Antibody-drug conjugates and their medical uses

Abstract

The invention relates to an antibody-drug conjugate and its medical use. Specifically, it relates to an anti-TROP-2 antibody-drug conjugate and its medical use. Further, it relates to an antibody-drug conjugate comprising an anti-TROP-2 antibody or an antigen-binding fragment thereof or a pharmaceutically acceptable salt or solvent compound thereof, and its use in preparing a drug for treating a TROP-2-mediated disease or condition and its use in tumor detection and diagnosis.

Description

Technical Field

[0001] The present invention relates to an anti-TROP-2 antibody-drug conjugate specifically having immunoreactivity to human TROP-2 receptor and a pharmaceutical composition thereof, as well as the use of the conjugate as an anticancer drug and for detecting or diagnosing tumors. Background Art

[0002] With the continuous deepening of research on tumor genomics, proteomics and signal transduction pathways, people have become increasingly clear about the interaction between oncogenes and tumor suppressor genes in tumor cells and their impact on the tumor microenvironment, which also makes it possible to design new anti-tumor treatment plans targeting specific molecular targets of tumors.

[0003] Molecularly targeted cancer 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 target sites, killing or inhibiting target cells by directly affecting the function of the target molecule or by the physical or chemical effector molecules it carries. Because of their well-defined targets, these drugs are typically highly selective, effectively killing or inhibiting target cells while producing minimal or no toxic side effects on normal tissue cells. Consequently, the development of molecularly targeted drugs has become a hot topic in clinical cancer research.

[0004] Human trophoblast cell surface antigen 2 (TROP-2) is a cell surface glycoprotein encoded by the TACSTD2 gene. TROP-2 consists of 323 amino acids, including 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 TROP-2 extracellular domain 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 has a characteristic thyroglobulin (TY) sequence, which is generally believed to be involved in cancer cell proliferation, invasion, and metastasis.

[0005] So far, the physiological ligand of TROP-2 has not been identified and its molecular function has not been elucidated. However, its intracellular serine residue 303 (S303) can be activated by Ca 2+ Trop2 is phosphorylated by the action of protein kinase C (PKC), which in turn promotes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to form inositol triphosphate IP3 related to the mitogen-activated protein kinase pathway (MAPK). This signaling pathway is closely related to cell proliferation, suggesting that Trop2 has the function of mediating signal transduction in tumor cells.

[0006] 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 epithelial cells, where it is expressed at lower levels than in cancer, suggesting a role for TROP-2 in tumorigenesis. Overexpression of TROP-2 in tumor tissue is closely associated with poor patient prognosis and metastasis, and also impacts overall survival. Therefore, TROP-2 has become a compelling target for molecularly targeted cancer therapy.

[0007] Several studies have reported the anti-tumor effects of anti-hTROP-2 antibodies:

[0008] US Patent No. 5,840,854 reports the cytotoxicity of an anti-hTROP-2 monoclonal antibody (BR110) conjugated with a cytotoxin against human cancer cell lines H3619, H2987, MCF-7, H3396, and H2981.

[0009] US Patent No. 6,653,104 discloses an antibody (RS7) that was tested in an in vivo model using a radioactively labeled antibody and showed antitumor activity in a nude mouse xenograft model. However, no antitumor effect of the naked antibody alone was reported.

[0010] US Patent No. 7,420,040 also reports that isolated monoclonal antibodies produced by hybridoma cell lines AR47A6.4.2 or AR52A301.5 obtained by immunizing mice with human ovarian cancer tissue bind to hTROP-2 and show anti-tumor activity in a nude mouse xenograft model.

[0011] CN102827282A discloses a humanized anti-TROP-2 genetically engineered antibody IgG and its application. In vitro test results show that the anti-TROP-2 antibody IgG has a significant inhibitory effect on the proliferation of pancreatic cancer cells.

[0012] 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 hybridoma that produces the antibody, a complex of the antibody and a pharmaceutical agent, a pharmaceutical composition for diagnosing or treating tumors, a method for detecting tumors, and a kit for detecting or diagnosing tumors. Summary of the Invention

[0013] 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 general formula (A):

[0014] Ab-(L2-L1-D) y

[0015] (A)

[0016] in:

[0017] D is a cytotoxic drug;

[0018] L1 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)-;

[0019] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group;

[0020] Or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0021] R 5 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;

[0022] R 6 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group or a heteroaryl group;

[0023] Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0024] Or, R aand R 6 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0025] m is an integer selected from 0 to 4;

[0026] y is a number selected from 1 to 10, and y is a decimal or an integer;

[0027] L2 is the connector unit;

[0028] Ab is an anti-TROP-2 antibody or an antigen-binding fragment thereof, which comprises an antibody light chain variable region and an antibody heavy chain variable region, 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; 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.

[0029] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof, wherein the antibody heavy chain variable region comprises:

[0030] HCDR1 shown in SEQ ID NO: 3,

[0031] HCDR2 shown in SEQ ID NO: 4 and

[0032] HCDR3 shown in SEQ ID NO:5.

[0033] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof, wherein the antibody light chain variable region comprises:

[0034] LCDR1 shown in SEQ ID NO: 6,

[0035] LCDR2 shown in SEQ ID NO: 7 and

[0036] LCDR3 shown in SEQ ID NO:8.

[0037] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof, wherein the antibody heavy chain variable region comprises:

[0038] HCDR1 shown in SEQ ID NO: 3,

[0039] HCDR2 shown in SEQ ID NO: 4 and

[0040] HCDR3 shown in SEQ ID NO:5; and

[0041] The antibody light chain variable region comprises:

[0042] LCDR1 shown in SEQ ID NO: 6,

[0043] LCDR2 shown in SEQ ID NO: 7 and

[0044] LCDR3 shown in SEQ ID NO:8.

[0045] In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof is selected from 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.

[0046] In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

[0047] 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.

[0048] 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.

[0049] In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody κ chain, λ chain, or a variant thereof.

[0050] 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;

[0051] 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.

[0052] In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof 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 heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequences.

[0053] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of 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, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared 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.

[0054] In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof contains 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, or a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity to the following sequences: SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, or SEQ ID NO: 47.

[0055] In a preferred embodiment of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the anti-TROP-2 antibody or antigen-binding fragment thereof contains 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, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity to the following sequences: SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44.

[0056] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises:

[0057] (1) the heavy chain variable region set forth in SEQ ID NO: 9 and the light chain variable region set forth in SEQ ID NO: 10; or

[0058] (2) the heavy chain variable region set forth in SEQ ID NO: 11 and the light chain variable region set forth in SEQ ID NO: 12; or

[0059] (3) the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 14; or

[0060] (4) the heavy chain variable region set forth in SEQ ID NO: 15 and the light chain variable region set forth in SEQ ID NO: 16; or

[0061] (5) the heavy chain variable region set forth in SEQ ID NO: 17 and the light chain variable region set forth in SEQ ID NO: 18; or

[0062] (6) the heavy chain variable region set forth in SEQ ID NO: 19 and the light chain variable region set forth in SEQ ID NO: 20; or

[0063] (7) the heavy chain variable region set forth in SEQ ID NO: 21 and the light chain variable region set forth in SEQ ID NO: 22; or

[0064] (8) the heavy chain variable region set forth in SEQ ID NO: 23 and the light chain variable region set forth in SEQ ID NO: 24; or

[0065] (9) The heavy chain variable region shown in SEQ ID NO: 25 and the light chain variable region shown in SEQ ID NO: 26.

[0066] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof, wherein the anti-TROP-2 antibody comprises:

[0067] (1) the heavy chain shown in SEQ ID NO: 27 and the light chain shown in SEQ ID NO: 28; or,

[0068] (2) the heavy chain shown in SEQ ID NO: 29 and the light chain shown in SEQ ID NO: 30; or,

[0069] (3) the heavy chain shown in SEQ ID NO: 31 and the light chain shown in SEQ ID NO: 32; or

[0070] (4) the heavy chain shown in SEQ ID NO: 33 and the light chain shown in SEQ ID NO: 34; or

[0071] (5) the heavy chain shown in SEQ ID NO: 35 and the light chain shown in SEQ ID NO: 36; or

[0072] (6) the heavy chain shown in SEQ ID NO: 37 and the light chain shown in SEQ ID NO: 38; or

[0073] (7) the heavy chain shown in SEQ ID NO: 39 and the light chain shown in SEQ ID NO: 40; or

[0074] (8) the heavy chain shown in SEQ ID NO: 41 and the light chain shown in SEQ ID NO: 42; or

[0075] (9) the heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 44; or

[0076] (10) the heavy chain shown in SEQ ID NO: 45 and the light chain shown in SEQ ID NO: 38; or

[0077] (11) The heavy chain represented by SEQ ID NO: 47 and the light chain represented by SEQ ID NO: 28.

[0078] In some embodiments of the present invention, according to the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, L1 is represented by general formula (B):

[0079]

[0080] in,

[0081] M1 is -CR1R2-;

[0082] R1 and R2 are the same or different, and R1 and R2 are each independently selected from hydrogen, alkyl, halogen, hydroxyl or amino;

[0083] n is an integer selected from 0 to 5, and preferably 1, 2 or 3.

