1. Patent Search
  2. Details

ANTI-HUMAN TROP-2 ANTIBODY AND APPLICATION THEREOF

20240092930 ยท 2024-03-21

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention provides an antibody binding a human tumor-associated calcium signal sensor 2 (Trop-2) protein or fragments thereof, and use of the antibody or fragments thereof in preventing or treating diseases. The antibody or fragments thereof of the present invention can effectively bind to the human Trop-2 protein, and have internalization activity, and the internalization activity is enhanced after ADC drug labeling, and the in vivo efficacy and safety of a mouse model are not lower than those of a control antibody.

    Claims

    1. A method for preparing an anti-Trop-2 monoclonal antibody, including steps as follows: (1) preparing hybridoma cells by immunizing an animal with recombinant Trop-2 protein as an immunogen; (2) screening positive hybridoma cells secreting anti-Trop-2 monoclonal antibodies using the recombinant Trop-2 protein as a coating antigen; (3) rescreening the positive hybridoma cells obtained in step (2) using cells positive for Trop-2 on cell membrane surface; wherein the anti-Trop-2 monoclonal antibody specifically recognizes and binds a natural epitope of Trop-2 extracellular domain.

    2. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 1, wherein the cells positive for Trop-2 on cell membrane surface in step (3) are recombinant animal cells from the same species as the animal immunized when the hybridoma cells are prepared in step (1).

    3. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 2, wherein in step (1) the hybridoma cells are prepared by immunizing a mouse, and in step (3) the cells positive for Trop-2 on cell membrane surface are recombinant mouse cells expressing exogenous Trop-2 protein.

    4. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 1, wherein in step (2) the positive hybridoma cells secreting anti-Trop-2 monoclonal antibodies are screened using enzyme-linked immunosorbent assay (ELISA); and in step (3) the rescreening is performed by flow cytometry (FACS) analysis to obtain a hybridoma secreting an antibody specifically recognizing and binding a natural epitope of Trop-2 extracellular domain.

    5. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 1, wherein the method further includes step (4): identifying the antibody specifically recognizing and binding a natural epitope of Trop-2 extracellular domain, and the identifying comprises affinity identification and specificity identification.

    6. The method for preparing an anti-Trop-2 monoclonal antibody according to claim 5, wherein in step (4) a monoclonal antibody having a specific binding ability to human Trop-2 and cynomolgus Trop-2 but not to murine Trop-2 is selected.

    7. An anti-Trop-2 monoclonal antibody or fragment thereof obtained by the method for preparing an anti-Trop-2 monoclonal antibody according to claim 6.

    8. The anti-Trop-2 monoclonal antibody or fragment thereof according to claim 7, wherein the monoclonal antibody binds to recombination human Trop-2 extracellular domain with an affinity as measured by a KD value less than 1?10.sup.?8 M, 5?10.sup.?9 M, 1?10.sup.?10 M or 5?10.sup.?10 M.

    9. An antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of CDRs (HCDR1, HCDR2, HCDR3; and LCDR1, LCDR2, LCDR3) selected from the group consisting of: TABLE-US-00035 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3[A1] 1 SYWMY EINPSNGRT EGHNYDG RSSQSLTN GISNR FQSTHQPYT NYNEKFKS SLGAMDH SYGNTFLS FS 2 DYVIG EIYLGSGTI GSIFPFD SASSSVSY DTSTL QQWSSYPYT YYTEKFKG Y MY AS 3 DYVIG EIYLGSGTI GSIFPFD SASSSVSY DTSTL QQWSSYPYT YYAEKFKG Y MY AS 4 DYVIG EIYLGSGTI GSIFPFD RASSSVSY DTSTL QQWSSYPYT YYTEKFKG Y MY AS 5 DYVIG EIYLGSGTI GSIFPFD RASSSVSY DTSTL QQWSSYPYT YYAEKFKG Y MY AS 6 DYVIG EIYLGSGTI GSIFPFD SASSSVSY DASTL QQWSSYPYT YYTEKFKG Y MY AS 7 DYVIG EIYLGSGTI GSIFPFD SASSSVSY DTSTL QQWSSYPYT YYTEKFKG Y MY QS 8 SYWIN NIYPSNSYT YRSDGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIH IS 9 SYWIN NIYPSNSYT YRSDGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIE IS 10 SYWIN NIYPSNSYT YRSEGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIE IS 11 SYWIN NIYPSNSYT YRSGGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIE IS 12 SYWIN NIYPSNSYT YRSDAFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIE IS 13 SYWIN NIYPSNSYT YRSDGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIS IS 14 SYWIN NIYPSNSYT YRSEGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SSI IS 15 SYWIN NIYPSNSYT YRSGGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIS IS 16 SYWIN NIYPSNSYT YRSDAFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIS IS 17 SYWIN NIYPSNSYT YRSDGFA RASQNIGT FASES QQSNSWPFT KNYNQKFD Y SIA IS 18 SYWIN NIYPSNSYT YRSEGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIA IS 19 SYWIN NIYPSNSYT YRSGGFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIA IS 20 SYWIN NIYPSNSYT YRSDAFA RASQNIGT FASES QQSNSWPFT NYNQKFKD Y SIA IS 21 DSAMS SISRGDDTY DRFGFAY KSGQSLLD LVSML WQGTHFPFT YPDSVKG SDGKTYFN DS 22 SYWMH EITPSDNYT GHGNYVS RASQDISN YTSRL QQGYTLPPY SYNQKFKG FDY YLN HS T 23 SYWMH EITPSDNYT GEGNYVS RASQDISN YTSRL QQGYTLPPY SYNQKFKG FDY YLN HS T 24 SYWMH EITPSDNYG GHGNYVS RASQDISN YTSRL QQGYTLPPY SYNQKFKG FDY YLN ES T 25 SYWMH EITPSDNYT GEGNYVS RASQDISN YTSRL QQGYTLPPY SYNQKFKG FDY YLN ES T 26 SYWMH EITPSDNYG GHGNYVS RASQDISN YTSRL QQGYTLPPY SYNQKFKG FDY YLN QS T 27 SYWMH EITPSDNYG GHGNYVS RASQDISN YTSRL QQYYTLPPY SYNQKFKG FDY YLN HS T 28 SYWMH EITPGDNYT GHGNYVS RASQDISN YTSRL QQGYSLPPY SYNQKFKG FDY YLN HS T 29 SYWMH EITPSDNYG GHGNYVS RASQDISN YTSRL QQGYSLPPY SYNQKFKG FDY YLN HS T 30 SYWMH EITPSDNYT GEGNYVS RASQDISN YTSRL QQGYSLPPY SYNQKFKG FDY YLN HS T

    10. The antibody or fragment thereof according to claim 9, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ TD NOs: 1 to 17 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown; and/or, the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 18 to 36 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown.

    11. The antibody or fragment thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region comprised by the antibody or fragment thereof comprise one of the following: (1) an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ TD NO: 1; and, an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 18; (2) an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 2; and, an amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 19; (3) an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 3; and, an amino acid sequence as shown in SEQ ID NO: 20 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 20; (4) an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 4; and, an amino acid sequence as shown in SEQ ID NO: 20 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 20; (5) an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 3; and, an amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 21; (6) an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 4; and, an amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 21; (7) an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 3; and, an amino acid sequence as shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 22; (8) an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 3; and, an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 23; (9) an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 5; and, an amino acid sequence as shown in SEQ ID NO: 24 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 24; (10) an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 6; and, an amino acid sequence as shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 25; (11) an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 7; and, an amino acid sequence as shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 26; (12) an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 7; and, an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 27; (13) an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 8; and, an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 27; (14) an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 9; and, an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 27; (15) an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 10; and, an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 27; (16) an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 7; and, an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 28; (17) an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 8; and, an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 28; (18) an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 9; and, an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 28; (19) an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 10; and, an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 28; (20) an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 7; and, an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 29; (21) an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 8; and, an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 29; (22) an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 9; and, an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 29; (23) an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 10; and, an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 29; (24) an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 11; and, an amino acid sequence as shown in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 30; (25) an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 12; and, an amino acid sequence as shown in SEQ ID NO: 31 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 31; (26) an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 13; and, an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 32; (27) an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 16; and, an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 32; (28) an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 14; and, an amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 33; (29) an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 16; and, an amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 33; (30) an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 14; and, an amino acid sequence as shown in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 34; (31) an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 14; and, an amino acid sequence as shown in SEQ ID NO: 35 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 35; (32) an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 15; and, an amino acid sequence as shown in SEQ ID NO: 36 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 36; (33) an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 14; and, an amino acid sequence as shown in SEQ ID NO: 36 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 36; and (34) an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 16; and, an amino acid sequence as shown in SEQ ID NO: 36 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown in SEQ ID NO: 36

    12. The antibody or fragment thereof according to claim 11, wherein the antibody or fragment thereof is in any form, e.g., a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody and the like; alternatively, the antibody or fragment thereof is a half-antibody or an antigen-binding fragment of the half-antibody, e.g., scFv, BsFv, dsFv, (dsFv).sub.2, Fab, Fab, F(ab).sub.2, or Fv; preferably, the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or heavy chain constant region (CH); more preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE and/or a kappa or lambda type light chain constant region.

    13. The antibody or fragment thereof according to claim 12, wherein the antibody is a monoclonal antibody, preferably a murine, chimeric, or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of IgG1 or IgG4 subtype and the light chain constant region of the monoclonal antibody is of kappa type; preferably, the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 37 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown; and preferably, the light chain constant region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO: 38 or an amino acid sequence having at least 75% identity to the amino acid sequence as shown.

    14. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or fragment thereof according claim 13, or encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain comprised in the antibody or fragment thereof.

    15. A vector comprising the nucleic acid molecule according to claim 14.

    16. A host cell comprising the nucleic acid molecule and/or the vector, or transformed or transfected with the nucleic acid molecule and/or the vector according to claim 15.

    17. A pharmaceutical composition comprising the antibody or fragment thereof, the nucleic acid molecule, the vector, or the host cell according to claim 16, and optionally a pharmaceutically acceptable excipient.

    18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition comprises a further antibody-based drug; preferably, the further antibody-based drug is an antibody against macrophage-related immune checkpoint, more preferably an anti-CD47 antibody.

    19. Use of the antibody or fragment thereof, the nucleic acid molecule, the vector, the host cell, and/or the pharmaceutical composition according to claim 18 in the manufacture of a medicament; preferably, the medicament is a medicament for treating a Trop-2 high expression cancer; preferably, the Trop-2 high expression cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple negative breast cancer, or cervical cancer.

    20. A kit comprising the antibody or fragment thereof, the nucleic acid molecule, the vector, the host cell, or the pharmaceutical composition according to claim 18.

    21. A fusion protein comprising the antibody or fragment thereof according to claim 13.

    22. A conjugate comprising the antibody or fragment thereof according to claim 13 and a drug conjugated thereto, wherein the drug is a cytotoxic agent.

