Anti-ALPP and / or Anti-ALPG antibody, antibody-drug conjugate, and use thereof

Abstract

An anti-ALPP and / or anti-ALPG antibody, an antibody-drug conjugate, and a use thereof, more specifically, an anti-ALPP and / or anti-ALPG antibody or an antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a host cell comprising the nucleic acid molecule or the vector, an antibody-drug conjugate and a pharmaceutical composition, and a use thereof for preventing and / or treating tumors.

Description

Anti-ALPP and / or ALPG antibodies, antibody-drug conjugates and their uses

[0001] This application claims priority to Chinese patent application No. 2025103826149, filed on March 27, 2025, entitled "Anti-ALPP and / or ALPG Antibodies, Antibody-Drug Conjugates and Their Uses", the contents of which are to be understood as incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biopharmaceutical technology. Specifically, this invention relates to anti-ALPP and / or ALPG antibodies, antibody-drug conjugates and their uses. More specifically, this invention relates to anti-ALPP and / or ALPG antibodies or their antigen-binding fragments, nucleic acid molecules encoding said antibodies or their antigen-binding fragments, vectors containing said nucleic acid molecules, host cells containing said nucleic acid molecules or said vectors, antibody-drug conjugates and pharmaceutical compositions, and their uses for the prevention and / or treatment of tumors. Background Technology

[0003] Antibody-drug conjugates (ADCs) are derivatives of traditional antibody drugs, formed by linking a monoclonal antibody targeting a specific antigen with a small-molecule cytotoxic drug via a linker. They combine the specific targeting of antibody drugs with the cytotoxic effects of traditional small-molecule drugs, and their primary indication is malignant tumors. Due to their excellent tumor-killing effects, ADCs have rapidly become a hot topic in anti-tumor drug development. However, ADCs are typically highly toxic, thus requiring high specificity of the target antigen. Suitable targets for ADC development are generally those highly expressed in tumors and low or almost unexpressed in healthy tissues; the antigen should be a surface receptor upregulated by tumor cells, promoting tumor growth or survival, and the target antigen should possess internalization properties.

[0004] Alkaline phosphatase (ALP or AKP) is a zinc-containing glycoprotein that catalyzes the removal of the 5' phosphate group from nucleic acids, ATP, ADP, and organophosphates. It is a group of isoenzymes. Based on tissue specificity, human ALP can be divided into at least four tissue-specific forms: placental alkaline phosphatase (ALPP), germ cell alkaline phosphatase (ALPG), intestinal alkaline phosphatase (ALPI), and non-tissue-specific alkaline phosphatase (ALPL) expressed in the liver, bone, and kidney. Among them, placental alkaline phosphatase (ALPP) and germ cell alkaline phosphatase (ALPG) share a high sequence homology of 97.47% in their extracellular regions. ALPP and ALPG are GPI-anchored membrane-bound glycoproteins that can form homodimers (between ALPPs or between ALPGs) or heterodimers (ALPP / ALPG) on the cell surface. In normal tissues, ALPP / ALPG is highly expressed in the placenta and lowly expressed in the lungs and reproductive tissues. ALPP / ALPG is highly expressed in a variety of cancers, including ovarian cancer, endometrial cancer, gastric cancer, lung cancer, and bladder cancer. Given the significant differences in ALPP / ALPG expression between normal and tumor tissues, it could serve as an ideal target for drug development.

[0005] Currently, there are no marketed drugs targeting ALPP / ALPG. The most advanced drug is TC-A101 from Tiankeya, a CAR-T cell therapy specifically targeting ALPP, for the indications of ovarian cancer and endometrial cancer, and it is currently in Phase I / II clinical trials. Another ADC drug targeting ALPP / ALPG that entered clinical trials was SGN-ALPV, developed by Seagen. It entered Phase I clinical trials in April 2022, for indications including ovarian cancer, endometrial cancer, NSCLC, gastric cancer, and other solid tumors. However, after Pfizer completed its acquisition of Seagen in December 2023, the SGN-ALPV clinical trial was terminated due to portfolio priorities.

[0006] Therefore, there is an urgent need in this field to develop antibody drugs and / or antibody-drug conjugates that target ALPP / ALPG for cancer treatment. Summary of the Invention

[0007] The antibodies of this invention specifically recognize / bind to human ALPP and / or ALPG, without cross-reactivity with other members of the alkaline phosphatase family (e.g., ALPI and ALPL). The antibodies of this invention exhibit good endocytic activity in tumor cells (e.g., human gastric cancer NCI-N87 cells), significantly superior to positive control antibodies. The antibodies of this invention can also induce the killing of ALPP-expressing cells (e.g., tumor cells) via ADCC and / or CDC. Furthermore, the antibodies of this invention possess cross-reactivity with human, monkey, and mouse ALPP, good hydrophilicity and thermostability, and favorable pharmacokinetic characteristics. The antibodies of this invention also exhibit significant antitumor function by inhibiting tumor growth. Therefore, the antibodies of this invention have the potential for the prevention and / or treatment of tumors and possess significant clinical value. The antibody-drug conjugates of the present invention exhibit good in vitro killing activity in tumor cells (e.g., NCI-N87 cells, Caov-3 cells, and COV644 cells); and good in vivo efficacy in mice (e.g., mice with subcutaneous xenograft of BALB / c Nude cells from NCI-N87 and HPAC cells, and mice with subcutaneous xenograft of NOD / SCID cells from human ovarian cancer cells (e.g., OV17054 cells, OV15209 cells, OV9419 cells, OV9422 cells, OV9418 cells, OV5308 cells, OV9534 cells, and OV9409 cells), which is superior to the positive control antibody-drug conjugates; furthermore, the antibody-drug conjugates of the present invention also exhibit good in vitro stability and low toxicity. Therefore, the antibody-drug conjugates of the present invention have the potential for the prevention and / or treatment of tumors and have significant clinical value.

[0008] The antibody of the present invention

[0009] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to ALPP and / or ALPG, said antibody or antigen-binding fragment thereof comprising complementarity-determining regions (CDRs) as follows:

[0010] (a) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 1; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 2;

[0011] (b) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 3; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 4;

[0012] (c) Having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 5; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) having the sequence shown in SEQ ID NO: 6;

[0013] (d) Having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 7; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 8;

[0014] (e) having CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 9; and / or having CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 10; or,

[0015] (f) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) below, and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) below, wherein, compared with the heavy chain variable region (VH) and / or the light chain variable region (VL) respectively, at least one CDR of the heavy chain variable region (VH) and / or the light chain variable region (VL) contains a mutation, and the CDR containing the mutation has at least [aspect] compared with the sequence from which it originates. 70%, 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, or the mutation being a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids); preferably, the substitution is a conservative substitution; preferably, the heavy chain variable region (VH) has a sequence selected from any of SEQ ID NOs: 114, 116, 118, 120, or 122; preferably, the light chain variable region (VL) has a sequence selected from any of SEQ ID NOs: 115, 117, 119, 121, or 123.

[0016] In some embodiments, the antibody or its antigen-binding fragment includes complementarity-determining regions (CDRs) as follows:

[0017] (a) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 1; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 2;

[0018] (b) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 3; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 4;

[0019] (c) Having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 5; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) having the sequence shown in SEQ ID NO: 6;

[0020] (d) Having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 7; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 8;

[0021] (e) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 9; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) having the sequence shown in SEQ ID NO: 10;

[0022] (f) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 114; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) having the sequence shown in SEQ ID NO: 115;

[0023] (g) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 116; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) having the sequence shown in SEQ ID NO: 117;

[0024] (h) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 118; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 119;

[0025] (i) having CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 120; and / or having CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 121; or,

[0026] (j) having CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 122; and / or having CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) having the sequence shown in SEQ ID NO: 123.

[0027] In some implementations, the CDR is defined by the Kabat, IMGT, Chothia, Contact, or AbM numbering system.

[0028] In some embodiments, the antibody or its antigen-binding fragment binds to human ALPP and / or human ALPG.

[0029] In some embodiments, an antibody or antigen-binding fragment thereof capable of specifically binding to ALPP and / or ALPG is provided, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0030] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined by the Kabat numbering system.

[0031] (1a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 12 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 13 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 15 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 16 or a variant thereof;

[0032] (1b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 32 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 33 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 34 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 35 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 36 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 37 or a variant thereof;

[0033] (1c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 53 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 54 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 55 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 56 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 57 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 58 or a variant thereof;

[0034] (1d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 74 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 75 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 76 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 77 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 78 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 79 or a variant thereof; or,

[0035] (1e) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 94 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 95 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 96 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 97 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 98 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 99 or a variant thereof;

[0036] The variant described in any one of (1a)-(1e) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0037] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined by the IMGT numbering system.

[0038] (2a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 17 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 18 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 19 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 20 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 21 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 16 or a variant thereof;

[0039] (2b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 38 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 39 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 124 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 41 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 42 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 37 or a variant thereof;

[0040] (2c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 59 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 60 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 61 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 62 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 63 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 58 or a variant thereof;

[0041] (2d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence SEQ ID NO: 80 or a variant thereof, CDR-H2 with the sequence SEQ ID NO: 81 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO: 82 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence SEQ ID NO: 83 or a variant thereof, CDR-L2 with the sequence SEQ ID NO: 84 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO: 79 or a variant thereof; or,

[0042] (2e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 100 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 101 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 102 or a variant thereof, and / or Light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 103 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 104 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 99 or a variant thereof;

[0043] The variant described in any of (2a)-(2e) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0044] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined by the Chothia numbering system.

[0045] (3a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 22 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 23 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 13 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 15 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 16 or a variant thereof;

[0046] (3b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 43 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 44 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 34 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 35 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 36 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 37 or a variant thereof;

[0047] (3c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 64 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 65 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 55 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 56 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 57 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 58 or a variant thereof;

[0048] (3d) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 43 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 85 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 76 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 77 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 78 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 79 or a variant thereof; or,

[0049] (3e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 64 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 105 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 96 or a variant thereof, and / or Light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 97 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 98 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 99 or a variant thereof;

[0050] The variant described in any of (3a)-(3e) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0051] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined by a Contact numbering system.

[0052] (4a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 24 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 25 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 26 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 27 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 28 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 29 or a variant thereof;

[0053] (4b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 45 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 46 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 47 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 48 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 49 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 50 or a variant thereof;

[0054] (4c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 66 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 67 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 68 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 69 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 70 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 71 or a variant thereof;

[0055] (4d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence SEQ ID NO: 86 or a variant thereof, CDR-H2 with the sequence SEQ ID NO: 87 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO: 88 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence SEQ ID NO: 89 or a variant thereof, CDR-L2 with the sequence SEQ ID NO: 90 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO: 91 or a variant thereof; or,

[0056] (4e) Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO: 106 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 107 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 108 or a variant thereof, and / or Light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO: 109 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 110 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 111 or a variant thereof;

[0057] The variant described in any one of (4a)-(4e) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0058] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined by the AbM numbering system.

[0059] (5a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 30 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 31 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 13 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 15 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 16 or a variant thereof;

[0060] (5b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 51 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 52 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 34 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 35 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 36 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 37 or a variant thereof;

[0061] (5c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 72 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 73 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 55 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 56 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 57 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 58 or a variant thereof;

[0062] (5d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 92 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 93 or a variant thereof, and CDR-H3 with sequence SEQ ID NO: 76 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 77 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 78 or a variant thereof, and CDR-L3 with sequence SEQ ID NO: 79 or a variant thereof; or,

[0063] (5e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 112 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 113 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 96 or a variant thereof, and / or Light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 97 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 98 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 99 or a variant thereof;

[0064] The variant described in any one of (5a)-(5e) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0065] In some embodiments, the CDR-H2 variant of the sequence SEQ ID NO: 46 is the CDR-H2 of the sequence SEQ ID NO: 125.

[0066] In some embodiments, the CDR-H2 variant of the sequence SEQ ID NO: 54 is the CDR-H2 variant of the sequence SEQ ID NO: 126.

[0067] In some embodiments, the CDR-H2 variant of the sequence SEQ ID NO: 60 is the CDR-H2 of the sequence SEQ ID NO: 127.

[0068] In some embodiments, the CDR-H2 variant of the sequence SEQ ID NO: 65 is the CDR-H2 variant of the sequence SEQ ID NO: 128.

[0069] In some embodiments, the CDR-H2 variant of the sequence SEQ ID NO: 67 is the CDR-H2 of the sequence SEQ ID NO: 129.

[0070] In some embodiments, the CDR-L2 of the variant with sequence SEQ ID NO: 70 is the CDR-L2 with sequence SEQ ID NO: 130.

[0071] In some embodiments, the CDR-H2 variant of the sequence SEQ ID NO: 73 is the CDR-H2 of the sequence SEQ ID NO: 131.

[0072] In some embodiments, the CDR-H3 variant of the sequence SEQ ID NO: 76 is the CDR-H3 of the sequence SEQ ID NO: 132.

[0073] In some embodiments, the CDR-H3 variant of the sequence SEQ ID NO: 82 is the CDR-H3 variant of the sequence SEQ ID NO: 133.

[0074] In some embodiments, the CDR-H2 variant of the sequence SEQ ID NO: 87 is the CDR-H2 of the sequence SEQ ID NO: 134.

[0075] In some embodiments, the CDR-H3 variant of the sequence SEQ ID NO: 88 is the CDR-H3 of the sequence SEQ ID NO: 135.

[0076] In some embodiments, the CDR-L2 of the variant of the sequence SEQ ID NO: 110 is the CDR-L2 of the sequence SEQ ID NO: 136.

[0077] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR is defined by the Kabat numbering system.

[0078] (1a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 11, CDR-H2 with sequence SEQ ID NO: 12, CDR-H3 with sequence SEQ ID NO: 13, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 14, CDR-L2 with sequence SEQ ID NO: 15 or SEQ ID NO: 16, and CDR-L3 with sequence SEQ ID NO: 16;

[0079] (1b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 32, CDR-H2 with sequence SEQ ID NO: 33, CDR-H3 with sequence SEQ ID NO: 34, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 35, CDR-L2 with sequence SEQ ID NO: 36, and CDR-L3 with sequence SEQ ID NO: 37;

[0080] (1c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 53, CDR-H2 with sequence SEQ ID NO: 54 or 126, CDR-H3 with sequence SEQ ID NO: 55, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 56, CDR-L2 with sequence SEQ ID NO: 57, and CDR-L3 with sequence SEQ ID NO: 58;

[0081] (1d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 74, CDR-H2 with sequence SEQ ID NO: 75, and CDR-H3 with sequence SEQ ID NO: 76 or 132; and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 77, CDR-L2 with sequence SEQ ID NO: 78, and CDR-L3 with sequence SEQ ID NO: 79; or,

[0082] (1e) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 94, CDR-H2 with sequence SEQ ID NO: 95, CDR-H3 with sequence SEQ ID NO: 96, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 97, CDR-L2 with sequence SEQ ID NO: 98, and CDR-L3 with sequence SEQ ID NO: 99.

[0083] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR is defined by the IMGT numbering system.

[0084] (2a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 17, CDR-H2 with sequence SEQ ID NO: 18, and CDR-H3 with sequence SEQ ID NO: 19, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 20, CDR-L2 with sequence SEQ ID NO: 21, and CDR-L3 with sequence SEQ ID NO: 16;

[0085] (2b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 38, CDR-H2 with sequence SEQ ID NO: 39, CDR-H3 with sequence 124, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 41, CDR-L2 with sequence SEQ ID NO: 42, and CDR-L3 with sequence SEQ ID NO: 37;

[0086] (2c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 59, CDR-H2 with sequence SEQ ID NO: 60 or 127, CDR-H3 with sequence SEQ ID NO: 61, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 62, CDR-L2 with sequence SEQ ID NO: 63, and CDR-L3 with sequence SEQ ID NO: 58;

[0087] (2d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 80, CDR-H2 with sequence SEQ ID NO: 81, and CDR-H3 with sequence SEQ ID NO: 82 or 133; and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 83, CDR-L2 with sequence SEQ ID NO: 84, and CDR-L3 with sequence SEQ ID NO: 79; or,

[0088] (2e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 100, CDR-H2 with sequence SEQ ID NO: 101, CDR-H3 with sequence SEQ ID NO: 102, and light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 103, CDR-L2 with sequence SEQ ID NO: 104, and CDR-L3 with sequence SEQ ID NO: 99.

[0089] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR is defined by the Chothia numbering system.

[0090] (3a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 22, CDR-H2 with sequence SEQ ID NO: 23, CDR-H3 with sequence SEQ ID NO: 13, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 14, CDR-L2 with sequence SEQ ID NO: 15, and CDR-L3 with sequence SEQ ID NO: 16;

[0091] (3b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 43, CDR-H2 with sequence SEQ ID NO: 44, CDR-H3 with sequence SEQ ID NO: 34, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 35, CDR-L2 with sequence SEQ ID NO: 36, and CDR-L3 with sequence SEQ ID NO: 37;

[0092] (3c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 64, CDR-H2 with sequence SEQ ID NO: 65 or 128, CDR-H3 with sequence SEQ ID NO: 55, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 56, CDR-L2 with sequence SEQ ID NO: 57, and CDR-L3 with sequence SEQ ID NO: 58;

[0093] (3d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 43, CDR-H2 with sequence SEQ ID NO: 85, and CDR-H3 with sequence SEQ ID NO: 76 or 132; and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 77, CDR-L2 with sequence SEQ ID NO: 78, and CDR-L3 with sequence SEQ ID NO: 79; or,

[0094] (3e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 64, CDR-H2 with sequence SEQ ID NO: 105, CDR-H3 with sequence SEQ ID NO: 96, and light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 97, CDR-L2 with sequence SEQ ID NO: 98, and CDR-L3 with sequence SEQ ID NO: 99.

[0095] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR is defined by a Contact numbering system.

[0096] (4a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 24, CDR-H2 with sequence SEQ ID NO: 25, and CDR-H3 with sequence SEQ ID NO: 26, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 27, CDR-L2 with sequence SEQ ID NO: 28, and CDR-L3 with sequence SEQ ID NO: 29;

[0097] (4b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 45, CDR-H2 with sequence SEQ ID NO: 46 or 125, CDR-H3 with sequence SEQ ID NO: 47, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 48, CDR-L2 with sequence SEQ ID NO: 49, and CDR-L3 with sequence SEQ ID NO: 50;

[0098] (4c) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 66, CDR-H2 with sequence SEQ ID NO: 67 or 129, CDR-H3 with sequence SEQ ID NO: 68, and light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 69, CDR-L2 with sequence SEQ ID NO: 70 or 130, and CDR-L3 with sequence SEQ ID NO: 71;

[0099] (4d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 86, CDR-H2 with sequence SEQ ID NO: 87 or 134, and CDR-H3 with sequence SEQ ID NO: 88 or 135; and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 89, CDR-L2 with sequence SEQ ID NO: 90, and CDR-L3 with sequence SEQ ID NO: 91; or,

[0100] (4e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 106, CDR-H2 with sequence SEQ ID NO: 107, CDR-H3 with sequence SEQ ID NO: 108, and light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 109, CDR-L2 with sequence SEQ ID NO: 110 or 136, and CDR-L3 with sequence SEQ ID NO: 111.

[0101] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR is defined by the AbM numbering system.

[0102] (5a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 30, CDR-H2 with sequence SEQ ID NO: 31, and CDR-H3 with sequence SEQ ID NO: 13, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 14, CDR-L2 with sequence SEQ ID NO: 15, and CDR-L3 with sequence SEQ ID NO: 16;

[0103] (5b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 51, CDR-H2 with sequence SEQ ID NO: 52, and CDR-H3 with sequence SEQ ID NO: 34, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 35, CDR-L2 with sequence SEQ ID NO: 36, and CDR-L3 with sequence SEQ ID NO: 37;

[0104] (5c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 72, CDR-H2 with sequence SEQ ID NO: 73 or 131, CDR-H3 with sequence SEQ ID NO: 55, and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 56, CDR-L2 with sequence SEQ ID NO: 57, and CDR-L3 with sequence SEQ ID NO: 58;

[0105] (5d) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 92, CDR-H2 with sequence SEQ ID NO: 93, and CDR-H3 with sequence SEQ ID NO: 76 or 132; and a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 77, CDR-L2 with sequence SEQ ID NO: 78, and CDR-L3 with sequence SEQ ID NO: 79; or,

[0106] (5e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 112, CDR-H2 with sequence SEQ ID NO: 113, CDR-H3 with sequence SEQ ID NO: 96, and light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 97, CDR-L2 with sequence SEQ ID NO: 98, and CDR-L3 with sequence SEQ ID NO: 99.

[0107] In some embodiments, the antibody or its antigen-binding fragment further includes a framework region (FR) derived from mammalian (e.g., mouse or human) immunoglobulin.

[0108] In some embodiments, the antibody or its antigen-binding fragment includes frame regions (FRs) of immunoglobulins derived from humans or mice in its VH and / or VL regions.

[0109] In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from mouse immunoglobulin, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from mouse immunoglobulin. Therefore, in some embodiments, the antibody or its antigen-binding fragment is murine in origin.

[0110] In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulins, and / or the VL region of the antibody or its antigen-binding fragment comprises a light chain variable region (VL) framework region (FR) derived from human immunoglobulins. Therefore, in some embodiments, the antibody or its antigen-binding fragment is humanized. In such embodiments, the heavy chain variable region (VH) and / or the light chain variable region (VL) of the antibody or its antigen-binding fragment may contain one or more non-human (e.g., murine) amino acid residues; for example, the heavy chain framework region (FR) and / or the light chain framework region (FR) may contain one or more amino acid reversion mutations containing corresponding murine amino acid residues.

[0111] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0112] (a) The heavy chain framework region of a human immunoglobulin or a variant thereof, said variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to the human germline antibody gene sequence from which it is derived; and / or

[0113] (b) The light chain framework region of a human immunoglobulin or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to the sequence of the human germline antibody gene from which it is derived.

[0114] In some embodiments, the degree of humanization of the antibody or antigen-binding fragment of the present invention is at least 75%, 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%.

