WO2021190480A1 - Antibody-drug conjugate and medical use thereof - Google Patents

Abstract

The present application relates to an antibody-drug conjugate and medical use thereof, specifically relates to an anti-TROP-2 antibody-drug conjugate and medical use thereof, and furthermore, relates to an antibody-drug conjugate containing an anti-TROP-2 antibody or an antigen-binding fragment thereof, or a pharmaceutically acceptable salt or solvent compound thereof, and use thereof in preparing medicines for treating diseases or disorders mediated by TROP-2 and use thereof in tumor detection and diagnosis.

Description

Antibody-drug conjugate and its medical use Technical field

The present invention relates to an anti-TROP-2 antibody-drug conjugate which is specifically immunoreactive to human TROP-2 receptor and its pharmaceutical composition, as well as its use as an anti-cancer drug and its use in detecting or diagnosing tumors .

Background technique

With the continuous in-depth research on tumor genomics, proteomics, and signal transduction pathways, people have become more and more aware of the interaction between oncogenes and tumor suppressor genes in tumor cells and their impact on the tumor microenvironment. It is possible to design new anti-tumor treatments for specific molecular targets of tumors.

Molecular targeted therapy of tumors is a new treatment model that is different from traditional surgery, radiotherapy, and chemotherapy. Its advantage is that drugs usually only bind to the corresponding target, and directly affect the function of its target molecule or carry Physical or chemical effector molecules to achieve the effect of killing or inhibiting target cells. Because the target site is clear, this type of drug usually has a high selectivity, which can effectively kill or inhibit the target cells, but also produces no or only minor toxic side effects on normal tissue cells. Therefore, the development of molecular targeted drugs has become a hot spot in tumor clinical research.

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

Up to now, the physiological ligand of TROP-2 has not been identified, and its molecular function has not been elucidated. However, because its intracellular Serine 303 (S303) can be ^{phosphorylated by the action of Ca 2+} -dependent protein kinase C (PKC) Promotes the hydrolysis of 4,5-bisphosphate phosphatidylinositol (PIP2) to form the mitogen-activated protein kinase pathway (MAPK) related inositol triphosphate IP3. This signaling pathway is closely related to cell proliferation, suggesting that Trop2 has Mediates the function of signal transmission in tumor cells.

A large number of clinical studies and literature reports have shown that TROP-2 is overexpressed in a variety of epithelial cancers such as gastric cancer, lung cancer, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, and esophageal cancer. In contrast, TROP-2 is rarely expressed or not expressed in normal adult tissues, and is limited to a small amount of expression in cells in the epithelial area, and the expression level is lower than that in cancer, indicating that TROP-2 is related to tumor formation. The overexpression of TROP-2 in tumor tissues is closely related to the poor prognosis of patients and the metastasis of cancer cells, and at the same time affects the overall survival rate of patients. Therefore, TROP-2 has become an attractive target in tumor molecular targeted therapy.

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

US Patent No. 5840854 reports the cytotoxicity of anti-hTROP-2 monoclonal antibody (BR110) bound to cytotoxin on human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981.

U.S. Patent No. 6653104 discloses an antibody (RS7), which was tested in an in vivo model using an antibody labeled with a radioactive substance. It showed anti-tumor activity in a nude mouse xenograft model, but there is no report on the naked antibody. Timely anti-tumor effect.

US Patent No. 7420040 also reported that the isolated monoclonal antibody produced by the hybridoma cell line AR47A6.4.2 or AR52A301.5 obtained from human ovarian cancer tissue immunized mice was bound to hTROP-2, and was used in a nude mouse xenograft model Shows anti-tumor activity in.

CN102827282A discloses a human anti-TROP-2 genetically engineered antibody IgG and its application. In vitro test results show that the anti-TROP-2 antibody IgG has a significant inhibitory effect on the proliferation of pancreatic cancer cells.

CN104114580A discloses an antibody (especially a humanized antibody) that specifically reacts with hTROP-2 and has anti-tumor activity in the body, as well as hybridomas that produce the antibody, a complex of the antibody and a drug, and diagnostic applications for tumors. Or therapeutic pharmaceutical composition, tumor detection method, tumor detection or diagnostic kit.

Summary of the invention

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

Ab- ⁽ L ₂ -L ₁ -D)y (A)

in:

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 are the} same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group Group, cycloalkyl or heterocyclic group;

Alternatively, R ^a and R ^b together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;

R ^{5 is} selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;

R ^{6 is} selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;

Alternatively, R ^a and R ⁶ together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;

m is selected from an integer from 0 to 4;

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

L ₂ is the joint unit;

Ab is an anti-TROP-2 antibody or an antigen-binding fragment thereof, which comprises an antibody light chain variable region and an antibody heavy chain variable region, and the antibody heavy chain variable region includes at least one HCDR selected from the following sequences : SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5; the antibody light chain variable region includes at least one LCDR selected from the following sequence: SEQ ID NO: 6, SEQ ID NO :7, SEQ ID NO: 8.

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

SEQ ID NO: HCDR1 shown in 3

SEQ ID NO: 4 HCDR2 and

The HCDR3 shown in SEQ ID NO: 5.

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

SEQ ID NO: LCDR1 shown in 6

SEQ ID NO: 7 LCDR2 and

The LCDR3 shown in SEQ ID NO: 8.

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

SEQ ID NO: HCDR1 shown in 3

SEQ ID NO: 4 HCDR2 and

SEQ ID NO: HCDR3 shown in 5; and

The antibody light chain variable region comprises:

SEQ ID NO: LCDR1 shown in 6

SEQ ID NO: 7 LCDR2 and

The LCDR3 shown in SEQ ID NO: 8.

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

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises human IgG1, The heavy chain constant region of IgG2, IgG3, or IgG4 or variants 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 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 shown in SEQ ID NO: 48 or SEQ ID NO: 49.

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

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 shown in SEQ ID NO:50.

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

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

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof contains a sequence selected from the following The heavy chain shown, or a heavy chain with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31. SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, or SEQ ID NO: 47.

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof contains a sequence selected from The light chain shown, or a light chain with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: 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 present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises:

(1) The heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 10; or,

(2) The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12; or,

(3) The heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 14; or,

(4) The heavy chain variable region shown in SEQ ID NO: 15 and the light chain variable region shown in SEQ ID NO: 16; or,

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

(6) The heavy chain variable region shown in SEQ ID NO: 19 and the light chain variable region shown in SEQ ID NO: 20; or,

(7) The heavy chain variable region shown in SEQ ID NO: 21 and the light chain variable region shown in SEQ ID NO: 22; or,

(8) The heavy chain variable region shown in SEQ ID NO: 23 and the light chain variable region shown in SEQ ID NO: 24; or,

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

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

(1) The heavy chain shown in SEQ ID NO: 27 and the light chain shown in SEQ ID NO: 28; or,

(2) The heavy chain shown in SEQ ID NO: 29 and the light chain shown in SEQ ID NO: 30; or,

(3) The heavy chain shown in SEQ ID NO: 31 and the light chain shown in SEQ ID NO: 32; or,

(4) The heavy chain shown in SEQ ID NO: 33 and the light chain shown in SEQ ID NO: 34; or,

(5) The heavy chain shown in SEQ ID NO: 35 and the light chain shown in SEQ ID NO: 36; or,

(6) The heavy chain shown in SEQ ID NO: 37 and the light chain shown in SEQ ID NO: 38; or,

(7) The heavy chain shown in SEQ ID NO: 39 and the light chain shown in SEQ ID NO: 40; or,

(8) The heavy chain shown in SEQ ID NO: 41 and the light chain shown in SEQ ID NO: 42; or,

(9) The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 44; or,

(10) The heavy chain shown in SEQ ID NO: 45 and the light chain shown in SEQ ID NO: 38; or,

(11) The heavy chain shown in SEQ ID NO: 47 and the light chain shown in SEQ ID NO: 28.

