Anti-LIV-1 antibodies and drug conjugates thereof

Abstract

The invention relates to an anti-LIV-1 antibody and a drug conjugate (ADC) thereof. The anti-LIV-1-antibody or the antigen binding fragment thereof comprises three light chain complementarity determining regions and / or three heavy chain complementarity determining regions of a specific amino acid sequence. Specifically, the invention also provides nucleic acid for coding the antibody or the antigen binding fragment thereof, a cell containing the nucleic acid, a pharmaceutical composition containing the antibody or the antigen binding fragment thereof, the nucleic acid and the cell, and application of the LIV-1 antibody or the antigen binding fragment thereof in prevention, treatment, detection or diagnosis of LIV-1 related diseases. The anti-LIV-1 antibody or the antigen binding fragment thereof provided by the invention has good affinity to LIV-1 and cells expressing LIV-1. The anti-LIV-1 antibody drug conjugate has a good tumor inhibition effect on an animal tumor transplantation model, and is small in side effect and toxicity and high in safety.

Description

Anti-LIV-1 antibody and drug conjugate Technical Field

[0001] The present invention relates to the technical field of biomedicine or biopharmaceuticals, and in particular to an anti-LIV-1 antibody and a drug conjugate thereof. Background Art

[0002] LIV-1 (also known as SLC39A6 or ZIP6) is a multi-spanning membrane protein composed of eight transmembrane domains with specific zinc transporter and metalloprotease activities. It is mainly located on the plasma membrane and transports zinc into the cell.

[0003] LIV-1 belongs to the SLC39A family of zinc transporters. Intracellular zinc levels are tightly regulated by specific zinc transporters. Two complementary families of zinc transporters (SLC39A and SLC30A) are known to be directly involved in intracellular zinc homeostasis in mammals. The SLC39A family has been shown to facilitate the transport of zinc ions from the extracellular or intracellular organelles into the cytoplasm. The SLC30A family (ZnT family) has the opposite function of the SLC39A family, with multiple family members facilitating the efflux of zinc ions from the cytoplasm to the extracellular space or the influx of zinc ions into organelles.

[0004] LIV-1 is highly expressed in a variety of tumor cells, including breast cancer, prostate cancer, pancreatic cancer, cervical cancer, and liver cancer, but is limitedly expressed in normal tissues. Breast cancer is the most common malignant tumor in women. Compared with normal breast tissue, zinc ions are more highly enriched in breast tumor cells. LIV-1 is the first zinc ion transporter discovered in breast cancer cells whose expression can be induced by estrogen, so it is considered to be closely related to the occurrence and development of breast cancer. In addition to breast cancer, recent research results have shown that LIV-1 may also have a certain connection with the occurrence and development of other cancers (such as prostate cancer and pancreatic cancer). Pancreatic cancer is a highly malignant digestive system tumor that is extremely prone to metastasis. The expression of LIV-1 in pancreatic cancer tissue is significantly higher than that in normal pancreatic tissue.

[0005] Antibody-drug conjugates (ADCs) combine antibodies with small molecule chemotherapy drugs via a linker. They combine the highly targeted properties of antibodies with the cytotoxicity of chemotherapy drugs, effectively killing tumor cells. ADC technology has matured, with several ADCs successfully marketed and several more in late-stage clinical development. Given the variability in LIV-1 expression, LIV-1 may be an ideal target for developing ADCs.

[0006] In view of the above, LIV-1 antibody-drug conjugates are of great significance as anti-cancer drugs, especially in cancers with high LIV-1 expression. How to provide a drug for cancers with high LIV-1 expression has become an urgent problem to be solved.

[0007] Summary of the Invention

[0008] The present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof that can bind to LIV-1. The present invention also provides a nucleic acid encoding the antibody or the antigen-binding fragment thereof; a cell containing the nucleic acid; an anti-LIV-1 antibody conjugate or an anti-LIV-1 antibody-drug conjugate containing the anti-LIV-1 antibody or the antigen-binding fragment thereof; a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof, the nucleic acid, the cell, the anti-LIV-1 antibody conjugate, or the anti-LIV-1 antibody-drug conjugate; a kit containing the antibody or the antigen-binding fragment thereof, the nucleic acid, the anti-LIV-1 antibody conjugate, the anti-LIV-1 antibody-drug conjugate, and the pharmaceutical composition; use of the antibody or the antigen-binding fragment thereof, the nucleic acid, the anti-LIV-1 antibody conjugate, the anti-LIV-1 antibody-drug conjugate, or the pharmaceutical composition in preventing, treating, detecting, or diagnosing diseases associated with LIV-1, and use of the anti-LIV-1 antibody or the antigen-binding fragment thereof in preparing an antibody-drug conjugate (ADC).

[0009] One aspect of the present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises the following three light chain complementary determining regions and / or three heavy chain complementary determining regions:

[0010] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:23, and LCDR3 of SEQ ID NO:41, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 of SEQ ID NO:25, HCDR2 of SEQ ID NO:40, and HCDR3 of SEQ ID NO:27; preferably, the antibody is numbered CA365NQ;

[0011] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:23, and LCDR3 of SEQ ID NO:24, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 of SEQ ID NO:25, HCDR2 of SEQ ID NO:40, and HCDR3 of SEQ ID NO:27; preferably, the antibody is numbered CA365;

[0012] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:22, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:24, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:25, HCDR2 set forth in SEQ ID NO:26, and HCDR3 set forth in SEQ ID NO:27; preferably, the antibody is numbered CA317;

[0013] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:28, LCDR2 set forth in SEQ ID NO:29, and LCDR3 set forth in SEQ ID NO:30, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:31, HCDR2 set forth in SEQ ID NO:32, and HCDR3 set forth in SEQ ID NO:33; preferably, the antibody is numbered CA332;

[0014] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:34, LCDR2 set forth in SEQ ID NO:35, and LCDR3 set forth in SEQ ID NO:36, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:37, HCDR2 set forth in SEQ ID NO:38, and HCDR3 set forth in SEQ ID NO:39; preferably, the antibody is numbered CA336;

[0015] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:42, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:43, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:25, HCDR2 set forth in SEQ ID NO:40, and HCDR3 set forth in SEQ ID NO:27; preferably, the antibody is numbered CA406;

[0016] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 of SEQ ID NO:44, LCDR2 of SEQ ID NO:23, and LCDR3 of SEQ ID NO:45, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 of SEQ ID NO:46, HCDR2 of SEQ ID NO:40, and HCDR3 of SEQ ID NO:27; preferably, the antibody is numbered CA454;

[0017] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:44, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:45, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:47, HCDR2 set forth in SEQ ID NO:40, and HCDR3 set forth in SEQ ID NO:27; preferably, the antibody is numbered CA478;

[0018] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 of SEQ ID NO:44, LCDR2 of SEQ ID NO:23, and LCDR3 of SEQ ID NO:45, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 of SEQ ID NO:48, HCDR2 of SEQ ID NO:40, and HCDR3 of SEQ ID NO:27; preferably, the antibody is numbered CA672;

[0019] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:45, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:48, HCDR2 shown in SEQ ID NO:40, and HCDR3 shown in SEQ ID NO:27; preferably, the antibody is numbered CA789; or

[0020] The three light chain complementary determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:49 and LCDR3 shown in SEQ ID NO:50, and / or the three heavy chain complementary determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:38 and HCDR3 shown in SEQ ID NO:52; preferably, the antibody is numbered CA1328.

[0021] In one embodiment of the present invention, the present invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof, characterized in that:

[0022] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 9, and / or a heavy chain variable region of SEQ ID NO: 8; more preferably, the antibody is numbered CA365NQ;

[0023] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 7, and / or a heavy chain variable region of SEQ ID NO: 8; more preferably, the antibody is numbered CA365;

[0024] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 1, and / or a heavy chain variable region of SEQ ID NO: 2; more preferably, the antibody is numbered CA317;

[0025] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 3, and / or a heavy chain variable region of SEQ ID NO: 4; more preferably, the antibody is numbered CA332;

[0026] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 6; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 5, and / or a heavy chain variable region of SEQ ID NO: 6; more preferably, the antibody is numbered CA336;

[0027] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 10, and / or a heavy chain variable region of SEQ ID NO: 11; more preferably, the antibody is numbered CA406;

[0028] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 12, and / or a heavy chain variable region of SEQ ID NO: 13; more preferably, the antibody is numbered CA454;

[0029] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 14, and / or a heavy chain variable region of SEQ ID NO: 15; more preferably, the antibody is numbered CA478;

[0030] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 16, and / or a heavy chain variable region of SEQ ID NO: 17; more preferably, the antibody is numbered CA672;

[0031] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO: 18, and / or a heavy chain variable region of SEQ ID NO: 19; more preferably, the antibody is numbered CA789; or

[0032] The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region as shown in SEQ ID NO: 20, and / or a heavy chain variable region as shown in SEQ ID NO: 21; more preferably, the antibody is numbered CA1328.