[0084] In some embodiments of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, L2 is represented by general formula (C):

[0085]

[0086] in,

[0087] M2 is -CR4R5-;

[0088] R3 is selected from hydrogen, halogen, hydroxy, amino, alkyl, alkoxy and cycloalkyl:

[0089] R4 and R5 are the same or different and are independently selected from hydrogen, alkyl, halogen, hydroxyl or amino;

[0090] m is an integer selected from 0 to 5, and preferably 1, 2 or 3.

[0091] In a further preferred embodiment of the present invention, the O end of L1 is connected to the linker unit L2.

[0092] In a further preferred embodiment of the present invention, the O end of L1 is connected to the S end of the linker unit L2.

[0093] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof, wherein:

[0094] L1 is -O-(CR a R b ) m -CR 5 R 6 -C(O)-;

[0095] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;

[0096] R 5 is a halogenated alkyl or C 3-6 Cycloalkyl;

[0097] R 6 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl;

[0098] Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0099] m is selected from 0 or 1.

[0100] In a preferred embodiment of the present invention, according to the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof of the present invention, the L1 is represented by the general formula (E):

[0101]

[0102] Among them, R 5 is a haloalkyl group or a cycloalkyl group,

[0103] R 6 is selected from hydrogen, haloalkyl or cycloalkyl,

[0104] Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms a cycloalkyl group;

[0105] Preferably,

[0106] R 5 Selected from C 1-6 Haloalkyl or C 3-6 Cycloalkyl,

[0107] R 6 Selected from hydrogen, C 1-6 Haloalkyl or C 3-6 Cycloalkyl,

[0108] Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0109] m is selected from integers of 0 to 4.

[0110] In a further preferred embodiment of the present invention, the general formula (E) is selected from the following substituents:

[0111]

[0112] In some embodiments of the present invention, according to the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, L2 is represented by the following general formula (D):

[0113] -K 1 -K 2 -K 3 -K 4 -

[0114] (D)

[0115] in,

[0116] K 1 for s is an integer selected from 2 to 8;

[0117] K 2 Selected from-NR 1 (CH2CH2O) p CH2CH2C(O)-、-NR 1 (CH2CH2O) p CH2C(O)-、-S(CH2) p C(O)- or a single bond, p is selected from an integer from 1 to 20, preferably an integer from 1 to 6;

[0118] R 1 is selected from the group consisting of hydrogen, deuterium, hydroxy, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl;

[0119] K 3 It is a tetrapeptide residue, preferably, the tetrapeptide residue is selected from a peptide residue formed by two or more amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; more preferably, it is a tetrapeptide residue of GGFG;

[0120] K 4 -NR 2 (CR 3 R 4 )t-,R 2 、R 3 or R 4 Each is independently hydrogen, deuterium, hydroxy, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl, and t is selected from 1 or 2.

[0121] 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, wherein the linker unit -L2-, its K 1 The end is connected to Ab, K 4 The end is connected to L1.

[0122] 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, wherein -L2-L1- is the following structure:

[0123]

[0124] Among them, K 2 is the key;

[0125] K3 is the tetrapeptide residue of GGFG;

[0126] R 5 Selected from haloalkyl or C 3-6 Cycloalkyl;

[0127] R 6 Selected from hydrogen, haloalkyl or C 3-6 Cycloalkyl;

[0128] Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0129] R 2 、R 3 or R 4 are each independently selected from hydrogen or alkyl;

[0130] s is an integer selected from 2 to 8;

[0131] m is selected from integers of 0 to 4.

[0132] In a further preferred embodiment of the present invention, the -L2-L1- is selected from the following structures:

[0133]

[0134]

[0135] In some embodiments of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug is selected from toxins, chemotherapeutic drugs, antibiotics, radioisotopes and nucleolytic enzymes.

[0136] In a preferred embodiment of the present invention, the cytotoxic drug is selected from microtubule inhibitors or DNA topoisomerase inhibitors that inhibit cell division; preferably camptothecin derivatives, DM1, DM3, DM4, SN-38, MMAF or MMAE; more preferably exotecan or exotecan derivatives, SN-38, MMAE or MMAF.

[0137] In a preferred embodiment of the present invention, the cytotoxic drug is selected from:

[0138]

[0139] In a preferred embodiment of the present invention, the cytotoxic drug is selected from exotecan derivatives, preferably, the exotecan derivative is compound 2-A:

[0140]

[0141] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent thereof is a compound represented by general formula (I) or a pharmaceutically acceptable salt or solvent thereof:

[0142]

[0143] Among them, L1 and L2 are linker units;

[0144] y is a number selected from 1 to 10, preferably a number from 2 to 8, more preferably a number from 2 to 4;

[0145] The Ab is selected from the TROP-2 antibodies or antigen-binding fragments thereof as described above.

[0146] In a preferred embodiment of the present invention, the general formula (I) is as shown in general formula (IA):

[0147]

[0148] wherein L2 is as defined above; preferably, L2 is as defined above in general formula (C).

[0149] In a preferred embodiment of the present invention, the general formula (I) is as shown in the general formula (IB):

[0150]

[0151] wherein L1 is as defined above; preferably, L2 is as defined above in general formula (B).

[0152] In some embodiments of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent thereof is a compound represented by general formula (II) or a pharmaceutically acceptable salt or solvent thereof:

[0153]

[0154] Among them, L1 and L2 are linker units;

[0155] y is a number selected from 1 to 10, preferably a number from 2 to 8, more preferably a number from 2 to 4;

[0156] Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof as described above;

[0157] In a preferred embodiment of the present invention, the general formula (II) is as shown in general formula (II-A):

[0158]

[0159] wherein L2 is as defined above; preferably, L2 is as defined above in general formula (C).

[0160] In a preferred embodiment of the present invention, the general formula (II) is as shown in general formula (II-B):

[0161]

[0162] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent thereof is a compound represented by general formula (III) or a pharmaceutically acceptable salt or solvent thereof:

[0163]

[0164] Among them, L1 and L2 are linker units;

[0165] y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 4 to 8;

[0166] Ab is selected from the aforementioned TROP-2 antibody or antigen-binding fragment thereof.

[0167] In a further preferred embodiment of the present invention, the antibody-drug conjugate described in general formula (III) or a pharmaceutically acceptable salt or solvent compound thereof, wherein L1 is as defined in general formula (E) and L2 is as defined in general formula (D).

[0168] In a further preferred embodiment of the present invention, the antibody-drug conjugate described in general formula (III) or a pharmaceutically acceptable salt or solvent compound thereof, wherein the linker unit -L2-, its K1 end is connected to Ab, and its K4 end is connected to L1.

[0169] In a further preferred embodiment of the present invention, in the antibody-drug conjugate of general formula (III) or a pharmaceutically acceptable salt or solvent thereof, the -L2-L1- is selected from the following structures:

[0170]

[0171] K 2 is the key;

[0172] K 3 is the tetrapeptide residue of GGFG;

[0173] R 5 Selected from haloalkyl or C 3-6 Cycloalkyl;

[0174] R 6 Selected from hydrogen, haloalkyl or C 3-6 Cycloalkyl;

[0175] Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0176] R 2 、R 3 or R 4 are each independently hydrogen or alkyl;

[0177] s is an integer selected from 2 to 8;

[0178] m is an integer selected from 0 to 4;

[0179] Preferably, -L2-L1- is selected from the following structures:

[0180]

[0181]

[0182] In a preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to the present invention is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof represented by general formula (IV):

[0183]

[0184] in,

[0185] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0186] K 2 Selected from-NR 1 (CH2CH2O) p1 CH2CH2C(O)-、-NR 1 (CH2CH2O) p1 CH2C(O)-、-S(CH2) p1 C(O)- or bond, R 1 is selected from the group consisting of 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;

[0187] K 3A peptide residue selected from 2 to 7 amino acids, wherein the amino acids may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available attachment point, wherein the substituent is one or more independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0188] R 2 are independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0189] R 3 and R 4 are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl;

[0190] R 5 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;

[0191] R 6 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0192] Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0193] m is an integer selected from 0 to 4;

[0194] y is a number selected from 1 to 10, and y is a decimal or an integer;

[0195] Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof described in the present invention.

[0196] In a further preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent thereof is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent thereof represented by the general formula (IV-A):

[0197]

[0198] In a further preferred embodiment of the present invention, the general formula (IV-A) is selected from the following structures:

[0199]

[0200]

[0201]

[0202] In some embodiments of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent thereof is selected from the following compounds:

[0203]

[0204]

[0205] Wherein Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof described in the present invention; preferably, Ab is selected from HU1-HU10 and HU6DL of the present invention.

[0206] In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent thereof is selected from the following compounds:

[0207]

[0208]

[0209]

[0210]

[0211] Wherein, y is selected from 2-10, preferably 4-8, more preferably 6-8, further preferably 7-8, and most preferably 8, and y is a decimal or an integer.

[0212] 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:

[0213]

[0214] After Ab is reduced, it is coupled with the compound of formula (F) to obtain the compound of formula (IV);

[0215] wherein Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof as described above;

[0216] W, K 2 , K 3 、R 2 ~R 6 , m and y are as defined in the general formula (IV).

