WO2023241621A1 - Anti-liv-1-antibody and antibody-drug conjugate - Google Patents

Abstract

The present invention provides an anti-LIV-1-antibody and an antibody-drug conjugate (ADC). The anti-LIV-1-antibody or an antigen-binding fragment thereof comprises three light-chain complementary determining regions and/or three heavy-chain complementary determining regions of specific amino acid sequences. Specifically, the present invention also provides a nucleic acid encoding the antibody or the antigen-binding fragment thereof, a cell comprising the nucleic acid, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, the nucleic acid or the cell, and use of the LIV-1 antibody or the antigen-binding fragment thereof in preventing, treating, detecting or diagnosing a disease related to LIV-1. The anti-LIV-1-antibody or the antigen-binding fragment thereof provided herein has a high affinity for LIV-1 and cells expressing LIV-1. The anti-LIV-1-antibody-drug conjugate has a good tumor inhibitory effect in an animal tumor transplantation model, and features mild adverse effects, low toxicity and thus high safety.

Description

Anti-LIV-1 antibodies and drug conjugates Technical field

The present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to an anti-LIV-1 antibody and its drug conjugate.

Background technique

LIV-1 (also known as SLC39A6 or ZIP6) is a multi-transmembrane protein consisting of eight transmembrane domain proteins with specific zinc transporter and metalloprotease activities. It is mainly located on the plasma membrane and transports zinc to in cells.

LIV-1 belongs to the SLC39A family of zinc transporters. Intracellular zinc levels are tightly regulated by specific zinc transporters. It is currently known that mammals have two complementary zinc transporter families (SLC39A and SLC30A) that are directly involved in the steady-state metabolism of intracellular zinc ions. The SLC39A family has been shown to promote the transport of zinc ions from extracellular or intracellular organelles into the cytoplasm. The SLC30A family (ZnT family) has the opposite function to the SLC39A family. Multiple family members can assist the efflux of zinc ions from the cytoplasm to the outside of the cell or into the cell. within the cell organelle.

LIV-1 is highly expressed in various tumor cells such as breast cancer, prostate cancer, pancreatic cancer, cervical cancer, and liver cancer, but has limited expression in normal tissues. Breast cancer is the most common malignant tumor in women. Compared with normal breast tissue, zinc ions are highly enriched in breast tumor cells. LIV-1 is the first estrogen-induced expression found in breast cancer cells. Zinc ion transporter, 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 be related to the occurrence and development of other cancers (prostate cancer, pancreatic cancer, etc.). Pancreatic cancer is a highly malignant digestive system tumor and is prone to metastasis. The expression of LIV-1 in pancreatic cancer tissue is significantly higher than that in normal pancreatic tissue.

Antibody-drug conjugates (antibody-drug conjugates, ADC) conjugate antibodies and small molecule chemotherapy drugs through linkers, which not only have the high targeting properties of antibodies, but also can give full play to chemotherapy drugs Cytotoxicity, achieving the effect of killing tumor cells efficiently. ADC technology has gradually matured. Currently, multiple antibody drug conjugates have been successfully launched, and multiple products under development are in the late clinical stage. Based on the difference in LIV-1 expression levels, LIV-1 may be an ideal target for the development of antibody-conjugated drugs.

In view of the above, LIV-1 antibody-drug conjugates are of great significance as an anti-cancer drug, especially in cancers that highly express LIV-1. How to provide a drug targeting cancers that highly express LIV-1 has become an urgent problem to be solved.

Contents of the invention

The invention provides an anti-LIV-1 antibody or an antigen-binding fragment thereof, which can bind to LIV-1. The invention also provides nucleic acids encoding the antibodies or antigen-binding fragments thereof; cells containing the nucleic acids; anti-LIV-1 antibody conjugates and anti-LIV-1 antibodies comprising the anti-LIV-1 antibodies or antigen-binding fragments thereof. 1 Antibody drug conjugate; a pharmaceutical combination containing the antibody or antigen-binding fragment thereof, the nucleic acid, the cell, the anti-LIV-1 antibody conjugate, and the anti-LIV-1 antibody drug conjugate object; a kit containing the antibody or antigen-binding fragment thereof, the nucleic acid, the anti-LIV-1 antibody conjugate, the anti-LIV-1 antibody drug conjugate, and the pharmaceutical composition; the Antibodies or antigen-binding fragments thereof, the nucleic acids, the anti-LIV-1 antibody conjugates, the anti-LIV-1 antibody drug conjugates, and the pharmaceutical compositions are used in the prevention, treatment, detection or diagnosis of LIV- 1-related diseases, and the use of anti-LIV-1 antibodies or antigen-binding fragments thereof in the preparation of antibody-drug conjugates (ADCs).

One aspect of the invention provides an anti-LIV-1-antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof The segment contains the following 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions,

The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:41, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; Preferably, The antibody number is CA365NQ;

The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:24, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; Preferably, The antibody number is CA365;

The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:24, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26 and HCDR3 shown in SEQ ID NO:27; Preferably, The antibody number is CA317;

The three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof include LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32 and HCDR3 shown in SEQ ID NO:33; Preferably, The antibody number is CA332;

The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:37, HCDR2 shown in SEQ ID NO:38 and HCDR3 shown in SEQ ID NO:39; Preferably, The antibody number is CA336;

The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:42, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:43, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; Preferably, The antibody number is CA406;

The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:45, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:46, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; Preferably, The antibody number is CA454;

The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:45, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:47, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; Preferably, The antibody number is CA478;

The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:45, and/or The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include 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 number is CA672;

The three light chain complementarity 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/or The three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof include HCDR1 shown in SEQ ID NO:48 and HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; preferably, the antibody number is CA789; or

The three light chain complementarity 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 complementarity 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 number is CA1328.

In a specific embodiment of the invention, the invention provides an anti-LIV-1 antibody or antigen-binding fragment thereof, which is characterized by:

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 set forth in SEQ ID NO: 9, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 8; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 9, and/or the heavy chain variable region shown in SEQ ID NO: 8; more preferably, the antibody number is CA365NQ;

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 set forth in SEQ ID NO: 7, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 8; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO:7, and/or the heavy chain variable region shown in SEQ ID NO:8; more preferably, the antibody number is CA365;

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 set forth in SEQ ID NO: 1, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 2; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 1, and/or the heavy chain variable region shown in SEQ ID NO: 2; more preferably, the antibody number is CA317;

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 set forth in SEQ ID NO: 3, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 4; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 3, and/or the heavy chain variable region shown in SEQ ID NO: 4; more preferably, the antibody number is CA332;

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 set forth in SEQ ID NO: 5, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 6; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 5, and/or the heavy chain variable region shown in SEQ ID NO: 6; more preferably, the antibody number is CA336;

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 set forth in SEQ ID NO: 10, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 11; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 10, and/or the heavy chain variable region shown in SEQ ID NO: 11; more preferably, the antibody number is CA406;

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 set forth in SEQ ID NO: 12, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence set forth in SEQ ID NO: 13; preferably, The antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO: 12, and/or the heavy chain variable region shown in SEQ ID NO: 13; more preferably, the antibody number is CA454 ;

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 set forth in SEQ ID NO: 14, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 15; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 14, and/or the heavy chain variable region shown in SEQ ID NO: 15; more preferably, the antibody number is CA478;

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 set forth in SEQ ID NO: 16, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 17; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 16, and/or the heavy chain variable region shown in SEQ ID NO: 17; more preferably, the antibody number is CA672;

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 set forth in SEQ ID NO: 18, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 19; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 18, and/or the heavy chain variable region shown in SEQ ID NO: 19; more preferably, the antibody number is CA789; 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 set forth in SEQ ID NO: 20, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 21; preferably, the antibody or its The antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 20, and/or the heavy chain variable region shown in SEQ ID NO: 21; more preferably, the antibody number is CA1328.