[0115] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0116] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0117] (a) A heavy chain variable region (VH) comprising a sequence or a variant thereof as shown in SEQ ID NO: 1 or 114, and / or a light chain variable region (VL) comprising a sequence or a variant thereof as shown in SEQ ID NO: 2 or 115;

[0118] (b) A heavy chain variable region (VH) comprising a sequence or a variant thereof as shown in SEQ ID NO: 3 or 116, and / or a light chain variable region (VL) comprising a sequence or a variant thereof as shown in SEQ ID NO: 4 or 117;

[0119] (c) A heavy chain variable region (VH) comprising a sequence or a variant thereof as shown in SEQ ID NO: 5 or 118, and / or a light chain variable region (VL) comprising a sequence or a variant thereof as shown in SEQ ID NO: 6 or 119;

[0120] (d) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 7 or 120 or a variant thereof, and / or, containing a light chain variable region (VL) of the sequence shown in SEQ ID NO: 8 or 121 or a variant thereof; or,

[0121] (e) A heavy chain variable region (VH) comprising a sequence or a variant thereof as shown in SEQ ID NO: 9 or 122, and / or a light chain variable region (VL) comprising a sequence or a variant thereof as shown in SEQ ID NO: 10 or 123;

[0122] The variant has at least 70%, 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 sequence from which it originates; or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0123] In some exemplary embodiments, the antibody or its antigen-binding fragment comprises:

[0124] (a) VH having the sequence shown in SEQ ID NO: 1 and VL having the sequence shown in SEQ ID NO: 2;

[0125] (b) VH having the sequence shown in SEQ ID NO: 114 and VL having the sequence shown in SEQ ID NO: 115;

[0126] (c) VH having the sequence shown in SEQ ID NO: 3 and VL having the sequence shown in SEQ ID NO: 4;

[0127] (d) VH having the sequence shown in SEQ ID NO: 116 and VL having the sequence shown in SEQ ID NO: 117;

[0128] (e) VH having the sequence shown in SEQ ID NO: 5 and VL having the sequence shown in SEQ ID NO: 6;

[0129] (f) VH having the sequence shown in SEQ ID NO: 118 and VL having the sequence shown in SEQ ID NO: 119;

[0130] (g) VH having the sequence shown in SEQ ID NO: 7 and VL having the sequence shown in SEQ ID NO: 8;

[0131] (h) VH having the sequence shown in SEQ ID NO: 120 and VL having the sequence shown in SEQ ID NO: 121;

[0132] (i) VH having the sequence shown in SEQ ID NO: 9 and VL having the sequence shown in SEQ ID NO: 10; or,

[0133] (j) VH having the sequence shown in SEQ ID NO: 122 and VL having the sequence shown in SEQ ID NO: 123.

[0134] In some embodiments, the antibody or its antigen-binding fragment further comprises a constant region (CH) or a variant thereof derived from a mammalian (e.g., mouse or human) immunoglobulin, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence. In some embodiments, the variant has one or more conserved amino acid substitutions compared to its derived wild-type sequence.

[0135] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or, the light chain of the antibody or its antigen-binding fragment comprises the light chain constant region (CL) of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids).

[0136] In some embodiments, the antibody or its antigen-binding fragment comprises a variant of the heavy chain constant region (CH) of a human immunoglobulin, which has altered (e.g., enhanced, reduced, or eliminated) effector functions compared to its derived wild-type sequence. In such embodiments, the variant typically has at least one amino acid substitution compared to its derived wild-type sequence. The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc. In some cases, these effector functions are desired for therapeutic antibodies; however, in other cases, these effector functions may be unnecessary or even detrimental, depending on the intended purpose. Therefore, in some embodiments, the antibody or its antigen-binding fragment has reduced or even eliminated effector functions (e.g., ADCC and / or CDC activity). In such embodiments, the antibody or its antigen-binding fragment comprises a variant of the human IgG heavy chain constant region having at least one of the following substitutions compared to its derived wild-type sequence: S228P, L234A, L235A, G237A, S239D, A330L, I332E, N297A, D356E, L358M, and K447 deletion (the amino acid positions mentioned above are based on the EU numbering system, Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969).

[0137] In some embodiments, the antibody or its antigen-binding fragment comprises a variant of the human IgG1 heavy chain constant region, said variant having at least one of the following substitutions compared to its derived wild-type sequence: L234A, L235A, G237A, S239D, I332E, D356E, L358M, and K447 deletion, wherein the amino acid positions of said substitutions are according to the EU numbering system. In such embodiments, the antibody or its antigen-binding fragment has reduced ADCC and CDC activity.

[0138] In some embodiments, the variant of the heavy chain constant region (CH) may have one or more conserved substitutions of amino acids compared to its derived wild-type sequence. In such embodiments, the variant of the heavy chain constant region (CH) may have the same or substantially the same effector function compared to its derived wild-type sequence.

[0139] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) of mouse immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or, the light chain of the antibody or its antigen-binding fragment comprises the light chain constant region (CL) of mouse immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids).

[0140] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is a mouse IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is a human IgG1 heavy chain constant region.

[0141] In some embodiments, the light chain constant region is a κ or λ light chain constant region. In some embodiments, the light chain constant region is a mouse κ light chain constant region. In some embodiments, the light chain constant region is a human κ light chain constant region.

[0142] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region selected from the following:

[0143] (1) Human IgG1 heavy chain constant region; or,

[0144] (2) A variant of the constant region of the human IgG1 heavy chain, the variant having altered effector function compared to the wild-type sequence from which it is derived, preferably having at least one of the following substitutions compared to the wild-type sequence from which it is derived: L234A, L235A, G237A, S239D, I332E, D356E, L358M and K447 deletion; wherein the amino acid position of the substitution is according to the EU numbering system.

[0145] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain constant region (CH) or a variant thereof comprising the sequence shown in SEQ ID NO: 137, the variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 137; and / or, a light chain constant region (CL) or a variant thereof comprising the sequence shown in SEQ ID NO: 138, the variant having up to 20 conserved substitutions (e.g., up to 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 138.

[0146] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) having a sequence as shown in SEQ ID NO: 137, 159, 172 or 218 and a light chain constant region (CL) having a sequence as shown in SEQ ID NO: 138.

[0147] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain containing a heavy chain variable region (VH) and a heavy chain constant region (CH), and / or a light chain containing a light chain variable region (VL) and a light chain constant region (CL).

[0148] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0149] (a) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 1 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 172, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 2 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0150] (b) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 114 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 137, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 115 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138;

[0151] (c) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 114 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 159, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 115 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0152] (d) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 3 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 172, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 4 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0153] (e) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 116 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 137, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 117 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0154] (f) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 116 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 159, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 117 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0155] (g) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 5 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 137, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 6 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0156] (h) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 118 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 137, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 119 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0157] (i) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 118 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 159, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 119 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138;

[0158] (j) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 7 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 172, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 8 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0159] (k) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 120 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 137, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 121 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138;

[0160] (l) a heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 120 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 159, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 121 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138;

[0161] (m) a heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 9 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 172, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 10 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0162] (n) a heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 122 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 137, and / or, a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 123 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138; or,

[0163] (o) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 122 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 159, and / or, a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 123 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138; or,

[0164] (p) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 114 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 218, and / or, a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 115 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138; or,

[0165] (q) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 116 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 218, and / or, a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 117 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138; or,

[0166] (r) a heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 118 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 218, and / or, a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 119 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138; or,

[0167] (s) a heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 120 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 218, and / or, a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 121 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138; or,

[0168] (t) A heavy chain comprising a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 122 and a heavy chain constant region (CH) having the sequence shown in SEQ ID NO: 218, and / or a light chain comprising a light chain variable region (VL) having the sequence shown in SEQ ID NO: 123 and a light chain constant region (CL) having the sequence shown in SEQ ID NO: 138.

[0169] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0170] (a) A heavy chain comprising a sequence as shown in SEQ ID NO: 139 or a variant thereof, and / or a light chain comprising a sequence as shown in SEQ ID NO: 140 or a variant thereof;

[0171] (b) A heavy chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 149, 160 or 219, and / or a light chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 150;

[0172] (c) A heavy chain comprising the sequence shown in SEQ ID NO: 141 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 142 or a variant thereof;

[0173] (d) A heavy chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 151, 161 or 220, and / or a light chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 152;

[0174] (e) a heavy chain comprising a sequence as shown in SEQ ID NO: 143 or a variant thereof, and / or a light chain comprising a sequence as shown in SEQ ID NO: 144 or a variant thereof;

[0175] (f) a heavy chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 153, 162 or 221, and / or a light chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 154;

[0176] (h) a heavy chain comprising a sequence as shown in SEQ ID NO: 145 or a variant thereof, and / or a light chain comprising a sequence as shown in SEQ ID NO: 146 or a variant thereof;

[0177] (i) a heavy chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 155, 163 or 222, and / or a light chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 156;

[0178] (j) a heavy chain comprising the sequence shown in SEQ ID NO: 147 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 148 or a variant thereof; or,

[0179] (k) a heavy chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 157, 164 or 223, and / or a light chain comprising a sequence or a variant thereof as shown in SEQ ID NO: 158;

[0180] The variant has at least 70%, 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 sequence from which it originates; or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0181] In some exemplary embodiments, the antibody or its antigen-binding fragment comprises:

[0182] (a) A heavy chain having a sequence as shown in SEQ ID NO: 139 and a light chain having a sequence as shown in SEQ ID NO: 140;

[0183] (b) Heavy chains having sequences as shown in SEQ ID NO: 149, 160 or 219 and light chains having sequences as shown in SEQ ID NO: 150;

[0184] (c) A heavy chain having the sequence shown in SEQ ID NO: 141 and a light chain having the sequence shown in SEQ ID NO: 142;

[0185] (d) Heavy chains having sequences as shown in SEQ ID NO: 151, 161 or 220 and light chains having sequences as shown in SEQ ID NO: 152;

[0186] (e) A heavy chain having a sequence as shown in SEQ ID NO: 143 and a light chain having a sequence as shown in SEQ ID NO: 144;

[0187] (f) Heavy chains having sequences as shown in SEQ ID NO: 153, 162 or 221 and light chains having sequences as shown in SEQ ID NO: 154;

[0188] (h) a heavy chain having the sequence shown in SEQ ID NO: 145 and a light chain having the sequence shown in SEQ ID NO: 146;

[0189] (i) a heavy chain having a sequence as shown in SEQ ID NO: 155, 163 or 222 and a light chain having a sequence as shown in SEQ ID NO: 156;

[0190] (j) A heavy chain having the sequence shown in SEQ ID NO: 147 and a light chain having the sequence shown in SEQ ID NO: 148; or,

[0191] (k) A heavy chain having a sequence as shown in SEQ ID NO: 157, 164 or 223 and a light chain having a sequence as shown in SEQ ID NO: 158.

[0192] In some embodiments, the antigen-binding fragment is selected from ScFv, Fab, Fab', (Fab')2, Fv fragment, disulfide-linked Fv (dsFv), single-domain antibody (sdAb), or nanobody (VHH); the antibody is selected from diabody, bispecific antibody, or multispecific antibody; and / or, the antibody is a murine antibody, chimeric antibody, or humanized antibody.

[0193] In some embodiments, the antibody or its antigen-binding fragment has one or more of the following biological functions:

[0194] (1) Binding of human ALPP with a KD of less than about 35 nM, for example less than about 15 nM, 10 nM, 5 nM, 1 nM or lower; and / or binding of monkey ALPP with a KD of less than about 75 nM, for example less than about 70 nM, 20 nM, 10 nM, 5 nM or lower; and / or binding of human ALPG with a KD of less than about 25 nM, for example less than about 20 nM, 10 nM, 5 nM, 1 nM or lower; preferably, the KD is measured by surface plasmon resonance (SPR) technology (e.g. in Biacore);

[0195] (2) With EC less than about 1 nM, for example less than about 0.8 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM or smaller 50 In combination with human ALPP; preferably, the EC50 is measured by ELISA;

[0196] (3) It has ADCC activity, such as inducing the killing of cells expressing human ALPP (e.g., tumor cells, such as tumor cells expressing ALPP) through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0197] (4) It has CDC activity, for example, by inducing the killing of cells expressing human ALPP (e.g., tumor cells, such as tumor cells expressing ALPP) through complement-dependent cytotoxicity (CDC).

[0198] (5) It has cross-reactivity with human, monkey and mouse ALPP;

[0199] (6) Does not bind to ALPI and ALPL; for example, as determined by ELISA;

[0200] (7) It does not bind to rat ALPP and ALPG, and basically does not bind to mouse ALPG, exhibiting good species specificity; for example, it can be detected by ELISA.

[0201] (8) It induces ALPP internalization and has good endocytic activity;

[0202] (9) It has good hydrophilicity; for example, it can be determined by analytical hydrophobic interaction chromatography (HIC).

[0203] (10) It has good thermal stability; for example, as determined by differential scanning fluorescence (DSF).

[0204] (11) It has good pharmacokinetic characteristics;

[0205] (12) It has a significant anti-tumor function that inhibits tumor growth.

[0206] Derived antibodies

[0207] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to ALPP or ALPG (particularly human ALPP and / or ALPG). Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or a multihistidine tag). Furthermore, the antibodies or antigen-binding fragments of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.

[0208] In some embodiments, the antibodies or antigen-binding fragments of the present invention are labeled. In some embodiments, the antibodies or antigen-binding fragments of the present invention are labeled with detectable markers. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750), acridine esters, magnetic beads (e.g., ), calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of such markers include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). Detectable markers as described above can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing an enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers. In some embodiments, such markers can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0209] Antibody preparation

[0210] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0211] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10:163-167 (1992)). Additionally, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture media. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0212] Nucleic acid molecules

[0213] In a second aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof described in the first aspect, or the variable region of its heavy chain and / or the variable region of its light chain, or the nucleotide sequence of its heavy chain and / or light chain.

[0214] In some embodiments, the isolated nucleic acid molecule encodes the antibody or antigen-binding fragment thereof described in the first aspect, or the variable region of its heavy chain and / or the variable region of its light chain, or its heavy chain and / or light chain, wherein the isolated nucleic acid molecule is DNA or RNA.

[0215] In some embodiments, the DNA comprises a first nucleotide sequence encoding the heavy chain variable region of the antibody described in the first aspect, and / or a second nucleotide sequence encoding the light chain variable region of the antibody described in the first aspect.

[0216] In some embodiments, the first nucleotide sequence encoding the heavy chain variable region of the antibody described in the first aspect has a sequence selected from the following:

[0217] (a) A nucleotide sequence as shown in any of SEQ ID NOs: 173, 175, 177, 179, 181, 193, 195, 197, 199 or 201;

[0218] (b) A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions); or,

[0219] (c) A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0220] In some embodiments, the second nucleotide sequence encoding the light chain variable region of the antibody described in the first aspect has a sequence selected from:

[0221] (a) A nucleotide sequence as shown in any of SEQ ID NOs: 174, 176, 178, 180, 182, 194, 196, 198, 200 or 202;

[0222] (b) A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions); or,

[0223] (c) A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0224] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 173, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 174.

[0225] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 175, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 176.

[0226] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 177, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 178.

[0227] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 179, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 180.

[0228] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 181, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 182.

[0229] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 193, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 194.

[0230] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 195, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 196.

[0231] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 197, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 198.

[0232] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 199, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 200.

[0233] In some embodiments, the DNA encoding the heavy chain variable region of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 201, and the DNA encoding the light chain variable region of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 202.

[0234] In some embodiments, the DNA comprises a first nucleotide sequence encoding the heavy chain of the antibody described in the first aspect, and / or a second nucleotide sequence encoding the light chain of the antibody described in the first aspect.

[0235] In some embodiments, the first nucleotide sequence of the heavy chain encoding the antibody of the first aspect has a sequence selected from the following:

[0236] (a) A nucleotide sequence as shown in any of SEQ ID NOs: 183, 185, 187, 189, 191, 203, 205, 207, 209, 211, 213-217 or 224-228;

[0237] (b) A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions); or,

[0238] (c) A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0239] In some embodiments, the second nucleotide sequence encoding the light chain of the antibody described in the first aspect has a sequence selected from the following:

[0240] (a) A nucleotide sequence as shown in any of SEQ ID NO: 184, 186, 188, 190, 192, 204, 206, 208, 210 or 212;

[0241] (b) A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions); or,

[0242] (c) A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0243] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 183, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 184.

[0244] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 185, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 186.

[0245] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 187, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 188.

[0246] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 189, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 190.

[0247] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 191, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 192.

[0248] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 203, 213 or 224, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 204.

[0249] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 205, 214 or 225, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 206.

[0250] In some embodiments, the DNA encoding the heavy chain of the antibody of the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 207, 215 or 226, and the DNA encoding the light chain of the antibody of the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 208.

[0251] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 209, 216 or 227, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 210.

[0252] In some embodiments, the DNA encoding the heavy chain of the antibody described in the first aspect consists of a first nucleotide sequence as shown in SEQ ID NO: 211, 217, or 228, and the DNA encoding the light chain of the antibody described in the first aspect consists of a second nucleotide sequence as shown in SEQ ID NO: 212. Based on codon degeneracy in the art, in some embodiments, the nucleotide sequence can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0253] Antibody expression

[0254] Thirdly, the present invention provides a vector comprising the isolated nucleic acid molecule described in the second aspect. In some embodiments, the vector is a cloning vector or an expression vector. In some embodiments, the vector is, for example, a plasmid, a granulosome, a bacteriophage, a lentivirus, etc. In some embodiments, the vector is capable of expressing the antibody or its antigen-binding fragment described in the first aspect in a subject (e.g., a mammal, such as a human).

[0255] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment of the present invention and a second nucleotide sequence encoding a light chain or a light chain variable region thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector containing the first nucleotide sequence and a second vector containing the second nucleotide sequence.

[0256] Fourthly, the present invention provides a host cell comprising the isolated nucleic acid molecule described in the second aspect or the vector described in the third aspect. Such host cells can be prokaryotic cells (e.g., *E. coli* cells) or eukaryotic cells (e.g., yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.)). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell is a mammalian cell, such as Chinese hamster ovary CHO cells (e.g., CHO-K1, CHO-S, CHO DG 44) or human embryonic kidney HEK293 cells.

[0257] Fifthly, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect, the method comprising culturing the host cell of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0258] Antibody-drug conjugates and their preparation methods

[0259] In a sixth aspect, the present invention provides an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof conjugated to one or more drugs as described in the first aspect.

[0260] In some embodiments, the drug is a cytotoxic drug.

[0261] In some embodiments, the antibody-drug conjugate has a structure represented by general formula (I):

[0262] Ab-(LD)p(I);

[0263] Wherein, Ab is the antibody or its antigen-binding fragment described in this invention; L is a linker; D is a cytotoxic drug; p is selected from an integer or decimal between 1 and 10; preferably, p is selected from an integer or decimal between 1 and 8.

[0264] In some embodiments, the connector L has the structure shown in formula (II):

[0265] L 1 -L 2 -L 3 -L 4 (II)

[0266] Wherein, the L 1The terminal is connected to Ab, L 4 The terminal is connected to D;

[0267] L 1 It is succinimide or maleimide; preferably, the L 1 It is maleimide;

[0268] L 2 For (PEG) 1-10 C 1-10 alkylene acyl or (PEG) 1-10 C 1-10 Alkyl group; preferably, the L 2 It is (PEG)2, hexanoyl, or (PEG)2 acetylated;

[0269] L 3 It is a peptide residue or chemical bond composed of 1 to 7 amino acid residues; preferably, the amino acid is selected from alanine, glycine, valine, phenylalanine, lysine, citrulline, serine, cysteine, glutamic acid, aspartic acid, histidine, cysteine, isoleucine, leucine, methionine, asparagine, proline, glutamine, arginine, threonine, tryptophan, tyrosine, pentanediol, leucine, pyrrolidone, homoserine, homocysteine, or demethylpyrrolidone; preferably, the L 3 It is a peptide residue composed of 2 to 4 amino acid residues; preferably, the amino acid is selected from alanine, glycine, valine, phenylalanine, or citrulline; preferably, the L 3 It is a dipeptide residue of valine-citrulline or a tetrapeptide residue of glycine-glycine-phenylalanine-glycine.

[0270] L 4 p-Aminobenzyloxycarbonyl, p-aminobenzyloxy, NR 1 (CR 2 R 3 ) t Or chemical bond; R 1 R 2 and R 3 They may be the same or different, and each is independently selected from hydrogen atoms or C atoms. 1-10 Alkyl group, where t is an integer from 1 to 10; preferably, L 4 It is p-aminobenzyloxycarbonyl or NHCH2.

[0271] In some embodiments, the linker L is 6-maleiminohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl, with the following structure:

[0272] In some embodiments, the linker L is 6-maleiminohexanoyl-glycine-glycine-phenylalanine-glycine-aminomethyl, with the following structure:

[0273] In some embodiments, the linker L is 6-maleimino-diethylene glycol acetyl-valine-citrulline-p-aminobenzyloxycarbonyl, with the following structure:

[0274] In some embodiments, the linker L is 6-maleiminohexanoyl-valine-alanine-p-aminobenzyloxycarbonyl, with the following structure:

[0275] In some embodiments, the linker L is 6-maleimino-diethylene glycol formyl-valine-citrulline-p-aminobenzyloxycarbonyl, with the following structure:

[0276] In some embodiments, the cytotoxic drug D is selected from microtubule inhibitors, DNA topoisomerase inhibitors, or DNA damaging agents.

[0277] In some embodiments, the microtubule inhibitors include orisstatin compounds and their derivatives (e.g., MMAE or MMAF), maytansine compounds and their derivatives (e.g., DM1 or DM4), or eribulin compounds and their derivatives.

[0278] In some embodiments, the DNA topoisomerase inhibitor includes DNA topoisomerase I inhibitors and their derivatives (e.g., camptothecin, 7-ethyl-10-hydroxycamptothecin (SN38), eczema, or Dxd) or topoisomerase II inhibitors and their derivatives (e.g., doxorubicin, doxorubicin, or PNU-159682).

[0279] In some embodiments, the DNA damaging agent includes PBD (pyrrolobenzodiazepine) compounds and their derivatives, calicheamicin compounds and their derivatives, or duocarmycin compounds and their derivatives.

[0280] In some embodiments, the cytotoxic drug D is selected from MMAE, MMAF, DM1, DM4, eribulin, SN38, eczema, or Dxd.

