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WO2025039809A1 - 抗ror1抗体及其用途 - Google Patents

抗ror1抗体及其用途 Download PDF

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Publication number
WO2025039809A1
WO2025039809A1 PCT/CN2024/106172 CN2024106172W WO2025039809A1 WO 2025039809 A1 WO2025039809 A1 WO 2025039809A1 CN 2024106172 W CN2024106172 W CN 2024106172W WO 2025039809 A1 WO2025039809 A1 WO 2025039809A1
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Prior art keywords
seq
sequence
antibody
amino acid
deletions
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PCT/CN2024/106172
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English (en)
French (fr)
Inventor
潘荣
王晓艳
李良维
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上海麦科思生物医药有限公司
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Publication of WO2025039809A1 publication Critical patent/WO2025039809A1/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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present disclosure belongs to the field of medicine, and specifically, the present disclosure relates to antibodies targeting ROR1, antibody-drug conjugates containing the same, nucleic acid molecules encoding the same, and their use in the preparation of antibody-drug conjugates.
  • the antibody targeting ROR1 or its antigen-binding fragment has a heavy chain variable region, a light chain variable region, and a complementary determining region disclosed herein.
  • the present disclosure further relates to an antibody-drug conjugate containing the antibody or its antigen-binding fragment, and a preparation method and use thereof.
  • Cancer is one of the leading causes of illness and death in the world. As the population ages, the global cancer burden will continue to increase. The AACR report points out that by 2040, the total number of cancer patients in the world will reach about 28 million, and an estimated 16.2 million patients will die from it.
  • Receptor tyrosine kinase-like orphan receptor is a member of the type I receptor tyrosine kinase (RTK) family and a type of membrane receptor, mainly including ROR1 and ROR2.
  • ROR1 is highly expressed during early embryonic development and plays an important role in a variety of physiological processes, including regulating cell division, proliferation, migration, etc., and participating in the formation of organs such as nerves, bones and blood vessels. In the subsequent fetal development, the expression of ROR1 gradually decreases, and there is basically no expression of ROR1 on the surface of normal children and adult tissue cells (Semin Cancer Biol 2014 Vol.29,21-31).
  • ROR1 is greatly increased in a variety of tumor cells, including solid tumor cells such as lung adenocarcinoma, breast cancer, ovarian cancer, melanoma, kidney cancer, gastric cancer, colorectal cancer, and pancreatic cancer (BMC Cancer. 2021 Nov 11; 21(1): 1199.) and certain blood malignancies such as chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL).
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • the expression of ROR1 is closely related to the progression of the disease and the therapeutic effect (Clin Cancer Res. 2017 Jun 15; 23(12): 3061-3071; Front Oncol. 2021 May 28; 11: 680834).
  • ROR1 can play an important role in various physiological processes by mediating signal transduction of non-canonical Wnt pathways, including regulating cell division, proliferation, migration, and cell chemotaxis, especially It is Wnt5a.
  • Wnt5a is a typical activator of the non-classical Wnt signaling pathway, involved in the phosphorylation of the NF- ⁇ B subunit p65, activating the NF- ⁇ B pathway in tumor cells, promoting cell migration and invasion, epithelial-mesenchymal transition (EMT), cancer metastasis, etc.
  • EMT epithelial-mesenchymal transition
  • ROR1 plays an important role in the development and EMT process of cancer stem cells.
  • Cancer stem cells are cancer cells with more stem cell characteristics in tumors, and they usually have higher resistance to chemotherapy drugs.
  • the EMT process transforms the morphology of cells from epithelial cells to mesenchymal cells, making them more invasive and promoting cancer metastasis.
  • ROR1 is involved in the activation of proto-oncogenes (c-Src) and MET, leading to the inhibition of tumor cell apoptosis.
  • c-Src proto-oncogenes
  • MET proto-oncogenes
  • ROR1 has also been found to be a scaffold protein for cavin-1 and caveolin-1, which can activate AKT in lung adenocarcinoma.
  • ROR1 promotes the activation of the PI3K/AKT pathway, and high ROR1 expression is associated with more severe disease progression.
  • aberrant expression of ROR1 and associated growth-promoting signaling events have been observed in many types of malignancies, making ROR1 an attractive therapeutic target for anticancer drug development
  • ROR1 is highly expressed in many cancers, but not expressed or expressed at a low level in normal tissues
  • drug developers have developed a variety of anti-tumor treatment strategies targeting ROR1, among which ADC drugs have made rapid progress and shown good application prospects.
  • ADC drugs Due to the dual advantages of high targeting of monoclonal antibody drugs and high activity of cytotoxins in tumor tissues, ADC drugs can efficiently kill tumor cells, have fewer side effects than chemotherapy drugs, and have better efficacy than traditional antibody tumor drugs. They are called "biological missiles" in the field of tumor treatment.
  • ROR1 ADC drugs in the clinical stage include: VLS-101 (WO2018237335) jointly developed by Merck (MSD) and VelosBio, which consists of a monoclonal antibody targeting ROR1 and monomethyl auristatin E (MMAE). It is currently in Phase II clinical development for the treatment of patients with hematological malignancies and solid tumors. However, due to the limited therapeutic window of MMAE, it only shows weak tumor inhibitory activity in preclinical efficacy models for solid tumors with low ROR1 expression.
  • LCB71/CS5001 (CN202010852078.1), an antibody-drug conjugate for ROR1, was jointly developed by CStone Pharmaceuticals and LegoChem Biosciences.
  • LCB71 carries a tumor-activated pyrrolobenzodiazepine (PBD) protoxin.
  • PBD tumor-activated pyrrolobenzodiazepine
  • NBE-002 (WO2017127664), an ADC candidate drug targeting ROR1 developed by Boehringer Ingelheim/NBE Therapeutics, couples PNU-159682 to the C-terminus of the heavy and light chains of the antibody in a site-specific and quantitative manner through a specific reaction mediated by Sortase A.
  • PNU-159682 has the same safety risks as PBD toxins.
  • ADC drugs have entered a harvest period, and the number of approved drugs has increased rapidly. As of December 2022, a total of 15 ADC drugs have been approved for marketing worldwide. The world's first ADC drug Mylotarg was approved for marketing in 2000, but due to fatal liver damage It was withdrawn from the market in 2010, and was re-listed in 2017 after optimizing the dosing regimen to improve efficacy and safety. Between 2001 and 2010, no ADC drugs were approved for marketing worldwide, and the industry entered a trough period. With the continuous advancement of technology, ADC drugs have entered a concentrated period of marketing. As of now, a total of 10 have been approved.
  • ROR1 is different from other ADC antigens.
  • HER2 has hundreds of thousands of copies on tumor cells, and the expression level of ROR1 in most tumor cells is not very high, with no more than 5,000 copies per cell.
  • An ADC molecule that is more effective for extremely low expression and has an improved safety window is needed.
  • the first aspect of the present disclosure provides an antibody or an antigen-binding fragment thereof capable of specifically binding to ROR1, comprising:
  • VH variable region
  • CDRs complementarity determining regions
  • VH CDR1 having the sequence of CDR1 contained in a VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in the VH;
  • VH CDR2 having the sequence of CDR2 contained in VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR2 contained in said VH; and
  • VH CDR3 having the sequence of the CDR3 contained in the VH as shown in SEQ ID NO: 37 or 41, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;
  • VL CDR1 having the sequence of the CDR1 contained in the VL as shown in SEQ ID NO: 38 or 42, or having one or more amino acid substitutions, deletions or additions compared to the sequence of the CDR1 contained in the VL (e.g. 1 or 2 amino acid substitutions, deletions or additions);
  • VL CDR2 having the sequence of CDR2 contained in VL as shown in SEQ ID NO: 38 or 42, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR2 contained in said VL;
  • VL CDR3 having the sequence of the CDR3 contained in the VL as shown in SEQ ID NO: 38 or 42, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VL;
  • VH CDR2 having the sequence of CDR2 contained in VH as shown in SEQ ID NO:39, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of CDR2 contained in said VH; and
  • VH CDR3 having the sequence of the CDR3 contained in the VH as shown in SEQ ID NO: 39, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;
  • VL CDR1 which has the sequence of CDR1 contained in VL as shown in SEQ ID NO:40, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of CDR1 contained in VL;
  • VL CDR2 which has the sequence of CDR2 contained in VL as shown in SEQ ID NO:40, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of CDR2 contained in said VL;
  • VL CDR3 which has the sequence of CDR3 contained in VL as shown in SEQ ID NO:40, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of CDR3 contained in said VL.
  • substitutions described in any of (i)-(vi) above are conservative substitutions.
  • the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are numbered according to the Kabat, Chothia or IMGT numbering system. System definition.
  • the antibody or antigen-binding fragment thereof comprises:
  • the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and/or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 1,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 2, or which consists of the following sequence: SEQ ID NO: 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 45, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 3;
  • VL CDR1 which consists of the following sequence: SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO:4, or it consists of the following sequence: SEQ ID NO:46, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO:46,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 5, and
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 6.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 1,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 2, or which consists of the following sequence: SEQ ID NO: 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 45, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 3;
  • VL CDR1 which consists of the following sequence: SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO:4,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 5, and
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 6, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 6.
  • substitutions described in any of (i)-(vi) above are conservative substitutions.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:2; and VH CDR3 as shown in SEQ ID NO:3; and/or, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:45; and VH CDR3 as shown in SEQ ID NO:3; and/or, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:2; and VH CDR3 as shown in SEQ ID NO:3; and/or, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:46; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.
  • the VH of the antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 1 as shown in SEQ ID NO:45; VH CDR2; and, VH CDR3 as shown in SEQ ID NO:3; and/or, the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO:46; VL CDR2 as shown in SEQ ID NO:5; and, VL CDR3 as shown in SEQ ID NO:6.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:2; and VH CDR3 as shown in SEQ ID NO:3; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:45; and VH CDR3 as shown in SEQ ID NO:3; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:2; and VH CDR3 as shown in SEQ ID NO:3; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:46; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:45; and VH CDR3 as shown in SEQ ID NO:3; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:46; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 7,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 8, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 9;
  • VL CDR1 consisting of the following sequence: SEQ ID NO: 10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 10;
  • the sequence is, or consists of, SEQ ID NO: 47, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 47,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 11, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 11, and
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 12, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 12.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 7,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 8, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 9;
  • VL CDR1 which consists of the following sequence: SEQ ID NO: 10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 10,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 11, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 11, and
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 12, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 12.
  • substitutions described in any of (i)-(vi) above are conservative substitutions.
  • the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO: 7; a VH CDR2 as shown in SEQ ID NO: 8; and a VH CDR3 as shown in SEQ ID NO: 9; and/or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as shown in SEQ ID NO: 10 CDR1; VL CDR2 as shown in SEQ ID NO:11; and, VL CDR3 as shown in SEQ ID NO:12.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:7; VH CDR2 as shown in SEQ ID NO:8; and VH CDR3 as shown in SEQ ID NO:9; and/or, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:47; VL CDR2 as shown in SEQ ID NO:11; and VL CDR3 as shown in SEQ ID NO:12.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:7; VH CDR2 as shown in SEQ ID NO:8; and VH CDR3 as shown in SEQ ID NO:9; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:10; VL CDR2 as shown in SEQ ID NO:11; and VL CDR3 as shown in SEQ ID NO:12.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:7; VH CDR2 as shown in SEQ ID NO:8; and VH CDR3 as shown in SEQ ID NO:9; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:47; VL CDR2 as shown in SEQ ID NO:11; and VL CDR3 as shown in SEQ ID NO:12.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 13,
  • VH CDR2 consisting of the following sequence: SEQ ID NO: 14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 14, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 15, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 15;
  • VL CDR1 which consists of the following sequence: SEQ ID NO: 16, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 16,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 17, and
  • VL CDR3 consisting of the following sequence: SEQ ID NO: 18, or having a A sequence having one or several amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions).
  • substitutions described in any of (i)-(vi) above are conservative substitutions.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:13; VH CDR2 as shown in SEQ ID NO:14; and VH CDR3 as shown in SEQ ID NO:15; and/or, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:16; VL CDR2 as shown in SEQ ID NO:17; and VL CDR3 as shown in SEQ ID NO:18.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:13; VH CDR2 as shown in SEQ ID NO:14; and VH CDR3 as shown in SEQ ID NO:15; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:16; VL CDR2 as shown in SEQ ID NO:17; and VL CDR3 as shown in SEQ ID NO:18.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 19,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 20, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 20, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 21;
  • VL CDR1 which consists of the following sequence: SEQ ID NO: 22, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 22,
  • VL CDR3 consisting of the following sequence: SEQ ID NO: 24, or a sequence having one or several amino acid substitutions, deletions or additions (eg, 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 24.