[0217] In a preferred embodiment of the present invention, according to the preparation method of the general formula (IV) of the present invention, the general formula (F) is a compound represented by the general formula (F-1) or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

[0218]

[0219] Among them, K 2 , K 3 、R 2 ~R 6 , s and m are as defined in the general formula (IV).

[0220] In some embodiments of the present invention, the compound represented by general formula (F) or general formula (F-1) according to the present invention is selected from:

[0221]

[0222]

[0223] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0224] 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.

[0225] In a more preferred embodiment of the present invention, the disease associated with human TROP-2 is the use of the invention in the preparation of a medicament for treating a cancer in which TROP-2 is highly expressed, wherein the cancer is selected from triple-negative breast cancer, small cell lung cancer, urothelial carcinoma, human brain astrocytoblastoma, 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 body tumor, 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 of bone, gallbladder or bile duct carcinoma. carcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, 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, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic renal cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, thymoma, metastatic thyroid cancer, and uterine cancer.

[0226] 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 endocytosis efficiency, and a long half-life in vivo, and can significantly kill tumors while ensuring safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0227] Figure 1 : Antibody ELISA in vitro binding assay showed the binding activity of 11 humanized anti-TROP-2 antibodies to human TROP-2 antigen. DETAILED DESCRIPTION Detailed Description of the Invention

[0229] 1. Terminology

[0230] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0231] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).

[0232] 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 order of the constant region of the heavy chains 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 their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class, Ig can be further divided into subclasses based on the amino acid composition of the hinge region and the number and location of 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 based on differences in the constant region. Each of the five classes of Ig can have either kappa or lambda chains.

[0233] 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 murine κ, λ chain or a variant thereof.

[0234] 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.

[0235] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as 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 of the present invention conform to the known Kabat numbering convention and Kabat or ABM definition rules (http: / / bioinf.org.uk / abs / ).

[0236] The term "antigen presenting cell" or "APC" is a cell that displays a foreign antigen in complex with MHC on its surface. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.

[0237] 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.

[0238] 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 obtained from non-human species. Alternatively, the antibody 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 that naturally express them, or produced by recombinant technology using techniques commonly used in the art and those described herein. Preferably, the anti-TROP-2 antibody targets human TROP-2 with a normal glycosylation pattern.

[0239] The term "recombinant human antibody" includes human antibodies prepared, expressed, created or isolated by recombinant means, and the techniques and methods involved are well known in the art, such as:

[0240] 1. Antibodies isolated from transgenic or transchromosomal animals (e.g., mice) expressing human immunoglobulin genes or hybridomas prepared therefrom;

[0241] 2. Antibodies isolated from host cells transformed to express the antibody, such as transfectomas;

[0242] 3. Antibodies isolated from a recombinant combinatorial human antibody library; and

[0243] 4. Antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.

[0244] Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by the germline genes, but also include subsequent rearrangements and mutations that occur, such as during antibody maturation.

[0245] 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. This antibody is prepared by injecting a test subject with a TROP-2 antigen, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional properties. In a preferred embodiment of the present invention, the murine TROP-2 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0246] The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences (i.e., "humanized antibodies").

[0247] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework. Humanized antibodies can overcome the disadvantage of chimeric antibodies, which often induce strong immune responses due to the presence of large amounts of mouse protein components. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the human antibody variable region to maintain activity.

[0248] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, it is necessary to select a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed. The mouse variable region genes and the human constant region genes are connected to form a chimeric gene and inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or using an IgG1 heavy chain constant region whose toxicity is enhanced by amino acid mutations in ADCC (antibody-dependent cell-mediated cytotoxicity).

[0249] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody 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, an 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.

[0250] A "Fab fragment" consists of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0251] The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.

[0252] A "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH domain and the CH1 domain as well as the region between the CH1 and CH2 domains, such that an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0253] A "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, such that an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0254] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0255] The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has 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 domains, each binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that are covalently or non-covalently linked together, etc.

[0256] 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 connecting peptide. It is the smallest antibody fragment with a complete antigen binding site.

[0257] The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.

[0258] The term "binding to TROP-2" in the present invention means being able to interact with human TROP-2.

[0259] The term "antigen binding site" of the present invention refers to the three-dimensional site recognized by the antibody or antigen-binding fragment of the present invention.

[0260] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by the 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 generally 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 in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0261] The terms "specific binding" and "selective binding" used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human TROP-2 is used as an analyte and an antibody is used as a ligand, the antibody binds to the epitope at a specific binding rate of less than 10 -7M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen, and its affinity for binding to the predetermined antigen is at least twice that of its affinity for binding to a nonspecific antigen (such as BSA, etc.) other than the predetermined antigen or a closely related antigen. The term "antibody that recognizes an antigen" is used interchangeably with the term "specifically binding antibody" herein.

[0262] The term "cross-reactivity" refers to the ability of an antibody of the present invention to bind to TROP-2 from different species. For example, an antibody of the present invention that binds to human TROP-2 may also bind to TROP-2 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens 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.

[0263] 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 blocking. 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.

[0264] The term "inhibit growth" (eg, with respect to cells) is intended to include any measurable decrease in cell growth.

[0265] The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to the stimulation of an immune response (ie, passive or adaptive) to a specific antigen. The term "induce" with respect to inducing CDC or ADCC refers to the stimulation of specific direct cell killing mechanisms.

[0266] The term "ADCC," or antibody-dependent cell-mediated cytotoxicity, as used herein, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the antibody's Fc region. Modification of the Fc region of IgG can enhance, reduce, or eliminate the antibody's ADCC effector function. Such modification involves mutations in the heavy chain constant region of the antibody.

[0267] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques, Chapters 5-8 and 15. For example, mice can be immunized with human TROP-2 or fragments thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in the present invention are engineered to have one or more human FR regions added to the non-human CDR regions using genetic engineering methods. 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.

[0268] The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended 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 clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones 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 by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product must be immediately frozen, such as at -70°C, or freeze-dried.

[0269] The antibodies of the present invention are monoclonal antibodies. Monoclonal antibodies (mAbs) described herein are antibodies derived from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clones. Monoclonal antibodies or antigen-binding fragments can be recombinantly obtained using hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR-grafting), or other existing technologies.

[0270] "Administer," "give," and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0271] "Treatment" means administering an internal or external therapeutic agent, such as any of the antibodies of the present invention, to a patient who has one or more symptoms of a disease, and it is known that the therapeutic agent has a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent that effectively alleviates any specific 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, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. While an embodiment of the invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in every patient, it should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0272] The term "consisting essentially of" or variations thereof as used throughout the specification and claims means that all recited elements or groups of elements are included, and optionally other elements of similar or different properties from the recited elements, which do not significantly alter the basic or novel properties of a specified dosage regimen, method, or composition.

[0273] As used herein, the term "naturally occurring" as applied to an object 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 source in nature and that has not been intentionally modified by man in a laboratory is naturally occurring.

[0274] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or signs 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 overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0275] "Exogenous" refers to substances produced outside the body of an organism, cell, or human body, depending on the context.

[0276] "Endogenous" refers to substances that are produced within a cell, organism, or human body on a background basis.

[0277] "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 percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, 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 speaking, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0278] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny that possess the same function or biological activity as that screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.

[0279] "Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and 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 the antibody heavy chain variable region of the specified sequence can but need not be present.

[0280] 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 excipients. 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.

[0281] The following examples further illustrate the present invention, but these examples are not intended to limit the scope of the present invention. Experimental methods in the examples without specifying specific conditions generally followed conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents without specifying their sources were commercially available.

[0282] Example 1 Antigen preparation

[0283] The His-tagged human TROP-2 (TROP-2-His) protein was synthesized by SinoBiologics (10428-H08H).

[0284] TROP-2-His sequence:

[0285] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPPDGPGGRCQCRA

[0286] LGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFK

[0287] ARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLD

[0288] AELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKG

[0289] ESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGM

[0290] AVLVITNRRKSGKYKKVEIKELGELRKEPSLHHHHHHHH

[0291] SEQ ID NO: 46

[0292] Example 2 Obtaining Mouse Hybridomas and Antibody Sequences

[0293] Animals were immunized with the human antigen TROP-2-His. A total of 5 female Balb / c and 5 A / J mice, 10 weeks old, were immunized using Sigma complete Freund's adjuvant (CFA) and Sigma incomplete Freund's adjuvant (IFA). The immunogen and immune adjuvant were thoroughly mixed and emulsified in a 1:1 ratio to prepare a stable "water-in-oil" liquid; the injection dose was 25 μg / 200 μL / mouse.

[0294] Table 1. Immunization schedule

[0295] Day 1 The first immunization was with complete Freund's adjuvant. Day 21 The second immunization was with incomplete Freund's adjuvant. Day 35 The third immunization was with incomplete Freund's adjuvant. Day 42 Blood collection and serum titer test (3 free blood) Day 49 The fourth immunization was with incomplete Freund's adjuvant. Day 56 Blood collection and serum titer test (4 free blood)

[0296] The serum of the immunized mice was evaluated for serum titer and ability to bind to cell surface antigens using the indirect ELISA method as described in Example 3, and the titer detection result (greater than 100,000-fold dilution) was used to determine the initiation of cell fusion. Immune mice with strong serum titer, affinity and FACS binding were selected for one final immunization and then killed. Splenocytes were fused with SP2 / 0 myeloma cells and plated to obtain hybridomas. The target hybridomas were screened by indirect ELISA and established as monoclonal cell lines by limiting dilution. The positive antibody strains obtained were further screened by indirect ELISA to select hybridomas that bind to recombinant proteins. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig primer set (Novagen, TB326 Rev. B 0503) and then sequenced to obtain the sequence of the mouse antibody.

