WO2023104080A1 - 抗trop-2抗体或其抗原结合片段 - Google Patents

抗trop-2抗体或其抗原结合片段 Download PDF

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WO2023104080A1
WO2023104080A1 PCT/CN2022/137192 CN2022137192W WO2023104080A1 WO 2023104080 A1 WO2023104080 A1 WO 2023104080A1 CN 2022137192 W CN2022137192 W CN 2022137192W WO 2023104080 A1 WO2023104080 A1 WO 2023104080A1
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seq
heavy chain
light chain
variable region
identity
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PCT/CN2022/137192
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English (en)
French (fr)
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甄子朋
黄梁敏
马元婧
吕彩云
张正平
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正大天晴药业集团股份有限公司
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Publication of WO2023104080A1 publication Critical patent/WO2023104080A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the present application relates to an anti-TROP-2 antibody or an antigen-binding fragment thereof, a murine antibody comprising the CDR region of the anti-TROP-2 antibody, a chimeric antibody, a humanized antibody, a nucleic acid encoding the same, a pharmaceutical composition comprising the same, As well as the use of such antibodies or antigen-binding fragments for the preparation of medicines for treating cancer.
  • human Trop-2 is human trophoblast cell surface glycoprotein antigen 2, also known as tumor-associated calcium ion signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1) , surface marker 1 (M1S1), is a transmembrane cell surface glycoprotein encoded and expressed by the Tacstd2 gene in the region of chromosome 1p32, belonging to the GA733 protein family, and has a relationship with epithelial cell adhesion molecule (EpCAM, also known as Trop-1, TACSTD1) High structural sequence similarity, homology up to 49%.
  • EpCAM epithelial cell adhesion molecule
  • Human Trop-2 protein (323 amino acid residues) has a molecular weight of about 36kD, including a hydrophilic signal peptide (amino acids at positions 1-26), an extracellular domain (amino acids at positions 27-274), a cell span Membrane domain (amino acids 275-297) and intracellular domain (amino acids 298-323).
  • the extracellular domain is generally considered to be related to the proliferation, invasion and metastasis of cancer cells.
  • the physiological ligand of human Trop-2 has not yet been identified, and its molecular function has not yet been fully elucidated, but it has been disclosed that it transmits calcium signals in tumor cells, and because the Serine residue at position 303 in Trop2 cells is Ca 2+ dependent Phosphorylation of protein kinase C (PKC) and the intracellular domain has a PIP2 binding sequence, indicating that it has a signaling function in tumor cells.
  • PPC protein kinase C
  • Trop-2 is overexpressed in various epithelial cancers such as gastric cancer, lung cancer, colorectal cancer, ovarian cancer, breast cancer, and prostate cancer.
  • its expression in normal tissues is limited to cells in the epithelial region, and the expression level is lower than that in tumor cells. This suggests its involvement in tumor formation. Therefore, the research on anti-tumor drugs targeting Trop-2 is of great significance.
  • the present application provides an isolated antibody or antigen-binding fragment thereof that binds TROP-2 with high affinity, high selectivity and high biological activity.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 23 , SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 50, SEQ ID NO: 51, SEQ The heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the variable region sequence shown in ID NO:52, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:65, said light Chain variable regions comprising SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO :66 or the light chain CDR1, light chain CDR2 and light chain CDR3 of the variable
  • the application provides an isolated antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising:
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 23
  • the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the variable region sequence shown the light chain variable region comprises the light chain CDR1, light chain CDR2 and light chain CDR3 of the variable region sequence shown in SEQ ID NO:24
  • the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the variable region sequence shown in SEQ ID NO:25, and the light chain variable region comprises the variable shown in SEQ ID NO:26
  • Said heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of variable region sequence shown in SEQ ID NO:27, said light chain
  • the chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3 of the variable region sequence shown
  • the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the variable region sequence shown in SEQ ID NO:51, the light chain variable region Comprising light chain CDR1, light chain CDR2 and light chain CDR3 of the variable region sequence shown in SEQ ID NO:55;
  • the heavy chain variable region includes heavy chain CDR1, heavy chain variable region sequence shown in SEQ ID NO:52 Chain CDR2 and heavy chain CDR3, described light chain variable region comprises the light chain CDR1 of variable region sequence shown in SEQ ID NO:55, light chain CDR2 and light chain CDR3;
  • Described heavy chain variable region comprises SEQ ID NO Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the variable region sequence shown in SEQ ID NO:53, described light chain variable region comprises the light chain CDR1 of the variable region sequence shown in SEQ ID NO:55, light chain CDR2 and light chain Chain CDR3;
  • the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR1, heavy chain
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain chain CDR2 and heavy chain CDR3, said heavy chain CDR1 having at least 80% identity to an amino acid sequence selected from SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:48 or SEQ ID NO:17, said Heavy chain CDR2 and amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:49, SEQ ID NO:12, SEQ ID NO:62 or SEQ ID NO:18 Having at least 80% identity, said heavy chain CDR3 has at least 80% identity to an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:19, said light chain variable region comprising Light chain CDR1, light chain CDR2 and light chain CDR3, said light chain CDR1 has at least 80%
  • the application provides an isolated antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising:
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, described heavy chain CDR1 comprises the aminoacid sequence shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:48 or SEQ ID NO:17 or has at least therewith A sequence of 80% identity, the heavy chain CDR2 comprising SEQ ID NO: 6, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 49, SEQ ID NO: 12, SEQ ID NO: 62 or SEQ ID NO: 62 or SEQ ID NO: The amino acid sequence shown in ID NO: 18 or a sequence having at least 80% identity therewith, the heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 19 or with it Sequences with at least 80% identity; and/or
  • Light chain CDR1, light chain CDR2 and light chain CDR3, described light chain CDR1 comprises the aminoacid sequence shown in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:14 or SEQ ID NO:20 or has at least therewith A sequence of 80% identity, said light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:15 or SEQ ID NO:21 or having at least 80% identity thereto Sequence, the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 16 or SEQ ID NO: 22 or a sequence having at least 80% identity therewith.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain chain CDR2 and heavy chain CDR3, said heavy chain CDR1 has at least 80% identity, at least 81 % identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity Sex, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, At least 98% identity, or at least 99% sequence identity.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain chain CDR2 and heavy chain CDR3, described heavy chain CDR2 is selected from SEQ ID NO:6, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:49, SEQ ID NO:12, SEQ ID NO:62 or the amino acid sequence of SEQ ID NO: 18 has at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, At least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% sequence identity.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain chain CDR2 and heavy chain CDR3, described heavy chain CDR3 has at least 80% identity, at least 81% identity, at least 82% identity to the amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:19 % identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity sex, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, Or at least 99% sequence identity.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the light chain variable region comprising a light chain CDR1, a light chain chain CDR2 and light chain CDR3, said light chain CDR1 has at least 80% identity, at least 81 % identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity Sex, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, At least 98% identity, or at least 99% sequence identity.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the light chain variable region comprising a light chain CDR1, a light chain chain CDR2 and a light chain CDR3, said light chain CDR2 having at least 80% identity, at least 81% identity to an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:15 or SEQ ID NO:21 % identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity Sex, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, At least 98% identity, or at least 99% sequence identity.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the light chain variable region comprising a light chain CDR1, a light chain chain CDR2 and light chain CDR3, said light chain CDR3 has at least 80% identity, at least 81 % identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity Sex, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, At least 98% identity, or at least 99% sequence identity.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain Chain CDR2 and heavy chain CDR3, described heavy chain CDR1 is the aminoacid sequence selected from SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:48 or SEQ ID NO:17, and described heavy chain CDR2 is selected from The amino acid sequence of SEQ ID NO:6, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:49, SEQ ID NO:12, SEQ ID NO:62 or SEQ ID NO:18, said heavy chain CDR3 Be selected from the amino acid sequence of SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:19, said light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3, said light chain CDR1 is Amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:8,
  • the application provides an isolated antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising:
  • Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, described heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO:48 or 17, and described heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO:49,18 or 62 Sequence, the heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO: 13 or 19; and
  • Light chain CDR1, light chain CDR2 and light chain CDR3 said light chain CDR1 has the amino acid sequence shown in SEQ ID NO:14 or 20, said light chain CDR2 has the amino acid sequence shown in SEQ ID NO:15 or 21, The light chain CDR3 has the amino acid sequence shown in SEQ ID NO: 16 or 22.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain chain CDR2 and heavy chain CDR3, said light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein said heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 and light chain CDR1, light chain CDR2 and The light chain CDR3s are respectively selected from the following sequences or sequences having at least 80% identity thereto:
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO: 51.
  • the amino acid sequence of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:65 has at least 80% identity, at least 81% identity, at least 82% Identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity , at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or At least 99% identity, and wherein said light chain variable region is selected from SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID
  • the amino acid sequence of NO:54, SEQ ID NO:55, SEQ ID NO:66 or SEQ ID NO:67 has at least 80% identity, at least 81% identity, at least 8
  • the application provides an isolated antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising:
  • a heavy chain variable region comprising SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:65 an amino acid sequence, or an amino acid sequence at least 80% identical thereto; and/or
  • a light chain variable region comprising SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:54, The amino acid sequence shown in SEQ ID NO: 55, SEQ ID NO: 66 or SEQ ID NO: 67, or an amino acid sequence having at least 80% identity thereto.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:50, SEQ ID NO: 51.
  • amino acid sequence of SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:65 and wherein said light chain variable region is selected from the group consisting of SEQ ID NO :24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:66 or SEQ ID NO:67 amino acid sequence.
  • the application provides an isolated antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising:
  • a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 50, 63, 64 or 65;
  • a light chain variable region has the amino acid sequence shown in SEQ ID NO: 55, 66 or 67.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are respectively A sequence selected from or having at least 80% identity to:
  • Heavy chain variable region SEQ ID NO:36 light chain variable region SEQ ID NO:40; or,
  • heavy chain variable region SEQ ID NO:37 light chain variable region SEQ ID NO:41; or,
  • Heavy chain variable region SEQ ID NO:51 light chain variable region SEQ ID NO:54; or,
  • Heavy chain variable region SEQ ID NO:52 light chain variable region SEQ ID NO:54; or,
  • Heavy chain variable region SEQ ID NO:53 light chain variable region SEQ ID NO:54; or,
  • Heavy chain variable region SEQ ID NO:51 light chain variable region SEQ ID NO:55; or,
  • Heavy chain variable region SEQ ID NO:53 light chain variable region SEQ ID NO:55; or,
  • Heavy chain variable region SEQ ID NO:63 Heavy chain variable region SEQ ID NO:66; or,
  • Heavy chain variable region SEQ ID NO:65 light chain variable region SEQ ID NO:66; or,
  • Heavy chain variable region SEQ ID NO:63 Heavy chain variable region SEQ ID NO:67; or,
  • Heavy chain variable region SEQ ID NO:65 light chain variable region SEQ ID NO:67; or,
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain Chain CDR2 and heavy chain CDR3, described light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein said heavy chain CDR1 comprises the aminoacid sequence shown in SEQ ID NO:48, and heavy chain CDR2 comprises SEQ ID NO:48
  • the amino acid sequence shown in ID NO:49, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO:13, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO:14, and the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO:15
  • the amino acid sequence shown in the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 16, and the heavy chain variable region has at least 80% identity and at least 81% identity with the amino acid sequence shown in SEQ ID NO
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain Chain CDR2 and heavy chain CDR3, described light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein said heavy chain CDR1 comprises the aminoacid sequence shown in SEQ ID NO:17, and heavy chain CDR2 comprises SEQ ID NO:17
  • the amino acid sequence shown in ID NO:18, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO:19, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO:20, and the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO:21
  • the amino acid sequence shown, the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:22, and the heavy chain variable region has at least 80% identity and at least 81% identity with the amino acid sequence shown in SEQ ID
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain Chain CDR2 and heavy chain CDR3, described light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein said heavy chain CDR1 comprises the aminoacid sequence shown in SEQ ID NO:17, and heavy chain CDR2 comprises SEQ ID NO:17
  • the amino acid sequence shown in ID NO:18, the heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO:19, the light chain CDR1 includes the amino acid sequence shown in SEQ ID NO:20, and the light chain CDR2 includes the amino acid sequence shown in SEQ ID NO:21
  • the amino acid sequence shown, the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:22, and the heavy chain variable region has at least 80% identity and at least 81% identity with the amino acid sequence shown in SEQ ID
  • the human TROP-2-binding antibody or antigen-binding fragment thereof provided herein is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, wherein the Trop-2 antibody comprises a heavy chain constant region and a light chain constant region, preferably, the heavy chain constant region
  • the constant region selected from human IgG1, IgG2, IgG3 and IgG4 is selected from the constant region of human antibody kappa and lambda chain; more preferably, the antibody comprises a sequence as shown in SEQ ID NO:29
  • the heavy chain constant region and the light chain constant region whose sequence is shown in SEQ ID NO:30.
  • the application provides a chimeric antibody binding to human TROP-2, which comprises a heavy chain and a light chain, wherein the heavy chain is selected from SEQ ID NO:73, SEQ ID NO:75 or
  • the amino acid sequence set forth in SEQ ID NO: 77 has at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity Identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity , at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, and wherein the light chain is selected from SEQ ID NO:74, SEQ ID NO :76 or the amino acid sequence shown in SEQ ID NO:78 has at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 99%
  • the application provides a humanized antibody binding to human TROP-2, which comprises a heavy chain and a light chain, wherein the heavy chain is selected from the group consisting of SEQ ID NO:42, SEQ ID NO:43 , SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:68, SEQ ID NO:69, or SEQ
  • the amino acid sequence shown in ID NO:70 has at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, At least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, and wherein said light chain
  • amino acid sequence shown in SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:71 or SEQ ID NO:72 has at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% Identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity sex.
  • the application provides an isolated antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising:
  • a heavy chain comprising SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, The amino acid sequence shown in SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:68, SEQ ID NO:69 or SEQ ID NO:70, or a combination thereof Amino acid sequences of at least 80% identity; and/or
  • a light chain comprising SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:60, SEQ ID NO:61, The amino acid sequence shown in SEQ ID NO: 71 or SEQ ID NO: 72, or an amino acid sequence having at least 80% identity thereto.
  • the application provides a humanized antibody binding to human TROP-2, which comprises a heavy chain and a light chain, wherein the heavy chain is selected from SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID
  • the amino acid sequence shown in NO:70, and wherein said light chain is selected from SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:71 or SEQ ID NO : the amino acid sequence shown in 72.
  • the application provides an isolated humanized antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising
  • Heavy chain described heavy chain has the aminoacid sequence shown in SEQ ID NO:56,68,69 or 70
  • the application provides a humanized antibody or an antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain and a light chain, wherein the heavy chain and the light chain are respectively selected from the following sequences or Sequences with at least 80% identity to:
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, the The light chain comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein said heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:48, and heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:49, and heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:13, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:14, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:15, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO The amino acid sequence shown in: 16, and the heavy chain has at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85%
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, the The light chain comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:17, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:18, and the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:19, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:21, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO The amino acid sequence shown in: 22, and the heavy chain has at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least
  • the application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2, comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, the The light chain comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:17, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:18, and the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:19, the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:20, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:21, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO The amino acid sequence shown in: 22, and the heavy chain has at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least
  • the antibody or antigen-binding fragment thereof provided herein is selected from monoclonal antibodies, fusion proteins, multispecific antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated CDR regions, scFv and Nanobodies.
  • the antibody or antigen-binding fragment thereof provided herein is of the IgG1, IgG2, IgG3 or IgG4 type.
  • the application provides an isolated antibody or antigen-binding fragment thereof that binds to Trop-2, wherein the antibody or antigen-binding fragment thereof exhibits one or more of the following properties in combination:
  • the application provides an isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on Trop-2 as any of the exemplary antibodies or antigen-binding fragments thereof provided herein.
  • the application provides a TROP-2-binding antibody or antigen-binding fragment thereof that binds to the same epitope on TROP-2 as any of the exemplary antibodies provided herein, for example, binds to the same epitope as the hz-Ab2 series of antibodies For example, it binds to the same epitope as hz-Ab21 series antibodies, for example, it binds to the same epitope as hz-Ab35 series antibodies.
  • the antibody or antigen-binding fragment thereof provided herein competes for binding to TROP-2 with any exemplary antibody provided herein for binding to TROP-2, for example, competes with hz-Ab2 series antibodies for binding to TROP-2, for example, it competes with hz-Ab21 series antibodies for TROP-2 binding, for example, it competes with hz-Ab35 series antibodies for TROP-2 binding. Binding to TROP-2 can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art.
  • SPR surface plasmon resonance
  • the heavy chain variable region and the light chain variable region described herein comprise the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 described herein sequence features.
  • an isolated Trop-2-binding antibody provided herein comprises a heavy chain and a light chain comprising, in addition to the heavy and light chain variable regions described herein, constant region.
  • the constant regions are humanized.
  • both the constant region and the framework regions (FR) of the variable region are humanized. Immunogenicity is reduced by constructing chimeric antibodies (eg, humanized constant regions and non-human variable regions) or humanized antibodies (eg, humanized constant regions and FR regions).
  • the light chain constant region of the antibody is a human kappa chain constant region. In some embodiments, the light chain constant region of the antibody is a human lambda chain constant region.
  • the heavy chain constant region of an antibody can be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotype, such as IgGl, IgG2, IgG3, and IgG4.
  • the isolated antibody that binds Trop-2 is of the IgGl isotype.
  • the isolated antibody that binds Trop-2 is of the IgG4 isotype.
  • antibodies comprise modified constant regions.
  • the hinge region within the human IgG4 constant region is modified to avoid or reduce chain exchange, eg, an IgG4 type antibody has a Ser228Pro (S228P) mutation indexed according to EU numbering.