In some embodiments of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, L _{1 is represented by} general formula (B):

in,

M ₁ is -CR ₁ R ₂ -;

R ₁ and R _{2 are the} same or different, and R ₁ and R ₂ are each independently selected from hydrogen, alkyl, halogen, hydroxyl, or amino;

n is selected from an integer of 0-5, preferably 1, 2 or 3.

In some embodiments of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, L _{2 is represented by} general formula (C):

in,

M ₂ is -CR ₄ R ₅ -;

R _{3 is} selected from hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy and cycloalkyl:

R ₄ and R _{5 are the} same or different, and are independently selected from hydrogen, alkyl, halogen, hydroxyl or amino;

m is selected from an integer of 0-5, preferably 1, 2 or 3.

In a further preferred embodiment of the present invention, the _{O end of the L 1} is connected to the joint unit L ₂ .

In a further preferred embodiment of the present invention, the O end of _{the L 1} is connected to the S end of the _{joint unit L 2.}

In a preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, 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 cycloalkyl;

R ^{6 is} selected from a hydrogen atom, a halogenated alkyl group or a C _3-6 cycloalkyl group;

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

m is selected from 0 or 1.

In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the L _{1 is represented by the} general formula (E):

Among them, R ⁵ is haloalkyl or cycloalkyl,

R ^{6 is} selected from hydrogen, haloalkyl or cycloalkyl,

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a cycloalkyl group;

Preferably,

R ^{5 is} selected from C _1-6 haloalkyl or C _3-6 cycloalkyl,

R ^{6 is} selected from hydrogen, C _1-6 haloalkyl or C _3-6 cycloalkyl,

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

m is selected from 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 present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the L _{2 is represented by the} following general formula (D):

-K ¹ -K ² -K ³ -K ^4- (D)

in,

K ¹ is

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 an integer from 1 to 6;

R ^{1 is} selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl;

K ³ is a tetrapeptide residue, preferably, the tetrapeptide residue is selected from two or more of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and Peptide residues formed by amino acids in 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 are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkane Base, t is selected from 1 or 2.

In a preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein the linker unit -L ₂ -, its K ¹ end is connected to Ab Connected, K ⁴ terminal is connected to L ₁ .

In a preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein the -L ₂ -L ₁ -has the following structure:

Among them, K ² is a key;

K ³ is the tetrapeptide residue of GGFG;

R ^{5 is} selected from haloalkyl or C _3-6 cycloalkyl;

R ^{6 is} selected from hydrogen, haloalkyl or C _3-6 cycloalkyl;

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

R ² , R ³ or R ⁴ are each independently selected from hydrogen or alkyl;

s is selected from an integer from 2 to 8;

m is selected from an integer from 0 to 4.

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

In some embodiments of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein the cytotoxic drug is selected from the group consisting of toxins, chemotherapeutics, antibiotics, radioactive Isotope and nucleolytic enzyme.

In a preferred embodiment of the present invention, the cytotoxic drug is selected from tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division; preferably camptothecin derivatives, DM1, DM3, DM4, SN- 38. MMAF or MMAE; more preferably exenotecan or exenotecan derivatives, SN-38, MMAE or MMAF.

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

In a preferred embodiment of the present invention, the cytotoxic drug is selected from exenotecan derivatives, preferably, the exenotecan derivative is compound 2-A:

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

Among them, L ₁ and L ₂ are joint units;

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

Ab is selected from the TROP-2 antibody or antigen-binding fragment thereof as described above.

In a preferred embodiment of the present invention, the general formula (I) is as shown in the general formula (I-A):

Wherein L _{2 is} as defined in the foregoing; preferably, L 2 is as defined in the foregoing general formula (C).

In a preferred embodiment of the present invention, the general formula (I) is as shown in the general formula (I-B):

Wherein L _{1 is} as defined in the foregoing; preferably, L2 is as defined in the foregoing general formula (B).

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

Among them, L ₁ and L ₂ are joint units;

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

Ab is selected from the aforementioned anti-TROP-2 antibody or antigen-binding fragment thereof;

In a preferred embodiment of the present invention, the general formula (II) is as shown in the general formula (II-A):

Wherein L _{2 is} as defined in the foregoing; preferably, L 2 is as defined in the foregoing general formula (C).

In a preferred embodiment of the present invention, the general formula (II) is as shown in the general formula (II-B):

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

Among them, L ₁ and L ₂ are joint units;

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

Ab is selected from the aforementioned TROP-2 antibody or antigen-binding fragment thereof.

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 solvent compound thereof, wherein L _{1 is} as defined by the general formula (E) , L ₂ is defined by general 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 solvent compound thereof, wherein the linker unit -L2-, its K1 end is connected to Ab is connected, and the K4 terminal is connected to L1.

In a further preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof described in the general formula (III), and the -L ₂ -L ₁ -is selected from The following structure:

K ² is the key;

K ³ is the tetrapeptide residue of GGFG;

R ^{5 is} selected from haloalkyl or C _3-6 cycloalkyl;

R ^{6 is} selected from hydrogen, haloalkyl or C _3-6 cycloalkyl;

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

R ² , R ³ or R ⁴ are each independently hydrogen or alkyl;

s is selected from an integer from 2 to 8;

m is selected from 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 or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by general formula (IV) or Its pharmaceutically acceptable salt or solvate:

in,

W is selected from a C _1-8 alkyl group, a C _1-8 alkyl-cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O or S The heteroatoms, wherein the C _1-8 alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, Substituted by one or more substituents of 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 bond, R 1 is} selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group, and p ₁ is an integer from 1 to 20;

K ^{3 is} selected from peptide residues consisting of 2 to 7 amino acids. The amino acids can be substituted or unsubstituted. When substituted, the substituents can be substituted at any available point of attachment, and the substituents are one Or more are independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

R ^{2 is} independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group;

R ³ and R ⁴ are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

R ^{5 is} selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;

R ^{6 is} selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;

m is selected from an integer from 0 to 4;

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

Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof of the present invention.

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

In a further preferred embodiment of the present invention, the general formula (IV-A) is selected from the following structures:

In some embodiments of the present invention, the antibody-drug conjugate or pharmaceutically acceptable salt or solvent compound thereof according to the present invention is selected from the following compounds:

Wherein Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof of the present invention; preferably, Ab is selected from HU1-HU10, HU6DL of the present invention.

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

Among them, y is selected from 2-10, preferably 4-8, more preferably 6-8, still more preferably 7-8, 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 general formula (IV) or a pharmaceutically acceptable salt or solvate thereof, which comprises the following steps:

After Ab is reduced, it is coupled and reacted with the general formula (F) to obtain the compound represented by the general formula (IV);

Wherein, Ab is selected from the aforementioned anti-TROP-2 antibody or antigen-binding fragment thereof;

W, K ² , K ³ , R ² to R ⁶ , m and y are as defined in the general formula (IV).

