CN115298220A

CN115298220A - Antibody-drug conjugates and their medical uses

Info

Publication number: CN115298220A
Application number: CN202180021931.8A
Authority: CN
Inventors: 花海清; 包如迪
Original assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Current assignee: Jiangsu Hansoh Pharmaceutical Group Co Ltd; Shanghai Hansoh Biomedical Co Ltd
Priority date: 2020-03-24
Filing date: 2021-03-23
Publication date: 2022-11-04
Also published as: TW202144012A; WO2021190480A1

Abstract

Relates to an antibody-drug conjugate and medical application thereof. In particular to an anti-TROP-2 antibody-drug conjugate and medical application thereof. Further, it relates to an antibody-drug conjugate comprising an anti-TROP-2 antibody or an antigen-binding fragment thereof, or a pharmaceutically acceptable salt or solvate thereof, and its use in the manufacture of a medicament for the treatment of a TROP-2 mediated disease or condition, and for use in tumor detection and diagnosis.

Description

Antibody-drug conjugates and their medical uses

Technical Field

The present invention relates to an anti-TROP-2 antibody-drug conjugate which specifically has immunoreactivity to a human TROP-2 receptor, a pharmaceutical composition thereof, and uses thereof as an anticancer drug and for detecting or diagnosing tumors.

Background

With the continuous and deep research on tumor genomics, proteomics and signal transduction pathways, the interaction of cancer genes and cancer suppressor genes of tumor cells and the influence of the cancer genes and the cancer suppressor genes on the tumor microenvironment become clearer, so that the design of a new anti-tumor treatment scheme aiming at the specific molecular target of the tumor becomes possible.

The molecular target 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 function of target site molecule is directly influenced or the carried physical or chemical effector molecule is used to kill or inhibit target cell. Because the target position is clear, the medicine has high selectivity, can effectively kill or inhibit target cells, and does not generate or only generates less toxic and side effects on normal tissue cells. Therefore, the development of molecular targeted drugs is a hot spot for clinical research on tumors.

Human trophoblast cell surface antigen 2 (human trophoblast cell surface antigen 2, trop-2) is a cell surface glycoprotein encoded by the tactd 2 gene. TROP-2 is composed of 323 amino acids, wherein 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 by 11 to 13kD upon addition of sugar chains. The extracellular domain of the tactd gene family has a characteristic Thyroglobulin (TY) sequence, which is generally thought to be involved in the proliferation, infiltration, and metastasis of cancer cells.

Until now, no physiological ligand of TROP-2 has been identified, and the molecular function has not been elucidated, but since the intracellular 303 th residue serine (S303) can pass Ca ²⁺ Phosphorylation is carried out by the action of dependent Protein Kinase C (PKC), so that hydrolysis of 4, 5-diphosphatidylphosphatidylethyl-inositol (PIP 2) is promoted, and mitogen-activated protein kinase pathway (MAPK) -related inositol triphosphate IP3 is formed, and the signal pathway is closely related to cell proliferation, so that the Trop2 has the function of mediating signal transmission in tumor cells.

A large number of clinical studies and literature reports indicate that TROP-2 is over-expressed in various epithelia cancers such as gastric cancer, lung cancer, large intestine cancer, ovarian cancer, breast cancer, prostatic cancer, pancreatic cancer, liver cancer, esophageal cancer and the like. In contrast, TROP-2 was expressed only in a small amount in cells in the epithelial region, and was not expressed in normal tissues of adults, and the expression level was lower than that in cancer, indicating that TROP-2 is involved in tumor formation. The overexpression of TROP-2 in tumor tissues is closely related to poor prognosis of patients and metastasis of cancer cells, and affects the overall survival rate of patients. Thus, TROP-2 has become an attractive target for molecular targeted therapy of tumors.

Several studies of the anti-tumor effect of anti-hTROP-2 antibodies have been reported:

U.S. Pat. No. 5,5840854 reports the cytotoxicity of anti-hTROP-2 monoclonal antibody (BR 110) bound to cytotoxin against human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981.

U.S. Pat. No. 6653104 discloses an antibody (RS 7) which has been tested in an in vivo model using an antibody labeled with a radioactive substance and shows an antitumor activity in a nude mouse xenograft model, but an antitumor effect is not reported only in a nude antibody.

U.S. Pat. No. 7420040 also reports that isolated monoclonal antibodies produced by hybridoma cell lines AR47A6.4.2 or AR52A301.5, derived from mice immunized with human ovarian cancer tissue, bind to hTROP-2 and show anti-tumor activity in a nude mouse xenograft model.

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 a remarkable inhibitory effect on proliferation of pancreatic cancer cells.

CN104114580A discloses an antibody (particularly humanized antibody) specifically reacting with hTROP-2 and having anti-tumor activity in vivo, as well as a hybridoma producing the antibody, a complex of the antibody and a drug, a pharmaceutical composition for diagnosis or treatment of tumor, a method for detecting tumor, and a kit for detection or diagnosis of tumor.

Disclosure of Invention

According to some embodiments of the present invention, there is provided an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, represented by general formula (a):

Ab- ⁽ L ₂ -L ₁ -D)y (A)

wherein:

d is a cytotoxic drug;

L ₁ is selected from-O- (CR) ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-、-O-CR ⁵ R ⁶ -(CR ^a R ^b ) _m -、-O-CR ⁵ R ⁶ -、-NH-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C (O) -or-S- (CR) ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-；

R ^a And R ^b The same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxyl, aminoCyano, nitro, hydroxyalkyl, cycloalkyl or heterocyclyl;

or, R ^a And R ^b Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

R ⁵ selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, or heteroaryl;

R ⁶ selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, or heteroaryl;

or, R ⁵ And R ⁶ Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

or, R ^a And R ⁶ Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

m is an integer selected from 0 to 4;

y is selected from a number from 1 to 10, y is a decimal or an integer;

L ₂ is a joint unit;

ab is an anti-TROP-2 antibody or antigen-binding fragment thereof comprising an antibody light chain variable region comprising at least 1 HCDR selected from the group consisting of seq id nos: 3, SEQ ID NO; the antibody light chain variable region comprises at least 1 LCDR selected from the following sequences: 6,7, 8 of SEQ ID NO.

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

HCDR1 shown in SEQ ID NO. 3,

HCDR2 and SEQ ID NO. 4

HCDR3 shown in SEQ ID NO. 5.

In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present 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 LCDR2 shown in SEQ ID NO. 7

LCDR3 shown in SEQ ID NO. 8.

HCDR1 shown in SEQ ID NO. 3,

HCDR2 and SEQ ID NO. 4

HCDR3 as shown in SEQ ID NO. 5; and

the antibody light chain variable region comprises:

LCDR1 shown in SEQ ID NO. 6,

LCDR2 and LCDR2 as shown in SEQ ID NO. 7

LCDR3 shown in SEQ ID NO. 8.

In a preferred embodiment of the present invention, 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, according to the present invention, or a pharmaceutically acceptable salt or solvate thereof.

In a 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, igG3 or IgG4, or a variant thereof, according to the antibody-drug conjugate of the invention, or a pharmaceutically acceptable salt or solvate 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 present 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 as set forth in SEQ ID NO. 49.

In a preferred embodiment of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof, according to the antibody-drug conjugate of the invention, or a pharmaceutically acceptable salt or solvate thereof, further comprises a light chain constant region derived from a human antibody kappa chain, lambda chain, 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 present 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 present invention, the antibody-drug conjugate according to the present 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 those shown in seq id no, or those having at least 70%,75%,80%,85%,90%,95% or 99% identity as compared to seq id no:9, 11, 13,15, 17, 19, 21, 23 or 25 SEQ ID NO.

In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present 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 those shown in seq id no, or a light chain variable region having at least 70%,75%,80%,85%,90%,95% or 99% identity compared to seq id no:10, 12, 14, 16, 18, 20, 22, 24 or 26 SEQ ID NO.