  • substitutions described in any of (i)-(vi) above are conservative substitutions.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:19; VH CDR2 as shown in SEQ ID NO:20; and VH CDR3 as shown in SEQ ID NO:21; and/or, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:22; VL CDR2 as shown in SEQ ID NO:23; and VL CDR3 as shown in SEQ ID NO:24.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:19; VH CDR2 as shown in SEQ ID NO:20; and VH CDR3 as shown in SEQ ID NO:21; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:22; VL CDR2 as shown in SEQ ID NO:23; and VL CDR3 as shown in SEQ ID NO:24.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 25, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 25,
  • VH CDR2 consisting of the following sequence: SEQ ID NO: 26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 26, and
  • VL CDR1 consisting of the following sequence: SEQ ID NO: 28, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 28,
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 30, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 30.
  • substitutions described in any of (i)-(vi) above are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH CDR1 consisting of the following sequence: SEQ ID NO: 31, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 31,
  • VH CDR2 consisting of the following sequence: SEQ ID NO: 32, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 32, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 33, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 33;
  • VL CDR1 consisting of the following sequence: SEQ ID NO: 34, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 34,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 35, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 35, and
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 36, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to SEQ ID NO: 36.
  • substitutions described in any of (i)-(vi) above are conservative substitutions.
  • the VH of the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as shown in SEQ ID NO: 31; a VH CDR2 as shown in SEQ ID NO: 32; and a VH CDR3 as shown in SEQ ID NO: 33; and/or the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR3 as shown in SEQ ID NO: 34.
  • the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:31; VH CDR2 as shown in SEQ ID NO:32; and VH CDR3 as shown in SEQ ID NO:33; and, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:34; VL CDR2 as shown in SEQ ID NO:35; and VL CDR3 as shown in SEQ ID NO:36.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • substitutions described in (ii) or (v) above are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • the antibody or antigen-binding fragment thereof comprises:
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) above are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain variable region comprising a sequence as shown in SEQ ID NO:39 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:39, and a light chain variable region (VL) comprising a sequence as shown in SEQ ID NO:40 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:40.
  • the antibody or antigen-binding fragment thereof comprises:
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) above are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain variable region comprising a sequence as shown in SEQ ID NO:41 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:41, and a light chain variable region (VL) comprising a sequence as shown in SEQ ID NO:42 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:X.
  • the antibody or antigen-binding fragment thereof comprises:
  • the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is humanized.
  • the antibody or antigen-binding fragment thereof further comprises a framework region of a human immunoglobulin.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), and/or a light chain framework region of a human immunoglobulin (e.g., a light chain framework region contained in the amino acid sequence encoded by a human light chain germline antibody gene).
  • a human immunoglobulin e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene
  • a light chain framework region of a human immunoglobulin e.g., a light chain framework region contained in the amino acid sequence encoded by a human light chain germline antibody gene
  • the heavy chain framework region and/or light chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from human residues to murine residues.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).
  • a human immunoglobulin eg, IgG1, IgG2, IgG3, or IgG4.
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • a human immunoglobulin eg, kappa or lambda
  • the monoclonal antibody comprises non-CDR regions, and the non-CDR regions are from a species other than murine, such as from a human antibody.
  • the antibody or antigen-binding fragment thereof binds to human or monkey ROR1 with a KD of less than or equal to 1.0 ⁇ 10 -8 M.
  • the second aspect of the present disclosure provides an isolated nucleic acid molecule, which comprises a nucleic acid sequence encoding the antibody or its antigen-binding fragment, its heavy chain and/or light chain, or its heavy chain variable region and/or light chain variable region, or it encodes the antibody or its antigen-binding fragment, its heavy chain and/or light chain, or its heavy chain variable region and/or light chain variable region.
  • the third aspect of the present disclosure provides a vector comprising the isolated nucleic acid molecule.
  • the fourth aspect of the present disclosure provides a host cell comprising the nucleic acid molecule or the vector.
  • the fifth aspect of the present disclosure provides the use of the antibody or its antigen-binding fragment in the preparation of an antibody-drug conjugate, or in the preparation of a kit for diagnosing a disease, especially a ROR1-mediated disease or condition, or in the preparation of a medicament for preventing and/or treating a disease, especially a ROR1-mediated disease or condition.
  • the present disclosure also provides the antibody or antigen-binding fragment thereof, which is used as an antibody-drug conjugate, or for diagnosing, preventing and/or treating a disease, especially a ROR1-mediated disease or condition.
  • the present disclosure also provides a method for diagnosing, preventing and/or treating a disease, especially a disease or condition mediated by ROR1, comprising administering an effective amount of the antibody or antigen-binding fragment thereof to an individual in need thereof.
  • the sixth aspect of the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in formula (I),
  • Q 1 -L 1 -L 2 -L 3 is a linker
  • Ab is the antibody or antigen-binding fragment thereof, which is coupled to Q 1 in the linker via the S atom in the antibody or antigen-binding fragment thereof,
  • T is a drug unit or a diagnostic agent unit, which is coupled to L 3 in the linker via an N atom or an O atom in the drug unit or the diagnostic agent unit,
  • n is a number between 1 and 9, for example, m is a number between 2 and 9, for example, about 4, about 5, about 6, about 7, about 8, or about 9,
  • Q 1 is selected from: Replaced Replaced Replaced and replaced by
  • L2 is a polypeptide residue or a substituted polypeptide residue
  • L 1a is selected from: an alkylene group, a polyethylene glycol group, an alkenylene group, an alkynylene group, and an alicyclic group.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein L2 and/or L3 may be Substituted with CH 2 NHCH 3 , a polysarcosine residue, a glycosylated polyethylene glycol fragment, a substituted polysarcosine residue or a substituted glycosylated polyethylene glycol fragment.
  • the substituted polysarcosine residue is wherein n2 is a number between 4 and 18, for example, a number between 4 and 12, and R is selected from the group consisting of: C 1 -C 6 alkyl, C 1 -C 6 cycloalkyl, and C 1 -C 6 alkoxy,
  • the antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein m is a number between 7 and 9, e.g., 7.5-9, e.g., about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the drug unit is selected from:
  • Tubulin inhibitors such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubulysin or its analogs (e.g. tubulysin A, tubulysin B, tubulysin D, tubulysin H, tubulysin U, tubulysin V), eribulin or its analogs, vinblastine or its analogs (e.g. vincristine, vindesine, vinblastine, DAVLBH), Aur0101;
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • tubulysin or its analogs e.g. tubulysin A, tubulysin B, tubulysin D, tubulysin H, tubulysin U, tubulysin V
  • eribulin or its analogs e.g. vincristine, vindesine, vinblastine, DAVLBH
  • Camptothecin topoisomerase inhibitors such as Exatecan (CAS171335-80-1), DXd (CAS1599440-33-1), 7-ethyl-10-hydroxycamptothecin (SN38, CAS 86639-52-3), Belotecan (CAS213819-48-8), (4- NH2 )-Exatecan (AZD'0132, CAS2495742-21-5), 7-MAD-MDCPT (CAS 765871-81-6), 7-Aminomethyl-10-methyl-11-fluoro camptothecin (CAS2378616-23-8);
  • Immune activators such as TLR7/8 agonists and STING agonists.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is selected from:
  • a seventh aspect of the present disclosure provides a method for preparing the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, comprising:
  • the compound represented by formula L-D is selected from compounds L-D-3 to L-D-11.
  • a typical preparation method includes: diluting the antibody stock solution to 2-30 mg/mL with a reaction buffer, adding 140-200 times excess molar ratio of dithiothreitol (DTT), or adding 6.0-20 times excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and stirring the reaction solution at 35-39° C. for 2-48 hours.
  • DTT dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the above reaction buffer can be a buffer prepared according to the following ratio: 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH)/150 mM sodium chloride (NaCl) /1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50 mM disodium hydrogen phosphate-citric acid/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50 mM boric acid-borax/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50 mM histidine-sodium hydroxide/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; PBS/1 mM diethyltriaminepent
  • the above reaction solution is cooled to 0-10°C. If DTT reduction is used, excess DTT needs to be removed by desalting column or ultrafiltration after the reduction reaction is completed, and then the substituted maleimide compound (preliminarily dissolved in acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethylacetamide (DMA) at a concentration of 10 mg/ml) is added, and the volume proportion of the organic solvent in the total reaction solution is ensured not to exceed 15%, and the coupling reaction is stirred at 0-37°C for 2-4 hours. If TCEP reduction is used, it is also possible to directly add the substituted maleimide compound for coupling without removing the remaining TCEP.
  • ACN acetonitrile
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • DMA diethylacetamide
  • the coupling reaction mixture was purified by filtration using a desalting column with histidine-acetate buffer containing sodium chloride or a phosphate buffer containing sodium chloride, centrifuged, and the flow-through fraction was collected.
  • the DAR value of the obtained antibody drug conjugate is relatively uniform.
  • the ADC product uniformity is very high (usually DAR-dominant products (such as DAR values of about 4, 5, 6, 7, 8 or 9) account for at least 60%, at least 70%, at least 80%, at least 90% or more of all ADCs).
  • DAR-dominant products such as DAR values of about 4, 5, 6, 7, 8 or 9
  • the following methods can be further used for separation and purification, but are not limited to: hydrophobic interaction chromatography (HIC), molecular exclusion chromatography (SEC), ion exchange chromatography (IEC).
  • the eighth aspect of the present disclosure provides a pharmaceutical composition, which contains the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and/or excipients.
  • the antibody-drug conjugate is present in the pharmaceutical composition in an effective amount for treating a disease.
  • the antibody-drug conjugate can be administered as a single active pharmaceutical ingredient or in combination with other drugs, such as other anticancer drugs.
  • the pharmaceutical composition also contains other drugs, such as other anticancer drugs.
  • the other anticancer drugs include, but are not limited to, anti-PD-1 antibodies, anti-PDL-1 antibodies, and/or anti-CTLA-4 antibodies.
  • the non-small cell lung cancer is selected from lung adenocarcinoma and lung squamous cell carcinoma.
  • the term "about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies at an acceptable level in the art. In some embodiments, such variation may be as much as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% by weight or length.
  • the term "about” modifies the range by extending the boundaries above and below the numerical values set forth.
  • ROR1 mediates signal transduction of the non-canonical Wnt pathway by binding to the ligand Wnt5a, which is involved in the occurrence and development of various cellular physiological processes and various cancers.
  • ROR1 is highly expressed during early embryonic development and is involved in regulating cell division, proliferation, migration, etc., and is involved in the generation of organs such as nerves, bones, and blood vessels. Along with the process of fetal development, the expression of ROR1 gradually decreases. In children and adults, the expression of ROR1 is low or absent in almost all normal tissues.
  • ROR1 is expressed in a variety of cancers, including hematological cancers such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and solid tumors (ovarian, breast, prostate, lung, melanoma, and colorectal cancer, among others).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • solid tumors ovarian, breast, prostate, lung, melanoma, and colorectal cancer, among others.
  • High expression of ROR1 is usually determined by comparing its expression levels in cancer tissues and normal tissues. Usually, this is determined by using various experimental techniques such as immunohistochemistry (IHC), Western blotting, fluorescence quantitative PCR (qPCR), and gene chips. Generally speaking, for a certain tumor antigen, its expression level above a certain threshold can be considered to be high expression. The specific threshold may vary depending on factors such as the purpose of the study, experimental techniques, and research background. In clinical studies, researchers usually determine the threshold based on the analysis of large sample data to determine whether the expression level of the tumor antigen is higher than the normal level. It should be noted that the high expression of tumor antigens may vary in different cancer types and may also vary in different patients.
  • antibody refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of three domains ( CH1 , CH2 , and CH3 ).
  • Each light chain consists of a light chain variable region ( VL ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three regions arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDR and 4 FRs.
  • the variable regions ( VH and VL ) of each heavy chain/light chain pair form antibody binding sites, respectively.
  • the allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
  • antibody is not limited by any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be antibodies of different types, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG e.g., IgG1, IgG2, IgG3 or IgG4 subtypes
  • IgA1, IgA2, IgD, IgE or IgM antibodies are antibodies of different types, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • CDR complementarity determining region
  • Kabat numbering system Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991
  • Chothia numbering system Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al.
  • the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure are preferably identified by the Kabat, Chothia or IMGT numbering systems.
  • antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.
  • the term “Fd fragment” means an antibody fragment consisting of VH and CH1 domains;
  • the term “Fv fragment” means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody;
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 (1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains;
  • the term “F(ab') 2 fragment” means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region;
  • the term “Fab'fragment” means an antibody fragment obtained by reducing a F(ab') 2 fragment. One of the two Fab' fragments formed by cleaving the disulfide bond in the hinge region of F(ab') 2 .
  • the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which the VL and VH domains are paired to form a monovalent molecule via a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al., Science 242:423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 5883 (1988)).