    23. The conjugate according to claim 22, wherein the conjugate is an antibody drug conjugate (ADC) represented by the formula: (an antibody or fragment thereof according to any one of claims 1 to 13)-(linker)-(a cytotoxic agent); preferably, the cytotoxic agent is a tubulin inhibitor (such as paclitaxel, docetaxel, etc.) or a DNA replication inhibitor (such as irinotecan or its active metabolite SN-38, etc.).

    24. Use of the antibody or fragment thereof, the nucleic acid molecule, the vector, or the host cell according to claim 16 in the manufacture of an antibody drug conjugate (ADC); preferably, the ADC is an ADC for treating a Trop-2 high expression cancer; preferably, the Trop-2 high expression cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple negative breast cancer, or cervical cancer.

    25. A method for preventing and/or treating a disease, including administering to a subject in need thereof the antibody or fragment thereof, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition, the fusion protein, or the conjugate according to claim 23, and optionally other drug or means; preferably, the disease is a Trop-2 high expression cancer; preferably, the Trop-2 high expression cancer is gastric cancer, pancreatic cancer, intestinal cancer, ovarian cancer, squamous lung cancer, non-small cell lung cancer, small cell lung cancer, urothelial cancer, triple negative breast cancer, or cervical cancer.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0115] Embodiments of the present invention are described in detail below with reference to the attached figures, in which:

    [0116] FIG. 1 shows the screening results of binding of positive hybridoma supernatants to Trop-2 on the surface of CHO cells.

    [0117] FIG. 2 shows the screening results of binding of positive hybridoma supernatants to Trop-2 on the surface of CHO cells.

    [0118] FIG. 3 shows the detection results via ELISA of cross-reactivity of positive hybridoma supernatants with recombinant Trop-2 proteins of different species.

    [0119] FIG. 4 shows the detection results via ELISA of binding activity of anti-human Trop-2 chimeric antibodies to recombinant Trop-2 protein, in which panel 4A: ch3-11; panel 4B: ch4-3; panel 4C: ch23-12; panel 4D: ch11-4; panel 4E: ch17-1.

    [0120] FIG. 5 shows the detection results via FACS of binding activity of anti-human Trop-2 chimeric antibodies to recombinant Trop-2 protein on the surface of cells, in which panel 5A: ch3-11; panel 5B: ch23-12; panel 5C: ch11-4; panel 5D: ch4-3; panel 5E: ch17-1.

    [0121] FIG. 6 shows the detection results via ELISA of species-specific binding of anti-human Trop-2 antibodies to Trop-2, in which panel 6A: h23-12; panel 6B: h4-3; panel 6C: Sacituzumab.

    [0122] FIG. 7 shows the analysis results of affinity of anti-human Trop-2 antibodies for recombinant human Trop-2 extracellular domain, in which panel 7A: Sacituzumab; panel 7B: h23-12; panel 7C: h4-3.

    [0123] FIG. 8 shows the observations of internalization of anti-Trop-2 humanized antibodies after binding to Trop-2 on the surface of N87 cells.

    [0124] FIG. 9 shows the drug concentration versus time curves after single doses of anti-Trop-2 humanized antibodies in nude mice (detected with Trop-2), in which panel 9A: h23-12; panel 9B: h4-3.

    [0125] FIG. 10 shows the inhibitory rates of anti-Trop-2-ADCs on cell growth.

    [0126] FIG. 11 shows the curves of changes in body weight of BALB/c nu mice bearing tumors in a mouse model of N87 gastric cancer, in which panel 11A: after the administration of ch4-3-SN38; panel 11B: after the administration of h23-12-SN38; panel 11C: after the administration of isotype control antibody; panel 11D: after the high dosage administration of ch4-3-SN38 and h23-12-SN38.

    [0127] FIG. 12 shows the curves of changes in tumor volume of BALB/c nu mice bearing tumors in a mouse model of N87 gastric cancer, in which panel 12A: after the administration of ch4-3-SN38; panel 12B: after the administration of h23-12-SN38; panel 12C: after the administration of isotype control antibody; panel 12D: after the high dosage administration of ch4-3-SN38 and h23-12-SN38.

    [0128] FIG. 13 shows the curves of changes in body weight of mice in SKOV3 subcutaneous xenograft mouse model with the co-administration of anti-Trop-2 antibody and anti-CD47 antibody.

    [0129] FIG. 14 shows the curves of changes in tumor volume of mice in SKOV3 subcutaneous xenograft mouse model with the co-administration of anti-Trop-2 antibody and anti-CD47 antibody.

    [0130] FIG. 15 shows the curves of changes in tumor volume of BALB/c nu mice bearing tumors in a subcutaneous xenograft mouse model of NCI-N87 gastric cancer.

    [0131] FIG. 16 shows the curves of changes in body weight of BALB/c nu mice bearing tumors in a subcutaneous xenograft mouse model of NCI-N87 gastric cancer.

    DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

    [0132] The present invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way.

    [0133] Experimental procedures in the following examples are all conventional ones, unless otherwise specified. Raw materials and reagents used in the following examples are all commercially available products, unless otherwise specified.

    Example 1 Preparation of Hybridoma Cells Secreting Anti-Human Trop-2 Antibodies

    [0134] Immunization: Balb/c mice were immunized with recombinant human Trop-2 protein (ACCESSION NO.: NP_002344.2, AA 1-274), and serum titers were detected via ELISA in 96-well ELISA plates coated with recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274). Mice with serum titers meeting the requirements for cell fusion were used for cell fusion of the next step.

    [0135] Cell fusion and hybridoma preparation: mice with required titers were selected to perform rush immunization, and spleens were collected aseptically from the mice 3 days later. B lymphocyte suspensions were prepared and mixed with SP2/0 myeloma cells at a ratio of 4:1, to make the cells fuse in the presence of PEG. The fused cells were resuspended in HAT medium, inoculated into 96-well cell culture plates, and cultured in an incubator at 37? C., 5% CO.sub.2.

    Example 2 Screening of Positive Hybridoma Cell Lines Secreting Anti-Human Trop-2 Antibodies

    [0136] 1. Screening of Binding of Positive Hybridomas

    [0137] 10-14 days after fusion, ELISA plates were coated with recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274) (20 ng/ml) overnight at 4? C.; washed with PBS 3 times, the plates were blocked with 4% skimmed milk powder-PBS at room temperature for 1 h; then the plates were washed with PBS 3 times, and culture supernatants of hybridoma clones were added into the plates and incubated at room temperature for 1 h. Controls as follows were set: (1) Positive Control (PC): serum from mice post immunization (1:1000 diluted in PBS); (2) Negative Control (NC): fusion well without cell growth. The plates were washed with PBST (0.05% Tween-PBS) 3 times, and washed with PBS 2 times, and then HRP-goat anti-mouse IgG (Fc gamma) was added into the plates for incubation at 37? C. for 0.5 h. Afterwards, the plates were washed with PBST (0.05% Tween 20-PBS) 3 times again, and TMB substrate was added for color development in dark for 15-30 min. Then ELISA stop solution was added to stop reaction; and A450 values were read using a microplate reader.

    [0138] The clones were ordered from highest reading to lowest reading, and the top 95 clones with high readings were selected for double-checking via ELISA, and a pool of 25 positive cells secreting antibodies was subcloned by limiting dilution. 10 days after plating, culture supernatants of monoclonal cells were selected for further screening for positive clones via ELISA which was performed as above described. The clones were ordered from highest reading to lowest reading again, and the top 21 clones with high readings, i.e., mi-1, m3-11, m4-3, m5-5, m6-6, m7-13, m11-4, m12-2, m12-4, m13-2, m14-2, m15-3, m16-7, m17-1, m18-4, m19-5, m20-4, m21-1, m22-1, m23-12 and m24-3, were selected for the next step screening of binding via FACS.

    [0139] 2. Screening of Binding of Positive Hybridomas to Trop-2 on the Surface of CHO Cells

    [0140] The reading frame of Trop-2 gene was cloned from a vector containing Trop-2 cDNA (Cat.: HG10428-M, Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.) by PCR, and cloned into a stable expression vector containing Glutamine Synthetase (GS) gene by enzyme digestion for screening. Suspension cultured CHO-K1 cells were electrotransfected (Nucleofector IIb, Lonza) and the transfected cells were transferred into CD CHO AGT? medium (Cat.: 12490-025, Gibco) containing 50 ?M MSX (Cat.: M5379, Sigma), and inoculated in 96-well cell culture plates. After being allowed to stand at 37? C., 5% CO.sub.2 for 2-3 weeks, 22 wells containing cells were obtained through prescreening with MSX pressure screening, and the cells were expanded in 24-well cell culture plates, and finally the clone No. 1-T-21 (CHO/Trop-2 cells) was selected via flow cytometry (FACS) analysis. Scale-up culture of the clone was performed and the cells were cryopreserved and used for FACS detection.

    [0141] According to the above ELISA results, supernatants of the 21 hybridoma clones selected were diluted 100 times and incubated with suspensions of the constructed CHO cells (CHO/Trop-2 cells) at 37? C. for 30 min. Controls as follows were set: (1) Positive Control (PC): Sacituzumab, a version having murine IgG constant region, 1 ?g/ml; (2) Negative Control (NC): irrelevant murine antibody, 1 ?g/ml. The cells were washed 3 times with PBS, and 1:200 dilution of goat anti-mouse IgG-FITC (Cat.: F9006, Sigma) was added into the cells which then were incubated for 30 min. Then the cells were washed 3 times with PBS, and the Mean Fluorescence Intensity (MFI) of the cells was measured by a flow cytometer (model no. B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by each hybridoma could bind Trop-2 on the surface of CHO cells. The results are shown in FIG. 1.

    [0142] Based on FIG. 1 and states of the cells, clones ml-1, m3-11, m4-3, m6-6, m7-13, m11-4, m12-2, m12-4, m13-2, m14-2, m16-7, m17-1, m19-5, m21-1, and m23-12 were selected, supernatants of those hybridomas were purified through ProA affinity chromatography column, and binding of the purified murine antibodies was confirmed again. The antibodies were diluted to 13 nM and 0.66 nM respectively, and then incubated with a suspension of CHO cells recombinantly expressing human Trop-2 (CHO/Trop-2 cells) for 30 min at 37? C. Controls as follows were set: (1) Positive Control (PC): Sacituzumab, a version having murine IgG constant region, 1 ?g/ml; (2) Negative Control (NC): irrelevant murine antibody, 1 ?g/ml. The cells were washed 3 times with PBS, and 1:200 dilution of goat anti-mouse IgG-FITC (Cat.: F9006, Sigma) was added into the cells which then were incubated for 30 min. Then, the cells were washed 3 times with PBS, and the Mean Fluorescence Intensity (MFI) of the cells was measured by a flow cytometer (model no. B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by each hybridoma could bind Trop-2 on the surface of CHO cells. As shown in FIG. 2, all the antibodies from the supernatants of 15 clones bound to Trop-2 on the surface of CHO cells well.