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 those shown in seq id no, or those having at least 80%,85%,90%,95% or 99% identity compared to seq id no: SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 33. the amino acid sequence of 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:47.

in a preferred embodiment of the present invention, the anti-TROP-2 antibody or antigen-binding fragment thereof, according to the antibody-drug conjugate of the invention, or a pharmaceutically acceptable salt or solvate thereof, contains a light chain selected from the group consisting of those shown in seq id no: the amino acid sequence of 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, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate compound thereof, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises:

(1) SEQ ID NO:9 and SEQ ID NO:10, a light chain variable region; or,

(2) The amino acid sequence of 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,

(3) SEQ ID NO:13 and SEQ ID NO:14, a light chain variable region; or the like, or a combination thereof,

(4) The amino acid sequence of SEQ ID NO:15 and SEQ ID NO:16, a light chain variable region shown in seq id no; or,

(5) The amino acid sequence of SEQ ID NO:17 and SEQ ID NO:18, the light chain variable region shown in; or,

(6) SEQ ID NO:19 and the heavy chain variable region of SEQ ID NO:20, a light chain variable region; or the like, or a combination thereof,

(7) The amino acid sequence of SEQ ID NO:21 and the heavy chain variable region shown in SEQ ID NO:22, a light chain variable region shown in seq id no; or the like, or a combination thereof,

(8) The amino acid sequence of SEQ ID NO:23 and SEQ ID NO:24, a light chain variable region; or,

(9) The amino acid sequence of SEQ ID NO:25 and the heavy chain variable region of SEQ ID NO:26, or a light chain variable region as shown.

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

(1) The amino acid sequence of SEQ ID NO:27 and the heavy chain of SEQ ID NO:28, a light chain; or,

(2) SEQ ID NO:29 and the heavy chain of SEQ ID NO:30, a light chain; or,

(3) The amino acid sequence of SEQ ID NO:31 and the heavy chain of SEQ ID NO:32, a light chain; or the like, or a combination thereof,

(4) SEQ ID NO:33 and SEQ ID NO:34, a light chain; or the like, or a combination thereof,

(5) SEQ ID NO:35 and SEQ ID NO:36, a light chain; or the like, or a combination thereof,

(6) The amino acid sequence of SEQ ID NO:37 and SEQ ID NO: 38; or,

(7) SEQ ID NO:39 and SEQ ID NO:40, a light chain; or,

(8) SEQ ID NO:41 and the heavy chain of SEQ ID NO: 42; or the like, or a combination thereof,

(9) The amino acid sequence of SEQ ID NO:43 and the heavy chain of SEQ ID NO:44, a light chain; or the like, or a combination thereof,

(10) SEQ ID NO:45 and SEQ ID NO: 38; or,

(11) SEQ ID NO:47 and the heavy chain of SEQ ID NO:28, or a light chain as shown.

In some embodiments of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, L ₁ As shown in the general formula (B):

wherein,

M ₁ is-CR ₁ R ₂ -；

R ₁ And R ₂ Identical or different, R ₁ And R ₂ Each independently selected from hydrogen, alkyl, halogen, hydroxy or amino;

n is selected from integers from 0 to 5, preferably 1,2 or 3.

In some embodiments of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, L ₂ As shown in the general formula (C):

wherein,

M ₂ is-CR ₄ R ₅ -；

R ₃ Selected from hydrogen, halogen, hydroxy, amino, alkyl, alkoxy, and cycloalkyl:

R ₄ and R ₅ The same or different, each independently selected from hydrogen, alkyl, halogen, hydroxyl or amino;

m is an integer from 0 to 5, preferably 1,2 or 3.

In a further preferred embodiment of the invention, L is ₁ And the O terminal and the joint unit L of ₂ Are connected.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate compound thereof, wherein:

L ₁ is-O- (CR) ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-；

R ^a And R ^b Are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, or an alkyl group;

R ⁵ is haloalkyl or C _3-6 A cycloalkyl group;

R ⁶ selected from hydrogen atoms, haloalkyl radicals or C _3-6 A cycloalkyl group;

or, R ⁵ And R ⁶ Together with the carbon atom to which they are attached form C _3-6 A cycloalkyl group;

m is selected from 0 or 1.

In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention, or a pharmaceutically acceptable salt or solvent compound thereof, the L ₁ As shown in the general formula (E):

wherein R is ⁵ Is a halogenated alkyl group or a cycloalkyl group,

R ⁶ selected from hydrogen, haloalkyl or cycloalkyl,

or, R ⁵ And R ⁶ Together with the carbon atom to which they are attached form a cycloalkyl group;

preferably, the first and second electrodes are formed of a metal,

R ⁵ is selected from C _1-6 Haloalkyl or C _3-6 A cycloalkyl group,

R ⁶ selected from hydrogen, C _1-6 Haloalkyl or C _3-6 A cycloalkyl group,

m is an integer from 0 to 4.

In a further preferred embodiment of the present invention, the general formula (E) is selected from the following substituents:

in some embodiments of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, the L ₂ As shown in the following general formula (D):

-K ¹ -K ² -K ³ -K ⁴ - (D)

wherein,

K ¹ is composed of

s is an integer from 2 to 8;

K ² is selected from-NR ¹ (CH ₂ CH ₂ O) _p CH ₂ CH ₂ C(O)-、-NR ¹ (CH ₂ CH ₂ O) _p CH ₂ C(O)-、-S(CH ₂ ) _p C (O) -or a single bond, p is selected from an integer from 1 to 20, preferably from 1 to 6;

R ¹ selected from the group consisting of hydrogen, deuterium, hydroxy, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl;

K ³ is a tetrapeptide residue, preferably selected from the group consisting of peptide residues formed from two or more amino acids of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid; more preferably the tetrapeptide residues of GGFG;

K ⁴ is-NR ² (CR ³ R ⁴ )t-，R ² 、R ³ Or R ⁴ Each independently hydrogen, deuterium, hydroxy, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl, and t is selected from 1 or 2.

In a preferred embodiment of the invention, an antibody-drug couple according to the inventionA conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit-L ₂ -, K thereof ¹ End is connected to Ab, K ⁴ Terminal and L ₁ Are connected.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention or a pharmaceutically acceptable salt or solvate thereof, wherein said-L ₂ -L ₁ -is of the structure:

wherein, K ² Is a bond;

K ³ is a tetrapeptide residue of GGFG;

R ⁵ selected from haloalkyl or C _3-6 A cycloalkyl group;

R ⁶ selected from hydrogen, haloalkyl or C _3-6 A cycloalkyl group;

R ² 、R ³ or R ⁴ Each independently selected from hydrogen or alkyl;

s is selected from an integer from 2 to 8;

m is an integer from 0 to 4.

In a further preferred embodiment of the present invention, said-L ₂ -L ₁ -is selected from the following structures:

in some embodiments of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug is selected from the group consisting of a toxin, a chemotherapeutic drug, an antibiotic, a radioisotope and a nucleolytic enzyme.

In a preferred embodiment of the invention, the cytotoxic drug is selected from the group consisting of a tubulin inhibitor or a DNA topoisomerase inhibitor that inhibits cell division; preferably camptothecin derivatives, DM1, DM3, DM4, SN-38, MMAF or MMAE; more preferably, irinotecan or an irinotecan derivative, SN-38, MMAE or MMAF.

In a preferred embodiment of the invention, the cytotoxic drug is selected from the group consisting of:

in a preferred embodiment of the invention, the cytotoxic drug is selected from an irinotecan derivative, preferably the irinotecan derivative is compound 2-a:

in a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention, or a pharmaceutically acceptable salt or solvate thereof, is a compound represented by general formula (I):

wherein L is ₁ 、L ₂ Is a joint unit;

y is a number selected from 1 to 10, preferably a number from 2 to 8, more preferably a number from 2 to 4;

ab is selected from the TROP-2 antibodies or antigen-binding fragments thereof as described above.

In a preferred embodiment of the present invention, the general formula (I) is represented by the general formula (I-A):

wherein L is ₂ As defined above; preferably, L2 is as defined for formula (C) above.

In a preferred embodiment of the present invention, the general formula (I) is represented by the general formula (I-B):

wherein L is ₁ As defined above; preferably, L2 is as defined for formula (B) above.

In some embodiments of the invention, the antibody-drug conjugate according to the present invention, or a pharmaceutically acceptable salt or solvate thereof, is a compound represented by general formula (II):

wherein L is ₁ 、L ₂ Is a joint unit;

ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof as described previously;

in a preferred embodiment of the present invention, the general formula (II) is represented by the general formula (II-A):

In a preferred embodiment of the present invention, the general formula (II) is represented by the general formula (II-B):

in a preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to the present invention is a compound represented by formula (III):

wherein L is ₁ 、L ₂ Is a joint unit;

y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8;

ab is selected from the group consisting of TROP-2 antibodies or antigen-binding fragments thereof as described previously.

In a further preferred embodiment of the invention, the antibody-drug conjugate described by the general formula (III) or a pharmaceutically acceptable salt or solvate thereof, wherein L ₁ As defined by formula (E), L ₂ As defined by formula (D).

In a further preferred embodiment of the present invention, the antibody-drug conjugate described in the general formula (III) or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit-L2-, is linked to Ab at the K1 terminal and L1 at the K4 terminal.