  • scFv molecules may have the general structure: NH2 - VL-linker-VH-COOH or NH2 - VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 49) amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 50) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody, which is a small antibody fragment with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) connected thereto in the same polypeptide chain (VH-VL or VL-VH), wherein the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993), and Poljak R.J. et al., Structure 2:1121 1123 (1994)).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • Antibody antigen-binding fragments can be obtained from a given antibody (e.g., the monoclonal antibodies ch517, ch704 and Hu517 provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.
  • a given antibody e.g., the monoclonal antibodies ch517, ch704 and Hu517 provided herein
  • conventional techniques known to those skilled in the art e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
  • Monoclonal antibodies have high specificity for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, which usually recognize different epitopes on the antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, U.S.P 4,816,567).
  • monoclonal antibodies can be prepared as follows. First, an immunogen (with an adjuvant added when necessary) is injected into an immunized mouse or other suitable host animal.
  • the immunogen or adjuvant is usually injected subcutaneously at multiple points or intraperitoneally.
  • the immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the antigen's ability to be expressed in the host. Immunogenicity in vivo.
  • the adjuvant can be Freund's adjuvant or MPL-TDM, etc.
  • lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body.
  • lymphocytes can also be obtained by in vitro immunization.
  • the target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996).
  • a suitable fusion agent such as PEG
  • the hybridoma cells prepared above can be inoculated into a suitable culture medium for growth, and the culture medium preferably contains one or more substances that can inhibit the growth of unfused, parent myeloma cells.
  • myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT)
  • adding substances such as hypoxanthine, aminopterin and thymine (HAT culture medium) to the culture medium will inhibit the growth of HGPRT-deficient cells.
  • Preferred myeloma cells should have characteristics such as high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium.
  • myeloma cells are preferably mouse myeloma, such as MOP-21 or MC-11 mouse tumor derivatives (THE Salk Institute Cell Distribution Center, San Diego, Calif.
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the affinity of monoclonal antibodies can be determined by the Scatchard analysis method described by Munson et al. in Anal. Biochem. 107: 220 (1980).
  • the target cell line can be subcloned by the standard limiting dilution method described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).
  • Suitable culture fluids can be DMEM or RPMI-1640, etc.
  • hybridoma cells can also grow in animals in the form of ascites tumors.
  • immunoglobulin purification methods such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by subcloned cells can be separated from cell culture fluid, ascites or serum.
  • Monoclonal antibodies can also be obtained through genetic engineering recombination technology. Using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody for PCR amplification, DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells. The obtained DNA molecules are inserted into expression vectors, and then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins), and cultured under appropriate conditions to obtain recombinantly expressed target antibodies.
  • host cells such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins
  • chimeric antibody refers to an antibody in which a portion of the light chain and/or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and/or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class). or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • humanized antibody refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • the term “isolated” or “isolated” refers to that obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated.
  • isolated or “isolated” does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • multispecific antibody refers to an antibody that has binding specificity for at least two different epitopes on the same antigen or different antigens.
  • the multispecific antibody can be a full-length antibody or a fragment of the antibody.
  • bispecific antibody refers to an antibody that has binding specificity for two different antigens or two different epitopes that can recognize one antigen.
  • human antibody or “fully human antibody” includes antibodies with variable regions and constant regions (if present) derived from human germline immunoglobulin sequences.
  • Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo).
  • human antibody does not include antibodies (i.e., humanized antibodies) in which CDR sequences derived from another mammalian species germline (e.g., mouse) are grafted to human framework sequences.
  • Fully human antibodies or human antibodies may be derived from transgenic mice carrying human antibody genes or from human cells.
  • an antibody that specifically binds to an antigen means that the antibody binds to the antigen with an affinity ( KD ) of less than about 10-5 M, such as less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less.
  • KD refers to the dissociation equilibrium constant for a specific antibody-antigen interaction, which is expressed as The affinity between an antibody and an antigen is described in terms of the equilibrium dissociation constant. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.
  • polypeptide refers to a chain of at least two consecutively linked amino acid residues, with no upper limit on the length of the chain.
  • One or more amino acid residues in a polypeptide may contain modifications, such as, but not limited to, optionally substituted polysarcosine residue modifications, optionally substituted glycosylated polyethylene glycol modifications, glycosylation modifications, phosphorylation modifications, or disulfide bond modifications.
  • a "protein” may contain one or more polypeptides.
  • anti-ROR1 antibody As used herein, the terms “anti-ROR1 antibody,” “anti-ROR1,” “ROR1 antibody,” “ROR1 monoclonal antibody,” “antibody that binds to ROR1,” and “antibody that targets ROR1” have the same meaning and refer to an antibody that is capable of binding to a ROR1 protein or a fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic agent and/or therapeutic agent targeting ROR1.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also referred to as an "antigenic determinant” in the art.
  • An epitope or antigenic determinant is usually composed of chemically active surface groups of a molecule, such as amino acids or carbohydrates or sugar side chains, and usually has specific three-dimensional structural characteristics and specific charge characteristics.
  • an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear” or “conformational”. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • the term "vector” refers to a nucleic acid carrier into which a polynucleotide can be inserted.
  • a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; Artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as ⁇ phage or M13 phage and animal viruses, etc.
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosomes
  • PAC P1-derived artificial chromosomes
  • bacteriophages such as ⁇ phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses such as SV40
  • a vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain a replication initiation site.
  • the term "host cell” refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine)
  • the molecules are identical at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • two sequences are compared when they are aligned to produce maximum identity.
  • Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.).
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the essential properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Replacement. Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Pharmaceutically acceptable carriers and/or excipients include, but are not limited to, pH adjusters, surfactants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives, stabilizers.
  • pH adjusters include, but are not limited to, phosphate buffers.
  • Stabilizers have the meanings commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.
  • sugars such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dried whey, albumin or casein
  • degradation products such as lactalbumin hydrolysate
  • prevention refers to a method implemented to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a tumor or infection) in a subject or to minimize its effect.
  • treatment refers to a method implemented to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving prognosis, whether detectable or undetectable.
  • the amount of the therapeutic agent that effectively alleviates any particular disease symptom can vary according to factors such as the patient's disease state, age and weight, and the ability of the drug to cause a desired response in the subject. Whether the disease symptoms are alleviated can be assessed by any clinical measurement, which is usually used by a doctor or other skilled health care provider to assess the severity or progression of the symptom.
  • the terms “patient” and “subject”, “individual”, “object” refer to any human or non-human animal, especially a human, that is receiving prophylactic or therapeutic treatment.
  • the antibodies, methods and compositions described herein can be used to treat a subject with cancer.
  • non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, horses, cats, chickens, mice, rats, amphibians, reptiles, etc.
  • IC50 half maximal inhibitory concentration
  • IC50 can measure the sensitivity of the antibody. The lower the IC50 , the higher the sensitivity of the antibody.
  • mAb517 and mAb704 obtained in the present invention have better affinity with human ROR1 than the clinical antibody UC-961, and ch517, ch704 and Hu517 obtained based on mAb517 and mAb704 can obtain better efficacy when coupled with the same drugs; and mAb517 and mAb704 obtained in the present invention can specifically bind to human ROR1, and ch517, ch704 and Hu517 obtained based on mAb517 and mAb704 can also specifically bind to human ROR1.
  • the antibody-drug conjugate obtained by the present disclosure in particular, the antibody-drug conjugate comprising ch517 and humanized Hu517 thereof
  • the antibody-drug conjugate obtained in the present invention especially the antibody-drug conjugate containing Hu517, has a significant bystander effect.
  • Figure 1 shows representative binding curves of mAb517, mAb704 and UC-961 antibody (positive control antibody) binding to human ROR1;
  • Figure 2 shows the results of mAb517 and mAb704 antibodies binding to human ROR1 and human ROR2 as well as blank control plasmid transfected cells;
  • FIG3 shows a size exclusion (SEC) chromatogram of naked anti-ch517 prepared in Example 2 of the present disclosure
  • FIG4 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate ch517-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG5 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate ch517-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG6 shows a size exclusion chromatography (SEC) chromatogram of naked antibody ch704 prepared in Example 2 of the present disclosure
  • FIG7 shows a hydrophobic chromatography (HIC) chromatogram of naked anti-ch704 prepared in Example 2 of the present disclosure
  • FIG8 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate ch704-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG9 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate ch704-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG10 shows a size exclusion (SEC) chromatogram of naked antibody Hu517 prepared in Example 2 of the present disclosure
  • FIG11 shows a hydrophobic chromatography (HIC) chromatogram of naked anti-Hu517 prepared in Example 2 of the present disclosure
  • FIG12 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate Hu517-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG13 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate Hu517-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG14 shows a size exclusion (SEC) chromatogram of naked antibody UC-961 prepared in the present disclosure
  • FIG15 shows a hydrophobic chromatography (HIC) chromatogram of naked anti-UC-961 prepared in the present disclosure
  • FIG16 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate UC-961-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG17 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate UC-961-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG18 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate UC-961-vc-MMAE prepared in Example 5 of the present disclosure
  • FIG19 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate UC-961-vc-MMAE prepared in Example 5 of the present disclosure
  • FIG20 shows a size exclusion (SEC) chromatogram of the antibody-drug conjugate HuIgG-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG21 shows a hydrophobic chromatography (HIC) chromatogram of the antibody-drug conjugate HuIgG-T1000-exatecan prepared in Example 5 of the present disclosure
  • FIG29 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against triple-negative breast cancer MDA-MB-231;
  • FIG. 33 shows the in vivo anti-tumor activity of the disclosed antibody-drug conjugates against human triple-negative breast cancer PDX LD1-2009-362263.
  • amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 1-3; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 4-6, or
  • amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NO:13-15; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NO:16-18.
  • mouse ROR1 monoclonal antibody (mAb704) is as follows:
  • amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 19-21; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 22-24, or
  • amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 25-27; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 28-30, or
  • the amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NOs: 31-33; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NOs: 34-36.
  • the hybridoma sequences were humanized to obtain chimeric ROR1 antibodies (abbreviated as “ch517” and “ch704") and humanized ROR1 antibodies (abbreviated as "Hu517”), the sequences of which are shown in Table 3.
  • amino acid sequence of CDR1-3 of the heavy chain variable region is shown in SEQ ID NO: 1, 45, 3; amino acid sequence of CDR1-3 of the light chain variable region is shown in SEQ ID NO: 46, 5, 6, or
  • amino acid sequences of CDR1-3 of the heavy chain variable region are shown in SEQ ID NO:13-15; the amino acid sequences of CDR1-3 of the light chain variable region are shown in SEQ ID NO:16-18.
  • the heavy chain and light chain variable region sequences of ch517 (see SEQ ID NO: 37, SEQ ID NO: 38) and the heavy chain and light chain variable region sequences of ch704 (see SEQ ID NO: 39, SEQ ID NO: 40) were cloned into a vector containing the human IgG1 heavy chain constant region and the Kappa chain constant region by gene recombination technology. After sequencing, the constructed chimeric antibodies were expressed by transfection technology and a mammalian expression system (FreeStyle TM 293T cells), and then purified. The obtained human-mouse chimeric antibodies were named ch517 and ch704, and the sequences of their heavy chain and light chain variable regions are shown in Table 1.
  • the positive control antibody UC-961 was prepared by the same method.
  • the antibody sequence can be found in SEQ ID NO.2 and SEQ ID NO.4 in patent WO2021159029 A1.
  • the full-length sequences of the heavy chain and light chain of UC-961 (the full-length sequences described in Ab1 light chain and Ab1 heavy chain in WO2018237335A1) were used to construct pCDNA3.1 expression vectors, respectively.
  • the antibody was expressed in 293F suspension cell culture and purified by protein A/G gravity column.
  • the humanized template that best matches the non-CDR region of ch517 was searched and selected from the Germline database.
  • the CDR region of the antibody was then transplanted to the selected humanized template to replace the CDR region of the human template, and then recombined with the IgG1 constant region.
  • back mutations were performed on the buried residues, residues that directly interacted with the CDR region, and residues that had an important impact on the conformation of VL and VH.
  • the designed humanized variable region sequence was cloned into the human IgG1 heavy chain constant region and After the kappa chain constant region vector was sequenced, the constructed humanized antibody was expressed using transfection technology and a mammalian expression system (FreeStyle TM 293 cells), and then purified. Finally, a humanized antibody Hu517 was obtained, and the sequences of its heavy chain and light chain variable regions are shown in Table 1.
  • Human ROR1 protein (Catalog No.: RO1-H522y, Acrobiosystems) was fixed to a 96-well plate by incubation overnight at 4°C. The plate was then blocked by incubation with 1% BSA in PBS for 1 hour at 37°C. After blocking, the plate was washed 3 times with PBST (PBS containing 0.05% Tween20). Serial dilutions of anti-ROR1 antibodies were prepared in binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) and incubated with the fixed protein at 37°C for 1 hour.
