CN115998900A

CN115998900A - anti-TROP-2 antibody drug conjugate and medical application thereof

Info

Publication number: CN115998900A
Application number: CN202211164813.5A
Authority: CN
Inventors: 花海清; 毛东杰
Original assignee: Changzhou Hengbang Pharmaceutical Co ltd; Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Current assignee: Changzhou Hengbang Pharmaceutical Co ltd; Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Priority date: 2021-09-23
Filing date: 2022-09-23
Publication date: 2023-04-25

Abstract

The invention relates to an antibody drug conjugate and medical application thereof. The invention relates to an anti-TROP-2 antibody drug conjugate and medical application thereof, in particular to an antibody drug conjugate which is formed by connecting an anti-TROP-2 antibody or an antigen binding fragment thereof and an irinotecan derivative through a joint.

Description

anti-TROP-2 antibody drug conjugate and medical application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to an anti-TROP-2 antibody conjugate and medical application thereof.

Background

With the continuous and intensive research of tumor genomics, proteomics and signal transduction pathways, the interactions of oncogenes and tumor suppressor genes of tumor cells and their effects on tumor microenvironment have become more and more clear, which also makes it possible to design new anti-tumor therapeutic strategies for specific molecular targets of tumors.

The molecular targeting therapy of tumor is a new therapeutic mode different from traditional operation, radiotherapy and chemotherapy, and has the advantages that the medicine is usually combined with corresponding target site only, and the effect of killing or inhibiting target cell is achieved by directly affecting the function of target molecule or carried physical or chemical effector molecule. Because the target position is clear, the medicine has high selectivity, not only can effectively kill or inhibit target cells, but also does not produce or only produces less toxic and side effects on normal tissue cells. Therefore, the development of molecular targeted drugs has become a hotspot in clinical research of tumors.

Human trophoblast cell surface antigen 2 (human trophoblast cell surface antigen, trop-2) is a cell surface glycoprotein encoded by the TACSTD2 gene. TROP-2 is composed of 323 amino acids, of which the signal peptide is 26 amino acids, the extracellular region is 248 amino acids, the transmembrane region is 23 amino acids and the cytoplasmic region is 26 amino acids. There are 4 heterogeneous N-binding glycosylation sites in the TROP-2 extracellular domain, and the apparent molecular weight increases 11 to 13KD upon addition of sugar chains. In the TACSTD gene family, the extracellular domain has a characteristic Thyroglobulin (TY) sequence, which is generally thought to be involved in proliferation, infiltration, and metastasis of cancer cells.

A large number of clinical studies and literature reports indicate that TROP-2 is over-expressed in a plurality of epithelial cancers such as gastric cancer, lung cancer, large intestine, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, esophagus cancer and the like. In contrast, TROP-2 is expressed little or no in normal adult tissues, and cells limited to the epithelial region are expressed little, and the expression level is lower than in cancerous tumors, indicating that TROP-2 is involved in tumor formation. Overexpression of TROP-2 in tumor tissue is closely related to patient prognosis and metastasis of cancer cells, while affecting the overall survival of the patient. Thus, TROP-2 has become an attractive target in tumor molecular targeted therapies.

Several studies of the anti-tumor effects of anti-httrop-2 antibodies have been reported:

U.S. patent No. 5840854 reports cytotoxicity of anti-httrop-2 monoclonal antibodies (BR 110) that bind to cytotoxins against human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981.

An antibody (RS 7) was disclosed in us patent No. 6653104, which was tested in an in vivo model using an antibody labeled with a radioactive substance, and showed antitumor activity in a nude mouse xenograft model, but no antitumor effect was reported when only nude antibodies were used.

The isolated monoclonal antibodies produced by hybridoma cell lines ar47a6.4.2 or ar52a301.5 derived from mice immunized with human ovarian cancer tissue have also been reported by U.S. patent No. 7420040 to bind to httrop-2 and exhibit anti-tumor activity in nude mice xenograft models.

CN102827282A discloses a humanized anti-TROP-2 genetic engineering antibody IgG and application thereof, and in-vitro test results show that the anti-TROP-2 antibody IgG has remarkable inhibition effect on proliferation of pancreatic cancer cells.

CN104114580a discloses an antibody (particularly a humanized antibody) which specifically reacts with httrop-2 and has an anti-tumor activity in vivo, and a hybridoma producing the antibody, a complex of the antibody and a pharmaceutical agent, a pharmaceutical composition for diagnosis or treatment of tumor, a method for detecting tumor, a kit for detection or diagnosis of tumor.

Disclosure of Invention

The invention aims to provide an antibody drug conjugate shown in a general formula (I) or pharmaceutically acceptable salt or solvent compound thereof,

wherein:

w is selected from- (CR) ^e R ^f ) _g -[X ₁ -(CR ^e R ^f ) _u- X ₂ ] _v -(CR ^e R ^f ) _h -，

R ^e Or R is ^f Each independently selected from hydrogen, deuterium, hydroxy, amino, alkyl, halo, haloalkyl, deuterated alkyl, or hydroxyalkyl; preferably, R ^e Or R is ^f Each independently selected from hydrogen, deuterium, more preferably hydrogen,

X ₁ or X ₂ Each independently selected from N, O or S; preferably X ₁ Or X ₂ Each independently selected from S or O; more preferably it is an O-ring,

g. u, v or h are each independently selected from 1, 2, 3 or 4; preferably, g, u or h are each independently selected from 1, 2, 3; more preferably 2; v is preferably 1 or 2, more preferably 1;

y is 1 to 20, preferably 1 to 10, more preferably 2 to 8, still more preferably 4, 6 or 8;

the mAb is an anti-TROP-2 antibody or antigen-binding fragment thereof.

In a further preferred embodiment of the invention, the mAb antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising at least 1 HCDR selected from the group consisting of: 3,SEQ ID NO:4,SEQ ID NO:5 of SEQ ID NO; the antibody light chain variable region comprises at least 1 LCDR described by a sequence selected from the group consisting of seq id nos: SEQ ID NO. 6,SEQ ID NO:7,SEQ ID NO:8.

In a preferred embodiment of the invention, an antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody heavy chain variable region comprises:

HCDR1 shown in SEQ ID NO 3,

HCDR2 and shown in SEQ ID NO. 4

HCDR3 shown in SEQ ID NO. 5.

In a preferred embodiment of the invention, an antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody light chain variable region comprises:

LCDR1 shown in SEQ ID NO. 6,

LCDR2 and shown in SEQ ID NO. 7

LCDR3 as shown in SEQ ID NO. 8.

HCDR1 shown in SEQ ID NO 3,

HCDR2 and shown in SEQ ID NO. 4

HCDR3 shown in SEQ ID NO. 5; and

the antibody light chain variable region comprises:

LCDR1 shown in SEQ ID NO. 6,

LCDR2 and shown in SEQ ID NO. 7

LCDR3 as shown in SEQ ID NO. 8.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen binding fragment thereof is selected from the group consisting of a murine antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a human antibody or antigen binding fragment thereof, a humanized antibody or antigen binding fragment thereof.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2, igG3 or IgG4, or a variant thereof.

In a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, igG2 or IgG4 or a variant thereof.

In a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as set forth in SEQ ID NO. 48, or SEQ ID NO. 49.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen binding fragment thereof further comprises a light chain constant region derived from a kappa chain, a lambda chain of a human antibody, or a variant thereof.

In a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen binding fragment thereof further comprises a light chain constant region derived from a kappa chain of a human antibody;

in a further preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region as set forth in SEQ ID NO. 50.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen binding fragment thereof comprises a heavy chain variable region selected from the group consisting of the heavy chain variable regions shown in the following sequences, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95% or 99% identity to the following sequences: SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23 or SEQ ID NO. 25.