[0033] In a specific embodiment of the present invention, the sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 53.

[0034] Furthermore, the sequence of the light chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 54.

[0035] In the embodiments of the present invention, the antibodies or antigen-binding fragments thereof include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, scFv or dsFv fragments, etc.

[0036] A second aspect of the present invention provides a nucleic acid, including a recombinant nucleic acid or an isolated nucleic acid, encoding the antibody or antigen-binding fragment thereof.

[0037] A third aspect of the present invention provides a vector comprising the nucleic acid encoding the antibody or antigen-binding fragment thereof. The vector can be used to express the antibody or antigen-binding fragment thereof. Preferably, the vector can be a viral vector; preferably, the viral vector includes but is not limited to a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector; preferably, the vector can be a non-viral vector; preferably, the vector can be a mammalian cell expression vector; preferably, the expression vector can be a bacterial expression vector; preferably, the expression vector can be a fungal expression vector.

[0038] A fourth aspect of the present invention provides a cell comprising the nucleic acid or the vector, wherein the cell can express the antibody or antigen-binding fragment thereof. Preferably, the cell is a bacterial cell; preferably, the bacterial cell is an Escherichia coli cell; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast cell is a Pichia pastoris cell; preferably, the cell is a mammalian cell; preferably, the mammalian cell is a Chinese hamster ovary cell (CHO), a human embryonic kidney cell (293), a B cell, a T cell, a DC cell, or a NK cell.

[0039] The fifth aspect of the present invention provides an anti-LIV-1 antibody conjugate, characterized in that the anti-LIV-1 antibody conjugate comprises (a) the LIV-1 antibody or its antigen-binding fragment, and (b) a conjugated portion conjugated to the antibody portion, wherein the conjugated portion is selected from the following group: a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0040] In another preferred embodiment, the drug is selected from the following group: chemotherapy drugs, radiotherapy drugs, hormone therapy drugs or immunotherapy drugs.

[0041] In another preferred embodiment, the drug or toxin is selected from the group consisting of taxanes, maytansinoids, auristatins, calicheamicin, anthracyclines, docetaxel, cathepsin, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, RNase, and radioisotope.

[0042] The sixth aspect of the present invention provides an anti-LIV-1 antibody drug conjugate (antibody drug conjugate is also called antibody-drug conjugate, or antibody conjugate, or antibody-conjugate, or ADC), wherein the antibody drug conjugate ADC is shown in Formula 1 below:

[0043] Wherein: Ab is an anti-LIV-1 antibody or an antigen-binding fragment thereof of the present invention,

[0044] LU is a linker (also called a connector);

[0045] D is for medicine;

[0046] Furthermore, the subscript p corresponds to the average DAR value of the antibody-drug conjugate, and p is a value selected from 1-10, preferably 1-8, more preferably 1-4 or 4-8, and most preferably p is 4.

[0047] Furthermore, the linker LU consists of the general formula R'-L1-L2-L3;

[0048] In this general formula, L3 is:

[0049] Wherein the a end of L3 is connected to the drug D, and the b end is connected to the L2;

[0050] R1 is hydrogen, carboxyl, ester, nitro, sulfonyl, or halogen; or R1 is R2-R6 are each independently hydrogen, n is 0-8;

[0051] In this general formula, L2 is: wherein A is independently a phenylalanine residue, a glycine residue, an alanine residue, a glutamic acid residue, an aspartic acid residue, a cysteine ​​residue, a histidine residue, a lysine residue, a proline residue, or a valine residue, a guanine residue, a β-glycine residue, or a β-alanine residue; and X is: n is 0-8;

[0052] In this general formula, L1 is: and / or

[0053] In this general formula, R' is: Among them, the c-terminus of R' is connected to L1, and the d-terminus is connected to Ab;

[0054] In another preferred embodiment, the LU-D is vcMMAE, vc is a valine-citrulline linker, and MMAE is monomethyl auristatin E; vcMMAE can also be written as MC-Val-Cit-PAB-MMAE or mc-vc-PAB-MMAE.

[0055] In another preferred embodiment, LU-D in the antibody-drug conjugate (ADC) formula 1 is BNLD11, wherein LU is MC-β-Ala-(glucuronide)PAB, D is MMAE, and the structure of BNLD11 is as follows:

[0056] , the Exact Mass of the BNLD11 is 1322.690; the BNLD11 is synthesized using conventional methods in the prior art; in a preferred example, BNLD11 is obtained using the synthesis route shown in FIG23 .

[0057] Furthermore, the present invention provides an anti-LIV-1 antibody conjugate, wherein:

[0058] The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:41, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40, and HCDR3 shown in SEQ ID NO:27, and the LU-D is BNLD11;

[0059] The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:41, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40, and HCDR3 shown in SEQ ID NO:27, and the LU-D is vcMMAE;

[0060] The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40, and HCDR3 shown in SEQ ID NO:27, and the LU-D is vcMMAE;

[0061] The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:45, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO:48, HCDR2 shown in SEQ ID NO:40, and HCDR3 shown in SEQ ID NO:27, and the LU-D is vcMMAE;

[0062] The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:45, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO:48, HCDR2 shown in SEQ ID NO:40, and HCDR3 shown in SEQ ID NO:27, and the LU-D is vcMMAE; or,

[0063] The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:50, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:38, and HCDR3 shown in SEQ ID NO:52, and the LU-D is vcMMAE;

[0064] Furthermore, the present invention provides an anti-LIV-1 antibody conjugate, which is CA365NQ-BNLD11, CA365NQ-vcMMAE, CA365-vcMMAE, CA789-vcMMAE, CA672-vcMMAE, or CA1328-vcMMAE, wherein CA365NQ, CA365, CA789, CA672, and CA1328 are the above-mentioned anti-LIV-1 antibodies.

[0065] The seventh aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate or anti-LIV-1 antibody drug conjugate; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, preferably, the pharmaceutically acceptable carrier includes one or more of the following: a pharmaceutically acceptable solvent, a dispersant, an additive, a plasticizer, and a pharmaceutical excipient.

[0066] The eighth aspect of the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention, or a nucleic acid encoding the antibody or antigen-binding fragment thereof, or a cell, or an anti-LIV-1 antibody conjugate, or an anti-LIV-1 antibody-drug conjugate, or a pharmaceutical composition.

[0067] The ninth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, anti-LIV-1 antibody drug conjugate, or pharmaceutical composition of the present invention in the preparation of a pharmaceutical composition for treating or preventing a disease.

[0068] The tenth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, anti-LIV-1 antibody drug conjugate, or pharmaceutical composition of the present invention in the preparation of diagnostic and detection kits.

[0069] In an eleventh aspect, the present invention provides a method for treating or preventing a disease, comprising administering to a subject in need thereof an antibody, antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, anti-LIV-1 antibody-drug conjugate, or pharmaceutical composition of the present invention. Furthermore, the disease is an LIV-1-related disease; further, the LIV-1-related disease includes cancer; further, the cancer is an LIV-1-positive solid tumor; further, the cancer includes one or more of breast cancer, prostate cancer, pancreatic cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer, esophageal squamous cell carcinoma, gastric cancer, and melanoma.

[0070] The twelfth aspect of the present invention provides a method for diagnosis and detection, comprising administering the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, anti-LIV-1 antibody-drug conjugate, or pharmaceutical composition of the present invention to a subject or sample in need.

[0071] The thirteenth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, anti-LIV-1 antibody-drug conjugate, or pharmaceutical composition of the present invention for treating or preventing a disease.

[0072] A fourteenth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, anti-LIV-1 antibody drug conjugate, kit or pharmaceutical composition of the present invention for detection and diagnosis.

[0073] A fifteenth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, anti-LIV-1 antibody-drug conjugate, kit or pharmaceutical composition of the present invention for preventing, treating, detecting or diagnosing diseases associated with LIV-1.

[0074] In the embodiments of the present invention, the disease associated with LIV-1 includes cancer.

[0075] In the embodiments of the present invention, the LIV-1-related disease includes LIV-1-positive solid tumors.

[0076] In the embodiments of the present invention, the diseases associated with LIV-1 include one or more of melanoma, mesothelioma, pancreatic cancer, non-small cell lung cancer, breast cancer and colon cancer.