  • PBST PBS containing 0.05% Tween20
  • the plate was washed 3 times with PBST, incubated with peroxidase-labeled goat anti-mouse IgG or goat anti-human IgG (Jackson Immuno Research) diluted 1/10,000 in binding buffer for 1 hour at 37°C, washed again, developed with TMB and terminated with 1M H 2 SO 4 . The absorbance at 450nm-620nm was measured.
  • Example 4 Using overexpression plasmid transfection flow cytometry to determine the binding specificity of antibodies to human ROR1 and human ROR2
  • 293T human embryonic kidney cells, cell bank of the Chinese Academy of Sciences, catalog number: SCSP-502
  • SCSP-502 Chinese Academy of Sciences, catalog number: SCSP-502
  • 3 mL was plated per well.
  • Plasmid transfection was performed on the second day.
  • 3 ⁇ g of human ROR1 plasmid and 3 ⁇ g of human ROR2 plasmid were mixed with 9 ⁇ L PEI respectively, and allowed to stand for 20 minutes, during which serum-free medium was replaced. The plasmid that had been allowed to stand was added to the cells, mixed, and returned to the incubator. After 6 hours, the complete medium was replaced and cultured overnight.
  • the cells were removed and digested with trypsin. After centrifugation at 1000 rpm for 5 minutes, the digested cells were blocked with 10% goat serum (purchased from Beijing Solebow Technology Co., Ltd., catalog number SL038) for 30 minutes.
  • Anti-ROR1 antibodies mAb517 and mAb704 were added to the cells at 100 nM and incubated for 1 h. After washing twice with PBS, APC-labeled goat anti-mouse secondary antibody (purchased from Jackson ImmunoResarch, diluted 1:500) was added and incubated for 1 h. Finally, the cells were washed twice with PBS and the results were analyzed by flow cytometry.
  • results of mAb517 and mAb704 antibodies binding to human ROR1 and human ROR2 as well as blank control plasmid transfected cells As shown in Figure 2, the results showed that both mAb517 and mAb704 specifically bound to human ROR1 transfected cells and did not bind to human ROR2 transfected cells. At the same time, mAb517 and mAb704 antibodies also had no binding reaction in blank control plasmid transfected cells.
  • the prepared antibody-drug conjugate (Formula (I)) can be concentrated, liquid-changed, purified, and the antibody concentration measured and the average number of drug molecules carried by each antibody calculated according to the common operations described below to identify the antibody-drug conjugate.
  • the absorption of the antibody was measured using an ELISA reader (Multiskan GO, Thermo Fisher Scientific) according to the method defined by the manufacturer, and the ratio of the absorption to the absorption coefficient of the antibody at that wavelength was the concentration of the antibody.
  • the characteristic absorption wavelength of the antibody was 280 nm, and the absorption coefficient of the antibody at this wavelength is usually between 1.3 mL mg -1 cm -1 and 1.8 mL mg -1 cm -1 .
  • a zeba desalting column (5 mL, 40K MWCO) was pre-equilibrated with phosphate buffer (50 mM, pH 7.0) containing sodium chloride (50 mM) and EDTA (2 mM). 2 mL was loaded on each zeba desalting column (5 mL), centrifuged (1000 g, 4 min), and the flow-through fraction was collected. This fraction was concentrated by operation A, and the antibody concentration was determined by operation B, and the antibody concentration was adjusted with PBS7.0/EDTA.
  • a zeba desalting column (5 mL, 40K MWCO) was pre-equilibrated with any suitable storage buffer.
  • Storage buffers include, but are not limited to, histidine-acetate buffer (20 mM histidine, pH 5.5) containing 150 mM sodium chloride and phosphate buffer (50 mM, pH 7.0) containing 50 mM sodium chloride.
  • the reaction solution approximately 2 mL
  • the reaction solution was added to a 5 mL zeba desalting column, centrifuged (1000 g, 4 minutes), and the flow-through portion (approximately 2 mL) was collected.
  • the method uses a gel filtration purification process, and the collected components are applied to the desalting column for centrifugal elution, and the elution process is repeated twice to remove unbound drug linkers and low molecular weight compounds such as reducing agent tris (2-carboxyethyl) phosphine hydrochloride (TCEP), N-acetyl-(L)-cysteine (NAC), dimethyl sulfoxide, etc., to obtain the antibody-drug conjugate.
  • TCEP (2-carboxyethyl) phosphine hydrochloride
  • NAC N-acetyl-(L)-cysteine
  • dimethyl sulfoxide etc.
  • Procedure E Measure the concentration of the antibody in the antibody-drug conjugate and the average number of drug molecules attached to each antibody (DAR value)
  • the concentration of the drug coupled to the antibody-drug conjugate can be obtained by measuring the ultraviolet absorption value of the antibody-drug conjugate aqueous solution at 280 nm and 370 nm and calculating it using the following formula.
  • the total absorbance of a system is equal to the sum of the absorbances of all light-absorbing chemicals present in the system (additivity of absorbance). Therefore, assuming that the molar absorption coefficients of the antibody and drug remain unchanged before and after conjugation, the antibody concentration and drug concentration in the antibody-drug conjugate can be expressed by the following formula.
  • A280 represents the total absorbance of the antibody-drug conjugate aqueous solution at 280 nm
  • A370 represents the total absorbance of the antibody-drug conjugate aqueous solution at 370 nm
  • AA,280 represents the absorbance of the antibody at 280 nm
  • AA,370 represents the absorbance of the antibody at 370 nm
  • AD,280 represents the absorbance of the drug molecule at 280 nm
  • AD,370 represents the absorbance of the drug molecule at 370 nm
  • ⁇ A,280 represents the molar extinction coefficient of the antibody at 280 nm
  • ⁇ A,370 represents the molar extinction coefficient of the antibody at 370 nm
  • ⁇ D,280 represents the molar extinction coefficient of the drug molecule at 280 nm
  • ⁇ D,370 represents the molar extinction coefficient of the drug molecule at 370 nm
  • CA represents the concentration of the antibody in the antibody-drug conjugate
  • CD represents the
  • ⁇ A,280, ⁇ A,370, ⁇ D,280 and ⁇ D,370 are all known values (calculated from the antibody sequence or measured by UV absorbance of the compound).
  • ⁇ A,280 can be calculated from the amino acid sequence of the antibody using a known method (Protein Science, 1995, Vol. 4, pp. 2411-2423).
  • Antibodies usually have no absorption at 370 nm, so ⁇ A,370 is usually 0.
  • CA and CD can be obtained by measuring the absorbance values of the antibody-drug conjugate at 280 nm and 370 nm, A280 and A370, and then solving the two-variable linear equation system (Equations (1) and (2)).
  • equation (1) and (2) the average number of drug molecules attached to each antibody
  • Aggregates in antibody-drug conjugates can be measured by using size exclusion chromatography in high performance liquid chromatography.
  • the analytical method is as follows:
  • the size exclusion chromatogram of the quality control (QC, Hu517 naked antibody) is shown in Figure 10. It is worth noting that under this analytical method, the retention time of the main peak (single peak) of the 150 kDa quality control is between 9.5 and 10.5 minutes. At the same time, the retention time of the aggregate should be earlier than the retention time of the above monomer.
  • the hydrophobicity of antibody-drug conjugates can be analyzed on a high performance liquid chromatography column using a hydrophobic column.
  • the analysis method is as follows:
  • Mobile phase A 1 mol/L (NH 4 ) 2 SO 4 , 50 mmol/L KHPO 4 , pH 7.0
  • Mobile phase B 50mmol/L KHPO 4 , 25% (v/v) isopropanol, pH 7.0
  • the hydrophobic chromatography chromatogram of the quality control (QC, naked Hu517 antibody) is shown in Figure 11. Compared with naked antibodies that are not conjugated to drug molecules, antibody-drug conjugates have higher hydrophobicity, so their retention time is longer. It is well known that the shorter the retention time, the weaker the hydrophobicity of the sample. The DAR value of the antibody-drug conjugate can be obtained by calculation.
  • Antibody reduction The antibody was replaced with PBS 7.0 buffer containing EDTA by the above-mentioned operation B (the extinction coefficient of the antibody at 280 nm was 1.462 mL mg -1 cm -1 ) and operation C. The antibody concentration after replacement was 8.6 mg/mL. 0.184 mL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added to 2.3 mL of the ch517 protein aqueous solution to ensure that the pH value of the reaction solution was 7.0 ⁇ 0.1. The mixture was placed in a 37 degree Celsius environment for 2 hours.
  • Conjugation between antibody and drug linker Incubate the above mixture at 4 degrees Celsius for 10 minutes.
  • the drug linker LD-6 T1000-exatecan
  • DMA N,N-dimethylacetamide
  • the concentration of the antibody-drug conjugate was calculated to be 7.85 mg/mL by operation E, and the average number of small molecules coupled to each antibody was 8.86 by operation E, that is, the DAR value was about 8.86.
  • the aggregate content of naked anti-ch517 was measured to be 2.55% by operation F ( Figure 3), and the aggregate content of antibody-drug conjugate ch517-T1000-exatecan was 6.58% ( Figure 4).
  • the retention time of antibody-drug conjugate ch517-T1000-exatecan was measured to be 6.718 min by operation G ( Figure 5).
  • Antibody reduction The antibody was replaced with PBS 7.0 buffer containing EDTA by the above-mentioned operation B (the extinction coefficient of the antibody at 280 nm was 1.48 mL mg -1 cm -1 ) and operation C.
  • the antibody concentration after the replacement was 7.2 mg/mL.
  • 0.184 mL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added to 2.75 mL of the ch704 protein aqueous solution to ensure that the pH value of the reaction solution was 7.0 ⁇ 0.1.
  • the mixture was placed in a 37 degree Celsius environment for 2 hours.
  • Coupling between antibody and drug linker Incubate the above mixture at 4 degrees Celsius for 10 minutes. Then add the drug linker L-D-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) to the mixture, with a volume of 0.66 mL (equivalent to 25 times the antibody content). Place the mixture at 22 degrees Celsius and continue to react for 30 minutes.
  • L-D-6 T1000-exatecan
  • DMA N,N-dimethylacetamide
  • the concentration of the antibody-drug conjugate was calculated to be 8.01 mg/mL by operation E, and the average number of small molecules coupled to each antibody was 8.68, that is, the DAR value was about 8.68.
  • the aggregate content of naked anti-ch704 was measured to be 13.21% by operation F ( Figure 6), and the aggregate content of antibody-drug conjugate ch704-T1000-exatecan was 1.54% (Figure 8).
  • the retention time of naked anti-ch704 was measured to be 3.527 min by operation G ( Figure 7), and the retention time of antibody-drug conjugate ch704-T1000-exatecan was 6.423 min ( Figure 9). 9).
  • Antibody reduction The antibody was replaced with PBS 7.0 buffer containing EDTA by the above-mentioned operation B (the extinction coefficient of the antibody at 280 nm was 1.444 mL mg -1 cm -1 ) and operation C.
  • the antibody concentration after the replacement was 29.47 mg/mL.
  • 0.307 mL of 5 mM TCEP solution (equivalent to 9 times the antibody content) was added to 0.87 mL of the aqueous solution of Hu517 protein to ensure that the pH value of the reaction solution was 7.0 ⁇ 0.1.
  • the mixture was placed in an environment of 37 degrees Celsius for 2 hours.
  • Coupling between antibody and drug linker Incubate the above mixture at 4 degrees Celsius for 10 minutes. Then add the drug linker L-D-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) to the mixture, with a volume of 0.24 mL (equivalent to 14 times the antibody content). Place the mixture at 22 degrees Celsius and continue to react for 30 minutes.
  • L-D-6 T1000-exatecan
  • DMA N,N-dimethylacetamide
  • the concentration of the antibody drug conjugate measured and calculated by operation E was 7.21 mg/mL, and the number of small molecules coupled on each antibody was 7.91, that is, the DAR value was about 7.91.
  • the aggregate content of naked anti-Hu517 measured by operation F was 0.26% (Figure 10), and the aggregate content of antibody-drug conjugate Hu517-T1000-exatecan was 1.08% ( Figure 12).
  • the retention time of naked anti-Hu517 measured by operation G was 3.707 min ( Figure 11), and the retention time of antibody-drug conjugate Hu517-T1000-exatecan was 6.653 min (Figure 13).
  • Antibody reduction The antibody was replaced with PBS 7.0 buffer containing EDTA by the above-mentioned operation B (the extinction coefficient of the antibody at 280 nm was 1.505 mL mg -1 cm -1 ) and operation C.
  • the antibody concentration after the replacement was 6.71 mg/mL.
  • 0.24 mL of 5 mM TCEP solution (equivalent to 9 times the antibody content) was added to 2.98 mL of the aqueous solution of UC-961 protein to ensure that the pH value of the reaction solution was 7.0 ⁇ 0.1.