  • an isolated antibody and/or antigen-binding fragment thereof that binds Trop-2 binds Trop-2 with a KD value of 1.00E-08 or less.
  • the antibody and/or antigen-binding fragment thereof binds human Trop-2.
  • the antibody and/or antigen-binding fragment thereof binds Cynomolgus Trop-2.
  • the antibody and/or antigen-binding fragment thereof binds human Trop-2 and cynomolgus Trop-2.
  • the antibody and/or antigen-binding fragment thereof has a binding affinity (K D ) for human TROP-2 of about 1.00E-08M or less, about 1.16E-08M or less.
  • the binding affinity KD of the antibodies and antigen-binding fragments thereof that bind TROP-2 provided herein is measured by Biacore.
  • the antibody and/or antigen-binding fragment thereof that binds TROP-2 has a binding EC50 for human TROP-2 of about 0.035 nM to about 1.078 nM. In some embodiments, the antibody and/or antigen-binding fragment thereof that binds TROP-2 has a binding EC50 for human TROP-2 of about 1.078 nM or less, about 0.359 nM or less, about 0.035 nM or less. In some embodiments, the EC50 of the antibodies and antigen-binding fragments thereof provided herein that bind TROP-2 are measured by ELISA or FACS.
  • the antibody or/and antigen-binding fragment thereof that binds to TROP-2 shows strong endocytic activity in cell lines with different Trop-2 expression levels, and can continuously accumulate endocytic activity with increasing endocytic time.
  • the amount of antibody was uptake, and there was no obvious saturation at a higher concentration of antibody (20nM).
  • antibodies or/and antigen-binding fragments thereof that bind TROP-2 are shown to induce antibody-dependent cell-mediated cytotoxicity against Trop-2-expressing cells in different Trop-2-expressing cell lines (ADCC) activity.
  • ADCC Trop-2-expressing cell lines
  • antibodies and/or antigen-binding fragments thereof that bind TROP-2 do not bind to tumor cells that do not express Trop-2.
  • the isolated antibodies provided herein that bind TROP-2 are monoclonal antibodies.
  • an isolated antibody provided herein that binds TROP-2 is a monospecific antibody.
  • the isolated antibodies provided herein that bind TROP-2 are multispecific antibodies, eg, bispecific antibodies or trispecific antibodies, etc.
  • an isolated antibody provided herein that binds TROP-2 is a fusion protein.
  • the present application provides an antibody-drug conjugate, which comprises the human TROP-2-binding antibody or antigen-binding fragment thereof provided in the present application linked to a therapeutic agent.
  • the application provides an antibody drug conjugate, which comprises the antibody or antigen-binding fragment thereof that binds to human TROP-2 provided by the application linked to a therapeutic agent, wherein the therapeutic agent is a cytotoxic drug , radioisotopes, immunomodulators or antibodies.
  • Said cytotoxic drugs can be selected from alkaloids, anti-metabolites, antitumor antibiotics, alkylating agents and platinums, etc.
  • preferred cytotoxic drugs are microtubule inhibitors cytotoxic drugs (including maytansine class, orlistatins) or cytotoxic drugs that act on DNA (including calicheamicins, duocarmycins, PBDs (pyrrolobenzodiazepines), topoisomerase I inhibitors, etc.).
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the TROP-2 antibody or antigen-binding fragment thereof provided herein and a pharmaceutically acceptable carrier.
  • the present application provides a pharmaceutical composition, which comprises the drug conjugate of the TROP-2 antibody or antigen-binding fragment thereof provided herein and a pharmaceutically acceptable carrier.
  • the present application provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof provided herein that binds to human TROP-2.
  • the present application provides an expression vector comprising an isolated nucleic acid molecule encoding the human TROP-2-binding antibody or antigen-binding fragment thereof provided herein.
  • the application provides an expression vector comprising an isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that binds to human TROP-2 provided by the application or integrated with the isolated nucleic acid described herein in its genome Molecular host cells.
  • the present application provides the use of an antibody binding to human TROP-2 or an antigen-binding fragment thereof or a pharmaceutical composition thereof in the preparation of a medicament for treating a TROP-2-mediated disease. In one aspect, the present application provides the use of the antibody-drug conjugate of the present application in the preparation of a medicament for treating a TROP-2-mediated disease.
  • the present application provides an antibody or antigen-binding fragment thereof that binds to human TROP-2 or a pharmaceutical composition thereof for use in the treatment of a TROP-2-mediated disease.
  • the application provides a method of treating a TROP-2-mediated disease, the method comprising administering to a patient in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the application that binds to human TROP-2 or A pharmaceutical composition comprising the human TROP-2-binding antibody or antigen-binding fragment thereof provided in the present application and a pharmaceutically acceptable carrier.
  • the TROP-2-mediated disease described above is cancer.
  • the application provides a method of producing an antibody or antigen-binding fragment thereof that binds to human TROP-2, the method comprising culturing a host cell under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering Expressed antibody or antigen-binding fragment thereof.
  • the new anti-TROP-2 antibody or antigen-binding fragment thereof provided by this application can bind to different epitopes of TROP-2, has high affinity and excellent endocytic activity, and exhibits strong cell killing activity and animal xenograft tumor inhibitory activity, Moreover, it has good safety and is suitable for clinical application.
  • antibody or antigen-binding fragment thereof uses the antibody in its broadest sense, specifically covering intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed from at least two intact antibodies, Multifunctional antibodies, nanobodies, and fusion proteins, as long as they have the desired biological activity.
  • antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated CDR regions, single-chain Fv molecules (scFv) and other antibody fragments.
  • the TROP-2 antibody or antigen-binding fragment thereof of the present application may be of IgG1, IgG2, IgG3 or IgG4 subtype.
  • the TROP-2 antibody or antigen-binding fragment thereof of the present application can be derived from any species, including but not limited to mice, rats, rabbits, non-human primates (such as chimpanzees, cynomolgus monkeys, spider monkeys, macaques), Llamas and people.
  • the TROP-2 antibody or antigen-binding fragment thereof can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • Fab fragment consists of the CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • F(ab') 2 fragment contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain chain between the two heavy chains. disulfide bond.
  • the F(ab') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • Fv fragment comprises variable regions from both the heavy and light chains, but lacks the constant regions.
  • single-chain Fv molecule refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains, which linker enables the scFv to form the desired structure for antigen binding.
  • CDR complementarity determining region
  • hypervariable region Native four-chain antibodies typically contain six CDRs, three in the heavy chain variable region and three in the light chain variable region.
  • variable region The structural unit of an antibody is composed of two pairs of polypeptide chains, each pair having a heavy chain and a light chain, the N-terminal domain of each chain defining a major region of about 100 to 110 or more amino acids.
  • the regions responsible for antigen recognition are the variable regions.
  • chimeric antibody refers to antibodies and fragments of said antibodies that exhibit the desired biological activity in which portions of the heavy and/or light chains correspond to antibodies derived from a specific species or belonging to a specific antibody class or subclass while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass.
  • Humanized antibody forms are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • the majority of humanized antibodies are human immunoglobulins (recipient antibodies) in which hypervariable region residues of the recipient antibody are replaced by hypervariable region residues of a non-human species (donor antibody) having the desired specificity, affinity, and capacity. Residue substitution, non-human species such as mouse, rat, rabbit or non-human primate.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance.
  • humanized antibodies will comprise at least one, and usually substantially all of two, variable domains, of which all or substantially all hypervariable regions correspond to those of a non-human immunoglobulin, all or substantially all of FR regions FR regions of human immunoglobulin sequences.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region.
  • Anti-TROP-2 antibodies disclosed herein having one or more amino acid substitutions, insertions, deletions, or combinations thereof in a CDR or variable region retain the biological activity of a corresponding anti-TROP-2 antibody without amino acid substitutions, insertions, or deletions . Accordingly, the anti-TROP-2 antibody variants provided herein retain binding to TROP-2.
  • variants of said CDR or variable region amino acid sequences are provided, wherein said variants comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Amino acid substitutions, insertions or deletions or combinations thereof.
  • the amino acid substitutions are conservative substitutions.
  • the term "identity” is also called consistency.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as illustrated in the following non-limiting examples.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl.
  • the term "derived" when used in reference to a molecule or polypeptide relative to a reference antibody or other binding protein means a molecule or polypeptide capable of specifically binding the same epitope as the reference antibody or other binding protein.
  • isolated refers to a compound of interest (eg, antibody or nucleic acid) that has been separated from its natural environment.
  • EC50 refers to the effective concentration, 50% of the maximal response of an antibody.
  • IC50 refers to the inhibitory concentration, 50% of the maximal response of an antibody. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.
  • KD refers to the dissociation constant expressed in molarity (M).
  • M molarity
  • cancer refers to the physiological condition in mammals, usually characterized by unregulated cell growth.
  • treating means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) preventing a disease or disease state in breast-feeding in animals, especially when such mammals are susceptible to the disease state but have not been diagnosed as having the disease state; (ii) inhibiting the disease or disease state, that is, arresting its development; (iii) ameliorating the disease state or disease state, even if the disease or disease state subsides; (iv) reducing any direct or indirect pathological consequences of the disease or disease state.
  • terapéuticaally effective amount means (i) treating or preventing a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or delaying The amount of a compound of the application for the onset of one or more symptoms of a particular disease, condition or disorder described herein.
  • the amount of a compound of the present application that constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by a person skilled in the art according to its own knowledge and this disclosure.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive Toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • salts for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. .
  • solvate refers to a substance formed by the association of a compound with solvent molecules.
  • composition refers to a mixture of one or more compounds of the present application or their salts and a pharmaceutically acceptable carrier.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound of the present application to an organism.
  • pharmaceutically acceptable carrier refers to those excipients that have no obvious stimulating effect on the organism and will not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.
  • subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, ovines, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.
  • the subject according to the present application is a human.
  • the terms "patient” or “subject” are used interchangeably.
  • the terms “comprise, comprises, and comprising” or their equivalents are open-ended expressions meaning that all In addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.
  • Figure 1 ELISA (enzyme-linked immunosorbent assay) detection of Ab2 humanized antibody, Ab21 chimeric and humanized antibody, and Ab35 chimeric and humanized antibody combined with human Trop-2-His protein level .
  • Figure 2 Flow cytometry detection of humanized antibody of Ab2, humanized antibody of Ab21, and humanized antibody of Ab35 binding to A549 lung tumor cells.
  • Fig. 3 The endocytosis of Ab2 humanized antibody and Ab21 humanized antibody in HCC38 cells detected by flow cytometry (cultured for 2 hours).
  • Figure 6 The endocytosis of positive control datopotamab in HCC38 cells detected by flow cytometry (cultivated for 2 hours, 4 hours or 6 hours).
  • Figure 7 Detection of ADCC activity of chimeric and humanized antibodies of Ab2, chimeric and humanized antibodies of Ab21, and chimeric and humanized antibodies of Ab35 based on reporter gene method, in which Jurkat-CD16a v158-NFAT- Luciferase cells were effector cells, and NCI-H322 or HCC1806 cells were target cells.
  • Figure 8 Detection of ADCC activity of chimeric and humanized antibodies of Ab2, chimeric and humanized antibodies of Ab21, and chimeric and humanized antibodies of Ab35 based on PBMC killing effect, wherein PBMCs are effector cells, NCI- H322 or HCC1806 cells were the target cells.
  • the positive control drug TINA (also known as datopotamab) used in the examples of this application is the TROP-2 antibody developed by Daiichi Sankyo Co., Ltd., its heavy chain sequence corresponds to CN105849126A sequence number 12, and the light chain sequence corresponds to CN105849126A sequence number 18, according to Antibodies are prepared by conventional methods, first constructing vectors, transfecting eukaryotic cells, and then purifying and expressing them.
  • the application provides immunoconjugates of antibodies or antigen-binding fragments thereof that bind TROP-2.
  • the immunoconjugate is an antibody drug conjugate comprising an antibody that binds to human TROP-2 or an antigen-binding fragment thereof provided herein that is linked or conjugated to a therapeutic agent.
  • Therapeutic agents that can be linked or conjugated to the TROP-2-binding antibody or antigen-binding fragment thereof can include, but are not limited to, cytotoxic drugs, radioisotopes, immunomodulators, or antibodies.
  • the antibody or antigen-binding fragment thereof that binds TROP-2 is directly conjugated to the therapeutic agent.
  • an antibody or antigen-binding fragment thereof that binds TROP-2 is conjugated to a therapeutic agent via a linker.
  • the present application also provides a chimeric antigen receptor comprising the antibody of the present application that binds to human TROP-2 or an antigen-binding fragment thereof.
  • the CAR that binds to human TROP-2 may comprise: (a) an extracellular antigen-binding domain comprising an antibody or antigen-binding fragment thereof that binds to human TROP-2; (b) a transmembrane domain; (c) an intracellular signaling structure area.
  • CARs can be further added with elements that enhance T cell expansion, persistence, and antitumor activity, such as cytokines and costimulatory ligands.
  • the present application also provides engineered immune effector cells comprising the CAR provided in the present application.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated TROP-2-binding antibody or antigen-binding fragment thereof, or a nucleic acid encoding the antibody or fragment, or an immunoconjugate as described herein, and also Contains one or more pharmaceutically acceptable carriers.
  • the pharmaceutical composition includes any monoclonal antibody among the chimeric antibodies and humanized antibodies described herein, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, stabilizers, excipients, diluents, encapsulating materials, fillers, buffers, vehicles or other agents.
  • the Trop-2-binding antibody or antigen-binding fragment thereof provided herein is produced by a hybridoma. Accordingly, this application also includes any hybridoma that produces an antibody or antigen-binding fragment thereof disclosed herein.
  • the application provides an isolated nucleic acid comprising a sequence encoding a TROP-2-binding antibody or antigen-binding fragment thereof described herein.
  • the nucleic acid may encode an amino acid sequence comprising a heavy chain variable region or/and a light chain variable region of an antibody or antigen-binding fragment thereof described herein.
  • the nucleic acid may encode an amino acid sequence comprising a heavy chain or/and light chain of an antibody described herein.
  • the nucleotide sequences of some variable regions, antibody heavy chains and light chains are exemplarily listed in the sequence listing.
  • the present application provides a vector comprising said isolated nucleic acid.
  • the vector is a cloning vector; in other embodiments, the vector is an expression vector.
  • the expression vector is optionally any expression vector capable of expressing the antibodies or antigen-binding fragments thereof described herein.
  • the present application provides a host cell comprising the vector, which is a suitable host cell for cloning or encoding an antibody or antigen-binding fragment thereof that binds TROP-2.
  • the host cell is a prokaryotic cell.
  • the host cell is a eukaryotic cell.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • Mammalian cells are, for example, Chinese Hamster Ovary (CHO) cells.
  • the present application provides a method for preparing an isolated antibody or antigen-binding fragment thereof that binds TROP-2, the method comprising: cultivating the host cells of the nucleic acid of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the host cell or host cell culture medium.
  • nucleic acid encoding the antibody or antigen-binding fragment thereof is isolated and inserted into one or more vectors for further cloning or/and expression in host cells.
  • the nucleic acid can be obtained by various methods well known in the art, such as gene cloning, gene splicing, and chemical synthesis.
  • the application provides the use of an isolated antibody or an antigen-binding fragment thereof that binds TROP-2, or a method of using the antibody or an antigen-binding fragment thereof to treat cancer.
  • a therapeutically effective amount of the present invention is administered to a subject.
  • the isolated TROP-2-binding antibody, or antigen-binding fragment thereof can reduce or inhibit tumor cell growth.
  • administering to a subject a therapeutically effective amount of an isolated TROP-2-binding antibody or antigen-binding fragment thereof herein treats cancer.
  • the application provides the use of the pharmaceutical composition of the application, or a method of using the pharmaceutical composition to treat cancer.
  • administering a therapeutically effective amount of the pharmaceutical composition of the application to a subject can reduce or inhibit Tumor cells grow.
  • administering a therapeutically effective amount of the pharmaceutical composition of the present application to a subject can treat cancer.
  • the present application provides the use of the antibody drug conjugate of the present application, or the method of using the antibody drug conjugate to treat cancer.
  • a therapeutically effective amount of the antibody drug conjugate of the present application is administered to the subject. substances that can reduce or inhibit the growth of tumor cells.
  • cancer can be treated by administering a therapeutically effective amount of the antibody drug conjugate of the present application to a subject.
  • cancer refers to a physiological condition in mammals that is often characterized by unregulated cell growth.
  • cancers include, but are not limited to: leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, urothelial cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer Carcinoma, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, esophagus cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, endocrine gland cancer, adrenal gland cancer, pituitary gland cancer, skin cancer, soft tissue cancer , vascular cancer, brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, and uterine cancer, glioblastoma, medulloblastoma, astrocytoma, glue glioma, meningioma, gastrinoma, neuroblastoma, melanoma
  • Embodiment 1 Trop-2 high expression cell line construction
  • the DNA sequence of the full-length open reading frame of human Trop-2 was synthesized by General Biosystems (Anhui) Co., Ltd., and was subcloned into the expression vector pcDNA3.1 (Invitrogen catalog number: V-79020), and Lipofectamine3000 (Gibco ) transfection reagent into blank CHO-K1 cells, after geneticin selection for 2-3 weeks, the limited dilution method was used to isolate monoclonal clones, and the monoclonal cell lines obtained by subcloning the limited dilution method were detected by FACS Trop-2 expression on the surface of CHO-K1 cells, and CHO-K1/hTrop-2 monoclonal cell lines with high Trop-2 expression were selected.
  • the full-length amino acid sequence of human Trop-2 is shown in SEQ ID NO:1.
  • mice use human Trop-2-hFc to immunize mice, specifically, the extracellular domain of human Trop-2 is recombined with human IgG Fc to form TROP2-hFc (SEQ ID NO: 2).