In a preferred embodiment of the present invention, according to the preparation method of the general formula (IV) of the present invention, the general formula (F) is a compound represented by the general formula (F-1) or its tautomerism Forms, meso, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof:

Wherein, K ² , K ³ , R ² to R ⁶ , s and m are as defined in the general formula (IV).

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

In another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate, and one or A variety of pharmaceutically acceptable excipients, diluents or carriers.

On the other hand, the present invention also provides the antibody-drug conjugate of the general formula (A) or the pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate or its pharmaceutical composition in preparation for Application in medicines for the treatment of diseases related to human TROP-2.

In a more preferred embodiment of the present invention, the disease related to human TROP-2 is an application in the preparation of a medicament for the treatment of cancers with high TROP-2 expression, and the cancer is selected from the group consisting of triple-negative breast cancer, small Cell lung cancer, urothelial carcinoma, human brain astroblastoma, human pharynx cancer, adrenal tumors, AIDS-related cancers, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain And spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferation small circle Cell tumor, ependymoma, Juventus tumor, extraosseous mucinoid chondrosarcoma, bone fibrous hypoplasia, osteofibrous dysplasia, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, liver Cell carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma, malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine tumor disease , Multiple Myeloma, Myelodysplastic Syndrome, Neuroblastoma, Neuroendocrine Tumor, Ovarian Cancer, Pancreatic Cancer, Papillary Thyroid Cancer, Parathyroid Adenoma, Pediatric Cancer, Peripheral Neurilemmoma, Pheocytoma, Pituitary tumors, prostate cancer, posterior uveal melanoma, renal metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, Guwan cancer, thymic carcinoma, thymoma , Metastatic thyroid cancer and uterine cancer.

The antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound of the present invention can specifically bind to the target antigen, has high endocytosis efficiency, and has a long in vivo half-life time, and it can significantly kill tumors while ensuring safety.

Description of the drawings

Figure 1: ELISA in vitro binding experiment of antibodies, showing the binding activity of 11 humanized anti-TROP-2 antibodies to human TROP-2 antigen.

Detailed ways

Detailed description of the invention

1. Terminology

To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

The three-letter codes and one-letter codes of amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, and γ chain. , Α chain and ε chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into a kappa chain or a lambda chain by the difference of the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further include a light chain constant region, and the light chain constant region includes human or murine κ, λ chains or variants thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or its variants. body.

The sequence of about 110 amino acids near the N-terminus of antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions. The sequence from the amino terminus to the carboxy 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 number and position of the CDR amino acid residues of the VL and VH regions of the antibody or antigen-binding fragment of the present invention comply with the known Kabat numbering rules and Kabat or ABM definition rules (http://bioinf.org.uk/abs /).

The term "antigen presenting cell" or "APC" is a cell that displays foreign antigen complexed with MHC on its surface. T cells use the T cell receptor (TCR) to recognize this complex. Examples of APCs include, but are not limited to, dendritic cells (DC), topical blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.

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

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

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

1. Antibodies isolated from transgenes of human immunoglobulin genes, transchromosomal animals (such as mice) or hybridomas prepared therefrom;

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

3. Antibodies isolated from the recombinant combinatorial human antibody library; and

4. Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.

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

The term "murine antibody" in the present invention refers to a monoclonal antibody to human TROP-2 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with TROP-2 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated. In a preferred embodiment of the present invention, the murine TROP-2 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1 , IgG2, IgG3 or IgG4 or variants of the heavy chain constant region.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as 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 (ie, "humanized antibodies") .

The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework. Humanized antibodies can overcome the shortcomings of strong immune responses induced by chimeric antibodies that carry a large amount of mouse protein components. In order to avoid the decrease of immunogenicity and the decrease of activity at the same time, the variable region of the human antibody can be subjected to minimal reverse mutation to maintain activity.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and change the mouse variable region gene. The region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations to enhance ADCC (antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1 heavy chain constant region.

The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a mole basis, the antibody fragment retains at least 10% of the parental binding activity. 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 chain and one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

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

The "Fab' fragment" contains a light chain and a portion of a heavy chain that contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that it can be between the two heavy chains of the two Fab' fragments The formation of interchain disulfide bonds to form F(ab')2 molecules.

The "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.

The "Fv region" contains 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 with polyepitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH regions Antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable regions, each single variable region with a different target Or binding to different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together Wait.

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

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

The term "binding to TROP-2" in the present invention refers to the ability to interact with human TROP-2.

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

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

The terms "specific binding" and "selective binding" as used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human TROP-2 is used as an analyte and an antibody is used as a ligand, the antibody dissociates at an equilibrium of ^{about less than 10 -7 M or even smaller when measured by surface plasmon resonance (SPR) technology in the instrument} The constant (K _D ) binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to non-specific antigens (such as BSA, etc.) other than the predetermined antigen or closely related antigens. The term "antibody that recognizes an antigen" can be used interchangeably with the term "antibody that specifically binds" herein.

The term "cross-reactivity" refers to the ability of the antibodies of the present invention to bind to TROP-2 from different species. For example, the antibody of the present invention that binds to human TROP-2 can also bind to TROP-2 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or by binding or functional interaction with cells that physiologically express TROP-2. Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

The terms "inhibition" or "blocking" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking 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 "inhibition of growth" (eg, in relation to cells) is intended to include any measurable decrease in cell growth.

The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation (ie, passive or adaptive) of the immune response to a specific antigen. The term "induction" for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.

In the present invention, "ADCC", namely antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors are directly killed by recognizing the Fc segment of antibodies and are coated with antibodies. The target cell. The ADCC effect function of the antibody can be enhanced or reduced by modifying the Fc segment of IgG. The modification refers to mutations in the constant region of the heavy chain of the antibody.

The methods of producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human TROP-2 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions. The human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImmunoGeneTics (IMGT), or from the Journal of Immunoglobulin, 2001ISBN012441351.

The engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies. The antibody-secreted culture medium can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.

"Administration", "administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact. "Administration", "administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells. "Administration", "administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent, such as containing any one of the antibodies of the present invention, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in the treated patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Through any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated. As far as the embodiments of the present invention (such as treatment methods or products) may be ineffective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.

The term "essentially composed of" or its variants used throughout the specification and claims means that it includes all the elements or element groups, and optionally includes other elements similar to or different in nature from the elements. The element does not significantly change the basic or new properties of a given dosing regimen, method, or composition.

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

"Effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of medical conditions. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.

"Exogenous" refers to substances that are produced outside organisms, cells, or humans according to the background.

"Endogenous" refers to a substance produced in a cell, organism, or human body according to the background.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the 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 × 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Therefore, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.

"Optional" or "optionally" means that the event or environment described later can but does not have to occur, and the description includes the occasion where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.

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

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention. The experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. The reagents without specific sources are the conventional reagents purchased on the market.

Example 1 Antigen preparation

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

TROP-2-His sequence:

Example 2 Obtaining Murine Hybridoma and Antibody Sequence

Animals were immunized with human antigen TROP-2-His, 5 Balb/c and 5 A/J mice, female, 10 weeks old, using Sigma Complete Freund’s Adjuvant (CFA) and Sigma Incomplete Freund’s Adjuvant (IFA), immunogen and immune adjuvant are fully mixed and emulsified at a ratio of 1:1 to make a stable "water-in-oil" liquid; the injection dose is 25μg/200μL/mouse.