[0077] A sixteenth aspect of the present invention provides use of an LIV-1 antibody or an antigen-binding fragment thereof in the preparation of an antibody-drug conjugate.

[0078] The anti-LIV-1 antibodies and drug conjugates provided by the present invention have one or more of the following advantages:

[0079] 1. The anti-LIV-1 antibody or antigen-binding fragment thereof provided by the present invention has good affinity for LIV-1 and cells expressing LIV-1.

[0080] 2. The anti-LIV-1 antibody-drug conjugate provided by the present invention has good killing ability against human breast cancer cell lines.

[0081] 3. The anti-LIV-1 antibody-drug conjugate provided by the present invention has a good tumor inhibition effect on the breast cancer cell / mouse transplant tumor model. For example, the tumor volume growth inhibition rates of CA365-vcMMAE and CA672-vcMMAE were 86.08% and 79.73%, respectively. The tumor volume growth inhibition rate TGI (%) of CA365NQ-vcMMAE was 58.3%, and the tumor weight inhibition rate was 56.82%.

[0082] 4. The efficacy of LIV-1-BNLD11 was evaluated in Balb / c-nude mice transplanted with MCF7-hLIV1 human breast cancer cells. The CA365NQ-BNLD11 group achieved complete tumor clearance.

[0083] 5. The anti-LIV-1 antibody drug conjugate provided by the present invention has a good tumor inhibition effect on the small cell lung cancer cell transplantation mouse model. For example, the tumor weight inhibition rate of the CA365NQ-vcMMAE group was 56.3%.

[0084] 6. The anti-LIV-1 antibody-drug conjugate provided by the present invention has good pharmacokinetics in mice.

[0085] 7. The anti-LIV-1 antibody-drug conjugate provided by the present invention has low side effects and toxicity and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIG1 shows the results of serum antibody titer detection in mice after the third LIV-1 booster immunization;

[0087] Figures 2A-2B show the binding activity curves of various LIV-1 antibodies to human LIV-1 at the protein level;

[0088] Figures 3A-3B show the binding activity curves of each LIV-1 antibody on MCF-7 / LIV-1 cells;

[0089] Figures 4A-4B show the binding activity curves of various LIV-1 antibodies to cynomolgus monkey LIV-1 protein;

[0090] Figures 5A-5B show the binding activity curves of various LIV-1 antibodies to mouse LIV-1 antigen;

[0091] FIG6 shows the internalization detection results of each LIV-1 antibody;

[0092] FIG7 shows the results of ADCC activity of each LIV-1 antibody based on the human breast cancer MCF-7 / LIV-1 cell line;

[0093] FIG8 shows the results of the in vitro killing effect of each LIV-1 antibody conjugate on MCF-7 / LIV-1 cells;

[0094] FIG9 shows the results of the in vitro killing effect of each LIV-1 antibody conjugate on MDA-MB-231 / LIV-1 cells;

[0095] Figures 10A-10B show the tumor growth curves and tumor photos of each LIV-1 antibody conjugate at a high dose of 5 mg / kg in the MCF-7 / LIV-1 breast cancer cell / mouse xenograft tumor model;

[0096] FIG11 shows the tumor growth curves of each LIV-1 antibody conjugate at a low dose of 2.5 mg / kg in the MCF-7 / LIV-1 breast cancer cell / mouse xenograft tumor model;

[0097] FIG12 shows the drug-dose time curves of various LIV-1 antibodies in Balb / c mice.

[0098] FIG13 shows the tumor growth curves of 2.5 mg / kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, solvent control group (PBS), and negative control group in M-NSG mice transplanted with human MCF-7 human breast cancer cells.

[0099] FIG14 shows a graph showing the inhibition of tumor weight in M-NSG mice transplanted with human MCF-7 human breast cancer cells by a 2.5 mg / kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, a solvent control group (PBS), and a negative control group.

[0100] FIG15 shows the body weight curves of M-NSG mice transplanted with human MCF-7 human breast cancer cells in a 2.5 mg / kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, a solvent control group (PBS), and a negative control group.

[0101] FIG16 shows the tumor growth curves of 2.5 mg / kg doses of LIV-1 antibody conjugates CA365NQ-vcMMAE, hLiv22-vcMMAE, and solvent control group (PBS) in M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells.

[0102] FIG17 shows a graph showing the inhibition of tumor weight in M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells by a 2.5 mg / kg dose of LIV-1 antibody conjugates CA365NQ-vcMMAE, hLiv22-vcMMAE, and a solvent control group (PBS).

[0103] FIG18 shows the body weight curves of M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells and treated with 2.5 mg / kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, hLiv22-vcMMAE, and solvent control group (PBS).

[0104] Figure 19 shows the tumor growth curves of the 2.5 mg / kg dose LIV-1 antibody conjugate CA365NQ-BNLD11 group, CA365NQ-vcMMAE group, solvent control group (PBS), and negative control group on Balb / c-nude mice transplanted with MCF7-hLIV1 human breast cancer cells.

[0105] FIG20 shows the body weight curves of Balb / c-nude mice transplanted with MCF7-hLIV1 human breast cancer cells in the 2.5 mg / kg dose LIV-1 antibody conjugate CA365NQ-BNLD11 group, CA365NQ-vcMMAE group, solvent control group (PBS), and negative control group.

[0106] FIG21 shows the body weight curves of Balb / c mice in a single-dose toxicity experiment of the LIV-1 antibody conjugate CA365NQ-vcMMAE group at a dose of 50 mg / kg and the solvent control group (PBS).

[0107] FIG22 shows the body weight curves of Balb / c mice in a single-dose toxicity experiment of a 50 mg / kg dose of the LIV-1 antibody conjugate CA1328-vcMMAE group and a solvent control group (PBS).

[0108] FIG23 shows a schematic diagram of the synthesis route of BNLD11. DETAILED DESCRIPTION

[0109] The present invention will be further described below in conjunction with specific examples. The described embodiments are some embodiments of the present invention, rather than all embodiments. It should be understood that the following examples are provided to provide a complete disclosure and description of how to utilize the methods and compositions of the present invention to those skilled in the art, and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0110] The statistical analysis in the examples of the present invention was performed using mean ± SEM. Graphpad 8.0 software was used for data analysis and processing. Tumor volume and body weight were compared between the groups at each time point using two-way analysis of variance. Tumor weight was analyzed using one-way analysis of variance. T-test was used to compare the two groups. P < 0.05 indicated that the difference was statistically significant.

[0111] Example 1. Generation of anti-LIV-1 monoclonal antibodies

[0112] 1.1 Mouse immunization method

[0113] Transgenic mice with fully human antibodies for immune experiments (Developed and produced by Shandong Boan Biotechnology Co., Ltd.) Two antigenic proteins were used for immunization in two batches, for a total of 13 mice. The first batch used the His-Tag-fused LIV-1-His antigen protein (manufacturer ACRO, catalog number LV1-H5223) to immunize five fully human antibody transgenic mice (Q06-Q10). The second batch used human IgG1Fc-fused LIV-1-Fc (manufacturer ACRO, catalog number LV1-H5254) and His-Tag-fused LIV-1-His (manufacturer ACRO, catalog number LV1-H5223) antigen proteins, respectively, for a total of eight fully human antibody transgenic mice. Five mice, numbered Q11-Q15, were immunized with human IgG1Fc-fused LIV-1-Fc (manufacturer ACRO, catalog number LV1-H5254), and three mice, numbered Q16-Q18, were immunized with His-Tag-fused LIV-1-His (ACRO, LV1-H5223). All mice were immunized using multiple subcutaneous and inguinal injections at a dose of 20 μg per mouse. The initial immunization used antigen emulsified in Freund's complete adjuvant, while subsequent immunizations used antigen emulsified in Freund's incomplete adjuvant. Fully human antibody transgenic mice received one immunization and four booster immunizations, each with a 14-day interval.

[0114] Starting with the second immunization, peripheral blood serum was collected 7 days after each immunization for antibody titer analysis, and mice with substandard titers were removed. The results of serum antibody titer analysis after the third booster immunization are shown in Figure 1, where 500X, 2500X, 12500X, and 62500X represent dilution ratios. Three days after the fourth booster immunization, mice were euthanized, and spleens were harvested for single cell banking.

[0115] 1.2 Establishment of phage library

[0116] Mice were sacrificed, spleens were removed by dissection, and cDNA was obtained after RNA extraction. Phage libraries were constructed according to conventional methods. The library capacity data are shown in Table 1.