[0281] In some embodiments, the cytotoxic drug D is selected from MMAE, erribulin, or Dxd.

[0282] In some implementations, the LD is mc-vc-PAB-MMAE (MCE, Cat#HY-15575, CAS No.: 646502-53-6), and its structure is as follows:

[0283] In some embodiments, the linker-drug conjugate (LD) is Mal-PEG2-VCP-Eribulin (MCE, Cat#HY-128870, CAS No.:2130869-18-8), with the following structure:

[0284] In some embodiments, the linker-drug conjugate (LD) is MC-GGFG-DXD (MCE, Cat#HY-13631E, CAS No.: 1599440-13-7), with the following structure:

[0285] In some embodiments, the linker-drug conjugate (LD) is MC-VC-PAB-SN38 (MCE, Cat#HY-131057, CAS No.: 1801838-28-7), with the following structure:

[0286] In some embodiments, the linker-drug conjugate (LD) is MC-VA-PAB-Exatecan (MCE, Cat#HY-HY-147270, CAS No.: 2680543-57-9), with the following structure:

[0287] In a seventh aspect, the present invention provides a method for preparing the antibody-drug conjugate described in the sixth aspect, comprising the following steps:

[0288] (1) The antibody described in the first aspect is reacted with a reducing agent in a buffer solution to obtain the reduced antibody;

[0289] (2) The linker-drug conjugate (LD) described in the sixth aspect is cross-linked with the reduced antibody obtained in step (1) in a mixture of buffer and organic solvent to obtain the antibody-drug conjugate.

[0290] The generation of antibody-drug conjugates can be accomplished by any technique known to those skilled in the art. In some embodiments, the conjugation of the linker-drug conjugate (LD) to the antibody is accomplished by reacting with amino acid residues of the antibody. In some embodiments, the linker L is used to conjugate the drug D to a cysteine ​​residue of the antibody to prepare the antibody-drug conjugate of formula (I) of the present invention. In some embodiments, the interchain disulfide bonds of the antibody can be disrupted and free thiol groups exposed for conjugation with the linker-drug conjugate by controlling the conditions of treating the antibody with a reducing agent such as tris(2-hydroxyethyl)phosphine (TCEP). For IgG1 type antibodies, up to four linker disulfide bonds can be reduced, thereby generating up to eight reactive thiol groups for conjugation. Conjugates prepared by this method can contain one, two, three, four, five, six, seven, or eight drugs in each antibody molecule.

[0291] When the prepared antibody-drug conjugate is a composition of conjugates with different drug conjugation sites and / or numbers, the drug loading of the conjugate is expressed as the average DAR, which is the average number of drugs per antibody. The average number of drugs per antibody in the prepared antibody-drug conjugate can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. In some embodiments, the quantitative distribution of the antibody-drug conjugate, expressed as p (an abbreviation for the number of drug molecules carried on the ADC molecule, or drug loading), can also be determined. This can be achieved by methods such as reversed-phase HPLC or electrophoresis, where p is a certain value, for the separation, purification, and characterization of homogeneous antibody-drug conjugates with different drug loadings.

[0292] Treatment methods and pharmaceutical compositions

[0293] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the isolated nucleic acid molecule as described in the second aspect, the carrier as described in the third aspect, the host cell as described in the fourth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0294] In some embodiments, the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof described in the first aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0295] In some embodiments, the pharmaceutical composition comprises the carrier described in the third aspect or the host cell described in the fourth aspect, as well as a pharmaceutically acceptable carrier and / or excipient.

[0296] In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate described in the sixth aspect, as well as a pharmaceutically acceptable carrier and / or excipient.

[0297] In some embodiments, the pharmaceutical composition may further comprise additional pharmaceutically active agents. In some embodiments, the additional pharmaceutically active agents are drugs with antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, antiangiogenic agents, cytokines, antibodies that specifically target tumor cells, immune checkpoint inhibitors, or immunomodulators.

[0298] In some embodiments, the antibody or antigen-binding fragment thereof described in the first aspect of the pharmaceutical composition is provided as a separate component or as a component of the same composition as the other pharmaceutically active agent. Therefore, the antibody or antigen-binding fragment thereof described in the first aspect and the other pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0299] In a ninth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the carrier described in the third aspect, the host cell described in the fourth aspect, the antibody-drug conjugate described in the sixth aspect, or the pharmaceutical composition described in the eighth aspect in the preparation of a medicament for use in the prevention and / or treatment of tumors in a subject.

[0300] In some implementations, the tumor expresses ALPP and / or ALPG.

[0301] In some embodiments, the tumor is selected from tumors of the genitourinary system or tumors of the digestive tract. In some embodiments, the genitourinary system tumor is selected from ovarian cancer or endometrial cancer. In some embodiments, the digestive tract tumor is selected from gastric cancer or pancreatic cancer. In some embodiments, the tumor is selected from ovarian cancer, endometrial cancer, gastric cancer, or pancreatic cancer.

[0302] In some implementations, the subject is a mammal, such as a human, monkey, or mouse.

[0303] In a tenth aspect, the present invention provides a method for preventing and / or treating tumors in a subject, the method comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in the first aspect, the antibody-drug conjugate described in the sixth aspect, or the pharmaceutical composition described in the eighth aspect.

[0304] In some embodiments, the tumor expresses ALPP and / or ALPG. In some embodiments, the tumor is selected from genitourinary tumors or gastrointestinal tumors. In some embodiments, the genitourinary tumor is selected from ovarian cancer or endometrial cancer. In some embodiments, the gastrointestinal tumor is selected from gastric cancer or pancreatic cancer. In some embodiments, the tumor is selected from ovarian cancer, endometrial cancer, gastric cancer, or pancreatic cancer. In some embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0305] The antibodies or antigen-binding fragments or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The antibodies or antigen-binding fragments or pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody or antigen-binding fragment of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0306] Furthermore, the antibody or its antigen-binding fragment of the present invention may be present in the pharmaceutical composition in unit dose form for ease of administration.

[0307] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions thereof, may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the antibodies or antigen-binding fragments thereof or the pharmaceutical compositions thereof of the present invention are administered by intravenous injection or bolus.

[0308] The pharmaceutical compositions of the present invention may include, in a “therapeutic effective amount” or “preventative effective amount”, an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, an isolated nucleic acid molecule as described in the second aspect, a carrier as described in the third aspect, a host cell as described in the fourth aspect, or an antibody-drug conjugate as described in the sixth aspect. A “preventative effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutic effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments, etc.

[0309] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0310] Detection methods and kits

[0311] In one aspect, the present invention provides a kit containing the antibody or antigen-binding fragment thereof described in the first aspect or the antibody-drug conjugate described in the sixth aspect.

[0312] In some embodiments, the antibody or its antigen-binding fragment, or the antibody-drug conjugate, carries a detectable label.

[0313] In some embodiments, the kit further includes a second antibody that specifically recognizes the antibody of the present invention or its antigen-binding fragment, or the antibody-drug conjugate described in the sixth aspect. In some embodiments, the second antibody further includes a detectable label.

[0314] The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750), acridine esters, magnetic beads (e.g., ), calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of such markers include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). Detectable markers as described above can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing an enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers. In some embodiments, such markers can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0315] In a twelfth aspect, the present invention provides a method for detecting the presence or amount of ALPP and / or ALPG in a sample, the method comprising the following steps:

[0316] (1) Contact the sample with the antibody or its antigen-binding fragment as described in the first aspect, or the antibody-drug conjugate as described in the sixth aspect;

[0317] (2) Detect the formation of a complex between the antibody or its antigen-binding fragment and ALPP and / or ALPG.

[0318] The formation of the complex indicates the presence of ALPP and / or ALPG or cells expressing ALPP and / or ALPG.

[0319] In some embodiments, the sample is a cell sample, i.e., a sample containing cells (e.g., tumor cells). In such embodiments, preferably, the complex is formed between the antibody or antigen-binding fragment and ALPP and / or ALPG expressed by the cells in the sample.

[0320] In some embodiments, the antibody or its antigen-binding fragment or antibody-drug conjugate is further labeled with a detectable marker. In some embodiments, in step (2), a reagent labeled with a detectable marker is used to detect the antibody or its antigen-binding fragment or antibody-drug conjugate of the present invention.

[0321] The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient). In some preferred embodiments, the ALPP and / or ALPG are human ALPP and / or ALPG.

[0322] In some embodiments, the tumor is selected from tumors of the genitourinary system or tumors of the digestive tract.

[0323] In some embodiments, the urogenital tumor is selected from ovarian cancer or endometrial cancer.

[0324] In some implementations, the gastrointestinal tumor is selected from gastric cancer or pancreatic cancer.

[0325] In some implementations, the tumor is selected from ovarian cancer, endometrial cancer, gastric cancer, or pancreatic cancer.

[0326] In a thirteenth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, or the antibody-drug conjugate described in the sixth aspect, in the preparation of a kit for detecting the presence or amount of ALPP and / or ALPG in a sample, and / or diagnosing tumors associated with ALPP and / or ALPG. In some embodiments, the ALPP and / or ALPG are human ALPP and / or ALPG.

[0327] In some embodiments, the tumor is selected from tumors of the genitourinary system or tumors of the digestive tract.

[0328] In some embodiments, the urogenital tumor is selected from ovarian cancer or endometrial cancer.

[0329] In some implementations, the gastrointestinal tumor is selected from gastric cancer or pancreatic cancer.

[0330] In some implementations, the tumor is selected from ovarian cancer, endometrial cancer, gastric cancer, or pancreatic cancer.

[0331] In a fourteenth aspect, the present invention provides a diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the isolated nucleic acid molecule as described in the second aspect, the carrier as described in the third aspect, the host cell as described in the fourth aspect, the antibody-drug conjugate as described in the sixth aspect, or the pharmaceutical composition as described in the eighth aspect, and optionally, instructions for use. The kit may also include a drug delivery device for local administration. The drug delivery device includes a drug-loaded syringe or a needleless device.

[0332] In vitro cytotoxicity assay

[0333] The in vitro cytotoxicity mentioned in this invention includes antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The in vitro cells (i.e., target cells) mentioned in this invention can include various tumor cell lines, including but not limited to human ovarian cancer cell lines Caov-3, RMUG-S, COV644, EF027; human embryonic kidney cell line HEK293; human endometrial cancer cell lines Caski, ARK2, RL95-2, AN3CA; human gastric cancer cell lines AGS, MKN1, MKN45, BGC823, NCI-N87; human pancreatic cancer cell lines HPAC, Capan-1, MIA PaCa-2; and human breast cancer cell line MDA-MB-468, etc.

[0334] There are many methods for detecting antibody-dependent cell-mediated cytotoxicity (ADCC), including but not limited to methods using primary PBMC / NK cell killing and bioluminescent reporter gene assays using genetically engineered Jurkat cells as effector cells. For example, a bioluminescent reporter gene assay using genetically engineered Jurkat cells as effector cells involves first expanding the culture of Jurkat effector cells and resuspending the cells in culture medium. Next, target cells are resuspended in culture medium. The target cell suspension is then mixed thoroughly with the Jurkat effector cells, and serially diluted samples containing the antibody to be tested are added. After incubation, the cells are removed, detection reagents are added, and the fluorescence signal values ​​are read using a multi-functional microplate reader to evaluate the ADCC effect.

[0335] The complement-dependent cytotoxicity (CDC) assay involves serially diluting a recombinant antibody and incubating it with target cells. In the presence of complement, the antibody drug forms a membrane attack complex on the surface of the target cells, leading to cell lysis. The efficacy of CDC is evaluated using reagents that detect cell viability.

[0336] Tumor cell line models

[0337] The tumor cell line models mentioned in this invention may include various tumor cell lines, including but not limited to human ovarian cancer cell lines Caov-3, RMUG-S, COV644, EF027, OV17054, OV15209, OV9419, OV9422, OV9418, OV5308, OV9534, OV9409, human embryonic kidney cell line HEK293, human endometrial cancer cell lines Caski, ARK2, RL95-2, AN3CA, human gastric cancer cell lines AGS, MKN1, MKN45, BGC823, NCI-N87, human pancreatic cancer cell lines HPAC, Capan-1, MIAPaCa-2, and human breast cancer cell line MDA-MB-468, etc.

[0338] There are various ways to establish a model, the purpose of which is to simulate the tumor microenvironment. As an example, a tumor cell line model can be constructed by inoculating human tumor cell lines on an immunodeficient animal model. The animals include, but are not limited to, monkeys, mice, and rats. Monkeys include, but are not limited to, cynomolgus monkeys and rhesus monkeys.

[0339] A tumor inhibition experiment was conducted to detect tumor size and observe tumor growth. The administration routes are subcutaneous (SC) and / or intraperitoneal (IP), with a recommended dosing frequency of 5-30 mg / kg, including 10 mg / kg / Q3D, 20 mg / kg / Q6D, 10 mg / kg / Q7D, and 20 mg / kg / Q3D.

[0340] It should be noted that mg / kg / QnD refers to a dosage that is administered once every n days. For example, 10 mg / kg / Q3D means that the dosage is 10 mg / kg, administered once every three days.

[0341] In vivo efficacy verification

[0342] The antibody molecules involved in this invention are used to verify in vivo efficacy. This involves using, but is not limited to, human ovarian cancer cell lines Caov-3, RMUG-S, COV644, EF027, OV17054, OV15209, OV9419, OV9422, OV9418, OV5308, OV9534, OV9409; human endometrial cancer cell lines Caski, ARK2, RL95-2, AN3CA; human gastric cancer cell lines AGS, MKN1, MKN45, BGC823, NCI-N87; human pancreatic cancer cell lines HPAC, Capan-1, MIA PaCa-2; and human breast cancer cell line MDA-MB-468. These are inoculated into animals, including but not limited to mice, rats, cynomolgus monkeys, and rhesus monkeys, to establish animal models, test anti-tumor activity, and verify the effect on tumors by detecting indicators such as tumor volume and survival rate.

[0343] Terminology Definition

[0344] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as cell culture, molecular biology, biochemistry, nucleic acid chemistry, and immunology, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0345] As used herein, the term "antibody" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant and can include a variety of antibody structures, provided they exhibit the desired antigen-binding activity. Typically, an antibody can be an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0346] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0347] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, IMGT, Chothia, Contact, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat numbering system.

[0348] As used herein, the term "framework region" or "FR" residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0349] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0350] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0351] As used herein, the term “Fd” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0352] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0353] As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0354] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL of the scFv. In some embodiments of the present invention, scFv can form di-scFv, which refers to two or more individual scFvs linked together to form an antibody. In some embodiments of the present invention, scFv can form (scFv)2, which refers to two or more individual scFvs linked together in parallel to form an antibody.

[0355] As used herein, the term “diabody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0356] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0357] As used in this article, the term "total antibody" refers to the antibody portion carried by the antibody-drug conjugate (ADC) formed after conjugation, as well as the sum of unconjugated free naked antibodies.

[0358] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0359] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0360] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0361] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case. In some embodiments, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0362] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with human antibodies. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and 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 the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, and reactivity. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity).

[0363] As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to a sequence in the genome of an organism that encodes an immunoglobulin, which has not undergone a maturation process involving genetic rearrangements and mutations that would lead to the expression of a specific immunoglobulin. In this invention, the term "heavy chain germline gene" refers to a germline antibody gene or gene segment encoding the heavy chain of immunoglobulins, including the V (variable), D (diversity), J (joining), and C (constant) genes; similarly, the term "light chain germline gene" refers to a germline antibody gene or gene segment encoding the light chain of immunoglobulins, including the V (variable), J (joining), and C (constant) genes. In this invention, the amino acid sequence encoded by the germline antibody gene or germline antibody gene segment is also referred to as a "germline sequence." Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from specialized databases (e.g., IMGT, UNSWIg, NCBI, or VBASE2).

[0364] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that interaction. In this invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0365] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of KD, kon, and kdis can be measured using any effective method. In some embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR) technology. Alternatively, bioluminescent interferometry or Kinexa can be used to measure the dissociation constant.

[0366] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; granules; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0367] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells, as well as immune cells (such as T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells). Host cells can include single cells or cell populations.

[0368] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0369] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Sci USA 94:412-417 (1997), which are incorporated herein by reference).

[0370] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0371] In the amino acid sequences of the heavy chain constant region listed in this article, if the terminal amino acid is "K (lysine)," except for special cases of the antibody heavy chain constant region amino acid sequences, it can usually be explained as follows: at the mRNA level, in the gene sequence encoding this constant region, there is a codon encoding lysine before the stop codon; in mature secreted antibody proteins, this lysine will be selectively deleted during post-translational modification under different cell line environments.

[0372] As used herein, the terms "antibody-drug conjugate," "ADC," "ADC conjugate," or "ADC molecule" all refer to the same thing: an antibody or antibody fragment covalently coupled to a therapeutic active substance or active pharmaceutical ingredient, thereby enabling the therapeutic active substance or active pharmaceutical ingredient to target the antibody's binding target and exert its pharmacological function. The therapeutic active substance or active pharmaceutical ingredient can be a drug capable of killing cells (preferably cancer cells) targeted by the ADC, and is preferably a cytotoxic drug. The covalent linking of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxic drug can be performed using a linker in a non-site-specific manner or in a site-specific manner.

[0373] As used in this article, the term "connector" refers to a chemical module that covalently links an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC, i.e., a drug.

[0374] As used herein, the term “linker-drug” (LD) refers to the portion of an “antibody-drug conjugate” consisting of a linker and a drug.

[0375] As used herein, the terms "cytotoxic drug," "toxin," or "payload" all refer to the same thing: a toxic drug that can be a chemical molecule that strongly disrupts the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations.

[0376] As used herein, the term "succinimide" has the following structure:

[0377] As used herein, the term "maleimide" has the following structure:

[0378] As used herein, the term "alkylene" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, wherein no unsaturated bonds are present in the group. As used herein, the term "C 1-10 "Alkylene" refers to a divalent straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, having one to ten carbon atoms. 1-10Non-limiting examples of "alkylene" include methylene (C1 alkylene), ethylene (C2 alkylene), propylene (C3 alkylene), butylene (C4 alkylene), pentylene (C5 alkylene), and hexylene (C6 alkylene).

[0379] As used in this article, the term "acyl" has the following structure: C=O.

[0380] As used herein, the term "PEG" refers to a straight-chain or branched structure consisting of (OCH2CH2) groups. In some embodiments, (PEG) 1-10 The functional group is -(OCH2CH2)n*- or Where n is 1-10, and "-" and "*-" are respectively related to L. 1 or L 3 The connection point.

[0381] As used herein, the term "peptide residue" refers to a structural segment consisting of one or more amino acids linked by an amide bond. The one or more amino acid residues are selected from alanine (Ala), glycine (Gly), valine (Val), phenylalanine (Phe), lysine (Lys), citrulline (Cit), cysteine ​​(Ser), glutamic acid (Glu), aspartic acid (Asp), histidine (His), cysteine ​​(Cys), isoleucine (Ile), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), pentaneous (Nva), ortholeucine (Nle), pyrrolidone (Pyl), homoserine, homocysteine, and demethylpyrrolidone. Non-limiting embodiments of “peptide residues” include dipeptide residues, tripeptide residues, or tetrapeptide residues, wherein non-limiting embodiments of dipeptide residues include valine-citrulline (Val-Cit) and valine-alanine (Val-Ala); and non-limiting embodiments of tetrapeptide residues include (Gly-Gly-Phe-Gly, GGFG).

[0382] As used herein, the term "aminobenzyloxycarbonyl" or "PAB" has the following structure:

[0383] As used herein, the term "aminobenzyloxy" has the following structure:

[0384] As used herein, the term "drug loading," or drug-to-antibody ratio or DAR, refers to the average number of drugs conjugated to each antibody in an ADC. This can range, for example, from about 1 to about 10 drugs per antibody. In some embodiments, the range is from about 1 to about 8 drugs per antibody, preferably 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, or 6-8. For example, DAR can be 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 6, 7, 8, 9, or 10. In some embodiments, DAR is the average DAR, which is the ratio of the drug (D) coupled to the Ab portion of the product to the total Ab portion, as determined by detection methods (e.g., by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, electrophoresis, and / or HPLC).

[0385] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0386] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0387] As used herein, the term "subject" refers to a mammal, including but not limited to humans, monkeys (e.g., cynomolgus monkeys, rhesus monkeys), rabbits, and mice (e.g., mice, rats). In some embodiments, the subject (e.g., a human) has a tumor. Beneficial effects

[0388] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0389] The antibodies of this invention specifically recognize / bind to human ALPP and / or ALPG, without cross-reactivity with other members of the alkaline phosphatase family (e.g., ALPI and ALPL). The antibodies of this invention exhibit good endocytic activity in tumor cells (e.g., human gastric cancer NCI-N87 cells), significantly superior to positive control antibodies. The antibodies of this invention can also induce the killing of ALPP-expressing cells (e.g., tumor cells) via ADCC and / or CDC. The antibodies of this invention also possess cross-reactivity with human, monkey, and mouse ALPP, good hydrophilicity and thermostability, and favorable pharmacokinetic characteristics. Furthermore, the humanized antibodies of this invention not only retain the function and properties of the parent antibodies but also have a high degree of humanization, thus allowing safe administration to human subjects without inducing immunogenic reactions. The antibodies of this invention also possess significant antitumor function by inhibiting tumor growth. Therefore, the antibodies of this invention have the potential for the prevention and / or treatment of tumors (especially those expressing ALPP) and have significant clinical value.

[0390] The antibody-drug conjugates of the present invention exhibit good in vitro killing activity in tumor cells (e.g., NCI-N87 cells, Caov-3 cells, and COV644 cells); and good in vivo efficacy in mice (e.g., mice with subcutaneous xenograft of BALB / c Nude cells from NCI-N87 and HPAC cells, and mice with subcutaneous xenograft of NOD / SCID cells from human ovarian cancer cells (e.g., OV17054 cells, OV15209 cells, OV9419 cells, OV9422 cells, OV9418 cells, OV5308 cells, OV9534 cells, and OV9409 cells), which is superior to the positive control antibody-drug conjugates; furthermore, the antibody-drug conjugates of the present invention also exhibit good in vitro stability and low toxicity. Therefore, the antibody-drug conjugates of the present invention have the potential for the prevention and / or treatment of tumors and have significant clinical value.