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.

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

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

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

A third aspect of the invention provides a vector comprising said nucleic acid encoding said 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 lentiviral vector, adenoviral vector, adeno-associated virus vector or retroviral vector, etc.; Preferably, the vector can be 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.

A fourth aspect of the invention provides a cell, said cell comprising said nucleic acid or said vector, said cell expressing said antibody or antigen-binding fragment thereof. Preferably, the cells are bacterial cells; Preferably, the bacterial cells are E. coli cells, etc.; Preferably, the cells are fungal cells; Preferably, the fungal cells are yeast cells; Preferably, the yeast The cells are Pichia pastoris cells, etc.; preferably, the cells are mammalian cells; preferably, the mammalian cells are Chinese hamster ovary cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells or NK cells etc.

The fifth aspect of the present invention provides an anti-LIV-1 antibody conjugate, characterized in that the anti-LIV-1 antibody conjugate includes the LIV-1 antibody or antigen-binding fragment thereof described in (a), and (b) a coupling moiety coupled to the antibody moiety, the coupling moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In another preferred embodiment, the drug is selected from the group consisting of chemotherapy drugs, radiotherapy drugs, hormone therapy drugs or immunotherapy drugs.

In another preferred embodiment, the drug or toxin is selected from the following group: taxanes, maytansinoid, auristatin, calicheamicin, Anthracycline, docetaxel, cathepsin, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin toxin), ribonuclease (RNase) and radioactive isotope (radioisotope).

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) , the antibody drug conjugate ADC is represented by the following formula 1:

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

LU is a linker (also called a connector);

D is drug;

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

Further, the linker LU is composed of the general formula R’-L1-L2-L3;

In this general formula, the L3 is:

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

_R1 is hydrogen, carboxyl, ester, nitro, sulfonyl, halogen group; or R1 is R ₂ to R ₆ are each independently hydrogen, n is 0-8;

In this general formula, the L2 is: Among them, 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, and a lysine residue. amino acid residue, proline residue, or valine, guanidine residue, β-glycine residue, β-alanine residue; X is: n is 0-8;

In this general formula, the L1 is: and / or

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

In another preferred example, the LU-D is vcMMAE, vc is the 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.

In another preferred example, LU-D in formula 1 of the antibody drug conjugate (ADC) is BNLD11, where LU is MC-β-Ala-(glucuronide)PAB, D is MMAE, and the structure of BNLD11 is as follows Shown:

, 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 Figure 23.

Further, the present invention provides an anti-LIV-1 antibody conjugate, wherein,

The three light chain complementarity 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 complementarity 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, the LU-D is BNLD11;

The three light chain complementarity 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 complementarity 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, the LU-D is vcMMAE;

The three light chain complementarity determining regions of the Ab include LCDR1 shown in SEQ ID NO:22, SEQ ID NO:23 LCDR2 and LCDR3 shown in SEQ ID NO:24, the three heavy chain complementarity 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 , the LU-D is vcMMAE;

The three light chain complementarity 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 complementarity 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, the LU-D is vcMMAE;

The three light chain complementarity 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 complementarity 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, the LU-D is vcMMAE; or,

The three light chain complementarity 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 complementarity 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, the LU-D is vcMMAE;

Further, 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.

The seventh aspect of the present invention provides a pharmaceutical composition, which includes 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 also includes a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvents, dispersants, additional agents, plasticizers, pharmaceutical excipients.

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

The ninth aspect of the present invention provides the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells, anti-LIV-1 antibody conjugates, anti-LIV-1 antibody drug conjugates, or pharmaceutical compositions of the present invention. Application in pharmaceutical compositions for treating or preventing diseases.

The tenth aspect of the present invention provides the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells, anti-LIV-1 antibody conjugates, anti-LIV-1 antibody drug conjugates, or pharmaceutical compositions of the present invention. Application in diagnostic and detection kits.

The eleventh aspect of the present invention provides a method for treating or preventing diseases, including using the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, anti-LIV-1 The antibody drug conjugate, or pharmaceutical composition is administered to a subject in need. Further, the disease is a LIV-1 related disease; further, the LIV-1 related disease includes cancer; further, the cancer is a LIV-1 positive solid tumor; further, the cancer includes breast One or more of prostate cancer, pancreatic cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer, esophageal squamous cell carcinoma, gastric cancer, and melanoma.

The twelfth aspect of the present invention provides a method for diagnosis and detection, which includes combining the antibody of the present invention or its antigen-binding fragment, nucleic acid, vector, cell, anti-LIV-1 antibody conjugate, and anti-LIV-1 antibody. The drug conjugate, or pharmaceutical composition is administered to a subject or sample in need.

The thirteenth aspect of the present invention provides the antibodies of the present invention or antigen-binding fragments thereof, nucleic acids, vectors, cells, anti-LIV-1 antibody conjugates, anti-LIV-1 antibody drug conjugates, or pharmaceutical compositions. For the treatment and prevention of diseases.

The fourteenth aspect of the present invention provides the antibodies of the present invention or antigen-binding fragments thereof, nucleic acids, vectors, cells, anti-LIV-1 antibody conjugates, anti-LIV-1 antibody drug conjugates, kits or pharmaceutical combinations Materials are used for detection and diagnosis purposes.

The fifteenth aspect of the present invention provides the antibodies of the present invention or antigen-binding fragments thereof, nucleic acids, vectors, cells, anti-LIV-1 antibody conjugates, anti-LIV-1 antibody drug conjugates, kits or pharmaceutical combinations Applications of substances for preventing, treating, detecting or diagnosing diseases related to LIV-1.

In aspects of the present invention, the disease associated with LIV-1 includes cancer.

In aspects of the present invention, the LIV-1-related diseases include LIV-1-positive solid tumors.

In the scheme of the present invention, the LIV-1-related diseases include one or more of melanoma, mesothelioma, pancreatic cancer, non-small cell lung cancer, breast cancer and colon cancer.

A sixteenth aspect of the present invention provides the use of LIV-1 antibodies or antigen-binding fragments thereof in the preparation of antibody-drug conjugates.

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

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

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

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

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

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

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

7. The anti-LIV-1 antibody drug conjugate provided by the present invention has low side effects and toxicity, and is highly safe.