[0117] Table 1 Phage library capacity of each immunized mouse

[0118] 1.3 Screening by plate screening and magnetic bead screening

[0119] 1.3.1 Plate Screening: Coat plates with His-tagged LIV-1-His protein (manufacturer: Acro, Catalog No. LV1-H5223) or human IgG1Fc-fused LIV-1-Fc protein (manufacturer: Acro, Catalog No. LV1-H5254). The next day, add the phage library and incubate for 2 hours. Wash 4-10 times and elute specifically bound phage with elution buffer.

[0120] 1.3.2 Magnetic bead screening: LIV-1-His protein (manufacturer Acro, catalog number LV1-H5223) or LIV-1-Fc protein (manufacturer Acro, catalog number LV1-H5254) was biotinylated according to the kit instructions, then bound to Thermo magnetic beads, incubated with the phage library, washed 4-10 times, and eluted specifically bound phage with Elution Buffer.

[0121] Table 2. Sources of LIV-1 antibodies obtained through screening

[0122] Based on the CA365 clone, a point mutation was performed on one of the amino acid sites to obtain the CA365NQ clone (the amino acid sequence of the variable region is shown in Table 3).

[0123] Example 2. Molecular construction and production of complete antibodies

[0124] A total of 175 positive cloned IgG1 antibodies were constructed and sequenced, of which the amino acid sequences of the variable regions of 11 lead antibodies are shown in Table 3 (the CDR regions are underlined and the analysis system was the IMGT system).

[0125] Table 3 Variable region sequences of 11 lead antibody amino acid sequences

[0126] Among them, CA365NQ has an amino acid mutation compared with CA365.

[0127] The antibody variable region genes were amplified by conventional molecular biology techniques PCR (2×Phanta Max Master Mix, manufacturer Vazyme, catalog number P515-P1-AA). The antibody heavy chain variable region genes were connected to the vector pCDNA3.4 (Life Technology) containing the nucleic acid sequence of the antibody heavy chain constant region sequence by homologous recombination. The antibody light chain variable region genes were connected to the vector pCDNA3.4 containing the nucleic acid sequence of the antibody light chain constant region sequence. The control antibody (also called reference antibody) hLiv22 (also called Ladiratuzumab) variable region sequence (see SEQ ID NO: 55, SEQ ID NO: 56 below) is from patent US 2020 / 0165335 A1, IgG1 / Kappa.

[0128] Except for the control antibody hLiv22, the variable region sequences of the antibodies in the examples of this application are shown in Table 3, and the heavy chain and light chain constant region sequences are shown in Table 4.

[0129] Table 4 Amino acid sequences of the heavy and light chain constant regions of the 11 lead antibodies

[0130] The amino acid sequence of the hLiv22 heavy chain is shown in SEQ ID NO: 55, and is specifically shown below:

[0131] The amino acid sequence of the hLiv22 light chain is shown in SEQ ID NO: 56, and is specifically as follows:

[0132] The plasmids of the positive clones after sequencing were extracted and co-transfected into HEK293 cells and cultured in a shaker at 37°C\8% CO2\125rpm. After 7 days of transient expression, the supernatant was purified by Protein A affinity chromatography to obtain the antibody, and the antibody concentration was determined by UV280 binding theoretical extinction coefficient.

[0133] Example 3. Characterization of LIV-1 monoclonal antibody molecules

[0134] 3.1 Binding activity of LIV-1 antibodies to human LIV-1 protein

[0135] Human LIV-1 protein (0.2 μg / mL) was coated with carbonate buffer at 100 μL / well overnight at 4°C. Plates were blocked with 3% skim milk powder at 300 μL / well, incubated at 37°C for 1 hour, and then washed twice. Whole antibody was serially diluted in PBST (phosphate buffered saline + 0.05 Tween-20) to 0.2 μg / mL in eight four-fold dilutions, with 100 μL per well added. Plates were incubated at 37°C for 1 hour, and then washed twice. Goat anti-human IgG (H+L) / HRP (1:5000 dilution) was added at 100 μL / well, incubated at 37°C for 1 hour, and washed five times. 100 μL of TMB was added to each well, and after 10 minutes of color development, the reaction was terminated by adding 50 μL of 2 M H2SO4 to each well. OD450 values ​​were read on a multi-functional microplate reader. Figure 2A (for the newly screened antibodies CA332, CA317, CA365, CA406, CA672, CA336, and the reference antibody hLiv22) and Figure 2B (for the newly screened antibodies CA1328, CA454, CA478, CA789, and the reference antibody hLiv22) show the binding curves of each LIV-1 antibody to human LIV-1 at the protein level. EC50 values ​​were calculated from the curves in Figures 2A-2B. Table 5 shows the EC50 values ​​for binding of each antibody to human LIV-1 protein, demonstrating that the newly screened antibodies exhibit higher binding sensitivity than the reference antibodies.

[0136] Table 5 EC50 values ​​of LIV-1 antibody binding to LIV-1 antigen

[0137] 3.2 Binding affinity of LIV-1 antibodies to cells

[0138] The LIV-1-overexpressing cell line, MCF-7 / LIV-1 (breast cancer cell line), was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. and cultured in RPMI 1640 medium supplemented with 10% FBS and 0.5 μg / mL puromycin at 37°C and 5% CO2. Antibodies were diluted to the appropriate concentrations and added to a round-bottom 96-well plate. Then, 100,000 digested MCF-7 / LIV-1 cells were added per well. After incubation at 4°C for 1 hour, the cells were washed once with PBS, and a secondary antibody, FITC-anti-human IgG, was added at a 1:500 ratio. After incubation at 4°C for 0.5 hours, the cells were washed once with PBS, and cell fluorescence intensity was measured by flow cytometry. The binding activity of each LIV-1 antibody on MCF-7 / LIV-1 cells is shown in Figure 3A (for antibodies CA317, CA365, CA478, CA672, CA454, CA789, and hLiv22) and Figure 3B (for antibodies CA336, CA1328, and hLiv22). The EC50 values ​​for each antibody binding on MCF-7 / LIV-1 cells are shown in Table 6. It can be seen that these newly screened antibodies all have strong cell binding activity, with CA317, CA365, CA454, CA478, CA672, and CA789 showing stronger binding activity than the reference antibody.

[0139] Table 6 EC50 values ​​of LIV-1 antibody binding on MCF-7 / LIV-1 cells

[0140] 3.3 Cross-species reactivity of LIV-1 antibodies with cynomolgus monkey and mouse LIV-1 proteins

[0141] Cynomolgus monkey LIV-1 protein (manufacturer Acro, Catalog No. LV1-C52H5) at 0.2 μg / mL and mouse LIV-1 protein (manufacturer Acro, Catalog No. LV1-M5223) at 1 μg / mL were coated with carbonate buffer at 100 μL / well and incubated overnight at 4°C. The plate was blocked with 3% nonfat dry milk at 300 μL / well and incubated at 37°C for 1 hour, followed by two washes. To detect binding to mouse LIV-1 protein, the intact antibody was serially diluted in PBST (phosphate buffered saline + 0.05 M Tween-20) using six four-fold dilutions to 4 μg / mL. To detect binding to cynomolgus monkey LIV-1 protein, the intact antibody was serially diluted in PBST (phosphate buffered saline + 0.05 M Tween-20) using six four-fold dilutions to 0.25 μg / mL, with 100 μL per well. The plate was incubated at 37°C for 1 hour, followed by two washes. Add 100 μL / well of goat anti-human IgG (H+L) / HRP (1:5000 dilution), incubate at 37°C for 1 hour, and wash the plate five times. Add 100 μL of TMB to each well, develop for 10 minutes, and then terminate the reaction by adding 50 μL of 2M H2SO4 to each well. Finally, read the OD450 value on a multifunctional microplate reader. Figure 4A (involving antibodies CA317, CA332, CA365, CA478, and hLiv22) and Figure 4B (involving antibodies CA672, CA406, CA454, CA789, and hLiv22) show the binding curves of each LIV-1 antibody to the cynomolgus monkey LIV-1 protein. Table 7 shows the corresponding EC50 values. It can be seen that all antibodies bind to the cynomolgus monkey LIV-1 antigen.

[0142] Table 7 EC50 values ​​of binding of LIV-1 antibodies to cynomolgus monkey LIV-1 protein

[0143] Figure 5A (involving antibodies CA317, CA332, CA365, CA478, and hLiv22) and Figure 5B (involving antibodies CA672, CA406, CA454, CA789, and hLiv22) show the binding activity of each LIV-1 antibody to the mouse LIV-1 antigen. It can be seen that the CA478 antibody, CA454, and CA672 bind to the mouse protein, while the remaining antibodies do not bind to the mouse LIV-1 protein.