    [0143] Clones m3-11, m4-3, m11-4, m17-1, and m23-12 were selected as candidate clones for further screening.

    [0144] 3. Screening Via ELISA of Cross-Reactivity of Positive Hybridoma Clones

    [0145] Plates were coated with recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274), recombinant cynomolgus Trop-2-his protein (ACCESSION NO.: UniProtKB-A0A2K5UE71, AA 1-272), and recombinant mouse Trop-2-his protein (Cat.: 50922-M08H, Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.) respectively overnight at 4? C., each protein having a coating concentration of 0.2, and 1 ?g/ml respectively. Washed with PBS 3 times, the plates were blocked with the added 5% BSA PBS at 37? C. for 60 min, and then washed 3 times with PBST. The 15 purified murine antibodies were diluted to 1 ?g/ml with PBS. Controls as follows were set: (1) Positive Control (PC): Sacituzumab (WHO Drug Information (Vol. 31, No. 1, 2017), SEQ ID NO: 39 and SEQ ID NO: 40), a version having murine IgG constant region, 1 g/ml; (2) Negative Control (NC): an antibody from an irrelevant hybridoma, 1 ?g/ml; (3) blank control: PBS. After incubation at 37? C. for 60 min, the plates were washed 4 times with PBST. 1:5000 dilution of HRP-goat anti-mouse IgG (Fc gamma) (Cat: 115-035-071; Jackson ImmunoResearch) was added into the plates for incubation at 37? C. for 30 min. Afterwards, the plates were washed with PBST 4 times, and TMB substrate was added for color development at 37? C. for 10 min. Then 2M HCl was added to stop reaction; and absorbances at 450 nm and at 630 nm (as reference wavelength) were read, and A450 nm-630 nm values of the wells in the plates were recorded. Except that the antibodies from clones m12-4, m17-1, m19-5, and m21-1 exhibited cross-reactivity with mouse Trop-2, other antibodies exhibited no cross-reactivity with mouse Trop-2; and all the antibodies of the hybridomas could specifically bind recombinant human and cynomolgus Trop-2 (FIG. 3).

    Example 3 Sequencing of Murine Anti-Human Trop-2 Antibodies

    [0146] After expanded culture of hybridomas m3-11, m4-3, m11-4, m17-1, and m23-12 secreting anti-human Trop-2 antibodies, subtypes of the antibodies were detected using Mouse Monoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo) and Mouse Monoclonal Antibody Light/Heavy Chain Test Card (Cat.: A12401, VicNovo) according to Reagent Protocol. Subtypes were identified as: the antibodies were IgG1 for heavy chains, and were Kappa for light chains. The results provided a basis for gene cloning of the antibodies from m3-11, m4-3, m11-4, m17-1, and m23-12.

    [0147] Total RNA was extracted from the hybridoma cells m3-11, m4-3, ml 1-4, m17-1, and m23-12 using TRIzol (Cat: 15596026, Invitrogen) following the steps described in the instructions. The total RNA of the hybridoma cells was reversely transcribed into cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB). The sequences of light chain variable region IgVL (?) and heavy chain variable region VH of the antibodies were amplified using degenerate primers (see Zhiwei DONG and Yan Wang, Antibody Engineering (2nd Edition), Peking University Medical Press, 2001, pages 313-314) and Phusion kit (Cat: E0553L, NEB). PCR products were purified with gel extraction kit (Cat: AP-GX-250, Axygen) and the purified PCR products were ligated to T vector following the instructions of a T vector cloning Kit (Cat: ZC205, Beijing Zoman Biotechnology Co., Ltd.) and then the obtained vectors were transformed into competent E. coli cells for amplification. The plasmids were then extracted for DNA sequencing to obtain variable region sequences of the monoclonal antibodies.

    [0148] Sequencing Results Showed:

    [0149] The nucleotide sequence (DNA) of the heavy chain variable region of the murine antibody from clone m3-11 is as shown in SEQ ID NO: 41, and the amino acid sequence of the heavy chain variable region of the murine antibody from clone m3-11 deduced from the nucleotide sequence is as shown in SEQ ID NO: 1; and the nucleotide sequence (DNA) of the light chain variable region of the murine antibody from clone m3-11 is as shown in SEQ ID NO: 42, and the amino acid sequence of the light chain variable region of the murine antibody from clone m3-11 deduced from the nucleotide sequence is as shown in SEQ ID NO: 18.

    TABLE-US-00002 SEQIDNO:1: QVQLQQPGAELVKPGSSVKLSCKASGYTFTSYWMYWVKQRPGQGLEWIG EINPSNGRTNYNEKFKSKATLTVDKSSSTAYMQFSSLTSEDSAVYYCTR EGHNYDGSLGAMDHWGQGTSVTVSS SEQIDNO:18: DVVVTQTPLSLPVSFGDQVSISCRSSQSLTNSYGNTFLSWYLHKPGQSP QLLLYGISNRFSGVPDRFSGSGSGTDFTLKINTIKPEDLGMYYC FQSTHQPYTFGGGTKLEIK

    [0150] The nucleotide sequence (DNA) of the heavy chain variable region of the murine antibody from clone m4-3 is as shown in SEQ ID NO: 43, and the amino acid sequence of the heavy chain variable region of the murine antibody from clone m4-3 deduced from the nucleotide sequence is as shown in SEQ ID NO: 2; and the nucleotide sequence (DNA) of the light chain variable region of the murine antibody from clone m4-3 is as shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the murine antibody from clone m4-3 deduced from the nucleotide sequence is as shown in SEQ ID NO: 19.

    TABLE-US-00003 SEQIDNO:2: QVQLQQSGPELVKPGASVKMSCKASGFTFTDYVIGWVKQRTGQGLEWIG EIYLGSGTIYYTEKFKGKATLTADTSSNTAYMQLSSLTSEDSAVYFCAR GSIFPFDYWGQGTTLTVSS SEQIDNO:19: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIY DTSTLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYC QQWSSYPYTFGGGTKLEIK

    [0151] The nucleotide sequence (DNA) of the heavy chain variable region of the murine antibody from clone m11-4 is as shown in SEQ ID NO: 47, and the amino acid sequence of the heavy chain variable region of the murine antibody from clone m11-4 deduced from the nucleotide sequence is as shown in SEQ ID NO: 5; and the nucleotide sequence (DNA) of the light chain variable region of the murine antibody from clone m11-4 is as shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region of the murine antibody from clone ml 1-4 deduced from the nucleotide sequence is as shown in SEQ ID NO: 24.

    TABLE-US-00004 SEQIDNO:5: QVQLQQPGAELVRPGASVNLSCKASGYTFTSYWINWVKQRPGQGLEWIG NIYPSNSYTNYNQKFKDTATLTVDKSSSTAYMQLSSPTSEDSAVYFCSS YRSDGFAYWGQGTLVTVSA SEQIDNO:24: DILLTQSPAILSVSPGEKVSFSCRASQNIGTSIHWYQQRINGSPRLLIE FASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYC QQSNSWPFTFGGGTKLEIK

    [0152] The nucleotide sequence (DNA) of the heavy chain variable region of the murine antibody from clone m17-1 is as shown in SEQ ID NO: 51, and the amino acid sequence of the heavy chain variable region of the murine antibody from clone m17-1 deduced from the nucleotide sequence is as shown in SEQ ID NO: 11; and the nucleotide sequence (DNA) of the light chain variable region of the murine antibody from clone m17-1 is as shown in SEQ ID NO: 52, and the amino acid sequence of the light chain variable region of the murine antibody from clone m17-1 deduced from the nucleotide sequence is as shown in SEQ ID NO: 30.

    TABLE-US-00005 SEQIDNO:11: EVKLVESGGVLVKPGGSLKLSCAASGFTFSDSAMSWVRQTPEKRLEWVA SISRGDDTYYPDSVKGRITISRDFARNILYLQMTSLRSEDTAMYYCTR DRFGFAYWGQGTLVTVSA SEQIDNO:30: DIVMTQSPLTLSVTIGQPASISCKSGQSLLDSDGKTYFNWLLQRPGQSP KRLIYLVSMLDSGVPDRFTGSGSGTDFTLKISRVETEDLGVYYC WQGTHFPFTFGSGTKLEIK

    [0153] The nucleotide sequence (DNA) of the heavy chain variable region of the murine antibody from clone m23-12 is as shown in SEQ ID NO: 53, and the amino acid sequence of the heavy chain variable region of the murine antibody from clone m23-12 deduced from the nucleotide sequence is as shown in SEQ ID NO: 12; and the nucleotide sequence (DNA) of the light chain variable region of the murine antibody from clone m23-12 is as shown in SEQ ID NO: 54, and the amino acid sequence of the light chain variable region of the murine antibody from clone m23-12 deduced from the nucleotide sequence is as shown in SEQ ID NO: 31.

    TABLE-US-00006 SEQIDNO:12: QVQLQQPGAELVKPGASVKLSCKADGYIFTSYWMHWVKQRPGQGLEWIG EITPSDNYTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTR GHGNYVSFDYWGQGTTLTVSS SEQIDNO:31: DIQMTQITSSLSASLGDRVTITCRASQDISNYLNWYQQKPDGTVKLLIY YTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGYTLPPYTFGGGTKLEIK

    Example 4 Preparation of Anti-Human Trop-2 Chimeric Antibodies and Control Antibody

    [0154] The light and heavy chain sequences of control antibody (Sacituzumab) were fully synthesized, and cloned into a eukaryotic transient-expression vector respectively to obtain expression plasmids expressing the light chain and heavy chain of the control antibody. The expression plasmids were transformed into E. coli cells for amplification, and a large number of plasmids containing the light chain and heavy chain of the control antibody respectively were obtained through plasmid recovery. The plasmids containing the light chain and heavy chain of the control antibody were in turn transfected into HEK293 cells respectively using 293fectin (Cat.: 12347019, Gibco) transfection reagent following the manufacturer's instructions for recombinant expression. 5-6 days after cell transfection, culture supernatant was taken and purified through ProA affinity chromatography column to obtain the control antibody. The amino acid sequences of the control antibody Sacituzumab were derived from WHO Drug Information (Vol. 31, No. 1, 2017), and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 39, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 40.