  • DNA sequences were synthesized by General Biosystems (Anhui) Co., Ltd., and subcloned into expression vector pcDNA3.1 (Invitrogen catalog number: V-79020), respectively. After transient expression in FreeStyle293 cells, human Trop-2-hFc was purified with protein A column (GE healthcare).
  • mice were subcutaneously immunized with recombinant human Trop-2-hFc protein (100 ⁇ g/mouse) emulsified in an equal volume of complete/incomplete Freund's adjuvant every 2 weeks (Shanghai Xipuer-Bikay Experiment Animal Limited), for 6 weeks. Three days prior to fusion, mice were boosted by intravenous injection of antigen without adjuvant. Splenocytes (1 ⁇ 10 8 ) from immunized mice were fused with SP2/0 myeloma cells (1 ⁇ 10 7 ) using an electrofusion apparatus (BTX, 2001).
  • BTX electrofusion apparatus
  • cells were dispensed into 96-well plates at 0.1 ml/well and incubated in an incubator at 37°C, 5% CO 2 .
  • cells were fed by adding an additional 0.1 ml of HAT medium containing serum and 2x methotrexate to each well.
  • days 3 and 7 replace 0.1 ml of medium from each well with 0.1 ml of fresh HT medium. Screening is typically performed between days 9-14, and hybridoma culture supernatants are screened by ELISA for reaction with human Trop-2-His (SEQ ID NO:3).
  • Embodiment 3 Anti-Trop-2 antibody cDNA sequence cloning
  • RNA isolated from a human Trop-2 antibody-producing hybridoma cell line by RNeasy Mini Kit (Qiagen, catalog number: 74104) was used as a template.
  • First-strand cDNA was then synthesized according to the manufacturer's cDNA synthesis kit PrimeScript 1st strand cDNA Synthesis kit (TAKARA, catalog number: D6110A) instructions.
  • the cDNA product was then subjected to PCR using degenerate mouse IgG primers in a reaction mixture of 50 ⁇ l volume (Kettleborough CA et al., European Journal of Immunology 23:206-211 (1993), Strebe N et al., Antibody Engineering 1:3-14 (2010)) .
  • Reactions were performed in a S1000TM thermal cycler (Bio-Rad, catalog number: 184-2000) with 25 cycles of: 94°C, denaturation for 1.5 minutes; 50°C, annealing for 1 minute; and 72°C for 1 minute extension. At the end of the 25th cycle, the reaction mixture was incubated for an additional 5 minutes at 72°C to allow for topping up.
  • the PCR mixture was subjected to electrophoresis in a 1% agarose/Tris-borate gel containing 0.5 ⁇ g/ml ethidium bromide. DNA fragments of the expected size were excised from the gel and purified.
  • 3 ⁇ l of the purified PCR product was cloned into pMD-18T vector (Takara, catalog number: D101A) and transformed into OneTOP10 chemically competent E. coli (Invitrogen, catalog number: C4040-03). Clones were screened by colony PCR using universal M13 forward and reverse primers, and 6 positive clones were selected from each reaction for DNA sequencing in both directions using M13 forward and M13 reverse primers.
  • the heavy and light chain variable region sequences of antibodies comprising Ab2, Ab21 and Ab35 were amplified from the corresponding hybridoma clones. These antibodies exhibit desirable properties such as binding to human Trop-2-expressing tumor cells, species cross-binding ability, and high endocytosis rates.
  • Ab2 heavy chain variable region (SEQ ID NO: 23):
  • Ab21 heavy chain variable region (SEQ ID NO:25):
  • Ab35 heavy chain variable region (SEQ ID NO:27):
  • the cDNA of the mouse VL region cloned by PCR was respectively connected to the constant region of the human ⁇ chain (the amino acid sequence is shown in SEQ ID NO: 30) to construct ch-Ab2, ch-Ab21 and ch-Ab35 chimeric light chains (amino acid The sequences are respectively shown in SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78).
  • the chimeric heavy chains of ch-Ab2, ch-Ab21 and ch-Ab35 were constructed by linking the cDNA of the mouse VH region cloned by PCR to the human IgG1 constant region (the amino acid sequence is shown in SEQ ID NO: 29) (the amino acid sequences were respectively As shown in SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77).
  • the 5' end of the mouse cDNA sequence was modified using PCR primers designed to add leader sequences to both the light and heavy chains.
  • ExpiCHO cells (100 mL, 6.3 ⁇ 10 6 cells/mL) were transfected with 40 ⁇ g of each chimeric heavy chain expression plasmid and light chain expression plasmid and cultured for 10-12 days.
  • the chimeric antibody in the supernatant was then purified using a protein A column (GE healthcare).
  • the binding of the chimeric antibody to Trop-2 was measured by ELISA and Biacore, and the results showed that the chimeric antibody bound Trop-2 with an affinity comparable to that of the murine antibody.
  • the Ab2, Ab21 and Ab35 antibodies were humanized using the CDR grafting method (see eg, US Patent No. 5,225,539).
  • the homology modeling of murine antibodies Ab2, Ab21 and Ab35 was carried out by MOE (Molecular Operating Environment), and the protein structure model of the Fv domain was generated.
  • MOE Molecular Operating Environment
  • the complementarity determining regions (CDRs) in the VH and VL of the murine Ab2, Ab21 and Ab35 antibodies were grafted onto a template human antibody.
  • the CDR boundaries of antibodies can be named or identified by Kabat, Chothia, IMGT or Al-Lazikani nomenclature (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987); Chothia, C.
  • the selected template human VH was the combination of IGHV3-53*01, IGHV4-4*08 and IGHJ4*01, and the selected template human VL was the combination of IGKV1-39*01 and IGKJ1*01.
  • the selected template human VH was the combination of IGHV3-15*01 and IGHJ4*01, and the selected template human VL was the combination of IGKV3-11*01 and IGKJ1*01.
  • the selected template human VH was the combination of IGHV1-46*01 and IGHJ4*01, and the selected template human VL was the combination of IGKV3-11*01 and IGKJ1*01.
  • the CDR amino acid sequences of the above-mentioned template human antibody were substituted with the CDRs of the mouse antibody.
  • the essential amino acid sequences of VH and VL from mouse Ab2, Ab21 and Ab35 antibodies were used to graft the VH and VL frameworks of the above-mentioned template human germline antibodies to obtain functional humanized antibodies.
  • For the VH and VL of the Ab2, Ab21 and Ab35 antibodies several positions of the framework amino acids of the above-mentioned template human antibodies were back-mutated to the corresponding amino acid sequences in the mouse Ab2, Ab21 and Ab35 antibodies.
  • Embodiment 6 Antibody protein level binding experiment
  • Human Trop-2-His (SEQ ID NO: 3) protein was diluted to 2 ⁇ g/mL with the PBS (SmartBuffer, PBS-2050) buffer solution of pH 7.4, and was added in the 96-well microtiter plate with the volume of 100 ⁇ L/ hole, 4 Incubate overnight at °C. After the liquid was discarded, 300 ⁇ L of 2% BSA (SIGMA, B2064-100G) diluted with PBS was added to each well for blocking, and incubated at 37° C. for 2 hours.
  • PBS SmartBuffer, PBS-2050
  • PBS-2050 pH 7.4
  • 300 ⁇ L of 2% BSA (SIGMA, B2064-100G) diluted with PBS was added to each well for blocking, and incubated at 37° C. for 2 hours.
  • PBST buffer P7.4PBS containing 0.1% tween-20
  • 100 ⁇ L of gradiently diluted antibody solution was added to each well, and incubated at 37°C for 1 hour.
  • the plate was washed 3 times with PBST, 100 ⁇ L of goat anti-human IgG (H+L) (Jackson ImmunoResearch, 109-035-098, diluted 1:5000) was added to each well, and incubated at 37°C for 1 hour.
  • Table 5-2 Affinity EC 50 (nM) of chimeric and humanized Ab21 antibodies to Trop-2-His protein
  • the cells were prepared with FACS buffer (Miltenyi Biotec, 130-091-221) to a cell suspension of 0.5 ⁇ 10 6 -1 ⁇ 10 6 cells/mL, and added to a 96-well V-bottom plate at 100 ⁇ L/well. After the supernatant was removed by centrifugation, 50 ⁇ L/well of TINA and human IgG1 control antibody diluted to 3 ⁇ g/ml with FACS buffer were added, and incubated in a refrigerator at 4°C in the dark for 1 hour.
  • FACS buffer Miltenyi Biotec, 130-091-221
  • Embodiment 8 antibody cell level binding experiment
  • Tumor cells expressing Trop-2 were prepared into a cell suspension of 0.5-1 ⁇ 10 6 cells/mL with FACS buffer (Miltenyi Biotec, 130-091-221), and added to a 96-well V-bottom plate at 100 ⁇ L/well. After the supernatant was removed by centrifugation, 50 ⁇ L/well of different concentrations of the antibody to be tested (0.006 nM-100 nM) diluted with FACS buffer was added, and incubated in a refrigerator at 4° C. in the dark for 1 hour.
  • FACS buffer Miltenyi Biotec, 130-091-221
  • Table 7-1 Affinities (EC 50 ) of chimeric and humanized antibodies of Ab2 to different tumor cells
  • Antibody HCC38 (EC 50 , nM) HCC1806 (EC 50 , nM) ch-Ab2 0.59 0.78 hz-Ab2-1.2 0.76 0.90 hz-Ab2-1.3 0.65 1.22 hz-Ab2-1.4 1.20 1.07 hz-Ab2-2.4 0.45 1.23 TINA 1.21 1.44
  • Table 7-2 Affinities (EC 50 ) of chimeric and humanized antibodies of Ab21 to different tumor cells
  • Antibody HCC38 (EC 50 , nM) HCC1806 (EC 50 , nM) OVCAR-3 (EC 50 ,nM) ch-Ab21 0.64 0.65 1.02 hz-Ab21-1.1 0.55 0.64 0.62 hz-Ab21-1.2 0.66 1.23 0.67 hz-Ab21-2.1 0.64 0.63 0.68 hz-Ab21-2.2 0.66 1.18 0.80 TINA 0.79 0.84 1.29
  • Table 7-3 Affinities (EC 50 ) of chimeric and humanized antibodies of Ab35 to different tumor cells
  • Antibody HCC38 (EC 50 , nM) HCC1806 (EC 50 , nM) OVCAR-3 (EC 50 ,nM) ch-Ab35 0.45 0.38 0.37 hz-Ab35-1.1 0.77 0.80 0.83 hz-Ab35-1.2 0.71 0.62 0.57 hz-Ab35-1.3 0.70 0.54 0.64 hz-Ab35-2.1 0.89 0.76 0.90 hz-Ab35-2.2 0.91 0.58 0.88 hz-Ab35-2.3 0.91 0.61 0.74 TINA 1.04 0.73 1.28
  • Embodiment 9 antibody and human Trop-2-His affinity detection data
  • the detection of antibody affinity to human Trop-2-His protein adopts the form of capture antibody.
  • CM5 Cat. #29149603, GE
  • an anti-human IgG antibody from Human antibody capture kit, Cat. #29234600, GE
  • Biacore 8K instrument to detect the reaction signal in real time to obtain the binding and dissociation curves.
  • the chip was washed and regenerated with regeneration buffer 3M MgCl 2 (from Human antibody capture kit, Cat. #29234600, GE).
  • Biacore 8K Control Software 3.0 collects the binding dissociation curve in real time, Biacore Insight Evaluation Software 3.0 data analysis, after the data is double subtracted (that is, the signal of the experimental channel is subtracted from the signal of the control channel in each cycle, and the signal of the sample is subtracted from the blank signal ) Langmuir 1:1 model fitting to calculate the association rate constant Ka, dissociation rate constant Kd, and equilibrium constant K D values.
  • Ka association rate constant
  • Kd dissociation rate constant Kd
  • equilibrium constant K D values equilibrium constant
  • Embodiment 10 Antibody and monkey source protein cross activity detection
  • Macaque Trop-2-His (specifically, the extracellular domain of monkey Trop-2 is recombined with His, SEQ ID NO: 4) was diluted to 2 ⁇ g/mL with PBS (SmartBuffer, PBS-2050) buffer at pH 7.4 , added to a 96-well ELISA plate at a volume of 100 ⁇ L/well, and incubated overnight at 4°C. After the liquid was discarded, 300 ⁇ L of 2% BSA (SIGMA, B2064-100G) diluted with PBS was added to each well for blocking, and incubated at 37° C. for 2 hours.
  • PBS SmartBuffer, PBS-2050
  • PBST buffer PH7.4 PBS containing 0.1% tween-20
  • 100 ⁇ L of gradiently diluted antibody solution was added to each well, and incubated at 37° C. for 1 hour.
  • the plate was washed 3 times with PBST, 100 ⁇ L of goat anti-human IgG (H+L) (Jackson ImmunoResearch, 109-035-098, diluted 1:5000) was added to each well, and incubated at 37°C for 1 hour.
  • TMB chromogenic substrate TIANGEN, PA107-01
  • 1M H 2 SO 4 1M H 2 SO 4
  • Example 11 Non-specific binding of antibodies in tumor cells that do not express Trop-2
  • the tumor cell A549 (lung cancer tumor cell) not expressing Trop-2 was prepared into a cell suspension of 0.5-1 ⁇ 106 cells/mL with FACS buffer (MiltenyiBiotec, 130-091-221), and 96 cells were added at 100 ⁇ L/well. Hole V in the bottom plate. After the supernatant was removed by centrifugation, 50 ⁇ L/well of different concentrations of the antibody to be tested (0.0006 nM-50 nM) diluted with FACS buffer was added, and incubated in a refrigerator at 4° C. in the dark for 1 hour.
  • Embodiment 12 Antibody tumor cell endocytosis experiment
  • the antibody internalization kit (Sartoius, 90564) to detect the endocytosis of the antibody in the tumor cells, first dilute the labeled antibody in the kit and the humanized or chimeric antibody to be tested to 40nM with the complete medium used for the detection cells Afterwards, the first gradient was mixed in a 96-well V-bottom cell culture plate (Corning, catalog number 3894) at a ratio of 1:1, and after 2-fold dilution, placed at 37°C for different time (2 hours, 4 hours or 6 hours), the geometric mean fluorescence intensity (MFI) was detected on a Sartorius iQue3 flow cytometer, and the stronger the geometric mean fluorescence intensity (MFI), the stronger the endocytosis of the antibody in the cell.
  • MFI geometric mean fluorescence intensity
  • NCI-H322 and HCC1806 were used as target cells, and Jurkat cells stably transfected with CD16a v158 and NFAT-luciferase (Jurkat-CD16a v158-NFAT-luciferase) (BPS Bioscience, catalog number 60541) were used as effectors cells to measure antibody-dependent cell-mediated cytotoxicity (ADCC) activity of anti-Trop-2 antibodies.
  • TINA was used as a positive control
  • hIgG1 isotype control antibody (Baiying Biotechnology, catalog number: B117901) was used as a negative control.
  • NCI-H322 and HCC1806 cells Dilute NCI-H322 and HCC1806 cells with culture medium to 3 ⁇ 105 cells/mL, distribute each cell suspension into 96-well plates at 30 ⁇ L/well, and then add different concentrations of antibodies to be tested at 40 ⁇ L/well In a 96-well plate, the final concentrations of the antibodies to be tested are 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.013 nM, 0.0026 nM and 0.0005 nM.
  • the ADCC activity of the anti-Trop-2 antibody is shown in Figure 7, and the EC 50 data is shown in Table 10.
  • the chimeric antibodies ch-Ab2, ch-Ab21, ch- Ab35 and humanized antibodies hz-Ab2-1.2, hz-Ab2-1.4, hz-Ab2-1.3, hz-Ab21-2.1, hz-Ab21-2.2, hz-Ab35-1.1, hz-Ab35-1.2, hz- Ab35-1.3, hz-Ab35-2.1, hz-Ab35-2.2 and hz-Ab35-2.3 can all significantly induce the activation of NFAT signal in Jurkat-CD16a v158-NFAT-luciferase cells, and it is better than TINA.
  • ADCC activity of table 10 anti-Trop-2 antibody (effector cell is Jurkat-CD16a v158-NFAT-luciferase)
  • NCI-H322 and HCC1806 were used as target cells and PBMCs were used as effector cells to detect the ADCC activity of anti-Trop-2 antibody.
  • TINA was used as a positive control
  • hIgG1 isotype control antibody (Baiying Biotechnology, catalog number: B117901) was used as a negative control.
  • anti-Trop-2 antibodies ch-Ab2, ch-Ab21, ch-Ab35, humanized antibody hz-Ab2-1.2 , hz-Ab2-1.3, hz-Ab2-1.4, hz-Ab35-1.1, hz-Ab35-1.2, hz-Ab35-1.3, hz-Ab35-2.1, hz-Ab35-2.2 ADCC activity is better than TINA or with TINA quite.

