TWI891749B - Antibody-drug conjugates and medical use thereof - Google Patents

Abstract

The present invention relates a kind of antibody-drug conjugates and medical use thereof. Concretely, the present invention relates a kind of anti-TROP-2 antibody-drug conjugates and medical use thereof. Furtherly, the present invention relates antibody-drug conjugates containing anti-TROP-2 antibody or its antigen-binding fragment, their medicinal salts or solvates thereof, the use in the preparation of medicine for the treatment of TROP-2 mediated diseases or conditions thereof, and the use in tumor detection and diagnosis.

Description

Antibody-drug conjugates and their medical uses

The present invention relates to an anti-TROP-2 antibody-drug conjugate that is specifically immunoreactive with the human TROP-2 receptor and its pharmaceutical composition, as well as its use as an anticancer drug and for detecting or diagnosing tumors.

With the continuous deepening of research on tumor genomics, proteomics, and signal transduction pathways, we have a clearer understanding of the interactions between oncogenes and tumor suppressor genes in tumor cells and their impact on the tumor microenvironment. This has also made it possible to design new anti-tumor treatments targeting specific molecular targets in tumors.

Molecularly targeted tumor therapy is a novel treatment modality distinct from traditional surgery, radiotherapy, and chemotherapy. Its advantage lies in the fact that drugs typically bind only to their corresponding targets, killing or inhibiting target cells by directly affecting the function of the target molecule or by carrying physical or chemical effectors. Because of their well-defined targets, these drugs are typically highly selective, effectively killing or inhibiting target cells while causing minimal or no toxic side effects on normal tissue cells. Therefore, the development of molecularly targeted drugs has become a hot topic in clinical oncology research.

Human trophoblast cell surface antigen 2 (TROP-2) is a cell surface glycoprotein encoded by the TACSTD2 gene. TROP-2 is composed of 323 amino acids, including a 26-amino acid signal peptide, a 248-amino acid extracellular domain, a 23-amino acid transmembrane domain, and a 26-amino acid cytoplasmic domain. The extracellular domain of TROP-2 contains four heterogeneous N-linked glycosylation sites, and the addition of sugar chains increases the apparent molecular weight by 11 to 13 kDa. Within the TACSTD gene family, the extracellular domain possesses a characteristic thyroglobulin (TY) sequence, which is generally believed to be involved in cancer cell proliferation, invasion, and metastasis.

To date, the physiological ligand of TROP-2 has not been identified, and its molecular function remains unclear. However, its intracellular residual serine 303 (S303) can be phosphorylated by Ca2 ⁺ -dependent protein kinase C (PKC), thereby promoting the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to form inositol triphosphate (IP3) associated with the mitogen-activated protein kinase pathway (MAPK). This signaling pathway is closely related to cell proliferation, suggesting that Trop2 has a function in mediating signaling in tumor cells.

Numerous clinical studies and literature reports have demonstrated that TROP-2 is overexpressed in a variety of epithelial cancers, including gastric, lung, colorectal, ovarian, breast, prostate, pancreatic, liver, and esophageal cancers. In contrast, TROP-2 is rarely or not expressed in normal adult tissues, with expression limited to small amounts in epithelial cells, and at lower levels than in cancer, suggesting a role for TROP-2 in tumorigenesis. Overexpression of TROP-2 in tumor tissues is closely associated with poor patient prognosis and cancer cell metastasis, while also impacting overall survival. Therefore, TROP-2 has become a compelling target in molecularly targeted tumor therapy.

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

U.S. Patent No. 5,840,854 reports the cytotoxicity of an anti-hTROP-2 monoclonal antibody (BR110) conjugated to a cytotoxin against human cancer cell lines H3619, H2987, MCF-7, H3396, and H2981.

U.S. Patent No. 6,653,104 discloses an antibody (RS7) that was tested in an in vivo model using a radioactively labeled antibody. The antibody demonstrated anti-tumor activity in a nude mouse xenograft model, but no reports were found regarding the anti-tumor effect of the naked antibody alone.

U.S. Patent No. 7,420,040 also reports that isolated monoclonal antibodies produced by fusion tumor cell lines AR47A6.4.2 or AR52A301.5, derived from mice immunized with human ovarian cancer tissue, bind to hTROP-2 and exhibit anti-tumor activity in a nude mouse xenograft model.

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

CN104114580A discloses an antibody (particularly a humanized antibody) that specifically reacts with hTROP-2 and has anti-tumor activity in vivo, as well as a fusion tumor that produces the antibody, a complex of the antibody and a drug, a pharmaceutical composition for diagnosing or treating tumors, a method for detecting tumors, and a kit for detecting or diagnosing tumors.

According to some embodiments of the present invention, an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided, wherein 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)-;

^Ra and ^Rb 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, a cycloalkyl group, or a heterocyclic group;

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

^R5 is selected from halogen, halogenalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl or heteroaryl;

^R6 is selected from a hydrogen atom, a halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group;

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

Alternatively, ^Ra and ^R6 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group;

m is an integer selected from 0 to 4;

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

_L2 is the bridge unit;

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

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

HCDR1 shown in SEQ ID NO: 3,

HCDR2 shown in SEQ ID NO: 4 and

HCDR3 shown in SEQ ID NO: 5.

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

LCDR1 shown in SEQ ID NO: 6,

LCDR2 shown in SEQ ID NO: 7 and

LCDR3 shown in SEQ ID NO: 8.

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

HCDR1 shown in SEQ ID NO: 3,

HCDR2 shown in SEQ ID NO: 4 and

HCDR3 shown in SEQ ID NO: 5; and

The antibody light chain variable region comprises:

LCDR1 shown in SEQ ID NO: 6,

LCDR2 shown in SEQ ID NO: 7 and

LCDR3 shown in SEQ ID NO: 8.

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 thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof is selected from a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

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 heavy chain constant region derived from human IgG1, IgG2, IgG3 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 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, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody κ chain or λ chain, or a variant thereof, in the antibody-drug conjugate or pharmaceutically acceptable salt or solvent compound of the present invention.

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 κ 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 comprises an anti-TROP-2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region selected from the following sequences, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the following sequences: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25.

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

In a preferred embodiment of the present invention, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the antibody-drug conjugate of the present invention comprises a heavy chain selected from the following sequences, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity with 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.

In a preferred embodiment of the present invention, the anti-TROP-2 antibody or antigen-binding fragment thereof according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent thereof comprises a light chain selected from the following sequences, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity 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.

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

(1) the heavy chain represented by SEQ ID NO: 27 and the light chain represented by 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 represented by SEQ ID NO: 31 and the light chain represented by 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 represented by SEQ ID NO: 45 and the light chain represented by 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, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent thereof, _L1 is represented by the general formula (B):

in,

_{M1 is -CR1R2-} ;

_R1 and _R2 are the same or different, and _R1 and _R2 are each independently selected from hydrogen, alkyl, halogen, hydroxyl or amino;

n is an integer between 0 and 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 thereof, _L2 is represented by the general formula (C):

in,

M ₂ is -CR ₄ R ₅ -;

_R3 is selected from hydrogen, halogen, hydroxy, amino, alkyl, alkoxy and cycloalkyl:

_R4 and _R5 are the same or different and are independently selected from hydrogen, alkyl, halogen, hydroxyl or amino;

m is an integer between 0 and 5, preferably 1, 2, or 3.

In a further preferred embodiment of the present invention, the O terminal of _L1 is connected to the bridge unit _L2 .

In a further preferred embodiment of the present invention, the O terminal of _L1 is connected to the S terminal of the bridge unit _L2 .