    [0155] The light chain variable region and heavy chain variable region genes of the murine antibodies 3-11, 4-3, 11-4, 17-1 and 23-12 obtained from the clones as above, with cleavage sites of restriction enzymes introduced by PCR, were respectively cloned into the upstream of gene encoding human-kappa light chain constant region and the upstream of gene encoding human IgG1 heavy chain constant region carried in eukaryotic transient-expression vectors. The obtained expression plasmids expressing human-murine chimeric light chain (pKN019-ch3-11L, pKN019-ch4-3L, pKN019-ch11-4L, pKN019-ch17-1L, pKN019-ch23-12L) and expression plasmids expressing human-murine chimeric heavy chain (pKN 041-ch3-11H, pKN019-ch4-3H, pKN019-ch11-4H, pKN019-ch17-1H, pKN019-ch23-12H) were transformed into E. coli cells for amplification, and a large number of plasmids containing human-murine chimeric light chain and heavy chain respectively were obtained through plasmid recovery. The plasmids containing the light chains and heavy chains of chimeric antibodies ch3-11, ch4-3, ch11-4, ch17-1 and ch23-12 were in turn transfected into HEK293 cells respectively using 293fectin (Cat.: 12347019, Gibco) transfection reagent following the manufacturer's instructions for recombinant expression. 5-6 days after cell transfection, culture supernatants were taken and purified through ProA affinity chromatography columns to obtain chimeric antibodies ch3-11, ch4-3, ch11-4, ch17-1 and ch23-12.

    Example 5 Detection Via ELISA of Binding Activity of Anti-Human Trop-2 Chimeric Antibodies to Recombinant Trop-2 Protein

    [0156] Plates were coated with recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274) at a concentration of 0.2 ?g/ml overnight at 4? C. and then blocked with 5% BSA for 60 min in a constant temperature incubator at 37? C. Chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 and control antibody Sacituzumab (serial dilutions of 8 concentrations in total obtained through 3-fold serially diluting a solution with an initial concentration of 2 ?g/ml) were added into the plates which were then incubated in a constant temperature incubator at 37? C. for 60 min. The plates were washed with PBST 4 times, and 1:5000 dilution of HRP-anti-human Fc (Cat.: 109-035-098, Jackson ImmunoResearch) was added into the plates for reaction for 45 min. TMB (Cat.: ME142, GalaxyBio, Beijing) substrate was added for color development for 15 min, and then 2M HCl was added to stop reaction. Absorbances at 450 nm and at 630 nm (as reference wavelength) were read, and A450 nm-630 nm values of the wells in the plates were recorded.

    [0157] The binding ability of each of ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 and control antibody Saituzumab to recombinant human Trop-2 protein was determined by ELISA, and the half maximal effective concentration (EC50) values about the binding of the antibodies were 0.3147 nM, 0.3195 nM, 0.3278 nM, 0.2366 nM, 0.4581 nM and 0.271 nM, respectively (FIG. 4), which were roughly equivalent. The results showed that chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4 and ch23-12 had high affinity to recombinant human Trop-2 protein, and the sequences of the murine antibodies 3-11, 4-3, 11-4, 17-1 and 23-12 were correctly cloned.

    Example 6 Detection Via FACS of Binding Activity of Anti-Human Trop-2 Chimeric Antibodies to Recombinant Human Trop-2 Protein on the Surface of CHO Cells

    [0158] Suspensions of CHO cells recombinantly expressing human Trop-2 (CHO/Trop-2 cells) were incubated with chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 (dilutions at concentrations of 30 ?g/ml and 10 ?g/ml, and serial dilutions of 9 concentrations obtained through 3-fold serially diluting a solution with an initial concentration of 5 ?g/ml, 11 concentrations in total) for 30 min at 37? C. Controls as follows were set: (1) Positive Control (PC): control antibody Sacituzumab; (2) Negative Control (NC): IgG1 isotype control antibody NC-IgG1. The cells were washed 3 times with PBS, and 1:100 dilution of goat anti-human IgG-FITC (Cat.: F9512, Sigma) was added into the cell which then were incubated for 30 min. Then the cells were washed 3 times with PBS again, and the Mean Fluorescence Intensity (MFI) of the cells was measured by a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to detect binding ability of the chimeric antibodies to human Trop-2 on the surface of CHO cells.

    [0159] The binding ability of each of ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 and control antibody Sacituzumab to recombinant human Trop-2 protein on the surface of CHO cells was determined by FACS, and the half maximal effective concentration (EC50) values about binding of the antibodies were 0.993 nM, 3.326 nM, 2.918 nM, 1.154 nM, 2.748 nM, and 2.316 nM, respectively (FIG. 5). Compared with control antibody Sacituzumab, ch3-11 and ch11-4 had a better binding activity, and ch4-3, ch17-1 and ch23-12 had similar binding activity. The results showed that anti-human Trop-2 chimeric antibodies ch3-11, ch4-3, ch17-1, ch11-4, and ch23-12 could effectively bind recombinant human Trop-2 protein on the surface of CHO cells.

    Example 7 Internalization Activity of Anti-Human Trop-2 Chimeric Antibodies to Trop-2 on the Surface of Cells

    [0160] BxPC-3 human pancreatic cancer cells were added in an amount of 5?10.sup.5 cell/tube into tubes containing chimeric antibodies ch3-11, ch4-3, ch11-4, ch23-12, and positive control antibody Sacituzumab respectively, and each of the antibodies was diluted to 10 ?g/ml. Four groups were set for each antibody (experiment groups incubated for 1 h, 3 h, and 5 h respectively and control group) and each group included two tubes. The experimental groups were placed into an electric heating constant temperature incubator at 37? C., incubated for 1 h, 3 h, and 5 h respectively, and then placed on ice; and the control group was incubated always on ice as a negative control. When the incubation of all samples was completed, centrifugation was performed at 1,500 rpm at 4? C. for 3 min, and the supernatants were discarded. Cell pellets were washed with ice-cold PBS once, a secondary antibody, anti-human IgG (Fc-specific)-FITC antibody (Cat.: F9512, Sigma) was added into the cell which then were incubated on ice for 30 min. Afterwards, centrifugation was performed at 1,500 rpm for 3 min, and the supernatants were discarded. Cell pellets were washed with ice-cold PBS, and resuspended in 200 ?l of ice-cold PBS, and then FACS was performed to detect the mean fluorescence intensity (MFI). Internalization efficiency was calculated by the formula: % of MFI tx=MFI of sample incubated at 37? C./MFI of control sample incubated at 4? C.?100; and Internalization percentage (% tx)=100?% of MFI tx.

    [0161] The results are shown in Table 1, and as shown, ch4-3 and ch23-12 had internalization percentages similar to that of control antibody Sacituzumab, while no obvious internalization was observed for ch3-11 and ch11-4.

    TABLE-US-00007 TABLE 1 Internalization percentages of anti-human Trop-2 chimeric antibodies mediated by Trop-2 on the surface of cells Internalization percentage (%) 1 h 3 h 5 h ch3-11 1.12 ?5.13 ?8.48 ch11-4 ?2 ?7.4 ?9.38 ch23-12 16.24 21.12 27.15 ch4-3 17.05 23.15 28.1 Sacituzumab 17.16 21.84 20.35

    Example 8 Stability of Intolerance of Anti-Human Trop-2 Chimeric Antibodies to Disruption

    [0162] Chimeric antibodies ch3-11, ch4-3, ch11-4 and ch23-12 were placed in PBS, PBS containing 10% N,N-Dimethylacetamide (DMA) (Cat.: ARK2190, Shanghai Feibo Chemical Technology Co., Ltd.) and PBS containing 20% DMA, at a concentration of 5 mg/ml respectively, at 37? C. for 2 h. Afterwards, the samples were freed from DMA using an ultrafiltration centrifuge tube with PBS used for buffer exchange. The samples were analyzed for purity by size exclusion-high-performance liquid chromatography (SEC-IPLC) using G3000WXL liquid chromatography column (Cat.: SEC-0046, Tosoh Corporation). The analysis results of purity are shown in Table 2.

    [0163] The results showed that all the four antibodies could well tolerate DMA, with purity less obviously reduced in 10% DMA and slightly reduced in 20% DMA, suggesting the antibodies will be possibly well tolerant to the subsequent ADC conjugation process.

    TABLE-US-00008 TABLE 2 Purity of the antibodies analyzed by HPLC before and after DMA treatment ch23-12 ch3-11 ch4-3 ch11-4 PBS 97.52% 97.57% 99.47% 99.25% 10% DMA 97.18% 96.29% 98.44% 99.18% 20% DMA 93% 96.27% 94% 98.96%

    Example 9 Humanization and Recombinant Expression of Anti-Human Trop-2 Monoclonal Antibodies

    [0164] 1. Humanization of Murine Monoclonal Antibody 23-12

    [0165] (1) CDR Grafting

    [0166] Firstly, the heavy chain sequence of the murine antibody was comprehensively analyzed, and complementarity-determining regions (CDRs) of the antibody accounting for antigen binding and framework regions of the antibody supporting the conserved three-dimensional conformation of the antibody were determined. Subsequently, the most similar human template was searched for in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) based on homology alignment results, CDR grafting was performed according to full-sequence BLAST results in combination with sequence characteristics of the heavy chain CDR3, and the heavy chain variable region (VH) of the murine antibody 23-12 was fully humanized in the framework regions. The most similar human template was searched for in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) based on homology alignment results, CDR grafting was performed according to full-sequence BLAST results in combination with sequence characteristics of the light chain CDR3, and the light chain variable region (VL) of the murine antibody 23-12 was highly humanized in the framework regions.

    [0167] The nucleotide sequence and the amino acid sequence of the humanized heavy chain variable region (h23-12_VH1) of CDR grafted antibody 23-12 are as shown in SEQ ID NO: 55 and SEQ ID NO: 13 respectively; and the nucleotide sequence and the amino acid sequence of the humanized light chain variable region (h23-12_VL1) of CDR grafted antibody 23-12 are as shown in SEQ ID NO: 56 and SEQ ID NO: 32 respectively.

    TABLE-US-00009 SEQIDNO:13: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMG EITPSDNYTSYNQKFKGRVTITRDTSTSTAYMELSSLRSEDTAVYYCAR GHGNYVSFDYWGQGTLVTVSS SEQIDNO:32: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIY YTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQGYTLPPYTFGQGTKLEIKRTVAAP

    [0168] (2) Mutation Design in CDRs

    [0169] According to sequence characteristics of the murine antibody 23-12, mutations in CDR sequences in the CDR grafted humanized light and heavy chain variable regions were designed and the mutation sites are shown in Table 3.

    TABLE-US-00010 TABLE 3 Design of humanized sequences of murine antibody 23-12 Mutations relative to h23-12_VL1 Mutations relative to h23-12_VH1 h23-12_VL2 T51A h23-12_VH2 S54G h23-12_VL3 R53S h23-12_VH3 N56G h23-12_VL4 H55E h23-12_VH4 T58G h23-12_VL5 H55Q h23-12_VH5 N61A h23-12_VL6 G91Y h23-12_VH6 H100E h23-12_VL7 T93S h23-12_VH7 H100Q Note: amino acid residue positions were numbered according to the Kabat numbering system.