In the invention further preferredIn one embodiment, the antibody-drug conjugate of formula (III), or a pharmaceutically acceptable salt or solvate thereof, said-L ₂ -L ₁ -is selected from the following structures:

K ² is a bond;

K ³ tetrapeptide residues that are GGFG;

R ⁵ selected from haloalkyl or C _3-6 A cycloalkyl group;

R ⁶ selected from hydrogen, haloalkyl or C _3-6 A cycloalkyl group;

R ² 、R ³ or R ⁴ Each independently hydrogen or alkyl;

s is selected from an integer from 2 to 8;

m is an integer from 0 to 4;

preferably, -L ₂ -L ₁ -is selected from the following structures:

in a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention, or a pharmaceutically acceptable salt or solvate thereof, is an antibody-drug conjugate represented by general formula (IV):

wherein,

w is selected from C _1-8 Alkyl radical, C _1-8 Alkyl-cycloalkyl or a linear heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C _1-8 Alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

K ² is selected from-NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-、-NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-、-S(CH ₂ ) _p1 C (O) -or a bond, R ¹ Selected from the group consisting of hydrogen atoms, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl radicals, p ₁ Is an integer from 1 to 20;

K ³ selected from the group consisting of peptide residues consisting of 2 to 7 amino acids, which may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents being one or more independently selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

R ² independently selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups;

R ³ and R ⁴ Each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl;

R ⁵ selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroAn aryl group;

R ⁶ selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;

m is an integer from 0 to 4;

y is selected from a number from 1 to 10, y is a decimal or an integer;

ab is selected from the anti-TROP-2 antibodies or antigen-binding fragments thereof described herein.

In Sup>A further preferred embodiment of the present invention, the antibody-drug conjugate or Sup>A pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate or Sup>A pharmaceutically acceptable salt or solvent compound thereof represented by the general formulSup>A (IV-Sup>A):

in Sup>A further preferred embodiment of the present invention, the general formulSup>A (IV-A) is selected from the following structures:

in some embodiments of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, is selected from the following compounds:

wherein Ab is selected from the anti-TROP-2 antibodies or antigen-binding fragments thereof described herein; preferably, ab is selected from HU1-HU10, HU6DL of the present invention.

In a preferred embodiment of the invention, the antibody-drug conjugate according to the invention, or a pharmaceutically acceptable salt or solvate thereof, is selected from the following compounds:

wherein y is selected from 2 to 10, preferably 4 to 8, more preferably 6 to 8, even more preferably 7 to 8, most preferably 8, y is a decimal or an integer.

In another aspect, the present invention provides a method for preparing a ligand-drug conjugate represented by the general formula (IV) or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:

ab is subjected to coupling reaction with a general formula (F) after being reduced to obtain a compound shown in a general formula (IV);

wherein Ab is selected from the group consisting of an anti-TROP-2 antibody or antigen-binding fragment thereof as described previously;

W、K ² 、K ³ 、R ² ～R ⁶ m and y are as defined in formula (IV).

In a preferred embodiment of the present invention, the process according to the general formula (IV) of the present invention, wherein the general formula (F) is a compound represented by the general formula (F-1) or a tautomer, mesomer, ectomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein, K ² 、K ³ 、R ² ～R ⁶ S and m are as defined in formula (IV).

In some embodiments of the invention, the compound according to formula (F) or formula (F-1) of the present invention is selected from:

in another aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate of the present 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 also provides an application of the antibody-drug conjugate described in the general formula (a) or a pharmaceutically acceptable salt or solvate of the antibody-drug conjugate or a pharmaceutical composition thereof in preparing a medicament for treating a disease related to human TROP-2.

The antibody-drug conjugate or the pharmaceutically acceptable salt or the solvent compound thereof can be specifically bound with a target antigen, has high endocytosis efficiency and long in-vivo half-life period, and obviously kills tumors while ensuring safety.

Drawings

FIG. 1: ELISA in vitro binding experiments of the antibodies showed the binding activity of 11 humanized anti-TROP-2 antibodies to human TROP-2 antigen.

Detailed Description

1. Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three letter codes and the one letter codes for amino acids used in the present invention are as described in j.biol.chem,243, p3558 (1968).

The term "antibody" as used herein refers to an immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., igM, igD, igG, igA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of Ig can be divided into different subclasses according to the differences of amino acid composition of the hinge region and the number and position of disulfide bonds of heavy chains, for example, igG can be divided into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. The second of the five classes of Ig may have either a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human-or murine-derived kappa or lambda chain or a variant thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising IgG1, igG2, igG3, igG4 or a variant thereof of human or murine origin.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence-conserved. 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 the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxyl terminus is: 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 refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues in the VL and VH regions of the antibodies or antigen-binding fragments of the invention conform in number and position to the known Kabat numbering convention and the Kabat or ABM definition convention (http:// bio if. Org. Uk/abs /).

The term "antigen presenting cell" or "APC" is a cell that displays foreign antigens complexed with MHC on its surface. 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 lymphoblastoid 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 an MHC-I/MHC-II conjugate.

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

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

1. antibodies isolated from transgenic, transchromosomal animals (e.g., mice) of human immunoglobulin genes or hybridomas prepared therefrom;

2. antibodies isolated from host cells transformed to express the antibodies, such as transfectomas;

3. antibodies isolated from a library of recombinant combinatorial human antibodies; and

4. antibodies produced, expressed, created or isolated by methods such as splicing of human immunoglobulin gene sequences to other DNA sequences.

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

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

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting a mouse CDR sequence into a human antibody variable region framework. The humanized antibody can overcome the disadvantage of strong immune response induced by the chimeric antibody due to carrying a large amount of mouse protein components. To avoid a decrease in activity associated with a decrease in immunogenicity, the human antibody variable regions may be subjected to minimal back-mutation to maintain activity.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing a chimeric antibody, selecting and establishing a hybridoma secreting a mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to needs, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing a chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the human antibody may be selected from the heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or a variant thereof, preferably comprising human IgG1, igG2 or IgG4 heavy chain constant region, or IgG1 heavy chain constant region that enhances ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. 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 as activity 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.

The "Fab fragment" consists of the CH1 and variable regions of one light and one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond 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 interaction of the CH3 domains.

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

An "F (ab') 2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

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

The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These 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 unit has polyepitopic specificity; an antibody 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; an antibody 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 non-covalently linked together, and the like.

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

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

The term "in conjunction with TROP-2" in the context of the present invention means capable of interacting with human TROP-2.

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

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

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

The term "cross-reactive" refers to the ability of an antibody of the invention to bind to TROP-2 from a different species. For example, an antibody of the invention that binds to human TROP-2 may also bind to TROP-2 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens, or binding or functional interactions with cells that physiologically express 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 upon ligand binding in the absence of inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-TROP-2 antibody compared to a ligand not contacted with an anti-TROP-2 antibody.

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

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

The term "ADCC", i.e., antibody-dependent cell-mediated cytotoxicity, as used herein refers to the direct killing of antibody-coated target cells by Fc fragments of cells expressing Fc receptors through recognition of the antibody. The ADCC effector function of an antibody may be enhanced or reduced or eliminated by modification of the Fc-fragment of the 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 prior art, such as the antibody test technical guide of cold spring harbor, chapters 5-8 and 15. For example, mice can be immunized with human TROP-2 or a fragment thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to incorporate one or more human FR regions in a CDR region of non-human origin. Human FR germline sequences can be obtained from the website http:// IMGT. Cines. FR of ImmunoGeneTiCs (IMGT) or from the immunoglobulin journal, 2001ISBN 012441351.

The engineered antibodies or antigen binding fragments of the invention can be prepared and purified using conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into the GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the FC region. Stable clones were obtained by expressing antibodies that specifically bind to antigens of human origin. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified and collected by conventional techniques. The antibody can be concentrated by filtration by a conventional method. 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 antibody of the present invention refers to a monoclonal antibody. The monoclonal antibodies (mAbs) of the present invention 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 recombination using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering," "administering," and "treating" also mean treating, e.g., a cell in vitro and ex vivo, by an agent, a diagnostic, a 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.

By "treating" is meant administering a therapeutic agent, such as any one of the antibodies of the invention, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in 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 useful degree. The amount of therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") may 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 commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in every patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, kruskal-Wallis test (H-test), jonckhere-Terpsra test, and Wilcoxon test.