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

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

^Ra and ^Rb are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;

^R5 is a halogen group or a _C3-6 cycloalkyl group;

^R6 is selected from a hydrogen atom, a halogenated alkyl group or a _C3-6 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 or a pharmaceutically acceptable salt or solvent compound thereof, _L1 is represented by the general formula (E):

Wherein, R ⁵ is a halogen alkyl group or a cycloalkyl group,

^R6 is selected from hydrogen, halogen, or cycloalkyl,

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

Preferably,

^R5 is selected from _C1-6 halogen alkyl or _C3-6 cycloalkyl,

^R6 is selected from hydrogen, _C1-6 halogen alkyl or _C3-6 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 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 or a pharmaceutically acceptable salt or solvent compound thereof, 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 an integer selected from 2 to 8;

^K2 is selected from ^-NR1 ( _CH2CH2O )pCH2CH2C _(O ) _- , -NR1( _CH2CH2O ) _pCH2C ( ^O ₎ -, _-S ( _CH2 ) _pC (O)-, or a single bond, and p is selected from an integer from 1 to ₂₀ , preferably an integer from 1 to 6 _;

^R1 is selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, halogenated alkyl, deuterated alkyl and hydroxyalkyl;

^K3 is a tetrapeptide residue, preferably, the tetrapeptide residue is selected from peptide residues formed by two or more amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate, and aspartic acid; more preferably, it is a tetrapeptide residue 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, halogenated alkyl, deuterated alkyl and hydroxyalkyl, and t is selected from 1 or 2.

In a preferred embodiment of the present invention, in the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent thereof, the bridging unit -L ₂ - has its K ¹ end connected to Ab and its K ⁴ end connected to L ₁ .

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

Among them, K ² is the key;

^K3 is the tetrapeptide residue of GGFG;

^R5 is selected from a haloalkyl group or a _C3-6 cycloalkyl group;

^R6 is selected from hydrogen, halogenated alkyl or _C3-6 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 an integer selected from 2 to 8;

m is 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 toxins, chemotherapeutic drugs, antibiotics, radioactive isotopes and nucleolytic enzymes.

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 alkaloid derivatives, DM1, DM3, DM4, SN-38, MMAF or MMAE; more preferably exitecan or an exitecan derivative, 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 exotecan derivatives, preferably, the exotecan derivative is compound 2-A:

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

Among them, _L1 and _L2 are bridge units;

y is a number selected from 1 to 10, preferably 2 to 8, and more preferably 2 to 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 _L2 is as defined above; preferably, L2 is as defined above in formula (C).

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

wherein _L1 is as defined above; preferably, L2 is as defined above in formula (B).

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

Among them, _L1 and _L2 are bridge units;

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

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

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

wherein _L2 is as defined above; preferably, L2 is as defined above in 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 its pharmaceutically acceptable salt or solvent compound according to the present invention is a compound represented by general formula (III) or its pharmaceutically acceptable salt or solvent compound:

Among them, _L1 and _L2 are bridge units;

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

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

In a further preferred embodiment of the present invention, the antibody-drug conjugate of formula (III) or a pharmaceutically acceptable salt or solvent thereof, wherein L ₁ is as defined in formula (E) and L ₂ is as defined in formula (D).

In a further preferred embodiment of the present invention, the antibody-drug conjugate of general formula (III) or a pharmaceutically acceptable salt or solvent thereof, wherein the bridging unit -L2- has its K1 end connected to Ab and its K4 end connected to L1.

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

K ² is the key;

^K3 is the tetrapeptide residue of GGFG;

^R5 is selected from a haloalkyl group or a _C3-6 cycloalkyl group;

^R6 is selected from hydrogen, halogenated alkyl or _C3-6 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 an integer selected from 2 to 8;

m is an integer selected 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 its pharmaceutically acceptable salt or solvent compound according to the present invention is an antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound represented by the general formula (IV):

in,

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

^K2 is selected from ^-NR1 ( _CH2CH2O ) _p1CH2CH2C (O) _- , _-NR1 ( _CH2CH2O ) _p1CH2C (O) ^- , _-S ( _CH2 ) _p1C (O)-, or a bond; ^R1 is selected from a hydrogen atom, an alkyl group, _a halogenated alkyl group, _a deuterated alkyl group, and a hydroxyalkyl group; _{and p1} is an integer from 1 to 20;

^K3 is selected from a peptide residue consisting of 2 to 7 amino acids, which may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point, and the substituent is one or more independently selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

^R2 is independently selected from hydrogen atom, alkyl, halogenated alkyl, deuterated alkyl and hydroxyalkyl;

^R3 and ^R4 are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

^R5 is selected from halogen, halogenated alkyl, deuterated alkyl, cycloalkyl, heterocyclo, aryl or heteroaryl;

^R6 is selected from a hydrogen atom, a halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group;

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

m is an integer selected from 0 to 4;

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

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

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

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 according to the present invention or its pharmaceutically acceptable salt or solvent compound is selected from the following compounds:

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

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

and

Wherein, y is selected from 2-10, preferably 4-8, more preferably 6-8, further preferably 7-8, and most preferably 8. y can be a decimal or an integer.

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

After Ab is reduced, it is coupled with the compound of general formula (F) to obtain the compound of general formula (IV);

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

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 described in general formula (IV) of the present invention, the general formula (F) is a compound represented by general formula (F-1) or its tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or a pharmaceutically acceptable salt 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 the general formula (F) or general formula (F-1) of the present invention is selected from:

On the other hand, 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 more pharmaceutically acceptable excipients, diluents or carriers.

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

In a more preferred embodiment of the present invention, the disease associated with human TROP-2 is the use of the drug in the preparation of a drug for treating a cancer with high expression of TROP-2, wherein the cancer is selected from triple-negative breast cancer, small cell lung cancer, urothelial carcinoma, human brain astrocytoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, Cancer of the kidneys, bone cancer, brain and spinal cord cancer, metastatic brain tumors, breast cancer, carotid artery tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia, fibrous dysplasia, gallbladder or bile duct cancer, Gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma, malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myeloproliferative syndrome, neuroblastoma, neuroendocrine tumor Tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, thyroid metastatic carcinoma, and uterine cancer.

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

Figure 1 shows an in vitro ELISA binding assay, demonstrating the binding activity of 11 humanized anti-TROP-2 antibodies to human TROP-2 antigen.

[Invention Details]

1. Terminology

To facilitate understanding of the present invention, certain technical and scientific terms are 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 this invention belongs.

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

The term "antibody" as used herein refers to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the heavy chain of immunoglobulins vary, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of their heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains depending on the invariant region. Each of the five Ig classes can have either a kappa or lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region, wherein the light chain constant region comprises a human or mouse κ, λ chain or a variant thereof.

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

The approximately 110 amino acids near the N-terminus of the antibody heavy and light chains exhibit significant sequence variation, forming the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region (C region). The variable region consists of three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions, also known as complementarity-determining regions (CDRs), determine the antibody's specificity. Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the VL and VH regions of the antibodies or antigen-binding fragments described in the present invention conform to the well-known Kabat numbering rules and Kabat or ABM definition rules (http://bioinf.org.uk/abs/).

The term "antigen presenting cell" or "APC" refers to a cell that displays a foreign antigen in complex with MHC on its surface. T cells recognize this complex using a T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.

The term "antigen presentation" refers to the process by which APCs capture antigens and make them available for recognition by T cells, for example as components of MHC-I/MHC-II conjugates.

The term "TROP-2" includes any variant or isoform of TROP-2 naturally expressed by cells. The antibodies of the present invention may cross-react with TROP-2 from non-human species. Alternatively, the antibodies may be specific for human TROP-2 and may not exhibit cross-reactivity with other species. TROP-2 or any variant or isoform thereof may be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques commonly used in the art and those described herein. Preferably, the anti-TROP-2 antibodies target 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 transgenic or transchromosomal animals (e.g., mice) expressing human immunoglobulin genes or fusion tumors derived therefrom;

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

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

4. Antibodies prepared, expressed, created, or isolated by splicing human immunoglobulin gene sequences into other DNA sequences.