    [0170] 2. Recombinant Expression of Humanized Monoclonal Antibody 23-12

    [0171] The light chain variable region and heavy chain variable region (h23-12_VL1 and h23-12_VH1) genes of CDR grafted antibody 23-12 were fully synthesized. The humanized h23-12_VH1 gene was cloned by enzyme digestion into eukaryotic transient-expression vector pKN041 at the upstream of the coding gene of the heavy chain constant region of human IgG1, and the nucleotide sequence and the amino acid sequence of the heavy chain constant region are as shown in SEQ ID NO: 59 and SEQ ID NO: 37 respectively. The humanized h23-12_VL1 gene was cloned by enzyme digestion into eukaryotic transient-expression vector pKN019 at the upstream of the coding gene of human C? light chain, and the nucleotide sequence and the amino acid sequence of the light chain constant region are as shown in SEQ ID NO: 60 and SEQ ID NO: 38 respectively. As described, expression plasmids containing light and heavy chains of the CDR grafted antibody 23-12 were constructed and the obtained expression plasmids expressing light chain (pKN019-h23-12L1) and heavy chain (pKN019-h23-12H1) were transformed into E. coli cells for amplification, and then plasmids expressing the light chain h23-12L1 and the heavy chain h23-12H1 of the CDR grafted antibody 23-12 were isolated and obtained.

    [0172] According to the mutation design shown in Table 3, site-directed mutagenesis was performed in the expression plasmids expressing light chain (pKN019-h23-12L1) and heavy chain (pKN019-h23-12H1) respectively using StarMut gene Site-directed Mutagenesis Kit (Cat.: T111-01, GenStar). The mutated plasmids were transformed into E. coli cells for amplification, and expression plasmids expressing the light and the heavy chains of the humanized monoclonal antibodies with CDR mutations (h23-12H2 . . . h23-12H7; h23-12L2 . . . h23-12L7) were obtained, corresponding to the humanized sequences of murine antibody 23-12 shown in Table 3. All the plasmids containing various humanized heavy and light chain sequences of murine antibody 23-12 were combined as shown in Table 4 and transfected into HEK293 cells using 293fectin (Cat.: 12347019, Gibco) transfection reagent following the manufacturer's instructions for recombinant expression.

    TABLE-US-00011 TABLE 4 Combinations of humanized heavy and light chain sequences of murine antibody 23-12 h23-12H1 h23-12H2 h23-12H3 h23-12H4 h23-12H5 h23-12H6 h23-12H7 h23-12L1 h23-12-1 h23-12-2 h23-12-3 h23-12-4 h23-12-5 h23-12-6 h23-12-7 h23-12L2 h23-12-8 h23-12-9 h23-12-10 h23-12-11 h23-12-12 h23-12-13 h23-12-14 h23-12L3 h23-12-15 h23-12-16 h23-12-17 h23-12-18 h23-12-19 h23-12-20 h23-12-21 h23-12L4 h23-12-22 h23-12-23 h23-12-24 h23-12-25 h23-12-26 h23-12-27 h23-12-28 h23-12L5 h23-12-29 h23-12-30 h23-12-31 h23-12-32 h23-12-33 h23-12-34 h23-12-35 h23-12L6 h23-12-36 h23-12-37 h23-12-38 h23-12-39 h23-12-40 h23-12-41 h23-12-42 h23-12L7 h23-12-43 h23-12-44 h23-12-45 h23-12-46 h23-12-47 h23-12-48 h23-12-49 Note: Table 4 shows antibodies obtained from the combinations of various heavy and light chains derived from murine antibody 23-12. For example, h23-12-1 refers to an antibody composed of the humanized light chain h23-12L1 and humanized heavy chain h23-12H1 of the murine antibody 23-12, and so on.

    [0173] 5-6 days after cell transfection, culture supernatants were purified through ProA affinity chromatography column to obtain different humanized antibodies. Affinities of the obtained antibodies were determined by an assay including capturing Fc fragments of the antibodies with anti-human IgG Fc capture (AHC) biosensors using Octet QKe system instrument from Fortebio. For the assay, each of the humanized antibodies and control antibody Sacituzumab was diluted to 4 ?g/ml in PBS, and was allowed to flow through the surface of an AHC biosensor (Cat.: 18-0015, PALL) for 120 s. Recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274) was used as a mobile phase, and its concentration was 60 nM. The binding time was 100 s and the dissociation time was 300 s. When the assay was finished, data from which the response values of blank control had been deducted were fitted to a 1:1 Langmuir binding model using software, and then kinetic constants for antigen-antibody binding were calculated.

    [0174] Affinities of the antibodies obtained from the combinations of mutants of murine antibody 23-12, chimeric antibody ch23-12 and control antibody Sacituzumab to recombinant human Trop-2-his protein were determined by ForteBijo (Table 5).

    TABLE-US-00012 TABLE 5 Detection results of the affinities of the antibodies to recombinant human Trop-2 extracellular domain Antibody KD value (M) Sacituzumab 7.23E?10 ch23-12 7.15E?10 h23-12-1 2.72E?10 h23-12-2 8.93E?10 h23-12-3 1.50E?08 h23-12-4 8.27E?10 h23-12-5 3.20E?09 h23-12-6 2.98E?10 h23-12-7 6.80E?09 h23-12-8 9.00E?10 h23-12-9 1.10E?09 h23-12-10 9.62E?09 h23-12-11 8.81E?10 h23-12-12 2.62E?08 h23-12-13 9.16E?10 h23-12-14 2.39E?08 h23-12-15 1.43E?09 h23-12-16 2.87E?09 h23-12-17 2.80E?08 h23-12-18 9.91E?10 h23-12-19 1.00E?08 h23-12-20 1.46E?09 h23-12-21 2.28E?08 h23-12-22 1.30E?09 h23-12-23 7.70E?10 h23-12-24 5.21E?09 h23-12-25 5.02E?10 h23-12-26 5.43E?09 h23-12-27 4.96E?10 h23-12-28 4.13E?09 h23-12-29 6.72E?09 h23-12-30 9.76E?09 h23-12-31 6.39E?08 h23-12-32 6.01E?10 h23-12-33 8.91E?09 h23-12-34 5.57E?09 h23-12-35 9.77E?09 h23-12-36 8.81E?09 h23-12-37 8.93E?10 h23-12-38 7.86E?09 h23-12-39 4.92E?10 h23-12-40 2.16E?09 h23-12-41 7.82E?10 h23-12-42 3.09E?09 h23-12-43 9.80E?10 h23-12-44 7.17E?10 h23-12-45 4.09E?09 h23-12-46 3.12E?10 h23-12-47 2.31E?09 h23-12-48 4.05E?10 h23-12-49 2.99E?09

    [0175] Antibody h23-12-25 having an affinity measured by a KD value of 5.02E-10M was selected and was named as h23-12, which was used for further functional verification. The nucleotide sequence and the amino acid sequence of the heavy chain variable region of the antibody are as shown in SEQ ID NO: 57 and SEQ ID NO: 14 respectively; and the nucleotide sequence and the amino acid sequence of the light chain variable region of the antibody are as shown in SEQ ID NO: 58 and SEQ ID NO: 33 respectively.

    TABLE-US-00013 SEQIDNO:14: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMG EITPSDNYGSYNQKFKGRVTITRDTSTSTAYMELSSLRSEDTAVYYCAR GHGNYVSFDYWGQGTLVTVSS SEQIDNO:33: DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQGYTLPPYTFGQGTKLEIK

    [0176] 3. Humanization of Murine Monoclonal Antibody 4-3

    [0177] (1) CDR Grafting

    [0178] Firstly, the heavy chain sequence of the murine antibody was comprehensively analyzed, and complementarity-determining regions (CDRs) of the antibody accounting for antigen binding and framework regions of the antibody supporting the conserved three-dimensional conformation of the antibody were determined. Subsequently, the most similar human template was searched for in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) based on homology alignment results, CDR grafting was performed according to full-sequence BLAST results in combination with sequence characteristics of the heavy chain CDR3, and the heavy chain variable region (VH) of the murine antibody 4-3 was fully humanized in the framework regions. The most similar human template was searched for in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) based on homology alignment results, CDR grafting was performed according to full-sequence BLAST results in combination with sequence characteristics of the light chain CDR3, and the light chain variable region (VL) of the murine antibody 4-3 was fully humanized in the framework regions.

    [0179] The nucleotide sequence and the amino acid sequence of the humanized heavy chain variable region (h4-3_VH1) of CDR grafted antibody 4-3 are as shown in SEQ ID NO: 45 and SEQ ID NO: 3 respectively; and the nucleotide sequence and the amino acid sequence of the humanized light chain variable region (h4-3_VL1) of CDR grafted antibody 4-3 are as shown in SEQ ID NO: 46 and SEQ ID NO: 20 respectively.

    TABLE-US-00014 SEQIDNO:3: EVQLVQSGPEVKKPGASVKVSCKASGFTFTDYVIGWVRQAPGQGLEWIG EIYLGSGTIYYTEKFKGRVTMTADTSTSTAYMELSSLRSEDTAVYYCAR GSIFPFDYWGQGTLVTVSS SEQIDNO:20: DIQLTQSPSSLSASVGDRVTITCSASSSVSYMYWYQQKPGKAPKLLIY DTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSYPYTFGQGTKLEIK

    [0180] (2) Mutation Design in CDRs

    [0181] According to sequence characteristics of the murine antibody 4-3, mutations in CDR sequences in the CDR grafted humanized light and heavy chain variable regions were designed and the mutation sites are shown in Table 6.

    TABLE-US-00015 TABLE 6 Design of humanized sequences of murine antibody 4-3 Mutations relative to h4-3_VL1 Mutations relative to h4-3_VH1 h4-3_VL2 S24R h4-3_VH2 T61A h4-3_VL3 T50A h4-3_VH3 E62Q h4-3_VL4 A54Q h4-3_VH4 P103Y h4-3_VL5 S91N Note: amino acid residue positions were numbered according to the Kabat numbering system.

    [0182] 4. Recombinant Expression of Humanized Monoclonal Antibody 4-3

    [0183] The light chain variable region and heavy chain variable region (h4-3_VL1 and h4-3_VH1) genes of CDR grafted antibody 4-3 were fully synthesized. The humanized h4-3_VH1 gene was cloned by enzyme digestion into eukaryotic transient-expression vector pKN041 at the upstream of the coding gene of the heavy chain constant region of human IgG1, and the nucleotide sequence and the amino acid sequence of the heavy chain constant region are as shown in SEQ ID NO: 59 and SEQ ID NO: 37 respectively. The humanized h4-3_VL1 gene was cloned by enzyme digestion into eukaryotic transient-expression vector pKN019 at the upstream of the coding gene of human C? light chain, and the nucleotide sequence and the amino acid sequence of the light chain constant region are as shown in SEQ ID NO: 60 and SEQ ID NO: 38 respectively. As described, expression plasmids containing light and heavy chains of the CDR grafted antibody 4-3 were constructed and the obtained expression plasmids expressing light chain (pKN019-h24-3L1) and heavy chain (pKN019-h4-3H1) were transformed into E. coli cells for amplification, and then plasmids expressing the light chain h4-3L1 and the heavy chain h4-3H1 of the CDR grafted antibody 4-3 were isolated and obtained.