  • the mixture was placed in an environment of 37 degrees Celsius for 2 hours.
  • Coupling between antibody and drug linker Incubate the above mixture at 4 degrees Celsius for 10 minutes. Then add the drug linker LD-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) to the mixture at a volume of 0.186 mL (equivalent to 14 times the antibody content). The reaction was continued at 400 °C for 30 minutes.
  • drug linker LD-6 T1000-exatecan
  • DMA N,N-dimethylacetamide
  • the concentration of the antibody drug conjugate measured and calculated by operation E was 5.46 mg/mL, and the number of small molecules coupled on each antibody was 8.62, that is, the DAR value was about 8.62.
  • the aggregate content of naked anti-UC-961 measured by operation F was 7.15% ( Figure 14), and the aggregate content of antibody-drug conjugate UC-961-T1000-exatecan was 3.01% (Figure 16).
  • the retention time of naked anti-UC-961 measured by operation G was 6.293 min ( Figure 15), and the retention time of antibody-drug conjugate UC-961-T1000-exatecan was 7.534 min ( Figure 17).
  • Antibody reduction The antibody was replaced with PBS 7.0 buffer containing EDTA by the above-mentioned operation B (the extinction coefficient of the antibody at 280 nm was 1.505 mL mg -1 cm -1 ) and operation C.
  • the antibody concentration after replacement was 7.92 mg/mL.
  • 61.87 ⁇ L of 5 mM TCEP solution (equivalent to 3.2 times the antibody content) was added to 1.83 mL of the aqueous solution of UC-961 protein to ensure that the pH value of the reaction solution was 7.0 ⁇ 0.1.
  • the mixture was placed in a 37 degree Celsius environment for 2 hours.
  • Coupling between antibody and drug linker Incubate the above mixture at 4 degrees Celsius for 10 minutes. Then add the drug linker vc-MMAE (purchased from Shanghai Haoyuan Chemical, catalog number: HY-15575) dissolved in N,N-dimethylacetamide (DMA) to the mixture, with a volume of 135.33 ⁇ L (equivalent to 7 times the antibody content). Place the mixture at 22 degrees Celsius and continue to react for 30 minutes.
  • DMA N,N-dimethylacetamide
  • the concentration of the antibody-drug conjugate was calculated by operation E to be 4.08 mg/mL.
  • the aggregate content of the antibody-drug conjugate UC-961-vc-MMAE was measured by operation F to be 2.53% (Figure 18).
  • Figure 19 is a hydrophobic chromatography spectrum obtained by measuring the antibody-drug conjugate UC-961-vc-MMAE by operation G.
  • the average number of drug molecules connected to each antibody was calculated to be 4.49, that is, the DAR value was about 4.49 ( Figure 19).
  • Antibody reduction The antibody was replaced with PBS7.0 buffer containing EDTA by the above-mentioned operation B (the extinction coefficient of the antibody at 280nm was 1.35mL mg -1 cm -1 ) and operation C.
  • the antibody concentration after replacement was 15mg/mL.
  • 0.27mL of 5mM TCEP solution (equivalent to 10 times the antibody content) was added to the aqueous solution of 1.33mL HuIgG protein (Beijing Sino Biological, Cat: HG1K) to ensure that the pH value of the reaction solution was 7.0 ⁇ 0.1.
  • the mixture was placed in a 37°C environment for reaction for 2 hours.
  • Coupling between antibody and drug linker Incubate the above mixture at 4 degrees Celsius for 10 minutes. Then add the drug linker L-D-6 (T1000-exatecan) dissolved in N,N-dimethylacetamide (DMA) to the mixture in a volume of 0.2 mL (equivalent to 15 times the antibody content). Place the mixture at 22 degrees Celsius and continue to react for 30 minutes.
  • L-D-6 T1000-exatecan
  • DMA N,N-dimethylacetamide
  • the concentration of the antibody-drug conjugate measured and calculated by operation E was 12.09 mg/mL, and the average number of small molecules coupled to each antibody was 6.71, that is, the DAR value was about 6.71.
  • the aggregate content of the antibody-drug conjugate HuIgG-T1000-exatecan measured by operation F was 4.38% ( Figure 20).
  • the retention time of the antibody-drug conjugate HuIgG-T1000-exatecan measured by operation G was 6.213 min ( Figure 21).
  • Antibody reduction The antibody was replaced with PBS 7.0 buffer containing EDTA by the above-mentioned operation B (the extinction coefficient of the antibody at 280 nm was 1.35 mL mg -1 cm -1 ) and operation C. The antibody concentration after replacement was 15 mg/mL. 40 ⁇ L of 5 mM TCEP solution (equivalent to 2.5 times the antibody content) was added to 0.8 mL of the HuIgG protein aqueous solution to ensure that the pH value of the reaction solution was 7.0 ⁇ 0.1. The mixture was placed in a 37 degree Celsius environment for 2 hours.
  • Coupling between antibody and drug linker Incubate the above mixture at 4 degrees Celsius for 10 minutes. Then add the drug linker vc-MMAE dissolved in N,N-dimethylacetamide (DMA) to the mixture at a volume of 112 ⁇ L (equivalent to 7 times the antibody content). Place the mixture at 22 degrees Celsius and continue to react for 30 minutes.
  • DMA N,N-dimethylacetamide
  • Antibody-drug conjugates C-3, C-4, C-5, C-7, C-8, C-9, C-10, and C-11 were prepared by referring to the general operation method. Their structural formulas are shown in the following table, wherein m represents the DAR value, which is about 8 or about 4; Ab represents the antibody, which is ch517, ch704, or Hu517.
  • 6-week-old BALB/C nude mice female, weighing 18-21 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • the antibody-drug conjugate Hu517-T1000-exatecan prepared in Example 5 was intravenously injected at a dosage of 10 mg/kg, once a week, for a total of 1 dose.
  • mouse blood was collected at 15 minutes, 4 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, and 21 days for ELISA experiments to evaluate the drug concentration in the blood of mice at different time points, and the pharmacokinetic parameters were calculated.
  • Figure 24 shows the blood drug concentration-time change curve of the antibody-drug conjugate Hu517-T1000-exatecan
  • Table 5 shows its related pharmacokinetic parameters.
  • the cells were removed, digested with trypsin, and the cells were counted, and the total number of live cells in the ADC molecules and negative control wells was counted respectively. After centrifugation at 1000rpm for 5min, the digested cells were blocked with 10% goat serum (purchased from Beijing Solebow Technology Co., Ltd., catalog number SL038) for 30min.
  • Anti-ROR1 humanized antibody Hu517 was added to the cells at 100 nM as the primary antibody and incubated for 1 hour. After washing with PBS twice, FITC-labeled goat anti-human secondary antibody (purchased from Jackson ImmunoResarch) was added at 1:500 and incubated for 1 hour. Finally, after washing with PBS twice, the results were analyzed by flow cytometry. According to the test results, the number of PC9 and A549 live cells was calculated respectively, and a bar graph of cell survival was drawn.
  • Figure 25 is a bar graph of the number of surviving cells in a mixed cell line containing the antibody-drug conjugate Hu517-T1000-exatecan or HuIgG-T1000-exatecan prepared in Example 5.
  • the results show that compared with the negative antibody-drug conjugate HuIgG-T1000-exatecan, Hu517-T1000-exatecan not only has a strong killing effect on ROR1 positive cells PC9, but also has a killing effect on ROR1 negative cells A549, showing a significant bystander effect.
  • mice 6-week-old BALB/C nude mice, female, weighing 18-21 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Five million NCI-H1975 cells (cell bank of the Chinese Academy of Sciences Typical Culture Collection Committee, catalog number: SCSP-597) were subcutaneously inoculated into the mice.
  • the antibody-drug conjugates ch517-T1000-exatecan and ch704-T1000-exatecan prepared in Example 5 were intravenously injected at a dosage of 10 mg/kg; the antibody-drug conjugate HuIgG-T1000-exatecan prepared in Example 5 and a negative control Vehicle group were set up, and the same dose of PBS was intravenously injected.
  • mice Female, 6 weeks old, weighing 18-21 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Five million MDA-MB-231 cells (Cell Bank of Typical Culture Collection Committee of Chinese Academy of Sciences, Catalog No.: SCSP-5043) were subcutaneously inoculated into the mice.
  • mice 6-week-old NU/NU mice, female, weighing 18-21 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Five million NCI-H226 cells (Cell Bank of the Chinese Academy of Sciences Typical Culture Collection Committee, Catalog No.: TCHu235) were subcutaneously inoculated into the mice.
  • FIG30 shows the in vivo tumor volume-time curve of the lung squamous cell carcinoma mouse model.
  • the results show that, compared with the negative control, Hu517-T1000-exatecan can significantly inhibit tumor growth, while UC-961-vc-MMAE does not produce any tumor inhibitory effect.
  • FIG31 shows the in vivo tumor volume-time curve of the non-small cell lung cancer mouse model. The results show that, compared with the negative control, ch517-T1000-exatecan can significantly inhibit tumor growth.
  • 6-week-old female NU/NU mice weighing 18-21 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • the mice were subcutaneously inoculated with colorectal cancer tumors (specimen number 200730, from Xi'an Lidi Biotechnology Co., Ltd.).
  • the antibody-drug conjugate Hu517-T1000-exatecan and UC-961-vc-MMAE prepared in Example 5 were intravenously injected at a dosage of 2.5 mg/kg and 5 mg/kg, respectively, and a negative control Vehicle group was set up, and an equal dose of PBS was intravenously injected.
  • QW a total of 2 doses.
  • FIG32 shows the in vivo tumor volume-time curve of the non-small cell lung cancer mouse model.
  • the results show that, compared with the negative control, Hu517-T1000-exatecan can significantly inhibit tumor growth, while UC-961-vc-MMAE does not produce any tumor growth inhibitory effect.
  • Figure 33 shows the in vivo tumor volume-time curve of the triple-negative breast cancer mouse model.
  • the results show that, compared with the negative control, Hu517-T1000-exatecan can significantly inhibit tumor growth, while UC-961-vc-MMAE only produces a partial tumor growth inhibition effect.
  • T1000-exatecan has a higher tolerance dose, which is usually more than 5 times that of vc-MMAE. Therefore, Hu517-T1000-exatecan of the present invention has a larger therapeutic window.