[0391] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

[0392] Overview of the attached figures

[0393] Figure 1A shows the binding activity of three chimeric antibodies, HLP00564, HLP00601, and HLP00476, and their corresponding humanized antibodies, HLP00776, HLP00786, and HLP00879, to Caov-3 cells.

[0394] Figure 1B shows the binding activity of two chimeric antibodies, HLP00562 and HLP00556, and their corresponding humanized antibodies, HLP00891 and HLP00892, to Caov-3 cells.

[0395] Figure 2A shows the binding activity of three chimeric antibodies, HLP00564, HLP00601, and HLP00476, and their corresponding humanized antibodies, HLP00776, HLP00786, and HLP00879, to HEK293-CynoALPP cells.

[0396] Figure 2B shows the binding activity of two chimeric antibodies, HLP00562 and HLP00556, and their corresponding humanized antibodies, HLP00891 and HLP00892, to HEK293-CynoALPP cells.

[0397] Figure 3 shows the endocytic activity of five chimeric antibodies, HLP00564, HLP00601, HLP00476, HLP00562 and HLP00556, and their corresponding humanized antibodies HLP00776, HLP00786, HLP00879, HLP00891 and HLP00892, on NCI-N87 cells.

[0398] Figure 4 shows the binding activity of five chimeric antibodies, five humanized antibodies, and five humanized antibodies with Fc effect eliminated to human ALPI protein.

[0399] Figure 5 shows the binding activity of five chimeric antibodies, five humanized antibodies, and five humanized antibodies with Fc effect eliminated to human ALPL protein.

[0400] Figure 6 shows the binding activity of five chimeric antibodies, five humanized antibodies, and five humanized antibodies with Fc effect eliminated to rat ALPP protein.

[0401] Figure 7 shows the binding activity of five chimeric antibodies, five humanized antibodies, and five humanized antibodies with Fc effect eliminated to rat ALPG protein.

[0402] Figure 8 shows the binding activity of five chimeric antibodies, five humanized antibodies, and five humanized antibodies with Fc effect eliminated to mouse ALPG protein.

[0403] Figure 9 shows the binding activity of the chimeric antibody HLP00476 to mouse ALPG protein.

[0404] Figure 10 shows the binding activity of humanized antibody HLP00879 and humanized antibody HLP01058 after Fc effect elimination to mouse ALPG protein.

[0405] Figure 11 shows the pharmacokinetic data of humanized antibody HLP00776 and humanized antibodies HLP01056, HLP01060, and HLP01058 after Fc effect elimination in mice.

[0406] Figure 12 shows the results of the humanized antibody ADCC killing experiment.

[0407] Figure 13 shows the in vitro killing activity of the humanized antibody ADC conjugate on COV644 cells.

[0408] Figure 14 shows the in vitro killing activity of the humanized antibody ADC conjugate on Caov-3 cells.

[0409] Figure 15 shows the in vitro killing activity of the humanized antibody ADC conjugate on NCI-N87 cells.

[0410] Figure 16 shows the results of the mouse PK experiment of the ADC conjugate.

[0411] Figures 17A-17B show the in vivo efficacy of the ADC conjugate in the NCI-N87 subcutaneous xenograft BALB / c Nude mouse model; Figure 17A shows the rate of change in mean body weight of mice in each group, and Figure 17B shows the tumor volume in each group.

[0412] Figures 18A-18C show the in vivo efficacy of the ADC conjugate in the HPAC subcutaneous xenograft BALB / c Nude mouse model; Figure 18A shows the rate of change in mean body weight of mice in each group, Figure 18B shows the tumor volume in each group, and Figure 18C shows the tumor weight in each group.

[0413] Figures 19A-19B show the in vivo efficacy of the ADC conjugate in a female mouse model of subcutaneous xenograft of human ovarian cancer OV17054 using NOD / SCID; Figure 19A shows the mean tumor growth curves in each group; Figure 19B shows the mean rate of change in body weight of mice in each group.

[0414] Figures 20A-20B show the in vivo efficacy of the ADC conjugate in a female mouse model of subcutaneous xenograft of human ovarian cancer OV15209 in NOD / SCID; Figure 20A shows the mean tumor growth curves in each group; Figure 20B shows the mean change in body weight of mice in each group.

[0415] Figures 21A-21B show the in vivo efficacy of the ADC conjugate in a female mouse model of subcutaneous xenograft of human ovarian cancer OV9419 using NOD / SCID; Figure 21A shows the mean tumor growth curves in each group; Figure 21B shows the mean change in body weight of mice in each group.

[0416] Figures 22A-22B show the in vivo efficacy of the ADC conjugate in a female mouse model of subcutaneous xenograft of human ovarian cancer OV9422 using NOD / SCID; Figure 22A shows the mean tumor growth curves in each group; Figure 22B shows the mean change in body weight of mice in each group.

[0417] Figures 23A-23B show the in vivo efficacy of the ADC conjugate in a female mouse model of subcutaneous xenograft of human ovarian cancer OV9418 using NOD / SCID; Figure 23A shows the mean tumor growth curves in each group; Figure 23B shows the mean change in body weight of mice in each group.

[0418] Figures 24A-24B show the in vivo efficacy of the ADC conjugate in a female mouse model of subcutaneous xenograft of human ovarian cancer OV5308 using NOD / SCID; Figure 24A shows the mean tumor growth curves in each group; Figure 24B shows the mean change in body weight of mice in each group.

[0419] Figures 25A-25B show the in vivo efficacy of the ADC conjugate in a female mouse model of subcutaneous xenograft of human ovarian cancer OV9534 using NOD / SCID; Figure 25A shows the mean tumor growth curves in each group; Figure 25B shows the mean change in body weight of mice in each group.

[0420] Figures 26A-26B show the in vivo efficacy of the ADC conjugate in a female mouse model of subcutaneous xenograft of human ovarian cancer OV9409 using NOD / SCID; Figure 26A shows the mean tumor growth curves in each group; Figure 26B shows the mean change in body weight of mice in each group.

[0421] Figure 27 shows the dose-exploration trial after repeated intravenous administration for 8 weeks; where a: animal B36831 in the 10 / 30 mg / kg dose group, planned for euthanasia, no abnormal pathological changes were observed in the stomach in the image, HE staining, 4x objective lens; b: animal B36833 in the 60 mg / kg dose group, found dead, atrophy of the gastric mucosa is visible in the image, HE staining, 4x objective lens; c: animal B36831 in the 10 / 30 mg / kg dose group, planned for euthanasia, no abnormal pathological changes were observed in the jejunum in the image, HE staining, 1 0x objective; d: Animal B36833 in the 60mg / kg dose group, found dead. Inflammatory cell infiltration, crypt degeneration and regeneration are visible in the jejunum in the image. HE staining, 10x objective; e: Animal B36831 in the 10 / 30mg / kg dose group, planned for euthanasia. No abnormal pathological changes are seen in the cecum in the image. HE staining, 10x objective; f: Animal B36833 in the 60mg / kg dose group, found dead. Ulcers, crypt degeneration and visceral dilatation are visible in the cecal mucosa in the image. HE staining, 10x objective.

[0422] Figure 28 shows the weight and food intake of the monkeys in each group.

[0423] Figure 29 shows the concentration-time curves of toxin in monkey plasma after intravenous infusion of HLP01060-8D in each group of animals.

[0424] Figures 30A-30B show the in vitro stability experiment of monkey plasma; Figure 30A shows the toxin concentration (pg / mL); Figure 30B shows the toxin release rate.

[0425] In the figures, "IV" indicates intravenous injection; "PBS" indicates PBS was administered to the control group; "QW*2", "QW*3" or "QW*4" indicates administration once a week for a total of 2, 3 or 4 times; "NA" indicates no drug was administered to the experimental group; "1mg / kg", "3mg / kg" or "10mg / kg" indicates the dosage; "10μL / g" indicates the administration volume; in Figure 11, "hALPP+Fc" indicates the test method; in Figure 16, "Fc+Fc" indicates the detection of total antidote concentration, and "Fc+anti Dxd" indicates the detection of ADC drug concentration.

[0426] Detailed Explanation

[0427] To facilitate understanding of the present invention, the solutions of the present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the embodiments are merely illustrative of the present invention and should not be construed as limiting the scope of protection claimed by the present invention.

[0428] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. Where specific conditions are not specified in the embodiments, they should be performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0429] Example 1: Recombinant Protein and Stable Transfected Cell Lines

[0430] 1.1 Recombinant Protein

[0431] Recombinant human ALPP protein hALPP.his (purchased from ACROBiosystems, catalog number: ALP-H52H3), recombinant human ALPG protein hALPG.his (purchased from Kactus Biosystems, catalog number: APE-HM103), and recombinant monkey ALPP protein CynoALPP.his (purchased from Kactus Biosystems, catalog number: APE-CM102) were constructed using the amino acid sequences of the extracellular domain (ECD) of human ALPP protein (UniProt: P05187-1), human ALPG protein (UniProt: P10696), and monkey ALPP protein (UniProt: XP_045223825.1), respectively. These three recombinant proteins were used in the experiments described in the following examples or test cases. The specific sequence information of the three proteins is as follows:

[0432] Human ALPP protein (UniProt: P05187-1, where the underlined portion represents the extracellular domain, amino acid residues Ile23-Asp506) amino acid sequence:

[0433] Human ALPG protein (UniProt: P10696, where the underlined portion represents the extracellular domain, amino acid residues Ile20-Asp503) amino acid sequence:

[0434] Monkey ALPP protein (UniProt: XP_045223825.1, where the underlined portion represents the extracellular domain, amino acid residues Ile21-Thr503) amino acid sequence:

[0435] 1.2 Stable cell lines

[0436] CHO-K1-hALPP stable cell line (purchased from Jiman Biotechnology, catalog number: GM-C30339): is a cell line formed by transfecting the untagged human ALPP gene into CHO-K1 cells;

[0437] HEK293-hALPG stable transfection cell line (purchased from Jiman Biotechnology, catalog number: GM-C26399): is a cell line formed by transfecting untagged human ALPG gene into HEK293 cells;

[0438] HEK293-CynoALPP stable transgenic cell line (purchased from Jiman Biotechnology, catalog number: GM-C26410): is a cell line formed by transfecting untagged monkey ALPP gene into HEK293 cells;

[0439] These three stable cell lines were used in the following antibody screening and identification experiments.

[0440] Example 2: Preparation of hybridoma monoclonal antibodies

[0441] 2.1 Animal Immunization

[0442] To obtain monoclonal antibodies against human ALPP and human ALPG, they were produced by immunizing Balb / c mice (purchased from Slack) and SJL mice (purchased from Slack). Two immunization methods were used: routine immunization and rapid immunization. The specific immunization process is as follows:

[0443] Routine immunization: Two his-tagged recombinant human ALPP proteins hALPP.his (purchased from ACROBiosystems, catalog number: ALP-H52H3) and recombinant human ALPG protein hALPG.his (purchased from Kactus Biosystems, catalog number: APE-HM103) from Example 1.1 were mixed in equal volumes with the water-soluble immune adjuvant QUICK Antibody (purchased from Bio-Long, catalog number: KX0210041) and injected intraperitoneally every 3 weeks to immunize Balb / c mice (purchased from Slack) and SJL mice (purchased from Slack), for a total of 5 immunizations.

[0444] Rapid immunization: Two his-tagged recombinant human ALPP proteins hALPP.his (purchased from ACROBiosystems, catalog number: ALP-H52H3) from Example 1.1 were mixed in equal volumes with the water-soluble immunoadjuvant QUICK Antibody (purchased from Bio-Long, catalog number: KX0210041) and injected at intervals of days 1-4-7-10-13 to immunize Balb / c mice (purchased from Slack), for a total of 5 immunizations.

[0445] 2.2 Spleen cell fusion

[0446] Once the serum titer met the requirements, mouse spleen lymphocytes were fused with myeloma Sp2 / 0 cells (ATCC, CRL-2016). TM Hybridoma cells were obtained by electrofusion after mixing the samples at a 1:2 ratio. The fused hybridoma cells were transferred from the electrofusion dish to a 50 mL centrifuge tube, and 10 mL of RPMI Medium 1640 medium was added. The mixture was incubated at room temperature for 20 min. The fused hybridoma cell suspension was then added to RPMI Medium 1640 medium (10% FBS, 5% Hybridoma Feeder, 1×PS, 1×HAT, 1×GLUTAMAX-I, 5% Hybridomo) to adjust the hybridoma cell density to 1×10⁻⁶ cells / mL. 5 10 cells / mL were seeded into 60 96-well plates at 200 μL / well; the plates were then incubated at 37°C in a 5% CO2 incubator.

[0447] 2.3 Hybridoma cell screening

[0448] Based on hybridoma cell growth density, the hybridoma culture supernatant was analyzed using a human ALPP-binding ELISA method. Cells from positive wells bound to human ALPP protein were transferred to 24-well plates for culture. The supernatant from the 24-well plates was screened using human and monkey ALPP cell binding assays, ALPI and ALPL protein binding assays, and endocytosis assays on NCI-N87 cells. Mother clones that bound human and monkey ALPP cells, did not bind ALPI and ALPL proteins, and exhibited good endocytic activity were selected for amplification, cryopreservation, and seed culture, followed by subcloning to obtain single-cell clones.

[0449] 2.4 Sequence determination of hybridoma-positive clones

[0450] Log-growing hybridoma subclones were collected, and five murine antibody hybridoma clones (76C11A9, 05E3F1, 27D12C12, 02B2A8, and 60F7B3) were obtained through screening. These clones were then sent to a sequencing company for hybridoma sequencing. The CDR amino acid sequences of the five murine antibodies are shown in Table 1.

[0451] Table 1: Amino acid sequence of mouse antibody CDR

[0452] The variable region amino acid sequences of the five murine antibodies are shown below:

[0453] 76C11A9 heavy chain variable region amino acid sequence

[0454] 76C11A9 light chain variable region amino acid sequence

[0455] 05E3F1 Heavy Chain Variable Region Amino Acid Sequence

[0456] 05E3F1 light chain variable region amino acid sequence

[0457] 27D12C12 Heavy Chain Variable Region Amino Acid Sequence

[0458] 27D12C12 light chain variable region amino acid sequence

[0459] 02B2A8 Heavy Chain Variable Region Amino Acid Sequence

[0460] 02B2A8 light chain variable region amino acid sequence

[0461] The amino acid sequence of the variable region of the 60F7B3 heavy chain is: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWINTETGEPTYANDFKGRFAFSLETSASTAYLQINNLKNEDTATFFCAPIYYYAMDYWGQGTSVTVSS (SEQ ID NO: 9)

[0462] 60F7B3 light chain variable region amino acid sequence

[0463] The variable region nucleotide sequences of the five murine antibodies are shown below:

[0464] 76C11A9 heavy chain variable region nucleotide sequence

[0465] 76C11A9 light chain variable region nucleotide sequence

[0466] 05E3F1 Heavy Chain Variable Region Nucleotide Sequence

[0467] 05E3F1 light chain variable region nucleotide sequence

[0468] 27D12C12 Heavy Chain Variable Region Nucleotide Sequence

[0469] 27D12C12 light chain variable region nucleotide sequence

[0470] 02B2A8 Heavy Chain Variable Region Nucleotide Sequence

[0471] 02B2A8 light chain variable region nucleotide sequence

[0472] 60F7B3 heavy chain variable region nucleotide sequence

[0473] 60F7B3 light chain variable region nucleotide sequence

[0474] Example 3: Recombinant Expression and Purification of Chimeric Antibody and Control Antibody

[0475] 3.1 Molecular Cloning of Chimeric Antibodies

[0476] Based on the sequencing results of the five murine antibody variable region genes obtained in Example 2, the first and last primers were designed using the sequencing sequences. Using the sequenced genes as templates, the VH / VL gene fragments of each antibody were constructed by PCR. Then, homologous recombination was performed with the expression vector pCDNA3.4 (a fragment containing the signal peptide and the hIgG1 / hkappa constant region gene (CH1-FC / CL)) to construct the full-length recombinant antibody expression plasmid VH-CH1-Fc-pCDNA3.4 / VL-CL-pCDNA3.4. Five chimeric antibodies (Ch76C11A9, Ch05E3F1, Ch27D12C12, Ch02B2A8, and Ch60F7B3) were obtained and named HLP00556, HLP00564, HLP00476, HLP00562, and HLP00601, respectively. The CDR sequences of the five chimeric antibodies are identical to the sequences of the corresponding murine antibodies in Table 1. The amino acid and nucleotide sequences of the heavy chain variable region and light chain variable region of the five chimeric antibodies are identical to the sequences of the corresponding murine antibodies. Specific sequence information is shown in Table 2.

[0477] Table 2: Sequence information of the heavy chain variable region and light chain variable region of the five chimeric antibodies

[0478] The heavy chain constant region amino acid sequences of HLP00556, HLP00564, HLP00562 and HLP00601 are the heavy chain constant region amino acid sequences of the human IgG1 variant (EEM) as shown in SEQ ID NO: 172; the heavy chain constant region amino acid sequence of HLP00476 is the heavy chain constant region amino acid sequence of the human IgG1 (hIgG1) as shown in SEQ ID NO: 137; and the light chain constant region amino acid sequences of the five chimeric antibodies are the light chain constant region amino acid sequences of the human κ (hkappa) as shown in SEQ ID NO: 138.

[0479] Amino acid sequence of the constant region of the human IgG1 (hIgG1) heavy chain

[0480] Amino acid sequence of the heavy chain constant region of human IgG1 variant (EEM)

[0481] Human κ (hkappa) light chain constant region amino acid sequence

[0482] The full-length heavy chain and full-length light chain amino acid sequences of the five chimeric antibodies are shown below:

[0483] HLP00556 Heavy Chain Full-Length Amino Acid Sequence

[0484] HLP00556 Light Chain Full-Length Amino Acid Sequence

[0485] HLP00564 Heavy Chain Full-Length Amino Acid Sequence

[0486] HLP00564 light chain full-length amino acid sequence

[0487] HLP00476 Heavy Chain Full-Length Amino Acid Sequence

[0488] HLP00476 Light Chain Full-Length Amino Acid Sequence

[0489] HLP00562 Heavy Chain Full-Length Amino Acid Sequence

[0490] HLP00562 light chain full-length amino acid sequence

[0491] HLP00601 Heavy Chain Full-Length Amino Acid Sequence

[0492] HLP00601 Light Chain Full-Length Amino Acid Sequence

[0493] The full-length heavy chain and full-length light chain nucleotide sequences of the five chimeric antibodies are shown below:

[0494] HLP00556 heavy chain full-length nucleotide sequence

[0495] HLP00556 light chain full-length nucleotide sequence

[0496] HLP00564 heavy chain full-length nucleotide sequence

[0497] HLP00564 light chain full-length nucleotide sequence

[0498] HLP00476 Heavy Chain Full-Length Nucleotide Sequence

[0499] HLP00476 light chain full-length nucleotide sequence

[0500] HLP00562 heavy chain full-length nucleotide sequence

[0501] HLP00562 light chain full-length nucleotide sequence

[0502] HLP00601 Heavy Chain Full-Length Nucleotide Sequence

[0503] HLP00601 light chain full-length nucleotide sequence

[0504] 3.2 Design of control antibodies

[0505] The positive control antibody used in this invention is derived from the humanized antibody h12F3HGLF in the published PCT patent application WO2022197890A8, and is named HLP00082 in this invention. The IgG1 isotype control used in this invention is named HLP00686. The recombinant protein construction of the two control antibodies is the same as that of the recombinant protein construction of the chimeric antibody in section 3.1. The full-length amino acid sequences of the heavy and light chains of the two control antibodies are shown below.

[0506] HLP00082 Heavy Chain Full-Length Amino Acid Sequence

[0507] HLP00082 light chain full-length amino acid sequence

[0508] HLP00686 Heavy Chain Full-Length Amino Acid Sequence

[0509] HLP00686 Light Chain Full-Length Amino Acid Sequence

[0510] 3.3 Expression and purification of chimeric antibodies and control antibodies

[0511] Chimeric antibodies and plasmids containing the light and heavy chains of the control antibody were transfected into CHO cells at a 2:1 ratio. After 6 days, the expression supernatant was collected, impurities were removed by high-speed centrifugation, and purification was performed using a Protein A column. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0), neutralized with 1M Tris-HCl (pH 8.0), and the eluted sample was transferred to PBS. Further purification using Superdex 200 (GE) gel chromatography may be necessary if required.

[0512] Example 4 Humanization of Antibodies

[0513] 4.1 Humanization of chimeric antibodies

[0514] Humanization methods typically employ framework transplantation and protein surface amino acid humanization. The humanization steps are as follows: By comparing the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment) software, germline genes of the heavy and light chain variable regions with high homology to the maternal mouse antibody are selected as templates. Following the Kabat numbering system, the CDRs of the mouse antibody are transplanted into the corresponding human templates, forming a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0515] Studies have found that during antibody production, various physicochemical factors can easily lead to the generation of various post-translational modification (PTM) variants, such as glycosylation, oxidation, glycation, deamidation, isomerization, and terminal cyclization. Some PTM sites often exhibit potential modifications in the primary structure of the antibody. When PTMs occur in antibodies, they typically cause antibody heterogeneity; when PTMs occur in the CDR region of the antibody, they may lead to loss of antibody activity. In antibody development, to avoid the occurrence of PTMs, potentially risky sites are usually replaced with conserved amino acids.

[0516] After analyzing the sequences, the inventors discovered that both the CDR2 of the heavy chain variable region of chimeric antibody HLP00476 and the CDR3 of the heavy chain variable region of HLP00562 contain NG deamidation risk points. Therefore, the inventors modified the CDRs of the above antibodies by using a conservative amino acid substitution method to obtain the final humanized molecule.