    [0184] According to the mutation design shown in Table 6, site-directed mutagenesis was performed in the expression plasmids expressing light chain (pKN019-h4-3L1) and heavy chain (pKN019-h4-3H1) respectively using StarMut gene Site-directed Mutagenesis Kit (Cat.: T111-01, GenStar). The mutated plasmids were transformed into E. coli cells for amplification, and obtained expression plasmids expressing the light and the heavy chains of the humanized monoclonal antibodies with CDR mutations (h4-3H2 . . . h4-3H4; h4-3L2 . . . h4-3L5), corresponding to the humanized sequences of murine antibody 4-3 shown in Table 6. All the plasmids containing various humanized heavy and light chain sequences of murine antibody 4-3 were combined as shown in Table 7 and transfected into HEK293 cells using 293fectin (Cat.: 12347019, Gibco) transfection reagent following the manufacturer's instructions for recombinant expression.

    TABLE-US-00016 TABLE 7 Combinations of humanized heavy and light chain sequences of murine antibody 4-3 h4-3 H1 h4-3 H2 h4-3 H3 h4-3 H4 h4-3 L1 h4-3-1 h4-3-2 h4-3-3 h4-3-4 h4-3 L2 h4-3-5 h4-3-6 h4-3-7 h4-3-8 h4-3 L3 h4-3-9 h4-3-10 h4-3-11 h4-3-12 h4-3 L4 h4-3-13 h4-3-14 h4-3-15 h4-3-16 h4-3 L5 h4-3-17 h4-3-18 h4-3-19 h4-3-20 Note: Table 7 shows antibodies obtained from the combinations of various heavy and light chains derived from murine antibody 4-3. For example, h4-3-1 refers to an antibody composed of the humanized light chain h4-3L1 and humanized heavy chain h4-3H1 of the murine antibody 4-3, and so on.

    [0185] 5-6 days after cell transfection, culture supernatants were purified through ProA affinity chromatography column to obtain different humanized antibodies. Affinities of the obtained antibodies were determined by an assay including capturing Fc fragments of the antibodies with anti-human IgG Fc capture (AHC) biosensors using Octet QKe system instrument from Fortebio. For the assay, each of the humanized antibodies and control antibody Sacituzumab was diluted to 4 ?g/ml in PBS, and was allowed to flow through the surface of an AHC biosensor (Cat.: 18-0015, PALL) for 120 s. Recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274) was used as a mobile phase, and its concentration was 60 nM. The binding time was 100 s and the dissociation time was 300 s. When the assay was finished, data from which the response values of blank control had been deducted were fitted to a 1:1 Langmuir binding model using software, and then kinetic constants for antigen-antibody binding were calculated.

    [0186] Affinities of the antibodies obtained from the combinations of mutants of murine antibody 23-12, chimeric antibody ch4-3 and control antibody Sacituzumab to recombinant human Trop-2-his protein were determined by ForteBio (Table 8).

    TABLE-US-00017 TABLE 8 Detection results of the affinities of the antibodies to recombinant human Trop-2 extracellular domain Antibody KD value (M) Sacituzumab 7.23E?10 ch4-3 3.00E?10 h4-3-1 3.04E?10 h4-3-2 4.11E?10 h4-3-3 5.01E?10 h4-3-4 6.36E?10 h4-3-5 2.73E?10 h4-3-6 3.97E?10 h4-3-7 4.66E?10 h4-3-8 8.62E?10 h4-3-9 3.17E?10 h4-3-10 7.63E?10 h4-3-11 8.02E?10 h4-3-12 1.03E?09 h4-3-13 2.81E?10 h4-3-14 5.71E?10 h4-3-15 6.87E?10 h4-3-16 1.53E?09 h4-3-17 5.42E?10 h4-3-18 6.71E?10 h4-3-19 5.99E?10 h4-3-20 8.92E?10

    [0187] Antibody h4-3-1 having an affinity measured by a KD value of 3.04E-10M was selected and was named as h4-3, which was used for further functional verification. The nucleotide sequence and the amino acid sequence of the heavy chain variable region of the antibody are as shown in SEQ ID NO: 45 and SEQ ID NO: 3 respectively; and the nucleotide sequence and the amino acid sequence of the light chain variable region of the antibody are as shown in SEQ ID NO: 46 and SEQ ID NO: 20 respectively.

    TABLE-US-00018 SEQIDNO:3: EVQLVQSGPEVKKPGASVKVSCKASGFTFTDYVIGWVRQAPGQGLEWIG EIYLGSGTIYYTEKFKGRVTMTADTSTSTAYMELSSLRSEDTAVYYCAR GSIFPFDYWGQGTLVTVSS SEQIDNO:20: DIQLTQSPSSLSASVGDRVTITCSASSSVSYMYWYQQKPGKAPKLLIY DTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSYPYTFGQGTKLEIK

    [0188] 5. Humanization of Murine Monoclonal Antibody 11-4

    [0189] (1) CDR Grafting

    [0190] Firstly, the heavy chain sequence of the murine antibody was comprehensively analyzed, and complementarity-determining regions (CDRs) of the antibody accounting for antigen binding and framework regions of the antibody supporting the conserved three-dimensional conformation of the antibody were determined. Subsequently, the most similar human template was searched for in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) based on homology alignment results, CDR grafting was performed according to full-sequence BLAST results in combination with sequence characteristics of the heavy chain CDR3, and the heavy chain variable region (VH) of the murine antibody 11-4 was fully humanized in the framework regions. The most similar human template was searched for in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX) based on homology alignment results, CDR grafting was performed according to full-sequence BLAST results in combination with sequence characteristics of the light chain CDR3, and the light chain variable region (VL) of the murine antibody 11-4 was fully humanized in the framework regions.

    [0191] The nucleotide sequence and the amino acid sequence of the humanized heavy chain variable region (h11-4_VH1) of CDR grafted antibody 11-4 are as shown in SEQ ID NO: 49 and SEQ ID NO: 50 respectively; and the nucleotide sequence and the amino acid sequence of the humanized light chain variable region (h11-4_VL1) of CDR grafted antibody 4-3 are as shown in SEQ ID NO: 50 and SEQ ID NO: 25 respectively.

    TABLE-US-00019 SEQIDNO:6: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMG NIYPSNSYTNYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR YRSDGFAYWGQGTLVTVSS SEQIDNO:25: EIVLTQSPATLSLSPGERATLSCRASQNIGTSIHWYQQKPGQAPRLLIY FASESISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQSNSWPFTFGGGTKVEIK

    [0192] (2) Mutation Design in CDRs

    [0193] According to sequence characteristics of the murine antibody 11-4, mutations in CDR sequences in the CDR grafted humanized light and heavy chain variable regions were designed and the mutation sites are shown in Table 9.

    TABLE-US-00020 TABLE 9 Design of humanized sequences of murine antibody 11-4 Mutations relative to Mutations relative to h11-4_VL1 h11-4_VH1 h11-4_VL2 Y49E h11-4_VH2 A97S h11-4_VL3 H34E, Y49E h11-4_VH3 R98S h11-4_VL4 H34S, Y49E h11-4_VH4 Q1E, A97S, R98S h11-4_VL5 H34A, Y49E h11-4_VH5 Q1E, A97S, R98S, D102E h11-4_VH6 Q1E, A97S, R98S, D102G h11-4_VH7 Q1E, A97S, R98S, G103A Note: amino acid residue positions were numbered according to the Kabat numbering system.

    [0194] 6. Recombinant expression of humanized monoclonal antibody 11-4 The light chain variable region and heavy chain variable region (h11-4_VL1 and h11-4_VH1) genes of CDR grafted antibody 11-4 were fully synthesized. The humanized h11-4_VH1 gene was cloned by enzyme digestion into eukaryotic transient-expression vector pKN041 at the upstream of the coding gene of the heavy chain constant region of human IgG1, and the nucleotide sequence and the amino acid sequence of the heavy chain constant region are as shown in SEQ ID NO: 59 and SEQ ID NO: 37 respectively. The humanized h11-4_VL1 gene was cloned by enzyme digestion into eukaryotic transient-expression vector pKN019 at the upstream of the coding gene of human C? light chain, and the nucleotide sequence and the amino acid sequence of the light chain constant region are as shown in SEQ ID NO: 60 and SEQ ID NO: 38 respectively. As described, expression plasmids containing light and heavy chains of the CDR grafted antibody 11-4 were constructed and the obtained expression plasmids expressing light chain (pKN019-h11-4L1) and heavy chain (pKN019-h11-4H1) were transformed into E. coli cells for amplification, and then plasmids expressing the light chain h11-4L1 and the heavy chain h11-4H1 of the CDR grafted antibody 11-4 were isolated and obtained.

    [0195] According to the mutation design shown in Table 9, site-directed mutagenesis was performed in the expression plasmids expressing light chain (pKN019-h11-4L1) and heavy chain (pKN019-h11-4H1) respectively using StarMut gene Site-directed Mutagenesis Kit (Cat.: T111-01, GenStar). The mutated plasmids were transformed into E. coli cells for amplification, and expression plasmids expressing the light and the heavy chains of the humanized monoclonal antibodies with CDR mutations (h11-4H2 . . . h11-4H7; h11-4L2 . . . h11-4L5), corresponding to the humanized sequences of murine antibody 4-3 shown in Table 9. All the plasmids containing various humanized heavy and light chain sequences of murine antibody antibody 11-4 were combined as shown in Table 10 and transfected into HEK293 cells using 293fectin (Cat.: 12347019, Gibco) transfection reagent following the manufacturer's instructions for recombinant expression.

    TABLE-US-00021 TABLE 10 Combinations of humanized heavy and light chain sequences of murine antibody 11-4 h11-4 H1 h11-4 H2 h11-4 H3 h11-4 H4 h11-4 H5 h11-4 H6 h11-4 H7 h11-4 L1 h11-4-1 h11-4-2 h11-4-3 h11-4-4 h11-4 L2 h11-4-5 h11-4-6 h11-4-7 h11-4-8 h11-4 L3 h11-4-9 h11-4-10 h11-4-11 h11-4-12 h11-4 L4 h11-4-13 h11-4-14 h11-4-15 h11-4-16 h11-4 L5 h11-4-17 h11-4-18 h11-4-19 h11-4-20 Note: Table 10 shows antibodies obtained from the combinations of various heavy and light chains derived from murine antibody 11-4. For example, h11-4-1 refers to an antibody composed of the humanized light chain h11-4L1 and humanized heavy chain h11-4H1 of the murine antibody 11-4, and so on.