In a preferred embodiment of the invention, an antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody or antigen binding fragment thereof comprises a light chain variable region selected from the group consisting of the light chain variable regions shown in the following sequences, or a light chain variable region having at least 70%,75%,80%,85%,90%,95% or 99% identity to the following sequences: SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24 or SEQ ID NO. 26.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen binding fragment thereof, comprises a heavy chain selected from the group consisting of the heavy chains shown in the following sequences, or a heavy chain having at least 80%,85%,90%,95% or 99% identity compared to the sequences: SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 33. SEQ ID NO: 35. SEQ ID NO: 37. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 43. SEQ ID NO:45 or SEQ ID NO:46.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen binding fragment thereof, comprises a light chain selected from the group consisting of the light chains shown in the following sequences, or having at least 80%,85%,90%,95% or 99% identity to the sequences: SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO: 36. SEQ ID NO: 38. SEQ ID NO: 40. SEQ ID NO:42 or SEQ ID NO:44.

in a preferred embodiment of the invention, an antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody, or antigen binding fragment thereof, comprises:

(1) SEQ ID NO:9 and the heavy chain variable region shown in SEQ ID NO:10, a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

(2) SEQ ID NO:11 and the heavy chain variable region shown in SEQ ID NO:12, a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

(3) SEQ ID NO:13 and the heavy chain variable region shown in SEQ ID NO:14, a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

(4) SEQ ID NO:15 and the heavy chain variable region shown in SEQ ID NO:16, and a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

(5) SEQ ID NO:17 and the heavy chain variable region shown in SEQ ID NO:18, a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

(6) SEQ ID NO:19 and the heavy chain variable region shown in SEQ ID NO:20, and a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

(7) SEQ ID NO:21 and the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

(8) SEQ ID NO:23 and the heavy chain variable region shown in SEQ ID NO:24, and a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

(9) SEQ ID NO:25 and the heavy chain variable region shown in SEQ ID NO: 26.

In a preferred embodiment of the invention, an antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody comprises:

(1) SEQ ID NO:27 and SEQ ID NO:28, a light chain as shown in seq id no; or alternatively, the first and second heat exchangers may be,

(2) SEQ ID NO:29 and the heavy chain shown in SEQ ID NO:30, a light chain as shown in seq id no; or alternatively, the first and second heat exchangers may be,

(3) SEQ ID NO:31 and the heavy chain shown in SEQ ID NO:32, a light chain indicated by 32; or alternatively, the first and second heat exchangers may be,

(4) SEQ ID NO:33 and the heavy chain shown in SEQ ID NO:34, a light chain indicated by 34; or alternatively, the first and second heat exchangers may be,

(5) SEQ ID NO:35 and the heavy chain shown in SEQ ID NO:36, a light chain indicated by 36; or alternatively, the first and second heat exchangers may be,

(6) SEQ ID NO:37 and SEQ ID NO:38, a light chain indicated at 38; or alternatively, the first and second heat exchangers may be,

(7) SEQ ID NO:39 and the heavy chain shown in SEQ ID NO: 40. Or alternatively, the first and second heat exchangers may be,

(8) SEQ ID NO:41 and the heavy chain shown in SEQ ID NO: 42; or alternatively, the first and second heat exchangers may be,

(9) SEQ ID NO:43 and SEQ ID NO:44, a light chain indicated by 44; or alternatively, the first and second heat exchangers may be,

(10) SEQ ID NO:45 and the heavy chain shown in SEQ ID NO:38, a light chain indicated at 38; or alternatively, the first and second heat exchangers may be,

(11) SEQ ID NO:46 and the heavy chain shown in SEQ ID NO: 28.

In a further preferred embodiment of the invention, the antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof is selected from the group consisting of a prodrug conjugate of general formula (II):

y is selected from 2 to 10, preferably 4 to 10, more preferably 4, 6, 8 or 10.

In a further preferred embodiment of the invention, the antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof is selected from the group consisting of a bulk drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to the general formula (III):

the mAb is selected from the anti-TROP-2 antibodies described above or antigen-binding fragments thereof;

y is selected from 2 to 10, preferably 4 to 10, more preferably 4, 6, 8 or 10;

v is 1 or 2.

In a further preferred embodiment of the invention, the antibody drug conjugate or a pharmaceutically acceptable salt or a solvent compound thereof is selected from the following structures:

/>

/>

/>

wherein y is selected from 2 to 10, preferably 4 to 10, more preferably 4, 6, 8 or 10.

The present invention also provides a method for preparing an antibody-drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:

after mAb reduction, carrying out coupling reaction with a general formula (F) to obtain a compound shown in the general formula (I);

wherein W is as defined above.

y is 1-20; preferably 4-10; more preferably 4, 6, 8 or 10.

In another aspect, the invention provides a pharmaceutical composition comprising an antibody-drug conjugate of the invention or a pharmaceutically acceptable salt or solvate of said antibody-drug conjugate, and one or more pharmaceutically acceptable excipients, diluents or carriers.

In another aspect, the invention provides a pharmaceutical use, the invention relates to the use of an anti-TROP-2 antibody drug conjugate or a pharmaceutically acceptable salt or solvate of said antibody drug conjugate or a pharmaceutical composition thereof for the treatment or prophylaxis of a TROP-2 mediated disease or condition.

In another aspect, the invention also provides the use of an antibody-drug conjugate according to formula (I) or a pharmaceutically acceptable salt or solvate of the antibody-drug conjugate or a pharmaceutical composition thereof in the manufacture of a medicament for the treatment of a disease associated with human TROP-2.

In a more preferred embodiment of the invention, the disease associated with human TROP-2 is the use in the manufacture of a medicament for the treatment of a cancer in which TROP-2 is highly expressed, the cancer is selected from triple negative breast cancer, small cell lung cancer, urothelial cancer, human brain astrocytoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, acinar soft tissue sarcoma, astrocytoma, cancer of the breast, brain and spinal cord, metastatic brain tumor, breast cancer, carotid aneurysm, cervical cancer, chondrosarcoma, chordoma, renal chromocytoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymal tumor, ewing tumor, extraosseous mucoid chondrosarcoma, bone hypoplasia, bone fibrodysplasia, gall bladder or bile duct cancer, gastric cancer, gestational trophoblastoma, germ cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor, gastric cancer, renal cell carcinoma, and cervical cancer Kaposi's sarcoma, renal carcinoma, leukemia, liposarcoma, malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrinopathy, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, nameplate cell tumor, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdomyoma, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, innocent pill cancer, thymus cancer, thymoma, thyroid metastatic cancer, and uterine cancer.

The antibody-drug conjugate or pharmaceutically acceptable salt or solvent compound thereof can specifically bind to target antigen, has high endocytic efficiency and long in vivo half-life time, and can obviously kill tumors while ensuring safety.

The antibody drug conjugate and the pharmaceutically acceptable salt or solvent compound thereof have remarkable anti-tumor effect and good safety, and simultaneously have good in-vivo metabolic activity, long in-vivo drug effect time and wide clinical application prospect.

Detailed Description

1. Terminology

For easier understanding of the present application, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein within this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application belongs.

The amino acid three-letter codes and one-letter codes used in this application are as described in J.biol. Chem,243, p3558 (1968).

The term "antibody" as used herein refers to an immunoglobulin that is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, i.e., igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each class Ig of the five classes of Igs may have either a kappa chain or a lambda chain.

In the present application, the antibody light chain variable regions described herein may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin, or variants thereof.

In the present application, the antibody heavy chain variable region described herein may further comprise a heavy chain constant region comprising an IgG1, igG2, igG 3, igG 4 or variant thereof of human or murine origin.

The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the VL and VH regions of an antibody or antigen binding fragment described herein conform in number and position to known Kabat numbering rules and Kabat or ABM definition rules (http:// bioinf org. Uk/abs /).