[0517] According to the above method, the chimeric antibody obtained in Example 3 was humanized. Five humanized antibodies were thus obtained: hz76C11A9, hz05E3F1, hz27D12C12, hz02B2A8, and hz60F7B3, which are named HLP00892, HLP00776, HLP00879, HLP00891, and HLP00786 in this invention, respectively. The CDR sequences of the five humanized antibodies are shown in Table 3. The CDR-H2 sequence of the humanized antibody HLP00879 corresponding to HLP00476 is shown in Table 3 as shown in SEQ ID NO: 126-129, 131. The CDR-H3 sequence of the humanized antibody HLP00891 corresponding to HLP00562 is shown in Table 3 as shown in SEQ ID NO: 126-129, 131. The amino acid sequences shown in NO: 132, 133, and 135 are as follows. Additionally, since humanization is defined according to the Kabat numbering system, some CDRs may differ slightly from mouse CDRs when defined using other numbering systems. Specifically, the CDR-H2 sequence (defined by the Contact numbering system) of the humanized antibody HLP00776 corresponding to HLP00564 is shown in Table 3 as shown in SEQ ID NO: 125; the CDR-L2 sequence (defined by the Contact numbering system) of the humanized antibody HLP00879 corresponding to HLP00476 is shown in Table 3 as shown in SEQ ID NO: 130; and the CDR-H2 sequence (defined by the Contact numbering system) of the humanized antibody HLP00891 corresponding to HLP00562 is shown in Table 3 as shown in SEQ ID NO: 130. The amino acid sequence shown in NO: 134, and the CDR-L2 (Contact numbering system definition) sequence of the humanized antibody HLP00786 corresponding to HLP00601 are shown in Table 3 as shown in SEQ ID NO: 136.

[0518] Table 3: CDR amino acid sequence of humanized antibodies

[0519] The amino acid sequences of the heavy chain variable region and light chain variable region of the five humanized antibodies are shown below:

[0520] HLP00892 Heavy Chain Variable Region Amino Acid Sequence

[0521] HLP00892 light chain variable region amino acid sequence

[0522] HLP00776 Heavy Chain Variable Region Amino Acid Sequence

[0523] HLP00776 Light chain variable region amino acid sequence

[0524] HLP00879 Heavy Chain Variable Region Amino Acid Sequence

[0525] HLP00879 light chain variable region amino acid sequence

[0526] HLP00891 Heavy Chain Variable Region Amino Acid Sequence

[0527] HLP00891 Light chain variable region amino acid sequence

[0528] HLP00786 Heavy Chain Variable Region Amino Acid Sequence

[0529] HLP00786 Light chain variable region amino acid sequence

[0530] The nucleotide sequences of the heavy chain variable region and light chain variable region of the five humanized antibodies are shown below:

[0531] HLP00892 Heavy Chain Variable Region Nucleotide Sequence

[0532] HLP00892 light chain variable region nucleotide sequence

[0533] HLP00776 Heavy Chain Variable Region Nucleotide Sequence

[0534] HLP00776 light chain variable region nucleotide sequence

[0535] HLP00879 Heavy Chain Variable Region Nucleotide Sequence

[0536] HLP00879 light chain variable region nucleotide sequence

[0537] HLP00891 Heavy Chain Variable Region Nucleotide Sequence

[0538] HLP00891 light chain variable region nucleotide sequence

[0539] HLP00786 Heavy Chain Variable Region Nucleotide Sequence

[0540] HLP00786 light chain variable region nucleotide sequence

[0541] The amino acid sequence of the heavy chain constant region of the five humanized antibodies is SEQ ID NO: 137. The full-length amino acid sequences of the heavy and light chains of the five humanized antibodies are shown below:

[0542] HLP00892 Heavy Chain Full-Length Amino Acid Sequence

[0543] HLP00892 light chain full-length amino acid sequence

[0544] HLP00776 Heavy Chain Full-Length Amino Acid Sequence

[0545] HLP00776 Light Chain Full-Length Amino Acid Sequence

[0546] HLP00879 Heavy Chain Full-Length Amino Acid Sequence

[0547] HLP00879 light chain full-length amino acid sequence

[0548] HLP00891 Heavy Chain Full-Length Amino Acid Sequence

[0549] HLP00891 Light Chain Full-Length Amino Acid Sequence

[0550] HLP00786 Heavy Chain Full-Length Amino Acid Sequence

[0551] HLP00786 Light Chain Full-Length Amino Acid Sequence

[0552] The full-length nucleotides of the heavy and light chains of the five humanized antibodies are shown below:

[0553] HLP00892 heavy chain full-length nucleotide sequence

[0554] HLP00892 light chain full-length nucleotide sequence

[0555] HLP00776 Heavy Chain Full-Length Nucleotide Sequence

[0556] HLP00776 light chain full-length nucleotide sequence

[0557] HLP00879 Heavy Chain Full-Length Nucleotide Sequence

[0558] HLP00879 light chain full-length nucleotide sequence

[0559] HLP00891 heavy chain full-length nucleotide sequence

[0560] HLP00891 light chain full-length nucleotide sequence

[0561] HLP00786 Heavy Chain Full-Length Nucleotide Sequence

[0562] HLP00786 light chain full-length nucleotide sequence

[0563] 4.2 Humanized antibodies with Fc effect eliminated

[0564] Five humanized antibodies underwent L234A, L235A, and G237A mutations in the Fc region to reduce Fc effector function, resulting in five Fc-eliminating humanized antibodies named HLP01060, HLP01056, HLP01058, HLP01059, and HLP01057. The CDR sequences of the five Fc-eliminating humanized antibodies are identical to those of the corresponding humanized antibodies in Table 3. The amino acid and nucleotide sequences of the heavy chain variable region and light chain variable region of the five Fc-eliminating humanized antibodies are identical to those of the corresponding humanized antibodies. Specific sequence information is shown in Table 4.

[0565] Table 4: Sequence information of the heavy chain and light chain variable regions of five humanized antibodies that eliminated the Fc effect.

[0566] The amino acid sequence of the Fc segment, i.e., the constant region of the heavy chain of the human IgG1 variant (AAA), after the Fc effect elimination of the five humanized antibody mutations is SEQ ID NO: 159, as shown below:

[0567] Amino acid sequence of the heavy chain constant region of human IgG1 variant (AAA)

[0568] The full-length amino acid and nucleotide sequences of the light chain of the five humanized antibodies that eliminated the Fc effect are identical to those of the corresponding humanized antibodies. The specific sequence information of their full-length light and heavy chains is shown in Table 5.

[0569] Table 5: Sequence information of the full-length heavy and light chains of five humanized antibodies that eliminated the Fc effect.

[0570] The full-length amino acid sequences of the heavy chain of the five humanized antibodies that eliminated the Fc effect are as follows:

[0571] HLP01060 heavy chain full-length amino acid sequence

[0572] HLP01056 Heavy Chain Full-Length Amino Acid Sequence

[0573] HLP01058 Heavy Chain Full-Length Amino Acid Sequence

[0574] HLP01059 heavy chain full-length amino acid sequence

[0575] HLP01057 Heavy Chain Full-Length Amino Acid Sequence

[0576] The full-length heavy chain nucleotide sequences of the five humanized antibodies that eliminated the Fc effect are as follows:

[0577] HLP01060 heavy chain full-length nucleotide sequence

[0578] HLP01056 heavy chain full-length nucleotide sequence

[0579] HLP01058 heavy chain full-length nucleotide sequence

[0580] HLP01059 heavy chain full-length nucleotide sequence

[0581] HLP01057 heavy chain full-length nucleotide sequence

[0582] 4.3Fc enhanced humanized antibody with enhanced lethality

[0583] Five humanized antibodies and two control antibodies underwent S239D and I332E mutations in the Fc region to enhance their in vitro killing effect. This resulted in five humanized antibodies with enhanced Fc killing effect, named HLP01631, HLP01627, HLP01629, HLP01630, and HLP01628, and two control mutant antibodies, named HLP01625 and HLP01626. The CDR sequences of the five humanized antibodies with enhanced Fc killing effect are identical to the sequences of the corresponding humanized antibodies in Table 3. The amino acid and nucleotide sequences of the heavy chain variable region and light chain variable region of the five humanized antibodies with enhanced Fc killing effect are identical to the sequences of the corresponding humanized antibodies. The specific sequence information is shown in Table 6.

[0584] Table 6: Sequence information of the heavy chain and light chain variable regions of five humanized antibodies that enhance Fc killing effect

[0585] The amino acid sequence of the Fc segment, i.e., the constant region of the heavy chain of the human IgG1 variant (DE), five humanized antibodies with enhanced Fc killing effect is SEQ ID NO: 218, as shown below:

[0586] Amino acid sequence of the heavy chain constant region of human IgG1 variant (DE)

[0587] The full-length amino acid and nucleotide sequences of the light chain of the five humanized antibodies that enhance Fc killing effect are identical to those of the corresponding humanized antibodies. The specific sequence information of their light and heavy chains is shown in Table 7.

[0588] Table 7: Sequence information of the full-length heavy and light chains of five humanized antibodies and control mutant antibodies that enhance Fc killing effect.

[0589] The full-length heavy chain amino acid sequences of the five humanized antibodies and control mutant antibodies that enhance Fc killing effect are as follows:

[0590] HLP01631 Heavy Chain Full-Length Amino Acid Sequence

[0591] HLP01627 Heavy Chain Full-Length Amino Acid Sequence

[0592] HLP01629 heavy chain full-length amino acid sequence

[0593] HLP01630 heavy chain full-length amino acid sequence

[0594] HLP01628 Heavy Chain Full-Length Amino Acid Sequence

[0595] HLP01625 Heavy Chain Full-Length Amino Acid Sequence

[0596] HLP01626 Heavy Chain Full-Length Amino Acid Sequence

[0597] The full-length heavy chain nucleotide sequences of five humanized antibodies that enhance Fc killing effects are as follows:

[0598] HLP01631 heavy chain full-length nucleotide sequence

[0599] HLP01627 heavy chain full-length nucleotide sequence

[0600] HLP01629 heavy chain full-length nucleotide sequence

[0601] HLP01630 heavy chain full-length nucleotide sequence

[0602] HLP01628 heavy chain full-length nucleotide sequence

[0603] Test Example 1: Antibody Affinity Assay

[0604] Surface plasmon resonance (SPR) technology was used to determine the affinity of anti-ALPP antibodies for human ALPP, human ALPG, and monkey ALPP in a Biacore instrument. First, a certain amount of the antibody to be tested was captured by affinity bonding using a Protein A biosensor chip (GE, catalog number: 29127556). Then, a series of concentration gradients of human ALPP protein hALPP.his (ACROBiosystems, catalog number: ALP-H52H3), human ALPG protein hALPG.his (Kactus Biosystems, catalog number: APE-HM103), and monkey ALPP protein CynoALPP.his (Kactus Biosystems, catalog number: APE-CM102) were flowed onto the chip surface. The reaction signals were detected in real time using a Biacore instrument (GE, catalog number: Biacore T200) to obtain binding and dissociation curves. After each cycle of dissociation, the biochip was washed and regenerated using glycine-hydrochloric acid regeneration solution (pH 1.5) (purchased from GE, catalog number: BR-1003-54). The buffer used in the experiment was HBS-EP buffer solution (pH 7.4) (purchased from Cytiva, catalog number: BR-1001669). Finally, the experimental data were fitted using BIA T2000 evaluation version 3.0 software with a (1:1) Langmuir model to obtain affinity values. The binding rates (Ka), dissociation rates (Kd), and binding affinity (KD) of the chimeric antibodies and humanized antibodies with human ALPP protein, monkey ALPP protein, and human ALPG protein are shown in Tables 8-10. The results showed that the five chimeric antibodies and their corresponding humanized antibodies had strong affinity for human ALPP protein, monkey ALPP protein, and human ALPG protein, reaching nanomolar (10⁻⁶) values. -9 The order of magnitude M is reached, and some can even reach sub-nanomoles (10). -10 The order of magnitude (M) is at the level of [missing information].

[0605] Table 8: Reactivity affinity of different antibodies with human ALPP protein

[0606] Table 9: Reactivity affinity of different antibodies with monkey ALPP protein

[0607] Table 10: Reactivity affinity of different antibodies with human ALPG protein

[0608] Test Example 2: In vitro cell binding activity assay

[0609] This experiment evaluates antibody binding by detecting the fluorescence signal of antibodies on the cell surface and based on the intensity of the fluorescence signal. First, primary antibodies diluted at different folds were mixed with 2×10⁻⁶ antibodies. 5 Cav-3 (ATCC, HTB-75) or HEK293-CynoALPP (purchased from Jimon Biotechnology, catalog number: GM-C26410) cells were incubated on ice for 60 minutes, after which excess antibodies were washed away. The cells were then incubated with 647 anti-human IgG Fc (purchased from Jackson, catalog number: 109-605-098) at room temperature for 30 minutes. After washing away excess antibodies, the fluorescence signal on the cell surface was read using Attune NxT 4. The results are shown in Figures 1 and 2, Tables 11 and 12. As can be seen from the figures and tables, all five chimeric antibodies and their corresponding humanized antibodies could specifically bind to the Cav-3 tumor cell line and the monkey ALPP overexpressing cell line. Among them, the binding activity (EC50) of four humanized antibodies (HLP00776, HLP00879, HLP00891, and HLP00892) to Cav-3 cells was significantly higher than that of the others. 50 The activity of HLP00786 was comparable to that of the positive control antibody HLP00082 and to the corresponding chimeric antibody, but the binding activity of HLP00786 to Caov-3 cells (EC) was significantly lower. 50 The efficacy was reduced by 2 times compared to the chimeric antibody (HLP00601).

[0610] Table 11: Binding affinity of different antibodies to Caov-3 cells

[0611] Table 12: Binding affinity of different antibodies to Caov-3 cells

[0612] Test Example 3: Antibody endocytosis activity assay

[0613] The purpose of this experiment was to reflect the endocytosis of ALPP antibodies based on changes in fluorescence signal after dye internalization. The in vitro endocytic activity of the antibody was evaluated based on the intensity of the fluorescence signal. Anti-Human IgG antibodies (purchased from Jackson, catalog number 109-005-190) conjugated to pH-sensitive pHrodo iFL dye (Thermo, catalog number: P36010) could directly bind to the Fc region of ALPP antibodies without affecting antigen recognition. pHrodo iFL dye showed almost no fluorescence at neutral pH. During ALPP antibody endocytosis, the dye was simultaneously internalized, and the fluorescence signal gradually increased as the pH decreased. The endocytic activity of the antibody was evaluated based on the increase in fluorescence signal.

[0614] NCI-N87 cells (ATCC, CRL-5822) were cultured in RPMI-1640 Medium + 10% FBS. On the first day of the experiment, a cell suspension was prepared using culture medium containing fresh cells, and the cell density was 2 × 10⁶ cells / year. 5 / mL, add 100μL / well to a 96-well cell culture plate and incubate at 37°C for 24 hours with 5% carbon dioxide.

[0615] 4× Antibody preparation: Dilute the antibody to 80 nM using culture medium.

[0616] Preparation of 4×pHrodo labeling reagent: Dilute the IgG-pHrodo labeling reagent to 240 nM with culture medium. Mix the above 4× antibody solution and 4×IgG-pHrodo labeling reagent solution in equal volumes and incubate at 37°C for 30 minutes. Dilute the labeling mixture 3 times with culture medium to obtain 8 spots.

[0617] Aspirate 50 μL of cell culture from the culture plate and add 50 μL of antibody and pHrodo dye mixture to each well, setting up a dye-only group and a isotype IgG1 control group. After incubation for 16 hours, aspirate the culture medium, add 50 μL of trypsin to each well, digest for 2 minutes, and terminate digestion with 50 μL of fresh culture medium. Transfer the samples to a 96-well round-bottom plate using a pipette, centrifuge at 1500 rpm for 4 minutes, discard the culture medium, wash the cells once with FACS buffer (PBS + 2% FBS), and centrifuge at 1500 rpm for 4 minutes. Add 150 μL of FACS buffer (PBS + 2% FBS) to resuspend the cells, and read the fluorescence signal on the cell surface using an Attune NxT 4 instrument. The results are shown in Figure 3. As shown in the figure, all five chimeric antibodies and their corresponding humanized antibodies exhibited good endocytic activity on NCI-N87 cells, and the humanized antibodies HLP00776, HLP00891, and HLP00892 showed significantly better endocytic activity than the positive control antibody HLP00082.

[0618] Test Example 4: Determination of cross-reactivity with other members of the alkaline phosphatase family

[0619] Dilute the SA antibody (Jackson, catalog number 016000113) with PBS to a concentration of 1 μg / mL, and add 100 μL to each well of a 96-well microplate (Thermo, catalog number 442404). Incubate overnight at 4°C. Discard the liquid and wash the plate three times with PBST buffer (pH 7.4, containing 0.05% Tween-20). Add 250 μL of 2% BSA blocking buffer and incubate at 37°C for 1 hour. Discard the blocking buffer and wash the plate three times with PBST buffer (pH 7.4, containing 0.05% Tween-20). Biotinylated Human ALPI Protein (purchased from Jimon Biotechnology, catalog number: GM-85154RP) and Biotinylated Human ALPL Protein (purchased from Jimon Biotechnology, catalog number: GM-85157RP) were diluted to 1 μg / mL with blocking buffer, and 100 μL was added to each well. The mixture was incubated at 25°C for 1 hour. After washing three times with PBST buffer (pH 7.4, containing 0.05% Tween-20), the test antibody (100 nM), isotype control antibody HLP00686 (100 nM), human ALPI antibody Sino-ALPI Ab (1:1000, purchased from Sino, catalog number: 13225-R001), and human ALPL antibody R&D-ALPL Ab (1 μg / mL, purchased from R&D, catalog number: MAB1448) were added. Each sample was tested in duplicate, and the mixture was incubated at 25°C for 1 hour. After washing three times with PBST buffer (pH 7.4, containing 0.05% Tween-20), add the diluted secondary antibody of the corresponding species and incubate at 25°C in the dark for 1 hour. After washing six times with PBST buffer (pH 7.4, containing 0.05% Tween-20), add 100 μL / well TMB (purchased from SeraCare, catalog number: 5120-0077) and incubate at room temperature in the dark for about 10 minutes. Add 50 μL / well stop solution (purchased from Sangon Biotech, catalog number: E661006) to each well to stop the reaction, and measure the absorbance (OD450) at 450 nm using a microplate reader (purchased from PerkinElemer, catalog number: VICTOR X3). The results are shown in Table 13, Figure 4, and Figure 5. As can be seen from the table and figures, only the positive control antibodies, human ALPI antibody and human ALPL antibody, bound to human ALPI protein and human ALPL protein, respectively. However, the OD450 values ​​of the antibodies of the present invention reacting with human ALPI protein and human ALPL protein were all less than 0.2, indicating that the antibodies of the present invention do not bind to human ALPI protein and human ALPL protein, but only specifically bind to human ALPP protein and human ALPG protein, that is, they do not cross-react with other members of the alkaline phosphatase family.

[0620] Table 13: Results of antibody binding to human ALPI and human ALPL proteins Note: "-" indicates that no detection was performed.

[0621] Test Example 5: Determination of cross-reactivity with other species

[0622] Dilute mouse ALPG protein (Kactus, catalog number: APE-MM103), rat ALPP protein (ACROBiosystems, catalog number: ALP-R52-H3F001), and rat ALPG protein (Kactus, catalog number: APE-RM103) with PBS to a concentration of 1 μg / mL. Add 100 μL to each well of a 96-well microplate (Thermo, catalog number: 442404) and incubate overnight at 4°C. Discard the liquid and wash the plate three times with PBST buffer (pH 7.4, containing 0.05% Tween-20). Add 250 μL of 2% BSA blocking buffer and incubate at 37°C for 1 hour. Discard the blocking buffer and wash the plate three times with PBST buffer (pH 7.4, containing 0.05% Tween-20). Add the test antibody and ALPG antibody diluted at different folds, Solarbio Anti-ALPG Ab (1:1000, purchased from Solarbio, catalog number: K004825P), and incubate at 25°C for 1 hour. After washing three times with PBST buffer (pH 7.4, containing 0.05% Tween-20), add the diluted secondary antibody of the corresponding species and incubate at 25°C in the dark for 1 hour. After washing six times with PBST buffer (pH 7.4, containing 0.05% Tween-20), 100 μL / well TMB (purchased from SeraCare, catalog number: 5120-0077) was added, and the mixture was incubated at room temperature in the dark for approximately 10 minutes. 50 μL / well stop solution (purchased from Sangon Biotech, catalog number: E661006) was added to each well to terminate the reaction. The absorbance (OD450) at 450 nm was measured using a microplate reader (purchased from PerkinElemer, catalog number: VICTOR X3). The results are shown in Table 14 and Figures 6-10. Table 14, Figures 6 and 7 show that the OD450 values ​​of the antibodies of this invention are all less than 0.1, indicating that they do not bind to rat ALPP and rat ALPG proteins. Table 14 and Figure 8 show that HLP00476, HLP00879, and HLP01058 have OD450 values ​​greater than 1 when binding to mouse ALPG protein, indicating binding to mouse ALPG; the other antibodies have OD450 values ​​less than 0.1, indicating that they do not bind to mouse ALPG protein. Figures 9 and 10 show that HLP00476, HLP00879, and HLP01058 have weak binding activity to mouse ALPG protein.

[0623] Table 14: Results of antibody binding to rat ALPP, rat ALPG and mouse ALPG proteins Note: "-" indicates that no detection was performed.

[0624] Test Example 6: Analysis of protein purity and hydrophobicity using HIC-HPLC

[0625] Analytical hydrophobic interaction chromatography (HIC) was used to analyze the purity and hydrophobicity of protein samples. An analytical column, TSKge1Buty1-NPR (Tosoh Bioscience, 14947, 4.6 mm × 3.5 cm), was connected to an HPLC system (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. A linear gradient was set over 16 minutes from 100% mobile phase A (20 mM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0), with a flow rate of 0.7 mL / min, a protein sample concentration of 1 mg / mL, an injection volume of 20 μL, and a detection wavelength of 280 nm. After acquisition, the chromatograms were integrated and relevant data were calculated using ChemStation software to generate an analysis report, showing the residence times of different molecular sizes within the sample. The results are shown in Table 15. The data in the table show that all five humanized antibodies and the corresponding humanized antibodies with Fc effect elimination have good hydrophilicity.