[0297] The heavy chain and light chain variable region sequences of mouse monoclonal antibody M1 are as follows:

[0298] M1 HCVR

[0299] QVQLQQPGAELVRPGASVKLSCRASGYTFT NYWMN WVKQRPEQGLEWIG RIDPNDSE

[0300] THYNQKFKD RAILTVDKASNTAYMQLSGLTSEDSAVHYCAR SGFGSTYWFFDV WGAG

[0301] TTVTVSS

[0302] SEQ ID NO: 1

[0303] M1 LCVR

[0304] DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQQKPGQSPKLLIY SASYRYT GV

[0305] PDRFAGSGYGTDFTFTISSVQTEDLTVYHC QQHYSTPLT FGPGTRLELK

[0306] SEQ ID NO: 2

[0307] Table 2. CDR sequences of the heavy and light chain variable regions of mouse monoclonal antibody M1

[0308]

[0309]

[0310] Example 3 In vitro binding activity detection method of antibodies

[0311] (1) In vitro indirect ELISA binding assay:

[0312] 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 ELISA plate. Incubate overnight (16-20 hours) at 4°C. 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.

[0313] The antibody to be tested was diluted in PBST containing 3% BSA, starting at 1 μM and progressing through a 10-fold gradient of 10 doses. 100 μl / well of the plate was added and incubated at room temperature for 1 hour. After incubation, the plate was washed four times with PBST and 100 μl / well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted in PBST containing 3% BSA was added and incubated at room temperature for 1 hour. After washing the plate four times with PBST, 100 μl / well of TMB chromogenic 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 a microplate reader (BioTek, model Synergy H1) and the data analyzed. The concentration signal value curve analysis results are shown in the following table:

[0314] Table 3. Affinity of mouse antibodies to human TROP-2 antigen (EC 50 value)

[0315] Mouse antibodies <![CDATA[EC binding to human TROP-2His antigen 50 (nM)]]> M1 0.56

[0316] (2) In vitro cell binding assay:

[0317] The cultured TROP-2 high-expressing cells (CHO or 293 cells overexpressing TROP-2 and tumor cells expressing TROP-2, such as HCC-827, MDA-MB-468, etc.) were collected and plated in 96-well U-bottom plates after adjusting the cell density, with 1×10 cells per well. 5 to 2×10 5 Cells were centrifuged at 1200g for 5 minutes, the supernatant discarded, and 100µl of serially diluted antibody solution or mouse immune serum was added. The cells were incubated at 4°C for 60 minutes. After centrifugation at 1200g for 5 minutes, the supernatant discarded. The cells were washed twice with PBS. 100µl of fluorescently labeled secondary antibody (PE-GAM or PE-GAH) was added to each well and incubated at 4°C for 60 minutes. The cells were centrifuged at 1200g for 5 minutes, the supernatant discarded. The cells were washed twice with PBS and resuspended in PBS. Signals were detected using a flow cytometer, and concentration curve analysis was performed.

[0318] Example 4 Mouse Antibody Humanization Experiment

[0319] Humanization of murine anti-human TROP-2 monoclonal antibodies was performed using methods disclosed in numerous literatures in the art. Briefly, the murine antibody M1 was humanized by replacing the constant domains of the parent (murine antibody) with human constant domains and selecting human antibody sequences based on homology between murine and human antibodies.

[0320] Based on the typical structures of the obtained murine 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.

[0321] 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, backmutations were performed on buried residues, residues that directly interact with the CDR regions, and residues that have a significant impact on the VL and VH conformations. After expression testing and comparison of the number of backmutations, an antibody was selected that combines the humanized heavy chain variable region (HCVR) and light chain variable region (LCVR) sequences. The sequences are as follows:

[0322] HU1 HCVR

[0323] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGKGLEWVGRIDPND

[0324] SETHYNQKFKDRFTITRDNSKNTLYLQMNSLRAEDTAVYYCARSGFGSTYWFFDVWG

[0325] QGTTVTVSS

[0326] SEQ ID NO:9

[0327] HU1 LCVR

[0328] AIRMTQSPFSLSSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTRIPP

[0329] RFSGSGYGTDFTLTINNIESEDAAYYFCQQHYSTPLTFGQGTRLEIK

[0330] SEQ ID NO: 10

[0331] HU2 HCVR

[0332] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWIGRIDPNDSE

[0333] THYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQG

[0334] TTVTVSS

[0335] SEQ ID NO:11

[0336] HU2 LCVR

[0337] ETTLTQSPAFMSATPGDKVNISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVP

[0338] DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIK

[0339] SEQ ID NO:12

[0340] HU3 HCVR

[0341] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWMGRIDPNDS

[0342] ETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQ

[0343] GTMVTVSS

[0344] SEQ ID NO:13

[0345] HU3 LCVR

[0346] DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPS

[0347] RFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIK

[0348] SEQ ID NO:14

[0349] HU4 HCVR

[0350] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWIGRIDPNDSE

[0351] THYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQG

[0352] TMVTVSS

[0353] SEQ ID NO:15

[0354] HU4 LCVR

[0355] DIVMTQSPDSLAVSLGERATINCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVP

[0356] SRFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIK

[0357] SEQ ID NO:16

[0358] HU5 HCVR

[0359] EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWIGRIDPNDSE

[0360] THYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQG

[0361] TTVTVSS

[0362] SEQ ID NO:17

[0363] HU5 LCVR

[0364] DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVP

[0365] DRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK

[0366] SEQ ID NO:18

[0367] HU6 HCVR

[0368] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDS

[0369] ETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQ

[0370] GTTVTVSS

[0371] SEQ ID NO:19

[0372] HU6 LCVR

[0373] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVP

[0374] SRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIK

[0375] SEQ ID NO:20

[0376] HU7 HCVR

[0377] EVQLVESGGGLIQPGGSLKLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDS

[0378] ETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQ

[0379] GTTVTVSS

[0380] SEQ ID NO:21

[0381] HU7 LCVR

[0382] AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVP

[0383] DRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK

[0384] SEQ ID NO:22

[0385] HU8 HCVR

[0386] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGKGLEWMGRIDPND

[0387] SETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWG

[0388] QGTTVTVSS

[0389] SEQ ID NO:23

[0390] HU8 LCVR

[0391] AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVP

[0392] SRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK

[0393] SEQ ID NO:24

[0394] HU9 HCVR

[0395] EVQLVESGGGLIQPGGSLRLSCAASGFTVSNYWMNWVRQAPGQGLEWMGRIDPNDS

[0396] ETHYNQKFKDRLTITKDTSKNQVVLTTMNMDPVDTATYYCARSGFGSTYWFFDVWG

[0397] QGTTVTVSS

[0398] SEQ ID NO:25

[0399] HU9 LCVR

[0400] DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPD

[0401] RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIK

[0402] SEQ ID NO:26

[0403] The designed heavy chain and light chain variable region sequences were connected to the IgG heavy chain constant region and the human antibody light chain constant region sequences, respectively. Exemplary heavy chain constant region and light chain constant region sequences are as follows:

[0404] IgG1 C1

[0405] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

[0406] SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

[0407] GGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

[0408] EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0409] LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL

[0410] TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0411] SEQ ID NO:48

[0412] IgG1 C2

[0413] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

[0414] SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

[0415] GGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

[0416] EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

[0417] LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

[0418] LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0419] SEQ ID NO:49

[0420] Ig kappa C

[0421] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE

[0422] QDSKDSTYSLSSTLLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0423] SEQ ID NO:50

[0424] The heavy and light chain sequences were obtained as follows (wherein, the HU1-HU9 heavy chains were obtained by linking the sequences 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, and SEQ ID NO: 25 to the sequence of SEQ ID NO: 49, respectively; the HU6DL and HU10 heavy chains were obtained by linking the sequences of SEQ ID NO: 19 and SEQ ID NO: 9 to the sequence of SEQ ID NO: 48, respectively):

[0425] HU1 HC

[0426] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGKGLEWVGRIDPND

[0427] SETHYNQKFKDRFTITRDNSKNTLYLQMNSLRAEDTAVYYCARSGFGSTYWFFDVWG

[0428] QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0429] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0430] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

[0431] HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0432] QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0433] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0434] SEQ ID NO:27

[0435] HU1 LC

[0436] AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTRIPP

[0437] RFSGSGYGTDFTLTINNIESEDAAYYFCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPS

[0438] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0439] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0440] SEQ ID NO:28

[0441] HU2 HC

[0442] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWIGRIDPNDSE

[0443] THYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQG

[0444] TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

[0445] FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

[0446] CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

[0447] AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR

[0448] EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

[0449] FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0450] SEQ ID NO:29

[0451] HU2 LC

[0452] ETTLTQSPAFMSATPGDKVNISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVP

[0453] DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPP

[0454] SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0455] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0456] SEQ ID NO:30