Table 1. Immunization Scheme

Day 1	The first immunization, complete Freund's adjuvant.
Day 21	The second immunization, incomplete Freund's adjuvant.
Day 35	The third immunization, incomplete Freund's adjuvant.
Day 42	Blood sampling and serum titer test (3 blood exemption)
Day 49	The fourth immunization, incomplete Freund's adjuvant.
Day 56	Blood collection and serum titer test (4 blood exemption)

The indirect ELISA method as described in Example 3 was used for the immunized mouse serum to evaluate the serum titer and the ability to bind to cell surface antigens. The detection of the titer (larger than 100,000 times dilution) determines the start of cell fusion. The immunized mice with strong serum titer, affinity and FACS binding were selected for one final immunization and then sacrificed. The spleen cells and SP2/0 myeloma cells were fused and plated to obtain hybridomas. The target hybridomas were screened by indirect ELISA, and The strain was established as a monoclonal cell strain by the limiting dilution method. The obtained positive antibody strains are further screened using indirect ELISA to select hybridomas that bind to the recombinant protein. The logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript ^TM Reverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig primer set (Novagen, TB326 Rev. B 0503) and then sequenced to finally obtain the sequence of the mouse antibody.

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

Table 2. Murine monoclonal antibody M1 heavy chain and light chain variable region CDR sequences

name	sequence	serial number
HCDR1	NYWMN	SEQ ID NO: 3
HCDR2	RIDPNDSETHYNQKFKD	SEQ ID NO: 4
HCDR3	SGFGSTYWFFDV	SEQ ID NO: 5
LCDR1	KASQDVSTAVA	SEQ ID NO: 6
LCDR2	SASYRYT	SEQ ID NO: 7
LCDR3	QQHYSTPLT	SEQ ID NO: 8

Example 3 In vitro binding activity detection method of antibody

(1) In vitro indirect ELISA binding experiment:

Dilute TROP-2His protein (Sino Biological Inc., cat#10428-H08H) with pH7.4 PBS to a concentration of 1μg/ml, add 100μl/well to a 96-well high-affinity ELISA plate, and refrigerate at 4°C Incubate overnight (16-20 hours). After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), add 150 μl/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate for 1 hour at room temperature for blocking. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer.

Dilute the antibody to be tested with PBST containing 3% BSA, start with 1 μM, 10 times gradient, 10 doses, add 100 μl/well to the microtiter plate, and incubate at room temperature for 1 hour. After the incubation, the plate was washed 4 times with PBST, 100 μl/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with 3% BSA in PBST was added, and incubated for 1 hour at room temperature. After washing the plate 4 times with PBST, add 100μl/well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature and dark for 1 minute, and add 100μl/well of stop solution (Cell Signaling Technology, cat#7002S) The reaction was terminated, and the absorbance value was read at 450nm with a microplate reader (BioTek, model Synergy H1), and the data was analyzed. Do concentration signal value curve analysis results, as shown in the following table:

Table 3. Affinity of mouse antibodies to human TROP-2 antigen (EC ₅₀ value)

Mouse antibody	Binding EC 50 with human TROP-2His antigen (nM)
M1	0.56

(2) In vitro cell binding experiment:

Collect the cultured TROP-2 high-expressing cells (CHO or 293 cells overexpressing TROP-2 and tumor cells expressing TROP-2, such as HCC-827, MDA-MB-468, etc.), adjust the cell density and spread them 96-well U bottom plate, 1×10 ⁵ to 2×10 ⁵ cells per well. Centrifuge at 1200g for 5min, remove the supernatant, add 100ul of serially diluted antibody solution or mouse immune serum, incubate at 4°C for 60min; centrifuge at 1200g for 5min, remove the supernatant, and wash the cells twice with PBS, add a fluorescently labeled secondary antibody (PE-GAM or PE-GAH) 100ul per well, incubate at 4℃ for 60min. Centrifuge at 1200g for 5min to remove the supernatant. After washing the cells twice with PBS, resuspend them in PBS, use a flow cytometer to detect the signal, and make a concentration curve analysis result.

Example 4 Mouse antibody humanization experiment

The humanization of the murine anti-human TROP-2 monoclonal antibody is carried out according to the methods published in many documents in the field. In short, a human constant domain is used to replace the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody. The present invention humanizes the murine antibody M1.

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

The CDR region of the murine antibody M1 was transplanted to the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH were back-mutated. After expression testing and After comparing the number of back mutations, an antibody designed with a combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences was selected. The sequence is as follows:

The designed heavy chain and light chain variable region sequences are respectively connected with the IgG heavy chain constant region and human antibody light chain constant region sequences. Exemplary heavy chain constant region and light chain constant region sequences are as follows:

The heavy chain and light chain sequences are as follows (where the HU1-HU9 heavy chain is derived from the sequence 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, and SEQ ID NO: 25 are respectively linked to the sequence SEQ ID NO: 49; the HU6DL and HU10 heavy chains are derived from the sequence SEQ ID NO: 19, SEQ ID NO:9 are respectively connected to the sequence SEQ ID NO:48):

Table 4. Sequence numbers of antibodies and their heavy chains, light chains and variable regions

CDNA fragments were synthesized according to the amino acid sequences of the light and heavy chains of the above humanized antibodies, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and _{incubated in a CO 2} incubator 4- 5 days. Collect the cell culture fluid, centrifuge and filter, load the sample to the antibody purification affinity column, wash the column with phosphate buffer, elution with glycine hydrochloric acid buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0, and Dialysis with phosphate buffer to obtain the humanized antibody protein of the present invention, the concentration and purity of which are shown in the following table:

Table 5. Concentration and purity of each humanized antibody

Humanized antibody number	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 kinetic experiments

_{The affinity (EC 50} ) of each humanized antibody to the human TROP-2 antigen determined using the in vitro indirect ELISA binding experiment in Example 3 (1) is shown in the following table:

Table 6. each humanized antibody affinity for human TROP-2 antigen (EC ₅₀₎

In order to detect the binding ability of each humanized antibody to the target protein TROP-2 on tumor cells, each humanized antibody tested in Example 3 (2) in vitro cell binding experiment was used to test HCC827 tumor cells (non-small cell lung cancer), _{The affinity (EC 50} ) of MAB-MB-468 tumor cells (breast cancer, invasive ductal carcinoma) is shown in the following table:

Table 7. affinity of each humanized antibody HCC827 tumor cells (EC ₅₀₎

Example 6 Tumor cell killing effect mediated by humanized antibody

Humanized antibodies can kill tumor cells from many aspects, one of which is to mediate the killing effect of immune cells on tumor cells. In order to detect the killing effect of immune cells mediated by the humanized antibody of the present invention on tumor cells, a system in which human peripheral blood mononuclear cells (PBMC) and HCC827 tumor cells (non-small cell lung cancer) were co-cultured was used for evaluation. Collect HCC827 cells, after centrifugal counting, adjust the cell density to 0.44×10 ⁶ /mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate, each with 90 μL, and the number of cells is 4000. Collect commercialized human PBMC cells, after centrifugal counting, adjust the cell density to 2.2×10 ⁶ cells/mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate with HCC827 cells, 90μL per well, the number of cells is 20000. Add 200μL of PBS to the remaining side holes, and place the cell plate in a 37°C, 5% CO2 incubator overnight. On the second day of the experiment, the humanized antibody solution was prepared in a 96-well V-bottom plate with PBS, starting at a concentration of 1000 nM, diluted 3 times, and 9 concentrations. After the preparation was completed, added to the white 96-well plate, 20 μL per well, Two replicate wells, put the cell plate in a 37°C, 5% CO2 incubator and continue to incubate for 72 hours. On the fifth day of the experiment, test the reading: Take out the cell culture plate, after equilibrating to room temperature, add 50μL CTG solution (Promega G7573) to each well, shake and mix, place in the dark and stand for 10 minutes, then use the luminescence program of the microplate reader Perform testing. The experimental results are shown in Table 8 below:

Table 8. Humanized antibody-mediated killing effect on tumor cells

The same method was used to determine the killing effect of HU6 antibody on HCC827 tumor cells, and the results showed that the killing effect of the highest dose was 52.3%.