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Abstract

提供抗TROP-2 抗体或其抗原结合片段、包含其的药物组合物，以及所述抗TROP-2抗体或其抗原结合片段或者包含其的药物组合物在制备用于癌症治疗的药物中的用途。

Description

抗TROP-2抗体或其抗原结合片段 技术领域
本申请涉及抗TROP-2抗体或其抗原结合片段，包含所述抗TROP-2抗体CDR区的鼠源抗体、嵌合抗体、人源化抗体，编码其的核酸，包含其的药物组合物，以及这类抗体或抗原结合片段用于制备治疗癌症的药物的用途。
背景技术
人Trop-2全称为人滋养层细胞表面糖蛋白抗原2，又名肿瘤相关钙离子信号转导子2(TACSTD2)、表皮糖蛋白1(EGP-1)、胃肠肿瘤相关抗原(GA733-1)、表面标志物1(M1S1)，是由染色体1p32区域的Tacstd2基因编码表达的跨膜细胞表面糖蛋白，属于GA733蛋白家族，与上皮细胞黏附分子(EpCAM，又称为Trop-1、TACSTD1)有较高结构序列相似性，同源性达49％。
人Trop-2蛋白(323个氨基酸残基)具有约36kD分子量，包含亲水性的信号肽(第1-26位的氨基酸)、细胞外结构域(第27-274位的氨基酸)、细胞跨膜结构域(第275-297位的氨基酸)以及细胞内结构域(第298-323位的氨基酸)。其中细胞外结构域通常被认为与癌细胞的增殖、浸润、转移有关。
目前尚未鉴定出人Trop-2的生理学上的配体，分子功能尚未完全阐明，但已经公开了其在肿瘤细胞中传递钙信号，且由于Trop2细胞内303位丝氨酸残基通过属于Ca 2+依赖性蛋白激酶的蛋白激酶C(PKC)而磷酸化以及细胞内结构域具有PIP2结合序列，表明其在肿瘤细胞中有信号传递功能。
已经报道了人Trop-2在胃癌、肺癌、大肠癌、卵巢癌、乳腺癌、前列腺癌等多种上皮源性癌中过度表达。与此相对，其在正常组织中的表达则仅限于上皮区域的细胞，表达水平也比肿瘤细胞中低。这表明其与肿瘤形成有关。因此以Trop-2为靶点的抗肿瘤药物研究具有重要意义。
发明内容
本申请提供了一种高亲和力的、高选择性的、高生物活性的结合TROP-2的分离的抗体或其抗原结合片段。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:66或SEQ ID NO:67所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3。
或者，本申请提供了一种结合人TROP-2的分离的抗体或其抗原结合片段，包含：
SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65所示的重链可变区序列中包含的重链CDR1、重链CDR2和重链CDR3；和/或
SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:66或SEQ ID NO:67所示的轻链可变区序列中包含的轻链CDR1、轻链CDR2和轻链CDR3。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:23所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:24所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:25所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:26所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:27所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:28所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:36所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:40所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:37所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:40所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:38所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:40所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:39所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:40所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:36所示可变区 序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:41所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:37所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:41所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:38所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:41所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:39所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:41所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:50所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:54所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:51所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:54所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:52所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:54所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:53所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:54所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:50所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:55所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:51所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:55所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:52所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:55所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:53所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:55所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:63所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:66所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:64所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:66所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:65所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:66所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:63所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:67所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；所述重链可变区包含SEQ ID NO:64所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:67所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；或所述重链可变区包含SEQ ID NO:65所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:67所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述重链CDR1与选自SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:48或SEQ ID NO:17的氨基酸序列具有至少80％同一性，所述重链CDR2与选自SEQ ID NO:6、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:49、SEQ ID NO:12、SEQ ID NO:62或SEQ ID NO:18的氨基酸序列具有至少80％同一性，所述重链CDR3与选自SEQ ID NO:7、SEQ ID NO:13或SEQ ID NO:19的氨基酸序列具有至少80％同一性，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，所述轻链CDR1与选自SEQ ID NO:8、SEQ ID NO:33、SEQ ID NO:14或SEQ ID NO:20的氨基酸序列具有至少80％同一性，所述轻链CDR2与选自SEQ ID NO:9、SEQ ID NO:34、SEQ ID NO:15或SEQ ID NO:21的氨基酸序列具有至少80％同一性，所述轻链CDR3与选自SEQ ID NO:10、SEQ ID NO:35、SEQ ID NO:16或SEQ ID NO:22的氨基酸序列具有至少80％同一性。
或者，本申请提供了一种结合人TROP-2的分离的抗体或其抗原结合片段，包含：
重链CDR1、重链CDR2和重链CDR3，所述重链CDR1包含SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:48或SEQ ID NO:17所示的氨基酸序列或与其具有至少80％同一性的序列，所述重链CDR2包含SEQ ID NO:6、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:49、SEQ ID NO:12、SEQ ID NO:62或SEQ ID NO:18所示的氨基酸序列或与其具有至少80％同一性的序列，所述重链CDR3包含SEQ ID NO:7、SEQ ID NO:13或SEQ ID NO:19所示的氨基酸序列或与其具有至少80％同一性的序列；和/或
轻链CDR1、轻链CDR2和轻链CDR3，所述轻链CDR1包含SEQ ID NO:8、SEQ ID NO:33、SEQ ID NO:14或SEQ ID NO:20所示的氨基酸序列或与其具有至少80％同一性的序列，所述轻链CDR2包含SEQ ID NO:9、SEQ ID NO:34、SEQ ID NO:15或SEQ ID NO:21所示的氨基酸序列或与其具有至少80％同一性的序列，所述轻链CDR3包含SEQ ID NO:10、SEQ ID NO:35、SEQ ID NO:16或SEQ ID NO:22所示的氨基酸序列或与其具有至少80％同一性的序列。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述重链CDR1与选自SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:48或SEQ ID NO:17的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％的序列同一性。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述重链CDR2与选自SEQ ID NO:6、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:49、SEQ ID NO:12、SEQ ID NO:62或SEQ ID NO:18的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％的序列同一性。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述重链CDR3与选自SEQ ID NO:7、SEQ ID NO:13或SEQ ID NO:19的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％的序列同一性。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，所述轻链CDR1与选自SEQ ID NO:8、SEQ ID NO:33、SEQ ID NO:14或SEQ ID NO:20的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％的序列同一性。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，所述轻链CDR2与选自SEQ ID NO:9、SEQ ID NO:34、SEQ ID NO:15或SEQ ID NO:21的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％的序列同一性。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，所述轻链CDR3与选自SEQ ID NO:10、SEQ ID NO:35、SEQ ID NO:16或SEQ ID NO:22的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％的序列同一性。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述重链CDR1为选自SEQ ID NO:5、SEQ ID NO:11、SEQ ID NO:48或SEQ ID NO:17的氨基酸序列，所述重链CDR2为选自SEQ ID NO:6、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:49、SEQ ID NO:12、SEQ ID NO:62或SEQ ID NO:18的氨基酸序列，所述重链CDR3为选自SEQ ID NO:7、SEQ ID NO:13或SEQ ID NO:19的氨基酸序列，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，所述轻链CDR1为选自SEQ ID NO:8、SEQ ID NO:33、SEQ ID NO:14或SEQ ID NO:20的氨基酸序列，所述轻链CDR2为选自SEQ ID NO:9、SEQ ID NO:34、SEQ ID NO:15或SEQ ID NO:21的氨基酸序列，所述轻链CDR3为选自SEQ ID NO:10、SEQ ID NO:35、SEQ ID NO:16或SEQ ID NO:22的氨基酸序列。
在一个方面，本申请提供了一种结合人TROP-2的分离的抗体或其抗原结合片段，包含：
重链CDR1、重链CDR2和重链CDR3，所述重链CDR1具有SEQ ID NO:48或17所示的氨基酸序列，所述重链CDR2具有SEQ ID NO:49、18或62所示的氨基酸序列，所述重链CDR3具有SEQ ID NO:13或19所示的氨基酸序列；和
轻链CDR1、轻链CDR2和轻链CDR3，所述轻链CDR1具有SEQ ID NO:14或20所示的氨基酸序列，所述轻链CDR2具有SEQ ID NO:15或21所示的氨基酸序列，所述轻链CDR3具有SEQ ID NO:16或22所示的氨基酸序列。
在一些实施方案中，本申请提供了结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，其中所述重链CDR1、重链CDR2和重链CDR3以及轻链CDR1、轻链CDR2和轻链CDR3分别选自以下序列或与其具有至少80％同一性的序列：
i.重链CDR1 SEQ ID NO:5
重链CDR2 SEQ ID NO:31
重链CDR3 SEQ ID NO:7
轻链CDR1 SEQ ID NO:8
轻链CDR2 SEQ ID NO:34
轻链CDR3 SEQ ID NO:10；或，
ii.重链CDR1 SEQ ID NO:5
重链CDR2 SEQ ID NO:32
重链CDR3 SEQ ID NO:7
轻链CDR1 SEQ ID NO:8
轻链CDR2 SEQ ID NO:34
轻链CDR3 SEQ ID NO:10；或，
iii.重链CDR1 SEQ ID NO:5
重链CDR2 SEQ ID NO:31
重链CDR3 SEQ ID NO:7
轻链CDR1 SEQ ID NO:33
轻链CDR2 SEQ ID NO:34
轻链CDR3 SEQ ID NO:35；或，
iv.重链CDR1 SEQ ID NO:5
重链CDR2 SEQ ID NO:32
重链CDR3 SEQ ID NO:7
轻链CDR1 SEQ ID NO:33
轻链CDR2 SEQ ID NO:34
轻链CDR3 SEQ ID NO:35；或，
v.重链CDR1 SEQ ID NO:48
重链CDR2 SEQ ID NO:49
重链CDR3 SEQ ID NO:13
轻链CDR1 SEQ ID NO:14
轻链CDR2 SEQ ID NO:15
轻链CDR3 SEQ ID NO:16；或，
vi.重链CDR1 SEQ ID NO:48
重链CDR2 SEQ ID NO:12
重链CDR3 SEQ ID NO:13
轻链CDR1 SEQ ID NO:14
轻链CDR2 SEQ ID NO:15
轻链CDR3 SEQ ID NO:16；或，
vii.重链CDR1 SEQ ID NO:17
重链CDR2 SEQ ID NO:62
重链CDR3 SEQ ID NO:19
轻链CDR1 SEQ ID NO:20
轻链CDR2 SEQ ID NO:21
轻链CDR3 SEQ ID NO:22；或，
viii.重链CDR1 SEQ ID NO:17
重链CDR2 SEQ ID NO:18
重链CDR3 SEQ ID NO:19
轻链CDR1 SEQ ID NO:20
轻链CDR2 SEQ ID NO:21
轻链CDR3 SEQ ID NO:22；或，
ix.重链CDR1 SEQ ID NO:5
重链CDR2 SEQ ID NO:6
重链CDR3 SEQ ID NO:7
轻链CDR1 SEQ ID NO:8
轻链CDR2 SEQ ID NO:9
轻链CDR3 SEQ ID NO:10；或，
x.重链CDR1 SEQ ID NO:11
重链CDR2 SEQ ID NO:12
重链CDR3 SEQ ID NO:13
轻链CDR1 SEQ ID NO:14
轻链CDR2 SEQ ID NO:15
轻链CDR3 SEQ ID NO:16；或，
xi.重链CDR1 SEQ ID NO:17
重链CDR2 SEQ ID NO:18
重链CDR3 SEQ ID NO:19
轻链CDR1 SEQ ID NO:20
轻链CDR2 SEQ ID NO:21
轻链CDR3 SEQ ID NO:22。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区与选自SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，且其中所述轻链可变区与选自SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:66或SEQ ID NO:67的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
或者，本申请提供了一种结合人TROP-2的分离的抗体或其抗原结合片段，包含：
重链可变区，所述重链可变区包含SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65所示的氨基酸序列，或与其具有至少80％同一性的氨基酸序列；和/或
轻链可变区，所述轻链可变区包含SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:66或SEQ ID NO:67所示的氨基酸序列，或与其具有至少80％同一性的氨基酸序列。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区为选自SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65的氨基酸序列，且其中所述轻链可变区为选自SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:66或SEQ ID NO:67的氨基酸序列。
在一个方面，本申请提供了一种结合人TROP-2的分离的抗体或其抗原结合片段，包含：
重链可变区，所述重链可变区具有SEQ ID NO:50、63、64或65所示的氨基酸序列；和