The term "antigen presenting cell" or "APC" is a cell displaying on its surface a foreign antigen complexed with MHC. T cells recognize this complex using the T Cell Receptor (TCR). Examples of APCs include, but are not limited to, dendritic Cells (DCs), peripheral Blood Mononuclear Cells (PBMCs), monocytes, B lymphoblastic cells, and monocyte derived dendritic cells.

The term "antigen presentation" refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.

The term "TROP-2" includes any variant or isoform of TROP-2 that is naturally expressed by a cell. The antibodies of the invention can cross-react with TROP-2 from a non-human species. Alternatively, the antibody may be human TROP-2 specific and may not exhibit cross-reactivity with other species. TROP-2 or any variant or isoform thereof may be isolated from cells or tissues that naturally express them, or produced by recombinant techniques using techniques common in the art and those described herein. Preferably, the anti-TROP-2 antibody targets a human TROP-2 having a normal glycosylation pattern.

The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods, the techniques and methods involved are well known in the art, such as:

1. An antibody isolated from a transgene of a human immunoglobulin gene, a transchromosomal animal (e.g., mouse) or a hybridoma prepared therefrom;

2. an antibody isolated from a host cell transformed to express the antibody, such as a transfectoma;

3. an antibody isolated from a recombinant combinatorial human antibody library; and

4. antibodies are prepared, expressed, created or isolated by splicing human immunoglobulin gene sequences to other DNA sequences, and the like.

Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include rearrangements and mutations that occur later, such as during antibody maturation.

The term "murine antibody" is herein a monoclonal antibody to human TROP-2 prepared according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with a TROP-2 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine TROP-2 antibody or antigen binding fragment thereof may further comprise a light chain constant region of murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of murine IgG1, igG2, igG3 or IgG4 or variant thereof.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present application may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (i.e., a "humanized antibody").

The term "humanized antibody (humanized antibody)", also referred to as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting a mouse CDR sequence into the variable region framework of a human antibody. Humanized antibodies can overcome the disadvantage of strong immune response induced by chimeric antibodies, which carry a large number of mouse protein components. To avoid a decrease in activity while reducing immunogenicity, the human antibody variable region may be subjected to minimal reverse mutation to maintain activity.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. The method comprises the steps of establishing chimeric antibody, selecting hybridoma secreting murine specific monoclonal antibody, cloning variable region genes from mouse hybridoma cells, cloning constant region genes of human antibody according to requirements, connecting the mouse variable region genes and the human constant region genes into chimeric genes, inserting the chimeric genes into human vectors, and finally expressing chimeric antibody molecules in eukaryotic industrial systems or prokaryotic industrial systems. The constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or variants thereof, preferably comprising the heavy chain constant region of human IgG1, igG2 or IgG4, or the heavy chain constant region of IgG1 that enhances ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity using amino acid mutations.

The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen binding fragments include, but are not limited to: fab, fab ', F (ab') 2, fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson,2005, nat. Biotechnol.23: 1126-1136.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.

"Fab ' fragments" contain portions of one light chain and one heavy chain comprising the VH domain and CH1 domain and the region between the CH1 and CH2 domains, whereby an inter-chain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form a F (ab ') 2 molecule.

"F (ab') 2 fragments" contain two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. Such multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL units have polyepitopic specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, diabodies (diabodies), bispecific diabodies and triabodies (triabodies), antibody fragments that have been covalently or noncovalently linked together, and the like.

The term "single chain antibody" is a single chain recombinant protein formed by joining the heavy chain variable region VH and the light chain variable region VL of an antibody through a connecting peptide, which is the smallest antibody fragment with a complete antigen binding site.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment that contains only heavy chain variable region or light chain variable region chains. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment may target the same or different antigens.

The term "binds to TROP-2" as used herein refers to being capable of interacting with human TROP-2.

The term "antigen binding site" in this application refers to a three-dimensional spatial site recognized by an antibody or antigen binding fragment of the present application.

The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically maintained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection assays, among others. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and the like.

The terms "specifically bind", "selectively bind" and "selectively bind" as used herein refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when human TROP-2 is used as the analyte and antibodies are used as the ligand, the antibodies are present at about less than 10 as determined by Surface Plasmon Resonance (SPR) techniques in the instrument ^-7 M or even smaller equilibrium dissociation constant (K _D ) Binds to the predetermined antigen and has an affinity to bind to the predetermined antigen that is at least twice as great as its affinity to bind to non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, etc.). The term "antibody that recognizes an antigen" may be used interchangeably herein with the term "antibody that specifically binds".

The term "cross-reactive" refers to the ability of an antibody of the present application to bind to TROP-2 from a different species. For example, an antibody of the present application that binds to human TROP-2 can also bind to TROP-2 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen, or binding or functional interaction with cells physiologically expressing TROP-2, in binding assays (e.g., SPR and ELISA). Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking is also intended to include any measurable decrease in ligand binding affinity upon contact with an anti-TROP-2 antibody as compared to a ligand not contacted with an anti-TROP-2 antibody.

The term "inhibit growth" (e.g., involving a cell) is intended to include any measurable decrease in cell growth.

The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induce" with respect to inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.

The term "ADCC", namely, antibody-dependent cell-mediated cytotoxicity, as used herein, refers to antibody-dependent cell-mediated cytotoxicity, which means that cells expressing Fc receptors directly kill target cells coated with antibodies by recognizing the Fc segment of the antibodies. The ADCC effector function of an antibody may be reduced or decreased by modification of the Fc fragment on IgG. The modification refers to mutation in the heavy chain constant region of the antibody.

Methods for producing and purifying antibodies and antigen binding fragments are well known and can be found in the art, e.g., in the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. For example, mice can be immunized with human TROP-2 or fragments thereof, the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments of the invention are engineered to incorporate one or more human FR regions in the CDR regions of non-human origin. Human FR germline sequences can be obtained from the website http:// imgt. Cines. FR of ImMunoGeneTics (IMGT) or from the journal of immunoglobulins, 2001ISBN 012441351.

The engineered antibodies or antigen binding fragments of the present application can be prepared and purified using conventional methods. The cDNA sequences of the corresponding antibodies can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the FC region. Stable clones were obtained by expressing antibodies that specifically bound to human antigens. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The antibodies of the present application refer to monoclonal antibodies. Monoclonal antibodies (mabs) as described herein refer to antibodies derived from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen binding fragments can be obtained by recombinant techniques such as hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.

"administering," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administration," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

"treating" means administering a therapeutic agent, such as an antibody comprising any of the present application, to a patient for internal or external use, said patient having one or more symptoms of a disease for which said therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments (e.g., methods of treatment or articles of manufacture) of the present application may be ineffective in alleviating symptoms of the target disease with each patient, it should be determined according to any statistical test method known in the art, such as Student t test, chi-square test, U test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test, that the symptoms of the target disease should be alleviated in a statistically significant number of patients.

The term "consisting essentially of … …" or variations thereof as used throughout the specification and claims is meant to include all such elements or groups of elements, and optionally other elements of similar or different nature to those described, which do not significantly alter the basic or novel nature of a given dosing regimen, method or composition.

The term "naturally occurring" as used herein as applied to an object refers to the fact that the object may be found in nature. For example, polypeptide sequences or polynucleotide sequences that are present in organisms (including viruses) that can be isolated from natural sources and that have not been intentionally modified by man in the laboratory are naturally occurring.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"exogenous" refers to a substance that is to be produced by background outside an organism, cell or human.

"endogenous" refers to substances produced in cells, organisms or humans according to background.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the greatest percent homology.

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include their progeny. Thus, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell. "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may be, but need not be, present.