Example 7 Humanized antibody-mediated TROP-2 endocytosis

In order to study humanized antibody-mediated endocytosis of TROP-2 protein in tumor cells, SW780 cells were trypsinized, the cells were collected and resuspended in pre-cooled PBS, and the cell concentration was adjusted to 1×10 ⁶ cells/mL . Take the EP tube, add 1mL of cell suspension, centrifuge at 1500rpm for 5 minutes and remove the supernatant, add 1mL of the prepared antibody to be tested to resuspend the cells, the final concentration of the antibody is 20μg/ml, and incubate for 1h on a shaker at 4 degrees. The supernatant was discarded by centrifugation (4°C, 1500 rpm×5 min), washed twice with PBS, and the supernatant was removed. Add 100μL of fluorescent secondary antibody working solution to each tube to resuspend the cells, incubate for 30min in a shaker at 4°C, centrifuge to discard the supernatant (4°C, 1500rpm×5min), wash twice with PBS, and remove the supernatant. Add 1.0 mL of pre-warmed SW780 cell complete medium to each tube to resuspend the cells and mix them. Divide into 4 tubes, 200μL each, respectively for 0min group, blank group, 30min group and 2h group. Remove 0min and blank. Place the rest on ice, and place the rest in a 37°C incubator. Ingestion for 30min and 2h respectively. Take out 1 tube at different time points and place it on ice for 5min. Centrifuge and discard the supernatant in all treatment groups (4°C, 1500rpm×5min). ), wash once with PBS, and remove the supernatant. Add 250μL strip buffer to the tubes of all treatment groups except the 0min group, incubate for 8min at room temperature, centrifuge to discard the supernatant (4℃, 1500rpm×5min), wash twice with PBS, and remove the supernatant. All treatment groups were added with 100μL of immunostaining fixative, placed at 4°C for more than 30 minutes, and tested with flow cytometer DxFlex on the machine. Take 200μl from the tube of the 0min group and add the immunostaining fixative directly. Take 200μl from the blank group and add strip buffer and immunostaining fixative directly. On the machine, use the flow cytometer DxFlex for detection. Data statistics and analysis: 30min average endocytosis percentage (%)=(30min group MIF-blank group MFI)/(0min group MFI-blank group MFI), 2h average endocytosis percentage (%)=(2h group MIF-blank group MFI)/(0min group MFI-blank group MFI). Using the above method to detect the endocytosis percentage of humanized antibodies is shown in Table 9:

Table 9. Humanized antibody-mediated endocytosis of TROP-2 protein

Example 8 Competitive binding of humanized antibody to antigen

To study the binding modes and binding sites of different antibodies and antigens, competitive binding experiments are usually used. Dilute the hRS7 antibody protein to a concentration of 1 μg/ml with pH 7.4 PBS, add 100 μl/well to a 96-well high-affinity ELISA plate, and incubate overnight (16-20 hours) in a refrigerator at 4°C. After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 150 μl/well of 2% bovine serum albumin (BSA) blocking solution diluted with PBST was added, and the plate was incubated for 1 hour at room temperature for blocking. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer.

Dilute the antibody to be tested to 100 μg/ml with PBST containing 2% BSA, and add 50 μl/well to the microtiter plate. Dilute TROP-2His protein (Sino Biological Inc., cat#10428-H08H) with PBST containing 2% BSA, and add 50μl/well to the microtiter plate. Incubate the plate at room temperature for 1 hour. After the incubation, the plate was washed 4 times with PBST, and 100 μl/well of anti-His HRP-labeled secondary antibody (Abcam, cat#ab197049) diluted with PBST containing 2% BSA was added, and incubated for 1 hour at room temperature. After washing the plate 4 times with PBST, add 100μl/well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature and dark for 1 minute, add 100μl/well of Stop Solution (Cell Signaling Technology, cat#7002S) Terminate the reaction, read the absorbance value at 450nm with a microplate reader (BioTek, model Synergy H1), and analyze the data as shown in the table below. The humanized antibody of the present invention has a very low inhibition rate on the binding of hRS7 antibody and TROP2 protein, suggesting that the humanized antibody of the present invention and hRS7 antibody do not compete for binding to the same epitope.

Table 10. Humanized antibodies compete with hRS7 for antigen binding

Humanized antibody	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) was dissolved in 75mL of acetonitrile, and potassium carbonate (9.27g, 67.2mmol), benzyl bromide (20mL, 167.2mmol) and tetrabutylammonium iodide (620mg, 1.68mmol) were added in sequence. The reaction solution was stirred at room temperature for 48 hours, filtered through celite, the filter cake was rinsed with ethyl acetate (20ml), the combined filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with a developing solvent system C to obtain Product 5a (3.2g, yield: 90.1%).

In the second step, add 5a (181.3mg, 0.879mmol) and 4b (270mg, 0.733mmol) to the reaction flask, add 6mL of tetrahydrofuran, replace with argon three times, cool to 0-5℃ in an ice-water bath, add potassium tert-butoxide (164mg , 1.46 mmol), remove the ice bath, warm to room temperature and stir for 40 minutes, add 15 mL ice water, extract with ethyl acetate (40 mL×2) and chloroform (20 mL×5), combine the organic phases and concentrate. The obtained residue was dissolved in 6 mL of dioxane, 3 mL of water was added, sodium bicarbonate (73.8 mg, 0.879 mmol) and 9-fluorene methyl chloroformate (190 mg, 0.734 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 30 mL of water was added, extracted with ethyl acetate (20 mL×3), the organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography with developing solvent system C to obtain the product 5b 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-diox Benzyl hetero-4,7-diazaundec-11-acid (73 mg, yield: 19.4%).

MS m/z(ESI): 515.0[M+1].

The third step is to dissolve 5b (30mg, 0.058mmol) in 6.75mL of tetrahydrofuran and ethyl acetate (V:V=2:1) mixed solvent, add palladium on carbon (18mg, content 10%, dry type), replace with hydrogen Three times, the reaction was stirred at room temperature for 1 hour. The reaction solution was filtered with diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude product 5c 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo- 2,9-dioxa-4,7-diazaundec-11-acid (20mg), the product was directly subjected to the next reaction without purification.

MS m/z(ESI): 424.9[M+1].

In the fourth step, add 1b (15mg, 28.2μmol) into the reaction flask, add 1.5mL N,N-dimethylformamide, replace with argon three times, cool to 0-5℃ in an ice water bath, and add one drop of triethylamine. Add crude product 5c (20mg, 47.1μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine salt (25.4mg, 86.2μmol), the reaction was stirred in an ice bath for 40 minutes. Add 15 mL of water, extract with ethyl acetate (20 mL×3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by thin layer chromatography with the developing solvent system B to obtain 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]indolozino[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 (30 mg, 35.7 μmol) was dissolved in 3 mL of dichloromethane, 1.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 1.5 mL of toluene was added and concentrated under reduced pressure, repeated twice. Add 4.5 mL of n-hexane to the residue to make a slurry, and then pour out the supernatant after standing to keep the solid. The solid residue was concentrated under reduced pressure, and the 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]indolozino[1,2-b]quinolin-1-yl)acetamide (23mg), the product was directly used in the next reaction without purification.