[0144] 3.4 Affinity of LIV-1 Antibodies for Human and Rat LIV-1 Proteins

[0145] A ProA chip was used to capture LIV-1 antibodies at 2 μg / mL, and the binding activity of LIV-1 antibodies to human LIV-1 and rat LIV-1 was analyzed. LIV-1 protein was diluted two-fold with HBS-EP+ buffer into five concentrations, starting at a concentration of 50 nM. Biacore8K was used to analyze the binding kinetics of LIV-1 protein, and the affinity activity KD values ​​were calculated by fitting. Table 8 shows the affinity of LIV-1 antibodies to human LIV-1 protein and rat LIV-1 protein, among which CA317, CA365, CA406, CA672, CA789, and CA1328 can bind to rat LIV-1 antigen.

[0146] Table 8 Affinity of LIV-1 antibodies to human LIV-1 and rat LIV-1 proteins

[0147] 3.5 LIV-1 Antibody Epitope Competition Analysis

[0148] First, LIV-1 antibodies were grouped based on sequence similarity. Three groups were identified: Group 1 included CA332; Group 2 included CA336 / CA560 / CA1328; and Group 3 included CA317 / CA365 / CA406 / CA454 / CA478 / CA672 / CA789. One or two antibodies were selected for epitope analysis in each group: Group 1 included CA332, Group 2 included CA560, and Group 3 included CA454 and CA789. LIV-1 protein was captured using a His chip at 10 μg / mL. Saturation binding was first applied to the first LIV-1 antibody, 1, followed by the addition of another LIV-1 antibody, 2. The response of antibody 2 was measured on an Octet to determine if there was epitope competition between antibodies 1 and 2. A value above 75% indicated that the epitopes were related. Table 9 shows the results of the epitope analysis, demonstrating that the third group of antibodies overlapped with the reference antibody hLiv22, while the first and second groups of antibodies exhibited distinct epitopes.

[0149] Table 9 LIV-1 antibody epitope analysis

[0150] Example 4: Internalization experiment of LIV-1 monoclonal antibody molecules

[0151] First, the antibody to be tested was prepared at 24 μg / mL using complete culture medium (RPMI1640: manufacturer Gibco, product number 21870092; fetal bovine serum: manufacturer Gibco, product number 10099-141), and 25 μL / well was added to a 96-well U-bottom plate (manufacturer SARSTEDT, product number 83.3925.500). TM pHrodo TMiFL IgG labeling reagent (Invitrogen, catalog number Z25612) was prepared at 24 μg / mL, and 25 μL / well was added to the antibody in step (1). The mixture was incubated at room temperature to allow the antibody to form a complex with the labeling reagent. MCF-7 / LIV-1 (also known as MCF-7-LIV-1, Sanyou Bio, catalog number BA001-MCF-7) cells were then digested, washed twice with PBS, and the cell density was adjusted to 2×10 6 / mL, 50μL / well was added to the complex of step (2), and incubated at 37 degrees for 0 hours, 2 hours, 6 hours, and 24 hours. Finally, at different incubation times, the cells were washed twice with PBS and the MFI (mean fluorescence intensity) value was detected by flow cytometry (manufacturer ACEA, model NovoCyte). Figure 6 shows the internalization test results of various LIV-1 antibodies (CA317, CA365, CA478, CA406, CA454, CA789, CA336, CA672 and hLiv22). Isotype represents the isotype control of the antibody (an unrelated antibody with a different variable region and the same constant region as the LIV-1IgG1 antibody), and label represents the dye without labeled antibody. As can be seen from the results in Figure 6, CA336 antibody has the best internalization performance, CA478 antibody has the worst internalization performance, and the internalization abilities of the remaining antibodies are similar.

[0152] Example 5: Antibody-dependent cell-mediated cytotoxicity (ADCC) (Effector cells containing luciferase reporter gene)

[0153] Prepare ADCC reaction buffer, RPMI 1640 medium (RPMI1640: manufacturer Gibco, product number 21870092) containing 1% FBS (manufacturer Gibco, product number 10099-141); collect ADCC Bioassay effector cells (manufacturer Promega, product number G7011), and adjust the cell density to 2.4×10 6 / mL; collect target cells, human breast cancer MCF-7 / LIV-1 (manufacturer Sanyou Biotechnology, product number BA001-MCF-7), and adjust the cell density to 8×10 with ADCC buffer. 5 / mL; the test sample was diluted 4-fold with ADCC buffer to eight concentrations; 25 μL each of effector cells, target cells, and test sample were added to a white plate (Costar, Catalog No. 3917) for a total reaction volume of 75 μL and incubated at 37°C for 6 hours. Then, 75 μL of the Bio-Glo Luciferase System (Promega, Catalog No. G7940) was added to each well. After a 15-minute reaction, chemiluminescence was read using a microplate reader (BioTek, Synergy Neo2). Figure 7 shows the ADCC activity of various LIV-1 antibodies (including CA317, CA365, CA365NQ, CA406, CA672, CA789, CA1328, and hLiv22) against the human breast cancer MCF-7 / LIV-1 cell line. Figure 7 shows that CA365, CA672, and CA406 have superior ADCC activity.

[0154] Example 6 Preparation of anti-LIV-1 antibody conjugate

[0155] Prepare an antibody drug conjugate with the following molecular formula:

[0156] Wherein: Ab is the antibody against LIV-1, LU is the linker (also known as a connector); D is the drug; and the subscript p is the average DAR value of the antibody-drug conjugate.

[0157] 6.1 Preparation of anti-LIV-1 antibody-vcMMAE conjugate

[0158] For the anti-LIV-1 antibodies CA332, CA336, CA365, CA406, CA454, CA672, and CA789, as well as the control antibody hLiv22, VcMMAE (MCE, Catalog No. HY-15575 / CS-1242) was dissolved in DMSO to a 2 mM solution, stored at -80°C, removed before use, and thawed at room temperature. TCEP (Aldrich, Catalog No. C4706-10G, SLBT6121) was dissolved in purified water to a 10 mM solution, stored at -80°C, removed before use, and thawed at room temperature. DTPA (aladdin, Catalog No. D108512-100g) was dissolved in PBS to a 10 mM solution, adjusted to pH 8.3 with sodium hydroxide solution, and stored at 4°C. The antibody solution was replaced with PBS buffer, pH 7.5, and the antibody concentration was adjusted to 5-10 mg / mL. During conjugation, 1 / 10 volume of DTPA solution was added to the antibody solution, followed by 2-3 times the molar ratio of the antibody to TCEP solution. The reaction was allowed to reduce at 25°C for 2 hours. Then, 4-6 times the molar ratio of the antibody to VcMMAE solution was added and the reaction was allowed to proceed at 25°C for 1 hour. The buffer was then exchanged with PBS using an ultrafiltration tube (molecular weight cut-off 30 kD) at a rate of at least 10,000-fold, or with a desalting column (Sephadex G25) to remove free drug-linker and other additives. HIC-HPLC analysis showed that the average DAR value of the conjugated antibody-drug conjugates was 4±0.5. The proportions and averages of the DAR values ​​for CA332-vcMMAE, CA336-vcMMAE, CA365-vcMMAE, CA406-vcMMAE, CA454-vcMMAE, CA672-vcMMAE, and CA789-vcMMAE are shown in Table 10. The proportion of each DAR value of hLiv22-vcMMAE and the average value are shown in Table 11.

[0159] Table 10 DAR value distribution of anti-LIV-1 antibody-vcMMAE conjugate

[0160] Table 11 Distribution of DAR values ​​of hLiv22-vcMMAE conjugates

[0161] 6.2 Preparation of anti-LIV-1 antibody-BNLD11 conjugate

[0162] Taking the anti-LIV-1 antibody CA365NQ as an example, the antibody solution was exchanged into PBS buffer and concentrated by ultrafiltration. The antibody concentration was adjusted to 5-10 mg / ml. A 1 / 10 volume of DTPA stock [11 mM DTPA (Diethylenetriaminepentaacetic acid, CAS No. 67-43-6), dissolved in PBS, pH 7.2] was added to a final DTPA concentration of 1 mM. TCEP [Tris(2-carboxyethyl)phosphine, CAS No. 51805-45-9, dissolved in water to a 2 mM solution] was added at 2-3 times the molar ratio of the antibody. The reaction was allowed to proceed at 25°C for 2 hours. Six times the molar ratio of the antibody was then added to BN-LD-11. The coupling reaction was continued at 25°C for 1 hour. The remaining BNLD-11 was quenched by adding N-acetylcysteine ​​(CAS No. 38520-57-9) at 10 times the molar ratio of the antibody. The ADC solution was then dialyzed against PBS for at least 10,000 times or by gel filtration to remove small molecules. ADC concentrations were determined using the BSA method, and DAR distribution and mean DAR values ​​were determined using HIC-HPLC. The proportions of individual DAR values ​​for CA365NQ-BNLD11 and their mean values ​​are shown in Table 12.