[0626] Table 15: HIC detection results for different antibodies

[0627] Test Example 7: Determination of the thermal stability of protein molecules using DSF

[0628] Differential scanning fluorescence (DSF) is a commonly used high-throughput method for determining protein thermal stability. It uses a real-time quantitative PCR instrument to monitor changes in the fluorescence intensity of dyes binding to unfolded protein molecules, reflecting the protein denaturation process and thus the thermal stability of the protein molecule. This test example uses the DSF method to determine the thermal denaturation temperature (Tm) of a protein molecule. 10 μg of protein was added to a 96-well PCR plate (Thermo, catalog number: AB-0700 / W), followed by 2 μL of 100× diluted SYPROTM dye (Invitrogen, catalog number: 2008138), and then buffer was added to a final volume of 40 μL per well. The PCR plate was sealed and placed in a real-time quantitative PCR instrument (Bio-Rad, model: CFX96 PCR System). The plate was incubated at 25°C for 5 minutes, then gradually increased from 25°C to 95°C in increments of 0.2°C / 0.2 minutes. At the end of the test, the temperature was lowered to 25°C. The FRET scanning mode was used, and data analysis was performed using Bio-Rad CFX Maestro software to calculate the thermal stability of the samples. The results are shown in Table 16. As can be seen from the data in the table, all five humanized antibodies and their corresponding Fc-effect-eliminating humanized antibodies exhibit good thermal stability.

[0629] Table 16: Tm detection results for different antibodies Note: " / " indicates that it was not detected.

[0630] Test Example 8: Determination of pharmacokinetics (PK) of humanized antibodies in mice

[0631] To investigate the pharmacokinetic (PK) characteristics of humanized antibodies HLP00776, HLP01056, HLP01060, and HLP01058 in mice, six female BALB / c nude mice (purchased from Vitaliva) weighing 18–22 g were administered the drugs (humanized antibodies HLP00776, HLP01056, HLP01060, or HLP01058) via tail vein injection at a dose of 10 mg / kg. Whole blood was collected from one group of three mice before administration and at 30 minutes, 4 hours, and on days 3, 7, 12, and 21 after administration. Whole blood was collected from another group of three mice before administration and at 2 hours, and on days 1, 5, 10, and 14 after administration. The whole blood was allowed to stand for 30 minutes to coagulate, then centrifuged at 2000 rpm for 5 minutes at 4°C. The separated serum samples were then frozen at -80°C until analysis. ELISA was used to quantitatively determine drug concentrations in mouse serum. Recombinant human ALPP protein coated on 96-well plates was used to capture antibodies in mouse serum, followed by the addition of HRP-labeled goat anti-human Fc secondary antibody for detection. Phoenix WinNonlin software version 8.2 was used with a non-compartmental (NCA) model to analyze the blood drug concentration data and evaluate their pharmacokinetics. The results are shown in Table 17 and Figure 11. As can be seen from the table and figure, HLP00776, HLP01056, HLP01060, and HLP01058 all exhibited good pharmacokinetic characteristics.

[0632] Table 17: PK data of humanized antibodies Note: t 1 / 2 The values ​​represent the drug half-life, Tmax represents the time required for the drug to reach its maximum concentration after administration, Cmax represents the maximum concentration reached after administration, AUC 0-t represents the area under the concentration-time curve from 0 to time t after administration, AUC 0-inf represents the area under the concentration-time curve from 0 to positive infinity after administration, MRT 0-inf represents the average residence time from time zero to infinity, Vz represents the pseudo-distribution equilibrium volume of distribution or terminal phase volume of distribution, CL represents the drug clearance rate, and Vss represents the steady-state apparent volume of distribution.

[0633] Test Example 9: Antigen Binding Epitope Analysis

[0634] Tandem epitope competition assays for ALPP antibodies were performed using a Gator biomolecular interaction analyzer (Gator Prime). All consumables, including 96-well plates (Gator, #20-0110), Q Buffer (Gator, #20-5204), and the chip (Gator, #205066), were purchased from Gator. In the tandem epitope competition assay, an Anti-His sensor was used to capture human-ALPP His (Acro, #ALP-H52H3) antigen at a final concentration of 5 μg / mL, with a capture height limit of 0.5 nm. The sensor with captured antigen was immersed in the first antibody (10 μg / mL) for 180 s, and then in the second antibody (final concentration 10 μg / mL) for the same 180 s. The signal difference after immersion in the second antibody was recorded as Rc = Ab2 with Ab1, and the signal as the first antibody was recorded as R0 = Ab2 without Ab1.

[0635] Inhibition rate is calculated using the following formula: Inhibition Rate = (Rc / R0)%;

[0636] R0: This antibody serves as the 100% signal of the first antibody (obtained from the first step);

[0637] Rc: This antibody serves as a signal for the second antibody (obtained from step two).

[0638] If the inhibition rate is greater than 70%, it means that the two antibodies do not compete with each other; if the inhibition rate is between 30% and 70%, it means that the two antibodies partially compete; if the inhibition rate is less than 30%, it means that the two antibodies strongly compete. The inhibition rate results are shown in Table 18. As can be seen from the table, the epitopes of HLP00776 and HLP00879 are completely different from those of HLP00082; the epitopes of HLP00891, HLP00786, HLP00892 and HLP00082 are the same; the epitopes of HLP00776 partially overlap with those of HLP00892 and HLP00786; and the epitopes of HLP00879 are completely different from those of the other 5 antibodies.

[0639] Table 18: Inhibition rates between antibodies

[0640] Example 5: In vitro functional experiments of humanized antibodies

[0641] Test Example 1: Humanized Antibody ADCC Killing Experiment

[0642] Utilizing engineered Jurkat-Lucia TMAntibody-dependent cytotoxicity (ADCC) activity was assessed using NFAT-CD16A effector cells (purchased from Invivogen, Cat: JKTL-NFAT-CD16). 2E4 COV644 tumor cells / 50 μL were added to each well of a 96-well plate, followed by serially diluted target antibody. The plates were incubated at 37°C for 20 min, then 1E5 Jurkat-CD16a cells / 50 μL were added to each well, and the plates were incubated at 37°C for 5 h. Finally, 50 μL of Lumferserce reagent (GenScript L00877C-1000) was added to each well; the plates were shaken for 5 minutes, and the results were read using a microplate reader. The results are shown in Figure 12. As can be seen from the figure, the humanized antibodies provided in this invention all exhibit strong ADCC effects.

[0643] Example 6: Conjugation of humanized antibodies

[0644] The preparation process of the ADC conjugate is as follows: The antibodies (HLP00776, HLP00892, HLP01056, HLP01060, HLP00082, HLP00686) were placed in 0.05M PBS buffer solution at pH 7.4 (antibody concentration 5mg / mL), and a prepared 10mM aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (Sigma, Cat#C4706) was added. The mixture was placed in a constant temperature shaking incubator at 25℃ and reacted for 3 hours.

[0645] The linker-drug conjugates (LDs) (Mal-PEG2-VCP-Eribulin (MCE, Cat#HY-128870, CAS No.:2130869-18-8), mc-vc-PAB-MMAE (MCE, Cat#HY-15575, CAS No.: 646502-53-6), and MC-GGFG-DXD (MCE, Cat#HY-13631E, CAS No.: 1599440-13-7)) were dissolved in dimethyl sulfoxide and added to the above reaction solution. The reaction was carried out on a shaker at room temperature for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting centrifugation column (Thermo Cat#89889) (elution phase: 0.05M PBS buffered aqueous solution at pH 7.4 containing 0.001M EDTA) to obtain the target antibody-drug conjugate, i.e., the ADC conjugate.

[0646] By adjusting the antibody-to-drug ratio, reaction scale, and other conditions, ADC conjugates with different DAR values ​​(n) can be obtained, with an optimal DAR value of 4-8. Specific ADC conjugates prepared are shown in Table 19.

[0647] Table 19: List of ADC Conjugates

[0648] Example 7: In vitro cell-killing activity experiment of ADC conjugate

[0649] This experiment detects intracellular ATP levels and, based on IC50... 50 The inhibitory effects of the above-mentioned ADC conjugates on cell proliferation were evaluated. The tumor cell lines tested included: NCI-N87 (ATCC, CRL-5822), Caov-3 (ATCC, HTB-75), and COV644 (Shanghai Jining, JN-CC2025). The ADC conjugates were serially diluted 5-fold to eight concentrations (initial concentrations: 10 nM for NCI-N87 cells, 40 nM for Caov-3 and COV644). Samples were added to culture plates and incubated for 4 days (37°C, 5% CO2). Detection was performed using CellTiter-Glo reagent (Promega, G7572), with chemiluminescence signal values ​​read in Vector X3 luminescence. Data were processed using GraphPad software.

[0650] The cytotoxic activity results of the humanized antibody-ADC conjugate are shown in Figures 13-15. The measured IC50 values ​​are as follows: 50 The values ​​are shown in Table 20. As can be seen from the figures and tables, humanized antibodies and their Fc-effect-eliminating antibody-ADC conjugates, when having similar DAR values, exhibit similar inhibitory effects on the proliferation of various tumor cells, with similar IC50 values. 50 The maximum killing activity was comparable to that of the control HLP00082-4E, and superior to that of HLP00082-4M. The two humanized antibodies and their Fc-eliminated antibody-based ADC conjugates showed similar inhibitory effects on the proliferation of various tumor cells under similar DAR values, with IC50 values... 50 Its maximum killing activity was comparable to that of the control HLP00082-4E, and superior to that of HLP00082-4M.

[0651] Table 20: Inhibitory effects of ADC conjugates on cell proliferation

[0652] Example 8: Rapid druggability test of ADC conjugates

[0653] The stability of HLP01056-8D and HLP01060-8D under pressure screening conditions was evaluated. Samples were replaced with PBS buffer at pH 7.2, maintaining a concentration of approximately 5 mg / mL. Samples were processed and analyzed according to the experimental protocol in Table 21, and the results are shown in Table 22.

[0654] Table 21: Experimental Scheme Note: In the table, 1W, 2W, and 4W refer to weeks 1, 2, and 4; 3C and 5C indicate repetitions of 3 and 5 times, respectively.

[0655] As shown in the table, there was no significant difference in the concentration of samples HLP01056-8D and HLP0160-8D under various conditions, and the samples were colorless microemulsified liquids. SEC detection results showed good sample stability. CE_NR detection results showed no significant difference between the two samples under initial conditions, but with the extension of incubation temperature and time, some loads detached from the samples, with HLP01056-8D being more stable. RP_LC and reduced molecular weight detection results showed that some toxins detached from the samples under initial conditions, accounting for 10% and 7.5% of the total, respectively. The DAR of the samples detected by the two methods showed no significant difference, within the range of approximately 6.5±0.5. Mass spectrometry detection of the samples after incubation at 40℃ for 4 weeks showed the generation of an unknown peak of approximately 18D-20Da. iCIEF detection results showed a large difference in charge heterogeneity between the samples and the bare anti-charge, which is preliminarily speculated to be due to conformational changes during the coupling process. Under high temperature conditions, the acidic peaks increased, and the proportion of the main peak decreased.

[0656] Table 22: RP-LC and Mass Spectrometry Detection Results Note: T0 indicates that no freeze-thaw and temperature tests were conducted.

[0657] Peptide mapping analysis showed that the sample sequence was consistent with the theoretical sequence. Analysis of coupling sites showed that the coupling was located at the disulfide bonds between the light and heavy chains and at the hinge region disulfide bonds. Sample HLP01060-8D showed poor stability under pressure and was prone to load shedding, especially on the light chain. PTMs results showed that the main modifications of the sample were oxidation of methionine (M) and deamidation of asparagine (N). The modifications of sample HLP01056-8D mainly occurred at the Fc end of the heavy chain.

[0658] Example 9 Mouse PK Experiment of ADC Conjugate

[0659] The pharmacokinetic characteristics of HLP01056-8D and HLP01060-8D administered via a single tail vein dose of 5 mg / kg in BALB / c mice were evaluated.

[0660] 9.1 Grouping, drug administration, and blood collection

[0661] Twenty BALB / c female mice were ordered, with 16 enrolled and 4 reserved. At the start of the experiment, all mice were weighed and randomly grouped according to body weight. After grouping, drugs were administered via tail vein, with the day of administration designated as day 0. Blood collection: Mice were anesthetized with 5% isoflurane gas. Once the mice were deeply anesthetized (no response when the paws were pinched), approximately 30–50 μL of whole blood was collected from the ophthalmic venous plexus using a 0.5 mm capillary glass tube into an EDTA-K2 anticoagulant tube. After mixing, the tubes were centrifuged at 8000 rpm for 5 minutes at 4°C. The plasma was then transferred to a new centrifuge tube and stored at -80°C.

[0662] 9.2 Bioanalytical Detection

[0663] Total antibody: Dilute AffiniPure Goat Anti-Human IgG, FcγFragment Specific to 1 μg / mL with PBS and add 100 μL to each well of the ELISA plate, incubating overnight at 4°C. Discard the coating solution, wash three times with 0.05% PBST (pH 7.4, containing 0.05% Tween-20), 300 μL / well, and blot dry. Add 2% BSA and blocking buffer, 200 μL / well, and incubate at room temperature for 2–3 h. Discard the blocking solution, wash three times with 0.05% PBST (pH 7.4, containing 0.05% Tween-20), 300 μL / well, and blot dry. Add the test drug standard curve sample and diluted plasma sample, 100 μL / well, to the ELISA plate, cover, and incubate at room temperature for 1 h. Discard the supernatant, wash three times with 0.05% PBST (pH 7.4, containing 0.05% Tween-20), 300 μL / well, and blot dry. Add 100 μL / well of the detection antibody Goat Anti-human IgG-Fc[HRP] (diluted 1:10000) to the microplate and incubate at room temperature for 1 h. Discard the supernatant, wash three times with 0.05% PBST (pH 7.4, containing 0.05% Tween-20), 300 μL / well, and blot dry. Add 100 μL / well of TMB chromogenic solution, cover with a membrane, and incubate at room temperature in the dark for 5–8 min. Add 100 μL / well of stop solution (1M HCl) to terminate the reaction. Read the OD value of each well at a detection wavelength of 450 nm using a multi-functional optical microplate reader. Using the standard curve sample concentration as the x-axis and OD450nm as the y-axis, a four-parameter model was used for fitting, and the plasma drug concentration at different time points was calculated and measured using the standard curve.

[0664] ADC conjugate: Dilute AffiniPure Goat Anti-Human IgG, FcγFragment Specific to 1 μg / mL with PBS and add 100 μL to each well of the ELISA plate, incubating overnight at 4°C. Discard the coating solution, wash three times with 0.05% PBST (pH 7.4, containing 0.05% Tween-20), 300 μL / well, and blot dry. Add 2% BSA and blocking buffer, 200 μL / well, and incubate at room temperature for 2–3 h. Discard the blocking solution, wash three times with 0.05% PBST (pH 7.4, containing 0.05% Tween-20), 300 μL / well, and blot dry. Add the test drug standard curve sample and diluted plasma sample, 100 μL / well, to the ELISA plate, cover, and incubate at room temperature for 1 h. Discard the supernatant, wash three times with 0.05% PBST (pH 7.4, containing 0.05% Tween-20), 300 μL / well, and blot dry. Add 100 μL / well of the detection antibody HRP conjugated monoclonal Anti-DXD & Exatecan Antibody (0.2 μg / mL) to the microplate and incubate at room temperature for 1 h. Discard the supernatant, wash three times with 0.05% PBST, 300 μL / well, and blot dry. Add 100 μL / well of TMB chromogenic solution, cover with a membrane, and incubate at room temperature in the dark for 5–8 min. Add 100 μL / well of stop solution (1M HCl) to terminate the reaction. Read the OD value of each well at a detection wavelength of 450 nm using a multi-functional optical microplate reader. Using the standard curve sample concentration as the x-axis and OD450nm as the y-axis, a four-parameter model was used for fitting. The plasma drug concentration at different time points was calculated using the standard curve, and the results are shown in Figure 16 and Table 23.

[0665] 9.3 Results

[0666] No significant abnormalities were observed in any of the animals from the time of drug administration until the end of the experiment, and BALB / c mice tolerated the administered doses well. In BALB / c mice, HLP01056-8D and HLP01060-8D exhibited normal antibody pharmacokinetic characteristics. Systemic exposure to HLP01056-8D was slightly higher than that to HLP01060-8D. The total plasma antibody concentrations of each drug were consistent with the ADC conjugate concentrations.

[0667] Table 23: Pharmacokinetic Parameters Note: PK parameters were obtained by analyzing the average blood drug concentrations at various time points in four mice (with alternating blood collection). NCA fitting was performed using the concentrations at the end of the 72h-336h time period, and the analysis was conducted using a non-compartmental model from WinNonlin software.

[0668] Example 10: In vivo CDX model pharmacodynamic experiment of ADC conjugate

[0669] Test Example 1 NCI-N87 subcutaneous xenograft BALB / c Nude mouse model

[0670] NCI-N87 cells (AutoID: CRL-5822, Species: Human, Source: CRS-ATCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. NCI-N87 cells in the exponential growth phase were collected, resuspended in RPMI 1640 to an appropriate concentration, and subcutaneously inoculated into the subcutaneous right anterior back of mice at 2×10 6 NCI-N87 cells, and the cells were resuspended in a 1:1 mixture of RPMI-1640 and Matrigel (0.1 mL cell suspension + 0.1 mL Matrigel per mouse). Tumor growth was observed regularly. When the tumors grew to an average volume of approximately 160.00 mm 3 , mice were randomly grouped and administered with the test articles according to tumor size. After tumor cell inoculation, routine monitoring included the effects of tumor growth and treatment on the normal behavior of animals, specifically including the activity, food intake and water consumption of experimental animals, weight gain or loss, eyes, fur and other abnormal conditions. Abnormal clinical symptoms observed during the test were recorded in the original data. After the start of administration, the body weight and tumor volume of mice were measured twice a week, and the results are shown in Figures 17A-17B. Calculation formula of tumor volume: tumor volume (mm 3 ) = 1 / 2 × (a × b 2 ) (where a represents the long diameter and b represents the short diameter).

[0671] It can be seen from the figures that in the NCI-N87 human-derived gastric cancer model, the average body weight of mice in all groups showed an increasing trend. No abnormal conditions of mice were observed in the experiment, no mouse death was found, no severe weight loss of mice (15% < BWL < 20%) occurred, and mice generally tolerated the test articles well at the test doses.

[0672] Compared with the G1 control group, HLP00082-8D (10 mg / kg and 3 mg / kg), HLP01060-8D (10 mg / kg, 3 mg / kg and 1 mg / kg) and HLP01056-8D (10 mg / kg, 3 mg / kg and 1 mg / kg) all significantly inhibited tumor growth in a dose-related manner. At a dose of 10 mg / kg, compared with the HLP00686-8D isotype control group (TGI = 74.64%), HLP00082-8D, HLP01060-8D and HLP01056-8D all significantly inhibited tumor growth (P<0.01), with comparable efficacy and relative TGIs of 93.64%, 95.76% and 96.11%, respectively. At a dose of 3 mg / kg, compared with the HLP00686-8D isotype control group (TGI = 23.56%), HLP00082-8D, HLP01060-8D, and HLP01056-8D all significantly inhibited tumor growth (P < 0.01), with comparable efficacy and relative TGIs of 80.84%, 88.44%, and 84.04%, respectively. At a dose of 1 mg / kg, compared with the G1 control group, HLP01060-8D and HLP01056-8D both significantly inhibited tumor growth (P < 0.01), with comparable efficacy and relative TGIs of 67.51% and 66.59%, respectively.

[0673] Test Example 2: HPAC subcutaneous xenograft BALB / c Nude mouse model

[0674] HPAC (Auto ID: CL-00891, Species: Human) human pancreatic cancer cells were cultured in DMEM / Ham's F12 (1:1) + 1*ITS + 40 ng / mL Hydrocortisone + 10 ng / mL EGF medium containing 5% fetal bovine serum. HPAC cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice. 1×10⁻⁶ cells / mL 7 HPAC cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.2 mL / mouse) and subcutaneously seeded on the right anterior back of experimental mice. Tumor growth was observed regularly until the tumor reached an average volume of 174 mm². 3 Mice were randomly assigned to groups based on tumor size for drug administration. The drug used in this study was the same as that used in the test cases. After administration began, mouse body weight and tumor size were measured twice weekly. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2(where a represents the major axis and b represents the minor axis). Data were collected using StudyDirector™ (version 3.1.399.19, vendor Studylog System, Inc.) software. The results of the change rate of body weight, tumor volume and tumor weight of mice in each group from the start of the experiment to the end point are shown in Figures 18A-18C.

[0675] As shown in the figure, all mice generally tolerated the test drug well at the test dose. In the G1 control group, the average tumor volume reached 1218.39 mm at 23 days. 3 The average tumor volume in the G2 group mice at 23 days was 468.38 mm. 3 The absolute tumor inhibition rate (TGI) was 61.56%, which was not significantly different from the control group (p>0.05). The mean tumor volume in the G3 group at 23 days was 839.80 mm. 3 The absolute tumor inhibition rate (TGI) was 31.07%, which was not significantly different from the control group (p>0.05). The average tumor volume in the G4 group was 207.47 mm² at 23 days. 3 The absolute tumor inhibition rate (TGI) was 82.97%, which was significantly higher than that of the control group (p<0.001). The average tumor volume in the G5 group was 475.52 mm² at 23 days. 3 The absolute tumor inhibition rate (TGI) was 60.97%, which was significantly higher than that of the control group (p<0.05). The average tumor volume in the G6 group was 190.92 mm² at 23 days. 3 The absolute tumor inhibition rate (TGI) was 84.33%, which was significantly higher than that of the control group (p<0.001). The average tumor volume in the G7 group was 217.03 mm² at 23 days. 3 The absolute tumor inhibition rate (TGI) was 82.19%, which was significantly higher than that of the control group (p<0.001). The average tumor volume in the G8 group at 23 days was 185.53 mm. 3 The absolute tumor inhibition rate (TGI) was 84.77%, which was significantly higher than that of the control group (p<0.001). The average tumor volume in the G9 group at 23 days was 241.31 mm. 3 The absolute tumor inhibition rate (TGI) was 80.19%, which was significantly higher than that of the control group (p<0.001).