MS m/z(ESI): 638.0[M+18].

In the sixth step, the crude product 5e (20mg, 32.3μmol) was dissolved in 1mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5℃ in an ice water bath, and 4g (31.8mg, 67.3μmol) was added. 0.5mL N,N-dimethylformamide solution, add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine salt ( 27.8 mg, 94.3 μmol), the reaction was stirred in an ice bath for 10 minutes, the ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour to produce compound 5. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18OBD5um 19*250mm; mobile phase: A-water (10mmol NH ₄ OAc): B-acetonitrile, gradient elution, flow rate: 18mL/min) The corresponding components were collected and concentrated under reduced pressure to obtain 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 minutes, purity: 85% (chromatographic column: ACQUITY UPLC BEHC181.7um 2.1*50mm, mobile phase: A-water (5mmol 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 minutes, purity: 89% (chromatographic column: ACQUITY UPLC BEHC181.7um 2.1*50mm, mobile phase: A-water (5mmol 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).

Refer to Intermediate 5 for the preparation methods of other intermediates.

At 37℃, add the prepared tris (2-carboxyethyl) phosphine to the PBS buffered aqueous solution of antibody HU1 (0.05M PBS buffered aqueous solution with pH=6.5; 7.3ml, 13.8mg/ml, 0.681μmol) Aqueous solution (10mM, 0.347mL, 3.47μmol), place in a water bath shaker, shake the reaction at 37℃ for 3 hours, stop the reaction; cool the reaction solution to 25℃ with water bath, dilute to 14.0ml, and take out 3.3ml solution down reaction.

Compound 5-A (5.0 mg, 2.75 μmol) was dissolved in 0.15 mL of DMSO, added to the above 3.3 ml solution, placed in a water bath shaker, and reacted with shaking at 25° C. for 3 hours to stop the reaction. The reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffered aqueous solution, containing 0.001M EDTA), to obtain the exemplary product HU1-I of Ab-Isinotecan derivative The PBS buffer (1.45 mg/mL, 17 mL) of the Xitecan derivative was stored frozen at 4°C.

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

data processing:

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

The average drug load 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.

For the preparation method of compound 2-compound 11, refer to compound 1. Similarly, the same preparation method of compound 1 was used to prepare compound 12 (y=8) using the hRS7 antibody in the prior art:

Example 11 Antibody conjugated to MC-MMAF

In the first step, S-(3-aldehyde propyl) thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use. To the acetic acid/sodium acetate buffer (10.35mg/mL, 9.0mL, 0.97mol) of the antibody pH=4.3 was added the pre-prepared acetonitrile solution of S-(3-hydroxypropyl) thioacetate, and then 1.0mL was added dropwise An aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was shaken and reacted at 25° C. for 2 hours. After the reaction is over, use Sephadex G25 gel column for desalting and purification (elution phase: pH 6.5 0.05M PBS solution) to obtain product 1a solution, which is concentrated to 10 mg/mL and proceed directly to the next reaction.

In the second step, 0.35mL of 2.0M carboxyamine hydrochloride solution was added to 1a solution (11.0mL), and after shaking at 25°C for 30 minutes, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH6 .5 0.05M PBS solution) to obtain product 2a solution (concentration 6.17 mg/mL, 14.7 mL).

In the third step, the compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile, and 2a solution (concentration 6.17 mg/mL, 3.0 mL) was added to 25 After shaking and reacting at ℃ for 4 hours, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5), and filtered under sterile conditions with a filter to obtain the product Ab-MC -MMAF antibody-drug conjugate in PBS buffer (3.7 mg/mL, 4.7 mL), refrigerated at 4°C.

Example 12 Antibody coupling SN-38

In the first step, S-(3-aldehyde propyl) thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use. Add the pre-prepared acetonitrile solution of S-(3-hydroxypropyl) thioacetate to the acetic acid/sodium acetate buffer (10.35mg/mL, 9.0mL, 0.97mol) of antibody pH=4.3, and then add 1.0mL dropwise An aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was shaken and reacted at 25° C. for 2 hours. After the reaction is completed, use Sephadex G25 gel column for desalting and purification (elution phase: pH 6.5 0.05M PBS solution) to obtain a 1h solution of the product, which is concentrated to 10 mg/mL and directly proceed to the next reaction.

In the second step, 0.35mL of 2.0M carboxyamine hydrochloride solution was added to the 1h solution (11.0mL), and after shaking at 25°C for 30 minutes, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH6 .5 0.05M PBS solution), the product 2h solution (concentration 6.2mg/mL, 15.0mL) was obtained, which was concentrated to about 10mg/ml and used in the next reaction.

In the third step, the compound MC-VC-PAB-SN-38 (1.3mg, 1.2mol) was dissolved in 0.3ml of acetonitrile, added to the 2h solution (concentration 6.2mg/mL, 3.0mL), and shaken at 25°C After 4 hours of reaction, the reaction solution was desalted and purified with Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5), and filtered under sterile conditions with a filter to obtain the product Ab-SN-38 antibody -PBS buffer (3.7 mg/mL, 4.7 mL) of the drug conjugate, refrigerated at 4°C.

Example 13 The killing effect of antibody-conjugated drugs on tumor cells

To test the killing effect of the antibody-drug conjugate of the present invention on tumor cells, bladder cancer cell SW780 was used for evaluation. Collect SW780 cells, after centrifugal counting, adjust the cell density to 0.44×10 ⁶ cells/mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate, each with 90 μL, and the number of cells is 4000. Add 100 μL PBS to the remaining side holes. The cell plate was placed in a 37°C, 5% CO2 incubator and cultured overnight. On the second day of the experiment, prepare the antibody-drug conjugate solution in a 96-well V-bottom plate with PBS, starting with a concentration of 1000 nM, 3 times dilution, and 9 concentrations. After the preparation is completed, add it to the white 96-well plate, each well 10μL, two duplicate wells, put the cell plate in a 37°C, 5% CO2 incubator and continue to incubate for 72 hours. On the fifth day of the experiment, test the reading: Take out the cell culture plate, after equilibrating to room temperature, add 50μL CTG solution (Promega G7573) to each well, shake and mix, place in the dark and stand for 10 minutes, then use the luminescence program of the microplate reader Perform testing. The EC50 value was calculated by a four-parameter fitting method, and with reference to the above method, the killing effect of the antibody-drug conjugate of the present invention on bladder cancer cell RT4 and epidermal cancer cell A431 was evaluated. The experimental results are shown in Table 11:

Table 11. The killing effect of antibody-drug conjugates on tumor cells

Example 14 Pharmacokinetics of antibody-conjugated drugs

To further study the pharmacokinetics of the antibody-drug conjugate in vivo, the antibody-drug conjugate was injected intravenously into C57BL/6 mice (dose 10mg/kg). At 1h, 2h, 4h, 8h, 24h, 48h, 96h, 144h and 240h, 20 microliters of blood were drawn, and the concentration of the antibody-drug conjugate in the blood was determined by the ELISA method of Example 3(1) After that, WinNonlin software was used to analyze the pharmacokinetic data to obtain the pharmacokinetic parameters, as shown in Table 12 below.