[0163] Table 12 DAR value distribution of CA365NQ-BNLD11 conjugate

[0164] Example 7 Inhibitory activity of LIV-1 antibody conjugate on tumor cell proliferation in vitro

[0165] 7.1 Anti-LIV-1 Antibody Conjugate Inhibits Proliferation of MCF-7 / LIV-1 Breast Cancer Cells

[0166] The LIV-1 overexpressing cell line MCF-7 / LIV-1 was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. The culture conditions were RPMI-1640 medium supplemented with 10% FBS and 0.5 μg / mL puromycin, and cultured at 37°C and 5% CO2. MCF-7 / LIV-1 cells were plated at a density of 5000 cells / well on a 96-well flat plate. After overnight attachment, the corresponding concentrations of antibody conjugates were added. After 96 hours of continuous action, cell viability was measured using a Promega Cell Viability Assay Kit (G7570). Figure 8 shows the results of the inhibitory activity of various LIV-1 antibody conjugates (including CA332, CA789, CA672, CA454, CA336, CA406, CA365 antibodies, and the control antibody hLiv22) on MCF-7 / LIV-1 cell proliferation (nCov-CA521 is an antibody that does not bind to LIV-1, nCov-CA521-vcMMAE is an antibody conjugate that does not bind to LIV-1, i.e., the negative control group; hLiv22-vcMMAE is the experimental group prepared in Example 6). The EC50 values ​​of each LIV-1 antibody conjugate are shown in Table 13. FIG8 and Table 13 show that CA365-vcMMAE has the strongest proliferation inhibitory activity.

[0167] Table 13 Proliferation inhibition activity of LIV-1 antibody conjugates on MCF-7 / LIV-1 cells

[0168] 7.2 Anti-proliferation activity of LIV-1 antibody conjugate against MDA-MB231 / L1 breast cancer cells

[0169] The LIV-1-overexpressing cell line MDA-MB231 / LIV-1 (also referred to as MDA-MB-231 / LIV-1) was purchased from Nanjing GenScript Biotechnology Co., Ltd. and cultured in DMEM high-glucose medium supplemented with 10% FBS and 0.25 μg / mL puromycin at 37°C / 5% CO2. MDA-MB231 / LIV-1 cells were plated at a density of 5,000 cells / well in 96-well flatbed plates. After overnight adherence, the corresponding concentrations of antibody conjugates were added. After 96 hours of exposure, cell viability was assessed using a Promega Cell Viability Assay Kit (G7570). Figure 9 shows the results of the proliferation inhibition activity of various LIV-1 antibody conjugates (involving CA332, CA789, CA672, CA454, CA336, CA406, CA365 antibodies and the control antibody hLiv22) on MDA-MB231 / LIV-1 cells (nCov-CA521 is an antibody that does not bind to LIV-1, and nCov-CA521-vcMMAE is an antibody conjugate that does not bind to LIV-1, i.e., a negative control group). The EC50 values ​​of each LIV-1 antibody conjugate are shown in Table 14. Figure 9 and Table 14 show that CA365-vcMMAE has the strongest proliferation inhibition activity.

[0170] Table 14 Proliferation inhibition activity of LIV-1 antibody conjugates on MDA-MB231 / LIV-1 cells

[0171] Example 8 Evaluation of the efficacy of LIV-1 antibody conjugate in the MCF-7 / LIV-1 breast cancer cell / mouse transplant tumor model

[0172] Female M-NSG immunodeficient mice, 6-8 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and maintained by the Animal Experimental Center of Shandong Boan Biotechnology Co., Ltd. The LIV-1-overexpressing cell line MCF-7 / LIV-1 was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. and cultured in RPMI1640 medium supplemented with 10% FBS and 0.5 μg / mL puromycin at 37°C and 5% CO2. IRA SE-121-0.36 mg estradiol sustained-release tablets were purchased from Changzhou Xinlitai Biotechnology Co., Ltd.

[0173] Estradiol sustained-release tablets were inoculated into the left armpit of M-NSG mice using a trocar needle. Three days later, MCF-7 / LIV-1 cells were inoculated at a density of 1×10 7 The number of cells per mouse was inoculated into the subcutaneous area of ​​the right forelimb back of the mouse. When the average tumor volume reached about 100-150mm 3The LIV-1 antibody conjugate was administered in two batches, namely a 5 mg / kg high-dose batch and a 2.5 mg / kg low-dose batch. Each batch was equipped with a solvent control group and an antibody conjugate administration group, wherein the solvent control group was also called the Vehicle group, and its component was PBS solution. The solvent control groups in Examples 8-12 were all PBS solutions. The administration method was tail vein injection, and the administration was done once. The body weight and tumor volume of the mice were measured twice a week. Tumor volume (mm 3)=long diameter×short diameter×short diameter÷2. The efficacy results of a high dose of 5 mg / kg are shown in Figures 10A and 10B. Figure 10A is a 20-day tumor growth curve of 5 mg / kg high-dose LIV-1 antibody conjugates (involving the PBS group, as well as hLiv22, CA672, CA789, CA406, CA1328, CA365, and nCov-CA521 antibodies) in the MCF-7 / LIV-1 breast cancer cell / mouse transplant tumor model (wherein PBS represents the solvent control group, i.e., the blank control group; nCov-CA521 is a group not bound to LIV-1). Figure 10B shows a photo of a 5 mg / kg high-dose of each LIV-1 antibody (involving PBS, hLiv22, CA672, CA789, CA406, CA1328, CA365, and nCov-CA521 antibody) conjugate in an MCF-7 / LIV-1 breast cancer cell / mouse transplant tumor model. The numbers in the upper left corner of each cell in the photo, such as 925, 930, etc., are the numbers of the experimental mice. Figures 10A and 10B show that CA365-vcMMAE has the best tumor inhibition effect, with all 5 mice achieving tumor clearance. Three mice in each of the CA406-vcMMAE and CA789-vcMMAE groups achieved tumor clearance. Only two mice in the CA672-vcMMAE, CA1328-vcMMAE, and hLiv22-vcMMAE control groups achieved tumor clearance. The efficacy results of the 2.5 mg / kg low-dose are shown in Figure 11, which shows the 24-day tumor growth curve of the 2.5 mg / kg low-dose LIV-1 antibody conjugates (involving PBS, CA672, CA789, CA406, CA1328, CA365, and nCov-CA521 antibodies) in the MCF-7 / LIV-1 breast cancer cell / mouse transplant tumor model (where PBS represents the solvent control group, i.e., the blank control group; nCov-CA521 is an antibody that does not bind to LIV-1, and nCov-CA521-vcMMAE is an antibody conjugate that does not bind to LIV-1, i.e., the negative control group). Table 15 shows the tumor volume growth inhibition rates for CA365-vcMMAE, CA406-vcMMAE, CA672-vcMMAE, CA789-vcMMAE, and CA1328-vcMMAE at the end of the experiment, which were 86.08%, 55.22%, 79.73%, 89.22%, and 75.04%, respectively. Tumor weight inhibition rates were 72.97%, 42.57%, 70.27%, 72.97%, and 59.46%, respectively. Table 15 demonstrates that CA365-vcMMAE, CA672-vcMMAE, and CA789-vcMMAE have excellent tumor inhibition effects.