轻链可变区，所述轻链可变区具有SEQ ID NO:55、66或67所示的氨基酸序列。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中重链可变区和轻链可变区分别选自以下序列或与其具有至少80％同一性的序列：
i.重链可变区SEQ ID NO:36，轻链可变区SEQ ID NO:40；或，
ii.重链可变区SEQ ID NO:37，轻链可变区SEQ ID NO:40；或，
iii.重链可变区SEQ ID NO:38，轻链可变区SEQ ID NO:40；或，
iv.重链可变区SEQ ID NO:39，轻链可变区SEQ ID NO:40；或，
v.重链可变区SEQ ID NO:36，轻链可变区SEQ ID NO:41；或，
vi.重链可变区SEQ ID NO:37，轻链可变区SEQ ID NO:41；或，
vii.重链可变区SEQ ID NO:38，轻链可变区SEQ ID NO:41；或，
viii.重链可变区SEQ ID NO:39，轻链可变区SEQ ID NO:41；或，
ix.重链可变区SEQ ID NO:50，轻链可变区SEQ ID NO:54；或，
x.重链可变区SEQ ID NO:51，轻链可变区SEQ ID NO:54；或，
xi.重链可变区SEQ ID NO:52，轻链可变区SEQ ID NO:54；或，
xii.重链可变区SEQ ID NO:53，轻链可变区SEQ ID NO:54；或，
xiii.重链可变区SEQ ID NO:50，轻链可变区SEQ ID NO:55；或，
xiv.重链可变区SEQ ID NO:51，轻链可变区SEQ ID NO:55；或，
xv.重链可变区SEQ ID NO:52，轻链可变区SEQ ID NO:55；或，
xvi.重链可变区SEQ ID NO:53，轻链可变区SEQ ID NO:55；或，
xvii.重链可变区SEQ ID NO:63，轻链可变区SEQ ID NO:66；或，
xviii.重链可变区SEQ ID NO:64，轻链可变区SEQ ID NO:66；或，
xix.重链可变区SEQ ID NO:65，轻链可变区SEQ ID NO:66；或，
xx.重链可变区SEQ ID NO:63，轻链可变区SEQ ID NO:67；或，
xxi.重链可变区SEQ ID NO:64，轻链可变区SEQ ID NO:67；或，
xxii.重链可变区SEQ ID NO:65，轻链可变区SEQ ID NO:67；或，
xxiii.重链可变区SEQ ID NO:23，轻链可变区SEQ ID NO:24；或，
xxiv.重链可变区SEQ ID NO:25，轻链可变区SEQ ID NO:26；或，
xxv.重链可变区SEQ ID NO:27，轻链可变区SEQ ID NO:28。
在一些实施方案中，本申请提供了结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，其中所述重链CDR1包含SEQ ID NO:48所示的氨基酸序列，重链CDR2包含SEQ ID NO:49所示的氨基酸序列，重链CDR3包含SEQ ID NO:13所示的氨基酸序列，轻链CDR1包含SEQ ID NO:14所示的氨基酸序列，轻链CDR2包含SEQ ID NO:15所示的氨基酸序列，轻链CDR3包含SEQ ID NO:16所示的氨基酸序列，且所述重链可变区与SEQ ID NO:50所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，所述轻链可变区与SEQ ID NO:55所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在一些实施方案中，本申请提供了结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，其中所述重链CDR1包含SEQ ID NO:17所示的氨基酸序列，重链CDR2包含SEQ ID NO:18所示的氨基酸序列，重链CDR3包含SEQ ID NO:19所示的氨基酸序列，轻链CDR1包含SEQ ID NO:20所示的氨基酸序列，轻链CDR2包含SEQ ID NO:21所示的氨基酸序列，轻链CDR3包含SEQ ID NO:22所示的氨基酸序列，且所述重链可变区与SEQ ID NO:65所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％ 同一性、至少98％同一性、或至少99％同一性，所述轻链可变区与SEQ ID NO:66所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在一些实施方案中，本申请提供了结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，其中所述重链CDR1包含SEQ ID NO:17所示的氨基酸序列，重链CDR2包含SEQ ID NO:18所示的氨基酸序列，重链CDR3包含SEQ ID NO:19所示的氨基酸序列，轻链CDR1包含SEQ ID NO:20所示的氨基酸序列，轻链CDR2包含SEQ ID NO:21所示的氨基酸序列，轻链CDR3包含SEQ ID NO:22所示的氨基酸序列，且所述重链可变区与SEQ ID NO:65所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，所述轻链可变区与SEQ ID NO:67所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在一些实施方案中，本申请提供的结合人TROP-2的抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体或全人抗体。
在一个方面，本申请提供了一种结合人TROP-2的抗体或其抗原结合片段，其中所述Trop-2抗体包含重链恒定区和轻链恒定区，优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4的恒定区，所述轻链恒定区选自人抗体κ和λ链的恒定区；更优选地，所述抗体包含序列如SEQ ID NO:29所示的重链恒定区和序列如SEQ ID NO:30所示的轻链恒定区。
在一些实施方案中，本申请提供了一种结合人TROP-2的嵌合抗体，其包含重链和轻链，其中所述重链与选自SEQ ID NO:73、SEQ ID NO:75或SEQ ID NO:77所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，且其中所述轻链与选自SEQ ID NO:74、SEQ ID NO:76或SEQ ID NO:78所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在一些实施方案中，本申请提供了一种结合人TROP-2的人源化抗体，其包含重链和轻链，其中所述重链与选自SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:68、SEQ ID NO:69或SEQ ID NO:70所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，且其中所述轻链与选自SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:71或SEQ ID NO:72所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
或者，本申请提供了一种结合人TROP-2的分离的抗体或其抗原结合片段，包含：
重链，所述重链包含SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:68、SEQ ID NO:69或SEQ ID NO:70所示的氨基酸序列，或与其具有至少80％同一性的氨基酸序列；和/或
轻链，所述轻链包含SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:71或SEQ ID NO:72所示的氨基酸序列，或与其具有至少80％同一性的氨基酸序列。
在一些实施方案中，本申请提供了一种结合人TROP-2的人源化抗体，其包含重链和轻链，其中所述重链选自SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:68、SEQ ID NO:69或SEQ ID NO:70所示的氨基酸序列，且其中所述轻链选自SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:71或SEQ ID NO:72所示的氨基酸序列。
在一些实施方案中，本申请提供了一种结合人TROP-2的分离的人源化抗体或其抗原结合片段，包含
重链，所述重链具有SEQ ID NO:56、68、69或70所示的氨基酸序列
轻链，所述轻链具有SEQ ID NO:61、71或72所示的氨基酸序列。
在一些实施方案中，本申请提供了一种结合人TROP-2的人源化抗体或其抗原结合片段，其包含重链和轻链，其中所述重链和轻链分别选自以下序列或与其具有至少80％同一性的序列：
i)重链SEQ ID NO:42，轻链SEQ ID NO:46；或，
ii)重链SEQ ID NO:43，轻链SEQ ID NO:46；或，
iii)重链SEQ ID NO:44，轻链SEQ ID NO:46；或，
iv)重链SEQ ID NO:45，轻链SEQ ID NO:46；或，
v)重链SEQ ID NO:42，轻链SEQ ID NO:47；或，
vi)重链SEQ ID NO:43，轻链SEQ ID NO:47；或，
vii)重链SEQ ID NO:44，轻链SEQ ID NO:47；或，
viii)重链SEQ ID NO:45，轻链SEQ ID NO:47；或，
ix)重链SEQ ID NO:56，轻链SEQ ID NO:60；或，
x)重链SEQ ID NO:57，轻链SEQ ID NO:60；或，
xi)重链SEQ ID NO:58，轻链SEQ ID NO:60；或，
xii)重链SEQ ID NO:59，轻链SEQ ID NO:60；或，
xiii)重链SEQ ID NO:56，轻链SEQ ID NO:61；或，
xiv)重链SEQ ID NO:57，轻链SEQ ID NO:61；或，
xv)重链SEQ ID NO:58，轻链SEQ ID NO:61；或，
xvi)重链SEQ ID NO:59，轻链SEQ ID NO:61；或，
xvii)重链SEQ ID NO:68，轻链SEQ ID NO:71；或，
xviii)重链SEQ ID NO:69，轻链SEQ ID NO:71；或，
xix)重链SEQ ID NO:70，轻链SEQ ID NO:71；或，
xx)重链SEQ ID NO:68，轻链SEQ ID NO:72；或，
xxi)重链SEQ ID NO:69，轻链SEQ ID NO:72；或，
xxii)重链SEQ ID NO:70，轻链SEQ ID NO:72。
在一些实施方案中，本申请提供了结合人TROP-2的抗体或其抗原结合片段，其包含重链和轻链，所述重链包含重链CDR1、重链CDR2和重链CDR3，所述轻链包含轻链CDR1、轻链CDR2和轻链CDR3，其中所述重链CDR1包含SEQ ID NO:48所示的氨基酸序列，重链CDR2包含SEQ ID NO:49所示的氨基酸序列，重链CDR3包含SEQ ID NO:13所示的氨基酸序列，轻链CDR1包含SEQ ID NO:14所示的氨基酸序列，轻链CDR2包含SEQ ID NO:15所示的氨基酸序列，轻链CDR3包含SEQ ID NO:16所示的氨基酸序列，且所述重链与SEQ ID NO:56所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，所述轻链与SEQ ID NO:61所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在一些实施方案中，本申请提供了结合人TROP-2的抗体或其抗原结合片段，其包含重链和轻链，所述重链包含重链CDR1、重链CDR2和重链CDR3，所述轻链包含轻链CDR1、轻链CDR2和轻链CDR3，其中所述重链CDR1包含SEQ ID NO:17所示的氨基酸序列，重链CDR2包含SEQ ID NO:18所示的氨基 酸序列，重链CDR3包含SEQ ID NO:19所示的氨基酸序列，轻链CDR1包含SEQ ID NO:20所示的氨基酸序列，轻链CDR2包含SEQ ID NO:21所示的氨基酸序列，轻链CDR3包含SEQ ID NO:22所示的氨基酸序列，且所述重链与SEQ ID NO:70所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，所述轻链与SEQ ID NO:71所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在一些实施方案中，本申请提供了结合人TROP-2的抗体或其抗原结合片段，其包含重链和轻链，所述重链包含重链CDR1、重链CDR2和重链CDR3，所述轻链包含轻链CDR1、轻链CDR2和轻链CDR3，其中所述重链CDR1包含SEQ ID NO:17所示的氨基酸序列，重链CDR2包含SEQ ID NO:18所示的氨基酸序列，重链CDR3包含SEQ ID NO:19所示的氨基酸序列，轻链CDR1包含SEQ ID NO:20所示的氨基酸序列，轻链CDR2包含SEQ ID NO:21所示的氨基酸序列，轻链CDR3包含SEQ ID NO:22所示的氨基酸序列，且所述重链与SEQ ID NO:70所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，所述轻链与SEQ ID NO:72所示的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
在一些方案中，本申请提供的抗体或其抗原结合片段选自单克隆抗体、融合蛋白、多特异性抗体、Fab片段、Fab’片段、F(ab’)2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、scFv和纳米抗体。
在一些方案中，本申请提供的抗体或其抗原结合片段为IgG1、IgG2、IgG3或IgG4型。
在一个方面，本申请提供了结合Trop-2的分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段展现出下述性质中的一种或多种的组合：
(a)以1.00E-08M或更小的K D值与Trop-2结合；
(b)阻断Trop-2与配体的结合；
(c)在表达Trop-2的细胞中显示内吞活性；
(d)诱导针对表达Trop-2的细胞的抗体依赖的细胞介导的细胞毒性(ADCC)；
(e)与不表达Trop-2的肿瘤细胞不结合。
另一方面，本申请提供了分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段与本文提供的任何示例性抗体或其抗原结合片段结合Trop-2上的相同表位。
在一些实施方案中，本申请提供了与本文提供的任何示例性抗体结合TROP-2上的相同表位的结合TROP-2的抗体或其抗原结合片段，例如与hz-Ab2系列抗体结合相同表位，例如与hz-Ab21系列抗体结合相同表位，例如与hz-Ab35系列抗体结合相同表位。在一些实施方案中，本申请提供的抗体或其抗原结合片段与本文提供的用于结合TROP-2的任何示例性抗体竞争结合TROP-2，例如与hz-Ab2系列抗体竞争结合TROP-2，例如与hz-Ab21系列抗体竞争结合TROP-2，例如与hz-Ab35系列抗体竞争结合TROP-2。可以通过ELISA、流式细胞术、表面等离子体共振(SPR)测定或本领域已知的任何其他方法测量与TROP-2的结合。
在一些实施方案中，本文所述的重链可变区和轻链可变区中含有本文所述的重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2和轻链CDR3的序列特征。
在一些实施方案中，本文提供的结合Trop-2的分离的抗体包含重链和轻链，重链和轻链除了包含本文所述的重链可变区和轻链可变区外，还包括恒定区。在一些实施方案中，恒定区是人源化的。在一些实施方案中，恒定区与可变区的框架区(FR)都是人源化的。通过构建嵌合抗体(例如人源化的恒定区和非人源的可变区)或人源化抗体(例如人源化的恒定区和FR区)降低免疫原性。
在一些实施方案中，抗体的轻链恒定区是人κ链恒定区。在一些实施方案中，抗体的轻链恒定区是人 λ链恒定区。
抗体的重链恒定区可来自任何类型的恒定区，例如IgG、IgM、IgD、IgA和IgE；以及任何同种型，例如IgG1、IgG2、IgG3和IgG4。在一些实施方案中，结合Trop-2的分离的抗体是IgG1同种型。在一些实施方案中，结合Trop-2的分离的抗体是IgG4同种型。
在一些实施方案中，抗体包含经修饰的恒定区。在一些实施方案中，对人IgG4恒定区内的铰链区进行修饰以避免或减少链交换，例如IgG4型抗体具有根据EU编号索引的Ser228Pro(S228P)突变。
在一些实施方案中，结合Trop-2的分离的抗体或/和其抗原结合片段以1.00E-08或更小的K D值与Trop-2结合。在一些实施方案中，所述抗体或/和其抗原结合片段结合人Trop-2。在另一些实施方案中，所述抗体或/和其抗原结合片段结合猕猴Trop-2。在另一些实施方案中，所述抗体或/和其抗原结合片段结合人Trop-2以及食蟹猴Trop-2。在一些实施方案中，所述抗体或/和其抗原结合片段对人TROP-2具有下述结合亲和力(K D)：约1.00E-08M或更小、约1.16E-08M或更小。在一些实施方案中，通过Biacore来测量本文提供的结合TROP-2的抗体和其抗原结合片段的结合亲和力K D。
在一些实施方案中，结合TROP-2的抗体或/和其抗原结合片段对人TROP-2具有约0.035nM至约1.078nM的结合EC 50。在一些实施方案中，结合TROP-2的抗体或/和其抗原结合片段对人TROP-2具有约1.078nM或更小、约0.359nM或更小、约0.035nM或更小的结合EC 50。在一些实施方案中，通过ELISA或FACS来测量本文提供的结合TROP-2的抗体和其抗原结合片段的EC 50。
在一些实施方案中，结合TROP-2的抗体或/和其抗原结合片段在不同Trop-2表达量细胞系中显示了较强的内吞活性，同时能随着内吞时间的增加不断累积内吞抗体量，并且在较高浓度抗体(20nM)下没有表现出明显的饱和情况。
在一些实施方案中，结合TROP-2的抗体或/和其抗原结合片段在不同的表达Trop-2的细胞系中显示了诱导针对表达Trop-2的细胞的抗体依赖的细胞介导的细胞毒性(ADCC)活性。
在一些实施方案中，结合TROP-2的抗体或/和其抗原结合片段与不表达Trop-2的肿瘤细胞不结合。
在一些实施方案中，本文提供的结合TROP-2的分离的抗体是单克隆抗体。
在一些实施方案中，本文提供的结合TROP-2的分离的抗体是单特异性抗体。
在一些实施方案中，本文提供的结合TROP-2的分离的抗体是多特异性抗体，例如，双特异性抗体或者三特异性抗体等。
在一些实施方案中，本文提供的结合TROP-2的分离的抗体是融合蛋白。
在一个方面，本申请提供一种抗体药物偶联物，其包含与治疗剂相连接的本申请所提供的结合人TROP-2的抗体或其抗原结合片段。
在一个方面，本申请提供一种抗体药物偶联物，其包含与治疗剂相连接的本申请所提供的结合人TROP-2的抗体或其抗原结合片段，其中所述治疗剂是细胞毒药物、放射性同位素、免疫调节剂或抗体。所述细胞毒药物例如可以选自生物碱类、抗代谢类、抗肿瘤抗生素类、烷化剂类及铂类等，优选的细胞毒药物为微管抑制剂类细胞毒药物(包括美登素类、奥利斯他汀类)或者作用于DNA的细胞毒药物(包括卡奇霉素类、duocarmycin类、PBD(pyrrolobenzodiazepine)类、拓扑异构酶I抑制剂类等)。
在一方面，本申请提供一种药物组合物，其包含本文提供的TROP-2抗体或其抗原结合片段和药学上可接受的载体。
在一方面，本申请提供一种药物组合物，其包含本文提供的TROP-2抗体或其抗原结合片段的药物偶联物和药学上可接受的载体。
在一个方面，本申请提供一种分离的核酸分子，其编码本申请所提供的结合人TROP-2的抗体或其抗原结合片段。
在一个方面，本申请提供一种包含编码本申请所提供的结合人TROP-2的抗体或其抗原结合片段的分离的核酸分子的表达载体。
在一个方面，本申请提供一种包含编码本申请所提供的结合人TROP-2的抗体或其抗原结合片段的分离的核酸分子的表达载体或在其基因组中整合有本文所述的分离的核酸分子的宿主细胞。
在一个方面，本申请提供结合人TROP-2的抗体或其抗原结合片段或其药物组合物在制备用于治疗TROP-2介导的疾病的药物中的用途。在一个方面，本申请提供了本申请的抗体药物偶联物在制备用于治疗TROP-2介导的疾病的药物中的用途。
在一个方面，本申请提供了用于治疗TROP-2介导的疾病的结合人TROP-2的抗体或其抗原结合片段或其药物组合物。
在一个方面，本申请提供了一种治疗TROP-2介导的疾病的方法，该方法包含向有需要的患者施用治疗有效量的本申请的结合人TROP-2的抗体或其抗原结合片段或者包含本申请提供的结合人TROP-2的抗体或其抗原结合片段与药学上可接受的载体的药物组合物。
在一个方面，以上所述的TROP-2介导的疾病为癌症。
在一个方面，本申请提供了一种产生结合人TROP-2的抗体或其抗原结合片段的方法，所述方法包括在使得所述抗体或其抗原结合片段表达的条件下培养宿主细胞，和回收表达的抗体或其抗原结合片段。
本申请提供的全新抗TROP-2抗体或其抗原结合片段能结合TROP-2的不同表位，具有高的亲和力和优异的内吞活性，表现出强的细胞杀伤活性和动物移植瘤抑制活性，并且安全性好，适于临床应用。
定义和说明
术语“抗体或其抗原结合片段”中抗体以其最广义使用，特别涵盖完整的单克隆抗体、多克隆抗体、至少由两种完整抗体所形成的多特异性抗体(例如双特异性抗体)、多功能抗体、纳米抗体、以及融合蛋白，只要它们具有所需的生物学活性。抗原结合片段的实例包含但不限于Fab片段、Fab’片段、F(ab’)2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、单链Fv分子(scFv)等抗体片段。
本申请的TROP-2抗体或其抗原结合片段可以是IgG1、IgG2、IgG3或IgG4亚型。本申请的TROP-2抗体或其抗原结合片段可以衍生自任何物种，包含但不限于小鼠、大鼠、兔、非人灵长类动物(如黑猩猩、食蟹猴、蜘蛛猴、猕猴)、美洲驼和人。TROP-2抗体或其抗原结合片段可以是鼠类抗体、嵌合抗体、人源化抗体或全人抗体。