Table 12. Pharmacokinetic parameters of antibody-drug conjugates

Example 15 Anti-tumor effect of antibody conjugated drug in vivo

In order to further study the killing effect of the antibody-drug conjugate on the tumor formed in the body, the anti-tumor effect of the antibody-drug conjugate of the present invention was evaluated after N87 was used to form transplanted tumors in mice. 5× ^{10 6} N87 cells were injected subcutaneously into immunodeficient nude mice. After 2 weeks, the antibody-drug conjugate compound 10 (y=8) and compound 12 (y=8) were injected intravenously, and injected every 2 weeks. The dose is 2mg/kg. Human IgG1 protein was used as the control, and the dose was 2 mg/kg. There were 5 mice in each group in the control group or the administration group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate = 100%-(Tumor volume in the administration group on day 28-tumor volume in the administration group on day 0)/(Tumor volume in the control group on day 28-tumor volume in the control group on day 0). The experimental results are shown in Table 13 Shown:

Table 13. The killing effect of antibody-drug conjugates on tumors

Compound 10 (y=8) showed a tumor inhibition rate of more than 100%, which means that compound 10 (y=8) can not only inhibit the growth of tumors, but also has a killing effect on the formed tumors.

Example 16: Preparation of Exotecan Derivatives

Add 2mL ethanol and 0.4mL N,N-dimethylformamide to 2e (4mg, 7.53μmol), replace with argon three times, cool to 0-5℃ in an ice water bath, add 0.3mL N-methylmorpholine dropwise, Stir until the reaction solution becomes clear. Add 2-cyclopropyl-2-hydroxyacetic acid 1e (2.3mg, 19.8μmol, prepared by the method disclosed in the patent application "WO2013106717") and 1-hydroxybenzotriazole (3mg, 22.4μmol) to the reaction solution in sequence. ) And 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3mg, 22.4μmol), after the addition, the reaction was stirred at 0-5°C for 1 hour. Remove the ice-water bath, heat to 30°C and stir for 2 hours. The reaction solution was concentrated under reduced pressure, and the obtained crude compound 2-C was purified by high performance liquid chromatography (Separation conditions: Column: XBridge Prep C18 OBD 5um 19*250mm; Mobile phase: A-water (10mmol NH4OAc), B- Acetonitrile, gradient elution, flow rate: 18 mL/min), collect the corresponding components, and concentrate under reduced pressure to obtain the title product (2-A: 1.5 mg, 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 minutes, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*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 minutes, purity: 86% (column: ACQUITY UPLC BEHC18 1.7um 2.1*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: Inhibition test of exenotecan derivatives on tumor cell proliferation in vitro

The compounds 2-A and 2-B were tested for their inhibitory activity on the in vitro proliferation of U87MG cells (Cell Bank of Chinese Academy of Sciences, Catalog#TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30). The cells were treated in vitro with different concentrations of the compound, and after 6 days of culture, the proliferation of the cells was detected with CTG (Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent, and the in vitro activity of the compound was evaluated according to the IC50 value.

U87MG and SK-BR-3 cells were cultured with 10% FBS in EMEM medium (GE, article number SH30024.01) and McCoy's 5A medium (Gibco, article number 16600-108) containing 10% FBS, respectively.

Take the U87MG and SK-BR-3 cells in the logarithmic growth phase, wash them with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3ml trypsin (0.25% Trypsin-EDTA( 1x), Gibico, Life Technologies) digest for 2-3min, after the cells are digested completely, add 10-15ml cell culture solution, wash the digested cells, centrifuge at 1000rpm for 5min, discard the supernatant, and then add 10-20ml The cell culture fluid resuspends the cells to make a single cell suspension.

Mix the U87MG and SK-BR-3 single cell suspensions, adjust the viable cell density to 2.75×103cells/ml and 8.25×103cells/ml with cell culture medium, and mix the cell suspension after the density adjustment to 180μl /Well add 96-well cell culture plate. Only add 200ul medium to the peripheral wells of the 96-well plate. The culture plate was cultured in an incubator for 24 hours (37°C, 5% CO ₂ ).

The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution with an initial concentration of 10 mM.

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

Add 30μl of different samples to be tested into the first column of the 96-well U-bottomed dispensing plate, with a sample concentration of 100uM; add 20ul DMSO to each well in the second column to the 11th column. Take 10ul of the sample from the first column to 20ul in the second column and mix well, then take 10ul to the third column, and so on to the 10th column. Take 5ul to 95ul of EMEM medium from each well of the medicine in the dispensing plate, mix well, and set aside.

Adding samples: Add 20μl of the tested samples of different concentrations to the culture plate, and each sample has two duplicate wells. The culture plate was incubated in an incubator for 6 days (37°C, 5% CO ₂ ).

Color development: Take out the 96-well cell culture plate, add 90μl CTG solution to each well, and incubate at room temperature for 10 minutes.

Plate reading: Take out the 96-well cell culture plate, place it in a microplate reader (BMG labtech, PHERAstar FS), and measure chemiluminescence with the microplate reader.

Data analysis: Use Microsoft Excel, Graphpad Prism 5 to process and analyze the data. See Table 14 for the experimental results.

Table 14. Antitumor effects of ixitecan derivatives

Claims (24)

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

   Ab-(L ₂ -L ₁ -D) _y

   (A)

   in:

   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 are the} same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group Group, cycloalkyl or heterocyclic group;

   Alternatively, R ^a and R ^b together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;

   R ^{5 is} selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;

   R ^{6 is} selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;

   Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;

   Alternatively, R ^a and R ⁶ together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;

   m is selected from an integer from 0 to 4;

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

   L ₂ is the joint unit;

   Ab is an anti-TROP-2 antibody or an antigen-binding fragment thereof, which includes a light chain variable region and a heavy chain variable region, and the heavy chain variable region includes the HCDR1 shown in SEQ ID NO: 3 and SEQ ID NO: 4 The HCDR2 shown in SEQ ID NO: 5 and the HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes LCDR1 shown in SEQ ID NO: 6 and LCDR2 shown in SEQ ID NO: 7 and the LCDR2 shown in SEQ ID NO: 8 LCDR3 shown.
2. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof is selected from murine antibodies or antigen-binding fragments thereof, Integrating antibodies or antigen-binding fragments thereof, human antibodies or antigen-binding fragments thereof, or humanized antibodies or antigen-binding fragments thereof.
3. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a human IgG1, IgG2, IgG3 or IgG4 Heavy chain constant region or variants 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 shown in SEQ ID NO: 48 or SEQ ID NO: 49.
4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a human antibody kappa chain, lambda chain The light chain constant region of, or a variant thereof;

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

   More preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:50.
5. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequences , Or a heavy chain variable region with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25;

   And/or, selected from the light chain variable region shown in the following sequence, or light chain having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequence Variable region: SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 26.
6. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 5, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises:

   The heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 10; or,

   The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12; or,

   The heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 14; or,

   The heavy chain variable region shown in SEQ ID NO: 15 and the light chain variable region shown in SEQ ID NO: 16; or,

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

   The heavy chain variable region shown in SEQ ID NO: 19 and the light chain variable region shown in SEQ ID NO: 20; or,

   The heavy chain variable region shown in SEQ ID NO: 21 and the light chain variable region shown in SEQ ID NO: 22; or,