[0457] HU3 HC

[0458] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWMGRIDPNDS

[0459] ETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQ

[0460] GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0461] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0462] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

[0463] HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0464] QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0465] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0466] SEQ ID NO:31

[0467] HU3 LC

[0468] DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPS

[0469] RFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPS

[0470] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0471] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0472] SEQ ID NO:32

[0473] HU4 HC

[0474] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWIGRIDPNDSE

[0475] THYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQG

[0476] TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

[0477] FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

[0478] CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

[0479] AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR

[0480] EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

[0481] FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0482] SEQ ID NO:33

[0483] HU4 LC

[0484] DIVMTQSPDSLAVSLGERATINCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVP

[0485] SRFSGSGSGTDFTLTINSLEAEDAATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPP

[0486] SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0487] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0488] SEQ ID NO:34

[0489] HU5 HC

[0490] EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWIGRIDPNDSE

[0491] THYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQG

[0492] TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

[0493] FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

[0494] CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

[0495] AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR

[0496] EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

[0497] FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0498] SEQ ID NO:35

[0499] HU5 LC

[0500] DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVP

[0501] DRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFP

[0502] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0503] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0504] SEQ ID NO:36

[0505] HU6 HC

[0506] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDS

[0507] ETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQ

[0508] GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

[0509] TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP

[0510] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0511] NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP

[0512] REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0513] GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0514] SEQ ID NO:37

[0515] HU6 LC

[0516] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVP

[0517] SRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPS

[0518] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0519] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0520] SEQ ID NO:38

[0521] HU7 HC

[0522] EVQLVESGGGLIQPGGSLKLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDS

[0523] ETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWGQ

[0524] GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

[0525] TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP

[0526] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0527] NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP

[0528] REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0529] GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0530] SEQ ID NO:39

[0531] HU7 LC

[0532] AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVP

[0533] DRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFP

[0534] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

[0535] TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0536] SEQ ID NO:40

[0537] HU8 HC

[0538] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPGKGLEWMGRIDPND

[0539] SETHYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGFGSTYWFFDVWG

[0540] QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0541] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0542] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

[0543] HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0544] QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0545] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0546] SEQ ID NO:41

[0547] HU8 LC

[0548] AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVP

[0549] SRFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTRLEIK

[0550] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE

[0551] QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0552] SEQ ID NO:42

[0553] HU9 HC

[0554] EVQLVESGGGLIQPGGSLRLSCAASGFTVSNYWMNWVRQAPGQGLEWMGRIDPNDS

[0555] ETHYNQKFKDRLTITKDTSKNQVVLTMTNMDPVDTATYYCARSGFGSTYWFFDVWG

[0556] QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0557] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0558] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

[0559] HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0560] QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0561] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0562] SEQ ID NO:43

[0563] HU9 LC

[0564] DIVMTQTPLSLPVTPGEPASISCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTGVPD

[0565] RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPS

[0566] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0567] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0568] SEQ ID NO:44

[0569] HU6DL HC

[0570] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNYWMNWVRQAPGKGLEWMGRIDPNDS

[0571] ETHYNQKFKDRVTISVDKSKNQFSLKLSSVTAADTAVYYCARSGFGSTYWFFDVWGQ

[0572] GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

[0573] TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP

[0574] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0575] NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP

[0576] REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

[0577] SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0578] SEQ ID NO:45

[0579] HU6DL LC

[0580] DVVMTQSPLSLPVTLGQPASISCKASQDVSTAVAWYQQKPGKAPKLFIYSASYRYTGVP

[0581] SRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPS

[0582] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0583] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0584] SEQ ID NO:38

[0585] HU10 HC

[0586] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMNWVRQAPGKGLEWVGRIDPND

[0587] SETHYNQKFKDRFTITRDNSKNTLYLQMNSLRAEDTAVYYCARSGFGSTYWFFDVWG

[0588] QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0589] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0590] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

[0591] HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0592] QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0593] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0594] SEQ ID NO:47

[0595] HU10 LC

[0596] AIRMTQSPFSLSASVGDRVTITCKASQDVSTAVAWYLQKPGQSPQLLIYSASYRYTRIPP

[0597] RFSGSGYGTDFTLTINNIESEDAAYYFCQQHYSTPLTFGQGTRLEIKRTVAAPSVFIFPPS

[0598] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0599] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0600] SEQ ID NO: 28

[0601] Table 4. Sequence numbers of antibodies and their heavy chains, light chains, and variable regions

[0602]

[0603] According to the amino acid sequences of the light and heavy chains of each humanized antibody, cDNA fragments were synthesized and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2 and incubated in a CO2 incubator for 4-5 days. The cell culture medium was collected, centrifuged and filtered, and then loaded onto an antibody purification affinity column. The column was washed with phosphate buffer, eluted 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. Its concentration and purity are shown in the following table:

[0604] Table 5. Concentration and purity of each humanized antibody

[0605] Humanized antibody number Concentration (mg / ml) purity(%) HU1 0.72 98.2％ HU2 0.62 98.4％ HU3 0.75 96.2％ HU4 0.96 96.4％ HU5 1.17 97.1％ HU6 1.35 96.8％ HU7 1.26 98.5％ HU8 1.36 98.3％ HU6DL 1.25 97.4％ HU10 1.21 98.2％

[0606] Example 5 In vitro binding affinity and kinetics experiments

[0607] The affinity of each humanized antibody for human TROP-2 antigen (EC 50 ) are shown in the following table:

[0608] Table 6. Affinity of each humanized antibody for human TROP-2 antigen (EC 50 )

[0609]

[0610] To test the binding ability of each humanized antibody to the target protein TROP-2 on tumor cells, the affinity (EC) of each humanized antibody to HCC827 tumor cells (non-small cell lung cancer) and MAB-MB-468 tumor cells (breast cancer, invasive ductal carcinoma) determined by the in vitro cell binding assay in Example 3(2) was used. 50 ) are shown in the following table:

[0611] Table 7. Affinity of each humanized antibody for HCC827 tumor cells (EC 50 )

[0612]

[0613] Example 6 Humanized Antibody-Mediated Tumor Cell Killing

[0614] Humanized antibodies can exert their anti-tumor cell cytotoxicity in multiple ways, one of which is through mediating immune cell-mediated anti-tumor cell cytotoxicity. To test the anti-tumor cell cytotoxicity mediated by the humanized antibodies of the present invention, a system in which human peripheral blood mononuclear cells (PBMCs) were co-cultured with HCC827 tumor cells (non-small cell lung cancer) was used for evaluation. HCC827 cells were harvested, centrifuged, counted, and then adjusted to a cell density of 0.44×10 cells using complete culture medium. 6 / mL, plated in the middle 60 wells of a white 96-well plate, 90 μL per well, with a cell count of 4000. Commercial human PBMC cells were collected, centrifuged and counted, and then the cell density was adjusted to 2.2×10 6 / mL, spread in the middle 60 wells of a white 96-well plate with HCC827 cells, 90μL per well, and the number of cells is 20,000. 200μL PBS was added to the remaining side wells, and the cell plate was placed in a 37°C, 5% CO2 incubator for overnight culture. On the second day of the experiment, humanized antibody solution was prepared in a 96-well V-bottom plate with PBS, starting at a concentration of 1000nM, diluted 3 times, and 9 concentrations. After preparation, it was added to a white 96-well plate, 20μL per well, two replicates, and the cell plate was placed in a 37°C, 5% CO2 incubator for continued culture for 72 hours. On the fifth day of the experiment, the detection readings were taken out, equilibrated to room temperature, 50μL CTG solution (Promega G7573) was added to each well, shaken to mix, and then placed in the dark for 10 minutes, and then detected using the luminescence program of the microplate reader. The experimental results are shown in Table 8 below:

[0615] Table 8. Humanized antibody-mediated killing of tumor cells

[0616]

[0617]

[0618] The same method was used to determine the killing effect of HU6 antibody on HCC827 tumor cells. The results showed that the killing effect at the highest dose was 52.3%.