Description of the drawings

Figure 1 shows the results of mouse serum antibody titer detection after the third LIV-1 booster immunization;

Figure 2A-Figure 2B shows the binding activity curves of each LIV-1 antibody and human LIV-1 at the protein level;

Figure 3A-Figure 3B shows the binding activity curve of each LIV-1 antibody on MCF-7/LIV-1 cells;

Figure 4A-Figure 4B shows the binding activity curves of each LIV-1 antibody and cynomolgus monkey LIV-1 protein;

Figure 5A-Figure 5B shows the binding activity curves of each LIV-1 antibody and mouse LIV-1 antigen;

Figure 6 shows the internalization detection results of each LIV-1 antibody;

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

Figure 8 shows the results of the in vitro killing effect of each LIV-1 antibody conjugate on MCF-7/LIV-1 cells;

Figure 9 shows the results of the in vitro killing effect of each LIV-1 antibody conjugate on MDA-MB-231/LIV-1 cells;

Figure 10A-Figure 10B shows the tumor growth curve and tumor photos of each LIV-1 antibody conjugate at a high dose of 5 mg/kg on the MCF-7/LIV-1 breast cancer cell/mouse xenograft tumor model;

Figure 11 shows the tumor growth curve of each LIV-1 antibody conjugate at a low dose of 2.5 mg/kg on the MCF-7/LIV-1 breast cancer cell/mouse xenograft tumor model;

Figure 12 shows the drug-time curve of each LIV-1 antibody in Balb/c mice.

Figure 13 shows tumor growth of LIV-1 antibody conjugate CA365NQ-vcMMAE at 2.5 mg/kg dose, solvent control group (PBS), and negative control group on M-NSG mice transplanted with human MCF-7 human breast cancer cells. curve.

Figure 14 shows the tumor weight of LIV-1 antibody conjugate CA365NQ-vcMMAE at 2.5 mg/kg dose, solvent control group (PBS), and negative control group on M-NSG mice transplanted with human MCF-7 human breast cancer cells. Inhibition graph.

Figure 15 shows the body weight curve of M-NSG mice transplanted with human MCF-7 human breast cancer cells at a dose of 2.5 mg/kg of LIV-1 antibody conjugate CA365NQ-vcMMAE, solvent control group (PBS), and negative control group. .

Figure 16 shows tumor growth of LIV-1 antibody conjugates CA365NQ-vcMMAE, hLiv22-vcMMAE, vehicle control group (PBS) at 2.5 mg/kg dose on M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells. curve.

Figure 17 shows the tumor weight of LIV-1 antibody conjugate CA365NQ-vcMMAE, hLiv22-vcMMAE, vehicle control group (PBS) at 2.5 mg/kg dose on M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells. Inhibition graph.

Figure 18 shows the body weight curve of M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells using 2.5 mg/kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, hLiv22-vcMMAE, and solvent control group (PBS). .

Figure 19 shows the effects of 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 transplanted with MCF7-hLIV1 human breast cancer cells. -Nude tumor growth curve on nude mice.

Figure 20 shows the Balb/c transplantation of 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. -Body weight curve of nude mice.

Figure 21 shows the body weight curve of Balb/c mice in the single-dose toxicity experiment of the LIV-1 antibody conjugate CA365NQ-vcMMAE group and the solvent control group (PBS) at a dose of 50 mg/kg.

Figure 22 shows the body weight curve of Balb/c mice in the single-dose toxicity experiment of the 50 mg/kg LIV-1 antibody conjugate CA1328-vcMMAE group and the solvent control group (PBS).

Figure 23 shows a schematic diagram of the synthesis route of BNLD11.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. The described embodiments are some, but not all, of the embodiments of the present invention. It should be understood that the following examples are set forth to provide a complete disclosure and explanation of how to utilize the methods and compositions of the present invention to those skilled in the art to which the present invention belongs, 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 efforts fall within the scope of protection of the present invention.

For statistical analysis in the embodiments of the present invention, the result analysis is expressed as mean and standard error (Mean ± SEM), and Graphpad 8.0 software is used for data analysis and processing. Tumor volume and body weight are compared with each group at each time point using two-factor analysis of variance. The difference between the two groups; tumor weight was analyzed using one-way variance analysis; T-test was used to compare the two groups; P<0.05 indicated that the difference was statistically significant.

Example 1. Production of anti-LIV-1 monoclonal antibodies

1.1 Mouse immunization method

Fully human antibody transgenic mice for immune experiments (R&D and produced by Shandong Boan Biotechnology Co., Ltd.), 2 types of antigen proteins were used for immunization in two batches, and a total of 13 animals were immunized. The first batch used His Tag fused LIV-1-His (manufacturer ACRO, product number LV1-H5223) antigen protein to immunize 5 fully human antibody transgenic mice (Q06-Q10). The second batch was immunized with human IgG1Fc-fused LIV-1-Fc (manufacturer ACRO, product number LV1-H5254) and His Tag-fused LIV-1-His (manufacturer ACRO, product number LV1-H5223) antigen proteins, with a total of 8 immunizations. Fully human antibody transgenic mice, of which 5 were immunized with human IgG1Fc fused LIV-1-Fc (manufacturer ACRO, catalog number LV1-H5254), numbered Q11-Q15, and His Tag fused LIV-1-His (ACRO, LV1 -H5223) immunized 3 animals, numbered Q16-Q18. All mice were immunized by subcutaneous injection in the abdomen and multiple points in the groin, and the immunization dose was 20 μg/mouse. The antigen was emulsified in Freund's complete adjuvant for the first immunization, and the antigen was emulsified in Freund's incomplete adjuvant for subsequent immunizations. Fully human antibody transgenic mice underwent a total of 1 immunization and 4 booster immunizations, and the immunization intervals were all 14 days.

Starting from the second immunization, peripheral blood serum was collected on the 7th day after each immunization to detect antibody titers and mice with unqualified titers were eliminated. The results of mouse serum antibody titer detection after the third booster immunization are shown in Figure 1. 500X, 2500X, 12500X, and 62500X represent dilution ratios. Three days after the fourth booster immunization, the mice were euthanized, and the spleens were harvested to prepare single cells for bank construction.

1.2 Establishment of phage library

The mice were sacrificed, the spleens were removed by dissection, and RNA was extracted to obtain cDNA. The phage library was established according to the usual method. The storage capacity data of the built library is shown in Table 1.

Table 1 The phage library capacity constructed by each immunized mouse

1.3 Screening using two methods: plate screening and magnetic bead screening

1.3.1 Plate screening, coat the plate with His tag 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 then use elution buffer to elute the specifically bound phages.

1.3.2 Magnetic bead screening, biotinylate LIV-1-His protein (Manufacturer Acro, Catalog No. LV1-H5223) or LIV-1-Fc protein (Manufacturer Acro, Catalog No. LV1-H5254) according to the kit steps, and then use it with Thermo magnetic beads are combined, incubated with the phage library, washed 4-10 times, and then the specifically bound phages are eluted with Elution Buffer.