   The heavy chain variable region shown in SEQ ID NO: 23 and the light chain variable region shown in SEQ ID NO: 24; or,

   The heavy chain variable region shown in SEQ ID NO: 25 and the light chain variable region shown in SEQ ID NO: 26.
7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 5, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof contains a heavy chain selected from the following sequences, or Heavy chains with at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45 or SEQ ID NO: 47;

   And/or, selected from the light chains shown in the following sequences, or light chains with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequences: SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44.
8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 7, wherein the anti-TROP-2 antibody comprises:

   The heavy chain shown in SEQ ID NO: 27 and the light chain shown in SEQ ID NO: 28; or,

   The heavy chain shown in SEQ ID NO: 29 and the light chain shown in SEQ ID NO: 30; or,

   The heavy chain shown in SEQ ID NO: 31 and the light chain shown in SEQ ID NO: 32; or,

   The heavy chain shown in SEQ ID NO: 33 and the light chain shown in SEQ ID NO: 34; or,

   The heavy chain shown in SEQ ID NO: 35 and the light chain shown in SEQ ID NO: 36; or,

   The heavy chain shown in SEQ ID NO: 37 and the light chain shown in SEQ ID NO: 38; or,

   The heavy chain shown in SEQ ID NO: 39 and the light chain shown in SEQ ID NO: 40; or,

   The heavy chain shown in SEQ ID NO: 41 and the light chain shown in SEQ ID NO: 42; or,

   The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 44; or,

   The heavy chain shown in SEQ ID NO: 45 and the light chain shown in SEQ ID NO: 38; or,

   The heavy chain shown in SEQ ID NO: 47 and the light chain shown in SEQ ID NO: 28.
9. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to any one of claims 1-8, wherein the L _{1 is represented by the} general formula (E):

   

   in,

   R ⁵ is haloalkyl or cycloalkyl,

   R ^{6 is} selected from hydrogen, haloalkyl or cycloalkyl,

   Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a cycloalkyl group;

   Preferably,

   R ^{5 is} selected from C _1-6 haloalkyl or C _3-6 cycloalkyl,

   R ^{6 is} selected from hydrogen, C _1-6 haloalkyl or C _3-6 cycloalkyl,

   Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

   m is selected from an integer from 0 to 4,

   Preferably, the general formula (E) is selected from the following substituents:

   
10. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to any one of claims 1-8, wherein L _{2 is represented by the} general formula (D):

    -K ¹ -K ² -K ³ -K ^4-

    (D)

    in,

    K ¹ is

    

    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 an integer from 1 to 6;

    R ^{1 is} selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl;

    K ³ is a tetrapeptide residue, preferably, the tetrapeptide residue is selected from two or more of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and Peptide residues formed by amino acids in 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 are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkane Base, t is selected from 1 or 2.
11. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 10, wherein the linker unit L _{2 has} its K ¹ end connected to Ab and K ⁴ end connected to L ₁ .
12. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to any one of claims 1-11, wherein the -L ₂ -L ₁ -is selected from the following structures:

    

    Among them, K ² is a key;

    K ³ is the tetrapeptide residue of GGFG;

    R ^{5 is} selected from haloalkyl or C _3-6 cycloalkyl;

    R ^{6 is} selected from hydrogen, haloalkyl or C _3-6 cycloalkyl;

    Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

    R ² , R ³ or R ⁴ are each independently selected from hydrogen or alkyl;

    s is selected from an integer from 2 to 8;

    m is selected from an integer from 0 to 4;

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

    

    
13. The antibody-drug conjugate according to any one of claims 1-12, or a pharmaceutically acceptable salt or solvent compound thereof, wherein the cytotoxic drug is selected from the group consisting of toxins, chemotherapeutic drugs, antibiotics, radioisotopes and nucleolytic compounds. Enzymes; preferably tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division; more preferably camptothecin derivatives, DM1, DM3, DM4, SN-38, MMAF or MMAE; more preferably exenotecan or Exotecan derivative, SN-38, MMAE or MMAF.
14. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 13, wherein the cytotoxic drug is selected from the following structures:

    
15. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 14, wherein the antibody-drug conjugate is represented by the general formula (III):

    

    in,

    L ₁ and L ₂ are joint units;

    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 according to any one of claims 1-8.
16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 15, wherein the antibody-drug conjugate is represented by the general formula (IV):

    

    in,

    W is selected from a C _1-8 alkyl group, a C _1-8 alkyl-cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O or S The heteroatoms, wherein the C _1-8 alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, Substituted by one or more substituents of 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 bond, R 1 is} selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group, and p ₁ is an integer from 1 to 20;

    K ^{3 is} selected from peptide residues consisting of 2 to 7 amino acids. The amino acids can be substituted or unsubstituted. When substituted, the substituents can be substituted at any available point of attachment, and the substituents are one Or more are independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

    R ^{2 is} independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group;

    R ³ and R ⁴ are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

    R ^{5 is} selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;

    R ^{6 is} selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

    Alternatively, R ⁵ and R ⁶ together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;

    m is selected from an integer from 0 to 4;

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

    Ab is an anti-TROP-2 antibody or an antigen-binding fragment thereof.
17. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 16, wherein the antibody-drug conjugate is represented by the general formula (IV-A):

    

    Preferably, the general formula (IV-A) is selected from the following structures:

    

    

    
18. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, which is selected from the following compounds:

    

    

    

    

    Among them, y is selected from 2-10, preferably 4-8, more preferably 6-8, further preferably 7-8, and most preferably 8.
19. A method for preparing the antibody-drug conjugate represented by general formula (IV) or a pharmaceutically acceptable salt or solvate thereof, which comprises the following steps:

    

    After Ab is reduced, it is coupled and reacted with the general formula (F) to obtain the compound represented by the general formula (IV);

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

    W, K ² , K ³ , R ² to R ⁶ , m, and y are as defined in claim 16.
20. The method according to claim 19, wherein said general formula (F) is a compound represented by general formula (F-1) or its tautomer, meso, racemate, enantiomer Isomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof:

    

    Wherein, K ² , K ³ , R ² to R ⁶ , s and m are as defined in claim 12.
21. The compound represented by general formula (F) or general formula (F-1) is selected from:

    

    
22. A pharmaceutical composition comprising the antibody-drug conjugate or the pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate according to any one of claims 1-18, and one or more A pharmaceutically acceptable excipient, diluent or carrier.
23. The use of the antibody-drug conjugate according to any one of claims 1-18 or the pharmaceutical composition according to claim 27 in the preparation of a medicine for the treatment of human TROP-2 related diseases.
24. The use according to claim 23, wherein the disease related to human TROP-2 is a cancer with high TROP-2 expression, and the cancer is selected from the group consisting of triple-negative breast cancer, small cell lung cancer, and urothelial carcinoma , Human brain astroblastoma, human pharynx cancer, adrenal gland tumors, AIDS-related cancers, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain Tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferative small round cell tumor, ependymal cell Tumor, Juventus tumor, extraosseous mucinous chondrosarcoma, bone fibrous hypoplasia, osteofibrous dysplasia, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposine sarcoma, kidney cancer, leukemia, liposarcoma, malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, bone marrow Dysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral schwannoma, pheocytoma, pituitary tumor, prostate cancer, posterior Uveal melanoma, renal metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, Guwan carcinoma, thymic carcinoma, thymoma, metastatic thyroid carcinoma and uterus cancer.

Images