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Abstract

一种抗ROR1抗体或其抗原结合片段，编码它们的核酸分子以及它们在制备抗体偶联药物中的用途，还涉及包含所述抗体或其抗原结合片段的抗体偶联药物及其制备方法和用途。

Description

抗ROR1抗体及其用途
本申请是以CN申请号为202311056361.3，申请日为2023年8月21日的申请为基础，并主张其优先权，该CN申请的全部内容在此作为整体引入本申请中。
技术领域
本公开属于医药领域，具体而言，本公开涉及靶向ROR1的抗体、包含其的抗体偶联药物、编码它们的核酸分子以及它们在制备抗体偶联药物中的用途。该靶向ROR1的抗体或其抗原结合片段具有本文公开的重链可变区、轻链可变区和互补决定区。本公开进一步涉及包含所述抗体或其抗原结合片段的抗体偶联药物及其制备方法和用途。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
癌症是世界上致病和致死的首要原因之一，随着人口的老龄化，全球的癌症负担仍将不断增加，AACR的报告指出，到2040年，全球的癌症患者总数将达到约2800万，并且估计约1620万患者因此去世。
受体酪氨酸激酶样孤儿受体(receptor tyrosine kinase-like orphan receptor,ROR)是I型受体酪氨酸激酶(receptor tyrosine kinase,RTK)家族中的成员，是一类膜受体，主要包括ROR1和ROR2。ROR1在早期胚胎发育过程中高表达，并且在多种生理过程中发挥重要作用，包括调节细胞分裂、增殖、迁移等，参与神经、骨骼和血管等器官的生成。在随后的胎儿发育过程中，ROR1的表达量逐渐下降，正常的儿童和成人组织细胞表面基本没有ROR1的表达(Semin Cancer Biol 2014 Vol.29,21-31)。但在多种肿瘤细胞中，包括肺腺细胞癌、乳腺癌、卵巢癌、黑色素瘤、肾癌、胃癌、大肠癌、胰腺癌(BMC Cancer.2021 Nov 11；21(1):1199.)等实体瘤细胞以及某些血液恶性肿瘤如慢性淋巴细胞白血病(chronic lymphocytic leukemia，CLL)、急性淋巴细胞白血病(ALL)中ROR1的表达大幅提高，并且ROR1的表达和疾病的进展以及治疗效果密切相关(Clin Cancer Res.2017 Jun 15；23(12):3061-3071；Front Oncol.2021May 28；11:680834)，例如在CLL患者中ROR+的比例超过90％(Blood.2021Jun 17；137(24):3365-3377.)，ROR1的表达水平与CLL的进展密切相关，使其成为预后的良好生物标志物。
ROR1可通过介导非经典Wnt信号通路(non-canonical Wnt pathways)的信号传递，在多种生理过程中发挥重要作用，其中包括调节细胞分裂、增殖、迁移、和细胞趋化，尤 其是Wnt5a。Wnt5a是典型的非经典Wnt信号通路的激活因子，参与NF-κB亚基p65的磷酸化，激活肿瘤细胞中NF-κB通路，促进细胞迁移和侵袭，上皮间充质转化(Epithelial-Mesenchymal Transition，EMT)、癌转移等。作为Wnt5a的受体，ROR1参与激活肿瘤细胞NF-κB通路。ROR1在癌症干细胞的发育和EMT过程中起到重要作用。癌症干细胞是肿瘤中更具有干细胞特征的癌细胞，它们通常对化疗药物具有更高的耐药性。而EMT过程让细胞的形态从上皮细胞的形态转化为间质细胞的形态，让它们更具侵袭性，促进癌症的转移。在肺癌中，ROR1参与原癌基因(c-Src)和MET的激活，导致肿瘤细胞凋亡的抑制，ROR1还被发现是cavin-1和caveolin-1的支架蛋白，可激活肺腺癌中的AKT。在乳腺癌中，ROR1促进了PI3K/AKT通路的激活，高ROR1表达与疾病更严重的进展相关。综上，在许多类型的恶性肿瘤中观察到ROR1的异常表达和相关的促生长信号事件，使ROR1成为抗癌药物开发的一个有吸引力的治疗靶点。
鉴于ROR1在多种癌症中高表达，而在普通组织不表达或低表达的特性，药物研发人员开发了多种靶向ROR1的抗肿瘤治疗策略，其中ADC药物的进展较快，并且表现出良好的应用前景。由于兼具单抗药物的高靶向性以及细胞毒素在肿瘤组织中高活性的双重优点，ADC药物可高效杀伤肿瘤细胞，较化疗药物副作用更低，较传统抗体类肿瘤药物具有更好的疗效，被称为肿瘤治疗领域的“生物导弹”。截至目前，据悉全球已有14款ADC药物获批上市，7款用于血液肿瘤，7款用于治疗实体瘤，靶点涉及CD33、CD30、CD22、CD79b、HER2、Nectin-4、Trop-2、BCMA、CD19和TF。其中，4款ADC药物在中国上市。
目前尚未有靶向ROR1的抗体偶联药物上市，处于临床阶段的ROR1 ADC药物包括：默沙东(MSD)公司和VelosBio公司联合开发的VLS-101(WO2018237335)，由靶向ROR1的单克隆抗体和单甲基auristatin E(MMAE)组成，目前正处于临床II期开发用于治疗血液系统恶性肿瘤和实体瘤患者，但由于MMAE治疗窗口有限，对于ROR1表达量不高的实体瘤在临床前药效模型仅显示出弱的抑瘤活性。基石药业与LegoChem Biosciences公司共同开发的ROR1的抗体偶联药物LCB71/CS5001(CN202010852078.1)，LCB71携带肿瘤激活的吡咯并苯二氮卓(PBD)前毒素，而PBD由于其自身毒性，在实体瘤治疗过程中体现出安全窗口过窄(J Thorac Oncol.2021Sep；16(9):1429-1433.)；此外，勃林格殷格翰/NBE Therapeutics公司开发的靶向ROR1的ADC候选药物NBE-002(WO2017127664)，通过转肽酶Sortase A介导的特异性反应将PNU-159682定点、定量地偶联到抗体重链、轻链的C末端，PNU-159682与PBD类毒素存在同样的安全性隐患。
ADC药物进入收获期，获批数量快速增长。截至2022年12月，全球共有15款ADC药物获批上市。全球首款ADC药物Mylotarg于2000年获批上市，但由于致命性肝损伤 于2010年撤市，通过优化给药方案提高疗效和安全性后于2017年再次上市。2001-2010年间，全球无ADC药物获批上市，行业发展进入低谷期。随着技术的不断进步，ADC药物迎来上市集中期，截至目前累计获批10款，虽然以Enhertu为代表的新一代ADC取得了巨大成功，但仍有较大的改进空间，如耐药性与间质性肺炎副作用等问题。此外，ROR1与其他ADC抗原不同，如HER2在肿瘤细胞上有数十万个拷贝，ROR1在大多数肿瘤细胞中的表达水平并不很高，每个细胞不多于5000个拷贝，需要一种对极低表达更有效且安全窗口提高的ADC分子。
此外，抗体药物偶联物的作用机制看似简单，但一个抗体药物偶联物是否能成为安全有效的药物是非常复杂和不可预测的，依赖于多种因素，可以看出，ADC药物的研发需要大量的实验摸索和验证，其安全性和有效性是无法在实验前预测的。
因此，本领域迫切需要提供更为合适的新型的靶向ROR1的抗体偶联药物，一方面可以实现高效、简单、实用的化学制备与偶联，并且改善现有靶向ROR1抗体偶联药物的药学性质和药效，如综合提高ADC分子稳定性、提高半衰期、抗肿瘤耐药性能，以及治疗窗口等。
发明内容
为了解决现有技术中存在的问题，本公开的第一方面提供一种能够特异性结合ROR1的抗体或其抗原结合片段，其包含：
(1)(a)下述3个重链可变区(VH)互补决定区(CDR)：
(i)VH CDR1，其具有如SEQ ID NO:37或41所示的VH中含有的CDR1的序列，或者与所述VH中含有的CDR1的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
(ii)VH CDR2，其具有如SEQ ID NO:37或41所示的VH中含有的CDR2的序列，或者与所述VH中含有的CDR2的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；和
(iii)VH CDR3，其具有如SEQ ID NO:37或41所示的VH中含有的CDR3的序列，或者与所述VH中含有的CDR3的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其具有如SEQ ID NO:38或42所示的VL中含有的CDR1的序列，或者与所述VL中含有的CDR1的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如 1个或2个氨基酸的置换、缺失或添加)的序列；
(v)VL CDR2，其具有如SEQ ID NO:38或42所示的VL中含有的CDR2的序列，或者与所述VL中含有的CDR2的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；和
(vi)VL CDR3，其具有如SEQ ID NO:38或42所示的VL中含有的CDR3的序列，或者与所述VL中含有的CDR3的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
或者
(2)(a)下述3个重链可变区(VH)互补决定区(CDR)：
(i)VH CDR1，其具有如SEQ ID NO:39所示的VH中含有的CDR1的序列，或者与所述VH中含有的CDR1的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
(ii)VH CDR2，其具有如SEQ ID NO:39所示的VH中含有的CDR2的序列，或者与所述VH中含有的CDR2的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；和
(iii)VH CDR3，其具有如SEQ ID NO:39所示的VH中含有的CDR3的序列，或者与所述VH中含有的CDR3的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其具有如SEQ ID NO:40所示的VL中含有的CDR1的序列，或者与所述VL中含有的CDR1的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
(v)VL CDR2，其具有如SEQ ID NO:40所示的VL中含有的CDR2的序列，或者与所述VL中含有的CDR2的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；和
(vi)VL CDR3，其具有如SEQ ID NO:40所示的VL中含有的CDR3的序列，或者与所述VL中含有的CDR3的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，上述(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，所述重链可变区(VH)中含有的CDR1、CDR2及CDR3，和/或所述轻链可变区(VL)中含有的CDR1、CDR2及CDR3由Kabat、Chothia或IMGT编号系 统定义。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(1)如SEQ ID NO:37或41所示的VH中含有的CDR1、CDR2及CDR3的序列；和/或
如SEQ ID NO:38或42所示的VL中含有的CDR1、CDR2及CDR3序列；或者
(2)如SEQ ID NO:39所示的VH中含有的CDR1、CDR2及CDR3的序列；和/或
如SEQ ID NO:40所示的VL中含有的CDR1、CDR2及CDR3序列。
在某些实施方案中，所述重链可变区(VH)中含有的CDR1、CDR2及CDR3，和/或所述轻链可变区(VL)中含有的CDR1、CDR2及CDR3由Kabat、Chothia或IMGT编号系统定义。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)下述3个重链可变区(VH)CDR：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与SEQ ID NO:1相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与SEQ ID NO:2相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，或者其由下述序列组成：SEQ ID NO:45，或与SEQ ID NO:45相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与SEQ ID NO:3相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:4，或与SEQ ID NO:4相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，或者其由下述序列组成：SEQ ID NO:46，或与SEQ ID NO:46相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:5，或与SEQ ID NO:5相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:6，或与SEQ ID NO:6相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)下述3个重链可变区(VH)CDR：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与SEQ ID NO:1相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与SEQ ID NO:2相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，或者其由下述序列组成：SEQ ID NO:45，或与SEQ ID NO:45相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与SEQ ID NO:3相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:4，或与SEQ ID NO:4相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:5，或与SEQ ID NO:5相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:6，或与SEQ ID NO:6相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，上述(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:2所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:4所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:45所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:4所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:2所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:46所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示 的VH CDR1；如SEQ ID NO:45所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:46所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:2所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:4所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:45所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:4所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:2所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:46所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:45所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:46所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)下述3个重链可变区(VH)CDR：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:7，或与SEQ ID NO:7相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:8，或与SEQ ID NO:8相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:9，或与SEQ ID NO:9相比具有一个或几个氨基酸的置换、缺失或添加(例如1或2个个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:10，或与SEQ ID NO:10相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序 列，或者其由下述序列组成：SEQ ID NO:47，或与SEQ ID NO:47相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:11，或与SEQ ID NO:11相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:12，或与SEQ ID NO:12相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)下述3个重链可变区(VH)CDR：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:7，或与SEQ ID NO:7相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:8，或与SEQ ID NO:8相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:9，或与SEQ ID NO:9相比具有一个或几个氨基酸的置换、缺失或添加(例如1或2个个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:10，或与SEQ ID NO:10相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:11，或与SEQ ID NO:11相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:12，或与SEQ ID NO:12相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，上述(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:7所示的VH CDR1；如SEQ ID NO:8所示的VH CDR2；以及，如SEQ ID NO:9所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:10所示的VL  CDR1；如SEQ ID NO:11所示的VL CDR2；以及，如SEQ ID NO:12所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:7所示的VH CDR1；如SEQ ID NO:8所示的VH CDR2；以及，如SEQ ID NO:9所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:47所示的VL CDR1；如SEQ ID NO:11所示的VL CDR2；以及，如SEQ ID NO:12所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:7所示的VH CDR1；如SEQ ID NO:8所示的VH CDR2；以及，如SEQ ID NO:9所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:10所示的VL CDR1；如SEQ ID NO:11所示的VL CDR2；以及，如SEQ ID NO:12所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:7所示的VH CDR1；如SEQ ID NO:8所示的VH CDR2；以及，如SEQ ID NO:9所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:47所示的VL CDR1；如SEQ ID NO:11所示的VL CDR2；以及，如SEQ ID NO:12所示的VL CDR3。