Table 2 List of sources of LIV-1 antibodies obtained by screening

Based on the CA365 clone, a point mutation was performed on one of the amino acid sites to obtain the CA365NQ clone (see Table 3 for the amino acid sequence of the variable region).

Example 2. Molecular construction and production of intact antibodies

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

Table 3 Variable region sequences of 11 lead antibody amino acid sequences

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

The antibody variable region genes were amplified by conventional molecular biology technology PCR (2×Phanta Max Master Mix, manufacturer Vazyme, product number P515-P1-AA), and the antibody heavy chain variable region genes were connected into the band through homologous recombination. The vector pCDNA3.4 (Life Technology) has the nucleic acid sequence of the antibody heavy chain constant region sequence, and the antibody light chain variable region gene is 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) comes from patent US 2020/0165335 A1, IgG1/Kappa.

Except for the control antibody hLiv22, the variable region sequences of each antibody 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.

Table 4 Amino acid sequences of heavy chain and light chain constant regions of 11 lead antibodies

The amino acid sequence of hLiv22 heavy chain is shown in SEQ ID NO:55, specifically as follows:

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

The sequenced positive clones were extracted with plasmids and co-transfected into HEK293 cells, cultured in a shaker at 37°C\8% _CO2 \125rpm. After transient expression for 7 days, the supernatant was passed through Protein A affinity chromatography, and the antibody was purified and passed through UV280 combined with theoretical extinction coefficient determines antibody concentration.

Example 3. Characterization of LIV-1 monoclonal antibody molecules

3.1 Binding activity of LIV-1 antibody to human LIV-1 protein level

Use carbonate buffer to coat human LIV-1 protein at 0.2 μg/mL, 100 μL/well, and incubate at 4°C overnight. Block with 3% skimmed milk powder, 300 μL/well, incubate at 37°C for 1 hour and then wash the plate twice. Use PBST (phosphate buffer saline + 0.05 Tween-20) to dilute the complete antibody in 8 gradients four-fold at 0.2 μg/mL, 100 μL per well, incubate at 37°C for 1 hour, and wash the plate twice. Add goat anti-human IgG (H+L)/HRP (1:5000 dilution), 100 μL/well, incubate at 37°C for 1 hour, and wash the plate 5 times. Add 100 μL TMB to each well, and after 10 minutes of color development, add 50 μL 2M H ₂ SO ₄ to each well to terminate the reaction, and read the OD450 value on a multifunctional microplate reader. Figure 2A (involving the newly screened antibodies CA332, CA317, CA365, CA406, CA672, CA336 and the reference antibody hLiv22) and Figure 2B (involving the newly screened antibodies CA1328, CA454, CA478, CA789 and the reference antibody hLiv22) are for each LIV-1 Binding curve of antibody and human LIV-1 at protein level. The EC50 value was calculated from the curves in Figure 2A-2B. Table 5 shows the EC50 value of each antibody binding to human LIV-1 protein. It can be seen that the newly screened antibody has higher binding sensitivity than the reference antibody.

Table 5 EC50 value of LIV-1 antibody binding to LIV-1 antigen

3.2 Binding ability of LIV-1 antibody on cells

The LIV-1 overexpression cell line MCF-7/LIV-1 (breast cancer cell line) was purchased from Sanyou Biomedicine (Shanghai) Co., Ltd., and the culture conditions were RPMI 1640 medium plus 10% FBS and 0.5 μg/mL. Puromycin, culture at 37°C, 5% _CO2 . First, dilute the antibody to the corresponding concentration and add it to a round-bottom 96-well plate, then add digested MCF-7/LIV-1 cells, 100,000 cells/well. After incubation at 4°C for 1 hour, wash once with PBS and add secondary antibody FITC-anti-human IgG at a ratio of 1:500. After incubating at 4°C for 0.5 hours, wash once with PBS and detect the fluorescence intensity of the cells using a cell flow cytometer. The binding activity results of each LIV-1 antibody on MCF-7/LIV-1 cells are shown in Figure 3A (involving antibodies CA317, CA365, CA478, CA672, CA454, CA789 and hLiv22) and Figure 3B (involving antibodies CA336, CA1328 and hLiv22). The binding EC50 values of each antibody on MCF-7/LIV-1 cells are shown in Table 6. It can be seen that these newly screened antibodies have good cell-binding activity, among which CA317/CA365/CA454/CA478/CA672/CA789 have strong to the reference antibody.

Table 6 EC50 value of LIV-1 antibody binding on MCF-7/LIV-1 cells

3.3 Species cross-activity of LIV-1 antibodies and cynomolgus monkey and mouse LIV-1 proteins

Use carbonate buffer to coat cynomolgus monkey LIV-1 protein (Manufacturer Acro, Catalog No. LV1-C52H5) 0.2 μg/mL and mouse LIV-1 protein (Manufacturer Acro, Catalog No. LV1-M5223) 1 μg/mL, 100 μL/ well, overnight at 4°C. Block with 3% skimmed milk powder, 300 μL/well, incubate at 37°C for 1 hour and then wash the plate twice. When detecting binding to mouse LIV-1 protein, complete antibody was diluted in PBST (phosphate buffer saline + 0.05 Tween-20) gradient, 4 μg/mL was diluted four times in 6 gradients, and when binding to cynomolgus monkey LIV-1 protein was detected , PBST (phosphate buffer saline + 0.05 Tween-20) gradient dilution of complete antibody, 0.25 μg/mL four-fold dilution 6 gradients, 100 μL per well, incubate at 37°C for 1 hour, wash the plate twice. Add goat anti-human IgG (H+L)/HRP (1:5000 dilution), 100 μL/well, incubate at 37°C for 1 hour, and wash the plate 5 times. Add 100 μL TMB to each well, and after 10 minutes of color development, add 50 μL 2M H ₂ SO ₄ to each well to terminate the reaction. 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 is the corresponding EC50 value. It can be seen that all antibodies can bind to the cynomolgus monkey LIV-1 antigen.

Table 7 EC50 value of LIV-1 antibody binding to cynomolgus monkey LIV-1 protein

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 mouse LIV-1 antigen. It can be seen that CA478 antibody, CA454 and CA672 bind to mouse protein, while the remaining antibodies do not bind to mouse LIV-1 protein.

3.4 Affinity of LIV-1 antibodies to human and rat LIV-1 proteins

Use ProA chip to capture LIV-1 antibody 2μg/mL, and analyze the binding activity of LIV-1 antibody to human LIV-1 and rat LIV-1. LIV-1 protein was diluted twice to 5 concentrations with HBS-EP+ buffer, with a starting concentration of 50 nM. Biacore8K was used to analyze LIV-1 protein binding kinetics, and the affinity activity KD value was 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.