[0174] Table 15 Anti-LIV-1 ADC 2.5 mg / kg inhibition rate of tumor volume and tumor weight

[0175] Example 9 Pharmacokinetic Study of LIV-1 Antibody in Mice

[0176] Three Balb / c mice were subcutaneously injected with each antibody at a dose of 5 mg / kg. Blood was collected at 0 hour before dosing, and 5 minutes, 0.5 hours, 1 hour, 6 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, and 28 days after dosing. Serum antibody concentrations were determined using ELISA. Antigen protein solutions were coated with carbonate buffer. 200 μL of blocking solution (2% BSA / PBST) was added to each well and incubated for 2 hours. Standard solution concentrations ranged from 5 to 0.039 μg / mL. Samples were diluted 10-fold and 100-fold. Before loading, 100 μL / well of a 50-fold dilution in 1% BSA / PBST was added and incubated at 37°C, 180 rpm, for 1 hour. Then, 100 μL / well of 2WX goat anti-human IgG Fc-HRP was added and incubated at 37°C, 180 rpm, for 1 hour. Add 100 μL / well of TMA colorimetric solution and incubate at room temperature in the dark for 5-10 minutes. Finally, terminate the color development with 100 μL / well of 1M H3PO4. Using a microplate reader, read the visible light at 450 nm and 650 nm, and employ four-parameter fitting analysis. Table 16 shows the drug-dose curve parameters. Figure 12 shows the drug-dose curves of various LIV-1 antibodies (including CA365NQ, CA365, CA672, CA1328, and hLiv22 antibodies) in Balb / c mice. Figure 12 and Table 16 show that the half-life and blood drug exposure of CA365NQ, CA365, CA672, and CA1328 antibodies are significantly higher than those of the control antibody. In Table 16, mean represents the mean, and SD represents the standard deviation.

[0177] Table 16 Drug-time curve parameters

[0178] Example 10 Evaluation of the efficacy of LIV-1-vcMMAE in M-NSG mice transplanted with MCF-7 human breast cancer cells

[0179] MCF-7 human breast cancer cells were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator in RMPI-1640 medium containing 10% FBS. M-NSG mice were purchased from Shanghai Model Organisms Science Co., Ltd., and 17β-estradiol sustained-release tablets were purchased from Innovative Research of America. MCF-7 cells were cultured in serum-free RMPI-1640 medium containing 50% Matrigel to a concentration of 1.0 × 108 The cells / mL were inoculated subcutaneously on the right side of M-NSG mice at a volume of 0.1 mL / mouse. When the average tumor volume reached 130 mm 3 Mice were divided into three experimental groups based on tumor volume, with six mice in each group: CA365NQ-vcMMAE group, solvent control group (PBS), and negative control nCov-CA521-vcMMAE group. Dosing began on the day of grouping, with a single dose of 2.5 mg / kg. The experiment ended on the 24th day of dosing. Compared with the solvent control group, the CA365NQ-vcMMAE group significantly inhibited tumor volume growth (P=0.001), with a tumor volume growth inhibition rate (TGI) of 58.3%. There was a significant difference between the CA365NQ-vcMMAE group and the negative control nCov-CA521-vcMMAE group (P<0.05). The tumor growth curves of the 2.5 mg / kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, solvent control group, and negative control group in M-NSG mice transplanted with human MCF-7 human breast cancer cells are shown in Figure 13.

[0180] At the end of the experiment, the tumor weight inhibition rate in the CA365NQ-vcMMAE group was 56.82%, significantly different from the solvent control group and the negative control nCov-CA521-vcMMAE group (P<0.05). The inhibition of tumor weight in M-NSG mice transplanted with MCF-7 human breast cancer cells by the 2.5 mg / kg dose of the LIV-1 antibody conjugate CA365NQ-vcMMAE, the solvent control group, and the negative control group is shown in Figure 14.

[0181] Throughout the experiment, the animals were active and fed well during the dosing period, and all groups showed some weight gain, indicating good tolerance of the test article. No significant differences were found between the groups (P>0.05). Body weight curves for M-NSG mice transplanted with human MCF-7 breast cancer cells, treated with a 2.5 mg / kg dose of the LIV-1 antibody conjugate CA365NQ-vcMMAE, a solvent control group, and a negative control group are shown in Figure 15.

[0182] Example 11 Evaluation of the efficacy of LIV-1-VcMMAE in M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells

[0183] NCI-H82 human small cell lung cancer cells were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator in RMPI-1640 medium containing 10% FBS. M-NSG mice were purchased from Shanghai Model Organisms Science Co., Ltd. The NCI-H82 cell concentration was adjusted to 5.0 × 10 cells / mL using serum-free RMPI-1640 medium containing 50% Matrigel. 7 The cells / mL were inoculated subcutaneously on the right side of Balb / c nude mice at a volume of 0.1 mL / mouse. When the average tumor volume reached 174 mm 3 Mice were divided into three experimental groups (n=5) based on tumor volume: CA365NQ-vcMMAE, hLiv22-vcMMAE, and a solvent control group (PBS). Drug administration began on the day of grouping at a single dose of 2.5 mg / kg. The experiment ended on the 13th day of dosing. Compared with the solvent control group, both the hLiv22-vcMMAE and CA365NQ-vcMMAE groups significantly inhibited tumor growth (P = 0.0015, P = 0.0025). No statistical difference was observed between the hLiv22-vcMMAE and CA365NQ-vcMMAE groups (P > 0.05). The tumor growth inhibition rates (TGI) (%) of the hLiv22-vcMMAE and CA365NQ-vcMMAE groups were 70.6% and 76.1%, respectively. The tumor growth curves of 2.5 mg / kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, hLiv22-vcMMAE, and solvent control groups in M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells are shown in FIG16 .

[0184] At the end of the experiment, tumor weight inhibition rates in the hLiv22-vcMMAE and CA365NQ-vcMMAE groups were 54.2% and 56.3%, respectively. Tumor weights in both treatment groups were significantly lower than those in the vehicle control group (P2 = 0.0008, P3 = 0.0012). The inhibition of tumor weight in M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells at a dose of 2.5 mg / kg using the LIV-1 antibody conjugates CA365NQ-vcMMAE, hLiv22-vcMMAE, and the vehicle control group is shown in Figure 17.

[0185] During the experiment, the animals were active and ate well during the dosing period, and body weights remained stable across all groups, indicating good tolerance of the test article. There were no significant differences in the changes between groups (P>0.05). Body weight curves for M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells and treated with 2.5 mg / kg doses of the LIV-1 antibody conjugates CA365NQ-vcMMAE and hLiv22-vcMMAE, as well as the vehicle control group, are shown in Figure 18.

[0186] Example 12 Evaluation of the efficacy of LIV-1-VcMMAE and LIV-1-BNLD11 in Balb / c-nude mice transplanted with MCF7-hLIV1 human breast cancer cells

[0187] MCF7-hLIV1 cells are human breast cancer cells overexpressing hLIV1. Cells were cultured in a 37°C, 5% CO2 incubator in RMPI-1640 medium supplemented with 10% FBS. Balb / c nude mice were purchased from Nanjing Jicui Biotechnology Co., Ltd., and 17β-estradiol sustained-release tablets were purchased from Innovative Research of America. MCF-7 cells were cultured in serum-free RMPI-1640 medium supplemented with 50% Matrigel to a concentration of 1.0 × 10 8 The cells / mL were inoculated subcutaneously on the right side of M-NSG mice at a volume of 0.1 mL / mouse. When the average tumor volume reached 103 mm 3 Mice were divided into four experimental groups (n=6) based on tumor volume: CA365NQ-BNLD11, CA365NQ-vcMMAE, solvent control (PBS), and negative control nCov-CA521-vcMMAE. Drug administration began on the day of grouping at a single dose of 2.5 mg / kg.

[0188] The trial was terminated on day 40 of group-administered dosing. Because the CA365NQ-BNLD11 group achieved complete tumor clearance, this group was observed for an additional 64 days. Compared with the solvent control group, the CA365NQ-BNLD11 and CA365NQ-vcMMAE groups significantly inhibited tumor volume growth (P<0.05). However, there was no significant difference between the nCov-CA521-vcMMAE group and the solvent control group (P>0.05). The tumor volume growth inhibition rate (TGI) (%) in the CA365NQ-vcMMAE group was 83.2%. The CA365NQ-BNLD11 group achieved complete tumor clearance and experienced no recurrence within the next 25 days. The tumor growth curves of the 2.5 mg / kg dose of LIV-1 antibody conjugate CA365NQ-BNLD11 group, CA365NQ-vcMMAE group, solvent control group, and negative control group in Balb / c-nude mice transplanted with MCF7-hLIV1 human breast cancer cells are shown in FIG19 .

[0189] During the experiment, the animals were active and fed well during the dosing period, and all groups showed some weight gain, indicating good tolerance of the test article. There were no significant differences between the groups (P>0.05). Body weight curves for Balb / c nude mice transplanted with MCF7-hLIV1 human breast cancer cells in the 2.5 mg / kg dose group of the LIV-1 antibody conjugate CA365NQ-BNLD11, CA365NQ-vcMMAE, solvent control, and negative control groups are shown in Figure 20.