术语“Fab片段”由一条轻链和一条重链的C H1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
术语“F(ab’) 2片段”含有两条轻链和两条包含C H1和C H2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab’) 2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。
术语“Fv片段”包含来自重链和轻链二者的可变区，但缺少恒定区。
术语“单链Fv分子”(或“scFv抗体”)是指包含抗体的V H和V L结构域的抗体片段，其中这些结构域存在于单个多肽链中。一般而言，Fv多肽另外在V H和V L结构域之间包含多肽接头，该接头使得scFv能形成用于抗原结合的所需结构。
术语“CDR”(互补决定区)，也称为“高变区”。天然四链抗体通常包含六个CDR，三个在重链可变区中，三个在轻链可变区中。
术语“可变区”：抗体结构单元由两对多肽链构成，每一对具有一条重链和一条轻链，每条链的N端结构域限定约100至110个或更多个氨基酸的主要负责抗原识别的区域为可变区。
术语“嵌合抗体”指这样的抗体以及表现出所需生物学活性的所述抗体的片段：其中部分重链和/或轻链与来源于特定物种或属于特定抗体类别或亚类的抗体相应的序列等同或同源，同时所述链的其余部分与来源于另一物种或属于另一抗体类别或亚类的抗体相应的序列等同或同源。
“人源化抗体”形式为含有最小限度的来源于非人类免疫球蛋白序列的嵌合抗体。人源化抗体的大部分为人免疫球蛋白(受体抗体)，其中受体抗体的高变区残基被具有所需特异性、亲和力和能力的非人类物种(供体抗体)高变区的残基置换，非人类物种例如有小鼠、大鼠、兔或非人类灵长类。在某些情况下，人免疫球蛋白的Fv框架区(FR)残基被相应的非人类残基取代。此外，人源化抗体可包含不在受体抗体或供体抗体中存在的残基。进行这些修饰以进一步改进抗体性能。一般而言，人源化抗体包含至少一个且通常为两个可变结构域的几乎全部，其中全部或几乎全部高变区对应于非人类免疫球蛋白的高变区，全部或几乎全部FR区为人免疫球蛋白序列的FR区。人源化抗体还任选包含至少部分免疫球蛋白(通常为人免疫球蛋白)恒定区。
本文公开的在CDR或可变区具有一个或多个氨基酸取代、插入、缺失或其组合的抗TROP-2抗体保留了不具有氨基酸取代、插入或缺失的对应的抗TROP-2抗体的生物活性。因此，本文提供的抗TROP-2抗体变体保留与TROP-2的结合。在一些实施方案中，提供了所述CDR或可变区氨基酸序列的变体，其中所述变体包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代、插入或缺失或其组合。在另一个实施方案中，氨基酸取代为保守取代。
术语“同一性(identity)”，也称一致性。两序列间的百分比同一性为序列共有的相同位置数的函数(即％同一性＝相同位置数/位置总数×100)，其中需考虑产生两序列的最优比对需要引入的缺口数和每个缺口的长度。如下述非限制性实施例所示，可以使用数学算法完成序列的比较和两序列间百分比同一性的测定。可以使用E.Meyers和W.Miller的算法(Comput.Appl.Biosci.,4:11-17(1988))测定两氨基酸序列间的 百分比同一性，该算法已收入到ALIGN程序(版本2.0)中，其使用PAM120残基权重表，缺口长度罚分为12，缺口罚分为4。此外，可以使用Needleman和Wunsch的算法(J.Mol.Biol.484-453(1970))测定两氨基酸序列的百分比同一性，该算法已掺入到GCG软件包(可在www.gcg.com获得)中的GAP程序中，其使用Blossum 62矩阵或PAM250矩阵，缺口权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6。
如本文所用，术语“衍生的”当用于指相对于参考抗体或其他结合蛋白的分子或多肽时，意指能够与参考抗体或其他结合蛋白特异性地结合相同表位的分子或多肽。
术语“分离的”是指已经从其天然环境中分离的目标化合物(例如，抗体或核酸)。
如本文所用，术语“EC 50”是指有效浓度，抗体的50％最大应答。如本文所用，术语“IC 50”是指抑制浓度，抗体的50％最大应答。EC 50和IC 50两者均可以通过ELISA或FACS分析或本领域已知的任何其他方法进行测量。
术语“K D”在用于本文时指解离常数，以摩尔浓度(M)表示。抗体的K D值可以使用本领域公知的方法测定。一种优选的测定抗体K D的方法是使用表面等离振子共振(surface plasmon resonance)，更优选使用生物传感器系统，例如Biacore系统。
术语“癌症”：指哺乳动物中特征通常在于不受调控的细胞生长的生理病状。
术语“治疗”意为将本申请所述化合物或制剂进行给药以预防、改善或消除疾病或与所述疾病相关的一个或多个症状，且包含：(i)预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时；(ii)抑制疾病或疾病状态，即遏制其发展；(iii)缓解疾病或疾病状态，即使该疾病或疾病状态消退；(iv)降低疾病或疾病状态的任何直接或间接病理学后果。
术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本申请化合物的用量。构成“治疗有效量”的本申请化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
作为药学上可接受的盐，例如，可以提及金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。
术语“溶剂化物”是指化合物与溶剂分子缔合形成的物质。
术语“药物组合物”是指一种或多种本申请的化合物或其盐与药学上可接受的载体组成的混合物。药物组合物的目的是有利于对有机体给予本申请的化合物。
术语“药学上可接受的载体”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。
术语“受试者”包括任何人类或非人动物。术语“非人动物”包括所有的脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类、绵羊、犬、猫、马、牛、鸡、两栖动物、爬行动物等。优选地，根据本申请的受试者是人。除非标明，术语“患者”或“受试者”可以互换使用。
除非上下文另有明确指示，单数术语涵盖复数的指示对象，反之亦然。类似地，除非上下文另有明确指示，词语“或”意在包含“和”。
在本文中，除非另有说明，术语“包含、包含和含有(comprise、comprises和comprising)”或其等同物(contain、contains、containing、include、includes、including)为开放式表述，意味着除所列出的要素、组分和步骤外，还可涵盖其它未指明的要素、组分和步骤。
在本文中，除非另有说明，本文所使用的表示成分的量、测量值或反应条件的所有数字应理解为在所有情况下均由术语“约”修饰。如本文所用，“约”表示在本领域普通技术人员判定的对特定值可以接受的误差范围内，其部分取决于如何测量或测定该值，即测量系统的限制。例如，“约”按照本领域实践可表示1倍或超过1倍标准偏差以内。或者，“约”可以表示多至±5％的范围，例如在所给定的具体数值范围±2％范围内、±1％范围内或±0.5％范围内波动。当本申请或权利要求中给出特定值时，除非另有说明，“约”的含义应认为是在该特定值的可接受的误差范围内。在本文中，除非另有说明，步骤参数或条件的值默认均由“约”修饰。
附图说明
图1.ELISA(酶联免疫吸附法)检测Ab2的人源化抗体、Ab21的嵌合和人源化抗体、以及Ab35的嵌合和人源化抗体与人Trop-2-His蛋白水平结合情况。
图2.流式细胞术法检测Ab2的人源化抗体、Ab21的人源化抗体、以及Ab35的人源化抗体与A549肺肿瘤细胞的结合情况。
图3.流式细胞术法检测Ab2人源化抗体和Ab21的人源化抗体在HCC38细胞的内吞情况(培养2小时)。
图4.流式细胞术法检测Ab35的人源化抗体在OVCAR3细胞的内吞情况(培养2小时)。
图5.流式细胞术法检测人源化抗体hzAb2-2.4、hzAb21-2.1和hz-Ab35-2.3在HCC38细胞的内吞情况(培养2小时、4小时或6小时)。
图6.流式细胞术法检测阳性对照datopotamab在HCC38细胞的内吞情况(培养2小时、4小时或6小时)。
图7.基于报告基因法检测Ab2的嵌合和人源化抗体、Ab21的嵌合和人源化抗体、以及Ab35的嵌合和人源化抗体的ADCC活性，其中Jurkat-CD16a v158-NFAT-luciferase细胞为效应细胞，NCI-H322或HCC1806细胞为靶细胞。
图8.基于PBMC杀伤作用检测Ab2的嵌合和人源化抗体、Ab21的嵌合和人源化抗体、以及Ab35的嵌合和人源化抗体的ADCC活性，其中PBMC为效应细胞，NCI-H322或HCC1806细胞为靶细胞。
具体实施方式
尽管为了清楚理解的目的，已经通过举例说明和实施例相当详细地描述了前述发明，但是根据本申请的教义，本领域的普通技术人员将显而易见的是，可另外对本申请进行某些改变和修改而不背离所附权利要求的精神和范围。以下实施例仅以说明方式提供，而并不起限制作用。本领域的技术人员将容易地识别多种非关键性参数，所述参数可发生改变或修改以产生基本上类似的结果。
本申请实施例中采用的阳性对照药TINA(又名datopotamab)是第一三共株式会社开发的TROP-2抗体，其重链序列对应CN105849126A序列号12，轻链序列对应CN105849126A序列号18，按照抗体的常规方法进行制备，先进行载体构建，转染真核细胞之后纯化表达。
除非特殊说明，否则本申请的实践采用本领域熟知的并且在以下中描述的常规分子生物学、细胞生物学、生物化学和免疫学技术：例如，Methods in Molecular Biology，Humana Press；Molecular Cloning：A Laboratory Manual，第二版(Sambrook等，1989)；Current Protocols in Immunology(J.E.Coligan等编，1991)；Immunobiology(C.A.Janeway和P.Travers，1997)；Antibodies(P.Finch，1997)；Antibodies：a practical approach(D.Catty.编，IRL Press，1988-1989)；Monoclonal antibodies：a practical approach(P.Shepherd和C.Dean编，Oxford University Press，2000)；Phage display：a laboratory manual(C.Barbas III等Cold Spring Harbor Laboratory Press，2001)；以及Using antibodies：a laboratory manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press，1999)等。
免疫偶联物
在一些实施方案中，本申请提供了结合TROP-2的抗体或其抗原结合片段的免疫偶联物。在一些实施方案中，所述免疫偶联物是包含与治疗剂相连接或缀合的本申请所提供的结合人TROP-2的抗体或其抗原结合片段的抗体药物偶联物。可以与结合TROP-2的抗体或其抗原结合片段连接或缀合的治疗剂可以包括但不限于细胞毒药物、放射性同位素、免疫调节剂或抗体。在一些实施方案中，结合TROP-2的抗体或其抗原结合片段与治疗剂直接缀合。在一些实施方案中，结合TROP-2的抗体或其抗原结合片段通过接头与治疗剂缀合。
嵌合抗原受体(CAR)
本申请还提供了包含本申请的结合人TROP-2的抗体或其抗原结合片段的嵌合抗原受体。
结合人TROP-2的CAR可以包含：(a)包含结合人TROP-2的抗体或其抗原结合片段的胞外抗原结合结构域；(b)跨膜结构域；(c)胞内信号传导结构域。
CARs可以进一步添加增强T细胞扩增、持久性和抗肿瘤活性的要素，例如细胞因子和共刺激配体。
本申请还提供了工程化免疫效应细胞，其包含本申请提供的CAR。
药物组合物
本申请提供了药物组合物，所述药物组合物包含分离的结合TROP-2的抗体或其抗原结合片段，或编码所述抗体或片段的核酸，或本文所述的免疫偶联物，并且还包含一种或多种药学上可接受的载体。在一 些实施方案中，药物组合物包括本文所述的嵌合抗体及人源化抗体中的任一单克隆抗体，以及药学上可接受的载体。药学上可接受的载体包括例如稳定剂、赋形剂、稀释剂、包封材料、填充剂、缓冲剂、溶媒或其他试剂。
杂交瘤
本申请提供的结合Trop-2的抗体或其抗原结合片段由杂交瘤产生。因此，本申请还包括产生本文公开的抗体或其抗原结合片段的任何杂交瘤。
分离的核酸
本申请提供了分离的核酸，其包含编码本文所述结合TROP-2的抗体或其抗原结合片段的序列。在一些实施方案中，所述核酸可以编码包含本文所述抗体或其抗原结合片段重链可变区或/和轻链可变区的氨基酸序列。在另一些实施方案中，所述的核酸可以编码包含本文所述的抗体的重链或/和轻链的氨基酸序列。序列表中示例性的列举了一些可变区、抗体重链和轻链的核苷酸序列。
载体
本申请提供了包含所述的分离的核酸的载体。在一些实施方案中，所述的载体为克隆载体；在另一些实施方案中，所述的载体为表达载体。
所述表达载体可选地为能够表达本文所述抗体或其抗原结合片段的任意表达载体。
宿主细胞
在一些实施方案中，本申请提供包含所述载体的宿主细胞，宿主细胞为用于克隆或编码结合TROP-2的抗体或其抗原结合片段的适当宿主细胞。在一些实施方案中，宿主细胞为原核细胞。在另一些实施方案中，宿主细胞为真核细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳细胞或适用于制备抗体或其抗原结合片段的其他细胞。哺乳细胞例如为中国仓鼠卵巢(CHO)细胞。
制备结合TROP-2抗体或其抗原结合片段的方法
在一些实施方案中，本申请提供了制备结合TROP-2的分离的抗体或其抗原结合片段的方法，所述方法包括：在适合所述抗体或其抗原结合片段表达的条件下，培养包含编码所述抗体或其抗原结合片段的核酸的宿主细胞，从所述宿主细胞或宿主细胞培养基中回收所述抗体或其抗原结合片段。为了产生结合TROP-2的抗体或其抗原结合片段，分离编码所述抗体或其抗原结合片段的核酸，并插入一个或多个载体，用于在宿主细胞中进一步克隆或/和表达。所述核酸可以采用基因克隆、基因拼接、化学合成等多种本领域所熟知的方法获取。
用途/治疗方法
本申请提供了结合TROP-2的分离的抗体或其抗原结合片段的用途，或者使用所述抗体或其抗原结合片段治疗癌症的方法，一些实施方案中，向受试者施用治疗有效量的本文的分离的结合TROP-2的抗体或其抗原结合片段，可以减少或抑制肿瘤细胞生长。一些实施方案中，向受试者施用治疗有效量的本文的分离的结合TROP-2的抗体或其抗原结合片段，可以治疗癌症。
本申请提供了本申请的药物组合物的用途，或者使用所述药物组合物治疗癌症的方法，一些实施方案中，向受试者施用治疗有效量的本申请的药物组合物，可以减少或抑制肿瘤细胞生长。一些实施方案中，向受试者施用治疗有效量的本申请的药物组合物，可以治疗癌症。
本申请提供了本申请的抗体药物偶联物的用途，或者使用所述抗体药物偶联物治疗癌症的方法，一些实施方案中，向受试者施用治疗有效量的本申请的抗体药物偶联物，可以减少或抑制肿瘤细胞生长。一些实施方案中，向受试者施用治疗有效量的本申请的抗体药物偶联物，可以治疗癌症。
需要治疗的受试者包括那些已经患有疾病或病状的受试者，以及可能患疾病或病状并且其目的是预防、延迟或减弱疾病或病状的受试者。如本文所用，“癌症”是指哺乳动物中特征通常在于不受调控的细胞生长的生理病状。癌症的实例包括但不限于：白血病、淋巴瘤、卵巢癌、乳腺癌、子宫内膜癌、结肠癌、大肠癌、直肠癌、膀胱癌、尿路上皮癌、肺癌、支气管癌、骨癌、前列腺癌、胰腺癌、胃癌、肝细胞癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、宫颈癌、以及子宫癌、成胶质细胞瘤、成神经管细胞瘤、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、急性髓系白血病、骨髓增生异常综合征、以及肉瘤。
实施例1:Trop-2高表达细胞株构建
人Trop-2的全长开放阅读框的DNA序列通过通用生物系统(安徽)有限公司进行合成，并且被亚克隆到表达载体pcDNA3.1(Invitrogen目录号：V-79020)中，用Lipofectamine3000(Gibco)转染试剂转染至空 白的CHO-K1细胞中，经遗传霉素进行筛选2-3周后，进行有限稀释法分离得到单克隆，通过FACS检测有限稀释法亚克隆得到的单克隆细胞株CHO-K1细胞表面的Trop-2表达量，挑选出Trop-2表达量高的CHO-K1/hTrop-2单克隆细胞株。人源Trop-2氨基酸全长序列如SEQ ID NO:1所示。
实施例2：鼠源Trop-2单克隆抗体的产生
小鼠抗人Trop-2抗体的获得：使用人Trop-2-hFc对小鼠免疫，具体是将人Trop-2的细胞外结构域与人IgG Fc重组连接形成TROP2-hFc(SEQ ID NO:2)。DNA序列通过通用生物系统(安徽)有限公司进行合成，并且分别被亚克隆到表达载体pcDNA3.1(Invitrogen目录号：V-79020)中。在FreeStyle293细胞中瞬时表达后，用蛋白A柱(GE healthcare)纯化人Trop-2-hFc。使用等体积完全/不完全弗氏佐剂乳化的重组体人Trop-2-hFc蛋白(100μg/小鼠)每2周皮下免疫BALB/c和A/J小鼠(上海西普尔-必凯实验动物有限公司)，持续6周。融合前3天，通过静脉内注射不含佐剂的抗原来加强免疫小鼠。使用电融合仪(BTX,2001)将来自免疫小鼠的脾细胞(1×10 8)与SP2/0骨髓瘤细胞(1×10 7)融合。融合后，将细胞以0.1ml/孔分配到96孔板中，并且在37℃、5％CO 2的培养箱中进行孵育。在第1天，通过向每孔添加另外的0.1ml含有血清和2×甲氨蝶呤的HAT培养基来饲养细胞。在第3天和第7天，用0.1ml新鲜的HT培养基替换来自每个孔的0.1ml培养基。通常在第9-14天之间进行筛选，并且通过ELISA筛选针对与人Trop-2-His(SEQ ID NO:3)反应的杂交瘤培养物上清液。
实施例3：抗Trop-2抗体cDNA序列克隆
使用通过RNeasy微型试剂盒(Qiagen，目录号：74104)从产生人Trop-2抗体的杂交瘤细胞系中分离的总RNA作为模板。然后根据制造商的cDNA合成试剂盒PrimeScript1 ststrand cDNA Synthesis kit(TAKARA，目录号：D6110A)说明书合成第一链cDNA。然后使用简并小鼠IgG引物在50μl体积的反应混合物中使cDNA产物经受PCR(Kettleborough CA等，European Journal of Immunology23：206-211(1993)，Strebe N等，AntibodyEngineering1:3-14(2010))。反应在S1000TM热循环仪(Bio-Rad，目录号：184-2000)中进行，进行如下的循环25次：94℃，变性1.5分钟；50℃，退火1分钟；以及72℃延伸1分钟。在第25个循环结束时，将反应混合物在72℃下再温育5分钟以进行补齐。将PCR混合物在含有0.5μg/ml溴化乙锭的1％琼脂糖/Tris-硼酸盐凝胶中经受电泳。从凝胶上切下具有预期大小的DNA片段并且对其进行纯化。将3μl纯化的PCR产物克隆到pMD-18T载体(Takara，目录号：D101A)中，并且转化到OneTOP10化学感受态大肠杆菌(Invitrogen，目录号：C4040-03)中。通过菌落PCR使用通用M13正向引物和反向引物筛选克隆，并从每个反应中选择6个阳性克隆用于使用M13正向引物和M13反向引物在两个方向上进行DNA测序。
从对应的杂交瘤克隆扩增抗体的重链可变区序列和轻链可变区序列，这些抗体包含Ab2、Ab21和Ab35。这些抗体显示出所需的特性，诸如和表达人Trop-2的肿瘤细胞的结合、种属交叉结合能力以及较高的内吞率等。
Ab2重链可变区(SEQ ID NO:23)：
Figure PCTCN2022137192-appb-000001
Ab2轻链可变区(SEQ ID NO:24)：
Figure PCTCN2022137192-appb-000002
表1：Ab2的CDR序列
名称 序列 编号 名称 序列 编号
HCDR1 GFSLTYYGID SEQ ID NO:5 LCDR1 KASQDINSYLS SEQ ID NO:8
HCDR2 VIWGSGNTKYNSALMS SEQ ID NO:6 LCDR2 RANRLVD SEQ ID NO:9
HCDR3 FYEGFAY SEQ ID NO:7 LCDR3 LQYDEFPLT SEQ ID NO:10
Ab21重链可变区(SEQ ID NO:25):
Figure PCTCN2022137192-appb-000003
Ab21轻链可变区(SEQ ID NO:26):
Figure PCTCN2022137192-appb-000004
表2：Ab21的CDR序列
名称 序列 编号 名称 序列 编号
HCDR1 GFSLRNYGVH SEQ ID NO:11 LCDR1 RASKSVSTSAYSYIH SEQ ID NO:14
HCDR2 VIWSSGTTDYNAAFISR SEQ ID NO:12 LCDR2 LTSNLES SEQ ID NO:15
HCDR3 DGDYDYYTMDY SEQ ID NO:13 LCDR3 QHSRELPYT SEQ ID NO:16
Ab35重链可变区(SEQ ID NO:27)：
Figure PCTCN2022137192-appb-000005
Ab35轻链可变区(SEQ ID NO:28)：
Figure PCTCN2022137192-appb-000006
表3：Ab35的CDR序列
Figure PCTCN2022137192-appb-000007
实施例4：嵌合抗体的构建和表达