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

3.5 LIV-1 antibody epitope competition analysis

First, LIV-1 antibodies were sequence grouped based on sequence similarity. It was divided into three groups. The first group included antibody CA332; the second group included CA336/CA560/CA1328; and the third group included CA317/CA365/CA406/CA454/CA478/CA672/CA789. Each group selected 1-2 antibodies for epitope analysis. The first group selected antibody CA332, the second group selected antibody CA560, and the third group selected antibodies CA454 and CA789. LIV-1 protein 10μg/mL was captured using His chip. First, saturate the binding of the first LIV-1 antibody 1, and then add the other LIV-1 antibody 2. Observe the response value of antibody 2 on Octet and analyze whether there is epitope competition between antibody 1 and antibody 2. The value is higher than 75%, indicating that the epitopes are relevant. Table 9 shows the epitope analysis results. It can be seen that the epitopes of the third group of antibodies and the reference antibody hLiv22 overlap, and the first group of antibodies and the second group of antibodies have unique epitopes.

Table 9 LIV-1 antibody epitope analysis

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

First, use complete medium (RPMI1640: manufacturer gibco, product number 21870092; fetal bovine serum: manufacturer gibco, product number 10099-141) to prepare the antibody to be tested to 24 μg/mL, and add 25 μL/well to a 96-well U-shaped bottom plate (manufacturer SARSTEDT, item number 83.3925.500). Then use complete medium to prepare Zenon ^TM pHrodo ^TM iFL IgG labeling reagent (Manufacturer Invitrogen, Cat. No. Z25612) to 24 μg/mL, add 25 μL/well to the antibody in step (1), and incubate at room temperature to mix the antibody to be tested and the labeling reagent. Form a complex. Then digest the MCF-7/LIV-1 (also known as MCF-7-LIV-1, manufacturer Sanyou Biotech, product number BA001-MCF-7) cells, wash them twice with PBS, and adjust the cell density with complete culture medium to Add 2×10 ⁶ /mL, 50 μL/well to the complex in step (2), and incubate at 37 degrees for 0 hours, 2 hours, 6 hours, and 24 hours respectively. Finally, at different incubation times, the cells were washed twice with PBS and then the MFI (mean fluorescence intensity) value was detected by flow cytometry (manufacturer ACEA, model NovoCyte). Figure 6 shows the internalization detection results of each LIV-1 antibody (CA317, CA365, CA478, CA406, CA454, CA789, CA336, CA672 and hLiv22). isotype represents the isotype control of the antibody (different from the LIV-1 IgG1 antibody variable region, Irrelevant antibodies with the same constant region), label represents the dye without labeled antibody. from As can be seen from the results in Figure 6, the CA336 antibody has the best internalization performance, the CA478 antibody has the worst internalization performance, and the other antibodies have similar internalization capabilities.

Example 5: Antibody-dependent cell-mediated cytotoxicity (ADCC) (effector cells containing luciferase reporter gene)

Prepare ADCC reaction buffer, RPMI 1640 culture 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 use ADCC buffer Use ADCC buffer to adjust the cell density to 2.4×10 ⁶ /mL; collect target cells, human breast cancer MCF-7/LIV-1 (manufacturer Sanyou Biotech, product number BA001-MCF-7), and use ADCC buffer to adjust the cell density to 8× 10 ⁵ /mL; dilute the sample to be tested with ADCC buffer, dilute 4 times to 8 concentrations; add 25 μL each of effector cells, target cells and sample to be tested to the reaction white plate (Manufacturer Costar, Cat. No. 3917), the total reaction The volume is 75 μL and incubated at 37 degrees for 6 hours; then add 75 μL Bio-Glo Luciferase System (Manufacturer Promega, Cat. No. G7940) to each well, and after 15 minutes of reaction, use a microplate reader to read the chemiluminescence value (Manufacturer BioTek, model synergy neo2). Figure 7 shows the results of the ADCC activity of each LIV-1 antibody (involving CA317, CA365, CA365NQ, CA406, CA672, CA789, CA1328 and hLiv22) based on the human breast cancer MCF-7/LIV-1 cell line. Figure 7 shows that CA365, CA672, and CA406 have better ADCC effects.

Example 6 Preparation of anti-LIV-1 antibody conjugates

Prepare an antibody-drug conjugate represented by the following molecular formula:

Among them: Ab is the antibody of LIV-1, LU is the linker (also called linker); D is the drug; and the subscript p is the average DAR value of the antibody-drug conjugate.

6.1 Preparation of anti-LIV-1 antibody-vcMMAE conjugate

Taking anti-LIV-1 antibodies CA332, CA336, CA365, CA406, CA454, CA672, CA789 and control antibody hLiv22 as examples, VcMMAE (Manufacturer MCE, Cat. No. HY-15575/CS-1242) was dissolved in DMSO and prepared into a 2mM solution, - Store at 80°C, take out before use, and thaw at room temperature. TCEP (Manufacturer Aldrich, Cat. No. C4706-10G, SLBT6121) is dissolved in purified water, prepared into a 10mM solution, stored at -80°C, taken out before use, and melted at room temperature. DTPA (manufacturer aladdin, product number D108512-100g) was dissolved in PBS, prepared into a 10mM solution, adjusted to pH 8.3 with sodium hydroxide solution, and stored at 4°C. Replace the antibody solution with PBS buffer system, pH 7.5, and adjust the antibody concentration to 5-10 mg/mL. When coupling, add 1/10 volume of DTPA solution to the antibody solution, then add TCEP solution with 2-3 times the mole number of antibody, and perform a reduction reaction at 25°C for 2 hours, then add VcMMAE solution with 4-6 times the mole number of antibody. React for 1 hour at 25°C. Use an ultrafiltration tube (molecular weight cutoff 30KD) to replace the buffer with PBS at a replacement rate of not less than 10,000 times, or replace with a desalting column (Sephadex G25) to remove free drug-linkers and other components. kind of additives. After HIC-HPLC analysis, the average DAR value of the coupled antibody-drug conjugate was 4±0.5. The proportions and average values of each DAR value of 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.

Table 10 Distribution of DAR values of anti-LIV-1 antibody-vcMMAE conjugates

Table 11 DAR value distribution of hLiv22-vcMMAE conjugates

6.2 Preparation of anti-LIV-1 antibody-BNLD11 conjugate

Taking anti-LIV-1 antibody CA365NQ as an example, exchange the antibody solution to PBS buffer, concentrate by ultrafiltration, adjust the antibody concentration to 5-10mg/ml, and add 1/10 volume of DTPA stock [11mM DTPA (Diethylenetriaminepentaacetic acid, CAS No. 67-43-6), dissolved in PBS, pH 7.2], final concentration of DTPA 1mM. Add 2-3 times the mole number of antibody to TCEP [Tris(2-carboxyethyl)phosphine, CAS No.51805-45-9. Dissolve in water to make a 2mM solution], react at 25°C for 2 hours, then add 6 times the mole number of antibody. BN-LD-11, coupling reaction at 25°C for 1 hour, add 10 times the mole number of antibody N-acetylcysteine (CAS No. 38520-57-9) to quench the remaining BNLD11, ultrafiltrate and dialyze against PBS for 10,000 times or gel filtration to remove various small molecules to obtain the ADC solution. The BSA method was used to determine the ADC concentration, and HIC-HPLC was used to determine the DAR distribution and average DAR value. The proportion of each DAR value of CA365NQ-BNLD11 and the average value are shown in Table 12.