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)下述3个重链可变区(VH)CDR：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:13，或与SEQ ID NO:13相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:14，或与SEQ ID NO:14相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:15，或与SEQ ID NO:15相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:16，或与SEQ ID NO:16相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:17，或与SEQ ID NO:17相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:18，或与SEQ ID NO:18相比具有 一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，上述(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:13所示的VH CDR1；如SEQ ID NO:14所示的VH CDR2；以及，如SEQ ID NO:15所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:16所示的VL CDR1；如SEQ ID NO:17所示的VL CDR2；以及，如SEQ ID NO:18所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:13所示的VH CDR1；如SEQ ID NO:14所示的VH CDR2；以及，如SEQ ID NO:15所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:16所示的VL CDR1；如SEQ ID NO:17所示的VL CDR2；以及，如SEQ ID NO:18所示的VL CDR3。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)下述3个重链可变区(VH)CDR：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:19，或与SEQ ID NO:19相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:20，或与SEQ ID NO:20相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:21，或与SEQ ID NO:21相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:22，或与SEQ ID NO:22相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:23，或与SEQ ID NO:23相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:24，或与SEQ ID NO:24相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，上述(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:19所示的VH CDR1；如SEQ ID NO:20所示的VH CDR2；以及，如SEQ ID NO:21所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:22所示的VL CDR1；如SEQ ID NO:23所示的VL CDR2；以及，如SEQ ID NO:24所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:19所示的VH CDR1；如SEQ ID NO:20所示的VH CDR2；以及，如SEQ ID NO:21所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:22所示的VL CDR1；如SEQ ID NO:23所示的VL CDR2；以及，如SEQ ID NO:24所示的VL CDR3。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)下述3个重链可变区(VH)CDR：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:25，或与SEQ ID NO:25相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:26，或与SEQ ID NO:26相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:27，或与SEQ ID NO:27相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:28，或与SEQ ID NO:28相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:29，或与SEQ ID NO:29相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:30，或与SEQ ID NO:30相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，上述(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:25所示 的VH CDR1；如SEQ ID NO:26所示的VH CDR2；以及，如SEQ ID NO:27所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:28所示的VL CDR1；如SEQ ID NO:29所示的VL CDR2；以及，如SEQ ID NO:30所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:25所示的VH CDR1；如SEQ ID NO:26所示的VH CDR2；以及，如SEQ ID NO:27所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:28所示的VL CDR1；如SEQ ID NO:29所示的VL CDR2；以及，如SEQ ID NO:30所示的VL CDR3。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)下述3个重链可变区(VH)CDR：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:31，或与SEQ ID NO:31相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:32，或与SEQ ID NO:32相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:33，或与SEQ ID NO:33相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
和/或
(b)下述3个轻链可变区(VL)CDR：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:34，或与SEQ ID NO:34相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:35，或与SEQ ID NO:35相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:36，或与SEQ ID NO:36相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，上述(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:31所示的VH CDR1；如SEQ ID NO:32所示的VH CDR2；以及，如SEQ ID NO:33所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:34所示的VL  CDR1；如SEQ ID NO:35所示的VL CDR2；以及，如SEQ ID NO:36所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:31所示的VH CDR1；如SEQ ID NO:32所示的VH CDR2；以及，如SEQ ID NO:33所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:34所示的VL CDR1；如SEQ ID NO:35所示的VL CDR2；以及，如SEQ ID NO:36所示的VL CDR3。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO：37所示的序列；
(ii)与SEQ ID NO：37所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：37所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO：38所示的序列；
(v)与SEQ ID NO：38所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO：38所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，上述(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
重链可变区(VH)，其包含具有如SEQ ID NO:37所示的序列或与SEQ ID NO：37所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，和轻链可变区(VL)，其包含具有如SEQ ID NO:38所示的序列或与SEQ ID NO：38所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：
具有如SEQ ID NO:37所示的序列的VH和具有如SEQ ID NO:38所示的序列的VL。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO：39所示的序列；
(ii)与SEQ ID NO：39所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：39所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO：40所示的序列；
(v)与SEQ ID NO：40所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO：40所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，上述(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
重链可变区(VH)，其包含具有如SEQ ID NO:39所示的序列或与SEQ ID NO：39所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，和轻链可变区(VL)，其包含具有如SEQ ID NO:40所示的序列或与SEQ ID NO：40所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：
具有如SEQ ID NO:39所示的序列的VH和具有如SEQ ID NO:40所示的序列的VL。
在某些实施方案中，所述的抗体或其抗原结合片段，其包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO：41所示的序列；
(ii)与SEQ ID NO：41所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：41所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO：42所示的序列；
(v)与SEQ ID NO：42所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO：42所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，上述(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
重链可变区(VH)，其包含具有如SEQ ID NO:41所示的序列或与SEQ ID NO：41所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，和轻链可变区(VL)，其包含具有如SEQ ID NO:42所示的序列或与SEQ ID NO：X所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：
具有如SEQ ID NO:41所示的序列的VH和具有如SEQ ID NO:42所示的序列的VL。
在某些实施方案中，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是人源化的。
在某些实施方案中，所述抗体或其抗原结合片段进一步包含人免疫球蛋白的框架区。
在某些实施方案中，所述抗体或其抗原结合片段进一步包含人免疫球蛋白的重链框架区(例如，人重链胚系抗体基因所编码的氨基酸序列中所包含的重链框架区)，和/或，人免疫球蛋白的轻链框架区(例如，人轻链胚系抗体基因所编码的氨基酸序列中所包含的轻链框架区)。
在某些实施方案中，所述重链框架区和/或轻链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至鼠源残基的回复突变。
在某些实施方案中，所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段进一步包含来源于人免疫球蛋白的恒定区。
在某些实施方案中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区。
在某些实施方案中，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段选自单克隆抗体、小鼠抗体、兔抗体、人源化抗体、全人抗体、嵌合抗体(例如，人鼠嵌合抗体)、双特异性抗体、多特异性抗体、单链抗体、dAb、互补决定区片段、Fv、单链Fv(scFv)、Fd、Fab、Fab'和F(ab')2。
在某些实施方案中，所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在某些实施方案中，所述的抗体或其抗原结合片段，其以小于或等于1.0×10-8M的KD结合人或猴ROR1。
本公开的第二方面提供了一种分离的核酸分子，其包含编码所述的抗体或其抗原结合片段、其重链和/或轻链、或其重链可变区和/或轻链可变区的核酸序列，或者其编码所述的抗体或其抗原结合片段、其重链和/或轻链、或其重链可变区和/或轻链可变区。
本公开的第三方面提供了一种载体，其包含所述的分离的核酸分子。
本公开的第四方面提供了一种宿主细胞，其包含所述的核酸分子或所述的载体。
本公开的第五方面提供了所述的抗体或其抗原结合片段在制备抗体偶联药物中的用途，或在制备用于诊断疾病特别是ROR1介导的疾病或病症的试剂盒中的用途，或在制备用于预防和/或治疗疾病特别是ROR1介导的疾病或病症的药物中的用途。
本公开还提供了所述的抗体或其抗原结合片段，其用于作为抗体偶联药物，或用于诊断、预防和/或治疗疾病特别是ROR1介导的疾病或病症。
本公开还提供了诊断、预防和/或治疗疾病特别是ROR1介导的疾病或病症的方法，其包括给予有需要的个体有效量的所述的抗体或其抗原结合片段。
本公开的第六方面提供了一种如式(I)所示的抗体偶联药物或其药学上可接受的盐，
其中：
Q1-L1-L2-L3为连接子，
Ab为所述的抗体或其抗原结合片段，其通过所述的抗体或其抗原结合片段中的S原子偶联至连接子中的Q1，
T为药物单元或诊断试剂单元，其通过药物单元或诊断试剂单元中的N原子或O原子偶联至连接子中的L3，
m为1-9之间的数，例如m为2至9之间的数，例如约4，约5，约6，约7，约8，或约9，
Q1选自：取代的取代的取代的和取代的
L1为-L1a-C(＝O)-，其中，L1a选自：亚烷基、聚乙二醇基、亚烯基、亚炔基、亚脂环基、亚脂杂环基、亚芳基、亚杂芳基、取代的亚烷基、取代的聚乙二醇基、取代的亚烯基、取代的亚炔基、取代的亚脂环基、取代的亚脂杂环基、取代的亚芳基和取代的亚杂芳基，
L2为多肽残基或取代的多肽残基，
L3为或取代的
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中Q1选自：
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中Q1为
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中L1a选自：亚烷基、聚乙二醇基、亚烯基、亚炔基、亚脂环基。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中L1a为-(CH2)n1-，n1为1至10之间的数，例如1至8之间的数，1至6之间的数，1至4之间的数。在某些实施方案中，n1为1、2、3、4、5、6、7或8。
在某些实施方案中，L1为-(CH2)5-C(＝O)-。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中L2和/或L3可 被CH2NHCH3、聚肌氨酸残基、糖基化聚乙二醇片段、取代的聚肌氨酸残基或取代的糖基化聚乙二醇片段取代。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中所述的多肽残基包含选自以下的氨基酸：苯丙氨酸、异亮氨酸、亮氨酸、色氨酸、缬氨酸、甲硫氨酸、酪氨酸、丙氨酸、苏氨酸、组氨酸、丝氨酸、谷氨酰胺、精氨酸、赖氨酸、天冬酰胺、谷氨酸、脯氨酸、瓜氨酸、天冬氨酸和甘氨酸。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中所述的多肽残基选自：苯丙氨酸-赖氨酸(Phe-Lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-瓜氨酸(Val-Cit)、谷氨酸-缬氨酸-丙氨酸(Glu-Val-Ala)、谷氨酸-缬氨酸-瓜氨酸(Glu-Val-Cit)、缬氨酸-赖氨酸(Val-Lys)、丙氨酸-丙氨酸-丙氨酸(Ala-Ala-Ala)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)(SEQ ID NO:48)。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中所述的多肽残基选自：缬氨酸-丙氨酸(Val-Ala)、缬氨酸-瓜氨酸(Val-Cit)和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中L3为
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中L3为被-CH2NHCH3取代的