    [0196] 5-6 days after cell transfection, culture supernatants were purified through ProA affinity chromatography column to obtain different humanized antibodies. Affinities of the obtained antibodies were determined by an assay including capturing Fc fragments of the antibodies with anti-human IgG Fc capture (AHC) biosensors using Octet QKe system instrument from Fortebio. For the assay, each of the humanized antibodies and control antibody Sacituzumab was diluted to 4 ?g/ml in PBS, and was allowed to flow through the surface of an AHC biosensor (Cat.: 18-0015, PALL) for 120 s. Recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274) was used as a mobile phase, and its concentration was 60 nM. The binding time was 100 s and the dissociation time was 300 s. When the assay was finished, data from which the response values of blank control had been deducted were fitted to a 1:1 Langmuir binding model using software, and then kinetic constants for antigen-antibody binding were calculated.

    [0197] Affinities of the antibodies obtained from the combinations of mutants of murine antibody 11-4, chimeric antibody ch11-4 and control antibody Sacituzumab to recombinant human Trop-2-his protein were determined by ForteBio (Table 11).

    TABLE-US-00022 TABLE 11 Detection results of the affinities of the antibodies to recombinant human Trop-2 extracellular domain Antibody KD value (M) Sacituzumab 7.84E?10 ch11-4 2.64E?10 h11-4-1 2.16E?09 h11-4-2 9.91E?10 h11-4-3 8.68E?10 h11-4-4 8.11E?10 h11-4-5 1.08E?09 h11-4-6 9.52E?10 h11-4-7 8.03E?10 h11-4-8 2.22E?10 h11-4-9 3.01E?10 h11-4-10 3.51E?10 h11-4-11 3.75E?10 h11-4-12 2.82E?10 h11-4-13 2.11E?10 h11-4-14 3.52E?10 h11-4-15 3.89E?10 h11-4-16 2.31E?10 h11-4-17 2.38E?10 h11-4-18 3.94E?10 h11-4-19 2.54E?10 h11-4-20 2.10E?10

    Example 10 Detection Via ELISA of Species-Specific Binding of Anti-Trop-2 Humanized Antibodies to Trop-2

    [0198] Plates were coated with recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274), recombinant cynomolgus Trop-2-his protein (ACCESSION NO.: UniProtKB-A0A2K5UE71, AA 1-272), and recombinant mouse Trop-2-his protein (Cat.: 50922-M08H, Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.) respectively overnight at 4? C., each protein having a coating concentration of 1 ?g/ml respectively. Washed with PBS 3 times, the plates were blocked with the added 5% BSA PBS at 37? C. for 60 min, and then washed 3 times with PBST. Different concentrations of h23-12 (serial dilutions of 14 concentrations obtained through 3-fold serially diluting a solution with an initial concentration of 10 ?g/ml), h4-3 (serial dilutions of 12 concentrations obtained through 3-fold serially diluting a solution with an initial concentration of 3 ?g/ml), and Sacituzumab (serial dilutions of 12 concentrations obtained through 3-fold serially diluting a solution with an initial concentration of 3 ?g/ml) were added into the plates, with each concentration provided in a parallel well. The plates were incubated at 37? C. for 60 min, and then were washed 4 times with PBST. 1:5000 dilution of H1RP-anti-human Fc (Cat.: 109-035-098, Jackson ImmunoResearch) was added into the plates for incubation at 37? C. for 30 min. Afterwards, the plates were washed with PBST 4 times, and TMB substrate was added for color development at 37? C. for 10 min. Then 2M HCl was added to stop reaction; and absorbances at 450 nm and at 630 nm (as reference wavelength) were read, and A450 nm-630 nm values of the wells in the plates were recorded.

    [0199] The experiment results showed that h23-12, h4-3, and control antibody Sacituzumab could specifically bind recombinant human and cynomolgus Trop-2, but had no binding activity to mouse Trop-2 (FIG. 6, and Table 12), which provided a basis for pharmacological and toxicological experiments of the humanized antibodies.

    TABLE-US-00023 TABLE 12 EC50 of binding of anti-Trop-2 humanized antibodies to Trop-2 of different species EC50 (nM) Antibody human Trop-2 cynomolgus Trop-2 h23-12 0.04729 0.1245 h4-3 0.0469 0.04615 Sacituzumab 0.07032 0.07262

    Example 11 Analysis of Affinities of Anti-Human Trop-2 Humanized Antibodies

    [0200] Affinities of the antibodies were determined by an assay including capturing Fc fragments of the antibodies with anti-human IgG Fc capture (AHC) biosensors using Octet QKe system instrument from Fortebio.

    [0201] For the assay, each of the antibodies (h23-12, h4-3, and control antibody Sacituzumab) was diluted to 4 ?g/ml in PBS, and was allowed to flow through the surface of an AHC biosensor (Cat.: 18-0015, PALL) for 120 s. Recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274) was used as a mobile phase. The Trop-2-his protein was used in following concentrations which corresponded to different antibodies: in case of h23-12, the Trop-2 was used in concentrations of 23, 30, 45, and 75 nM; in case of h4-3, it was used in concentrations of 23, 30, 45, and 60 nM; and in case of Sacituzumab, it was used in concentrations of 23, 30, 45, and 75 nM. The binding time was 100 s and the dissociation time was 300 s. When the assay was finished, data from which the response values of blank control had been deducted were fitted to a 1:1 Langmuir binding model using software, and then kinetic constants for antigen-antibody binding were calculated.

    [0202] Reaction curves of h23-12, h4-3, and control antibody Sacituzumab with the recombinant human Trop-2 protein are shown in FIG. 7. The curves were fitted and the affinities were calculated, and it was shown that h23-12 had an affinity measured by a KD value of 6.40E-10M, h4-3 had an affinity measured by a KD value of 5.45E-10M, and Sacituzumab had an affinity measured by a KD value of 9.41E-10M. Kinetic parameters in detail are shown in Table 13. The results showed that h23-12 and h4-3 had high affinities to human Trop-2, equivalent to that of control antibody Sacituzumab, and h23-12 had a dissociation value superior to that of Sacituzumab.

    TABLE-US-00024 TABLE 13 Detection results of the affinities of the anti-human Trop-2 humanized antibodies to recombinant human Trop-2 extracellular domain KD value (M) kon (1/Ms) kdis (1/s) h23-12 6.40E?10 1.78E+05 1.14E?04 h4-3 5.45E?10 2.67E+05 1.46E?04 Sacituzumab 9.41E?10 2.08E+05 1.94E?04

    Example 12 Internalization Activities of Anti-Human Trop-2 Humanized Antibodies Binding to Trop-2 on the Surface of Cells

    [0203] NCI-N87 human gastric cancer cells naturally expressing human Trop-2 were inoculated at a density of 2?10.sup.3 cells/well into 96-well cell culture plates and cultured for 24 h. The cells were washed once with PBS and the supernatants were discarded. H23-12 and control antibody Sacituzumab labeled with Mix-n-Stain? CF? 488A (Cat.: MX488AS100, Sigma) were diluted to g/ml with RPMI 1640 (containing 10% FBS), and added to the NCI-N87 cells. The plates were divided into two groups, and one group was placed in an electric heating constant temperature incubator at 37? C., and one group was placed in a refrigerator at 4? C. as negative control. The plates as negative control were incubated for 30 min, washed with PBS 3 times, and observed and photographed with a fluorescence microscope. The plates of experiment groups were incubated at 37? C. for 5 h, and then observed and photographed with a fluorescence microscope.

    [0204] Experimental results (FIG. 8) showed that both the humanized h23-12 and control antibody Sacituzumab were able to be endocytosed through Trop-2 mediated endocytosis and distributed as spots in cytoplasm, suggesting that the internalization activities are maintained after antibody humanization.

    [0205] Internalization rate on BxPC cells at 3 h was measured via FACS using the procedure described in Example 7, and the results are shown in Table 14, suggesting that the internalization rates after humanization are comparable to that of Sacituzumab.

    TABLE-US-00025 TABLE 14 Internalization percentages of anti-human Trop-2 antibodies mediated by Trop-2 on the surface of BxPC cells Fluorescence intensity (MFI) Internalization Antibody 4? C. 37? C. percentage (%) h23-12 57771.6 30444.1 47.3 Sacituzumab 60612 31793.8 47.55 NC-IgG1 739.4 715.1 3.29

    Example 13 Pharmacokinetic Study in Balb/C Nude Mice Administered with Single Doses

    [0206] Healthy female 5 weeks old Balb/C nude mice were grouped, 2 in each group. The mice were intraperitoneally injected once with a single dose of h23-12 (15 mg/kg), and sera were collected respectively at 5 h, 25 h, 48 h, 96 h, 168 h and 240 h and stored at ?20? C. A control group was set, and the mice in the control group were intraperitoneally injected with control antibody Sacituzumab of the same dosage as h23-12 for comparison. Pharmacokinetic characteristics of the antibodies were observed.

    [0207] Healthy female 5 weeks old Balb/C nude mice were grouped, 4 in each group. The mice were intraperitoneally injected one time with a single dose of h4-3 (20 mg/kg), and sera were collected respectively at 4 h, 8 h, 24 h, 48 h, 96 h, 144 h, 192 h and 240 h and stored at ?20? C. Pharmacokinetic characteristics of the antibody were observed.

    [0208] Drug concentrations in serum were measured via ELISA using coated human Trop-2-his (ACCESSION NO.: NP_002344.2, AA 1-274); and in the meanwhile a standard curve was established. A linear curve was fitted by plotting the concentrations of standard antibody (Y axis) against OD values (X axis), and the content of an antibody in serum was obtained by substituting the OD value of the detected serum into the formula, and the half-life T.sub.1/2 of the antibody drug was calculated according to the formula T.sub.1/2=|0.693/k|.

    [0209] The results of drug concentration versus time curves showed that h23-12, h4-3, and control antibody Sacituzumab all had relatively long half-lives and exhibited comparable half-lives of drug metabolism (panel 9A, panel 9B, and Table 15), suggesting that the antibodies have no obvious inactivation phenomenon in vivo, and have good structural stability. The metabolism of the antibodies meets the basic characteristics of a monoclonal antibody drug, and T.sub.1/2 is about 170 h.

    TABLE-US-00026 TABLE 15 Pharmacokinetic parameters of anti-Trop-2 antibodies in nude mice administered with single doses T.sub.1/2 (detected by Trop-2) Sacituzumab (n = 2) 168 ? 14 h23-12(n = 2) 174 ? 12 h4-3 (n = 4) 180 ? 57

    Example 14 Analysis of Affinities of Anti-Trop-2 Naked Antibodies and ADCs Thereof

    [0210] Affinities of the antibodies were determined by an assay including capturing Fc fragments of the antibodies with anti-human IgG Fc capture (AHC) biosensors using Octet QKe system instrument from Fortebio.

    [0211] Firstly, an antibody labeled with SN38 used in ADC drugs was prepared. The antibody was reduced for 2 h in a sodium phosphate buffer, pH 7.0?0.5, using 20 equivalents of Dithiothreitol (DTT), and the reduced antibody was purified using an ultrafiltration centrifuge tube to remove excess DTT and exchanged into a sodium phosphate buffer, pH 7.0?0.5. The reduced antibody was incubated with CL2A-SN-38 for 30 min at ambient temperature using 7-15% (v/v) DMSO as a co-solvent. Finally, excess small molecules were removed through an ultrafiltration centrifuge tube. Molecular weight of the obtained antibody-drug conjugate was analyzed by mass spectrometry, and drug to antibody ratio (DAR) value of the ADC was calculated. It was confirmed eventually that 7.5 SN38 molecules were linked to one antibody.