The term "consisting essentially of 8230 \\8230%, \8230composition" or variations thereof, as used throughout the specification and claims, is meant to encompass all such elements or groups of elements, and optionally additional elements of similar or different nature to those described, which additional elements do not materially alter the basic or novel characteristics of a given dosing regimen, method or composition.

The term "naturally occurring" as applied to an object in accordance with the present invention refers to the fact that the object may be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow 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 method and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

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

"endogenous" refers to a substance produced in a cell, organism, or human body by background.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both compared sequences is occupied by the same base or amino acid monomer subunit, e.g., 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 matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, if there are 6 matches or homologies at 10 positions in two sequences when the sequences are optimally aligned, then the two sequences are 60% homologous. In general, the comparison is made when the two sequences are aligned to give the greatest percentage of homology.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny thereof. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.

"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 antibody heavy chain variable regions of a particular sequence may, but need not, be present.

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

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention. The experimental methods in the examples of the present invention, in which specific conditions are not specified, are generally performed under conventional conditions such as the antibody technique laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1 antigen preparation

The protein encoding His-tagged human TROP-2 (TROP-2-His) was synthesized by SinoBiologics corporation (10428-H08H).

TROP-2-His sequence:

example 2 murine hybridomas and obtaining antibody sequences

Animal immunization is carried out by using human antigen TROP-2-His, 5 Balb/c mice and 5A/J mice, female and 10 weeks old are used, sigma Complete Freund's Adjuvant (CFA) and Sigma Incomplete Freund's Adjuvant (IFA) are used, and immunogen and immunologic adjuvant are fully mixed and emulsified according to the proportion of 1; 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	And the third immunization, incomplete Freund's adjuvant.
Day 42	Blood sampling and serum titer test (3-serum free)
Day 49	Fourth immunization, incomplete Freund's adjuvant.
Day 56	Blood sampling and serum titer test (4 blood-free)

Serum titers and the ability to bind cell surface antigens were assessed using an indirect ELISA method as described in example 3 on immune mouse sera, with control titer measurements (greater than 10 ten thousand dilutions) determining the initiation of cell fusion. Selecting immunized mice with strong serum titer, affinity and FACS combination, performing one final immunization, killing the mice, and taking splenocytes and SP2/0 bone marrowAnd (3) after the tumor cells are fused, paving a plate to obtain hybridomas, screening the target hybridomas by indirect ELISA, and establishing strains as monoclonal cell strains by a limiting dilution method. The resulting positive antibody strains were further screened using indirect ELISA to select hybridomas that bind the recombinant protein. Hybridoma cells in the logarithmic growth phase were harvested, RNA extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript) ^TM Reverse Transcriptase, takara # 2680A). And (3) carrying out PCR amplification on cDNA obtained by reverse transcription by adopting a mouse Ig primer group (Novagen, TB326 Rev.B 0503) and then sequencing to finally obtain the sequence of the murine antibody.

The variable regions of the heavy chain and the light chain of the murine monoclonal antibody M1 have the following sequences:

TABLE 2 CDR sequences of the heavy and light chain variable regions of murine mAb M1

Name(s)	Sequence of	Number of
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 assay of antibodies

(1) In vitro indirect ELISA binding experiments:

TROP-2His protein (Nano 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 microplate 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 (pH7.4PBS containing 0.05% Tween-20), 150. Mu.l/well of 3% Bovine Serum Albumin (BSA) blocking solution diluted with PBST was added, and the plate was incubated at room temperature for 1 hour for blocking. After blocking was complete, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

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

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

Murine antibodies	Binds to human TROP-2His antigen EC50 (nM)
M1	0.56

(2) In vitro cell binding experiments:

collecting cultured TROP-2 high expression cells (CHO or 293 cells over-expressing TROP-2 and TROP-2-expressing tumor cells such as HCC-827, MDA-MB-468, etc.), adjusting cell density, and spreading on 96-well U-bottom plate with 1 × 10 cells per well ⁵ To 2X 10 ⁵ And (4) cells. 1200g,5min centrifugation, supernatant removal, addition of 100ul of antibody solution or mouse immune serum which is diluted in a gradient manner, and incubation for 60min at the temperature of 4 ℃;1200g,5min centrifugation, supernatant, PBS washing cells for 2 times, adding fluorescence labeling secondary antibody (PE-GAM or PE-GAH) 100ul each hole, 4 degrees C incubation for 60min.1200g, and 5min to remove the supernatant by centrifugation. After washing the cells 2 times with PBS, the cells were resuspended in PBS, and the signal was detected using a flow cytometer and analyzed by concentration profiling.

Example 4 mouse antibody humanization experiments

Humanization of murine anti-human TROP-2 monoclonal antibodies was performed as described in many publications in the art. Briefly, murine antibody M1 was humanized using human constant domains in place of the parent (murine antibody) constant domains, and human antibody sequences were selected based on the homology of the murine and human antibodies.

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

The CDR regions of murine antibody M1 were grafted onto the corresponding humanized templates that were selected. Then, based on the three-dimensional structure of the murine antibody, carrying out back mutation on the embedded residues, residues which directly interact with CDR regions, and residues which have important influence on the conformations of VL and VH, and selecting an antibody which is formed by designing the combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences through expression test and back mutation number comparison, wherein the sequences are as follows:

the heavy and light chain variable region sequences were designed to be linked to IgG heavy chain constant region and human antibody light chain constant region sequences, respectively, and exemplary heavy and light chain constant region sequences are as follows:

the heavy and light chain sequences were obtained as follows (wherein the HU1-HU9 heavy chains were derived from joining 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, and the HU10 heavy chains were derived from joining sequences SEQ ID NO:19, SEQ ID NO:9 to sequence SEQ ID NO:48, respectively):

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

cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of each of the humanized antibodies above, and inserted into pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). Expression vectors and transfection reagent PEI (Polysciences, inc. Cat.no. 23966) were transfected into HEK293 cells (Life Technologies cat.no. 11625019) at a ratio of 1 ₂ Incubate in incubator for 4-5 days. Collecting cell culture solution, loading the cell culture solution to an antibody purification affinity column after centrifugal filtration, washing the column by phosphate buffer, eluting by glycine-hydrochloric acid buffer solution (pH2.7.0.1M Gly-HCl), neutralizing by 1M Tris-hydrochloric acid pH 9.0, and dialyzing by phosphate buffer solution to obtain the humanized antibody protein of the invention, wherein the concentration and the purity of the humanized antibody protein are shown in the following table:

TABLE 5 concentration and purity of each humanized antibody

Humanized antibody numbering	Concentration (mg/ml)	Purity (%)
HU1	0.72	98.2％
HU2	0.62	98.4％
HU3	0.75	96.2％
HU4	0.96	96.4％
HU5	1.17	97.1％
HU6	1.35	96.8％
HU7	1.26	98.5％
HU8	1.36	98.3％
HU6DL	1.25	97.4％
HU10	1.21	98.2％

Example 5 in vitro binding affinity and kinetics experiments

Affinity (EC) of each humanized antibody to human TROP-2 antigen determined using the in vitro indirect ELISA binding assay of example 3 (1) ₅₀ ) As shown in the following table:

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

To examine the binding ability of each humanized antibody to the target protein TROP-2 on tumor cells, the affinity (EC) of each humanized antibody to HCC827 tumor cells (non-small cell lung cancer), MAB-MB-468 tumor cells (breast cancer, invasive ductal carcinoma) and to tumor cells (TROP-2) was determined using the in vitro cell binding experiment of example 3 (2) ₅₀ ) As shown in the following table:

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

Example 6 humanized antibody-mediated killing of tumor cells

The humanized antibody can exert the killing effect on the tumor cells from various aspects, and one of the effects is to mediate the killing effect of immune cells on the tumor cells. To test the killing effect of the humanized antibody-mediated immunocytes on tumor cells, human Peripheral Blood Mononuclear Cells (PBMC) were co-cultured with HCC827 tumor cells (non-small cell lung cancer) for evaluation. HCC827 cells were collected, and cell density was adjusted to 0.44X 10 with complete medium after centrifugal counting ⁶ Perml, spread in 60 wells of a white 96-well plate, 90. Mu.L per well, and the number of cells was 4000. Commercial human PBMC cells were collected and after centrifugation counted adjusted to a cell density of 2.2X 10 with complete medium ⁶ one/mL, spread in the middle 60 wells of a white 96-well plate with HCC827 cells, 90. Mu.L per well, and the number of cells is 20000. The remaining wells were filled with 200. Mu.L of PBS, and the cell plates were incubated at 37 ℃ in a 5% CO2 incubator overnight. The following day of the experiment, humanized antibody solutions were prepared in 96-well V-bottom plates with PBS at a concentration of 1000nM starting, 3-fold dilutions, 9 concentrations, added to white 96-well plates at 20 μ L per well, duplicate wells, and the plates were placed in 37 ℃ c, 5% co2 incubator for a further 72 hours. Fifth day of experiment, assay reading: the cell culture plate was removed, allowed to equilibrate to room temperature, and 50. Mu.L of CTG solution (Promega G7573) was added to each well and shakenAfter mixing uniformly, the mixture is placed in a dark place for 10 minutes, and then the detection is carried out by using a luminescence program of an enzyme-labeling instrument. The results of the experiment are shown in table 8 below:

TABLE 8 humanized antibody mediated killing of tumor cells

The same method is adopted to measure the killing effect of the HU6 antibody on HCC827 tumor cells, and the result shows that the highest dose killing effect is 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 trypsinized, harvested and resuspended in precooled PBS to a cell concentration of 1X 10 ⁶ One per mL. Adding 1mL of cell suspension into an EP tube, centrifuging at 1500rpm for 5 minutes, removing supernatant, adding 1mL of prepared antibody to be detected for heavy suspension of cells, wherein the final concentration of the antibody is 20 mu g/mL, incubating for 1h by a shaking table at 4 ℃, centrifuging, removing supernatant (4 ℃,1500rpm multiplied by 5 min), washing twice by PBS, and removing supernatant. Add 100. Mu.L of fluorescent secondary antibody working solution to each tube to resuspend the cells, incubate them in a shaker at 4 ℃ for 30min, centrifuge and discard the supernatant (4 ℃,1500 rpm. Times.5 min), wash them twice with PBS, and remove the supernatant. Adding 1.0mL of preheated SW780 cells into each tube, suspending the cells by a complete culture medium, uniformly mixing, subpackaging into 4 tubes, wherein 200 mu L of each tube is respectively a 0min group, a blank group, a30 min group and a 2h group, taking out 0min and blank, placing on ice, placing the rest in an incubator at 37 ℃, performing endocytosis for 30min and 2h respectively, taking out 1 tube at different corresponding time points, placing on ice for precooling for 5min, centrifuging all treatment groups, discarding supernatant (4 ℃,1500rpm multiplied by 5 min), washing once by PBS, and removing the supernatant. To the tubes of all treatment groups except the 0min group, 250. Mu.L strip buffer was added, incubated at room temperature for 8min, centrifuged to discard the supernatant (4 ℃,1500 rpm. Times.5 min), washed twice with PBS, and the supernatant was removed. All treatment groups were treated with 100. Mu.L of immunostaining fixative at 4 ℃ for more than 30min, and tested on the machine with a flow cytometer DxFlex. From the 0min group, 200. Mu.l of the tube was directly immunostainedAnd (4) fixing the color. From blank group, 200. Mu.l were taken, and strip buffer and immunostaining fixative were added directly. Detecting the DNA by a flow cytometer DxFlex on a computer. Data statistics and analysis: 30min mean percent of endocytosis (%) = (30 min group MIF-blank group MFI)/(0 min group MFI-blank group MFI), 2h mean percent of endocytosis (%) = (2 h group MIF-blank group MFI)/(0 min group MFI-blank group MFI). The percentage of endocytosis of the humanized antibody detected by the method is as follows 9:

TABLE 9 humanized antibody mediated endocytosis of TROP-2 protein

Example 8 competitive binding of humanized antibodies to antigen

The binding pattern and binding site of different antibodies to antigens are studied, usually by competitive binding experiments. The hRS7 antibody protein was diluted to a concentration of 1. Mu.g/ml with PBS, pH7.4, added to a 96-well high affinity microplate 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 (pH7.4PBS containing 0.05% Tween-20), 150. Mu.l/well of 2% Bovine Serum Albumin (BSA) blocking solution diluted with PBST was added, and the plate was incubated at room temperature for 1 hour for blocking. After blocking was complete, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

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

TABLE 10 humanized antibodies compete for binding to hRS7 antigen

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

EXAMPLE 9 preparation of Compound 1

In the first step, 2a (2g, 17.2mmol in 75mL acetonitrile was added potassium carbonate (9.27g, 67.2mmol), benzyl bromide (20mL, 167.2mmol) and tetrabutylammonium iodide (620mg, 1.68mmol) in this order, the reaction solution was stirred at room temperature for 48 hours, filtered through celite, the filter cake was rinsed with ethyl acetate (20 mL), the combined filtrates were concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system C to give product 5a (3.2 g, yield: 90.1%).

Second, 5a (181.3mg, 0.879mmol) and 4b (270mg, 0.733mmol) are added to the flask, 6mL tetrahydrofuran are added, replaced with argon three times, the ice bath is cooled to 0-5 deg.C, potassium tert-butoxide (164mg, 1.46mmol) is added, the ice bath is removed, the temperature is raised to room temperature and stirred for 40 minutes, 15mL ice water is added, extraction is performed with ethyl acetate (40 mL. Times.2) and chloroform (20 mL. Times.5), the organic phases are combined and concentrated. The resulting residue was dissolved in 6mL of dioxane, 3mL of water was added, sodium hydrogencarbonate (73.8mg, 0.879mmol) and 9-fluorenylmethyl chloroformate (190mg, 0.734mmol) were added, and the mixture was stirred at room temperature for 2 hours. 30mL of water was added, extraction was performed with ethyl acetate (20 mL. Times.3), and the organic phase was washed with a saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with the developer system C to give the product, benzyl 5b 10-cyclopropyl-1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diazaundec-11-ate (73 mg, yield: 19.4%).

MS m/z(ESI):515.0[M+1]。

Third, 5b (30mg, 0.058 mmol) was dissolved in 6.75mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V: V = 2. The reaction solution was filtered with celite, the filter cake was rinsed with ethyl acetate, the filtrate was concentrated to give crude product 5c 10-cyclopropyl-1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diazaundec-11-oic acid (20 mg), which was directly subjected to the next reaction without purification.

MS m/z(ESI):424.9[M+1]。

Step four, 1b (15mg, 28.2. Mu. Mol) was added to a reaction flask, 1.5mL of N, N-dimethylformamide was added, argon gas was substituted three times, the temperature was decreased to 0 to 5 ℃ in an ice water bath, one drop of triethylamine was added, crude 5c (20mg, 47.1. Mu. Mol) was added, 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylchloromorpholine salt (25.4mg, 86.2. Mu. Mol) was added, and the reaction was stirred for 40 minutes in an ice bath. 15mL of water was added, extraction was performed with ethyl acetate (20 mL. Times.3), and the organic phases were combined. The organic phase was washed with a saturated sodium chloride solution (20 mL. Times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developer system B to give the title product 5d (9H-fluoren-9-yl) methyl (2- (((1-cyclopropyl-2- (((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9,10,13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-B ] quinolin-1-yl) amino) -2-oxoethoxy) methyl) amino) -2-oxoethyl) carbamate (23.7 mg, yield: 78.9%).

MS m/z(ESI):842.1[M+1]。

In the fifth step, 5d (30mg, 35.7. Mu. Mol) was dissolved in 3mL of dichloromethane, and 1.5mL of diethylamine was added and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 1.5mL of toluene was added and concentrated under reduced pressure, and this was repeated twice. 4.5mL of n-hexane was added to the residue for beating, and after standing, the supernatant was poured off to retain the solid. The solid residue was concentrated under reduced pressure and oil-pumped to dryness to give crude product 5e 2- ((2-aminoacetamido) methoxy) -2-cyclopropyl-N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2, 3,9,10,13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) acetamide (23 mg) which was used in the next reaction without purification.

MS m/z(ESI):638.0[M+18]。

Sixthly, dissolving crude 5e (20mg, 32.3 mu mol) in 1mL of N, N-dimethylformamide, replacing the solution with argon for three times, cooling the solution in an ice-water bath to 0-5 ℃, adding 4g (31.8mg, 67.3 mu mol) of 0.5mL of N, N-dimethylformamide solution, adding 4- (4, 6-dimethoxy-1, 3, 5-triazine-2-yl) -4-methyl morpholine chloride salt (27.8mg, 94.3 mu mol), stirring the solution in an ice bath for 10 minutes, removing the ice bath, raising the temperature to room temperature, and stirring the solution for 1 hour to generate a compound 5. The reaction mixture was purified by high performance liquid chromatography (separation conditions: column: xbridge Prep C18OBD5um 19 × 250mm; mobile phase: A-water (10 mmol NH) ₄ OAc): b-acetonitrile, gradient elution, flow rate: 18 mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the products 5-A and 5-B (3.6 mg,2.6 mg).