"pharmaceutical composition" means a composition comprising one or more antibodies or antigen-binding fragments thereof as described herein, as well as other components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

By "pharmaceutically acceptable salt" is meant a salt of an antibody-drug conjugate of the invention which is safe and effective for use in a mammal, and which has the desired biological activity. The antibody-drug conjugates of the invention contain at least one amino group and thus can form salts with acids, non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pear, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.

"solvent compound" refers to an antibody-drug conjugate compound of the invention that forms a pharmaceutically acceptable solvent compound with one or more solvent molecules, non-limiting examples of which include: water, ethanol, acetonitrile, isopropanol, ethyl acetate.

"cytotoxic drug" as used herein refers to a substance that inhibits the function of cells and/or causes cell death or destruction.

"tubulin inhibitors" refer to a class of compounds that interfere with the mitotic process of cells by inhibiting the polymerization or promoting the aggregation of tubulin, thereby exerting an antitumor effect. Non-limiting examples of which include: maytansinoids, carbo Li Jimei, taxanes, vincristine, colchicine, dolastatin/oretinomycin/monomethyl oretinomycin E (MMAE)/monomethyl oretinostatin F (MMAF).

"linker" refers to a chemical moiety comprising a covalent bond or chain of atoms that refers to the covalent attachment of an antibody to a drug. Non-limiting examples of linkers include: arylene, heteroarylene, PEG, polymethyleneoxy, succinate, succinamide, diglycolate, malonate and caproamide.

"drug load" (DAR) is represented by y, the average number of cytotoxic drugs per antibody in formula (I). The drug loading in the present invention may range from 1-20 cytotoxic drugs per antibody. The antibody-drug conjugates of formula (I) are a collection of antibodies conjugated with a range of (1-20) cytotoxic drugs. The drug load (DAR) in the antibody-drug conjugate from the conjugation reaction can be characterized by conventional means, such as mass spectrometry, HPLC, ELISA, and the like. By these means the quantitative distribution of the antibody-drug conjugate over the y-value can be determined.

The invention also includes various deuterated forms of the compounds of formula (I). Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art are able to refer to the relevant literature for the synthesis of deuterated forms of the compounds of formula (I). Commercially available deuterated starting materials may be used in preparing the deuterated forms of the compounds of formula (I) or they may be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated boranes, trideuteroborane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

The term "pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity. The preparation of the conventional pharmaceutical composition is shown in Chinese pharmacopoeia.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to salts of the antibody drug conjugates of the invention, or salts of the compounds described herein, which are safe and effective when used in a mammal and which possess the desired biological activity, and the ligand drug conjugates of the invention contain at least one amino group and thus can form salts with acids, non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pear, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.

The term "drug loading" refers to the average amount of cytotoxic drug loaded per antibody or antigen fragment thereof in the molecule of formula (I), which may also be expressed as the ratio of the amount of drug to the amount of antibody, and the drug loading may range from 0 to 12, preferably 1 to 10, cytotoxic drugs per ligand. In embodiments of the invention, the drug loading is expressed as y, which may also be referred to as DAR value, and is illustratively the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The number of drug molecules per ADC molecule after the coupling reaction can be identified using conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays and HPLC characterization.

In some embodiments of the invention, the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the epsilon-amino group of the lysine residue via a linking unit, and in other embodiments of the invention, the cytotoxic drug is coupled to the sulfhydryl group of the ligand via a linking unit. Generally, the number of drug molecules that can be conjugated to an antibody in a coupling reaction will be less than the theoretical maximum.

The loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

(1) The molar ratio of the connecting reagent to the monoclonal antibody is controlled,

(2) The reaction time and the temperature are controlled,

(3) Different reagents are selected.

The antibody drug conjugate or the pharmaceutically acceptable salt or the solvent compound thereof has obvious anti-tumor effect and good safety.

Drawings

FIG. 1 is a diagram of Compound D ¹ H-NMR spectrum.

Detailed Description

The invention will be further described with reference to the following examples, which are not intended to limit the scope of the invention. The experimental method without specific conditions being noted in the embodiment of the invention is generally according to conventional conditions, such as an antibody technical laboratory manual and a molecular cloning manual of cold spring harbor; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1 antigen preparation

Human TROP-2 (TROP-2-His) protein encoding a His tag was synthesized by SinoBiologics (10428-H08H).

TROP-2-His sequence:

MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSLHHHHHHHH

SEQ ID NO：47

EXAMPLE 2 murine hybridomas and the acquisition of antibody sequences

Animal immunization is carried out by using human antigen TROP-2-His, 5 Balb/c and 5A/J mice are total, female is 10 weeks old, sigma Complete Freund's Adjuvant (CFA) and Sigma Incomplete Freund's Adjuvant (IFA) are used, and immunogen and immune adjuvant are fully mixed and emulsified in a ratio of 1:1 to prepare stable 'water-in-oil' liquid; the injection dose was 25. Mu.g/200. Mu.L/mouse.

TABLE 1 immunization protocol

Day 1	First immunization, complete Freund's adjuvant.
		Day 21	Second immunization, incomplete Freund's adjuvant.
Day 35	Third immunization, incomplete Freund's adjuvant.
		Day 42	Blood collection and serum titer detection (3 blood-free)
Day 49	Fourth immunization, incomplete Freund's adjuvant.
		Day 56	Blood collection and serum titer detection (4 blood-free)

Serum titers and the ability to bind cell surface antigens were assessed on immunized mouse serum using an indirect ELISA as described in example 4, with control titers detection (greater than 10-fold dilution) determining the initiation of cell fusion. The immunized mice with strong serum titer, affinity and FACS binding are selected for final immunization, the mice are killed, spleen cells and SP2/0 myeloma cells are fused and plated to obtain hybridomas, target hybridomas are screened by indirect ELISA, and strains are established into monoclonal cell strains by a limiting dilution method. The resulting positive antibody strains were further screened using an indirect ELISA to select hybridomas that bind the recombinant proteins. Hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript) ^TM Reverse Transcriptase, takara # 2680A). The cDNA obtained by reverse transcription is subjected to PCR amplification by using a mouse Ig primer group (Novagen, TB326 Rev.B 0503) and then is sequenced, and finally the sequence of the murine antibody is obtained.

The heavy and light chain variable region sequences of murine mab M1 are as follows:

M1 HCVR

QVQLQQPGAELVRPGASVKLSCRASGYTFTNYWMNWVKQRPEQGLEWIGRIDPNDSETHYNQKFKDRAILTVDKASNTAYMQLSGLTSEDSAVHYCARSGFGSTYWFFDVWGAGTTVTVSS

SEQ ID NO：1

M1 LCVR

DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFAGSGYGTDFTFTISSVQTEDLTVYHCQQHYSTPLTFGPGTRLELK

SEQ ID NO：2

TABLE 2 heavy and light chain variable region CDR sequences of murine monoclonal antibody M1

Name of the name	Sequence(s)	Numbering device
			HCDR1	NYWMN	SEQ ID NO：3
HCDR2	RIDPNDSETHYNQKFKD	SEQ ID NO：4
			HCDR3	SGFGSTYWFFDV	SEQ ID NO：5
LCDR1	KASQDVSTAVA	SEQ ID NO：6
			LCDR2	SASYRYT	SEQ ID NO：7
LCDR3	QQHYSTPLT	SEQ ID NO：8

Example 3 in vitro binding Activity detection method of antibodies

(1) In vitro indirect ELISA binding assay:

TROP-2His protein (Sino Biological Inc., cat# 10428-H08H) was diluted to a concentration of 1. Mu.g/ml with PBS pH7.4, added to a 96-well high affinity ELISA plate at a volume of 100. Mu.l/well and incubated overnight (16-20 hours) at 4℃in a refrigerator. After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 150. Mu.l/well of a blocking solution of 3% Bovine Serum Albumin (BSA) diluted with PBST was added, and incubated at room temperature for 1 hour for blocking. After blocking, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

