[0733] SEQ ID NO. 126:

[0734] AACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTACCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTCATCTATGCTGCATCCAGTTTGCAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGGATTTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGACTTTGCAACTTACTATTGTCAACAGGCTAGCAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0735] SEQ ID NO. 127:

[0736] NIQMTQSPSSVSASVGDRVTITCRASQGITSWLAWYQQKPGKAPKLLIYAASSLQNGVPSRFSGSGFGTDFTLTISSLQPEDFATYYCQQASSFPLTFGGGTKVEIK

[0737] SEQ ID NO. 128:

[0738] RASQGITSWLA

[0739] SEQ ID NO. 129:

[0740] AASSLQN

[0741] SEQ ID NO. 130:

[0742] QQASSFPLT

[0743] SEQ ID NO. 131:

[0744] CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGTCTAAGAGTGGGAGCCTTTGATATCTGGGGCCAGGGAACAACGGTCACCGTCTCATCA

[0745] SEQ ID NO. 132:

[0746] QVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLRVGAFDIWGQGTTVTVSS

[0747] SEQ ID NO. 133:

[0748] SYAMH

[0749] SEQ ID NO. 134:

[0750] VISYDGSNKYYADSVKG

[0751] SEQ ID NO. 135:

[0752] GLRVGAFDI

[0753] SEQ ID NO. 136:

[0754] GACATCCAGATGACCCAGTCTCCTTCCTTCCTGTATGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTACCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTCATCTATGCTGCATCCAGTTTGCAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGGATTTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGACTTTGCAACTTACTATTGTCAACAGGCTAGCAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0755] SEQ ID NO. 137:

[0756] DIQMTQSPSFLYASVGDRVTITCRASQGITSWLAWYQQKPGKAPKLLIYAASSLQNGVPSRFSGSGFGTDFTLTISSLQPEDFATYYCQQASSFPLTFGGGTKVEIK

[0757] SEQ ID NO. 138:

[0758] RASQGITSWLA

[0759] SEQ ID NO. 139:

[0760] AASSLQN

[0761] SEQ ID NO. 140:

[0762] QQASSFPLT

[0763] SEQ ID NO. 141:

[0764]

[0765] SEQ ID NO. 142:

[0766]

[0767] SEQ ID NO. 143:

[0768]

[0769] SEQ ID NO. 144:

[0770]

[0771] SEQ ID NO. 145:

[0772]

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the CTLA4 protein, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 3, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 4, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO:

5. and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

8. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 9, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 10; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 13, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 14, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 15; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

18. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 19, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 20; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 23, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 24, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 25; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

28. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 29, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 30; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 33, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 34, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 35; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

38. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 39, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 40; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 43, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 44, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 45; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

48. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 49, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 50; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 53, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 54, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 55; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

58. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 59, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 60; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 63, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 64, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 65; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

68. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 69, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 70; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 73, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 74, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 75; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

78. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 79, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 80; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 83, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 84, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 85; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

88. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 89, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 90; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 93, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 94, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 95; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

98. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 99, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 100; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 103, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 104, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 105; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

108. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 109, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 110; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 113, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 114, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 115; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

118. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 119, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 120; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 123, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 124, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 125; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

128. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 129, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO: 130; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) Heavy chain variable regions containing the following three complementary determinant regions: (i) VH CDR1, whose amino acid sequence is SEQ ID NO: 133, (ii) VH CDR2, whose amino acid sequence is SEQ ID NO: 134, and (iii) VH CDR3, whose amino acid sequence is SEQ ID NO: 135; and / or (b) Light chain variable regions containing the following three complementary determinant regions: (iv) VL CDR1, whose amino acid sequence is SEQ ID NO:

138. (v) VL CDR2, whose amino acid sequence is SEQ ID NO: 139, and (vi) VL CDR3, whose amino acid sequence is SEQ ID NO:

140.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The heavy chain variable region of the antibody or its antigen-binding fragment includes: VH CDR1 as shown in SEQ ID NO: 3, VH CDR2 as shown in SEQ ID NO: 4, and VH CDR3 as shown in SEQ ID NO: 5; and the light chain variable region of the antibody or its antigen-binding fragment includes: VL CDR1 as shown in SEQ ID NO: 8, VL CDR2 as shown in SEQ ID NO: 9, and VL CDR3 as shown in SEQ ID NO: 10; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 13, VH CDR2 as shown in SEQ ID NO: 14, and VH CDR3 as shown in SEQ ID NO: 15; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 18, VL CDR2 as shown in SEQ ID NO: 19, and VL CDR3 as shown in SEQ ID NO: 20; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 23, VHCDR2 as shown in SEQ ID NO: 24, and VHCDR3 as shown in SEQ ID NO: 25; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 28, VL CDR2 as shown in SEQ ID NO: 29, and VL CDR3 as shown in SEQ ID NO: 30; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 33, VHCDR2 as shown in SEQ ID NO: 34, and VHCDR3 as shown in SEQ ID NO: 35; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 38, VL CDR2 as shown in SEQ ID NO: 39, and VL CDR3 as shown in SEQ ID NO: 40; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 43, VHCDR2 as shown in SEQ ID NO: 44, and VHCDR3 as shown in SEQ ID NO: 45; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 48, VL CDR2 as shown in SEQ ID NO: 49, and VL CDR3 as shown in SEQ ID NO: 50; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 53, VHCDR2 as shown in SEQ ID NO: 54, and VHCDR3 as shown in SEQ ID NO: 55; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 58, VL CDR2 as shown in SEQ ID NO: 59, and VL CDR3 as shown in SEQ ID NO: 60; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 63, VHCDR2 as shown in SEQ ID NO: 64, and VHCDR3 as shown in SEQ ID NO: 65; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 68, VL CDR2 as shown in SEQ ID NO: 69, and VL CDR3 as shown in SEQ ID NO: 70; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 73, VHCDR2 as shown in SEQ ID NO: 74, and VHCDR3 as shown in SEQ ID NO: 75; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 78, VL CDR2 as shown in SEQ ID NO: 79, and VL CDR3 as shown in SEQ ID NO: 80; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 83, VHCDR2 as shown in SEQ ID NO: 84, and VHCDR3 as shown in SEQ ID NO: 85; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 88, VL CDR2 as shown in SEQ ID NO: 89, and VL CDR3 as shown in SEQ ID NO: 90; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 93, VHCDR2 as shown in SEQ ID NO: 94, and VHCDR3 as shown in SEQ ID NO: 95; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 98, VL CDR2 as shown in SEQ ID NO: 99, and VL CDR3 as shown in SEQ ID NO: 100; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 103, VH CDR2 as shown in SEQ ID NO: 104, and VH CDR3 as shown in SEQ ID NO: 105; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 108, VL CDR2 as shown in SEQ ID NO: 109, and VL CDR3 as shown in SEQ ID NO: 110; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 113, VH CDR2 as shown in SEQ ID NO: 114, and VH CDR3 as shown in SEQ ID NO: 115; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 118, VL CDR2 as shown in SEQ ID NO: 119, and VL CDR3 as shown in SEQ ID NO: 120; Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 123, VH CDR2 as shown in SEQ ID NO: 124, and VH CDR3 as shown in SEQ ID NO: 125; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 128, VL CDR2 as shown in SEQ ID NO: 129, and VL CDR3 as shown in SEQ ID NO:

130. Alternatively, the heavy chain variable region of the antibody or its antigen-binding fragment may include: VHCDR1 as shown in SEQ ID NO: 133, VHCDR2 as shown in SEQ ID NO: 134, and VHCDR3 as shown in SEQ ID NO: 135; and the light chain variable region of the antibody or its antigen-binding fragment may include: VL CDR1 as shown in SEQ ID NO: 138, VL CDR2 as shown in SEQ ID NO: 139, and VL CDR3 as shown in SEQ ID NO:

140.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment comprises or is any one of the amino acid sequences shown in SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122 and 132; and the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment comprises or is any one of the amino acid sequences shown in SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127 and 137; Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment comprises or is any one of the nucleotide sequences shown in SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, and 131; the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment comprises or is any one of the nucleotide sequences shown in SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, and 136.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The antibody or its antigen-binding fragment comprises: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 2; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 2; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 7; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 7; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 12; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 12; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 17; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 17; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 22; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 22; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 22; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 27; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 27; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 27; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 32; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 32; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 32; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 37; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 37; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 37; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 42; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 42; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 42; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 47; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 47; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 47; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 52; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 52; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 52; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 57; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 57; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 57; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 62; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 62; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 62; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 67; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 67; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 67; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 72; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 72; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 72; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 77; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 77; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 77; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 82; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 82; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 82; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 87; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 87; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 87; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 92; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 92; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 92; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 97; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 97; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 97; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 102; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 102; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 102; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 107; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 107; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 107; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 112; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 112; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 112; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 117; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 117; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 117; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 122; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 122; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 122; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 127; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 127; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 127; Alternatively, the antibody or its antigen-binding fragment may comprise: (a) The heavy chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (i) The amino acid sequence shown in SEQ ID NO: 132; (ii) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 132; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 132; And / or, (b) The light chain variable region, whose amino acid sequence comprises an amino acid sequence selected from the following: (iv) The amino acid sequence shown in SEQ ID NO: 137; (v) An amino acid sequence having one or more, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 137; or (vi) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 137; Preferably, the substitution described in (ii) or (v) is a conservative substitution.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes or is the amino acid sequence shown in SEQ ID NO: 7; Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

17. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:22, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

27. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:32, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

37. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

47. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

57. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may include or be the amino acid sequence shown in SEQ ID NO: 62, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may include or be the amino acid sequence shown in SEQ ID NO:

67. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:72, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

77. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO: 82, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

87. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO: 92, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

97. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO: 102, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

107. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO: 112, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

117. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO: 122, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

127. Alternatively, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO: 132, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment may contain or be the amino acid sequence shown in SEQ ID NO:

137.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein, The antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region of human immunoglobulin or a variant thereof, wherein the amino acid sequence of the variant has one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of the heavy chain constant region of the human immunoglobulin from which it is derived; and (b) The light chain constant region of a human immunoglobulin or a variant thereof, wherein the amino acid sequence of the variant has up to 20 conserved substitutions compared with the amino acid sequence of the light chain constant region of the human immunoglobulin from which it is derived; Preferably, the heavy chain constant region is the human IgG heavy chain constant region, more preferably the human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region, and even more preferably, it is the human IgG1 or human IgG4 heavy chain constant region. Preferably, the light chain constant region is the κ light chain constant region.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein, The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, bispecific antibodies, and single-domain antibodies; and / or, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a bispecific antibody, or a multispecific antibody; more preferably, the antibody is a fully human antibody.

8. A bispecific or multispecific molecule comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-7, wherein the bispecific or multispecific molecule specifically binds to the CTLA4 protein and additionally specifically binds to one or more other targets; Preferably, the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity against a target other than the CTLA4 protein, preferably a second antibody; Preferably, the bispecific or multispecific molecule further comprises other antibodies or antigen-binding fragments that specifically bind to the CTLA4 protein; Preferably, the bispecific or multispecific molecule is a bispecific T-cell conjugate; More preferably, the nucleotide sequence of the nucleic acid molecule encoding the bispecific T-cell connector is shown in SEQ ID NO: 142, 143, 144 or 145.

9. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-7; or a bispecific or multispecific molecule according to claim 8.