通过将PCR克隆的小鼠VL区的cDNA分别连接到人κ链恒定区(氨基酸序列如SEQ ID NO:30所示)来构建ch-Ab2、ch-Ab21和ch-Ab35嵌合轻链(氨基酸序列分别如SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78所示)。通过将PCR克隆的小鼠VH区的cDNA连接到人IgG1恒定区(氨基酸序列如SEQ ID NO:29所示)来构建ch-Ab2、ch-Ab21和ch-Ab35嵌合重链(氨基酸序列分别如SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77所示)。使用设计为向轻链和重链两者添加前导序列的PCR引物修饰小鼠cDNA序列的5'端。
将ExpiCHO细胞(100mL，6.3×10 6个/mL)用40μg的每种嵌合重链表达质粒和轻链表达质粒进行转染并且将其培养10-12天。然后用蛋白A柱(GE healthcare)纯化上清液中的嵌合抗体。通过ELISA和Biacore测量嵌合抗体与Trop-2的结合，结果表明嵌合抗体以与鼠类抗体相当的亲和力结合Trop-2。
实施例5：抗体人源化设计
Ab2、Ab21和Ab35抗体使用CDR移植方法进行人源化(参见例如，美国专利号5,225,539)。通过MOE(Molecular Operating Environment)对鼠源抗体Ab2、Ab21和Ab35进行同源建模，产生Fv结构域的蛋白质结构模型。分别将鼠源Ab2、Ab21和Ab35抗体的VH和VL氨基酸序列输入到国际免疫遗传信息系统相关工具网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)，从中筛选出与小鼠Ab2、Ab21和Ab35抗体具有高度同一性的人胚系抗体序列。将鼠源Ab2、Ab21和Ab35抗体的VH和VL中的互补决定区(CDR)嫁接至模板人抗体。抗体的CDR边界可通过Kabat、Chothia、IMGT或Al-Lazikani命名法命名或识别(Al-Lazikani,B.,Chothia,C.,Lesk,A.M.，J.Mol.Biol.，273(4)，927(1997)；Chothia,C.等，J Mol Biol.Dec 5；186(3):651-63(1985)；Chothia,C.和Lesk,A.M.，J.Mol.Biol.，196,901(1987)；Chothia,C.等，Nature.Dec 21-28；342(6252):877-83(1989)；Kabat E.A.等)。对于Ab2，所选择的模板人VH是IGHV3-53*01、IGHV4-4*08和IGHJ4*01的组合，所选择的模板人VL是IGKV1-39*01和IGKJ1*01的组合。对于Ab21，所选择的模板人VH是IGHV3-15*01和IGHJ4*01的组合，所选择的模板人VL是IGKV3-11*01和IGKJ1*01的组合。对于Ab35，所选择的模板人VH是IGHV1-46*01和IGHJ4*01的组合，所选择的模板人VL是IGKV3-11*01和IGKJ1*01的组合。
上述模板人抗体的CDR氨基酸序列被小鼠抗体的CDR取代。用来自小鼠Ab2、Ab21和Ab35抗体的VH和VL的必需氨基酸序列嫁接上述模板人胚系抗体VH和VL框架，以得到功能性人源化抗体。对于Ab2、Ab21和Ab35抗体的VH和VL，上述模板人抗体的框架氨基酸的数个位点被回复突变为小鼠Ab2、Ab21和Ab35抗体中对应的氨基酸序列。
表4：人源化抗体的序列编号
Figure PCTCN2022137192-appb-000008
Figure PCTCN2022137192-appb-000009
实施例6:抗体蛋白水平结合实验
用pH 7.4的PBS(SmartBuffer，PBS-2050)缓冲液将人Trop-2-His(SEQ ID NO:3)蛋白稀释至2μg/mL，以100μL/孔的体积加入96孔酶标板中，4℃过夜孵育。弃去液体后，每孔加入300μL用PBS稀释的2％BSA(SIGMA，B2064-100G)进行封闭，37℃孵育2小时。封闭结束后，弃去封闭液，并用PBST缓冲液(PH7.4PBS含0.1％tween-20)洗板3次后，每孔加入100μL梯度稀释的抗体溶液，于37℃孵育l小时。孵育结束后用PBST洗板3次，每孔加入100μL羊抗人IgG(H+L)(JacksonImmunoResearch，109-035-098，1:5000稀释)，37℃孵育l小时。用PBST洗板3次后，每孔加入100μL TMB显色底物(TIANGEN，PA107-01)，室温孵育10-15分钟，每孔加入50μL的1M H 2SO 4终止反应，用酶标仪读取在450nm处的吸收值，用软件拟合出抗体与抗原的结合曲线，计算出EC 50。抗体与蛋白的结合活性见表5-1、表5-2、表5-3以及图1。结果显示，人源化后的抗体和人Trop-2-His蛋白结合活性与亲本嵌合抗体相当。
表5-1：Ab2的嵌合和人源化抗体与Trop-2-His蛋白的亲和力EC 50(nM)
抗体 亲和力，EC 50(nM)
ch-Ab2 0.085
hz-Ab2-1.1 0.082
hz-Ab2-1.2 0.076
hz-Ab2-1.3 0.081
hz-Ab2-1.4 0.068
hz-Ab2-2.1 1.078
hz-Ab2-2.2 0.218
hz-Ab2-2.3 0.359
hz-Ab2-2.4 0.100
TINA 0.083
表5-2：Ab21的嵌合和人源化抗体与Trop-2-His蛋白的亲和力EC 50(nM)
抗体 亲和力，EC 50(nM)
ch-Ab21 0.165
hz-Ab21-1.1 0.162
hz-Ab21-1.2 0.248
hz-Ab21-2.1 0.214
hz-Ab21-2.2 0.223
TINA 0.166
表5-3：Ab35的嵌合和人源化抗体与Trop-2-His蛋白的亲和力EC 50(nM)
抗体 亲和力，EC 50(nM)
ch-Ab35 0.043
hz-Ab35-1.1 0.052
hz-Ab35-1.2 0.035
hz-Ab35-1.3 0.042
hz-Ab35-2.1 0.055
hz-Ab35-2.2 0.054
hz-Ab35-2.3 0.065
TINA 0.083
实施例7：不同细胞Trop-2表达水平
将细胞用FACS缓冲液(Miltenyi Biotec，130-091-221)制备成0.5×10 6-1×10 6个/mL的细胞悬液，以100μL/孔加入96孔V底板中。离心去除上清后加入50μL/孔用FACS缓冲液稀释至3μg/ml的TINA及人IgG1对照抗体，放于4℃冰箱中避光孵育l小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的AlexaFluor488羊抗人IgG(H+L)(Bio-legend，409304，1:500稀释)，放于4℃冰箱中避光孵育40分钟。 以FACS缓冲液300g离心洗涤3次后，在Sartorius iQue3流式细胞仪上检测几何平均数荧光强度(MFI)。根据TINA检测MFI值和IgG1对照抗体检测比值，认为NCI-H322、HCC38以及HCC1806细胞表面上的Trop-2表达量较高，Capan-1、PC-3、HUVEC和OVCAR-3细胞表面Trop-2为中等表达，而A549细胞表面无Trop-2表达。
表6：不同细胞上Trop-2的表达情况
细胞名称 组织来源 MFI(TINA) MFI(hIgG1) 阳性率(％) 比率
NCI-H322 非小细胞肺癌细胞 2826998 21265 99.97 132.9
Capan-1 人胰腺癌肿瘤细胞 1205346 65600 98.36 18.4
PC-3 人前列腺癌细胞 68507 8925 69.09 7.7
A549 人肺腺癌细胞 10105 9921 0.27 1.0
HUVEC 人脐静脉内皮细胞 125092 20475 22.86 6.1
OVCAR-3 人卵巢癌细胞 182074 11792 99.00 15.4
HCC38 人乳腺导管癌细胞 3853305 32418 99.30 118.9
HCC1806 人乳腺癌细胞 1655211 16247 99.87 101.9
实施例8:抗体细胞水平结合实验
将表达Trop-2的肿瘤细胞用FACS缓冲液(Miltenyi Biotec，130-091-221)制备成0.5-1×10 6个/mL的细胞悬液，以100μL/孔加入96孔V底板中。离心去除上清后加入50μL/孔用FACS缓冲液稀释的不同浓度待测抗体(0.006nM-100nM)，放于4℃冰箱中避光孵育l小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的AlexaFluor488羊抗人IgG(H+L)(Bio-legend，409304，1:500稀释)，放于4℃冰箱中避光孵育40分钟。以FACS缓冲液300g离心洗涤3次后，在Sartorius iQue3流式细胞仪上检测几何平均数荧光强度(MFI)，计算抗体对不同Trop-2表达水平的肿瘤细胞的结合EC 50值。
表7-1：Ab2的嵌合和人源化抗体对不同肿瘤细胞的亲和力(EC 50)
抗体 HCC38(EC 50,nM) HCC1806(EC 50,nM)
ch-Ab2 0.59 0.78
hz-Ab2-1.2 0.76 0.90
hz-Ab2-1.3 0.65 1.22
hz-Ab2-1.4 1.20 1.07
hz-Ab2-2.4 0.45 1.23
TINA 1.21 1.44
表7-2：Ab21的嵌合和人源化抗体对不同肿瘤细胞的亲和力(EC 50)
抗体 HCC38(EC 50,nM) HCC1806(EC 50,nM) OVCAR-3(EC 50,nM)
ch-Ab21 0.64 0.65 1.02
hz-Ab21-1.1 0.55 0.64 0.62
hz-Ab21-1.2 0.66 1.23 0.67
hz-Ab21-2.1 0.64 0.63 0.68
hz-Ab21-2.2 0.66 1.18 0.80
TINA 0.79 0.84 1.29
表7-3：Ab35的嵌合和人源化抗体对不同肿瘤细胞的亲和力(EC 50)
抗体 HCC38(EC 50,nM) HCC1806(EC 50,nM) OVCAR-3(EC 50,nM)
ch-Ab35 0.45 0.38 0.37
hz-Ab35-1.1 0.77 0.80 0.83
hz-Ab35-1.2 0.71 0.62 0.57
hz-Ab35-1.3 0.70 0.54 0.64
hz-Ab35-2.1 0.89 0.76 0.90
hz-Ab35-2.2 0.91 0.58 0.88
hz-Ab35-2.3 0.91 0.61 0.74
TINA 1.04 0.73 1.28
实施例9:抗体和人Trop-2-His亲和力检测数据
抗体对人Trop-2-His蛋白亲和力的检测，采用捕获抗体的形式。用偶联有抗人IgG抗体(来自Human antibody capture kit,Cat.#29234600,GE)的CM5(Cat.#29149603,GE)生物传感芯片亲和捕获抗体，然后于芯片表面流经抗原人Trop-2-His蛋白，用Biacore 8K仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用再生缓冲液3M MgCl 2(来自Human antibody capture kit,Cat.#29234600,GE)将芯片洗净再生。Biacore 8K Control Software 3.0实时采集结合解离曲线，Biacore Insight Evaluation Software 3.0数据分析，数据经双重扣减后(即每一个循环中实验通道信号扣减掉对照通道信号，样品信号再扣减掉空白 信号)Langmuir 1:1模型拟合，计算结合速率常数Ka、解离速率常数Kd、平衡常数K D值。结果显示本申请中所有人源化抗体都与人Trop-2-His蛋白有较高的亲和力(K D＜10 -8M)。具体见表8。
表8：人源化抗体亲和力
抗体 Ka(1/Ms) Kd(1/s) K D(M)
hz-Ab2-1.1 6.06E+04 2.90E-03 4.78E-08
hz-Ab2-1.2 3.56E+06 5.08E-03 1.43E-09
hz-Ab2-1.3 1.00E+06 1.65E-02 1.65E-08
hz-Ab2-1.4 1.22E+06 2.84E-03 2.32E-09
hz-Ab2-2.1 3.36E+05 2.30E-04 6.86E-10
hz-Ab2-2.2 2.45E+05 5.90E-04 2.41E-09
hz-Ab2-2.3 2.37E+05 6.43E-04 2.72E-09
hz-Ab2-2.4 4.15E+05 4.83E-03 1.16E-08
hz-Ab21-1.1 4.29E+05 5.14E-04 1.20E-09
hz-Ab21-1.2 4.50E+05 4.38E-04 9.73E-10
hz-Ab21-2.1 4.04E+05 5.59E-04 1.38E-09
hz-Ab21-2.2 4.60E+05 6.11E-04 1.33E-09
hz-Ab35-1.1 3.79E+05 2.38E-04 6.28E-10
hz-Ab35-1.2 4.01E+05 3.88E-03 9.66E-09
hz-Ab35-1.3 4.17E+05 3.10E-04 7.43E-10
hz-Ab35-2.1 4.36E+05 1.94E-04 4.44E-10
hz-Ab35-2.2 4.05E+05 3.57E-03 8.82E-09
hz-Ab35-2.3 4.16E+05 2.81E-04 6.75E-10
TINA 1.91E+05 2.39E-03 1.25E-08
实施例10:抗体和猴来源蛋白交叉活性检测
用pH 7.4的PBS(SmartBuffer，PBS-2050)缓冲液将猕猴Trop-2-His(具体是将猴Trop-2的细胞外结构域与His重组连接，SEQ ID NO:4)稀释至2μg/mL，以100μL/孔的体积加入96孔酶标板中，4℃过夜孵育。弃去液体后，每孔加入300μL用PBS稀释的2％BSA(SIGMA，B2064-100G)进行封闭，37℃孵育2小时。封闭结束后，弃去封闭液，并用PBST缓冲液(PH7.4 PBS含0.1％tween-20)洗板3次后，每孔加入100μL梯度稀释的抗体溶液，于37℃孵育l小时。孵育结束后用PBST洗板3次，每孔加入100μL羊抗人IgG(H+L)(JacksonImmunoResearch，109-035-098，1:5000稀释)，37℃孵育l小时。用PBST洗板3次后，每孔加入100μLTMB显色底物(TIANGEN，PA107-01)，室温孵育10-15分钟，每孔加入50μL的1M H 2SO 4终止反应，用酶标仪读取在450nm处的吸收值，用软件拟合出抗体与抗原的结合曲线，计算出EC 50。检测数据说明本申请的所有人源化抗体均和猴Trop-2有较好的结合活性。
表9-1.Ab2的嵌合和人源化抗体与猴Trop-2-His蛋白的结合活性
人源化抗体 亲和力，EC 50(nM)
ch-Ab2 0.048
hz-Ab2-1.1 0.133
hz-Ab2-1.2 0.064
hz-Ab2-1.3 0.068
hz-Ab2-1.4 0.053
hz-Ab2-2.1 1.188
hz-Ab2-2.2 0.341
hz-Ab2-2.3 0.830
hz-Ab2-2.4 0.070
TINA 0.106
表9-2.Ab21的嵌合和人源化抗体与猴Trop-2-His蛋白的结合活性
人源化抗体 亲和力，EC 50(nM)
ch-Ab21 0.093
hz-Ab21-1.1 0.098
hz-Ab21-1.2 0.086
hz-Ab21-2.1 0.064
hz-Ab21-2.2 0.081
TINA 0.063
表9-3.Ab35的嵌合和人源化抗体与猴Trop-2-His蛋白的结合活性
人源化抗体 亲和力，EC 50(nM)
ch-Ab35 0.048
hz-Ab35-1.1 0.047
hz-Ab35-1.2 0.038
hz-Ab35-1.3 0.033
hz-Ab35-2.1 0.056
hz-Ab35-2.2 0.050
hz-Ab35-2.3 0.031
TINA 0.046
实施例11:抗体在不表达Trop-2肿瘤细胞中的非特异结合
将不表达Trop-2的肿瘤细胞A549(肺癌肿瘤细胞)用FACS缓冲液(MiltenyiBiotec，130-091-221)制备成0.5-1×10 6个/mL的细胞悬液，以100μL/孔加入96孔V底板中。离心去除上清后加入50μL/孔用FACS缓冲液稀释的不同浓度待测抗体(0.0006nM-50nM)，放于4℃冰箱中避光孵育l小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的AlexaFluor488羊抗人IgG(H+L)(Bio-legend，409304，1:500稀释)，放于4℃冰箱中避光孵育40分钟。以FACS缓冲液300g离心洗涤3次后，在Sartorius iQue3流式细胞仪上检测几何平均数荧光强度(MFI)。图2的结果显示，本申请的人源化抗体和不表达Trop-2的肺癌肿瘤细胞A549没有非特异性结合。
实施例12:抗体肿瘤细胞内吞实验
使用抗体内化试剂盒(Sartoius，90564)检测抗体在肿瘤细胞的内吞情况，先用检测细胞所用的完全培养基将试剂盒内标记抗体以及待测的人源化或嵌合抗体稀释至40nM后，首梯度按照1:1的比例在96孔V底细胞培养板(Corning，目录号3894)中混合后，进行2倍倍稀后，放置37℃中培养不同的时间(2小时、4小时或6小时)，在Sartorius iQue3流式细胞仪上检测几何平均数荧光强度(MFI)，几何平均数荧光强度(MFI)越强，表明抗体在该细胞内的内吞越强。结果显示本申请的人源化抗体在不同Trop-2表达量细胞系中显示了较强的内吞活性，同时能随着内吞时间的增加不断累积内吞抗体量，并且在较高浓度抗体(20nM)下没有表现出明显的饱和情况。检测结果如图3-图6所示。
实施例13:抗Trop-2抗体的ADCC活性
基于报告基因的方法，分别以NCI-H322和HCC1806为靶细胞，以稳定转染CD16a v158和NFAT-luciferase的Jurkat细胞(Jurkat-CD16a v158-NFAT-luciferase)(BPS Bioscience，目录号60541)为效应细胞，测定抗Trop-2抗体的抗体依赖的细胞介导的细胞毒性(ADCC)活性。TINA作为阳性对照，hIgG1同型对照抗体(百英生物，货号：B117901)作为阴性对照。
用培养基将NCI-H322和HCC1806细胞分别稀释至3×10 5个/mL，每种细胞悬液以30μL/孔分配到96孔板中，然后将不同浓度的待测抗体以40μL/孔加到96孔板中，使待测抗体的终浓度为40nM、8nM、1.6nM、0.32nM、0.064nM、0.013nM、0.0026nM和0.0005nM。然后取出Jurkat-CD16a v158-NFAT-luciferase细胞，按照效应细胞数目:靶细胞数目＝10:1的比例，向96孔板的每孔加入30μL Jurkat-CD16a v158-NFAT-luciferase细胞悬液。低速离心使效应细胞和靶细胞充分混匀接触，于37℃、含5％CO 2的培养箱孵育5-6小时，然后按照萤火虫荧光素酶检测试剂盒(诺唯赞，目录号DD1203-03)说明书，每孔加入100μL检测试剂，静置15-30分钟，并使用酶标仪(Thermo，Varioskan Flash)检测荧光信号。
在报告基因实验中，抗Trop-2抗体的ADCC活性如图7所示，EC 50数据如表10所示，在两个不同的细胞上，嵌合抗体ch-Ab2、ch-Ab21、ch-Ab35及人源化抗体hz-Ab2-1.2、hz-Ab2-1.4、hz-Ab2-1.3、hz-Ab21-2.1、hz-Ab21-2.2、hz-Ab35-1.1、hz-Ab35-1.2、hz-Ab35-1.3、hz-Ab35-2.1、hz-Ab35-2.2和hz-Ab35-2.3均能显著诱导Jurkat-CD16a v158-NFAT-luciferase细胞NFAT信号的激活，并且优于TINA。
表10抗Trop-2抗体的ADCC活性(效应细胞为Jurkat-CD16a v158-NFAT-luciferase)
Figure PCTCN2022137192-appb-000010
Figure PCTCN2022137192-appb-000011
基于PBMC杀伤的作用，分别以NCI-H322和HCC1806为靶细胞，以PBMC为效应细胞，检测抗Trop-2抗体的ADCC活性。TINA作为阳性对照，hIgG1同型对照抗体(百英生物，货号：B117901)作为阴性对照。
用培养基将NCI-H322和HCC1806细胞分别稀释至3×10 5个/mL，每种细胞悬液以50μL/孔分配到96孔板中。然后将不同浓度的待测抗体加到96孔板的各孔中，使待测抗体的终浓度分别为66.7nM、13.34nM、2.67nM、0.53nM、0.11nM、0.021nM、0.004nM和0.0008nM。复苏PBMC细胞，按照效应细胞数目:靶细胞数目＝10:1的比例向96孔板的每孔加入100μL PBMC细胞悬液(细胞密度为1.5×10 6个/mL)。低速离心使效应细胞和靶细胞充分混匀接触，于37℃、含5％CO 2的培养箱孵育18小时。将裂解液组和最大释放组加入细胞裂解液20ul/孔，于37℃、含5％CO 2的培养箱孵育45min，然后将96孔板置于板式离心机中以250g/分钟离心5分钟，每孔取50μL上清液置于96孔检测板中。按照CytoTox96 Non-Radio试剂盒(Promega，目录号PR-G1781)说明书配制LDH工作液，96孔检测板中每孔加入50μL LDH工作液，混匀后避光室温孵育25-35分钟。每孔加入50μL终止液(Promega，目录号PR-G1782)，混匀，使用酶标仪(Tecan，Infinite F50)在492nm波长下读OD值。
Figure PCTCN2022137192-appb-000012
如图8和表11所示，在PBMC杀伤实验中，在两种不同的细胞水平上，抗Trop-2抗体ch-Ab2、ch-Ab21、ch-Ab35、人源化抗体hz-Ab2-1.2、hz-Ab2-1.3、hz-Ab2-1.4、hz-Ab35-1.1、hz-Ab35-1.2、hz-Ab35-1.3、hz-Ab35-2.1、hz-Ab35-2.2的ADCC活性优于TINA或与TINA相当。
表11抗Trop-2嵌合抗体的ADCC活性(效应细胞为PBMC)
Figure PCTCN2022137192-appb-000013