[0619] Example 7 Humanized Antibody-Mediated TROP-2 Internalization

[0620] To study the humanized antibody-mediated endocytosis of TROP-2 protein in tumor cells, SW780 cells were trypsinized, collected and resuspended in pre-cooled PBS to adjust the cell concentration to 1×10 6 Cells / mL. Add 1 mL of cell suspension to an EP tube, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Add 1 mL of the prepared antibody to be tested to resuspend the cells. The final concentration of the antibody is 20 μg / mL. Incubate on a shaker at 4°C for 1 hour. Centrifuge and discard the supernatant (4°C, 1500 rpm × 5 minutes). Wash twice with PBS and discard the supernatant. Add 100 μL of fluorescent secondary antibody working solution to each tube and resuspend the cells. Incubate on a shaker at 4°C for 30 minutes. Centrifuge and discard the supernatant (4°C, 1500 rpm × 5 minutes). Wash twice with PBS and discard the supernatant. 1.0 mL of prewarmed SW780 complete cell culture medium was added to each tube to resuspend the cells and mix thoroughly. The cells were then aliquoted into four tubes, each containing 200 μL, designated as the 0 min, blank, 30 min, and 2 h groups. The 0 min and blank tubes were removed and placed on ice, while the remaining tubes were placed in a 37°C incubator for 30 min and 2 h of endocytosis, respectively. At each corresponding time point, one tube was removed and pre-chilled on ice for 5 min. The supernatant from all treatment groups was centrifuged (4°C, 1500 rpm for 5 min), washed once with PBS, and discarded. 250 μL of strip buffer was added to all tubes except the 0 min tube, incubated at room temperature for 8 min, centrifuged (4°C, 1500 rpm for 5 min), discarded the supernatant, washed twice with PBS, and discarded the supernatant. 100 μL of immunostaining fixative was added to all treatment groups, incubated at 4°C for at least 30 min, and analyzed using a DxFlex flow cytometer. 200 μL was taken from the 0 min tube and directly added to the immunostaining fixative. Take 200 μl of the blank sample and directly add strip buffer and immunostaining fixative. Analyze using a DxFlex flow cytometer. Data statistics and analysis: 30-minute average endocytosis percentage (%) = (30-minute group MFI - blank group MFI) / (0-minute group MFI - blank group MFI); 2-hour average endocytosis percentage (%) = (2-hour group MFI - MIF - blank group MFI) / (0-minute group MFI - blank group MFI). The endocytosis percentage of the humanized antibody detected using the above method is shown in Table 9:

[0621] Table 9. Humanized antibody-mediated TROP-2 protein endocytosis

[0622]

[0623]

[0624] Example 8 Competitive Binding of Humanized Antibodies to Antigens

[0625] Competitive binding experiments are usually used to study the binding mode and binding site of different antibodies to antigens. Dilute the hRS7 antibody protein to a concentration of 1 μg / ml with PBS at pH 7.4 and add it to a 96-well high-affinity ELISA plate at a volume of 100 μl / well. Incubate in a 4°C refrigerator overnight (16-20 hours). After washing the plate four times with PBST (PBS at pH 7.4 containing 0.05% Tween-20), add 150 μl / well of 2% bovine serum albumin (BSA) blocking solution diluted with PBST and incubate at room temperature for 1 hour for blocking. After blocking, discard the blocking solution and wash the plate four times with PBST buffer.

[0626] Dilute the test antibody to 100 μg / ml in PBST containing 2% BSA and add 50 μl / well to the ELISA plate. Dilute TROP-2His 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 TMB chromogenic 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 Stop Solution (Cell Signaling Technology, cat#7002S). The absorbance was read at 450 nm using a microplate reader (BioTek, model Synergy H1). The data were analyzed as shown in the table below. The humanized antibody of the present invention had a very low inhibition rate on the binding of hRS7 antibody to TROP2 protein, indicating that the humanized antibody of the present invention does not compete with the hRS7 antibody for binding to the same epitope.

[0627] Table 10. Antigen competition between humanized antibodies and hRS7

[0628] Humanized antibodies Inhibition rate hRS7 94.7％ HU1 13.5％ HU6 6.7％ HU6DL 5.9％ HU10 10.2％

[0629] Example 9 Preparation of Compound 1

[0630]

[0631] In the first step, 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 solution was stirred at room temperature for 48 hours, filtered through celite, and the filter cake was rinsed with ethyl acetate (20 mL). The combined filtrates were concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography using solvent system C to obtain product 5a (3.2 g, yield: 90.1%).

[0632] In the second step, 5a (181.3 mg, 0.879 mmol) and 4b (270 mg, 0.733 mmol) were added to a reaction flask, followed by 6 mL of tetrahydrofuran. The atmosphere was replaced with argon three times, and the temperature was cooled to 0-5°C in an ice-water bath. 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, 3 mL of water was added, and sodium bicarbonate (73.8 mg, 0.879 mmol) and 9-fluorenylmethyl chloroformate (190 mg, 0.734 mmol) were added. 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 residue was purified by silica gel column chromatography using developing solvent System C to give product 5b, benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate (73 mg, yield: 19.4%).

[0633] MS m / z(ESI):515.0[M+1].

[0634] In the third step, 5b (30 mg, 0.058 mmol) was dissolved in 6.75 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (v:v = 2:1). Palladium on carbon (18 mg, 10% content, 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 solution was filtered through celite, 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-diazaundecane-11-oic acid (20 mg). The product was directly used in the next reaction without purification.

[0635] MS m / z(ESI):424.9[M+1].

[0636] In the fourth step, 1b (15 mg, 28.2 μmol) was added to a reaction flask, followed by 1.5 mL of N,N-dimethylformamide. The atmosphere was replaced with argon three times, and the temperature was cooled to 0-5°C in an ice-water bath. One drop of triethylamine was added dropwise, followed by the addition of crude product 5c (20 mg, 47.1 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium 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, 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 solvent 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%).

[0637] MS m / z(ESI):842.1[M+1].

[0638] In the fifth step, 5d (30 mg, 35.7 μmol) was dissolved in 3 mL of dichloromethane, and 1.5 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 1.5 mL of toluene was added and concentrated under reduced pressure twice. 4.5 mL of n-hexane was added to the residue, and the mixture was allowed to stand. The supernatant was decanted, retaining 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 in the next step without purification.

[0639] MS m / z(ESI):638.0[M+18].

[0640] Step 6: The crude product 5e (20 mg, 32.3 μmol) was dissolved in 1 mL of N,N-dimethylformamide, replaced with argon three times, and cooled to 0-5°C in an ice-water bath. 4 g (31.8 mg, 67.3 μmol) of a 0.5 mL N,N-dimethylformamide solution was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (27.8 mg, 94.3 μmol). The mixture was stirred in an ice bath for 10 minutes, the ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour to produce compound 5. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmolNH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding fractions were collected and concentrated under reduced pressure to obtain products 5-A and 5-B (3.6mg, 2.6mg).

[0641] MS m / z(ESI):1074.4[M+1].

[0642] Single configuration compound 5-A (shorter retention time):

[0643] 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).

[0644] 1H NMR (400MHz, DMSO-d6): δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H) ,7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,2 H),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).

[0645] Single configuration compound 5-B (longer retention time):

[0646] 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).

[0647] 1H NMR (400MHz, DMSO-d6): δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H), 7.31(s,1H),7.26-7.13(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.60- 5.50(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.78-4.68(m,1H),4.60- 4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H) ,3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13( m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52- 1.39(m,3H),1.34-1.12(m,5H),0.91-0.79(m,3H),0.53-0.34(m,4H).

[0648] The preparation methods of other intermediates refer to Intermediate 5.

[0649] At 37°C, a solution of antibody HU1 in PBS buffer (0.05 M PBS buffer at pH 6.5; 7.3 ml, 13.8 mg / ml, 0.681 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.347 mL, 3.47 μmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath, diluted to 14.0 ml, and 3.3 ml of the solution was removed for further reaction.

[0650] 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 placed in a water bath shaker and shaken at 25° C. for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of Ab-isotecan derivative, HU1-isotecan derivative in PBS buffer (1.45 mg / mL, 17 mL), which was stored frozen at 4° C.

[0651] The average value y was determined using the UV method. A cuvette containing sodium succinate buffer was placed in the reference absorption cell and the sample absorption cell, 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.

[0652] Data processing:

[0653] By establishing a standard curve, measuring the absorption at a wavelength of 280 nm, the antibody content Cmab is determined, and measuring the absorption at a wavelength of 370 nm, the small molecule content CDrug is determined.

[0654] The average drug loading value y = CDrug / Cmab.

[0655] 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.

[0656] The preparation methods of Compounds 2 to 11 refer to Compound 1. Similarly, Compound 12 (y=8) was prepared using the same preparation method of Compound 1 and the hRS7 antibody in the prior art:

[0657]

[0658] Example 11 Antibody Conjugation to MC-MMAF

[0659]

[0660] In the first step, S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile for later use. The pre-prepared acetonitrile solution of S-(3-hydroxypropyl)thioacetate was added to an acetic acid / sodium acetate buffer (10.35 mg / mL, 9.0 mL, 0.97 mol) at pH 4.3. Then, 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was added dropwise. The mixture was shaken at 25°C for 2 hours. After completion of the reaction, the product 1a was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M PBS at pH 6.5) to obtain a solution. This solution was concentrated to 10 mg / mL and directly used in the next step.

[0661] In the second step, 0.35 mL of 2.0 M carboxylamine hydrochloride solution was added to solution 1a (11.0 mL). After shaking at 25°C for 30 minutes, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5) to obtain a product 2a solution (concentration 6.17 mg / mL, 14.7 mL).

[0662] In the third step, the compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile and added to the 2a solution (concentration 6.17 mg / mL, 3.0 mL). After shaking at 25°C for 4 hours, the reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5). After aseptic filtration, the product Ab-MC-MMAF antibody-drug conjugate was obtained in PBS buffer (3.7 mg / mL, 4.7 mL), which was refrigerated at 4°C.

[0663] Example 12 Antibody Conjugation to SN-38

[0664]

[0665] In the first step, S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile for later use. The pre-prepared acetonitrile solution of S-(3-hydroxypropyl)thioacetate was added to an acetic acid / sodium acetate buffer (10.35 mg / mL, 9.0 mL, 0.97 mol) at pH 4.3. Then, 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was added dropwise. The mixture was shaken at 25°C for 2 hours. After completion of the reaction, the product was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M PBS at pH 6.5) to obtain a 1 h solution of the product. This was concentrated to 10 mg / mL and directly used in the next step.