[0190] Example 13 Single-dose toxicity study of LIV-1-vcMMAE in Balb / c mice

[0191] 13.1 CA365NQ-VcMMAE Single-Dose Toxicity Study

[0192] Balb / c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The mice were divided into two groups according to their weight, with 6 mice in each group, half male and half female. The experimental groups were: CA365NQ-vcMMAE group and solvent control group (PBS). One group was injected with CA365NQ-vcMMAE at a dose of 50 mg / kg through the tail vein, and the other group was injected with an equal volume of PBS as a solvent control. The weight changes of the mice were recorded every day thereafter. The results showed that the mice in the drug-treated group lost 6.2% of their weight only on the third day after administration, and then recovered rapidly. They were in good general condition and had no significant acute toxicity. The weight curves of Balb / c mice in the single-dose toxicity experiment of the 50 mg / kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE group and the solvent control group are shown in Figure 21.

[0193] 13.2 CA1328-VcMMAE Single-Dose Toxicity Study

[0194] Balb / c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The mice were divided into two groups according to their weight, with 6 mice in each group, half male and half female. The experimental groups were: CA1328-vcMMAE group and solvent control group (PBS). One group was injected with CA1328-vcMMAE, which can cross-react with mouse LIV1, at a dose of 50 mg / kg through the tail vein, and the other group was injected with an equal volume of PBS as a solvent control. The weight changes of the mice were recorded every day thereafter. The results showed that the weight of the mice in the drug-treated group decreased by 6.8% and 8.8% on the 2nd and 6th days after administration, respectively, and then recovered rapidly. They were in good general condition and had no significant acute toxicity. The weight curve of Balb / c mice in the single-dose toxicity experiment of the 50 mg / kg dose of LIV-1 antibody conjugate CA1328-vcMMAE group and the solvent control group is shown in Figure 22.

Claims

1. An anti-LIV-1 antibody or antigen-binding fragment thereof, comprising three light chain complementary determining regions and three heavy chain complementary determining regions, wherein The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:22, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:41, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:25, HCDR2 set forth in SEQ ID NO:40, and HCDR3 set forth in SEQ ID NO:27; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:22, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:24, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:25, HCDR2 set forth in SEQ ID NO:40, and HCDR3 set forth in SEQ ID NO:27; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:22, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:24, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:25, HCDR2 set forth in SEQ ID NO:26, and HCDR3 set forth in SEQ ID NO:27; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:28, LCDR2 set forth in SEQ ID NO:29, and LCDR3 set forth in SEQ ID NO:30, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:31, HCDR2 set forth in SEQ ID NO:32, and HCDR3 set forth in SEQ ID NO:33; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:34, LCDR2 set forth in SEQ ID NO:35, and LCDR3 set forth in SEQ ID NO:36, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:37, HCDR2 set forth in SEQ ID NO:38, and HCDR3 set forth in SEQ ID NO:39; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:42, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:43, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:25, HCDR2 set forth in SEQ ID NO:40, and HCDR3 set forth in SEQ ID NO:27; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:44, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:45, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:46, HCDR2 set forth in SEQ ID NO:40, and HCDR3 set forth in SEQ ID NO:27; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:44, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:45, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:47, HCDR2 set forth in SEQ ID NO:40, and HCDR3 set forth in SEQ ID NO:27; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:44, LCDR2 set forth in SEQ ID NO:23, and LCDR3 set forth in SEQ ID NO:45, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:48, HCDR2 set forth in SEQ ID NO:40, and HCDR3 set forth in SEQ ID NO:27; The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof include LCDR1 shown in SEQ ID NO: 22, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 45, and the antibody or antigen-binding fragment thereof The three heavy chain complementary determining regions of the segment include HCDR1 shown in SEQ ID NO:48, HCDR2 shown in SEQ ID NO:40, and HCDR3 shown in SEQ ID NO:27; or, The three light chain complementary determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:50, and the three heavy chain complementary determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:38, and HCDR3 shown in SEQ ID NO:

52.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:5, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:6; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17; The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19; or The antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 20, and / or a heavy chain variable region that is at least about 80%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

21.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The antibody comprises the heavy chain constant region shown in SEQ ID NO: 53, and / or the light chain constant region shown in SEQ ID NO:

54.

4. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A cell comprising the nucleic acid of claim 4.

6. An anti-LIV-1 antibody conjugate, characterized in that The anti-LIV-1 antibody conjugate comprises (a) a LIV-1 antibody or an antigen-binding fragment thereof, wherein the LIV-1 antibody or antigen-binding fragment thereof is the anti-LIV-1 antibody or antigen-binding fragment thereof described in any one of claims 1 to 3, and (b) a conjugated portion conjugated to the antibody portion; preferably, the conjugated portion is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

7. An anti-LIV-1 antibody drug conjugate, characterized in that The antibody drug conjugate is shown in Formula 1 below: wherein: Ab is the anti-LIV-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, LU is the linker; D is for medicine; The subscript p corresponds to the average DAR value of the antibody-drug conjugate, and p is a value selected from 1-10, preferably 1-8, more preferably 1-4 or 4-8, and most preferably p is 4.

8. The antibody-drug conjugate according to claim 7, characterized in that The LU-D is vcMMAE, vc is a valine-citrulline linker, and MMAE is monomethyl auristatin E.

9. The antibody-drug conjugate according to claim 7, characterized in that: The BNLD11 structure is shown below:

10. The antibody drug conjugate according to any one of claims 7 to 9, characterized in that: The antibody-drug conjugate is any one of the following (1)-(6): (1) The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:41, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40, and HCDR3 shown in SEQ ID NO:27, and the LU-D is BNLD11; (2) The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO: 22, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 41, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO: 25, HCDR2 shown in SEQ ID NO: 40, and HCDR3 shown in SEQ ID NO:

27. for vcMMAE; (3) the three light chain complementary determining regions of the Ab comprise LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:23, and LCDR3 of SEQ ID NO:24, the three heavy chain complementary determining regions comprise HCDR1 of SEQ ID NO:25, HCDR2 of SEQ ID NO:40, and HCDR3 of SEQ ID NO:27, and the LU-D is vcMMAE; (4) the three light chain complementary determining regions of the Ab comprise LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:23, and LCDR3 of SEQ ID NO:45, the three heavy chain complementary determining regions comprise HCDR1 of SEQ ID NO:48, HCDR2 of SEQ ID NO:40, and HCDR3 of SEQ ID NO:27, and the LU-D is vcMMAE; (5) the three light chain complementary determining regions of the Ab comprise LCDR1 of SEQ ID NO:44, LCDR2 of SEQ ID NO:23, and LCDR3 of SEQ ID NO:45, the three heavy chain complementary determining regions comprise HCDR1 of SEQ ID NO:48, HCDR2 of SEQ ID NO:40, and HCDR3 of SEQ ID NO:27, and the LU-D is vcMMAE; or, (6) The three light chain complementary determining regions of the Ab include LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:49, and LCDR3 shown in SEQ ID NO:50, and the three heavy chain complementary determining regions include HCDR1 shown in SEQ ID NO:51, HCDR2 shown in SEQ ID NO:38, and HCDR3 shown in SEQ ID NO:52, and the LU-D is vcMMAE.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid according to claim 4, or the cell according to claim 5, or the anti-LIV-1 antibody conjugate according to claim 6, or the antibody-drug conjugate according to any one of claims 7 to 10.

12. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid according to claim 4, or the anti-LIV-1 antibody conjugate according to claim 6, or the antibody-drug conjugate according to any one of claims 7 to 10, or the pharmaceutical composition according to claim 11.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid according to claim 4, or the anti-LIV-1 antibody conjugate according to claim 6, or the antibody-drug conjugate according to any one of claims 7 to 10, or the pharmaceutical composition according to claim 11 for preventing, treating, detecting or diagnosing a disease associated with LIV-1; preferably, the LIV-1-related disease includes cancer; more preferably, the cancer is an LIV-1-positive solid tumor; most preferably, the cancer includes one or more of breast cancer, prostate cancer, pancreatic cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer, esophageal squamous cell carcinoma, gastric cancer and melanoma.

14. A method for treating or preventing an LIV-1-related disease, comprising administering to a subject the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, or the nucleic acid of claim 4, or the anti-LIV-1 antibody conjugate of claim 6, or the antibody-drug conjugate of any one of claims 7 to 10, or the pharmaceutical composition of claim 11; preferably, the LIV-1-related disease comprises cancer; more preferably, the cancer is an LIV-1-positive solid tumor; most preferably, the cancer comprises one or more of breast cancer, prostate cancer, pancreatic cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer, esophageal squamous cell carcinoma, gastric cancer, and melanoma.

15. Use of the anti-LIV-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of an antibody-drug conjugate.

Images