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

The term "murine antibody" as used herein refers to a monoclonal antibody against human TROP-2 prepared according to the knowledge and skill in the art. To prepare the antibody, a test subject is injected with a TROP-2 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine TROP-2 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variants thereof.

The term "human antibody" includes antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic cell mutagenesis 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 mouse) have been grafted onto human framework sequences (i.e., "humanized antibodies").

The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences onto the human variable region framework. Humanized antibodies overcome the drawback of chimeric antibodies, which often induce a strong immune response due to the presence of large amounts of mouse protein. To avoid a decrease in immunogenicity and activity, minimal reverse mutations can be performed on the variable regions of the human antibody to maintain activity.

The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody. This antibody can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, a fusion tumor secreting a specific mouse monoclonal antibody is established. The variable region gene is then cloned from the mouse fusion tumor cells. Furthermore, the constant region gene of the human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are then ligated into a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell culture 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, it comprises a heavy chain constant region of human IgG1, IgG2, or IgG4, or an IgG1 heavy chain constant region with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar 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 parental antibody for 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.

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

The "Fc" region contains two heavy chain fragments that comprise the antibody's CH2 and CH3 domains. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions within the CH3 domains.

A "Fab' fragment" contains a light chain and a portion of a heavy chain including the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains. Interchain disulfide bonds can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

An "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, with interchain disulfide bonds forming between the two heavy chains. Thus, an F(ab')2 fragment consists of two Fab' fragments held together by the inter-heavy chain disulfide bonds.

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

The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. These 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 multiple epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, and antibody fragments that are covalently or non-covalently linked together, etc.

The term "single-chain antibody" refers to a single-chain recombinant protein composed of the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody, connected by a linker peptide. It is the smallest antibody fragment with a complete antigen-binding site.

The term "domain antibody fragment" refers to an immunologically functional immunoglobulin fragment containing only the variable heavy or light chain regions. In some cases, two or more VH domains are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH domains of a 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" in the present invention refers to the three-dimensional site recognized by the antibody or antigen binding fragment of the present invention.

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

As used herein, the terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when human TROP-2 is used as the analyte and the antibody as the ligand, the antibody binds to the predetermined antigen with an equilibrium dissociation constant ( _KD ) of approximately less than ^10-7 M or even less, as measured by surface plasmon resonance (SPR) technology. Furthermore, the antibody binds to the predetermined antigen with an affinity that is at least twice as high as its affinity for nonspecific antigens other than the predetermined antigen or closely related antigens (e.g., BSA). The term "antibody that recognizes an antigen" is used interchangeably herein with the term "specifically binding antibody."

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

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of a ligand preferably reduces or alters the normal level or type of activity that would occur upon ligand binding in the absence of inhibition or blockade. Inhibition and blocking are also intended to encompass any measurable decrease in ligand binding affinity when contacted with an anti-TROP-2 antibody, compared to the ligand not contacted with the anti-TROP-2 antibody.

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

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

The term "ADCC" as used in this invention, which stands for antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the antibody's Fc region. The ADCC effect of an antibody can be enhanced, reduced, or eliminated by modifying the Fc region of IgG. Such modification involves mutations in the heavy chain constant region of the antibody.

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory Manual of Antibody Experimental Techniques. For example, mice can be immunized with human TROP-2 or fragments thereof, and the resulting antibodies can be immunized, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described herein are constructed by genetically engineering non-human CDR regions with one or more human FR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website at http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001, ISBN 012441351.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified using 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 existing technology, the mammalian expression system will lead to glycosylation of the antibody, especially at the highly conserved N-terminus of the Fc region. Stable pure strains are obtained by expressing antibodies that specifically bind to human antigens. Positive pure strains are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibody can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular screening and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or freeze-dried.

The antibodies of the present invention refer to monoclonal antibodies. Monoclonal antibodies (mAbs) described herein refer to antibodies obtained from a single pure cell line, which is not limited to eukaryotic, prokaryotic, or phage pure cell lines. Monoclonal antibodies or antigen-binding fragments can be recombined using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

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

"Treatment" means administering a therapeutic agent, such as one comprising any of the antibodies of the present invention, to a patient experiencing one or more disease symptoms for which the therapeutic agent is known to have therapeutic effects. Generally, the therapeutic agent is administered in an amount effective to alleviate the one or more disease symptoms in the patient or population being treated, either by inducing regression of such symptoms or inhibiting their progression to any clinically relevant degree. The amount of a therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, as well as the drug's ability to produce the desired therapeutic effect in the patient. Whether disease symptoms have been alleviated can be assessed by any clinical test commonly used by physicians or other healthcare professionals to assess the severity or progression of such symptoms. Although an embodiment of the present invention (e.g., a treatment method or product) may not be effective in alleviating the target disease symptom in every patient, it should alleviate the target disease symptom in a statistically significant number of patients as determined by any statistical test known in the art, such as the Student t test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

The term "consisting essentially of..." or variations thereof used throughout the specification and claims is intended to include all recited elements or groups of elements, and optionally include other elements of similar or different properties from the recited elements, which other elements do not significantly alter the basic or novel properties of the specified dosage regimen, method, or composition.

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

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

"Exogenous" refers to substances that originate outside an organism, cell, or human body, depending on the context.

"Endogenous" refers to substances that are produced in cells, organisms, or the human body in a certain context.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage 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, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is performed when the two sequences are aligned to achieve the maximum percentage homology.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that possess the same function or biological activity as screened for in the originally transformed cell are included. Where a different designation is intended, this will be clear from the context.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a specified sequence may but need not be present.

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

The following examples are used to further describe the present invention, but these examples are not intended to limit the scope of the present invention. Experimental methods in the examples of the present invention where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Technology and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents whose specific sources are not specified are commercially available reagents.

Example 1 Antigen Preparation

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

TROP-2-His sequence:

SEQ ID NO: 46

Example 2 Obtaining Mouse Fusion Tumor and Antibody Sequences

Immunization of the human antigen TROP-2-His was performed in five female Balb/c and five A/J mice, 10 weeks of age, using Sigma Complete Freund's Adjuvant (CFA) and Sigma Incomplete Freund's Adjuvant (IFA). The immunogen and adjuvant were thoroughly mixed and emulsified at a 1:1 ratio to form a stable "water-in-oil" solution. The injection dose was 25 μg/200 μL/mouse.

Serum from immunized mice was assessed for titer and ability to bind to cell surface antigens using an indirect ELISA as described in Example 3. Cell fusion was initiated based on the titer (greater than 100,000-fold dilution) of the serum. Mice with high serum titer, affinity, and FACS binding were selected for a final immunization and sacrificed. Spleen cells were fused with SP2/0 myeloma cells and plated to obtain fusion tumors. Target fusion tumors were screened using an indirect ELISA and isolated as single cell lines using limiting dilution. Positive antibody strains were further screened using an indirect ELISA to identify fusion tumors that bind to the recombinant protein. Confluent tumor cells were harvested during logarithmic growth, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed using PrimeScript ^™ Reverse Transcriptase, Takara #2680A. The reverse-transcribed cDNA was amplified by PCR using a mouse Ig primer set (Novagen, TB326 Rev. B 0503) and then sequenced to obtain the mouse antibody sequence.