Table 12 Distribution of DAR values of CA365NQ-BNLD11 conjugates

Example 7 Proliferation inhibitory activity of LIV-1 antibody conjugates on tumor cells in vitro

7.1 Proliferation inhibitory activity of anti-LIV-1 antibody conjugates on MCF-7/LIV-1 breast cancer cells

The LIV-1 overexpression cell line MCF-7/LIV-1 was purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd., and the culture conditions were RPMI-1640 medium plus 10% FBS and 0.5 μg/mL puromycin. Culture at 37°C, 5% _CO2 . MCF-7/LIV-1 cells were spread on a 96-well flat plate at a density of 5000 cells/well. After adhesion overnight, corresponding concentration of antibody conjugate was added. After continuing for 96 hours, Promega cell viability detection kit was used ( G7570) to detect cell viability. Figure 8 shows the effects of each LIV-1 antibody (involving CA332, CA789, CA672, CA454, CA336, CA406, CA365 antibody and control antibody hLiv22) conjugates on MCF-7/LIV-1 cells. Result diagram of proliferation inhibitory activity (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, which is the negative control group; hLiv22-vcMMAE is Example 6 Prepare the experimental group obtained). The EC50 table for each LIV-1 antibody conjugate is shown in Table 13. Figure 8 and Table 13 show that CA365-vcMMAE has the strongest proliferation inhibitory activity.

Table 13 Proliferation inhibitory activity of LIV-1 antibody conjugates on MCF-7/LIV-1 cells

7.2 Proliferation inhibitory activity of LIV-1 antibody conjugates on MDA-MB231/L1 breast cancer cells

The LIV-1 overexpression cell line MDA-MB231/LIV-1 (also known as MDA-MB-231/LIV-1) was purchased from Nanjing GenScript Biotechnology Co., Ltd., and the culture conditions were DMEM high-glucose medium plus 10% FBS, add 0.25 μg/mL puromycin, and culture at 37°C/5% _CO2 . MDA-MB231/LIV-1 cells were spread on a 96-well flat plate at a density of 5000 cells/well. After adhesion overnight, corresponding concentrations of antibody conjugates were added. After continuing for 96 hours, Promega cell viability detection kit ( G7570) to detect cell viability. Figure 9 is a graph showing the results of the proliferation inhibitory activity of each LIV-1 antibody (involving CA332, CA789, CA672, CA454, CA336, CA406, CA365 antibody and control antibody hLiv22) conjugates on MDA-MB231/LIV-1 cells ( Among them, 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, which is the negative control group). The EC50 table for each LIV-1 antibody conjugate is shown in Table 14. Figure 9 and Table 14 show that CA365-vcMMAE has the strongest proliferation inhibitory activity.

Table 14 Proliferation inhibitory activity of LIV-1 antibody conjugates on MDA-MB231/LIV-1 cells

Example 8 Evaluation of the efficacy of LIV-1 antibody conjugates on the MCF-7/LIV-1 breast cancer cell/mouse xenograft tumor model

M-NSG immunodeficient mice, female, 6-8 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and raised by the Animal Experiment Center of Shandong Boan Biotechnology Co., Ltd. The LIV-1 overexpression cell line MCF-7/LIV-1 was purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd., and the culture conditions were RPMI1640 medium plus 10% FBS and 0.5 μg/mL puromycin at 37°C. , cultured in 5% _CO2 . IRA SE-121-0.36 mg estradiol sustained-release tablets were purchased from Changzhou Xinlitai Biotechnology Co., Ltd.

Estradiol sustained-release tablets were inoculated into the left armpit of M-NSG mice using a sleeve needle. Three days later, MCF-7/LIV-1 cells were inoculated into the right side of the mice at a cell number of 1×10 ⁷ /mouse. Subcutaneous area on the back of the forelimb. Group administration was started when the average tumor volume reached approximately 100-150 ^mm3 . LIV-1 antibody conjugates were produced in two batches, a 5 mg/kg high dose batch and a 2.5 mg/kg low dose batch. Dosage batches, each batch is provided with a solvent control group and an antibody conjugate administration group. The solvent control group is also called the Vehicle group, and its component is PBS solution. The solvent control groups in Examples 8-12 are all PBS. solution. The method of administration is tail vein injection, administered once. The body weight and tumor volume of the mice were measured twice a week. Tumor volume (mm ³⁾ = long diameter PBS group, and hLiv22, CA672, CA789, CA406, CA1328, CA365, nCov-CA521 antibody) conjugates on the MCF-7/LIV-1 breast cancer cell/mouse xenograft tumor model 20-day tumor growth curve (where PBS represents the solvent control group, which is 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, which is the negative control group), Figure 10B is 5mg /kg high dose of each LIV-1 antibody (involving PBS, hLiv22, CA672, CA789, CA406, CA1328, CA365, nCov-CA521 antibody) conjugates in the MCF-7/LIV-1 breast cancer cell/mouse xenograft tumor model On the tumor photo, the numbers in the upper left corner of each cell in the photo, such as 925, 930, etc., are the experimental mouse numbers. Figure 10A and Figure 10B show that CA365-vcMMAE has the best tumor inhibition effect, and all 5 mice achieved tumor clearance. Three mice in each of the CA406-vcMMAE and CA789-vcMMAE groups achieved tumor clearance. Only 2 mice in the CA672-vcMMAE, CA1328-vcMMAE and hLiv22-vcMMAE control groups achieved tumor clearance. The efficacy results of 2.5 mg/kg low dose are shown in Figure 11. Figure 11 shows the conjugation of various LIV-1 antibodies (involving PBS, CA672, CA789, CA406, CA1328, CA365, nCov-CA521 antibodies) at 2.5 mg/kg low dose. The 24-day tumor growth curve of the antibody on the MCF-7/LIV-1 breast cancer cell/mouse xenograft tumor model (where PBS represents the solvent control group, that is, the blank control group; nCov-CA521 is an antibody that does not bind LIV-1, nCov-CA521-vcMMAE is an antibody conjugate that does not bind LIV-1, which is the negative control group). Table 15 shows the tumor volume growth inhibition rates of 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 The inhibition rates were 72.97%, 42.57%, 70.27%, 72.97%, and 59.46% respectively. Table 15 shows that CA365-vcMMAE, CA672-vcMMAE, and CA789-vcMMAE have good tumor inhibitory effects.