The antibodies to be tested were diluted with 3% BSA in PBST, 1. Mu.M starting, 10-fold gradient, 10 doses, 100. Mu.l/well into the ELISA plate and incubated at room temperature for 1 hour. After incubation, plates were washed 4 times with PBST, 100. Mu.l/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab 97225) diluted with 3% BSA in PBST was added and incubated for 1 hour at room temperature. After washing the plate 4 times with PBST, 100. Mu.l/well TMB chromogenic substrate (Cell Signaling Technology, cat# 7004S) was added, incubated at room temperature for 1 minute in the absence of light, the reaction was stopped by adding 100. Mu.l/Kong Zhongzhi solution (Cell Signaling Technology, cat# 7002S), the absorbance was read at 450nm using a microplate reader (BioTek, model number Synergy H1), and the data was analyzed. The concentration signal value curve analysis results are shown in the following table:

TABLE 3 affinity of murine antibodies to human TROP-2 antigen (EC ₅₀ Value of

Murine antibodies	Binding to human TROP-2His antigen EC ₅₀ (nM)
		M1	0.56

EXAMPLE 4 mouse antibody humanization experiments

Humanization of murine anti-human TROP-2 monoclonal antibodies was performed as disclosed in many of the literature in the art. Briefly, murine antibody M1 is humanized according to the present invention using human constant domains in place of the parent (murine) constant domains, and selecting a humanized antibody sequence based on the homology of murine and human antibodies.

Based on the obtained typical structure of the VH/VL CDR of the murine antibody, comparing the heavy and light chain variable region sequences with a human antibody germline database to obtain a human germline template with high homology.

The CDR regions of the murine antibody M1 were grafted onto the selected corresponding humanized templates. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, residues with direct interaction with the CDR region and residues with important influence on the conformation of VL and VH are subjected to back mutation, and the antibody formed by combining the humanized heavy chain variable region HCVR and light chain variable region LCVR sequences is selected through expression test and comparison of the number of back mutations, wherein the sequences are as follows:

HU1 HCVR

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGKGLEWVGRIDPNDSETHYNQKFKDRFTITRDNSKNTLYLQMNSLRAEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

SEQ ID NO:9

HU1 LCVR

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTRIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:10

HU2 HCVR

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWIGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

SEQ ID NO:11

HU2 LCVR

ETTLTQSPAFMSATPGDKVNISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:12

HU3 HCVR

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTMVTVSS

SEQ ID NO:13

HU3 LCVR

DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:14

HU4 HCVR

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWIGRIDPNDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQGTMVTVSS

SEQ ID NO:15

HU4 LCVR

DIVMTQSPDSLAVSLGERATINCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:16

HU5 HCVR

EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWIGRIDPNDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

SEQ ID NO:17

HU5 LCVR

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:18

HU6 HCVR

EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

SEQ ID NO:19

HU6 LCVR

DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:20

HU7 HCVR

EVQLVESGGGLIQPGGSLKLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

SEQ ID NO:21

HU7 LCVR

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:22

HU8 HCVR

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSS

SEQ ID NO:23

HU8 LCVR

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPSRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:24

HU9 HCVR

EVQLVESGGGLIQPGGSLRLSCAASGFTVSNYWMNWVRQAPGQGLEWMGRIDPNDSETHYNQKFKDRLTITKDTSKNQVVLTMTNMDPVDTATYYCARSGFGSTYWFFDVWGQGTTVTVSS

SEQ ID NO:25

HU9 LCVR

DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIK

SEQ ID NO:26

designed heavy and light chain variable region sequences were joined to IgG heavy chain constant region and human antibody light chain constant region sequences, respectively, with exemplary heavy chain constant region and light chain constant region sequences as follows:

IgG1 C1

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:48

IgG1 C2

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:49

Ig kappa C

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:50

The heavy and light chain sequences were obtained as follows (wherein HU1-HU9 heavy chain was derived from the ligation of sequences SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 to sequence SEQ ID NO:49, respectively; HU6DL and HU10 heavy chain was derived from the ligation of sequences SEQ ID NO:19, SEQ ID NO:9 to sequence SEQ ID NO:48, respectively):

HU1 HC

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGKGLEWVGRIDPNDSETHYNQKFKDRFTITRDNSKNTLYLQMNSLRAEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：27

HU1 LC

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTRIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：28

HU2 HC

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWIGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：29

HU2 LC

ETTLTQSPAFMSATPGDKVNISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：30

HU3 HC

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：31

HU3 LC

DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：32

HU4 HC

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWIGRIDPNDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：33

HU4 LC

DIVMTQSPDSLAVSLGERATINCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：34

HU5 HC

EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWIGRIDPNDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：35

HU5 LC

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：36

HU6 HC

EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：37

HU6 LC

DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：38

HU7 HC

EVQLVESGGGLIQPGGSLKLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：39

HU7 LC

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：40

HU8 HC

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：41

HU8 LC

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPSRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：42

HU9 HC

EVQLVESGGGLIQPGGSLRLSCAASGFTVSNYWMNWVRQAPGQGLEWMGRIDPNDSETHYNQKFKDRLTITKDTSKNQVVLTMTNMDPVDTATYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：43

HU9 LC

DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：44

HU6DL HC

EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDSETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：45

HU6DL LC

SEQ ID NO：38

HU10 HC

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGKGLEWVGRIDPNDSETHYNQKFKDRFTITRDNSKNTLYLQMNSLRAEDTAVYYCARSGFGSTYWFFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：46

HU10 LC

SEQ ID NO：28

TABLE 4 sequence numbering of antibodies and their heavy, light and variable regions

cDNA fragments were synthesized from the amino acid sequences of the light and heavy chains of the above humanized antibodies, and inserted into pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the transfection reagent PEI (Polysciences, inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) in a 1:2 ratio and placed in CO ₂ Incubate in incubator for 4-5 days. Collecting cell culture solution, centrifugally filtering, loading the cell culture solution to an antibody purification affinity column, eluting by a phosphate buffer solution, eluting by glycine-hydrochloric acid buffer solution (pH2.7.1M Gly-HCl), neutralizing by 1M Tris-hydrochloric acid pH 9.0, and dialyzing by the phosphate buffer solution to obtain the humanized antibody protein, wherein the concentration and the purity of the humanized antibody protein are shown in the following table:

TABLE 5 concentration and purity of the humanized antibodies

Example 5 in vitro binding affinity and kinetic experiments

Affinity of each humanized antibody for human TROP-2 antigen (EC) as determined using the in vitro indirect ELISA binding assay of example 4 (1) ₅₀ ) The following table shows:

TABLE 6 affinity of each humanized antibody for human TROP-2 antigen (EC ₅₀ )

To examine the binding capacity of each humanized antibody to the target protein TROP-2 on tumor cells, the affinity (EC) of each humanized antibody for HCC827 tumor cells (non-small cell lung cancer), MAB-MB-468 tumor cells (breast cancer, invasive ductal carcinoma) was determined using the in vitro cell binding assay of example 4 (2) ₅₀ ) The following table shows:

TABLE 7 affinity of each humanized antibody for HCC827 tumor cells (EC ₅₀ )

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EXAMPLE 6 humanized antibody mediated tumor cell killing