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

M1 HCVR

SEQ ID NO: 1

M1 LCVR

SEQ ID NO: 2

Example 3 In vitro binding activity detection method of antibodies

(1) In vitro indirect ELISA binding experiment:

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

Dilute the test antibody in PBST containing 3% BSA, starting at 1 μM and progressing through a 10-fold gradient. Add 100 μl/well to the microplate and incubate at room temperature for 1 hour. After incubation, wash the plate four times with PBST and add 100 μl/well of HRP-conjugated goat anti-human secondary antibody (Abcam, cat#ab97225) diluted in PBST containing 3% BSA. Incubate at room temperature for 1 hour. After washing the plate four times with PBST, add 100 μl/well of TMB colorimetric substrate (Cell Signaling Technology, cat#7004S) and incubate at room temperature in the dark for 1 minute. Terminate the reaction by adding 100 μl/well of stop solution (Cell Signaling Technology, cat#7002S). Analyze the data using an enzyme-linked microplate reader (BioTek, model Synergy H1) at 450 nm. The concentration signal curve analysis results are shown in the following table:

(2) In vitro cell binding experiment:

Collect cultured TROP-2-overexpressing cells (CHO or 293 cells overexpressing TROP-2 and TROP-2-expressing tumor cells, such as HCC-827 and MDA-MB-468), adjust the cell density, and plate into 96-well U-bottom plates at 1× ^10⁵ to 2× ^10⁵ cells per well. Centrifuge at 1200g for 5 minutes, remove the supernatant, add 100µl of serially diluted antibody solution or mouse immune serum, and incubate at 4°C for 60 minutes. Centrifuge at 1200g for 5 minutes, remove the supernatant, wash the cells twice with PBS, and add 100µl of fluorescently labeled secondary antibody (PE-GAM or PE-GAH) per well. Incubate at 4°C for 60 minutes. Centrifuge at 1200g for 5 minutes, and remove the supernatant. After washing the cells twice with PBS, resuspend them in PBS and use a flow cytometer to detect the signal and analyze the results using a concentration curve.

Example 4 Mouse Antibody Humanization Experiment

Humanization of the mouse anti-human TROP-2 monoclonal antibody was performed using methods published in numerous literature in this field. Briefly, the human constant domain of the parental (mouse antibody) was replaced with a human constant domain, and the human antibody sequence was selected based on homology between the mouse and human antibodies. In this invention, the mouse antibody M1 was humanized.

Based on the typical structures of the obtained mouse antibody VH/VL CDRs, the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain human germline templates with high homology.

The CDR regions of the mouse antibody M1 were transplanted onto the selected humanized template. Then, based on the three-dimensional structure of the mouse antibody, back-mutations were performed on buried residues, residues directly interacting with the CDR regions, and residues with significant influence on VL and VH conformations. After expression testing and comparison of the number of back-mutations, an antibody design combining humanized heavy chain variable region (HCVR) and light chain variable region (LCVR) sequences was selected. The sequence is as follows:

HU1 HCVR

SEQ ID NO: 9

HU1 LCVR

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTRIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHYSTPLTFGQGTRLEIK SEQ ID NO: 10

HU2 HCVR

SEQ ID NO: 11

HU2 LCVR

ETTLTQSPAFMSATPGDKVNISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIK SEQ ID NO: 12

HU3 HCVR

SEQ ID NO: 13

HU3 LCVR

DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIK SEQ ID NO: 14

HU4 HCVR

SEQ ID NO: 15

HU4 LCVR

DIVMTQSPDSLAVSLGERATINCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIK SEQ ID NO: 16

HU5 HCVR

SEQ ID NO: 17

HU5 LCVR

SEQ ID NO: 18

HU6 HCVR

SEQ ID NO: 19

HU6 LCVR

DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK SEQ ID NO: 20

HU7 HCVR

SEQ ID NO: 21

HU7 LCVR

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK SEQ ID NO: 22

HU8 HCVR

SEQ ID NO: 23

HU8 LCVR

AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPSRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK SEQ ID NO: 24

HU9 HCVR

SEQ ID NO: 25

HU9 LCVR

DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIK SEQ ID NO: 26

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

IgG1 C1

SEQ ID NO: 48

IgG1 C2

SEQ ID NO: 49

Ig kappa C

SEQ ID NO: 50

The heavy and light chain sequences were obtained as follows (the HU1-HU9 heavy chains were derived by linking 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 to SEQ ID NO: 49, respectively; the HU6DL and HU10 heavy chains were derived by linking SEQ ID NO: 19 and SEQ ID NO: 9 to SEQ ID NO: 48, respectively):

HU1 HC

SEQ ID NO: 27

HU1 LC

SEQ ID NO: 28

HU2 HC

SEQ ID NO: 29

HU2 LC

SEQ ID NO: 30

HU3 HC

SEQ ID NO: 31

HU3 LC

SEQ ID NO: 32

HU4 HC

SEQ ID NO: 33

HU4 LC

SEQ ID NO: 34

HU5 HC

SEQ ID NO: 35

HU5 LC

SEQ ID NO: 36

HU6 HC

SEQ ID NO: 37

HU6 LC

SEQ ID NO: 38

HU7 HC

SEQ ID NO: 39

HU7 LC

SEQ ID NO: 40

HU8 HC

SEQ ID NO: 41

HU8 LC

SEQ ID NO: 42

HU9 HC

SEQ ID NO: 43

HU9 LC

SEQ ID NO: 44

HU6DL HC

SEQ ID NO: 45

HU6DL LC

SEQ ID NO: 38

HU10 HC

SEQ ID NO: 47

HU10 LC

SEQ ID NO: 28

cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of each humanized antibody and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). HEK293 cells (Life Technologies Cat. No. 11625019) were transfected with the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) at a 1:2 ratio and incubated in a _CO2 incubator for 4-5 days. The cell culture medium was collected, centrifuged, filtered, and loaded onto an antibody purification affinity column. The column was washed with phosphate buffer, extracted with glycine hydrochloride buffer (pH 2.7, 0.1M Gly-HCl), neutralized with 1M Tris hydrochloric acid, pH 9.0, and dialyzed against phosphate buffer to obtain the humanized antibody protein of the present invention. The concentration and purity are shown in the following table:

Example 5 In vitro binding affinity and kinetics experiments

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

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

Example 6 Humanized Antibody-Mediated Tumor Cell Killing

Humanized antibodies can exert their anti-tumor cell-killing effects in multiple ways, one of which is mediating immune cell-mediated anti-tumor cell cytotoxicity. To assess the anti-tumor cell-killing effect of the humanized antibodies of the present invention, a system was employed in which human peripheral blood mononuclear cells (PBMCs) were co-cultured with HCC827 tumor cells (non-small cell lung cancer). HCC827 cells were harvested, counted by centrifugation, and then adjusted to a cell density of 0.44 × 10 ⁶ /mL with complete medium. The cells were then plated in the center 60 wells of a white 96-well plate, with 90 μL per well, for a total of 4,000 cells. Commercially available human PBMCs were collected, counted by centrifugation, and adjusted to a cell density of 2.2 × ^10⁶ cells/mL with complete medium. The cells were plated in the center 60 wells of a white 96-well plate containing HCC827 cells, with 90 μL per well, for a total of 20,000 cells. 200 μL of PBS was added to the remaining wells, and the plate was incubated overnight at 37°C, 5% CO₂. On the second day of the experiment, a humanized antibody solution was prepared in PBS in a 96-well V-bottom plate. Starting at a concentration of 1000 nM, the solution was diluted three-fold to a total of nine concentrations. After preparation, 20 μL of the solution was added to each well of the white 96-well plate in duplicate. The plate was then incubated at 37°C, 5% CO₂ for 72 hours. On the fifth day of the experiment, the cells were cultured and read: 50 μL of CTG solution (Promega G7573) was added to each well after equilibration to room temperature. The plate was shaken to mix thoroughly and then placed in the dark for 10 minutes. The cells were then assayed using the luminescence program on an enzyme-linked microplate reader. The results are shown in Table 8 below:

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

Example 7 Humanized Antibody-Mediated TROP-2 Internalization

To investigate humanized antibody-mediated TROP-2 protein internalization in tumor cells, SW780 cells were trypsinized, harvested, and resuspended in pre-chilled PBS to a cell concentration of 1× ^10⁶ cells/mL. 1 mL of the cell suspension was added to an EP tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was then removed and 1 mL of the prepared test antibody was added to resuspend the cells. The final antibody concentration was 20 μg/mL. The tubes were incubated at 4°C on a rocking platform for 1 hour. The supernatant was discarded by centrifugation (4°C, 1500 rpm for 5 minutes), washed twice with PBS, and the supernatant removed. 100 μL of fluorescent secondary antibody working solution was added to each tube to resuspend the cells. The cells were incubated on a rocking platform at 4°C for 30 min, centrifuged, and the supernatant discarded (4°C, 1500 rpm for 5 min). The cells were washed twice with PBS, and the supernatant discarded. 1.0 mL of prewarmed SW780 complete cell culture medium was added to each tube to resuspend the cells and mix well. The cells were then aliquoted into four tubes, 200 μL each, designated as the 0 min, blank, 30 min, and 2 h groups. The 0 min and blank tubes were removed and placed on ice, and the remaining tubes were placed in a 37°C incubator. The cells were internalized for 30 min and 2 h, respectively. At each time point, one tube was removed and pre-chilled on ice for 5 min. All treatment groups were centrifuged (4°C, 1500 rpm for 5 min), the supernatant discarded, and the cells were washed once with PBS, and the supernatant discarded. Add 250 μL of strip buffer to the tubes of all treatment groups except the 0 min group, incubate at room temperature for 8 min, centrifuge and discard the supernatant (4°C, 1500 rpm for 5 min), wash twice with PBS, and discard the supernatant. Add 100 μL of immunostaining fixative to all treatment groups, incubate at 4°C for at least 30 min, and analyze on a DxFlex flow cytometer. Take 200 μL from the 0 min group tube and directly add the immunostaining fixative. Take 200 μL from the blank group tube and directly add strip buffer and immunostaining fixative. Analyze on a DxFlex flow cytometer. Data statistics and analysis: 30-minute average internalization percentage (%) = (30-minute MIF group - blank group MFI) / (0-minute MFI - blank group MFI); 2-hour average internalization percentage (%) = (2-hour MIF group - blank group MFI) / (0-minute MFI - blank group MFI). The internalization percentage of humanized antibodies detected using the above method is shown in Table 9 below:

Example 8 Competitive Binding of Humanized Antibodies to Antigens

Competitive binding assays are commonly used to investigate the binding mechanism and site of different antibodies to antigens. Dilute the hRS7 antibody protein to a concentration of 1 μg/ml in PBS (pH 7.4) and add 100 μl/well to a 96-well high-affinity microplate. Incubate at 4°C overnight (16-20 hours). Wash the plate four times with PBST (PBS (pH 7.4) containing 0.05% Tween-20). Then, add 150 μl/well of 2% bovine serum albumin (BSA) blocking buffer diluted in PBST and incubate at room temperature for 1 hour. After blocking, discard the blocking buffer and wash the plate four times with PBST buffer.

Dilute the test antibody to 100 μg/ml in PBST containing 2% BSA and add 50 μl/well to the ELISA plate. Dilute TROP-2 His protein (Sino Biological Inc., cat# 10428-H08H) in PBST containing 2% BSA and add 50 μl/well to the ELISA plate. Incubate the plate at room temperature for 1 hour. After incubation, wash the plate four times with PBST and add 100 μl/well of anti-His HRP-conjugated secondary antibody (Abcam, cat# ab197049) diluted in PBST containing 2% BSA and incubate at room temperature for 1 hour. After washing the plate four times with PBST, 100 μl/well of TMB colorimetric substrate (Cell Signaling Technology, cat#7004S) was added and incubated at room temperature in the dark for 1 minute. The reaction was terminated by adding 100 μl/well of Stop Solution (Cell Signaling Technology, cat#7002S). The absorbance was read at 450 nm using an ELISA reader (BioTek, model Synergy H1). The data were analyzed as shown in the table below. The humanized antibody of the present invention exhibited a very low inhibition rate on the binding of hRS7 antibody to TROP2 protein, indicating that the humanized antibody of the present invention and hRS7 antibody do not compete for binding to the same epitope.

Example 9 Preparation of Compound 1

In the first step, compound 2a (2 g, 17.2 mmol) was dissolved in 75 mL of acetonitrile. Potassium carbonate (9.27 g, 67.2 mmol), benzyl bromide (20 mL, 167.2 mmol), and tetrabutylammonium iodide (620 mg, 1.68 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 hours and filtered through celite. The filter cake was rinsed with ethyl acetate (20 mL). The combined filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography with developing agent System C to obtain product 5a (3.2 g, 90.1% yield).

In the second step, 5a (181.3 mg, 0.879 mmol) and 4b (270 mg, 0.733 mmol) were added to a reaction flask. 6 mL of tetrahydrofuran was added, and the atmosphere was replaced with hydrogen three times. The temperature was cooled to 0-5°C in an ice-water bath, and potassium tert-butoxide (164 mg, 1.46 mmol) was added. The ice bath was removed, and the mixture was warmed to room temperature and stirred for 40 minutes. 15 mL of ice water was added, and the mixture was extracted with ethyl acetate (40 mL x 2) and chloroform (20 mL x 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 6 mL of dioxane, and 3 mL of water was added. Sodium bicarbonate (73.8 mg, 0.879 mmol) and 9-fluorenylmethyl 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, and the mixture was extracted with ethyl acetate (20 mL x 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 resulting residue was purified by silica gel column chromatography using System C as the developer to obtain product 5b, 10-cyclopropyl-1-( 9H -fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazoundecanoate (73 mg, yield: 19.4%).

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

In the third step, 5b (30 mg, 0.058 mmol) was dissolved in 6.75 mL of a mixture of tetrahydrofuran and ethyl acetate (v:v = 2:1). Palladium on carbon (18 mg, 10% dry form) was added, and the atmosphere was replaced with hydrogen three times. The reaction was stirred at room temperature for 1 hour. The reaction mixture was filtered through 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-diazaundecanoic acid (20 mg). The product was carried on to the next reaction without purification.

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

In the fourth step, 1b (15 mg, 28.2 μmol) was added to a reaction flask, along with 1.5 mL of N , N -dimethylformamide. The atmosphere was replaced with hydrogen three times, cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added dropwise. Crude 5c (20 mg, 47.1 μmol) was then added, along with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylbenzonitrile chloride (25.4 mg, 86.2 μmol). The reaction was stirred in an ice-water bath for 40 minutes. 15 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined and washed with saturated sodium chloride solution (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography with developing agent System B to give the title product 5d ( 9H -fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-((( 1S , 9S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H , 12H -benzo[ de ]pyrano[3',4':6,7]indolizino[1,2- b ]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (23.7 mg, yield: 78.9%).

MS m/z (ESI): 842.1[M+1].

Step 5: Dissolve 5d (30 mg, 35.7 μmol) in 3 mL of dichloromethane, add 1.5 mL of diethylamine, and stir at room temperature for 2 hours. Concentrate the reaction mixture under reduced pressure, add 1.5 mL of toluene, and concentrate under reduced pressure. Repeat this process twice. Add 4.5 mL of n-hexane to the residue to slurry. After slurrying, pour off the supernatant and retain the solid. The solid residue was concentrated under reduced pressure and pumped dry to give 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]indolizino[1,2- b ]quinolin-1-yl)acetamide (23 mg), which was used directly in the next reaction without purification.