[0676] Example 11 Experimental Study of Human Ovarian Cancer PDX Model Using ADC Conjugates

[0677] Test Case 1: Subcutaneous xenograft of human ovarian cancer OV17054 into a NOD / SCID female mouse model

[0678] Tumor tissue was collected from OV17054-R3P7 tumor-bearing mice (ovarian cancer xenograft model), cut into tumor blocks with a diameter of 2-3 mm, and subcutaneously inoculated into the right anterior scapula of female NOD / SCID mice. When the average tumor volume of the tumor-bearing mice reached approximately 165 mm², the tumors were successfully transplanted. 3 Mice were randomly assigned to groups according to the experimental design and given the drug. Body weight and tumor size were measured twice weekly. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (Where a represents the major axis and b represents the minor axis). Data was collected using StudyDirector™ software (version 3.1.399.19, vendor: Studylog System, Inc.), including measurements of the tumor's major and minor axes and animal weight. Raw data, measured using a balance and calipers, was directly imported into the software; any changes in the data were recorded. The average tumor growth curves and weight changes for each group from the start of the experiment to the experimental endpoint are shown in Figures 19A-19B.

[0679] As shown in the figure, the OV17054 model mice generally tolerated the test drug well at the test dose. Mouse body weight remained stable or slightly increased during the experiment, and no mice died during the experiment. No mice experienced severe weight loss (BWL > 10%). The G1 control group reached an average tumor volume of 1396.98 mmHg at 23 days. 3 The average tumor volume in the G2 group mice at 23 days was 20.72 mm. 3 Two mice showed complete tumor disappearance, with an absolute tumor inhibition rate (TGI) of 98.52%, which was significantly better than the control group (p<0.001).

[0680] Test Example 2: Human Ovarian Cancer OV15209 Subcutaneous Xenograft NOD / SCID Female Mouse Model

[0681] Tumor tissue was collected from OV15209-R8P7 tumor-bearing mice (ovarian cancer xenograft model), cut into tumor blocks with a diameter of 2-3 mm, and subcutaneously inoculated into the right anterior scapula of female NOD / SCID mice. When the average tumor volume of the tumor-bearing mice reached approximately 165 mm², the tumors were successfully transplanted. 3 Mice were randomly assigned to groups according to the experimental design and given the drug. Body weight and tumor size were measured twice weekly. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2(where a represents the major axis and b represents the minor axis). Data was collected using StudyDirector™ software (version 3.1.399.19, vendor: Studylog System, Inc.), including measurements of the tumor's major and minor axes and animal weight. Raw data, measured using a balance and calipers, was directly imported into the software. Any changes in the data were recorded in the software. The results of the average tumor growth curves and weight changes for each group from the start of the experiment to the experimental endpoint are shown in Figures 20A and 20B.

[0682] As shown in the figure, the OV15209 model mice generally tolerated the test drug well at the test dose. Mouse body weight remained stable or slightly increased during the experiment, and no mice died. Mouse #71625 in the G2 group experienced severe weight loss (BWL > 10%) on day 14; this mouse was given supplemental hydrogel from day 14 until the end of the experiment. The G1 control group reached an average tumor volume of 1068.27 mmHg on day 35. 3 The average tumor volume in the G2 group mice at 35 days was 208.86 mm. 3 The absolute tumor inhibition rate (TGI) was 80.45%, which was significantly higher than that of the control group (p<0.01).

[0683] Test Case 3: Human Ovarian Cancer OV9419 Subcutaneous Xenograft NOD / SCID Female Mouse Model

[0684] Tumor tissue was collected from OV9419-R10P6 tumor-bearing mice (ovarian cancer xenograft model), cut into tumor blocks with a diameter of 2-3 mm, and subcutaneously inoculated into the right anterior scapula of female NOD / SCID mice. When the average tumor volume of the tumor-bearing mice reached approximately 165 mm², the tumors were successfully transplanted. 3 Mice were randomly assigned to groups according to the experimental design and given the drug. Body weight and tumor size were measured twice weekly. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). Data was collected using StudyDirector™ software (version 3.1.399.19, vendor: Studylog System, Inc.), including measurements of the tumor's major and minor axes and animal weight. Raw data, measured using a balance and calipers, was directly imported into the software. Any changes in the data were recorded in the software. The results of the average tumor growth curves and weight changes for each group from the start of the experiment to the experimental endpoint are shown in Figures 21A-21B.

[0685] As shown in the figure, the OV9419 model mice generally tolerated the test drug well at the test dose. Mouse body weight remained stable or slightly increased during the experiment; no mice died, and no mice experienced severe weight loss (BWL > 10%). In the G1 control group, three mice (#74251, #74272, #74275) developed tumor ulceration, and in the G2 group, two mice (#74252, #74266) developed tumor ulceration. The average tumor volume in the G1 control group reached 923.63 mm² at 32 days. 3 The average tumor volume in the G2 group mice at 32 days was 662.68 mm. 3 The absolute tumor inhibition rate (TGI) was 28.25%, which was comparable to the control group in terms of tumor inhibition effect.

[0686] Test Case 4: Subcutaneous xenograft of human ovarian cancer OV9422 into a NOD / SCID female mouse model

[0687] Tumor tissue was collected from OV9422-R9P9 tumor-bearing mice (ovarian cancer xenograft model), cut into tumor blocks with a diameter of 2-3 mm, and subcutaneously inoculated into the right anterior scapula of female NOD / SCID mice. When the average tumor volume of the tumor-bearing mice reached approximately 165 mm², the tumors were successfully transplanted. 3 Mice were randomly assigned to groups according to the experimental design and given the drug. Body weight and tumor size were measured twice weekly. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). Data was collected using StudyDirector™ (version 3.1.399.19, vendor: Studylog System, Inc.) software, including measurements of the tumor's major and minor axes and animal weight. Raw data, measured using a balance and calipers, was directly imported into the software. Any changes in the data were recorded in the software. The results of the average tumor growth curves and weight changes for each group from the start of the experiment to the experimental endpoint are shown in Figures 22A-22B.

[0688] As shown in the figure, the OV9422 model mice generally tolerated the test drug well at the test dose. Mouse body weight remained stable or slightly increased during the experiment; no mice died, and no mice experienced severe weight loss (BWL > 10%). A total of 12 mice were enrolled in the OV9422 model, divided into two groups of six mice each. Since 31 days was the last measurement period containing all mouse data, the tumor inhibition rate was analyzed based on the 31-day data. In the G1 control group, the average tumor volume reached 1247.41 mmHg at 31 days. 3 The average tumor volume in the G2 group mice at 31 days was 272.91 mm.3 The absolute tumor inhibition rate (TGI) was 78.12%, which was significantly higher than that of the control group (p<0.001).

[0689] Test Case 5: Human Ovarian Cancer OV9418 Subcutaneous Xenograft NOD / SCID Female Mouse Model

[0690] from Tumor tissue was collected from OV9418-R6P8 tumor-bearing mice (ovarian cancer xenograft model), cut into tumor blocks with a diameter of 2-3 mm, and subcutaneously inoculated into the right anterior scapula of female NOD / SCID mice. When the average tumor volume of the tumor-bearing mice reached approximately 172.03 mm²... 3 Mice were randomly assigned to groups according to the experimental design. After tumor cell inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior, specifically monitoring animal activity, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. After drug administration began, mouse weight and tumor size were measured twice weekly. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). Data was collected using StudyDirector™ software (version 3.1.399.19, vendor: Studylog System, Inc.), including measurements of the tumor's major and minor axes and animal weight. Raw data, measured using a balance and calipers, was directly imported into the software. Any changes in the data were recorded in the software. The results of the average tumor growth curves and weight changes for each group from the start of the experiment to the experimental endpoint are shown in Figures 23A-23B.

[0691] As shown in the figure, the OV9418 model mice generally tolerated the test drug well at the test dose. Mice gained a slight amount of body weight during the experiment, and no mice died. No mice experienced severe weight loss (BWL > 10%) during the experiment. A total of 12 mice were enrolled in the OV9418 model, divided into two groups of six mice each. Since 34 days was the last measurement period containing all mouse data, the tumor inhibition rate was analyzed based on the 34-day data. The G1 control group achieved an average tumor volume of 646.53 mmHg at 34 days. 3 The average tumor volume in the G2 group mice at 34 days was 23.99 mm. 3 The absolute tumor inhibition rate (TGI) was 96.29%, which was significantly higher than that of the control group (p<0.01).

[0692] Test Case 6: Human ovarian cancer OV5308 subcutaneous xenograft NOD / SCID female mouse model

[0693] from Tumor tissue was collected from OV5308-R10P8 tumor-bearing mice (ovarian cancer xenograft model), cut into tumor blocks with a diameter of 2-3 mm, and subcutaneously inoculated into the right anterior scapula of female NOD / SCID mice. When the average tumor volume of the tumor-bearing mice reached approximately 161.17 mm², [further details needed]. 3 Mice were randomly assigned to groups according to the experimental design. The coefficient of variation (CV) of tumor volume in the enrolled mice was 6.34%. After tumor cell inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior, specifically monitoring activity, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. After drug administration began, mouse weight and tumor size were measured twice weekly. The formula for calculating tumor volume is: Tumor volume (mm²) = (Tumor volume / (mm²)) / (Tumor volume / (mm²)) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). Data was collected using StudyDirector™ software (version 3.1.399.19, vendor: Studylog System, Inc.), including measurements of the tumor's major and minor axes and animal weight. Raw data, measured using a balance and calipers, was directly imported into the software. Any changes in the data were recorded in the software. The results of the average tumor growth curves and weight changes for each group from the start of the experiment to the experimental endpoint are shown in Figures 24A-24B.

[0694] As shown in the figure, the OV5308 model mice generally tolerated the test drug well at the test dose. Mouse body weight remained stable or slightly increased during the experiment, and no mice died. One mouse in the G2 group experienced severe weight loss (BWL > 10%). A total of 12 mice were enrolled in the OV5308 model, divided into two groups of six. Since 34 days was the last measurement period containing data from all mice, tumor inhibition rate was analyzed based on the 34-day data. The average tumor volume in the G1 control group reached 1032.88 mmHg at 34 days. 3 The average tumor volume in the G2 group mice at 34 days was 127.27 mm. 3 The absolute tumor inhibition rate (TGI) was 87.68%, which was significantly higher than that of the control group (p<0.001).

[0695] Test Case 7: Human Ovarian Cancer OV9534 Subcutaneous Xenograft NOD / SCID Female Mouse Model

[0696] from Tumor tissue was collected from OV9534-R4P5 tumor-bearing mice (ovarian cancer xenograft model), cut into tumor blocks with a diameter of 2-3 mm, and subcutaneously inoculated into the right anterior scapula of female NOD / SCID mice. When the average tumor volume of the tumor-bearing mice reached approximately 176.53 mm², [further details needed]. 3 Mice were randomly assigned to groups according to the experimental design. The coefficient of variation (CV) of tumor volume in the grouped mice was 12.79%. The changes in the mean tumor growth curve and body weight of each group from the start of the experiment to the end point are shown in Figures 25A-25B.

[0697] As shown in the figure, the OV9534 model mice generally tolerated the test drug well at the test dose. Mouse body weight remained stable or slightly increased during the experiment; no mice died, and no mice experienced severe weight loss (BWL > 10%). A total of 12 mice were enrolled in the OV9534 model, divided into two groups of six mice each. Since 28 days was the last measurement period containing all mouse data, tumor inhibition rate was analyzed based on the 28-day data. The G1 control group achieved an average tumor volume of 577.78 mmHg at 28 days. 3 The average tumor volume in the G2 group mice at 28 days was 11.10 mm. 3 The absolute tumor inhibition rate (TGI) was 98.08%, which was significantly higher than that of the control group (p<0.01).

[0698] Test Case 8: Human Ovarian Cancer OV9409 Subcutaneous Xenograft NOD / SCID Female Mouse Model

[0699] from Tumor tissue was collected from OV9409-R7P6 tumor-bearing mice (ovarian cancer xenograft model), cut into tumor blocks with a diameter of 2-3 mm, and subcutaneously inoculated into the right anterior scapula of female NOD / SCID mice. When the average tumor volume of the tumor-bearing mice reached approximately 163.13 mm², [further details needed]. 3 Mice were randomly grouped according to the experimental design. The coefficient of variation (CV) of tumor volume in the grouped mice was 6.45%. The changes in the mean tumor growth curve and body weight of each group from the start of the experiment to the end point are shown in Figures 26A-26B.

[0700] As shown in the figure, the OV9409 model mice generally tolerated the test drug well at the test dose. Mice body weight increased slightly during the experiment; however, one mouse in the G1 control group experienced severe weight loss (BWL > 10%) on day 10. This mouse died on day 32. A total of 12 mice were enrolled in the OV9409 model, divided into two groups of six. Since day 31 was the last measurement period containing data from all mice, tumor inhibition rate was analyzed based on data from day 31. The average tumor volume in the G1 control group reached 780.22 mmHg on day 31. 3 The average tumor volume in the G2 group mice was 0.00 mm at 31 days. 3 The tumors in 6 mice completely disappeared. The absolute tumor inhibition rate (TGI) was 100.00%, which was significantly higher than that in the control group (p<0.001).

[0701] Example 12: Exploratory experiment on the toxicology of ADC conjugates in monkeys

[0702] Test Example 1: Dose-exploration trial with repeated intravenous administration for 8 weeks

[0703] The purpose of this experiment was to obtain clinical and local observations of the animals after drug administration, including body weight, food intake, and gross anatomical reports. Specifically, three cynomolgus monkeys were used and randomly divided into three groups, with one animal in each group. Group G1 animals received 10 mg / kg on days 1 (D1) and 15 (D15), and 30 mg / kg on days 43 (D43) and 57 (D57). Group G2 animals received 30 mg / kg on days 1 (D1) and 15 (D15). Group G3 animals received 60 mg / kg on days 1 (D1) and 15 (D15). All animals received the drug via intravenous infusion. Day 1 was defined as the day of the first administration for groups G1-3. See Table 24 for details of the experimental groups.

[0704] Table 24: Experimental Design and Group Arrangement Note: 'a' indicates that animals in group G1 were given 10 mg / kg on day 1 (D1) and day 15 (D15), and 30 mg / kg on day 43 (D43) and day 57 (D57); animals in group G2 were given 30 mg / kg on day 1 (D1) and day 15 (D15); and animals in group G3 were given 60 mg / kg on day 1 (D1) and day 15 (D15).

[0705] All animals underwent systematic dissection and gross observation. Tissues were collected and preserved according to protocol requirements, and routine histological processing was performed on the stomach (fundus, body, and pylorus), large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), lungs, and bronchi, including paraffin embedding, sectioning, slide preparation, and HE staining. Diagnosis was performed using standard terminology, and lesions were graded using a 5-level system (mild, moderate, significant, and severe). Some lesions were not graded. The results are shown in Figure 27.

[0706] As shown in the figure, animals in the 60 mg / kg dose group died on day 10. Gross observation revealed rough skin on the back, emaciation, weakness, and lethargy. Since no histopathological examination was performed on the abnormal tissues according to the protocol, this lesion was not considered related. Additionally, microscopic observation revealed mucosal atrophy and crypt dilatation in the stomach (body / fundus / pylorus), mild atrophy and crypt degeneration in the duodenal mucosa, moderate degeneration / mild regeneration / moderate inflammatory cell infiltration in the jejunal crypts, mild ulceration / mild crypt degeneration / mild crypt dilatation in the cecal mucosa, mild degeneration and dilatation in the colonic crypts, and mild degeneration and mild dilatation in the rectal crypts. Since these lesions only appeared in the high-dose group, they are considered related to the test product. The 10 and 30 mg / kg dose groups were euthanized at the end of treatment (days 24 and 64), and no abnormalities related to the test product were observed in gross or microscopic examination (lungs and gastrointestinal tract).

[0707] Figure 28 shows the body weight and food intake of the three monkeys. The figure indicates that the preclinical safe dose (HLP01060-8D) of the molecule is higher than the highest non-toxic dose (HNSTD): HLP01060-8D >> SGNALPV (competitor) > Elahere (SOC). Elahere is only 4 mg / kg, and the competitor SGNALPV is only 5 mg / kg. It can be inferred that the highest non-toxic dose of HLP01060-8D can reach 30 mg / kg, comparable to the marketed drug DS-8201. In the monkey pre-toxicology exploration experiment, the low-dose groups (10 / 30 mg / kg) showed no abnormalities in clinical and local observations or biochemical indicators, with only local erythema observed at the injection site, which recovered quickly.

[0708] Test Example 2: Toxicokinetic (TK) Experiment of ADC Conjugate Toxins

[0709] The experimental grouping and animal dosing design were the same as in Test Example 1. The sampling time points for each group of animals were as follows: G1: before administration on D1, immediately after administration, 1h, 4h, 8h, 24h, 48h, 72h, 120h, 168h, 336h, and 1h, 168h, 336h after administration on D15; G2: before administration on D1, immediately after administration, 1h, 4h, 8h, 24h, 48h, 72h, 120h, 168h, 336h, and 1h, 168h after administration on D15; G3: before administration on D1, immediately after administration, 1h, 4h, 8h, 24h, 48h, 72h, 120h, 168h.

[0710] The concentration of toxin in monkey plasma samples was determined using LC-MS / MS, with a limit of quantitation (LLOQ) of 30 pg / mL. The concentration data were analyzed using the non-compartmental model (NCA) method with WinNonlin (version 8.3.5.340) to calculate the total kinematic index (TK) parameters and investigate the toxin exposure in monkeys after administration to each dose group. The main TK parameters of the toxin in each group after the first intravenous infusion are shown in Table 25.

[0711] Table 25: TK parameters of toxin in monkey plasma after intravenous infusion of HLP01060-8D in each group of animals Note: NA, due to insufficient elimination phase, t cannot be calculated. 1 / 2 .

[0712] The concentration-time relationship of toxins in animals after the first administration is shown in Figure 29 and Table 26, and the results of toxin exposure are shown in Table 27.

[0713] Table 26: Concentration-time data of toxin in monkey plasma after intravenous infusion of HLP01060-8D in each group of animals.

[0714] Table 27: Results of toxin exposure in animals Note: In the table, L represents low dose (10 mg / kg), M represents medium dose (30 mg / kg), and H represents high dose (60 mg / kg).

[0715] Conclusion: After intravenous infusion of HLP01060-8D at doses of 10, 30, and 60 mg / kg, the toxin was detectable in the plasma of the cynomolgus monkeys, and the exposure level increased with increasing dose.

[0716] Example 13 Plasma stability test of ADC conjugate

[0717] Test Example 1: In vitro stability test of monkey plasma

[0718] Diluent 1: (acetonitrile:water:formic acid = 50:50:0.1, v / v / v): Take 25 mL of acetonitrile, add 25 mL of ultrapure water and 50 μL of formic acid, and mix well.

[0719] Diluent 2: (acetonitrile:formic acid = 100:1, v / v): Take 50 mL of acetonitrile, add 0.5 mL of formic acid, and mix well.

[0720] Stability samples of HLP01060-8D were prepared to a final concentration of 150 μg / mL using cynomolgus monkey plasma and PBS (containing 1% BSA), respectively. Each HLP01060-8D stability sample was aliquoted into 18 portions, each 200 μL. The aliquots were placed in brown EP tubes and sealed with sealing film. The aliquoted samples were incubated at 37°C. Three aliquots were taken at each time point: 0 h (D1), 24 h (D2), 96 h (D5), 168 h (D8), 336 h (D15), and 504 h (D22). 10 μL of 0.5 M citric acid was added to each aliquot, and the mixture was vortexed and stored below -60°C.

[0721] Thaw the sample in an ice box containing crushed ice (if it is a frozen sample), and vortex to mix. Take 50 μL of blank matrix as Blank, Zero, and Carryover samples, and take 50 μL of standard curve sample, quality control sample, SST sample, and sample to be tested. Add 10 μL of internal standard working solution (replace Blank and Carryover samples with 10 μL of dilution 1), vortex to mix, and then add 600 μL of methyl tert-butyl ether, vortex to mix. Centrifuge at approximately 3200g for 10 min at 2-8℃, and add 400 μL of supernatant to the sample storage tube. Concentrate under vacuum at 45℃ until completely dry, add 100 μL of dilution 2, vortex to mix, and place in an autosampler for detection using LC-MS / MS. Use SCIEX Analyst software (Analyst 1.6.3 or later) to output raw spectra, concentration data, etc. The toxin release rate was calculated using Microsoft Office Excel (2007 and later versions), and the results are shown in Table 28 and Figures 30A-30B.

[0722] Among them, M HLP01060-8D The molecular weight of HLP01060-8D is W. HLP01060-8D The mass concentration of HLP01060-8D, M 毒素 This represents the molecular weight of the toxin.

[0723] As shown in the figure, after 21 days, the HLP01060-8D conjugate showed a stable free toxin release rate of 2.49% in monkey plasma, which is better than the 3.9% reported by the marketed drug DS-8201.