根据本申请所公开的内容，虽然根据优选实施方案对本申请的方法进行了描述，但对本领域技术人员而言，在不背离本申请的概念，精神和范围的情况下，可对在此所述的方法以及所述方法的步骤或步骤的顺序进行改变，这都落入了本公开的保护范围内。
本文所引用的所有文献的公开内容通过引用结合于此，引用程度为，他们提供示例性的，程序上和其他的细节补充本文所述内容。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本文中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。

Claims (19)

  1. 结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中，所述重链可变区包含SEQ ID NO:65、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:63或SEQ ID NO:64或所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:66、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:54、SEQ ID NO:55或SEQ ID NO:67所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3。
  2. 根据权利要求1所述的结合人TROP-2的抗体或其抗原结合片段，其中，
    所述重链可变区包含SEQ ID NO:65所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:66所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:23所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:24所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:25所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:26所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:27所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:28所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:36所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:40所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:37所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:40所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:38所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:40所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:39所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:40所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:36所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:41所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:37所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:41所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:38所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:41所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:39所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:41所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:50所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:54所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:51所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:54所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:52所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:54所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:53所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:54所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:50所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:55所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:51所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:55所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:52所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:55所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:53所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:55所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:63所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:66所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:64所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:66所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:63所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可 变区包含SEQ ID NO:67所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；
    所述重链可变区包含SEQ ID NO:64所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:67所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3；或者
    所述重链可变区包含SEQ ID NO:65所示可变区序列的重链CDR1、重链CDR2和重链CDR3，所述轻链可变区包含SEQ ID NO:67所示可变区序列的轻链CDR1、轻链CDR2和轻链CDR3。
  3. 结合人TROP-2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区包含重链CDR1、重链CDR2和重链CDR3，所述重链CDR1与选自SEQ ID NO:17、SEQ ID NO:5、SEQ ID NO:11或SEQ ID NO:48的氨基酸序列具有至少80％同一性，所述重链CDR2与选自SEQ ID NO:18、SEQ ID NO:6、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:49、SEQ ID NO:12或SEQ ID NO:62的氨基酸序列具有至少80％同一性，所述重链CDR3与选自SEQ ID NO:19、SEQ ID NO:7或SEQ ID NO:13的氨基酸序列具有至少80％同一性，所述轻链可变区包含轻链CDR1、轻链CDR2和轻链CDR3，所述轻链CDR1与选自SEQ ID NO:20、SEQ ID NO:8、SEQ ID NO:33或SEQ ID NO:14的氨基酸序列具有至少80％同一性，所述轻链CDR2与选自SEQ ID NO:21、SEQ ID NO:9、SEQ ID NO:34或SEQ ID NO:15的氨基酸序列具有至少80％同一性，所述轻链CDR3与选自SEQ ID NO:22、SEQ ID NO:10、SEQ ID NO:35或SEQ ID NO:16的氨基酸序列具有至少80％同一性。
  4. 根据权利要求3所述的抗体或其抗原结合片段，其中所述重链CDR1、重链CDR2和重链CDR3以及轻链CDR1、轻链CDR2和轻链CDR3分别选自以下序列或与其具有至少80％同一性的序列：
    i.重链CDR1 SEQ ID NO:17，
    重链CDR2 SEQ ID NO:18，
    重链CDR3 SEQ ID NO:19，
    轻链CDR1 SEQ ID NO:20，
    轻链CDR2 SEQ ID NO:21，
    轻链CDR3 SEQ ID NO:22；或，
    ii.重链CDR1 SEQ ID NO:5，
    重链CDR2 SEQ ID NO:32，
    重链CDR3 SEQ ID NO:7，
    轻链CDR1 SEQ ID NO:8，
    轻链CDR2 SEQ ID NO:34，
    轻链CDR3 SEQ ID NO:10；或，
    iii.重链CDR1 SEQ ID NO:5，
    重链CDR2 SEQ ID NO:31，
    重链CDR3 SEQ ID NO:7，
    轻链CDR1 SEQ ID NO:33，
    轻链CDR2 SEQ ID NO:34，
    轻链CDR3 SEQ ID NO:35；或，
    iv.重链CDR1 SEQ ID NO:5，
    重链CDR2 SEQ ID NO:32，
    重链CDR3 SEQ ID NO:7，
    轻链CDR1 SEQ ID NO:33，
    轻链CDR2 SEQ ID NO:34，
    轻链CDR3 SEQ ID NO:35；或，
    v.重链CDR1 SEQ ID NO:48，
    重链CDR2 SEQ ID NO:49，
    重链CDR3 SEQ ID NO:13，
    轻链CDR1 SEQ ID NO:14，
    轻链CDR2 SEQ ID NO:15，
    轻链CDR3 SEQ ID NO:16；或，
    vi.重链CDR1 SEQ ID NO:48，
    重链CDR2 SEQ ID NO:12，
    重链CDR3 SEQ ID NO:13，
    轻链CDR1 SEQ ID NO:14，
    轻链CDR2 SEQ ID NO:15，
    轻链CDR3 SEQ ID NO:16；或，
    vii.重链CDR1 SEQ ID NO:17，
    重链CDR2 SEQ ID NO:62，
    重链CDR3 SEQ ID NO:19，
    轻链CDR1 SEQ ID NO:20，
    轻链CDR2 SEQ ID NO:21，
    轻链CDR3 SEQ ID NO:22；或，
    viii.重链CDR1 SEQ ID NO:17，
    重链CDR2 SEQ ID NO:18，
    重链CDR3 SEQ ID NO:19，
    轻链CDR1 SEQ ID NO:20，
    轻链CDR2 SEQ ID NO:21，
    轻链CDR3 SEQ ID NO:22；或，
    ix.重链CDR1 SEQ ID NO:5，
    重链CDR2 SEQ ID NO:6，
    重链CDR3 SEQ ID NO:7，
    轻链CDR1 SEQ ID NO:8，
    轻链CDR2 SEQ ID NO:9，
    轻链CDR3 SEQ ID NO:10；或，
    x.重链CDR1 SEQ ID NO:11，
    重链CDR2 SEQ ID NO:12，
    重链CDR3 SEQ ID NO:13，
    轻链CDR1 SEQ ID NO:14，
    轻链CDR2 SEQ ID NO:15，
    轻链CDR3 SEQ ID NO:16；或，
    xi.重链CDR1 SEQ ID NO:5，
    重链CDR2 SEQ ID NO:31，
    重链CDR3 SEQ ID NO:7，
    轻链CDR1 SEQ ID NO:8，
    轻链CDR2 SEQ ID NO:34，
    轻链CDR3 SEQ ID NO:10。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，其中所述重链可变区与选自SEQ ID NO:65、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:63或SEQ ID NO:64的氨基酸序列具有至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性，且其中所述轻链可变区与选自SEQ ID NO:66、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:54、SEQ ID NO:55或SEQ ID NO:67的氨基酸序列至少80％同一性、至少81％同一性、至少82％同一性、至少83％同一性、至少84％同一性、至少85％同一性、至少86％同一性、至少87％同一性、至少88％同一性、至少89％同一性、至少90％同一性、至少91％同一性、至少92％同一性、至少93％同一性、至少94％同一性、至少95％同一性、至少96％同一性、至少97％同一性、至少98％同一性、或至少99％同一性。
  6. 根据权利要求5所述的抗体或其抗原结合片段，其中所述重链可变区和轻链可变区分别选自以下序列或与其具有至少80％同一性的序列：
    i)重链可变区SEQ ID NO:65，轻链可变区SEQ ID NO:66；或
    ii)重链可变区SEQ ID NO:37，轻链可变区SEQ ID NO:40；或
    iii)重链可变区SEQ ID NO:38，轻链可变区SEQ ID NO:40；或
    iv)重链可变区SEQ ID NO:39，轻链可变区SEQ ID NO:40；或
    v)重链可变区SEQ ID NO:36，轻链可变区SEQ ID NO:41；或
    vi)重链可变区SEQ ID NO:37，轻链可变区SEQ ID NO:41；或
    vii)重链可变区SEQ ID NO:38，轻链可变区SEQ ID NO:41；或
    viii)重链可变区SEQ ID NO:39，轻链可变区SEQ ID NO:41；或
    ix)重链可变区SEQ ID NO:50，轻链可变区SEQ ID NO:54；或
    x)重链可变区SEQ ID NO:51，轻链可变区SEQ ID NO:54；或
    xi)重链可变区SEQ ID NO:52，轻链可变区SEQ ID NO:54；或
    xii)重链可变区SEQ ID NO:53，轻链可变区SEQ ID NO:54；或
    xiii)重链可变区SEQ ID NO:50，轻链可变区SEQ ID NO:55；或
    xiv)重链可变区SEQ ID NO:51，轻链可变区SEQ ID NO:55；或
    xv)重链可变区SEQ ID NO:52，轻链可变区SEQ ID NO:55；或
    xvi)重链可变区SEQ ID NO:53，轻链可变区SEQ ID NO:55；或
    xvii)重链可变区SEQ ID NO:63，轻链可变区SEQ ID NO:66；或
    xviii)重链可变区SEQ ID NO:64，轻链可变区SEQ ID NO:66；或
    xix)重链可变区SEQ ID NO:36，轻链可变区SEQ ID NO:40；或
    xx)重链可变区SEQ ID NO:63，轻链可变区SEQ ID NO:67；或
    xxi)重链可变区SEQ ID NO:64，轻链可变区SEQ ID NO:67；或
    xxii)重链可变区SEQ ID NO:65，轻链可变区SEQ ID NO:67；或
    xxiii)重链可变区SEQ ID NO:23，轻链可变区SEQ ID NO:24；或
    xxiv)重链可变区SEQ ID NO:25，轻链可变区SEQ ID NO:26；或
    xxv)重链可变区SEQ ID NO:27，轻链可变区SEQ ID NO:28。
  7. 根据权利要求6所述的抗体或其抗原结合片段，其包含重链和轻链，其中所述重链和轻链分别选自以下序列或与其具有至少80％同一性的序列：
    i)重链SEQ ID NO:70，轻链SEQ ID NO:71；或，
    ii)重链SEQ ID NO:43，轻链SEQ ID NO:46；或，
    iii)重链SEQ ID NO:44，轻链SEQ ID NO:46；或，
    iv)重链SEQ ID NO:45，轻链SEQ ID NO:46；或，
    v)重链SEQ ID NO:42，轻链SEQ ID NO:47；或，
    vi)重链SEQ ID NO:43，轻链SEQ ID NO:47；或，
    vii)重链SEQ ID NO:44，轻链SEQ ID NO:47；或，
    viii)重链SEQ ID NO:45，轻链SEQ ID NO:47；或，
    ix)重链SEQ ID NO:56，轻链SEQ ID NO:60；或，
    x)重链SEQ ID NO:57，轻链SEQ ID NO:60；或，
    xi)重链SEQ ID NO:58，轻链SEQ ID NO:60；或，
    xii)重链SEQ ID NO:59，轻链SEQ ID NO:60；或，
    xiii)重链SEQ ID NO:56，轻链SEQ ID NO:61；或，
    xiv)重链SEQ ID NO:57，轻链SEQ ID NO:61；或，
    xv)重链SEQ ID NO:58，轻链SEQ ID NO:61；或，
    xvi)重链SEQ ID NO:59，轻链SEQ ID NO:61；或，
    xvii)重链SEQ ID NO:68，轻链SEQ ID NO:71；或，
    xviii)重链SEQ ID NO:69，轻链SEQ ID NO:71；或，
    xix)重链SEQ ID NO:42，轻链SEQ ID NO:46；或，
    xx)重链SEQ ID NO:68，轻链SEQ ID NO:72；或，
    xxi)重链SEQ ID NO:69，轻链SEQ ID NO:72；或，
    xxii)重链SEQ ID NO:70，轻链SEQ ID NO:72。
  8. 根据权利要求1-7中任一项所述的抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、人源化抗体或全人抗体。
  9. 根据权利要求1-8中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段选自单克隆抗体、融合蛋白、多特异性抗体、Fab片段、Fab’片段、F(ab’)2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、scFv和纳米抗体。
  10. 根据权利要求1-9中任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段为IgG1、IgG2、IgG3或IgG4型。
  11. 根据权利要求1-10中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段展现出下述性质中的一种或多种的组合：
    (a)以1.00E-08M或更小的K D值与Trop-2结合；
    (b)阻断Trop-2与配体的结合；
    (c)在表达Trop-2的细胞中显示内吞活性；
    (d)诱导针对表达Trop-2的细胞的ADCC；
    (e)与不表达Trop-2的肿瘤细胞不结合。
  12. 抗体药物偶联物，其包含与治疗剂相连接或缀合的权利要求1-11中任一项所述的抗体或其抗原结合片段；优选地，所述治疗剂是细胞毒药物、放射性同位素、免疫调节剂或抗体。
  13. 药物组合物，其包含药学上可接受的载体，以及i)权利要求1-11中任一项所述的抗体或其抗原结合片段，或ii)权利要求12所述的抗体药物偶联物。
  14. 分离的核酸分子，其编码权利要求1-11中任一项所述的抗体或其抗原结合片段。
  15. 包含权利要求14所述的核酸分子的表达载体。
  16. 包含权利要求15所述的表达载体或在其基因组中整合有权利要求14所述的分离的核酸分子的宿主细胞。
  17. 权利要求1-11中任一项所述的抗体或其抗原结合片段、权利要求12所述的抗体药物偶联物、或权利要求13所述的药物组合物在制备用于治疗TROP-2介导的疾病的药物中的用途。
  18. 权利要求17所述的用途，其中所述TROP-2介导的疾病为癌症。
  19. 产生权利要求1-10中任一项所述的抗体或其抗原结合片段的方法，所述方法包括在使得所述抗体或其抗原结合片段表达的条件下培养权利要求16所述的宿主细胞，和回收表达的抗体或其抗原结合片段。
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