MS m/z(ESI):1074.4[M+1]。

Single configuration compound 5-a (shorter retention time):

UPLC analysis, retention time 1.14 min, purity: 85% (column: ACQUITY UPLC BEHC181.7um 2.1 x 50mm, mobile phase: A-water (5 mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,2H),4.54-4.40(m,2H),3.76-3.64(m，4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,2H),2.80-2.62(m,2H),2.45-2.30(m,3H),2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H)。

Single configuration compound 5-B (longer retention time):

UPLC analysis: retention time 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC181.7um 2.1 x 50mm, mobile phase: A-water (5 mmol NH) ₄ OAc), B-acetonitrile).

¹ H NMR(400MHz,DMSO-d ₆ ):δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.13(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.60-5.50(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.78-4.68(m,1H),4.60-4.40(m,2H),3.76-3.58(m,4H),3.58-3.48 (m,1H),3.20-3.10(m,2H),3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13(m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.91-0.79(m,3H),0.53-0.34(m,4H)。

Preparation of other intermediates reference was made to intermediate 5.

To a PBS buffered aqueous solution of antibody HU1 (0.05M aqueous PBS buffered solution with pH = 6.5; 7.3ml,13.8mg/ml, 0.681. Mu. Mol), a prepared aqueous solution of tris (2-carboxyethyl) phosphine (10mM, 0.347mL, 3.47. Mu. Mol) was added, and the mixture was reacted in a water bath oscillator at 37 ℃ for 3 hours with shaking 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 and reacted.

Compound 5-A (5.0 mg, 2.75. Mu. Mol) was dissolved in 0.15mL of DMSO, added to the above 3.3mL of solution, placed in a water bath shaker, and reacted at 25 ℃ for 3 hours with shaking, and the reaction was stopped. The reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution at pH6.5, EDTA-0.001M) to obtain PBS buffer solution (1.45 mg/mL,17 mL) of an exemplary product of Ab-irinotecan derivative, HU 1-irinotecan derivative, and stored at 4 ℃ with refrigeration.

The average value y was determined by UV method. After the cuvette filled with the sodium succinate buffer solution is respectively placed in a reference absorption cell and a sample determination absorption cell, and the solvent blank is deducted, the cuvette filled with the test solution is placed in the sample determination absorption cell, and the absorbance at 280nm and 370nm is determined.

Data processing:

and (3) determining the content Cmab of the antibody by establishing a standard curve and measuring the absorption at the wavelength of 280nm, and determining the content CDrug of the micromolecule by measuring the absorption at the wavelength of 370 nm.

Drug load mean y = CDrug/Cmab.

The drug load of the exemplary product was determined by the above method and a sample of compound 1 (y = 8) was obtained by UV-HPLC purification.

Preparation of compound 2-compound 11 reference is made to compound 1. Similarly, compound 12 was prepared using the hRS7 antibody of the prior art using the same preparation method as compound 1 (y = 8):

example 11 antibody conjugation to MC-MMAF

In the first step, S- (3-Aldopropyl) thioacetate (0.7mg, 5.3mol) was dissolved in 0.9mL of acetonitrile and was used. To an acetic acid/sodium acetate buffer (10.35 mg/mL,9.0mL, 0.97mol) of antibody pH =4.3 was added the above-mentioned acetonitrile solution of S- (3-hydroxypropyl) thioacetate, followed by dropwise addition of 1.0mL of an aqueous solution of sodium cyanoborohydride (14.1mg, 224mol), and the reaction was stirred at 25 ℃ for 2 hours. After the reaction, the mixture was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution at pH 6.5) to obtain a solution of product 1a, which was concentrated to 10mg/mL and then directly subjected to the next reaction.

In the second step, 0.35mL of 2.0M carboxyamine hydrochloride solution was added to the 1a solution (11.0 mL), and after a reaction was carried out at 25 ℃ for 30 minutes with shaking, the reaction solution was purified by desalting with a Sephadex G25 gel column (elution phase: 0.05M PBS solution at pH 6.5), to obtain a product 2a solution (concentration 6.17mg/mL,14.7 mL).

Third, the compound MC-MMAF (1.1 mg,1.2mol, prepared by the method disclosed in PCT patent W02005081711) was dissolved in 0.3mL of acetonitrile, and the solution was added to a 2a solution (concentration 6.17mg/mL,3.0 mL) and reacted at 25 ℃ with shaking for 4 hours, and then the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution at pH 6.5), and then filtered by a filter under aseptic conditions to obtain a PBS buffer solution (3.7 mg/mL,4.7 mL) of the product Ab-MC-MMAF antibody-drug conjugate, which was refrigerated at 4 ℃.

EXAMPLE 12 antibody conjugation with SN-38

In the first step, S- (3-Aldopropyl) thioacetate (0.7mg, 5.3mol) was dissolved in 0.9mL of acetonitrile and was used. To an acetic acid/sodium acetate buffer (10.35 mg/mL,9.0mL, 0.97mol) of antibody pH =4.3 was added the above-mentioned acetonitrile solution of S- (3-hydroxypropyl) thioacetate, followed by dropwise addition of 1.0mL of an aqueous solution of sodium cyanoborohydride (14.1mg, 224mol), and the reaction was stirred at 25 ℃ for 2 hours. After the reaction, the mixture was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution at pH 6.5) to obtain 1h solution, and the solution was concentrated to 10mg/mL and directly subjected to the next reaction.

In the second step, 0.35mL of 2.0M carboxyamine hydrochloride solution was added to the 1h solution (11.0 mL), and after 30 minutes of shaking reaction at 25 ℃, the reaction solution was desalted and purified by a Sephadex G25 gel column (elution phase: 0.05M PBS solution at pH 6.5) to obtain a 2h solution (concentration 6.2mg/mL,15.0 mL) as a product, which was concentrated to about 10mg/mL and used for the next reaction.

Third, the compound MC-VC-PAB-SN-38 (1.3 mg,1.2 mol) was dissolved in 0.3mL of acetonitrile, added to a 2h solution (concentration 6.2mg/mL,3.0 mL) and reacted at 25 ℃ with shaking for 4 hours, and then the reaction mixture was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution at pH 6.5), filtered by a filter under aseptic conditions to obtain a PBS buffer (3.7 mg/mL,4.7 mL) of the product Ab-SN-38 antibody-drug conjugate, and refrigerated at 4 ℃.

EXAMPLE 13 killing of tumor cells by antibody-conjugated drugs

To examine the killing effect of the antibody-drug conjugate of the present invention on tumor cells, bladder cancer cells SW780 were used for evaluation. SW780 cells were collected, and after centrifugation, cell density was adjusted to 0.44X 10 with complete medium ⁶ one/mL of the cells was plated in 60 wells of a white 96-well plate at 90. Mu.L/well, the number of cells was 4000, 100. Mu.L of PBS was added to the remaining wells, the plate was incubated at 37 ℃ in a CO2 incubator at 5% by volume overnight. The next day of the experiment, antibody-drug conjugate solutions were prepared in 96-well V-bottom plates with PBS at a concentration of 1000nM starting, 3-fold dilution, 9 concentrations, added to white 96-well plates at 10 μ L per well after the preparation, duplicate wells, and the cell plates were placed in a 37 ℃, 5-cent co2 incubator for a further 72 hours. Fifth day of experiment, assay reading: the cell culture plate was removed, allowed to equilibrate to room temperature, 50. Mu.L of CTG solution (Promega G7573) was added to each well, shaken, mixed, allowed to stand in the dark for 10 minutes, and then detected using the luminescence protocol of the microplate reader. EC50 values are calculated by a four-parameter fitting method, and the killing effect of the antibody-drug conjugate on bladder cancer cells RT4 and epidermal cancer cells A431 is evaluated by referring to the method. The results of the experiment are shown in Table 11：

TABLE 11 killing of tumor cells by antibody-drug conjugates

Example 14 pharmacokinetics of antibody conjugate drugs

To further investigate the pharmacokinetics of the antibody-drug conjugates in vivo, the antibody-drug conjugates were injected intravenously into C57BL/6 mice (at a dose of 10 mg/kg). 20 μ l of blood was withdrawn at time points 1h,2h,4h,8h,24h,48h,96h,144h and 240h, and pharmacokinetic parameters were obtained by analyzing the pharmacokinetic data using WinNonlin software after measuring the concentration of the antibody-drug conjugate in the blood by the ELISA method of example 3 (1), as shown in Table 12 below.