10. A vector comprising the isolated nucleic acid molecule according to claim 9; Preferably, the vector is a cloning vector or an expression vector; More preferably, the vector is a cloning vector AbVec-hIgKappa, a cloning vector AbVec-hIgG1, or a recombinant viral vector; More preferably, the viral backbone of the recombinant viral vector is derived from vaccinia virus strains such as Tian Tan, New York, Copenhagen, Canary, and Ankara, as well as adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, varicella-zoster virus vectors, respiratory syncytial virus, Seneca Forest virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, smallpox virus, molluscum contagiosum virus, sheep pox virus, reovirus, rotavirus, enterovirus, Seneca virus, poliovirus, Coxsackie virus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, capsular virus, alphavirus, eastern equine encephalitis virus, Sindbis virus, rubella virus, coronavirus, flavivirus, hepatitis C virus, Japanese encephalitis virus, and St. Louis virus. Louisiana encephalitis virus, Murray Valley fever virus, yellow fever virus, West Nile virus, Zika virus, dengue virus, Ebola virus, Marburg virus, arena virus, Lassa virus, lymphocytic choriomeningitis virus, Pichend virus, Junin virus, Machupo virus, Hantavirus, Rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, simian virus 5, measles virus, vesicular stomatitis virus, rabies virus, respiratory syncytial virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, simian immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Laureus sarcoma virus, human T-cell leukemia virus type 1, simian foamy virus, hepatitis B virus, hepatitis E virus, human papillomavirus or polyomavirus; More preferably, the recombinant viral vector is the recombinant vaccinia virus Tian Tan strain; even more preferably, the recombinant vaccinia virus Tian Tan strain is a bispecific T-cell conjugate vaccinia virus Tian Tan strain; and even more preferably, the recombinant vaccinia virus Tian Tan strain comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NO: 142, 143, 144 or 145.

11. A host cell comprising the bispecific or multispecific molecule according to claim 8, the isolated nucleic acid molecule according to claim 9, or the vector according to claim 10; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing the antibody or antigen-binding fragment, bispecific antibody, or multispecific antibody; even more preferably, the host cell is a mammalian cell; even more preferably, the host cell is a human, mouse, sheep, horse, dog, or cat cell; most preferably, the host cell is a 293 cell, a 143TK-cell, or a CHO cell.

12. A method for preparing an antibody or an antigen-binding fragment thereof according to any one of claims 1-7, comprising culturing a host cell according to claim 11 under conditions allowing expression of the antibody or an antigen-binding fragment thereof according to any one of claims 1-7, and recovering the antibody or the antigen-binding fragment thereof from the cultured host cell culture.

13. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-7 and a therapeutic agent attached to said antibody or antigen-binding fragment thereof; Preferably, the therapeutic agent is a cytotoxic agent; Preferably, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive nuclides, and any combination thereof; Preferably, the immunoconjugate is an antibody-drug conjugate.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-7, a bispecific or multispecific molecule according to claim 8, or an immunoconjugate according to claim 13, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity; more preferably, the additional pharmaceutically active agent is an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus. Preferably, the antibody or its antigen-binding fragment, bispecific or multispecific molecule or immunoconjugate is provided as a separate component or as a component of the same composition with the additional pharmaceutically active agent.

15. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-7; Preferably, the antibody or its antigen-binding fragment carries a detectable label, radionuclide, fluorescent dye, luminescent substance, or biotin; more preferably, the detectable label is an enzyme, and even more preferably horseradish peroxidase; more preferably, the luminescent substance is a chemiluminescent substance. Preferably, the kit further includes a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof as described in any one of claims 1-7; Preferably, the second antibody further includes a detectable label, a radionuclide, a fluorescent dye, a luminescent substance, or biotin; more preferably, the detectable label is an enzyme, and even more preferably horseradish peroxidase; more preferably, the luminescent substance is a chemiluminescent substance.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the bispecific or multispecific molecule according to claim 8, or the immunoconjugate according to claim 13, or the pharmaceutical composition according to claim 14 in the preparation of a medicament for the prevention and / or treatment of tumors in a subject; Preferably, the drug further comprises additional pharmaceutically active agents; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity; more preferably, the additional pharmaceutically active agent is an alkylating agent, a mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an antiangiogenic agent, a cytokine, a molecularly targeted drug, an immune checkpoint inhibitor, or an oncolytic virus. Preferably, the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma (preferably high-grade glioma), blastoma (preferably neuroblastoma), osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer (preferably small cell lung cancer and non-small cell lung cancer), gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, colorectal cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or hematologic malignancies. Preferably, the subject is a mammal, more preferably a human.

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