Humanized antibodies can exert killing effects on tumor cells in a number of ways, one of which is to mediate killing effects of immune cells on tumor cells. To examine the tumor cell killing effect of the humanized antibody-mediated immune cells of the present invention, a system of co-culture of human Peripheral Blood Mononuclear Cells (PBMC) with HCC827 tumor cells (non-small cell lung cancer) was used for evaluation. HCC827 cells were collected and after centrifugation, the cell density was adjusted to 0.44X10 with complete medium ⁶ Per mL, spread in the middle 60 wells of a white 96-well plate, 90. Mu.L per well, cell number 4000. Commercial human PBMC cells were collected and after centrifugation, cell density was adjusted to 2.2X10 with complete medium ⁶ Each mL was plated on a white 96-well plate containing HCC827 cellsThe middle 60 wells, 90 μl per well, had a cell number of 20000. 200. Mu.L of PBS was added to the remaining wells, and the cell plates were incubated overnight at 37℃in a 5% CO2 incubator. The next day of the experiment, humanized antibody solutions were prepared in 96-well V-bottom plates with 1000nM initial concentration, 3-fold dilution, 9 concentrations, and after preparation, added to white 96-well plates with 20 μl per well, two-well, and the cell plates were placed in a 5% co2 incubator at 37 ℃ for a further 72 hours. Fifth day of experiment, detection reading: the cell culture plate was taken out, equilibrated to room temperature, 50. Mu.L of CTG solution (Promega G7573) was added to each well, mixed by shaking, left in the dark for 10 minutes, and then examined by a luminescence program of an enzyme-labeled instrument. The experimental results are shown in table 8 below:

TABLE 8 humanized antibody mediated killing of tumor cells

The killing effect of HU6 antibody on HCC827 tumor cells was measured by the same method, and the result showed that the highest dose killing effect was 52.3%.

EXAMPLE 7 humanized antibody mediated TROP-2 endocytosis

To study humanized antibody mediated endocytosis of TROP-2 protein in tumor cells, SW780 cells were digested with pancreatin, cells were collected and resuspended in pre-chilled PBS to adjust cell concentration to 1×10 ⁶ And each mL. Taking an EP tube, adding 1mL of cell suspension, centrifuging at 1500rpm for 5min, removing supernatant, adding 1mL of prepared antibody to be detected to re-suspend cells, incubating for 1h by a 4-degree shaking table, centrifuging, discarding supernatant (4 ℃,1500rpm multiplied by 5 min), washing twice by PBS, and removing supernatant. 100. Mu.L of a fluorescent secondary antibody working solution was added to each tube to resuspend the cells, incubated for 30min at 4℃in a shaker, the supernatant was centrifuged off (4℃at 1500 rpm. Times.5 min), washed twice with PBS, and the supernatant was removed. Adding 1.0mL of preheated SW780 cells per tube to fully culture medium, re-suspending the cells, mixing, and packaging4 tubes, 200. Mu.L each, respectively, of the 0min group, the blank group, the 30min group and the 2h group, taking out the 0min and blank groups, placing the rest on ice, placing the rest on a 37 ℃ incubator, endocytozing the rest for 30min and 2h respectively, taking out the 1 tube at different corresponding time points, placing on ice for precooling for 5min, centrifuging all the treatment groups, discarding the supernatant (4 ℃,1500rpm multiplied by 5 min), washing once with PBS, and removing the supernatant. 250. Mu.L of strip buffer was added to the tubes of all treatment groups except the 0min group, incubated at room temperature for 8min, the supernatant was centrifuged off (4 ℃ C., 1500 rpm. Times.5 min), washed twice with PBS, and the supernatant was removed. All treatment groups were added with 100. Mu.L of immunostaining fixative solution, left at 4℃for more than 30min, and tested on-machine with flow cytometer DxFlex. 200 μl of the mixture was taken from the 0min tube, and the immunostaining fixative solution was directly added. 200 μl was taken from the blank set, and strip buffer and immunostaining fixative were added directly. The machine was checked by flow cytometer DxFlex. Data statistics and analysis: average percentage of endocytosis (%) 30 min= (30 min group MIF-blanc group MFI)/(0 min group MFI-blanc group MFI), average percentage of endocytosis (%) 2 h= (2 h group MIF-blanc group MFI)/(0 min group MFI-blanc group MFI). The percentage endocytosis of the humanized antibodies was measured using the method described above as follows in table 9:

TABLE 9 humanized antibody mediated TROP-2 protein endocytosis

Example 8 competitive binding of humanized antibodies to antigen

The manner and binding sites of different antibodies to antigens are studied, usually using competitive binding experiments. hRS7 antibody protein was diluted to a concentration of 1. Mu.g/ml with PBS pH7.4, added to a 96-well high affinity ELISA plate at a volume of 100. Mu.l/well, and incubated overnight (16-20 hours) at 4℃in a refrigerator. After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 150. Mu.l/well of 2% Bovine Serum Albumin (BSA) blocking solution diluted with PBST was added, and incubated at room temperature for 1 hour for blocking. After blocking, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

The antibody to be tested was diluted to 100. Mu.g/ml with 2% BSA in PBST and added to the ELISA plate at 50. Mu.l/well. TROP-2His protein (Sino Biological Inc., cat# 10428-H08H) was diluted with 2% BSA in PBST and added to the ELISA plate at 50. Mu.l/well. The elisa plate was incubated at room temperature for 1 hour. After incubation, plates were washed 4 times with PBST, 100. Mu.l/well of anti-His HRP-labeled secondary antibody (Abcam, cat#ab 197049) diluted with PBST containing 2% BSA was added and incubated at room temperature for 1 hour. After washing the plate 4 times with PBST, 100. Mu.l/well TMB chromogenic substrate (Cell Signaling Technology, cat# 7004S) was added, incubated at room temperature for 1 minute in the absence of light, the reaction was stopped by adding 100. Mu.l/well Stop Solution (Cell Signaling Technology, cat# 7002S), and the absorbance was read at 450nm using a microplate reader (BioTek, model number Synergy H1) and the data was analyzed as shown in the following table. The humanized antibody has low binding inhibition rate to hRS7 antibody and TROP2 protein, which indicates that the humanized antibody does not compete with the hRS7 antibody for binding to the same epitope.

TABLE 10 antigen competitive binding of humanized antibodies to hRS7

Humanized antibodies	Inhibition rate
		hRS7	94.7％
HU1	13.5％
		HU6	6.7％
HU6DL	5.9％
		HU10	10.2％

EXAMPLE 9 Synthesis of Compound a

Raw material a-1 (4.1 g,9.71 mmol), raw material a-2 (4.3 g,8.09mmol, containing 4% amino isomer impurity) were taken in a 250mL reaction flask, DCM (54 mL) was added under nitrogen protection, meOH (18 mL) was stirred and cooled to 0 ℃, DMTMM (3.6 g,12.1 mmol), triethylamine (2.5 g,24.2 mmol) was added, and the reaction was kept at 0 ℃ for 1h with stirring, raw material a-2 was completely reacted in hplc, the reaction solution was evaporated under reduced pressure (< 25 ℃) and MTBE (120 mL) was added to stir and beaten (mud), the solution was poured out and filtered, the mud was added again with 120mL MTBE beaten (solid), filtered, the cake was washed with water (60 ml×2) and dried to give crude product, which was dissolved with dichloromethane and methanol, and wet column chromatography was carried out twice (eluent DCM: 40:1-20:1) to separate pure product a (6.2 g,7.37 mmol) purity: 99.3% and 91% yield.

EXAMPLE 9-2 Synthesis of Compound b

Putting a compound a (5.7 g,6.77 mmol) into a 500mL three-necked flask, adding dry THF (114 mL) under the protection of nitrogen, stirring for dissolving, cooling the mixture to about-10 ℃, adding DBU (3.09 g,20.31 mmol), keeping the internal temperature at-10 to-5 ℃ in the dripping process, after 5min of adding, keeping the internal temperature at-10 to-5 ℃ after dripping, reacting for 2.5h, and separating out solids.