TABLE 12 pharmacokinetic parameters of antibody-drug conjugates

Example 15 in vivo anti-tumor Effect of antibody-conjugated drugs

To further investigate the killing effect of the antibody-drug conjugate on tumors formed in vivo, the anti-tumor effect of the antibody-drug conjugate of the present invention was evaluated after forming a graft tumor with N87 in vivo in a mouse. Mixing 5x10 ⁶ N87 cells were injected subcutaneously into immunodeficient nude mice, beginning 2 weeks later by intravenous injection of antibody-drug conjugate compound 10 (y = 8) and compound 12 (y = 8), at a dose of 2mg/kg once every 2 weeks. Human IgG1 protein was used as a control at a dose of 2mg/kg. Control or dosing groups 5 mice per group. 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), and the results are shown in table 13:

TABLE 13 killing of tumors by antibody-drug conjugates

Compound 10 (y = 8) showed a tumor inhibition rate of over 100%, meaning that compound 10 (y = 8) not only inhibited tumor growth, but also had a killing effect on established tumors.

Example 16 preparation of an irinotecan derivative

2mL of ethanol and 0.4mL of N, N-dimethylformamide were added to 2e (4mg, 7.53. Mu. Mol), replaced with argon three times, cooled to 0 to 5 ℃ in an ice-water bath, and 0.3mL of N-methylmorpholine was added dropwise, and the mixture was stirred until the reaction solution became clear. To the reaction solution were added 2-cyclopropyl-2-hydroxyacetic acid 1e (2.3mg, 19.8. Mu. Mol, prepared by the method disclosed in patent application "WO 2013106717"), 1-hydroxybenzotriazole (3mg, 22.4. Mu. Mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.3mg, 22.4. Mu. Mol) in this order, and after completion of the addition, the reaction was stirred at 0 to 5 ℃ for 1 hour. The ice water bath was removed and the mixture was heated to 30 ℃ and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude compound 2-C was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18OBD5um 19 x 250mm; mobile phase: A-water (10mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min), and the corresponding fractions were collected and concentrated under reduced pressure to give the title product (2-A: 1.5mg,2-B:1.5 mg).

MS m/z(ESI):534.0[M+1]。

Single configuration Compound 2-B (shorter Retention time)

UPLC analysis: retention time 1.06 min, purity: 88% (column: ACQUITY UPLC BEHC 18.7um 2.1 x 50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

¹ HNMR(400MHz,DMSO-d6):δ8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H),5.58-5.56(m,1H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H),3.19-3.13(m,1H),2.38(s,3H),2.20-2.14(m,1H),1.98(q,2H),1.87-1.83(m,1H),1.50-1.40(m,1H),1.34-1.28(m,1H),0.86(t,3H),0.50-0.39(m,4H)。

Single configuration Compound 2-A (longer Retention time)

UPLC analysis: retention time 1.10 min, purity: 86% (column: ACQUITY UPLC BEHC 18.7um 2.1 x 50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

¹ HNMR(400MHz,DMSO-d6):δ8.35(d,1H),7.78(d,1H),7.31(s,1H),6.52(s,1H),5.58-5.53(m,1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3.20-3.15(m,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m,1H),1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H)。

Example 17 in vitro proliferation inhibition assay of tumor cells by an irinotecan derivative

Compounds 2-A and 2-B were tested for their inhibitory activity against proliferation of U87MG cells (Chinese academy of cells, catalog # TCTU 138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, cat # HTB-30) in vitro. Cells were treated with different concentrations of the compound in vitro, cultured for 6 days, and then proliferation of the cells was detected using CTG (luminescence Cell Viability Assay, promega, cat # G7573) reagent, and the in vitro activity of the compound was evaluated based on the IC50 value.

U87MG and SK-BR-3 cells were cultured in 10% FBS EMEM medium (GE, cat # SH 30024.01) and 10% FBS-containing McCoy's 5A medium (Gibco, cat # 16600-108), respectively.

Taking U87MG and SK-BR-3 cells in logarithmic growth phase, washing with PBS (phosphate buffered saline, shanghai culture Biotech Ltd.) for 1 time, adding 2-3ml trypsin (0.25% Trypsin-EDTA (1 x), gibico, life Technologies) for digestion for 2-3min, adding 10-15ml cell culture solution after cell digestion is completed, eluting digested cells, centrifuging at 1000rpm for 5min, discarding supernatant, and adding 10-20ml cell culture solution to resuspend cells to obtain single cell suspension.

The U87MG and SK-BR-3 single cell suspensions are mixed evenly, the viable cell density is adjusted to 2.75 × 103cells/ml and 8.25 × 103cells/ml respectively by using cell culture fluid, the cell suspensions after the density adjustment are mixed evenly, and the cell suspensions are added into a 96-hole cell culture plate at 180 μ l/hole. Only 200ul of medium was added to the peripheral wells of the 96-well plate. The plates were incubated in an incubator for 24 hours (37 ℃,5% CO) ₂ )。

The compound was dissolved in DMSO (dimethyl sulfoxide, shanghai Tantake technology Co., ltd.) to prepare a stock solution having an initial concentration of 10 mM.

The initial concentration of the small molecule compound is 500nM, and the dosing method is as follows:

respectively adding 30 mul of different samples to be detected into a first row of a 96-hole U-shaped bottom dispensing plate, wherein the concentration of the samples is 100uM; column 2 to column 11 each well was charged with 20ul DMSO. The first column of samples was taken 10ul to the second 20ul of DMSO, mixed well, 10ul to the third column, and so on to column 10. Taking 5ul to 95ul of EMEM culture medium per well of the medicines in the medicine preparation plate, and uniformly mixing for later use.

Sample adding: to the plates were added 20 μ l of each of two wells of different concentrations of the samples to be tested. Incubating the plates in the incubator for 6 days (37 ℃,5% ₂ )。

Color development: the 96-well cell culture plate was removed, 90. Mu.l of CTG solution was added to each well, and incubated at room temperature for 10 minutes.

Reading a plate: the 96-well cell culture plate was removed and placed in a microplate reader (BMG labtech, PHERAstar FS) and chemiluminescence was measured with the microplate reader.

And (3) data analysis: data were processed and analyzed using Microsoft Excel, graphpad Prism 5. The results are shown in Table 14.

TABLE 14 killing effect of irinotecan derivatives on tumors

Claims

An antibody-drug conjugate represented by the general formula (A) or a pharmaceutically acceptable salt or solvate thereof,

Ab-(L ₂ -L ₁ -D) _y

(A)

wherein:

d is a cytotoxic drug;

L ₁ is selected from-O- (CR) ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-、-O-CR ⁵ R ⁶ -(CR ^a R ^b ) _m -、-O-CR ⁵ R ⁶ -、-NH-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C (O) -or-S- (CR) ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-；

R ^a And R ^b The same or different, and each is independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, or a heterocyclic group;

or, R ^a And R ^b Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

R ⁵ selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, or heteroaryl;

R ⁶ selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, or heteroaryl;

or, R ⁵ And R ⁶ Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

or, R ^a And R ⁶ Together with the carbon atom to which they are attachedA cycloalkyl group or a heterocyclic group;

m is an integer selected from 0 to 4;

y is selected from a number from 1 to 10, y is a decimal or an integer;

L ₂ is a joint unit;

ab is an anti-TROP-2 antibody or antigen-binding fragment thereof comprising a light chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of 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.
The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof of 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.
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 derived from human IgG1, igG2, igG3, or IgG4, or a variant thereof,

preferably, 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;

further 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 as set forth in SEQ ID NO. 49.
The antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, said anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region or variant thereof further comprising a kappa chain, a lambda chain derived from a human antibody;

preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises the 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.
The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof of 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 those shown in seq id nos, or those having at least 70%,75%,80%,85%,90%,95%, or 99% identity compared to seq id nos: 9, 11, 13,15, 17, 19, 21, 23 or 25 SEQ ID NO;

and/or, is selected from the group consisting of the light chain variable region set forth in seq id no, or a light chain variable region having at least 70%,75%,80%,85%,90%,95%, or 99% identity compared to seq id no:10, 12, 14, 16, 18, 20, 22, 24 or 26 SEQ ID NO.
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:

the amino acid sequence of SEQ ID NO:9 and SEQ ID NO: 10; or the like, or a combination thereof,

SEQ ID NO:11 and SEQ ID NO: 12; or,

SEQ ID NO:13 and SEQ ID NO:14, a light chain variable region; or the like, or a combination thereof,

SEQ ID NO:15 and SEQ ID NO: 16; or,

SEQ ID NO:17 and SEQ ID NO:18, the light chain variable region shown in; or,

the amino acid sequence of SEQ ID NO:19 and the heavy chain variable region of SEQ ID NO:20, a light chain variable region; or the like, or a combination thereof,