Table 15 Growth inhibition rate of tumor volume and tumor weight inhibition rate of anti-LIV-1ADC 2.5mg/kg

Example 9 Study on pharmacokinetics of LIV-1 antibody in mice

Each antibody was administered subcutaneously to 3 Balb/c mice at a dose of 5 mg/kg. The blood collection points were set at 0 hours before administration, 5 minutes after administration, 0.5 hours, 1 hour, 6 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days and 28 days. ELISA method was used to detect the antibody concentration in serum. Coat the antigen protein solution with carbonate buffer. Add 200 μL of blocking solution (2% BSA/PBST) to each well and incubate for 2 hours. The concentration range of the standard solution was set to 5-0.039 μg/mL. The sample was diluted 10 times and 100 times. Before loading, it was diluted 50 times with 1% BSA/PBST, 100 μL/well, and incubated at 37°C at 180 rpm for 1 hour. Then add 2WX goat anti-human IgG Fc-HRP, 100μL/well, 37℃ 180rpm Incubate for 1 hour. Add 100 μL/well of TMA chromogenic solution, keep at room temperature in the dark for 5-10 minutes, and finally terminate color development with 100 μL/well of 1M H ₃ PO ₄ . Use a microplate reader to read visible light at 450nm and 650nm, and use four-parameter fitting analysis. Table 16 shows the drug-time curve parameters. Figure 12 is the drug-time curve of each LIV-1 antibody (involving CA365NQ, CA365, CA672, CA1328, and hLiv22 antibodies) in Balb/c mice. Figure 12 and Table 16 show the half-life and blood circulation of CA365NQ, CA365, CA672, and CA1328 antibodies. Drug exposure was significantly higher than control antibody. In Table 16, mean represents the mean value and SD represents the standard deviation.

Table 16 Drug time curve parameters

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

MCF-7 human breast cancer cells were purchased from ATCC. The cells were cultured in an incubator at 37°C and 5% _CO2 . The medium composition was RMPI-1640 medium containing 10% FBS. M-NSG mice were purchased from Shanghai Southern Model 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 containing 50% Matrigel to a concentration of 1.0×10 ⁸ cells/mL, and then inoculated subcutaneously into the right side of M-NSG mice at a volume of 0.1 mL/mouse. When the average tumor volume reached ^130mm3 , the mice were divided into 3 experimental groups according to the tumor volume, with 6 animals in each group. The experimental groups were: CA365NQ-vcMMAE group, solvent control group (PBS), and negative control nCov- CA521-vcMMAE group. Administration will begin on the day of grouping, and the dosage will be 2.5 mg/kg in a single dose. The trial ended on the 24th day of group administration. Compared with the solvent control group, the CA365NQ-vcMMAE group can significantly inhibit the growth of tumor volume (P=0.001), and the tumor volume growth inhibition rate TGI (%) is 58.3%; the CA365NQ-vcMMAE group and the negative control nCov-CA521-vcMMAE group have Significant difference (P<0.05). The tumor growth curves of 2.5 mg/kg LIV-1 antibody conjugate CA365NQ-vcMMAE, solvent control group, and negative control group on M-NSG mice transplanted with human MCF-7 human breast cancer cells are shown in Figure 13.

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

During the entire process of the experiment, the experimental animals were active and eating well during the administration period, and the weight of each group increased to a certain extent, indicating that the animals had good tolerance to the test product. There was no significant difference between each group (P>0.05). The body weight curves of M-NSG mice transplanted with human MCF-7 human breast cancer cells using 2.5 mg/kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, solvent control group, and negative control group are shown in Figure 15.

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

NCI-H82 human small cell lung cancer cells were purchased from ATCC. The cells were cultured in an incubator at 37°C and 5% _CO2 . The medium composition was RMPI-1640 medium containing 10% FBS. M-NSG mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd. The concentration of NCI-H82 cells was adjusted to 5.0 × 10 ⁷ cells/mL using serum-free RMPI-1640 medium containing 50% Matrigel, and then inoculated subcutaneously into the right side of Balb/c nude mice at a volume of 0.1 mL/mouse. When the average tumor volume reached ^174mm3 , the mice were divided into 3 experimental groups according to the tumor volume, with 5 animals in each group. The experimental groups were: CA365NQ-vcMMAE group, hLiv22-vcMMAE group, and solvent control group (PBS). . Administration started on the day of grouping, and the dosage was 2.5 mg/kg in a single dose. The trial ended on the 13th day of group administration. Compared with the vehicle control group, both the hLiv22-vcMMAE and CA365NQ-vcMMAE groups could significantly inhibit tumor volume growth, with statistical differences (P ₂ = 0.0015, P ₃ = 0.0025); No statistical difference was found (P>0.05); the tumor volume growth inhibition rate TGI (%) of hLiv22-vcMMAE and CA365NQ-vcMMAE groups were 70.6% and 76.1% respectively. The tumor growth curves of 2.5 mg/kg dose LIV-1 antibody conjugates CA365NQ-vcMMAE, hLiv22-vcMMAE, and solvent control groups on M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells are shown in Figure 16.

At the end of the trial, the tumor weight inhibition rates in the hLiv22-vcMMAE and CA365NQ-vcMMAE groups were 54.2% and 56.3% respectively. The tumor weights in the treatment groups were significantly smaller than those in the solvent control group (P ₂ =0.0008, P ₃ =0.0012). The inhibition of tumor weight by LIV-1 antibody conjugate CA365NQ-vcMMAE, hLiv22-vcMMAE, and solvent control group at 2.5 mg/kg dose in M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells is shown in Figure 17.

During the experiment, the experimental animals were active and eating well during the administration period, and the weights of each group remained stable, indicating that the animals had good tolerance to the test product. There was no significant difference in the amount of change between each group (P>0.05). The body weight curves of M-NSG mice transplanted with NCI-H82 human small cell lung cancer cells using 2.5 mg/kg dose of LIV-1 antibody conjugate CA365NQ-vcMMAE, hLiv22-vcMMAE, and solvent control groups are shown in Figure 18.

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

MCF7-hLIV1 cells are constructed human breast cancer cells that overexpress hLIV1. The cells are cultured in an incubator at 37°C and 5% _CO2 . The medium composition is RMPI-1640 medium containing 10% FBS. Balb/c-nude 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 containing 50% Matrigel to a concentration of 1.0×10 ⁸ cells/mL, and then inoculated subcutaneously into the right side of M-NSG mice at a volume of 0.1 mL/mouse. When the average tumor volume reaches ^103mm3 , the mice are divided into 4 experimental groups according to the tumor volume, with 6 animals in each group. The experimental groups are: CA365NQ-BNLD11 group, CA365NQ-vcMMAE group, and solvent control group (PBS). , negative control nCov-CA521-vcMMAE group. Administration started on the day of grouping, and the dosage was 2.5 mg/kg in a single dose.

The trial ended on the 40th day of group administration. Because the CA365NQ-BNLD11 group achieved complete tumor clearance, this group continued to be observed for 64 days. Compared with the solvent control group, the CA365NQ-BNLD11 and CA365NQ-vcMMAE groups could significantly inhibit tumor volume growth (P<0.05); while there was no significant difference between the nCov-CA521-vcMMAE group and the solvent control group (P>0.05); CA365NQ- The tumor volume growth inhibition rate TGI (%) in the vcMMAE group was 83.2%, and the CA365NQ-BNLD11 group achieved complete tumor clearance and no recurrence within the next 25 days. Tumor growth of 2.5 mg/kg LIV-1 antibody conjugate CA365NQ-BNLD11 group, CA365NQ-vcMMAE group, solvent control group, and negative control group on Balb/c-nude nude mice transplanted with MCF7-hLIV1 human breast cancer cells The curve is shown in Figure 19.