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

In the sixth step, crude product 5e (20 mg, 32.3 μmol) was dissolved in 1 mL of N , N -dimethylformamide and the atmosphere was replaced with hydrogen three times. The mixture was cooled to 0-5°C in an ice-water bath. 4 g (31.8 mg, 67.3 μmol) of N , N -dimethylformamide solution in 0.5 mL of N,N-dimethylformamide was added, along with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholinium 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 HPLC (separation conditions: chromatography column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give products 5-A and 5-B (3.6mg, 2.6mg).

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 BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO- d ₆ ): δ 8.60(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),7.2 6-7.15(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 BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO- d ₆ ): δ 8.68-8.60(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).

For the preparation of other intermediates, please refer to Intermediate 5.

At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.347 mL, 3.47 μmol) to a PBS-buffered solution of antibody HU1 (7.3 mL, 13.8 mg/mL, 0.681 μmol, pH 6.5, 0.05 M PBS). Place the solution in a shaker waterbath at 37°C for 3 hours to stop the reaction. Cool the reaction solution to 25°C in a waterbath, dilute to 14.0 mL, and remove 3.3 mL of the solution for the next reaction.

Compound 5-A (5.0 mg, 2.75 μmol) was dissolved in 0.15 mL of DMSO and added to the above 3.3 mL solution. The mixture was shaken in a waterbath at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the exemplary product of the Ab-isotecan derivative, HU1-isotecan derivative, in PBS buffer (1.45 mg/mL, 17 mL), which was stored frozen at 4°C.

The average value y was determined using the ultraviolet method. A cuvette containing sodium succinate buffer was placed in the reference and sample absorption cells, respectively. After subtracting the solvent blank, the cuvette containing the test solution was placed in the sample absorption cell and the absorbance at 280 nm and 370 nm was measured.

Data processing:

By establishing a standard curve, the antibody content (Cmab) is determined by measuring the absorbance at a wavelength of 280 nm, and the small molecule content (CDrug) is determined by measuring the absorbance at a wavelength of 370 nm.

Average drug loading value y = CDrug/Cmab.

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

The preparation methods of Compounds 2-11 refer to Compound 1. Similarly, Compound 12 (y=8) was prepared using the same preparation method as Compound 1 and the hRS7 antibody in the prior art:

Example 11 Antibody conjugated to MC-MMAF

In the first step, dissolve S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) in 0.9 mL of acetonitrile for later use. Add the pre-prepared S-(3-hydroxypropyl)thioacetate in acetonitrile to an acetic acid/sodium acetate buffer (10.35 mg/mL, 9.0 mL, 0.97 mol) buffered with the antibody at pH 4.3. Then, add 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) dropwise. The reaction is shaken at 25°C for 2 hours. After the reaction, desalination is performed using a Sephadex G25 gel column (elution phase: 0.05 M PBS, pH 6.5) to obtain a solution of product 1a. This solution is concentrated to 10 mg/mL and directly proceeds to the next step.

In the second step, 0.35 mL of 2.0 M carboxyamide hydrochloride solution was added to solution 1a (11.0 mL). The reaction was shaken at 25°C for 30 minutes. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5) to obtain a solution of product 2a (concentration 6.17 mg/mL, 14.7 mL).

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

Example 12 Antibody Conjugated to SN-38

In the first step, dissolve S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) in 0.9 mL of acetonitrile for later use. Add the pre-prepared S-(3-hydroxypropyl)thioacetate in acetonitrile to an acetic acid/sodium acetate buffer (10.35 mg/mL, 9.0 mL, 0.97 mol) solution of the antibody at pH 4.3. Then, add 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) dropwise. The reaction is shaken at 25°C for 2 hours. After the reaction is complete, desalination is performed using a Sephadex G25 gel column (elution phase: 0.05 M PBS at pH 6.5). The resulting product solution is concentrated to 10 mg/mL and directly used in the next step.

In the second step, 0.35 mL of 2.0 M carboxyamide hydrochloride solution was added to the 1h solution (11.0 mL). The reaction was shaken at 25°C for 30 minutes. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS, pH 6.5). This yielded a 2h solution (6.2 mg/mL, 15.0 mL) that was concentrated to approximately 10 mg/mL for use in the next reaction.

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

Example 13 Antibody-drug conjugates kill tumor cells

To test the tumor cell-killing effects of the antibody-drug conjugates of the present invention, bladder cancer SW780 cells were evaluated. SW780 cells were harvested, counted by centrifugation, and adjusted to a cell density of 0.44 × ^10⁶ cells/mL with complete culture medium. The cells were plated in the center 60 wells of a white 96-well plate, with 90 μL of solution per well and 4,000 cells per well. 100 μL of PBS was added to the remaining wells, and the plate was incubated overnight at 37°C in a 5% CO₂ incubator. On the second day of the experiment, the antibody-drug conjugate solution was prepared in PBS in a 96-well V-bottom plate. The concentration was initially 1000 nM and diluted three-fold to nine different concentrations. After preparation, 10 μL was added to each well of a white 96-well plate in duplicate. The plate was incubated in a 37°C, 5% CO2 incubator for 72 hours. On the fifth day of the experiment, the assay was performed: the plate was removed and equilibrated to room temperature. Then, 50 μL of CTG solution (Promega G7573) was added to each well. After vortexing to mix, the plate was incubated in the dark for 10 minutes before detection using an enzyme-linked microplate reader using the luminescence program. EC50 values were calculated using a four-parameter fitting method. Using the same method as above, the anti-drug conjugates of the present invention were evaluated for their ability to kill bladder cancer cells RT4 and epidermal cancer cells A431. The experimental results are shown in Table 11.

Example 14 Drug Metabolism Kinetics of Antibody-Drug Conjugates

To further study the drug metabolism kinetics of the antibody-drug conjugate in vivo, the antibody-drug conjugate was injected into C57BL/6 mice via intravenous injection (dose of 10 mg/kg). 20 μL of blood was drawn at 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h, 144 h, and 240 h. The concentration of the antibody-drug conjugate in the blood was determined by the ELISA method of Example 3 (1). The drug metabolism kinetics data were analyzed using WinNonlin software to obtain the drug metabolism kinetic parameters, as shown in Table 12 below.

Example 15 In vivo anti-tumor effect of antibody-drug conjugates

To further investigate the anti-tumor activity of the antibody-drug conjugate against in vivo tumors, the anti-tumor efficacy of the antibody-drug conjugate of the present invention was evaluated in mice using N87 xenografts. ^5x10⁶ N87 cells were injected subcutaneously into immunodeficient nude mice. Two weeks later, the antibody-drug conjugates Compound 10 (y=8) and Compound 12 (y=8) were administered intravenously every two weeks at a dose of 2 mg/kg. A control was administered with human IgG1 protein at a dose of 2 mg/kg. Five mice were assigned to each control or treatment group. Tumor inhibition rate was calculated by measuring tumor volume. Tumor inhibition rate = 100% - (tumor volume of the drug group on day 28 - tumor volume of the drug group on day 0) / (tumor volume of the control group on day 28 - tumor volume of the control group on day 0). The experimental results are shown in Table 13:

Compound 10 (y=8) showed a tumor inhibition rate exceeding 100%, indicating that compound 10 (y=8) not only inhibits tumor growth but also has a killing effect on established tumors.

Example 16: Preparation of Exitecan Derivatives

To 2e (4 mg, 7.53 μmol) was added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The atmosphere was replaced with hydrogen three times, cooled in an ice-water bath to 0-5°C, and 0.3 mL of N-methylmorpholine was added dropwise. The reaction mixture was stirred until clear. 2-Cyclopropyl-2-hydroxyacetic acid 1e (2.3 mg, 19.8 μmol, prepared using the method disclosed in patent application WO2013106717), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) were added sequentially. After addition, the mixture 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 mixture was concentrated under reduced pressure, and the resulting crude compound 2-C was purified by HPLC (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (2-A: 1.5mg, 2-B: 1.5mg).