Cooling the mixture to the internal temperature of-20 ℃, adding MTBE (114 mL), keeping the internal temperature at-20 to-10 ℃ during the period, completely separating out the product, filtering, washing a filter cake with MTBE (57 mL multiplied by 2), and pumping to obtain 6g of crude compound b, and preserving at-78 ℃ for later use.

Example 9-3 Synthesis of Compound e

Compound c (651 mg,1 mmol) was dissolved in 10mL of DCM and stirred under ice-cooling and DBU (266 mg,3 mmol) was added dropwise. After one hour of reaction in ice bath, the reaction was completed, compound d (257 mg,1 mmol) and HATU (420 mg,1.1 mmol) were added in this order, and after stirring in ice bath for 30 minutes, LCMS showed the reaction was completed, the reaction solution was concentrated at 25 ℃, and the residue was purified by column chromatography reverse phase column (ACN in H ₂ O,50% of the product) gives compound e as a reddish brown solid, 130mg, yield 19%.

MS:691.3[M+23]。

Examples 9-4 Synthesis of Compound f

Compound e (130 mg,0.19 mmol) was dissolved in DCM, anisole (62 mg,0.57 mmol) and dichloroacetic acid (245 mg,1.9 mmol) were added, the reaction was stirred overnight at room temperature for 16H, LC-MS was sampled for pilot control, the starting material was completely consumed, the reaction stopped, the reaction was concentrated at 25℃and the residue was purified by column chromatography on reverse phase (ACN/H) ₂ O,30% yield) to give compound f, as a pink solid, 53mg, 54% yield.

MS:519.2[M+1]。

Examples 9-5 Synthesis of Compound D

Compound f (23 mg,0.044 mmol) and compound b (27 mg,0.044 mmol) were dissolved in DCM (3 mL) and MeOH (1 mL) and cooled to-30℃under nitrogen. DMTMM (20 m) was addedg,0.067 mmol), the reaction is carried out at-20 ℃ to-10 ℃ for 1 hour, sampling LC-MS is carried out for central control, and the complete consumption of the raw materials is shown. The reaction was quenched by adding 10mL of water at-10deg.C, and separated by adding 30mL of DCM. The aqueous phase was extracted with DCM/meoh=10/1 (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure at 25℃and the residue was purified by prep. (ACN/H ₂ O/0.05% FA) to give compound D as a white solid, 5.8mg, yield 12%, HPLC purity 98.87%.

MS:1120.3[M+1]。

Example 10: preparation of antibody-conjugated drugs

Preparation of 1.1ADC1

To an aqueous solution of antibody HU6DL in PBS buffer (0.05M aqueous PBS buffer at ph=6.5; 7.3ml,13.8mg/ml,0.681 μmol) at 37 ℃ was added an aqueous solution of tris (2-carboxyethyl) phosphine (10 mm,0.239ml,1.70 μmol) prepared, and the mixture was placed in a water bath shaker and reacted for 3 hours with shaking at 37 ℃ to stop the reaction; the reaction solution was cooled to 25℃with a water bath, diluted to 14.0ml, and 3.3ml of the solution was taken out for reaction.

Compound D (3.0 mg, 3.72. Mu. Mol) was dissolved in 0.15mL of DMSO, added to the above 3.3mL solution, and placed in a water bath shaker to react for 3 hours at 25℃with shaking, and the reaction was stopped. The reaction was desalted and purified using Sephadex G25 gel column (eluent: 0.05M PBS buffer in water at pH 6.5, containing 0.001M EDTA) to give PBS buffer (1.35 mg/mL,13 mL) of an exemplary product ADC1 of mAb2 antibody conjugate, which was frozen at 4 ℃.

The average value y was determined by ultraviolet method. After placing the cuvette containing sodium succinate buffer solution in a reference absorption cell and a sample measuring absorption cell respectively, deducting the solvent blank, placing the cuvette containing the sample solution in the sample measuring absorption cell, and measuring the absorbance at 280nm and 370 nm.

And (3) data processing:

by establishing a standard curve, measuring the absorption at the wavelength of 280nm, determining the antibody content Cmab, measuring the absorption at the wavelength of 370nm, and determining the small molecule content CDrug.

Drug loading average y=cdrug/Cmab.

The y value of the product ADC1 was determined to be 4 by the above method. ADC1 (y=4) samples were obtained by UV-HPLC purification.

EXAMPLE 11 cell killing Activity of antibody drug conjugates

To examine the killing effect of the antibody-drug conjugate of the present disclosure on tumor cells, the Trop-2 positive cell line MDA-MB-468 (CBP 60387, south tokyo) was used for evaluation. The MDA-MB-468 monolayer cells cultured by pancreatin digestion are added into a culture medium to be resuspended, counted after centrifugation, and the cell density is adjusted to 4X 10 by using a complete culture medium ⁴ The cells are spread in the middle 60 holes of a white 96-well plate, 50 mu l of each hole, the cell number is 2000 cells/hole, 100 mu l/Kong Peiyang base is added to the edge hole, and the cell plate is placed in the space of 37 ℃ and 5% CO ₂ The incubator was incubated overnight. The next day, an antibody-drug conjugate working solution was prepared in 96-well V-bottom plates with a concentration of 10000nM, 10-fold diluted, 9 concentrations, and after preparation, added to a white 96-well plate with 50 μl per well, two wells, and the cell plates were placed in 5% co at 37 °c ₂ The incubator continued to incubate for 6 days. Day 6 of the experiment, detection reading: the cell culture plate was removed and after equilibration to room temperature, 50. Mu.l were added to each well

The cell viability detection reagent (Promega, cat#: G7573) was mixed with shaking, placed in the dark and allowed to stand for 20 minutes, and then detected by using the luminescence program of the enzyme-labeled instrument. Computing IC using GraphPad Prims software ₅₀ Values. The experimental results are shown in the following table: />

TABLE 11 killing of tumor cells by antibody conjugated drugs

By comparing cell killing IC ₅₀ The experimental results show that: ADC1 relative to negative control hIgG1-DHas very strong cell killing effect.

EXAMPLE 12 pharmacokinetics of antibody-conjugated drugs

The human FcRn transgenic mouse model was used to evaluate the drug metabolism of the anti-Trop-2 antibody drug conjugate ADC1 in mice. Human FcRn transgenic mice with average weight of 18-22g and 6-8 weeks age are randomly divided into 2 groups, 3 animals in each group are dosed with the drug conjugate in a single mode of 5mg/kg, I.V., PBS solvent is taken as a negative control group, blood is taken and separated from blood plasma at 1, 2, 4, 8, 24, 48, 96, 144, 240 and hours respectively, the blood plasma is frozen in a refrigerator at-20 ℃, then a high-affinity 96 Kong Pingde plate is coated by recombinant human Trop-2 protein, a diluted plasma sample to be detected is added, the Trop-2 ADC concentration in the plasma of the mice is detected by using HRP-labeled secondary antibodies, and PKSolver software is used for analyzing PK parameters by using a non-atrioventricular model and an intravascular dosing model. The experimental results are detailed in the following table

TABLE 12 pharmacokinetic parameters of antibody-drug conjugates

In combination with the above list of pharmacokinetic key parameters, ADC1 showed better metabolic properties.

EXAMPLE 13 in vivo anti-tumor Effect of antibody-conjugated drugs

In order to further study the killing effect of the antibody-drug conjugate on tumors formed in vivo, a Trop-2 positive tumor cell human triple negative breast cancer MDA-MB-468 tumor cell graft mouse model is adopted to evaluate the effect of inhibiting tumor proliferation in vivo of candidate molecules. Will be 5x10 ⁶ The MDA-MB-468 cells are injected into the subcutaneous of a Nude mouse (Balb/c Nude) with the age of 8 weeks and the weight of 18-20mg, and the tumor volume is up to 160mm after 10 days ³ Grouping, starting to inject the antibody-drug conjugate by intravenous injection, once a week, at a dose of 2mg/kg. Control with human IgG1 eggWhite, the dosage is 2mg/kg. Control or dosing groups each had 5 mice. Tumor inhibition was calculated by measuring tumor volume.