SEQ ID NO:21 and the heavy chain variable region of SEQ ID NO:22, a light chain variable region shown in seq id no; or the like, or a combination thereof,

SEQ ID NO:23 and SEQ ID NO: a light chain variable region shown at 24; or,

SEQ ID NO:25 and the heavy chain variable region of SEQ ID NO:26, or a light chain variable region as shown.
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 those shown in SEQ ID NO, or those having at least 80%,85%,90%,95% or 99% identity to SEQ ID NO: SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. the amino acid sequence of SEQ ID NO: 33. SEQ ID NO: 35. SEQ ID NO: 37. the amino acid sequence of SEQ ID NO: 39. the amino acid sequence of SEQ ID NO: 41. SEQ ID NO: 43. SEQ ID NO:45 or SEQ ID NO:47;

and/or, is selected from the group consisting of a light chain as represented by 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. the amino acid sequence of SEQ ID NO: 36. SEQ ID NO: 38. SEQ ID NO: 40. SEQ ID NO:42 or SEQ ID NO:44.
the antibody-drug conjugate of claim 7, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody comprises:

SEQ ID NO:27 and the heavy chain of SEQ ID NO:28, a light chain; or,

SEQ ID NO:29 and the heavy chain of SEQ ID NO:30, a light chain; or,

the amino acid sequence of SEQ ID NO:31 and the heavy chain of SEQ ID NO:32, a light chain; or,

SEQ ID NO:33 and SEQ ID NO:34, a light chain; or,

SEQ ID NO:35 and SEQ ID NO:36, a light chain; or the like, or a combination thereof,

SEQ ID NO:37 and SEQ ID NO: 38; or,

SEQ ID NO:39 and SEQ ID NO:40, a light chain; or,

the amino acid sequence of SEQ ID NO:41 and the heavy chain of SEQ ID NO:42, a light chain; or,

SEQ ID NO:43 and the heavy chain of SEQ ID NO:44, a light chain; or,

SEQ ID NO:45 and the heavy chain of SEQ ID NO: 38; or,

the amino acid sequence of SEQ ID NO:47 and SEQ ID NO:28, or a light chain as shown.
The antibody-drug conjugate of any one of claims 1 to 8, or a pharmaceutically acceptable salt or solvate thereof, wherein L is ₁ As shown in the general formula (E):

wherein,

R ⁵ is a halogenated alkyl group or a cycloalkyl group,

R ⁶ selected from hydrogen, haloalkyl or cycloalkyl,

or, R ⁵ And R ⁶ Together with the carbon atom to which they are attached form a cycloalkyl group;

preferably, the first and second electrodes are formed of a metal,

R ⁵ is selected from C _1-6 Haloalkyl or C _3-6 A cycloalkyl group, which is a cyclic alkyl group,

R ⁶ selected from hydrogen, C _1-6 Haloalkyl or C _3-6 A cycloalkyl group, which is a cyclic alkyl group,

or, R ⁵ And R ⁶ Together with the carbon atom to which they are attached form C _3-6 A cycloalkyl group;

m is selected from the group consisting of integers of 0 to 4,

preferably, the general formula (E) is selected from the following substituents:
the antibody-drug conjugate of any one of claims 1 to 8, or a pharmaceutically acceptable salt or solvate thereof, wherein L is ₂ As shown in general formula (D):

-K ¹ -K ² -K ³ -K ⁴ -

(D)

wherein,

K ¹ is composed of
s is selected from an integer from 2 to 8;

K ² is selected from-NR ¹ (CH ₂ CH ₂ O) _p CH ₂ CH ₂ C(O)-、-NR ¹ (CH ₂ CH ₂ O) _p CH ₂ C(O)-、-S(CH ₂ ) _p C (O) -or a single bond, p is selected from an integer from 1 to 20, preferably from 1 to 6;

R ¹ selected from the group consisting of hydrogen, deuterium, hydroxy, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl;

K ³ is a tetrapeptide residue, preferably selected from the group consisting of peptide residues formed from two or more amino acids of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid; more preferably tetrapeptide residues of GGFG;

K ⁴ is-NR ² (CR ³ R ⁴ )t-，R ² 、R ³ Or R ⁴ Each independently hydrogen, deuterium, hydroxy, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl, and t is selected from 1 or 2.
The antibody-drug conjugate of claim 10, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit L ₂ Of which K ¹ End is connected to Ab, K ⁴ Terminal and L ₁ Are connected.
The antibody-drug conjugate of any one of claims 1 to 11, or a pharmaceutically acceptable salt or solvate thereof, said-L ₂ -L ₁ -is selected from the following structures:

wherein, K ² Is a bond;

K ³ tetrapeptide residues that are GGFG;

R ⁵ selected from haloalkyl or C _3-6 A cycloalkyl group;

R ⁶ selected from hydrogen, haloalkyl or C _3-6 A cycloalkyl group;

or, R ⁵ And R ⁶ Together with the carbon atom to which they are attached form C _3-6 A cycloalkyl group;

R ² 、R ³ or R ⁴ Each independently selected from hydrogen or alkyl;

s is selected from an integer from 2 to 8;

m is an integer from 0 to 4;

preferably, -L ₂ -L ₁ -is selected from the following structures:
the antibody-drug conjugate of any one of claims 1 to 12, or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug is selected from the group consisting of a toxin, a chemotherapeutic drug, an antibiotic, a radioisotope, and a nucleolytic enzyme; preferably a tubulin inhibitor or a DNA topoisomerase inhibitor that inhibits cell division; further preferred is a camptothecin derivative, DM1, DM3, DM4, SN-38, MMAF or MMAE; more preferably, irinotecan or an irinotecan derivative, SN-38, MMAE or MMAF.
The antibody-drug conjugate of claim 13, or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug is selected from the following structures:
the antibody-drug conjugate of claim 14, which is represented by formula (III):

wherein,

L ₁ 、L ₂ is a joint unit;

y is a number from 1 to 10, preferably a number from 2 to 8, more preferably a number from 4 to 8;

ab is selected from the TROP-2 antibody or antigen-binding fragment thereof of any one of claims 1 to 8.
The antibody-drug conjugate according to claim 15, which is represented by the general formula (IV):

wherein,

w is selected from C _1-8 Alkyl radical, C _1-8 Alkyl-cycloalkyl or a linear 1 to 8 atom heteroalkyl containing 1 to 3 heteroatoms selected from N, O, or S, wherein said C is _1-8 Each of alkyl, cycloalkyl, and linear heteroalkyl is independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

K ² is selected from-NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C(O)-、-NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ C(O)-、-S(CH ₂ ) _p1 C (O) -or a bond, R ¹ Selected from the group consisting of hydrogen atoms, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl radicals, p ₁ Is an integer from 1 to 20;

K ³ selected from the group consisting of peptide residues consisting of 2 to 7 amino acids, which may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents being one or more independently selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

R ² independently selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups;

R ³ and R ⁴ Each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl;

R ⁵ selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl;

R ⁶ selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl;

or, R ⁵ And R ⁶ Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

m is an integer selected from 0 to 4;

y is selected from a number from 1 to 10, y is a decimal or an integer;

ab is an anti-TROP-2 antibody or antigen-binding fragment thereof.
The antibody-drug conjugate according to claim 16, which is represented by the general formulSup>A (IV-Sup>A):

preferably, formulSup>A (IV-A) is selected from the following structures:
the antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of:

wherein y is selected from 2 to 10, preferably 4 to 8, more preferably 6 to 8, further preferably 7 to 8, most preferably 8.
A method of preparing an antibody-drug conjugate of formula (IV) or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:

ab is reduced and then is subjected to coupling reaction with a general formula (F) to obtain a compound shown as a general formula (IV);

wherein Ab is an anti-TROP-2 antibody or antigen-binding fragment thereof;

W、K ² 、K ³ 、R ² ～R ⁶ m and y are as defined in claim 16.
The method of claim 19, wherein the compound of formula (F) is of formula (F-1) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein, K ² 、K ³ 、R ² ～R ⁶ S and m are as defined in claim 12.
The compound represented by the general formula (F) or the general formula (F-1) is selected from:
a pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 18, or a pharmaceutically acceptable salt or solvate of said antibody-drug conjugate, and one or more pharmaceutically acceptable excipients, diluents or carriers.
Use of an antibody-drug conjugate according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 27 in the manufacture of a medicament for the treatment of a disorder associated with human TROP-2.
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