[0724] Table 28: Release rate of HLP01060-8D in cynomolgus monkey plasma and PBS (containing 1% BSA)

[0725] The above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof, wherein, The antibody or its antigen-binding fragment contains the following complementarity-determining regions (CDRs): (a) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 1; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 2; (b) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 3; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 4; (c) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 5; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 6; (d) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 7; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 8; (e) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 9; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) of the sequence shown in SEQ ID NO: 10; or, (f) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) below, and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) below, wherein, compared with the heavy chain variable region (VH) and / or the light chain variable region (VL) respectively, at least one CDR contains a mutation, and the CDR containing the mutation has at least 70%, 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 sequence from which it originates, or the mutation is a substitution, deletion, or addition of one or more amino acids; preferably, the substitution is a conservative substitution; preferably, the heavy chain variable region (VH) is selected from SEQ ID NO. NOs: any of the sequences shown in SEQ ID NOs: 114, 116, 118, 120 or 122; preferably, the light chain variable region (VL) is selected from any of the sequences shown in SEQ ID NOs: 115, 117, 119, 121 or 123; Preferably, the CDR is defined by the Kabat, IMGT, Chothia, Contact, or AbM numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), where the CDR is defined by the Kabat numbering system: (1a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 12 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 13 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 15 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 16 or a variant thereof; (1b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 32 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 33 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 34 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 35 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 36 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 37 or a variant thereof; (1c) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 53 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 54 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 55 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 56 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 57 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 58 or a variant thereof; (1d) Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO: 74 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 75 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 76 or a variant thereof, and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO: 77 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 78 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 79 or a variant thereof; or, (1e) Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO: 94 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 95 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 96 or a variant thereof, and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO: 97 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 98 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 99 or a variant thereof; or, (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined by the IMGT numbering system: (2a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 17 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 18 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 19 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 20 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 21 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 16 or a variant thereof; (2b) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 38 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 39 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 124 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 41 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 42 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 37 or a variant thereof; (2c) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 59 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 60 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 61 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 62 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 63 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 58 or a variant thereof; (2d) Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO: 80 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 81 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 82 or a variant thereof, and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO: 83 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 84 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 79 or a variant thereof; or, (2e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 100 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 101 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 102 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 103 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 104 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 99 or a variant thereof; or, (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), where the CDR is defined by the Chothia numbering system: (3a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 22 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 23 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 13 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 15 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 16 or a variant thereof; (3b) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 43 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 44 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 34 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 35 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 36 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 37 or a variant thereof; (3c) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 64 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 65 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 55 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 56 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 57 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 58 or a variant thereof; (3d) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 43 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 85 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 76 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 77 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 78 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 79 or a variant thereof; or, (3e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 64 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 105 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 96 or a variant thereof, and / or Light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 97 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 98 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 99 or a variant thereof; or, (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined by the Contact numbering system: (4a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 24 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 25 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 26 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 27 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 28 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 29 or a variant thereof; (4b) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 45 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 46 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 47 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 48 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 49 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 50 or a variant thereof; (4c) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 66 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 67 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 68 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 69 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 70 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 71 or a variant thereof; (4d) Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO: 86 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 87 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 88 or a variant thereof, and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO: 89 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 90 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 91 or a variant thereof; or, (4e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 106 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 107 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 108 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 109 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 110 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 111 or a variant thereof; or, (5) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined by the AbM numbering system: (5a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 30 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO: 31 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO: 13 or a variant thereof, and / or a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO: 15 or a variant thereof, CDR-L3 with the sequence of SEQ ID NO: 16 or a variant thereof; (5b) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 51 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 52 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 34 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 35 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 36 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 37 or a variant thereof; (5c) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 72 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 73 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 55 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 56 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 57 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 58 or a variant thereof; (5d) Heavy chain variable region (VH) containing the following 3 CDRs: CDR-H1 with sequence SEQ ID NO: 92 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 93 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 76 or a variant thereof, and / or, light chain variable region (VL) containing the following 3 CDRs: CDR-L1 with sequence SEQ ID NO: 77 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 78 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 79 or a variant thereof; or, (5e) Heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO: 112 or a variant thereof, CDR-H2 with sequence SEQ ID NO: 113 or a variant thereof, CDR-H3 with sequence SEQ ID NO: 96 or a variant thereof, and / or, light chain variable region (VL) containing the following three CDRs: CDR-L1 with sequence SEQ ID NO: 97 or a variant thereof, CDR-L2 with sequence SEQ ID NO: 98 or a variant thereof, CDR-L3 with sequence SEQ ID NO: 99 or a variant thereof; The variants described in the heavy chain variable region (VH) and / or the light chain variable region (VL) and any of (1a)-(1e), (2a)-(2e), (3a)-(3e), (4a)-(4e) or (5a)-(5e) have one or more amino acid substitutions, deletions or additions compared to the sequence from which they are derived; preferably, the substitutions are conservative substitutions; Preferably, the CDR-H2 variant of the sequence SEQ ID NO: 46 is the CDR-H2 variant of the sequence SEQ ID NO: 125; Preferably, the CDR-H2 variant of the sequence SEQ ID NO: 54 is the CDR-H2 variant of the sequence SEQ ID NO: 126; Preferably, the CDR-H2 variant of the sequence SEQ ID NO: 60 is the CDR-H2 variant of the sequence SEQ ID NO: 127; Preferably, the CDR-H2 variant of the sequence SEQ ID NO: 65 is the CDR-H2 variant of the sequence SEQ ID NO: 128; Preferably, the CDR-H2 variant of the sequence SEQ ID NO: 67 is the CDR-H2 variant of the sequence SEQ ID NO: 129; Preferably, the CDR-L2 of the variant with sequence SEQ ID NO: 70 is the CDR-L2 with sequence SEQ ID NO: 130; Preferably, the CDR-H2 variant of the sequence SEQ ID NO: 73 is the CDR-H2 variant of the sequence SEQ ID NO: 131; Preferably, the CDR-H3 variant of the sequence SEQ ID NO: 76 is the CDR-H3 variant of the sequence SEQ ID NO: 132; Preferably, the CDR-H3 variant of the sequence SEQ ID NO: 82 is the CDR-H3 variant of the sequence SEQ ID NO: 133; Preferably, the CDR-H2 variant of the sequence SEQ ID NO: 87 is the CDR-H2 variant of the sequence SEQ ID NO: 134; Preferably, the CDR-H3 variant of the sequence SEQ ID NO: 88 is the CDR-L2 variant of the sequence SEQ ID NO: 135; Preferably, the CDR-L2 of the variant of the sequence SEQ ID NO: 110 is the CDR-L2 of the sequence SEQ ID NO:

136.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a framework region (FR) derived from mammalian immunoglobulins; Preferably, the VH and / or VL of the antibody or its antigen-binding fragment contain frame regions (FRs) of immunoglobulins derived from humans or mice; Preferably, the VH of the antibody or its antigen-binding fragment includes a heavy chain variable region (VH) framework region (FR) derived from mouse immunoglobulin, and / or the VL of the antibody or its antigen-binding fragment includes a light chain variable region (VL) framework region (FR) derived from mouse immunoglobulin. Preferably, the VH of the antibody or its antigen-binding fragment includes a heavy chain variable region (VH) framework region (FR) derived from human immunoglobulin, and / or the VL of the antibody or its antigen-binding fragment includes a light chain variable region (VL) framework region (FR) derived from human immunoglobulin.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein, The antibody or its antigen-binding fragment: (a) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1 or 114 or a variant thereof, and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 2 or 115 or a variant thereof; (b) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 3 or 116 or a variant thereof, and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 4 or 117 or a variant thereof; (c) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 5 or 118 or a variant thereof, and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 6 or 119 or a variant thereof; (d) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 7 or 120 or a variant thereof, and / or, containing a light chain variable region (VL) of the sequence shown in SEQ ID NO: 8 or 121 or a variant thereof; or, (e) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 9 or 122 or a variant thereof, and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 10 or 123 or a variant thereof; The variant has at least 70%, 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 sequence from which it originates; or has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein, The antibody or its antigen-binding fragment comprises: (a) VH having the sequence shown in SEQ ID NO: 1 and VL having the sequence shown in SEQ ID NO: 2; (b) VH having the sequence shown in SEQ ID NO: 114 and VL having the sequence shown in SEQ ID NO: 115; (c) VH having the sequence shown in SEQ ID NO: 3 and VL having the sequence shown in SEQ ID NO: 4; (d) VH having the sequence shown in SEQ ID NO: 116 and VL having the sequence shown in SEQ ID NO: 117; (e) VH having the sequence shown in SEQ ID NO: 5 and VL having the sequence shown in SEQ ID NO: 6; (f) VH having the sequence shown in SEQ ID NO: 118 and VL having the sequence shown in SEQ ID NO: 119; (g) VH having the sequence shown in SEQ ID NO: 7 and VL having the sequence shown in SEQ ID NO: 8; (h) VH having the sequence shown in SEQ ID NO: 120 and VL having the sequence shown in SEQ ID NO: 121; (i) VH having the sequence shown in SEQ ID NO: 9 and VL having the sequence shown in SEQ ID NO: 10; or, (j) VH having the sequence shown in SEQ ID NO: 122 and VL having the sequence shown in SEQ ID NO:

123.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, further comprising a constant region derived from human immunoglobulin; Preferably, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human immunoglobulin; Preferably, the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from human immunoglobulin; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region selected from the following: (1) Human IgG1 heavy chain constant region; or, (2) A variant of the human IgG1 heavy chain constant region, the variant having altered effector function compared to its derived wild-type sequence, preferably having at least one of the following substitutions compared to its derived wild-type sequence: deletion of L234A, L235A, G237A, S239D, I332E, D356E, L358M, and K447; wherein, The position of the replaced amino acid is based on its position in the EU numbering system; Preferably, the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 137 or a variant thereof, the variant having a conserved substitution of up to 20 amino acids compared to SEQ ID NO: 137; Preferably, the light chain constant region is the κ light chain constant region; Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) of the sequence shown in SEQ ID NO: 138 or a variant thereof, the variant having a conserved substitution of up to 20 amino acids compared to SEQ ID NO: 138; More preferably, the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 137, 159, 172 or 218 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein, The antibody or its antigen-binding fragment comprises: (a) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 172, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; (b) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 114 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 137, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 115 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (c) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 114 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 159, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 115 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (d) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 172, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (e) A heavy chain having a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 116 and a heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 137, and / or a light chain having a light chain variable region (VL) of the sequence shown in SEQ ID NO: 117 and a light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (f) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 116 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 159, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 117 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (g) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 5 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 137, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 6 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (h) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 118 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 137, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 119 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (i) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 118 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 159, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 119 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (j) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 7 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 172, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 8 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (k) A heavy chain having a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 120 and a heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 137, and / or a light chain having a light chain variable region (VL) of the sequence shown in SEQ ID NO: 121 and a light chain constant region (CL) of the sequence shown in SEQ ID NO:

138. (l) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 120 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 159, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 121 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; (m) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 9 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 172, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 10 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; (n) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 122 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 137, and / or, a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 123 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; or, (o) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 122 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 159, and / or, a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 123 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; or, (p) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 114 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 218, and / or, a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 115 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; or, (q) A heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 116 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 218, and / or, a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 117 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; or, (r) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 118 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 218, and / or, a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 119 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; or, (s) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 120 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 218, and / or, a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 121 and the light chain constant region (CL) of the sequence shown in SEQ ID NO: 138; or, (t) a heavy chain having the heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 122 and the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 218, and / or a light chain having the light chain variable region (VL) of the sequence shown in SEQ ID NO: 123 and the light chain constant region (CL) of the sequence shown in SEQ ID NO:

138.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein, The antibody or its antigen-binding fragment comprises: (a) A heavy chain comprising the sequence shown in SEQ ID NO: 139 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 140 or a variant thereof; (b) A heavy chain comprising the sequence shown in SEQ ID NO: 149, 160 or 219 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 150 or a variant thereof; (c) A heavy chain comprising the sequence shown in SEQ ID NO: 141 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 142 or a variant thereof; (d) A heavy chain comprising the sequence shown in SEQ ID NO: 151, 161 or 220 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 152 or a variant thereof; (e) a heavy chain comprising the sequence shown in SEQ ID NO: 143 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 144 or a variant thereof; (f) A heavy chain comprising the sequence shown in SEQ ID NO: 153, 162 or 221 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 154 or a variant thereof; (h) a heavy chain comprising the sequence shown in SEQ ID NO: 145 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 146 or a variant thereof; (i) a heavy chain comprising the sequence shown in SEQ ID NO: 155, 163 or 222 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 156 or a variant thereof; (j) a heavy chain comprising the sequence shown in SEQ ID NO: 147 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 148 or a variant thereof; or, (k) a heavy chain comprising the sequence shown in SEQ ID NO: 157, 164 or 223 or a variant thereof, and / or a light chain comprising the sequence shown in SEQ ID NO: 158 or a variant thereof; The variant has at least 70%, 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 sequence from which it originates; or has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein, The antigen-binding fragment is selected from ScFv, Fab, Fab', (Fab')2, Fv fragment, disulfide-linked Fv (dsFv), single-domain antibody (sdAb), or nanobody (VHH); the antibody is selected from diabody, bispecific antibody, or multispecific antibody; and / or, the antibody is a murine antibody, chimeric antibody, or humanized antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein, The antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to ALPP and / or ALPG; preferably, the antibody or its antigen-binding fragment is labeled; preferably, the antibody or its antigen-binding fragment is labeled with a detectable marker, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, or biotin.

11. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, or a variable region of its heavy chain and / or a variable region of its light chain, or a heavy chain and / or a light chain, as described in any one of claims 1-10, wherein the isolated nucleic acid molecule is DNA or RNA.

12. The isolated nucleic acid molecule according to claim 11, wherein, The DNA contains a first nucleotide sequence encoding the antibody heavy chain variable region and / or a second nucleotide sequence encoding the antibody light chain variable region; Preferably, the first nucleotide sequence encoding the antibody heavy chain variable region has a sequence selected from the following: (a) SEQ ID NOs: any of the nucleotide sequences shown in SEQ ID NOs: 173, 175, 177, 179, 181, 193, 195, 197, 199 or 201; (b) A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions); or, (c) A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; Preferably, the second nucleotide sequence encoding the variable region of the antibody light chain has a sequence selected from the following: (a) SEQ ID NOs: any of the nucleotide sequences shown in SEQ ID NOs: 174, 176, 178, 180, 182, 194, 196, 198, 200 or 202; (b) A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions); or, (c) A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides.

13. The isolated nucleic acid molecule according to claim 11 or 12, wherein, The DNA contains a first nucleotide sequence encoding the antibody heavy chain and / or a second nucleotide sequence encoding the antibody light chain; Preferably, the first nucleotide sequence encoding the antibody heavy chain has a sequence selected from the following: (a) SEQ ID NOs: any of the nucleotide sequences shown in SEQ ID NOs 183, 185, 187, 189, 191, 203, 205, 207, 209, 211, 213-217 or 224-228; (b) A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleotide sequence described in (a), or a sequence having one or more nucleotide substitutions); or, (c) A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; Preferably, the second nucleotide sequence encoding the antibody light chain has a sequence selected from the following: (a) SEQ ID NOs: any of the nucleotide sequences shown in SEQ ID NOs 184, 186, 188, 190, 192, 204, 206, 208, 210 or 212; (b) A sequence substantially identical to the nucleotide sequence described in (a); or, (c) A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides.

14. A vector comprising the isolated nucleic acid molecule of any one of claims 11-13; preferably, the vector is a cloning vector or an expression vector.

15. A host cell comprising the isolated nucleic acid molecule of any one of claims 11-13 or the vector of claim 14.

16. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1-10, comprising culturing a host cell according to claim 15 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

17. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-10 conjugated to one or more drugs; preferably, the drug is a cytotoxic drug.

18. The antibody-drug conjugate according to claim 17, having the structure shown in general formula (I): Ab-(LD)p(I); in, Ab is the antibody or antigen-binding fragment thereof as described in any one of claims 1-10; L stands for connector; D is a cytotoxic drug; p is selected from an integer or decimal between 1 and 10; preferably, p is selected from an integer or decimal between 1 and 8.

19. The antibody-drug conjugate according to claim 18, wherein, The connector L has the structure shown in formula (II): L 1 -L 2 -L 3 -L 4 (II) Wherein, the L 1 The terminal is connected to Ab, L 4 The terminal is connected to D; L 1 It is succinimide or maleimide; preferably, the L 1 It is maleimide; L 2 For (PEG) 1-10 C 1-10 alkylene acyl or (PEG) 1-10 C 1-10 Alkyl group; preferably, the L 2 It is (PEG)2, hexanoyl, or (PEG)2 acetylated; L 3 It is a peptide residue or chemical bond composed of 1 to 7 amino acid residues; preferably, the amino acid is selected from alanine, glycine, valine, phenylalanine, lysine, citrulline, serine, cysteine, glutamic acid, aspartic acid, histidine, cysteine, isoleucine, leucine, methionine, asparagine, proline, glutamine, arginine, threonine, tryptophan, tyrosine, pentanediol, leucine, pyrrolidone, homoserine, homocysteine, or demethylpyrrolidone; preferably, the L 3 It is a peptide residue composed of 2 to 4 amino acid residues; preferably, the amino acid is selected from alanine, glycine, valine, phenylalanine, or citrulline; preferably, the L 3 It is a dipeptide residue of valine-citrulline or a tetrapeptide residue of glycine-glycine-phenylalanine-glycine. L 4 p-Aminobenzyloxycarbonyl, p-aminobenzyloxy, NR 1 (CR 2 R 3 ) t Or chemical bond; R 1 R 2 and R 3 They may be the same or different, and each is independently selected from hydrogen atoms or C atoms. 1-10 Alkyl group, where t is an integer from 1 to 10; preferably, L 4 It is p-aminobenzyloxycarbonyl or NHCH2; Preferably, L is selected from 6-maleiminohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl, 6-maleiminohexanoyl-glycine-glycine-phenylalanine-glycine-aminomethyl, 6-maleimino-diethylene glycol acetyl-valine-citrulline-p-aminobenzyloxycarbonyl, 6-maleiminohexanoyl-valine-alanine-p-aminobenzyloxycarbonyl, or 6-maleimino-diethylene glycol formyl-valine-citrulline-p-aminobenzyloxycarbonyl.

20. The antibody-drug conjugate according to claim 18 or 19, wherein, The cytotoxic drug D is selected from microtubule inhibitors, DNA topoisomerase inhibitors, or DNA damaging agents. Preferably, the microtubule inhibitor includes olistatin compounds and their derivatives, maytansine compounds and their derivatives, or eribulin compounds and their derivatives; preferably, the olistatin compounds and their derivatives are MMAE or MMAF; the maytansine compounds and their derivatives are DM1 or DM4; Preferably, the DNA topoisomerase inhibitor includes a topoisomerase I inhibitor and its derivatives or a topoisomerase II inhibitor and its derivatives; the topoisomerase I inhibitor and its derivatives are camptothecin, 7-ethyl-10-hydroxycamptothecin, eczema, or Dxd; the topoisomerase II inhibitor and its derivatives are doxorubicin, doxorubicin, or PNU-159682. Preferably, the DNA damaging agent includes PBD-type compounds and their derivatives, kazimidic acid-type compounds and their derivatives, or pyruvic acid-type compounds and their derivatives; More preferably, the cytotoxic drug D is selected from MMAE, MMAF, DM1, DM4, eribulin, SN38, eczema, or Dxd.

21. A method for preparing an antibody-drug conjugate according to any one of claims 17-20, comprising the following steps: (1) The antibody according to any one of claims 1-10 is reacted with a reducing reagent in a buffer solution to obtain the reduced antibody; (2) Crosslink the LD of any one of claims 18-20 with the reduced antibody obtained in step (1) in a mixture of buffer and organic solvent to obtain an antibody-drug conjugate.

22. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-10, an isolated nucleic acid molecule as claimed in any one of claims 11-13, a carrier as claimed in claim 14, or a host cell as claimed in claim 15, or an antibody-drug conjugate as claimed in any one of claims 17-20, 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, wherein the drug with antitumor activity is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, antiangiogenic agents, cytokines, specific tumor cell-targeting antibodies, immune checkpoint inhibitors, or immunomodulators. Preferably, the antibody or its antigen-binding fragment is provided as a separate component or as a component of the same composition along with the additional pharmaceutically active agent.

23. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-10, the isolated nucleic acid molecule of any one of claims 11-13, the vector of claim 14, the host cell of claim 15, the antibody-drug conjugate of any one of claims 17-20, or the pharmaceutical composition of claim 22 in the preparation of a medicament for the prevention and / or treatment of tumors in a subject; Preferably, the tumor expresses ALPP and / or ALPG; Preferably, the tumor is selected from tumors of the genitourinary system or tumors of the digestive tract; Preferably, the urogenital tumor is selected from ovarian cancer or endometrial cancer; Preferably, the digestive tract tumor is selected from gastric cancer or pancreatic cancer; Preferably, the tumor is selected from ovarian cancer, endometrial cancer, gastric cancer, or pancreatic cancer; Preferably, the subject is a mammal, and more preferably, the subject is a human, monkey, or mouse.

24. A method for preventing and / or treating tumors in a subject, comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-10, an antibody-drug conjugate as described in any one of claims 17-20, or a pharmaceutical composition as described in claim 22; Preferably, the tumor expresses ALPP and / or ALPG; Preferably, the tumor is selected from tumors of the genitourinary system or tumors of the digestive tract; Preferably, the urogenital tumor is selected from ovarian cancer or endometrial cancer; Preferably, the digestive tract tumor is selected from gastric cancer or pancreatic cancer; Preferably, the tumor is selected from ovarian cancer, endometrial cancer, gastric cancer, or pancreatic cancer; Preferably, the subject is a mammal, and more preferably, the subject is a human, monkey, or mouse.

25. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-10, or the antibody-drug conjugate as described in any one of claims 17-20; Preferably, the antibody or its antigen-binding fragment, or antibody-drug conjugate, carries a detectable label, which is an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, or biotin. 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 1-10, or the antibody-drug conjugate as described in any one of claims 17-20; Preferably, the second antibody further includes a detectable marker, which is an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, or biotin.

26. A method for detecting the presence or amount of ALPP and / or ALPG in a sample, comprising the following steps: (1) Contact the sample with the antibody or antigen-binding fragment thereof as described in any one of claims 1-10, or the antibody-drug conjugate as described in any one of claims 17-20; (2) Detect the formation of a complex between the antibody or its antigen-binding fragment and ALPP and / or ALPG; Preferably, the antibody or its antigen-binding fragment carries a detectable label; Preferably, the ALPP and / or ALPG are human ALPP and / or ALPG; Preferably, the tumor is selected from tumors of the genitourinary system or tumors of the digestive tract; Preferably, the urogenital tumor is selected from ovarian cancer or endometrial cancer; Preferably, the digestive tract tumor is selected from gastric cancer or pancreatic cancer; Preferably, the tumor is selected from ovarian cancer, endometrial cancer, gastric cancer, or pancreatic cancer.

27. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-10, or the antibody-drug conjugate of any one of claims 17-20, in the preparation of a kit for detecting the presence or amount of ALPP and / or ALPG in a sample, and / or diagnosing tumors associated with ALPP and / or ALPG; Preferably, the tumor is selected from tumors of the genitourinary system or tumors of the digestive tract; Preferably, the urogenital tumor is selected from ovarian cancer or endometrial cancer; Preferably, the digestive tract tumor is selected from gastric cancer or pancreatic cancer; Preferably, the tumor is selected from ovarian cancer, endometrial cancer, gastric cancer, or pancreatic cancer.

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