Tumor inhibition = 100% - (day 28 dosing group tumor volume-day 0 dosing group tumor volume)/(day 28 control group tumor volume-day 0 control group tumor volume).

TABLE 13 killing of tumors by antibody-drug conjugates

Administration group	Tumor inhibition rate (%)
		ADC1(y＝4)	87.71

Experimental results show that ADC1 (y=4) shows better tumor inhibiting effect when injected once a week with a dose of 2mg/kg.

Claims

1. An antibody drug conjugate represented by the general formula (I) or a pharmaceutically acceptable salt or a solvent compound thereof,

wherein:

R ^e Or R is ^f Each independently selected from hydrogen, deuterium, hydroxy, amino, alkyl, halo, haloalkyl, deuterated alkyl, or hydroxyalkyl; preferably, R ^e Or R is ^f Each of which is a single pieceIndependently selected from hydrogen or deuterium, more preferably hydrogen,

X ₁ or X ₂ Each independently selected from N, O or S; preferably X ₁ Or X ₂ Is a compound of the formula O,

the mAb is an anti-TROP-2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising HCDR1 shown in SEQ ID NO. 3, HCDR2 shown in SEQ ID NO. 4 and HCDR3 shown in SEQ ID NO. 5; and the light chain variable region comprises LCDR1 shown in SEQ ID NO. 6, LCDR2 shown in SEQ ID NO. 7 and LCDR3 shown in SEQ ID NO. 8.

2. The antibody drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the anti-TROP-2 antibody or antigen binding fragment thereof is selected from a murine antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a human antibody or antigen binding fragment thereof, or a humanized antibody or antigen binding fragment thereof.

3. The antibody drug conjugate of claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, igG2, igG3, or IgG4, or variant thereof,

preferably, the anti-TROP-2 antibody or antigen binding fragment thereof further comprises a heavy chain constant region of human IgG1, igG2 or IgG 4;

more preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as set forth in SEQ ID NO. 48, or SEQ ID NO. 49.

4. The antibody drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody or antigen binding fragment thereof further comprises a light chain constant region of a human antibody kappa chain, lambda chain, or variant thereof;

preferably, the anti-TROP-2 antibody or antigen binding fragment thereof further comprises a light chain constant region of a human antibody kappa chain;

more preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region as set forth in SEQ ID NO. 50.

5. The antibody drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the anti-TROP-2 antibody or antigen binding fragment thereof comprises a heavy chain variable region selected from the group consisting of the heavy chain variable regions shown in the following sequences, or a heavy chain variable region having at least 70%,75%,80%,85%,90%,95% or 99% identity to the sequences: SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23 or SEQ ID NO. 25;

And/or a light chain variable region selected from the group consisting of the light chain variable regions shown in the following sequences, or a light chain variable region having at least 70%,75%,80%,85%,90%,95% or 99% identity to the following sequences: SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24 or SEQ ID NO. 26.

6. The antibody drug conjugate of claim 5, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody or antigen binding fragment thereof comprises:

SEQ ID NO:9 and the heavy chain variable region shown in SEQ ID NO:10, a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:11 and the heavy chain variable region shown in SEQ ID NO:12, a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:13 and the heavy chain variable region shown in SEQ ID NO:14, a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:15 and the heavy chain variable region shown in SEQ ID NO:16, and a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:17 and the heavy chain variable region shown in SEQ ID NO:18, a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:19 and the heavy chain variable region shown in SEQ ID NO:20, and a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:21 and the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:23 and the heavy chain variable region shown in SEQ ID NO:24, and a light chain variable region shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:25 and the heavy chain variable region shown in SEQ ID NO: 26.

7. The antibody drug conjugate of claim 5, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody or antigen binding fragment thereof comprises a heavy chain selected from the group consisting of the heavy chains shown in the following sequences, or a heavy chain having at least 80%,85%,90%,95% or 99% identity to the sequences shown in seq id no: SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 33. SEQ ID NO: 35. SEQ ID NO: 37. SEQ ID NO: 39. SEQ ID NO: 41. SEQ ID NO: 43. SEQ ID NO:45 or SEQ ID NO:46;

and/or a light chain selected from the group consisting of the light chains shown in the following sequences, or a light chain having at least 80%,85%,90%,95% or 99% identity compared to the following sequences: SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO: 36. SEQ ID NO: 38. SEQ ID NO: 40. SEQ ID NO:42 or SEQ ID NO:44.

8. the antibody drug conjugate of claim 7, or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody comprising:

SEQ ID NO:27 and SEQ ID NO:28, a light chain as shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:29 and the heavy chain shown in SEQ ID NO:30, a light chain as shown in seq id no; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:31 and the heavy chain shown in SEQ ID NO:32, a light chain indicated by 32; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:33 and the heavy chain shown in SEQ ID NO:34, a light chain indicated by 34; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:35 and the heavy chain shown in SEQ ID NO:36, a light chain indicated by 36; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:37 and SEQ ID NO:38, a light chain indicated at 38; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:39 and the heavy chain shown in SEQ ID NO: 40. Or alternatively, the first and second heat exchangers may be,

SEQ ID NO:41 and the heavy chain shown in SEQ ID NO: 42; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:43 and SEQ ID NO:44, a light chain indicated by 44; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:45 and the heavy chain shown in SEQ ID NO:38, a light chain indicated at 38; or alternatively, the first and second heat exchangers may be,

SEQ ID NO:46 and the heavy chain shown in SEQ ID NO: 28.

9. The antibody drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1-8, selected from a prodrug conjugate or pharmaceutically acceptable salt or solvate thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, according to formula (II):

Wherein mAb, y and v are as defined in claim 1.

10. The antibody drug conjugate or pharmaceutically acceptable salt or solvent compound thereof according to claim 9, selected from the group consisting of a bulk drug conjugate represented by general formula (III):

wherein v is 1 or 2.

11. The antibody drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 10, selected from the following structures:

/>

/>

/>

wherein y is as defined in claim 1.

12. A method of preparing an antibody-drug conjugate of general formula (I) or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:

after mAb reduction, the mAb is coupled with a general formula (F) to obtain a compound shown in a general formula (I),

wherein W, mAb, y are as defined in claim 1.

13. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1-11 or a pharmaceutically acceptable salt or solvate of the antibody drug conjugate, and one or more pharmaceutically acceptable excipients, diluents or carriers.

14. The antibody drug conjugate of any one of claims 1-11 or a pharmaceutically acceptable salt or solvate thereof or the pharmaceutical composition of claim 13 for use in the treatment of a TROP-2 related disease or disorder.

15. The use according to claim 14, wherein the disease associated with human TROP-2 is a cancer in which TROP-2 is highly expressed, the cancer is selected from triple negative breast cancer, small cell lung cancer, urothelial cancer, human brain astrocytoma, human pharyngeal head cancer, adrenal gland tumor, AIDS-related cancer, acinar soft tissue sarcoma, astrocytoma, cancer of the breast, brain and spinal cord, metastatic brain tumor, breast cancer, carotid aneurysm, cervical cancer, chondrosarcoma, chordoma, renal chromocytoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, ewing tumor, extraosseous mucoid chondrosarcoma, bone fibrodysplasia, gall bladder or bile duct cancer, gastric cancer, gestational trophoblastoma, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor Kaposi's sarcoma, renal carcinoma, leukemia, liposarcoma, malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrinopathy, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, nameplate cell tumor, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastatic cancer, rhabdomyoma, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, innocent pill cancer, thymus cancer, thymoma, thyroid metastatic cancer, and uterine cancer.