[0666] In the second step, 0.35 mL of 2.0 M carboxylamine hydrochloride solution was added to the 1h solution (11.0 mL), and the reaction was shaken at 25°C for 30 minutes. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5) to obtain the product 2h solution (concentration 6.2 mg / mL, 15.0 mL), which was concentrated to about 10 mg / ml and used in the next reaction.

[0667] 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 and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at 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), which was refrigerated at 4°C.

[0668] Example 13 Antibody-drug conjugates' killing effect on tumor cells

[0669] To test the killing effect of the antibody-drug conjugate of the present invention on tumor cells, bladder cancer cell line SW780 was used for evaluation. SW780 cells were collected, centrifuged and counted, and then the cell density was adjusted to 0.44×10 6 Cells were plated in the center 60 wells of a white 96-well plate at 90 μL per well, with a cell count of 4,000. 100 μL of PBS was added to the remaining side wells, and the plate was incubated overnight at 37°C, 5% CO2. On the second day of the experiment, an antibody-drug conjugate solution was prepared in PBS in a 96-well V-bottom plate. The solution was diluted three-fold to nine concentrations, starting at 1,000 nM. After preparation, 10 μL was added to each well of the white 96-well plate in duplicate, and the plate was incubated in a 37°C, 5% CO2 incubator for an additional 72 hours. On the fifth day of the experiment, the plate was removed from the plate, equilibrated to room temperature, and 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 a microplate reader with a luminescence program. EC50 values ​​were calculated using a four-parameter fitting method. The above method was also used to evaluate the killing effects of the antibody-drug conjugates of the present invention on bladder cancer cells RT4 and epidermal cancer cells A431. The experimental results are shown in Table 11:

[0670] Table 11. Antibody-drug conjugates' killing effects on tumor cells

[0671]

[0672] Example 14 Pharmacokinetics of Antibody-Drug Conjugates

[0673] To further investigate the pharmacokinetics of the antibody-drug conjugate in vivo, the antibody-drug conjugate was injected intravenously into C57BL / 6 mice (at a dose of 10 mg / kg). 20 μL of blood was drawn at time points of 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 pharmacokinetic data were analyzed using WinNonlin software to obtain the pharmacokinetic parameters shown in Table 12.

[0674] Table 12. Pharmacokinetic parameters of antibody-drug conjugates

[0675]

[0676] Example 15 In vivo anti-tumor effect of antibody-drug conjugates

[0677] To further study the killing effect of antibody-drug conjugates on tumors formed in vivo, N87 transplanted tumors were formed in mice to evaluate the anti-tumor effect of the antibody-drug conjugates of the present invention. 6 N87 cells were injected subcutaneously into immunodeficient nude mice. Two weeks later, the antibody-drug conjugate compound 10 (y=8) and compound 12 (y=8) were injected intravenously once every two weeks at a dose of 2 mg / kg. The control group used human IgG1 protein at a dose of 2 mg / kg. There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate = 100% - (tumor volume of the treatment group on day 28 - tumor volume of the treatment 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:

[0678] Table 13. Antibody-drug conjugates' tumor-killing effects

[0679]

[0680] Compound 10 (y=8) showed a tumor inhibition rate exceeding 100%, which means that compound 10 (y=8) can not only inhibit tumor growth but also has a killing effect on established tumors.

[0681] Example 16: Preparation of Exitecan Derivatives

[0682]

[0683] 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 argon three times, and the mixture was cooled to 0-5°C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise, and the reaction mixture was stirred until the solution became 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. The ice-water bath was removed, and the mixture was heated to 30°C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude compound 2-C was purified by high performance liquid chromatography (separation conditions: 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 give the title products (2-A: 1.5mg, 2-B: 1.5mg).

[0684] MS m / z(ESI):534.0[M+1].

[0685] Single configuration compound 2-B (shorter retention time)

[0686] 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).

[0687] 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).

[0688] Single configuration compound 2-A (longer retention time)

[0689] 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).

[0690] 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).

[0691] Example 17: In vitro inhibition test of tumor cell proliferation by exitecan derivatives

[0692] 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. In vitro activity of the compounds was evaluated based on IC50 values.

[0693] U87MG and SK-BR-3 cells were cultured with EMEM medium containing 10% FBS (GE, catalog number SH30024.01) and McCoy's 5A medium containing 10% FBS (Gibco, catalog number 16600-108), respectively.

[0694] U87MG and SK-BR-3 cells in the logarithmic growth phase were taken, washed once with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), and then 2-3 ml of trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) was added for digestion for 2-3 minutes. After the cells were completely digested, 10-15 ml of cell culture medium was added to elute the digested cells, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and then 10-20 ml of cell culture medium was added to resuspend the cells to make a single-cell suspension.

[0695] 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 of the suspension to a 96-well cell culture plate. Add only 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.

[0696] The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution with an initial concentration of 10 mM.

[0697] The starting concentration of the small molecule compound is 500 nM, and the dispensing method is as follows:

[0698] 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 the first column to the 20 μl DMSO in the second column, mix thoroughly, and then transfer 10 μl to the third column, 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 thoroughly, and set aside.

[0699] Sample addition: Add 20 μl of the sample to be tested at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37° C., 5% CO 2 ).

[0700] Color development: Take out the 96-well cell culture plate, add 90 μl CTG solution to each well, and incubate at room temperature for 10 minutes.

[0701] Plate reading: Take out the 96-well cell culture plate, place it in a microplate reader (BMG labtech, PHERAstar FS), and measure the chemiluminescence using a microplate reader.

[0702] Data analysis: Data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in Table 14.

[0703] Table 14. Tumor-killing effects of ixitecan derivatives

[0704] Sequence Listing <110> Shanghai Hansen Biopharmaceutical Technology 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> CHAIN <222> (1)..(121) <223> Heavy chain variable region of mouse monoclonal antibody M1 <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Ile Leu Thr Val Asp Lys Ala Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val His Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> Light chain variable region of mouse monoclonal antibody M1 <400> 2 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ala Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Thr 65 70 75 80 Glu Asp Leu Thr Val Tyr His Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Pro Gly Thr Arg Leu Glu Leu Lys 100 105 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> HCDR1 of murine monoclonal antibody M1 <400> 3 Asn Tyr Trp Met Asn 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> HCDR2 of mouse monoclonal antibody M1 <400> 4 Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(12) <223> HCDR3 of mouse monoclonal antibody M1 <400> 5 Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val 1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> LCDR1 of mouse monoclonal antibody M1 <400> 6 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> LCDR2 of mouse monoclonal antibody M1 <400> 7 Ser Ala Ser Tyr Arg Tyr Thr 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> LCDR3 of mouse monoclonal antibody M1 <400> 8 Gln Gln His Tyr Ser Thr Pro Leu Thr 1 5 <210> 9 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU1 HCVR <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU1 LCVR <400> 10 Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Arg Ile Pro Pro Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu Ser 65 70 75 80 Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 11 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU2 HCVR <400> 11 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU2 LCVR <400> 12 Glu Thr Thr Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Thr Pro Gly 1 5 10 15 Asp Lys Val Asn Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 13 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU3 HCVR <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU3 LCVR <400> 14 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 � Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 15 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU4 HCVR[[ID=^]] <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU4 LCVR <400> 16 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 17 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU5 HCVR <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU5 LCVR <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Phe Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 19 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU6 HCVR <400> 19 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU6 LCVR <400> 20 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Phe Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 21 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU7 HCVR <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU7 LCVR <400> 22 Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 23 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU8 HCVR <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU8 LCVR <400> 24 Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 25 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(121) <223> HU9 HCVR <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val 65 70 75 80 Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> HU9 LCVR <400> 26 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 27 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU1 HC <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 28 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU1 LC <400> 28 Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Arg Ile Pro Pro Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu Ser 65 70 75 80 Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 29 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU2 HC <400> 29 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 30 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU2 LC <400> 30 Glu Thr Thr Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Thr Pro Gly 1 5 10 15 Asp Lys Val Asn Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 31 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU3 HC <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 32 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU3 LC <400> 32 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 33 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU4 HC <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 34 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU4 LC <400> 34 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 35 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU5 HC <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 36 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU5 LC <400> 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Phe Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 37 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU6 HC <400> 37 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 38 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU6 LC <400> 38 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Phe Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 39 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU7 HC <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 40 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU7 LC <400> 40 Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 41 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU8 HC <400> 41 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 42 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU8 LC <400> 42 Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 43 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(43) <223> HU9 HC <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val 65 70 75 80 Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 44 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(214) <223> HU9 LC <400> 44 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190​​​​​​​​​​​​​​​​​​​​ <222> (1)..(451) <223> HU6DL HC <400> 45 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 46 <211> 331 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(331) <223> Human TROP-2 with His tag <400> 46 Met Ala Arg Gly Pro Gly Leu Ala Pro Pro Pro Leu Arg Leu Pro Leu 1 5 10 15 Leu Leu Leu Val Leu Ala Ala Val Thr Gly His Thr Ala Ala Gln Asp 20 25 30 Asn Cys Thr Cys Pro Thr Asn Lys Met Thr Val Cys Ser Pro Asp Gly 35 40 45 Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu Gly Ser Gly Met Ala Val 50 55 60 Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu Lys Ala Arg Met 65 70 75 80 Ser Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro Ser Glu His Ala 85 90 95 Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Pro Glu Gly 100 105 110 Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val Cys Trp Cys Val 115 120 125 Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly Asp Leu Ser Leu Arg 130 135 140 Cys Asp Glu Leu Val Arg Thr His His Ile Leu Ile Asp Leu Arg His 145 150 155 160 Arg Pro Thr Ala Gly Ala Phe Asn His Ser Asp Leu Asp Ala Glu Leu 165 170 175 Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu His Pro Lys Phe Val Ala 180 185 190 Ala Val His Tyr Glu Gln Pro Thr Ile Gln Ile Glu Leu Arg Gln Asn 195 200 205 Thr Ser Gln Lys Ala Ala Gly Asp Val Asp Ile Gly Asp Ala Ala Tyr 210 215 220 Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser Leu Phe Gln Gly Arg Gly 225 230 235 240 Gly Leu Asp Leu Arg Val Arg Gly Glu Pro Leu Gln Val Glu Arg Thr 245 250 255 Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro Lys Phe Ser Met Lys Arg 260 265 270 Leu Thr Ala Gly Leu Ile Ala Val Ile Val Val Val Val Val Ala Leu 275 280 285 Val Ala Gly Met Ala Val Leu Val Ile Thr Asn Arg Arg Lys Ser Gly 290 295 300 Lys Tyr Lys Lys Val Glu Ile Lys Glu Leu Gly Glu Leu Arg Lys Glu 305 310 315 320 Pro Ser Leu His His His His His His His His 325 330 <210> 47 <211> 451 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(451) <223> HU10 HC <400> 47 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Asp Pro Asn Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Phe Gly Ser Thr Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 48 <211> 330 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(330) <223> IgG1 C1 <400> 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys<00034​​​​​​​​​​​​​​​​​​​​​​​ Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 50 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(107) <223> Ig kappa C <400> 50 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims

1. An antibody-drug conjugate represented by general formula (A) or a pharmaceutically acceptable salt or solvent compound thereof, Ab-(L2-L1-D)y (A) in: D is a cytotoxic drug; L1 is selected from -O-(CRaR b )m-CR 5 R 6 -C(O)-, -O-CR 5 R 6 -(CRaR b )m-, -O-CR 5 R 6 -, -NH-(CRaR b )m-CR 5 R 6 -C(O)- or -S-(CRaR b )m-CR 5 R 6 -C(O)-; Ra and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; Or, Ra and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 5 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl; R 6 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Or, Ra and R 6 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; m is an integer selected from 0 to 4; y is a number selected from 1 to 10, and y is a decimal or an integer; L2 is the joint unit; Ab is an anti-TROP-2 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region and a heavy chain variable region, the 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 light chain variable region comprises LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:

8.

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

3. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, wherein the anti-TROP-2 antibody or its antigen-binding fragment 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 its antigen-binding fragment comprises a light chain constant region derived from a human antibody κ chain, λ chain or a variant thereof.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 3, wherein the anti-TROP-2 antibody or its antigen-binding fragment comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4 or a variant thereof, and the anti-TROP-2 antibody or its antigen-binding fragment comprises a light chain constant region of a human antibody κ chain.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 3, wherein the anti-TROP-2 antibody or its antigen-binding fragment 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 its antigen-binding fragment comprises a light chain constant region as shown in SEQ ID NO:

50.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of claim 1, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having at least 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; and a light chain variable region that is at least 99% identical to 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.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 6, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises: The heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 10; or, The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12; or, The heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 14; or, The heavy chain variable region shown in SEQ ID NO: 15 and the light chain variable region shown in SEQ ID NO: 16; or, The heavy chain variable region shown in SEQ ID NO: 17 and the light chain variable region shown in SEQ ID NO: 18; or, The heavy chain variable region shown in SEQ ID NO: 19 and the light chain variable region shown in SEQ ID NO: 20; or, The heavy chain variable region shown in SEQ ID NO: 21 and the light chain variable region shown in SEQ ID NO: 22; or, The heavy chain variable region shown in SEQ ID NO: 23 and the light chain variable region shown in SEQ ID NO: 24; or, The heavy chain variable region is shown in SEQ ID NO:25 and the light chain variable region is shown in SEQ ID NO:

26.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of claim 6, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain having at least 99% identity to the following sequences: SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45 or SEQ ID NO: 47; and, a light chain having at least 99% identity compared to 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.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 8, wherein the anti-TROP-2 antibody comprises: The heavy chain shown in SEQ ID NO: 27 and the light chain shown in SEQ ID NO: 28; or, The heavy chain shown in SEQ ID NO: 29 and the light chain shown in SEQ ID NO: 30; or, The heavy chain shown in SEQ ID NO: 31 and the light chain shown in SEQ ID NO: 32; or, The heavy chain shown in SEQ ID NO: 33 and the light chain shown in SEQ ID NO: 34; or, The heavy chain shown in SEQ ID NO: 35 and the light chain shown in SEQ ID NO: 36; or, The heavy chain shown in SEQ ID NO: 37 and the light chain shown in SEQ ID NO: 38; or, The heavy chain shown in SEQ ID NO: 39 and the light chain shown in SEQ ID NO: 40; or, The heavy chain shown in SEQ ID NO:41 and the light chain shown in SEQ ID NO:42; or, The heavy chain shown in SEQ ID NO:43 and the light chain shown in SEQ ID NO:44; or, The heavy chain shown in SEQ ID NO:45 and the light chain shown in SEQ ID NO:38; or, The heavy chain is shown in SEQ ID NO:47 and the light chain is shown in SEQ ID NO:

28.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to any one of claims 1 to 9, wherein L1 is represented by general formula (E): in, R 5 is a haloalkyl or cycloalkyl group, R 6 is selected from hydrogen, haloalkyl or cycloalkyl, Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms a cycloalkyl group.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 10, wherein: R 5 Selected from C 1-6 Haloalkyl or C 3-6 Cycloalkyl, R 6 Selected from hydrogen, C 1-6 Haloalkyl or C 3-6 Cycloalkyl, Or, R 5 and R 6 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl; m is selected from integers of 0-4.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 10, wherein the general formula (E) is selected from the following substituents:

13. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to any one of claims 1 to 9, wherein L2 is represented by general formula (D): -K 1 -K 2 -K 3 -K 4 - (D) in, K 1 for s is selected from an integer from 2 to 8; K 2 Selected from -NR 1 (CH2CH2O) p CH2CH2C(O)-、-NR 1 (CH2CH2O) p CH2C(O)-、-S(CH2) p C(O)- or a single bond, p is selected from an integer from 1 to 20; R 1 is selected from the group consisting of hydrogen, deuterium, hydroxy, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl; K 3 is a tetrapeptide residue; K 4 For-NR 2 (CR 3 R 4 )t-,R 2 , R 3 or R 4 Each is independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl, and t is selected from 1 or 2.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 13, wherein: Said p is selected from an integer from 1 to 6; The tetrapeptide residue is selected from the group consisting of peptide residues formed by two or more amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 13, wherein: The tetrapeptide residue is the tetrapeptide residue of GGFG.

16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 13, wherein the linker unit L2 has a K 1 The end is connected to Ab, K 4 The end is connected to L1.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to any one of claims 1 to 9, wherein the cytotoxic drug is selected from toxins, chemotherapeutic drugs, antibiotics, radioactive isotopes and nucleolytic enzymes.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 17, wherein the cytotoxic drug is selected from a microtubule inhibitor or a DNA topoisomerase inhibitor that inhibits cell division.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 17, wherein the cytotoxic drug is selected from exotecan or an exotecan derivative, SN-38, MMAE or MMAF.

20. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 17, wherein the cytotoxic drug is selected from the following structures:

21. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 20, wherein the antibody-drug conjugate is represented by the general formula (III): in, L1 and L2 are joint units; y is a number from 1 to 10; Ab is selected from the TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

22. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 21, wherein: y is a number from 2 to 8.

23. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 21, wherein: y is a number from 4 to 8.

24. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 21, wherein the antibody-drug conjugate is represented by the general formula (IV-A): s is selected from an integer from 2 to 8; R5 is selected from haloalkyl or C 3-6 Cycloalkyl; R6 is selected from hydrogen, haloalkyl or C 3-6 Cycloalkyl; Alternatively, R5 and R6 together with the carbon atom to which they are attached form C 3-6 Cycloalkyl; R2, R3 or R4 are each independently selected from hydrogen or alkyl.

25. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 24, wherein the general formula (IV-A) is selected from the following structures:

26. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, which is selected from the following compounds: in, y is selected from 2-10.

27. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 26, wherein: y is selected from 4-8.

28. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 26, wherein: y is selected from 6-8.

29. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 26, wherein: y is selected from 7-8.

30. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 26, wherein: y is selected from 8.

31. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 30 or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate, and one or more pharmaceutically acceptable carriers.

32. Use of the antibody-drug conjugate according to any one of claims 1 to 30 or the pharmaceutical composition according to claim 31 in the preparation of a drug for treating a human TROP-2-related disease, wherein the human TROP-2-related disease is bladder cancer, epidermal cancer, lung cancer, gastric cancer, breast cancer, or human brain astrocytoma.

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