During the experiment, the experimental animals were active and eating well during the administration period, and the weight of each group increased to a certain extent, indicating that the animals had good tolerance to the test product. There was no significant difference between each group (P>0.05). Body weight curve of Balb/c-nude nude mice transplanted with MCF7-hLIV1 human breast cancer cells using 2.5 mg/kg dose of LIV-1 antibody conjugate CA365NQ-BNLD11 group, CA365NQ-vcMMAE group, solvent control group, and negative control group As shown in Figure 20.

Example 13 Single dose toxicity experiment of LIV-1-vcMMAE Balb/c mice

13.1 CA365NQ-VcMMAE single dose toxicity test

Balb/c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The mice were evenly divided into 2 groups according to body weight, with 6 mice in each group, half male and half female. The experimental groups are: CA365NQ-vcMMAE group and solvent control group (PBS). One group was injected with CA365NQ-vcMMAE into the tail vein at a dose of 50 mg/kg, and the other group was injected with an equal volume of PBS as a solvent control. After that, the body weight changes of the mice were recorded daily. The results showed that the mice in the administration group were only on the 3rd day after administration. His body weight dropped by 6.2% on the first day, and he recovered quickly thereafter. He is in good general condition and has no significant acute toxicity. The body weight curve of Balb/c mice in the single-dose toxicity experiment of the 50 mg/kg LIV-1 antibody conjugate CA365NQ-vcMMAE group and the solvent control group is shown in Figure 21.

13.2 CA1328-VcMMAE single dose toxicity test

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

Claims (15)

An anti-LIV-1 antibody or an antigen-binding fragment thereof, said antibody or an antigen-binding fragment thereof comprising 3 light chain complementarity-determining regions and 3 heavy chain complementarity-determining regions, wherein The three light chain complementarity determining regions of the antibody or its antigen-binding fragment 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 The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; The three light chain complementarity determining regions of the antibody or its antigen-binding fragment 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 The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; The three light chain complementarity determining regions of the antibody or its antigen-binding fragment 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 The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26 and HCDR3 shown in SEQ ID NO:27; The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29 and LCDR3 shown in SEQ ID NO:30, and the The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32 and HCDR3 shown in SEQ ID NO:33; The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35 and LCDR3 shown in SEQ ID NO:36, and the The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:37, HCDR2 shown in SEQ ID NO:38 and HCDR3 shown in SEQ ID NO:39; The three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:42, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:43, and the The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; The three light chain complementarity determining regions of the antibody or its antigen-binding fragment 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 The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:46, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; The three light chain complementarity determining regions of the antibody or its antigen-binding fragment 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 The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:47, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; The three light chain complementarity determining regions of the antibody or its antigen-binding fragment 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 The three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:48, HCDR2 shown in SEQ ID NO:40 and HCDR3 shown in SEQ ID NO:27; The three light chain complementarity 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 Antibodies or antigen-binding fragments thereof The three heavy chain complementarity 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 complementarity 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 The three heavy chain complementarity 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. 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 set forth in SEQ ID NO: 9, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence set forth in 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 set forth in 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 set forth in 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 set forth in SEQ ID NO: 1, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence set forth in 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 set forth in SEQ ID NO: 3, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence set forth in 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 set forth in SEQ ID NO: 5, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence set forth in 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 set forth in SEQ ID NO: 10, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence set forth in 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 set forth in 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 set forth in 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 set forth in SEQ ID NO: 14, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence set forth in 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 set forth in SEQ ID NO: 16, and / Or a heavy chain variable region having at least about 80%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence set forth in 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 set forth in 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 set forth in 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 set forth 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 set forth in SEQ ID NO: 21. 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 shown in SEQ ID NO: 54 constant region. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-3. A cell comprising the nucleic acid of claim 4. An anti-LIV-1 antibody conjugate, characterized in that the anti-LIV-1 antibody conjugate includes (a) a LIV-1 antibody or an antigen-binding fragment thereof, the LIV-1 antibody or an antigen-binding fragment thereof Be the anti-LIV-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and (b) a coupling part coupled to the antibody part; preferably, the coupling part is selected from Lower group: Detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof. An anti-LIV-1 antibody drug conjugate, characterized in that the antibody drug conjugate is represented by the following formula 1:
Wherein: Ab is the anti-LIV-1 antibody or antigen-binding fragment thereof according to any one of claims 1-3, LU is a linker; D is drug; The subscript p corresponds to the average DAR value of the antibody-drug conjugate, p is a value selected from 1-10, preferably 1-8, more preferably 1-4 or 4-8, most preferably p is 4 . The antibody drug conjugate according to claim 7, wherein the LU-D is vcMMAE, vc is a valine-citrulline linker, and MMAE is monomethyl auristatin E. The antibody drug conjugate according to claim 7, wherein the BNLD11 structure is as follows:
The antibody drug conjugate according to any one of claims 7-9, characterized in that the antibody drug conjugate is any one of the following (1)-(6): (1) The three light chain complementarity 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 chains are complementary The decision area includes 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 complementarity 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 chains are complementary The determining region includes 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 for vcMMAE; (3) The three light chain complementarity 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 chains are complementary The decision area includes 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; (4) The three light chain complementarity 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 chains are complementary The decision area includes 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; (5) The three light chain complementarity 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 chains are complementary The decision area includes 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, (6) The three light chain complementarity 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 chains are complementary The decision area includes 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. A pharmaceutical composition containing 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 cell according to claim 6 Anti-LIV-1 antibody conjugate, or the antibody drug conjugate according to any one of claims 7-10. A kit containing 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 7-10, or the pharmaceutical composition according to claim 11. 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 any one of claims 7 to 10 Application of the antibody-drug conjugate of claim 11 or the pharmaceutical composition of claim 11 for preventing, treating, detecting or diagnosing diseases related to LIV-1; preferably, the diseases related to LIV-1 Including cancer; more preferably, the cancer is a LIV-1 positive solid tumor; most preferably, the cancer includes breast cancer, prostate cancer, pancreatic cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer, and esophageal squamous cell carcinoma , one or more of gastric cancer and melanoma. A method for treating or preventing LIV-1 related diseases, comprising combining 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 antibody according to claim 6 -1 antibody conjugate, or the antibody drug conjugate of any one of claims 7-10, or the pharmaceutical composition of claim 11 is administered to a subject; preferably, the LIV-1 related The disease includes cancer; more preferably, the cancer is a LIV-1 positive solid tumor; most preferably, the cancer includes breast cancer, prostate cancer, pancreatic cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer, esophageal squamous cell One or more of cancer, gastric cancer, and melanoma. 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 antibody-drug conjugates.