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% (chromatographic 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% (chromatographic 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,1 H),5.58-5.53(m,1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3 .20-3.15(m,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m ,1H),1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H).

Example 17: In vitro proliferation inhibition test of exotecan derivatives on tumor cells

Compounds 2-A and 2-B were tested for their inhibitory activity against the in vitro proliferation of U87MG cells (Cell Bank, Chinese Academy of Sciences, Catalog #TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Catalog #HTB-30). Cells were treated with various concentrations of the compounds in vitro. After 6 days of culture, cell proliferation was assessed using the CTG (Luminescent Cell Viability Assay, Promega, Catalog #G7573) reagent. The in vitro activity of the compounds was evaluated based on IC50 values.

U87MG and SK-BR-3 cells were cultured in EMEM medium containing 10% FBS (GE, cat. no. SH30024.01) and McCoy's 5A medium containing 10% FBS (Gibco, cat. no. 16600-108), respectively.

U87MG and SK-BR-3 cells in logarithmic growth were washed once with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.). The cells were then digested with 2-3 ml of trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) for 2-3 minutes. After complete digestion, 10-15 ml of cell culture medium was added, the digested cells were washed off, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in 10-20 ml of cell culture medium to prepare a single-cell suspension.

Mix the U87MG and SK-BR-3 single cell suspensions and adjust the viable cell density to 2.75 × 103 cells/ml and 8.25 × 103 cells/ml, respectively, using cell culture medium. Mix the adjusted cell suspensions and add 180 μl/well to a 96-well cell culture plate. Add 200 μl of culture medium to the outer wells of the 96-well plate. Incubate the plates in an incubator (37°C, 5% _CO2 ) for 24 hours.

Dissolve the compound in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a stock solution with an initial concentration of 10 mM.

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

Add 30 μl of each test sample to the first column of a 96-well U-bottomed plate at a 100 μM concentration. Add 20 μl of DMSO to each well in columns 2 through 11. Transfer 10 μl of the sample from column 1 to the 20 μl of DMSO in column 2, mix well, then transfer 10 μl to column 3, and so on through column 10. Transfer 5 μl of the drug from each well of the plate to 95 μl of EMEM medium, mix well, and set aside.

Sample addition: Add 20 μl of the test sample of different concentrations to the culture plate, with each sample plated in duplicate. Incubate the culture plate in an incubator for 6 days (37°C, 5% CO ₂ ).

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

Plate reading: Remove the 96-well cell culture plate and place it in an enzyme-labeled analyzer (BMG labtech, PHERAstar FS). Chemiluminescence is measured using an enzyme-labeled analyzer.

Data analysis: Data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in Table 14.

<110> Shanghai Hansoh BioMedical Co., Ltd. Jiangsu Hansoh Pharmaceutical Group Co., Ltd.

<120> Antibody-drug conjugates and their medical uses

<130> 721027CPCT

<150> 2020102153204

<151> 2020-03-24

<160> 50

<170> SIPOSequenceListing 1.0

<210> 1

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> Heavy chain variable region of mouse monoclonal antibody M1

<400> 1

<210> 2

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> Light chain variable region of mouse monoclonal antibody M1

<400> 2

<210> 3

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> Structural Domain

<222> (1)..(5)

<223> HCDR1 of mouse monoclonal antibody M1

<400> 3

<210> 4

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> Structural Domain

<222> (1)..(17)

<223> HCDR2 of mouse monoclonal antibody M1

<400> 4

<210> 5

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> Structural Domain

<222> (1)..(12)

<223> HCDR3 of mouse monoclonal antibody M1

<400> 5

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> Structural Domain

<222> (1)..(11)

<223> LCDR1 of mouse monoclonal antibody M1

<400> 6

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> Structural Domain

<222> (1)..(7)

<223> LCDR2 of mouse monoclonal antibody M1

<400> 7

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> Structural Domain

<222> (1)..(9)

<223> LCDR3 of mouse monoclonal antibody M1

<400> 8

<210> 9

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> HU1 HCVR

<400> 9

<210> 10

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU1 LCVR

<400> 10

<210> 11

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> HU2 HCVR

<400> 11

<210> 12

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU2 LCVR

<400> 12

<210> 13

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> HU3 HCVR

<400> 13

<210> 14

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU3 LCVR

<400> 14

<210> 15

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> HU4 HCVR

<400> 15

<210> 16

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU4 LCVR

<400> 16

<210> 17

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> HU5 HCVR

<400> 17

<210> 18

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU5 LCVR

<400> 18

<210> 19

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> HU6 HCVR

<400> 19

<210> 20

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU6 LCVR

<400> 20

<210> 21

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> HU7 HCVR

<400> 21

<210> 22

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU7 LCVR

<400> 22

<210> 23

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223>HU8 HCVR

<400> 23

<210> 24

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU8 LCVR

<400> 24

<210> 25

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(121)

<223> HU9 HCVR

<400> 25

<210> 26

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> HU9 LCVR

<400> 26

<210> 27

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU1 HC

<400> 27

<210> 28

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223> HU1 LC

<400> 28

<210> 29

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU2 HC

<400> 29

<210> 30

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223> HU2 LC

<400> 30

<210> 31

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU3 HC

<400> 31

<210> 32

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223> HU3 LC

<400> 32

<210> 33

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU4 HC

<400> 33

<210> 34

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223>HU4 LC

<400> 34

<210> 35

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU5 HC

<400> 35

<210> 36

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223> HU5 LC

<400> 36

<210> 37

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU6 HC

<400> 37

<210> 38

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223> HU6 LC

<400> 38

<210> 39

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU7 HC

<400> 39

<210> 40

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223> HU7 LC

<400> 40

<210> 41

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU8 HC

<400> 41

<210> 42

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223> HU8 LC

<400> 42

<210> 43

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(43)

<223> HU9 HC

<400> 43

<210> 44

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(214)

<223> HU9 LC

<400> 44

<210> 45

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU6DL HC

<400> 45

<210> 46

<211> 331

<212> PRT

<213> Artificial Sequence

<220>

<221> Peptide

<222> (1)..(331)

<223> His-tagged human TROP-2

<400> 46

<210> 47

<211> 451

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(451)

<223> HU10 HC

<400> 47

<210> 48

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(330)

<223> IgG1 C1

<400> 48

<210> 49

<211> 330

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(330)

<223> IgG1 C2

<400> 49

<210> 50

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> Link

<222> (1)..(107)

<223> Ig kappa C

<400> 50

Claims (35)