    [0212] For the assay, SN38-labeled antibodies prepared as described above: h23-12-SN38, ch4-3-SN38, ch11-4-SN38, and positive control antibody Sacituzumab-SN38, as well as naked antibodies h23-12, ch4-3, ch11-4, and positive control antibody Sacituzumab were diluted to 4 ?g/ml with PBS and allowed to flow through the surface of an AHC biosensor (Cat.: 18-0015, PALL) for 120 s. Recombinant human Trop-2-his protein (ACCESSION NO.: NP_002344.2, AA 1-274) was used as a mobile phase, and its concentration was 60 nM. The binding time was 300 s and the dissociation time was 300 s. When the assay was finished, data from which the response value of blank control had been deducted were fitted to a 1:1 Langmuir binding model using software, and then kinetic constants for antigen-antibody binding were calculated.

    [0213] As shown in Table 16, no significant changes in the affinities of the antibodies were observed when the antibodies were labeled with SN38 thereby forming ADCs, as compared with the affinities when they were naked.

    TABLE-US-00027 TABLE 16 Detection results of the affinities of the anti- Trop-2 naked antibodies and ADCs thereof KD value (M) Antibody Naked Labeled with SN38 h23-12 5.07E?10 4.41E?10 ch4-3 3.23E?10 1.77E?10 ch11-4 1.76E?10 7.34E?11 Sacituzumab 5.83E?10 4.31E?10

    Example 15 Detection Via FACS of Internalization of Anti-Trop-2 Naked Antibodies and ADCs Thereof Mediated by BXPC-3 Cells

    [0214] Internalization rates on BXPC-3 human pancreatic cancer cells and NCI-N87 human gastric cancer cell were detected respectively according to the procedure as described in Example 7. Antibodies to be detected included ADC drugs i.e. SN38 labeled antibodies prepared as described above: h23-12-SN38, ch4-3-SN38, ch11-4-SN38, positive control antibody Sacituzumab-SN38, as well as naked antibodies h32-12, ch4-3, ch11-4, positive control antibody Sacituzumab, and negative isotype control antibody NC-IgG1, and each was 10 ?g/ml.

    [0215] As shown in Table 17 and Table 18, h23-12 naked and h23-12 labeled with SN38 thereby forming an ADC had similar internalization percentages, which were similar to that of the control antibody; and ch4-3 and ch11-4 when labeled with SN38 to form ADCs had higher internalization percentages than when they were naked.

    TABLE-US-00028 TABLE 17 Internalization percentages of anti-human Trop-2 antibodies mediated by Trop-2 on the surface of NCI-N87 cells Internalization percentage, 1 h Antibody Naked Labeled with SN38 h23-12 54.04% 51.09% ch4-3 50.16% 64.56% Sacituzumab 49.33% 47.49% NC-IgG1 ?95.03%

    TABLE-US-00029 TABLE 18 Internalization percentages of anti-human Trop-2 antibodies mediated by Trop-2 on the surface of BXPC-3 cells Internalization percentage, 1 h Antibody Naked Labeled with SN38 h23-12 32.44% 20.12% ch4-3 30.70% 57.66% ch11-4 15.50% 51.14% Sacituzumab 37.39% 32.71% NC-IgG1 ?62.97%

    Example 16 Detection of Cell Killing Activity of Anti-Trop-2-ADCs

    [0216] BxPC-3 human pancreatic cancer cells were inoculated at 2?10.sup.3/well into 96-well cell culture plates, and cultured overnight in an incubator at 37? C., 5% CO.sub.2. Afterwards, samples of SN38-labeled anti-Trop-2 antibodies (with each concentration provided in two parallel wells) at different concentrations were added into the plates according to Table 19, and blank wells with cells only (without any treatment) were set. The cells were incubated for 3 h in an incubator at 37 C, 5 CO.sub.2 and then culture medium was replaced with fresh complete medium. Next day the treatment was repeated as the previous day; after 4 consecutive days of the treatment, cell killing activity of the SN38-labeled anti-Trop-2 antibodies was detected using Cell Counting Kit-8 (CCK-8).

    TABLE-US-00030 TABLE 19 Anti-Trop-2-ADCs and actual concentrations thereof Name Actual concentration (?g/ml) h23-12-SN38 100 33.33 11.11 3.704 1.852 0.926 0.463 0.231 0.077 0.026 0.009 0.003 Sacituzumab-SN38 100 33.33 11.11 3.704 1.852 0.926 0.463 0.231 0.077 0.026 0.009 0.003 Tocilizumab-SN38 100 33.33 11.11 3.704 1.852 0.926 0.463 0.231 0.077 0.026 0.009 0.003 (negative control ADC) Ch4-3-SN38 100 33.33 11.11 3.704 1.852 0.926 0.463 0.231 0.077 0.026 0.009 0.003 Ch11-4-SN38 100 33.33 11.11 3.704 1.852 0.926 0.463 0.231 0.077 0.026 0.009 0.003

    [0217] The results showed (FIG. 10, and Table 20) that each anti-Trop-2 ADC specifically killed target cells and had no significant difference in killing activity from control antibody Sacituzumab-SN38.

    TABLE-US-00031 TABLE 20 Inhibitory activity of anti-Trop-2 ADCs on cell growth Name IC.sub.50 (?g/ml) Blank cells / h23-12-SN38 1.409 Sacituzumab-SN38 4.311 ch4-3-SN38 3.299 ch11-4-SN38 2.879

    Example 17 Pharmacodynamic Evaluation of Anti-Trop-2-ADCs in N87 Subcutaneous Xenograft Tumor Model

    [0218] Five weeks old female BALB/c nude mice were subcutaneously inoculated with 3?10.sup.6 human gastric cancer cells (NCI-N87), and randomly grouped with 6 mice per group when tumors grew to around 150 mm.sup.3. Grouping as well as administration dosage and frequency for each group are shown in Table 21. Each group was injected intravenously twice a week, and at the same time, tumor volume and body weight of each mouse were measured. When a mouse lost weight more than 15%, or when an animal had a tumor volume exceeding 3000 mm.sup.3, or when a whole group of animals had an average tumor volume exceeding 2000 mm.sup.3, the experiment was stopped, and the mouse/mice was/were euthanized.

    TABLE-US-00032 TABLE 21 Grouping as well as administration dosage and frequency for the nude mice Administration Administration Group Drug dosage frequency 1 h23-12-SN38 10 mg/kg Biw 2 2 mg/kg Biw 3 0.4 mg/kg Biw 4 Sacituzumab-SN38 10 mg/kg Biw 5 2 mg/kg Biw 6 ch4-3-SN38 10 mg/kg Biw 7 2 mg/kg Biw 8 Negative control, hIgG1 10 mg/kg Biw 9 Control ADC, hIgG1-SN38 10 mg/kg Biw

    [0219] As shown in FIG. 11 and FIG. 12, the anti-Trop-2-ADCs had dose-dependent inhibitory effects on tumor growth, and no difference in efficacy was observed between the ADCs at high dosage (10 mg/kg). No significant toxic effect of the small molecule SN38 in the ADCs was observed, and the body weight of the animals in each experiment group steadily increased without significant difference from controls.

    Example 18 Pharmacodynamic Evaluation of Co-Administration of Anti-Trop-2 Antibody and Anti-CD47 Antibody in SKOV3 Subcutaneous Xenograft Tumor Model

    [0220] Five weeks old female BALB/c nude mice were subcutaneously inoculated with 3?10.sup.6 SKOV3 human ovarian cancer cells into the right flank of each mouse, and randomly grouped with 6 mice per group when tumors grew to around 150 mm.sup.3. Grouping as well as administration dosage and frequency for each group are shown in Table 22. Each group was injected intravenously twice a week, 5 times in total, and at the same time, tumor volume and body weight of each mouse were measured. States of the mice were observed, and after the last administration, the mice were euthanized. The anti-CD47 antibody used is described in patent application publication US2015/0183874A1, i.e. humanized 5F9, version 2.

    TABLE-US-00033 TABLE 22 Grouping as well as administration dosage and frequency for the nude mice Administration Administration Group Drug dosage frequency 1 anti-CD47 antibody 10 mg/kg Biw 2 h23-12 2 mg/kg Biw 3 h23-12 + anti-CD47 2 mg/kg + 10 mg/kg Biw antibody 4 Negative control hIgG4 2 mg/kg Biw

    [0221] As shown in FIG. 13 and FIG. 14, the co-administration group of h23-12+anti-CD47 antibody showed a certain inhibitory activity on tumor compared with the negative control hIgG4, and the group of anti-CD47 antibody and group of h23-12 showed no significant inhibitory effects on tumor. Therefore, it can be seen the anti-Trop-2 antibody of the present disclosure and the anti-CD47 antibody can synergistically promote phagocytosis of tumor cells by macrophages, and have a synergistic inhibitory effect on tumor.

    Example 19 Pharmacodynamic Evaluation of Anti-Trop-2-ADCs in N87 Subcutaneous Xenograft Tumor Model

    [0222] Five weeks old female BALB/c nude mice were subcutaneously inoculated with 3?10.sup.6 human gastric cancer cells (NCI-N87), and randomly grouped with 6 mice per group when tumors grew to around 100 mm.sup.3. Grouping as well as administration dosage and frequency for each group are shown in Table 23. Each group was injected intravenously twice a week, and at the same time, tumor volume and body weight of each mouse were measured. When a mouse lost weight more than 15%, or when an animal had a tumor volume exceeding 3000 mm.sup.3, or when a whole group of animals had an average tumor volume exceeding 2000 mm.sup.3, the experiment was stopped, and the mouse/mice was/were euthanized.

    TABLE-US-00034 TABLE 23 Grouping as well as administration dosage and frequency for the nude mice Administration Administration Group Drug dosage frequency 1 ch3-11-SN38 5 mg/kg Biw ? 6 2 ch11-4-SN38 5 mg/kg Biw ? 6 3 Sacituzumab-SN38 5 mg/kg Biw ? 6 4 Negative control, hIgG1 5 mg/kg Biw ? 6 5 Control ADC, hIgG1-SN38 5 mg/kg Biw ? 6

    [0223] As shown in FIG. 15 and FIG. 16, the anti-Trop-2-ADCs had dose-dependent inhibitory effects on tumor growth, and ADCs ch3-11-SN38 and ch11-4-SN38 at dosage of 5 mg/kg had a slight better efficacy than Sacituzumab-SN38; and no significant toxic effect of the small molecule SN38 in the ADCs was observed.

    [0224] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications to the present invention without departing from the spirit of the present invention, which should fall within the scope of the appended claims.