An antibody-drug conjugate represented by the general formula (A) or a pharmaceutically acceptable salt thereof, Ab-(L ₂ -L ₁ -D) _y (A) wherein D is a cytotoxic drug; L ₁ is selected from -O-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-, -O-CR ⁵ R ⁶ -(CR ^a R ^b ) _m -, -O-CR ⁵ R ⁶ -, -NH-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-, or -S-(CR ^a R ^b ) _m -CR ⁵ R ⁶ -C(O)-; R ^a and R ^R and R 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 hydroxyl alkyl group, a cycloalkyl group, or a heterocyclic group; or, ^Ra and ^R together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group; R ^is selected from a halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group; or, R and R are ^each independently selected from a hydrogen atom, a halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, or a ^heteroaryl group; ⁶ together with the carbon atom to which it is attached form a cycloalkyl or heterocyclic group; or, R ^a and R ⁶ together with the carbon atom to which it is attached form a cycloalkyl or heterocyclic group; m is an integer selected from 0 to 4; y is a number selected from 1 to 10, y is a decimal or an integer; L ₂ is a bridging unit, as shown in the general formula (D): -K ¹ -K ² -K ³ -K ⁴ - (D) wherein 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; R ¹ is selected from hydrogen, deuterium, hydroxyl, amine, alkyl, halogen, halogenated alkyl, deuterated alkyl and hydroxyalkyl; K ³ is a tetrapeptide residue; K ⁴ is -NR ² (CR ³ R ⁴ )t-, R ² , R ³ or R ⁴ are each independently hydrogen, deuterium, hydroxyl, amine, alkyl, halogen, halogenated alkyl, and hydroxyalkyl, and t is selected from 1 or 2; Ab is an anti-TROP-2 antibody or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising HCDR1 set forth in SEQ ID NO: 3, HCDR2 set forth in SEQ ID NO: 4, and HCDR3 set forth in SEQ ID NO: 5; and the light chain variable region comprises LCDR1 set forth in SEQ ID NO: 6, LCDR2 set forth in SEQ ID NO: 7, and LCDR3 set forth in SEQ ID NO: 8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof is selected from a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3, or IgG4, or a variant thereof, and the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody κ chain, or a variant thereof. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein 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, and the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO: 50. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of 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, or a light chain variable region selected from the group consisting of 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. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 5, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region set forth in SEQ ID NO: 9 and a light chain variable region set forth in SEQ ID NO: 10; or, a heavy chain variable region set forth in SEQ ID NO: 11 and a light chain variable region set forth in SEQ ID NO: 12; or, a heavy chain variable region set forth in SEQ ID NO: 13 and a light chain variable region set forth in SEQ ID NO: 14; or, a heavy chain variable region set forth in SEQ ID NO: 15 and a light chain variable region set forth in SEQ ID NO: 16; or, a heavy chain variable region set forth in SEQ ID NO: 17 and a light chain variable region set forth in SEQ ID NO: 18; or, a heavy chain variable region set forth in SEQ ID NO: 19 and a light chain variable region set forth 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. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 5, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of 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 a light chain selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 7, wherein the anti-TROP-2 antibody comprises: the heavy chain of SEQ ID NO: 27 and the light chain of SEQ ID NO: 28; or the heavy chain of SEQ ID NO: 29 and the light chain of SEQ ID NO: 30; or the heavy chain of SEQ ID NO: 31 and the light chain of SEQ ID NO: 32; or the heavy chain of SEQ ID NO: 33 and the light chain of SEQ ID NO: 34; or the heavy chain of SEQ ID NO: 35 and the light chain of SEQ ID NO: 36; or the heavy chain of SEQ ID NO: 37 and the light chain of SEQ ID NO: 38; or the heavy chain of SEQ ID NO: 39 and the light chain of SEQ ID NO: 40; or the heavy chain of SEQ ID NO: 41 and the light chain of 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. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 8, wherein L ₁ is represented by the general formula (E): wherein R ⁵ is a halogen group or a cycloalkyl group, and R ⁶ is selected from hydrogen, a halogen group or a cycloalkyl group; or, R ⁵ and R ⁶ together with the carbon atom to which they are connected form a cycloalkyl group. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 9, wherein: ^R5 is selected from _C1-6 haloalkyl or _C3-6 cycloalkyl, ^R6 is selected from hydrogen, _C1-6 haloalkyl or _C3-6 cycloalkyl, or, ^R5 and ^R6 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl; m is selected from an integer from 0 to 4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 9, wherein: the general formula (E) is selected from the following substituents: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 8, wherein: p is selected from an integer from 1 to 6; and the tetrapeptide residue is selected from a peptide residue formed by two or more amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate, and aspartic acid. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 12, wherein: the tetrapeptide residue is a tetrapeptide residue of GGFG. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 8, wherein the bridge unit L ₂ is connected to Ab at its K ¹ end and to L ₁ at its K ⁴ end. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 8, wherein -L ₂ -L ₁ - is selected from the following structures: wherein ^K2 is a bond; ^K3 is a tetrapeptide residue of GGFG; ^R5 is selected from a haloalkyl group or a _C3-6 cycloalkyl group; ^R6 is selected from a hydrogen group, a haloalkyl group or a _C3-6 cycloalkyl group; or, ^R5 and ^R6 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group; ^R2 , ^R3 or ^R4 are each independently selected from a hydrogen group or an alkyl group; s is selected from an integer from 2 to 8; and m is selected from an integer from 0 to 4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 15, wherein the -L ₂ -L ₁ - is selected from the following structures: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 8, wherein the cytotoxic drug is selected from toxins, chemotherapeutic drugs, antibiotics, radioactive isotopes and nucleolytic enzymes. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 17, wherein the cytotoxic drug is selected from: a microtubule inhibitor or a DNA topoisomerase inhibitor that inhibits cell division. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 18, wherein the cytotoxic drug is selected from: camptothecin derivatives, DM1, DM3, DM4, MMAF or MMAE. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 19, wherein the cytotoxic drug is selected from: exotecan, an exotecan derivative, SN-38, MMAE or MMAF. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 20, wherein the cytotoxic drug is selected from the following structures: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 21, wherein the antibody-drug conjugate is represented by the general formula (III): wherein L ₁ and L ₂ are bridging units; y is a number from 1 to 10; and Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 22, wherein y is a number from 2 to 8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 22, wherein y is a number from 4 to 8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 22, wherein the antibody-drug conjugate is represented by the general formula (IV): wherein W is selected from C _1-8 alkyl, C _1-8 alkyl-cycloalkyl or a straight-chain heteroalkyl group of 1 to 8 atoms, the heteroalkyl group containing 1 to 3 heteroatoms selected from N, O or S, wherein the C _1-8 alkyl, cycloalkyl and straight-chain heteroalkyl groups are each independently optionally further substituted with one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; K ² is selected from -NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ CH ₂ C (O) -, -NR ¹ (CH ₂ CH ₂ O) _p1 CH ₂ C (O) -, -S (CH ₂ ) _p1 C (O) - or a bond; R ¹ is selected from a hydrogen atom, an alkyl, a halogenated alkyl, a deuterated alkyl and a hydroxyl alkyl; p ₁ is an integer from 1 to 20; K ³ is selected from a peptide residue consisting of 2 to 7 amino acids, which may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point, and the substituent is one or more independently selected from halogen, hydroxyl, cyano, amine, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; R ² is independently selected from hydrogen atom, alkyl, halogenalkyl, deuterated alkyl and hydroxyalkyl; R ³ and R ⁴ are each independently selected from hydrogen atom, halogen, alkyl, halogenalkyl, deuterated alkyl and hydroxyalkyl; R ⁵ is selected from halogen, halogenalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; R ^{R 6} is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, or a heteroaryl group; or, 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 selected from a number from 1 to 10, and y is a decimal or an integer; Ab is an anti-TROP-2 antibody or an antigen-binding fragment thereof. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 25, wherein the antibody-drug conjugate is represented by the general formula (IV-A): The general formula (IV-A) is selected from the following structures: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 1, which is selected from the following compounds: Where y is selected from 2-10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 27, wherein y is selected from 4-8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 27, wherein y is selected from 6-8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 27, wherein y is selected from 7-8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 27, wherein y is selected from 8. A pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt of the antibody-drug conjugate as described in any one of claims 1 to 31, and one or more pharmaceutically acceptable excipients, diluents or carriers. Use of the antibody-drug conjugate according to any one of claims 1 to 31 or the pharmaceutical composition according to claim 32 in the preparation of a medicament for treating a human TROP-2-related disease. The use of claim 33, wherein the disease associated with human TROP-2 is a cancer with high expression of TROP-2, and the cancer is selected from urothelial carcinoma, human brain astrocytoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, Breast cancer, carotid artery tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, fibrofibrous dysplasia, fibrofibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, Germ cell carcinoma, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma, malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myeloproliferative syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, Pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, renal metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, thyroid metastatic carcinoma, and uterine cancer. The use as described in claim 34, wherein the breast cancer is triple-negative breast cancer and the lung cancer is non-small cell lung cancer.