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中L3为被聚肌氨酸残基、糖基化聚乙二醇片段、取代的聚肌氨酸残基或取代的糖基化聚乙二醇片段取代的其中聚肌氨酸残基通过结构单元-X-连接至苯环，
所述结构单元-X-为或取代的
所述取代的聚肌氨酸残基为其中n2为4至18之间的数，例如4至12之间的数，R选自：C1-C6烷基、C1-C6环烷基和C1-C6烷氧基，
所述取代的糖基化聚乙二醇片段为其中n3为4至18之间的数，例如4至12之间的数。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中L3为被聚肌氨酸残基、糖基化聚乙二醇片段取代的其中聚肌氨酸残基、糖基化聚乙二醇通过结构单元-X-连接至苯环，
所述结构单元-X-为
所述取代的聚肌氨酸残基为其中n2为4至18之间的数，例如4至12之间的数，R选自：C1-C6烷基、C3-C6环烷基和C1-C6烷氧基，
所述取代的糖基化聚乙二醇片段为其中n3为4至18之间的数，例如4至12之间的数。在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中n2为5、6、7、8、9、10、11、12。在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中n2为10。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中R选自：C1-C4烷基和C3-C6环烷基。在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中R选自：C1-C4烷基。在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中R选自：甲基、乙基、正丙基和异丙基。在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中R为甲基。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中n3为5、6、7、8、9、10、11、12。在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中n3为8、11。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中L3为
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中m为7至9之间的数，例如7.5-9，例如约7.5，约7.6，约7.7，约7.8，约7.9，约8.0，约8.1，约8.2，约8.3，约8.4，约8.5，约8.6，约8.7，约8.8，约8.9，约9.0。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中药物单元选自：
(i)微管蛋白抑制剂，如单甲基奥瑞他汀E(MMAE)、单甲基奥瑞他汀F(MMAF)、tubulysin或其类似物(例如tubulysin A、tubulysin B、tubulysin D、tubulysin H、tubulysin U、tubulysin V)、艾瑞布林(Eribulin)或其类似物、长春碱或其类似物(例如长春新碱、长春地辛、长春花碱、DAVLBH)、Aur0101；
(ii)喜树碱类拓扑异构酶抑制剂，如依沙替康(Exatecan，CAS171335-80-1)、DXd(CAS1599440-33-1)、7-乙基-10-羟基喜树碱(SN38，CAS 86639-52-3)、倍罗替康(Belotecan，CAS213819-48-8)、(4-NH2)-Exatecan(AZD'0132，CAS2495742-21-5)、7-MAD-MDCPT(CAS 765871-81-6)、7-Aminomethyl-10-methyl-11-fluoro camptothecin(CAS2378616-23-8)；
(iii)免疫激活剂，如TLR7/8激动剂、STING激动剂。
在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中Q1-L1-L2-L3-T选自：


在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中-Q1-L1-L2-L3-T选自：

在某些实施方案中，所述的抗体偶联药物或其药学上可接受的盐，其中所述的抗体偶联药物选自：

其中m、Ab的定义如本公开所述。
本公开的第七方面提供了一种制备所述抗体偶联药物或其药学上可接受的盐的方法，包括：
还原Ab，得到Ab’；
使Ab’与式L-D所示的化合物进行偶联反应，得到式(I)所示的抗体偶联药物，
其中：Ab、Q1、L1、L2、L3、T和m的定义如本公开所述。
在某些实施方案中，式L-D所示的化合物选自化合物L-D-3至L-D-11。
一种典型的制备方法包括：将抗体原液用反应缓冲液稀释至2-30mg/mL，加入140-200倍过量摩尔比的二硫苏糖醇(DTT)，或加入6.0-20倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于35-39℃搅动2-48小时。上述反应缓冲液可以是按以下比例制备的缓冲液：50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl) /1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM磷酸氢二钠-柠檬酸/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM硼酸-硼砂/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM组氨酸-氢氧化钠/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；PBS/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；PBS/EDTA(乙二胺四乙酸)，pH 6-9；和含有50mM氯化钠和2mM EDTA的磷酸盐缓冲液(50mM)，pH 6-9(例如pH 7.0)。
将上述反应液冷至0-10℃，若采用DTT还原，需在还原反应完成后过脱盐柱或超滤除去过量的DTT，再加入取代马来酰亚胺类化合物(预先按浓度10mg/ml将其溶在乙腈(ACN)、二甲亚砜(DMSO)、二甲基甲酰胺(DMF)或二乙基乙酰胺(DMA)中)，并保证总反应液中有机溶剂的体积占比不超过15％，偶联反应于0-37℃搅动2-4小时。若采用TCEP还原，也可不需除去剩余TCEP，直接加入取代马来酰亚胺类化合物进行偶联。
采用脱盐柱将偶联反应混合物用含有氯化钠的组氨酸-醋酸缓冲液或含有氯化钠的磷酸缓冲液过滤纯化，离心，收集流穿部分。
所得抗体药物偶联物的DAR值较为均一。采用本公开不同取代马来酰亚胺连接头(连接子片段)时，ADC产物均一性非常高(通常DAR优势产物(如DAR值约为4、5、6、7、8或9)，占所有ADC的至少60％，至少70％，至少80％，至少90％或更高)。对于DAR有一定差别的ADC，如需要获得均一性更好的样品，可进一步利用但不限于以下方法进行分离纯化：疏水作用层析方法(HIC)、分子排阻色谱法(SEC)、离子交换层析(IEC)。
本公开的第八方面提供了一种药物组合物，其含有所述的抗体偶联药物或其药学上可接受的盐，以及一种或多种药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述的抗体偶联药物在所述药物组合物中以治疗疾病的有效量存在。所述的抗体偶联药物可以作为单一的药物活性成分来施用，也可以与其他的药物，例如其他抗癌药物，联合施用。在一些实施方案中，所述的药物组合物中还含有其他的药物，例如其他抗癌药物。所述其他抗癌药物包括但不限于抗PD-1抗体、抗PDL-1抗体和/或抗CTLA-4抗体。
在某些实施方案中，所述的抗体偶联药物或包含其的药物组合物以有效量或有效剂量施用，例如每日1ng/kg至约20-100mg/kg体重的范围，可以每日一次、两次、三次或多次施用，持续1-4周或更长的时间。
所述的抗体偶联药物或包含其的药物组合物可以通过任一种常规途径施用，例如静脉内、动脉内、肌内、腹膜内施用，等等。
本公开的第九方面提供了所述的抗体偶联药物或其药学上可接受的盐或所述的药物组 合物在制备用于预防和/或治疗疾病特别是ROR1介导的疾病或病症的药物中的用途，或在制备用于治疗和/或预防肿瘤的药物中的用途，或在制备用于诊断疾病特别是ROR1介导的疾病或病症的试剂盒中的用途。
本公开还提供了所述的抗体偶联药物或其药学上可接受的盐或所述的药物组合物，其用于诊断、预防和/或治疗疾病特别是ROR1介导的疾病或病症，或者其用于治疗和/或预防肿瘤。
本公开的第十方面提供了一种试剂盒或组合物，其包含所述的抗体或其抗原结合片段，所述的分离的核酸分子，所述的载体，所述的宿主细胞，所述的抗体偶联药物或其药学上可接受的盐或所述的药物组合物。
在某些实施方案中，所述试剂盒包含所述的抗ROR1抗体或其抗原结合片段，以及使用说明。所述试剂盒可用于诊断疾病，特别是ROR1介导的疾病或病症。
这样的试剂盒可以例如包含一次性测试装置，所述测试装置被配置为产生与生物样品中ROR1的存在或量相关的可检测信号。或者，可以配制这样的试剂盒以在不利用一次性测试装置的临床分析仪中进行测定。优选地，所述试剂盒是体外诊断剂。如本文所用的，术语“体外诊断”是指医疗器械，其是试剂、试剂产品、校准品、对照材料、试剂盒、仪器、装置、设备或系统，无论是单独使用还是组合使用，制造商意图用于体外检查来自人体的标本(包括血液和组织捐赠)，专门提供或注意用于提供有关生理或病理状态或有关先天性异常的信息或确定安全性与潜在接受者的相容性，或监测治疗措施。
本公开的第十一方面提供了一种预防和/或治疗疾病，特别是ROR1介导的疾病或病症的方法，包括向受试者施用治疗有效量的所述的抗体或其抗原结合片段或所述的抗体偶联药物或所述的药物组合物。
本公开的第十二方面提供了一种用于诊断或预后疾病(特别是ROR1介导的疾病或病症)的方法，包括：
从受试者收集感兴趣的组织样品，
使组织样品与所述的抗体或其抗原结合片段接触。
如果检测到一定量的ROR1，受试者可能被诊断患有癌症。进一步，如果检测到的ROR1表达增加/减少，表明癌症发展/改善。
在某些实施方案中，所述ROR1介导的疾病或病症为ROR1中高表达的癌症。在某些实施方案中，所述ROR1中高表达的癌症包括但不限于：头颈鳞癌、肺癌(例如非小细胞肺癌，包括肺腺癌和肺鳞癌)、尿道癌、大肠癌、前列腺癌、卵巢癌、胰腺癌、乳腺癌(例如三阴乳腺癌)、膀胱癌、胃癌、胃肠道间质瘤、宫颈癌、食道癌、鳞状细胞癌、腹膜癌、肝癌、肝细胞癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌、子宫癌、唾液腺癌、 肾癌、外阴癌、甲状腺癌、阴茎癌、黑色素瘤、白血病(例如慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL))、恶性淋巴瘤(例如套细胞淋巴瘤(MCL))、浆细胞瘤、骨髓瘤、肉瘤。
在某些实施方案中，所述的肿瘤选自头颈鳞癌、肺癌、尿道癌、大肠癌、前列腺癌、卵巢癌、胰腺癌、乳腺癌、膀胱癌、胃癌、胃肠道间质瘤、宫颈癌、食道癌、鳞状细胞癌、腹膜癌、肝癌、肝细胞癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌、子宫癌、唾液腺癌、肾癌、外阴癌、甲状腺癌、阴茎癌、黑色素瘤、白血病、恶性淋巴瘤、浆细胞瘤、骨髓瘤和肉瘤。
在某些实施方案中，所述肿瘤或癌症选自头颈鳞癌、胰腺癌、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、套细胞淋巴瘤(MCL)、卵巢癌、乳腺癌、前列腺癌、肺癌、黑色素瘤和结肠直肠癌。
在某些实施方案中，所述肿瘤或癌症选自头颈鳞癌、胰腺癌、非小细胞肺癌和三阴乳腺癌。
在某些实施方案中，所述非小细胞肺癌选自肺腺癌和肺鳞癌。
本公开的其它特征和优点将在随后的说明书中阐述，并且，部分地从说明书中变得明显，或者通过实施本公开而了解。本公开的其他优点可通过在说明书所描述的方案来实现和获得。本公开全文引用的所有参考文献、Genbank条目、专利和公开的专利申请的内容通过引用明确并入本文。
术语定义
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
如本文中使用的，术语“和/或”应被认为是两个或更多个指定特征或要素中的每一个的具体公开，其具有或不具有其他特征或要素。因此，在短语中使用的术语“和/或”，例如本文的“A和/或B”旨在包括“A和B”、“A或B”、“A”(单独)和“B”(单独)。
应理解，无论在何处用语言“包括”或“包含”描述各方面，也提供了用术语“由组成”和/或“基本上由组成”描述的类似方面。
如本文中使用的，术语“约”意指在本领域中以可接受的水平变化的数量、水平、值、数目、频率、百分比、尺寸(dimension)、大小(size)、量、重量或长度。在一些实施方案中，这样的变化可以多达参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、 重量或长度的30％、25％、20％、15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％。当术语“约”与数值范围结合使用时，其通过将所述数值的上方和下方的边界延伸来修改该范围。
如本文中使用的，术语“ROR1”(receptor tyrosine kinase-like orphan receptor 1)是一种I型跨膜蛋白，包含细胞外部分、跨膜部分和细胞质区域，属于受体酪氨酸激酶样孤儿受体(ROR)家族。现在已知ROR家族成员是Wnt家族信号分子Wnt5a/b和Wnt16的受体，Wnt5a为主要配体。ROR1通过与配体Wnt5a结合，介导非经典Wnt信号通路(non-canonical Wnt pathways)的信号传递，该信号参与多种细胞生理过程和多种癌症的发生和发展。ROR1在早期胚胎发育过程中高表达，并参与调节细胞分裂、增殖、迁移等，参与神经、骨骼和血管等器官的生成。伴随着胎儿发育的过程，ROR1的表达量逐渐下降。在儿童和成人阶段，ROR1在几乎所有正常组织中的表达都很低或不表达。与正常组织相反，ROR1在多种癌症中表达，包括血液学癌症，如慢性淋巴细胞白血病(CLL)、套细胞淋巴瘤(MCL)和实体瘤(卵巢癌、乳腺癌、前列腺癌、肺癌、黑色素瘤和结肠直肠癌等)。
ROR1的高表达通常是通过比较其在癌症组织和正常组织中的表达水平来确定的。通常，这是通过使用各种实验技术，如免疫组织化学(IHC)、免疫印迹(Western blotting)、荧光定量PCR(qPCR)和基因芯片等技术来确定的。一般来说，对于某种肿瘤抗原，其表达水平高于一定阈值就可以被认为是高表达。具体的阈值可能会因研究目的、实验技术和研究背景等因素而有所不同。在临床研究中，研究人员通常会根据大样本数据的分析来确定阈值，以判断肿瘤抗原的表达水平是否高于正常水平。需要注意的是，肿瘤抗原的高表达在不同癌症类型中可能会有所不同，并且在不同患者中也可能存在差异。
如本文中使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为框架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个 CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。这些氨基酸残基的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat等人,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefrancetal.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc等人,Dev.Comparat.Immunol.27:55-77,2003)。
本公开的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本公开的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab')2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab'片段”意指通过还原F(ab')2片段 使F(ab')2铰链区上的二硫键断裂形成的2个Fab'片段之一。
在一些情况下，抗体的抗原结合片段是单链抗体(例如,scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird等人,Science 242:423 426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA 85:5879 5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS(SEQ ID NO:49)氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4(SEQ ID NO:50)的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其是具有两个抗原结合位点的小抗体片段，所述片段包含重链可变结构域(VH)和与其在同一多肽链中连接的轻链可变结构域(VL)(VH-VL或VL-VH)，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444 6448(1993)，和Poljak R.J.等人,Structure 2:1121 1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本公开提供的单克隆抗体ch517，ch704和Hu517)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
例如，可以如下来制备单克隆抗体。首先用免疫原(必要时候添加佐剂)注射免疫小鼠或其它合适的宿主动物。免疫原或佐剂的注射方式通常为皮下多点注射或腹腔注射。可将免疫原预先偶联到某些已知蛋白，如血清白蛋白或大豆胰酶抑制剂上，以增强抗原在宿 主内的免疫原性。佐剂可以是弗氏佐剂或MPL-TDM等。动物在接受免疫后，体内将产生分泌特异性结合免疫原的抗体的淋巴细胞。另外，淋巴细胞也可以利用体外免疫获得。收集目的淋巴细胞，并用合适的融合剂，如PEG，使其与骨髓瘤细胞融合以获得杂交瘤细胞(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)。上述制备的杂交瘤细胞可以接种到合适的培养液中生长，培养液中优选含有一种或多种能够抑制未融合的、母体骨髓瘤细胞生长的物质。例如，对于缺乏次黄嘌呤鸟嘌呤磷酸转移酶(HGPRT或HPRT)的母体骨髓瘤细胞，在培养液中添加次黄嘌呤、氨基喋呤和胸腺嘧啶(HAT培养基)等物质将可以抑制HGPRT-缺陷细胞的生长。优选的骨髓瘤细胞应该具有融合率高，抗体分泌能力稳定，对HAT培养液敏感等特征。其中，骨髓瘤细胞首选鼠源骨髓瘤，如MOP-21或MC-11小鼠肿瘤衍生株(THE Salk Institute Cell Distribution Center,San Diego,Calif.USA)，和SP-2/0或X63-Ag8-653细胞株(American Type Culture Collection,Rockville,Md.USA)。另外也有研究报道，利用人骨髓瘤和人鼠异源骨髓瘤细胞株制备人单抗(Kozbor,J.Immunol.,133:3001(1984)；Brodeur等人,Monoclonal Antibody Production Techniques and Applications,pp.51-63,Marcel Dekker,Inc.,New York,1987)。生长杂交瘤细胞的培养液用于检测针对特异抗原的单抗的产生。测定杂交瘤细胞产生的单抗的结合特异性的方法包括例如，免疫沉淀或体外结合试验，如放射免疫试验(RIA)、酶联免疫吸附试验(ELISA)。例如，可利用Munson等在Anal.Biochem.107:220(1980)描述的Scatchard分析法来测定单抗的亲和力。当确定了杂交瘤产生的抗体的特异性、亲和力和反应性之后，目的细胞株可以通过(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)所描述的标准的有限稀释法进行亚克隆化。合适的培养液可以是DMEM或RPMI-1640等。另外，杂交瘤细胞还可以腹水瘤的形式在动物体内生长。利用传统的免疫球蛋白纯化方法，如蛋白A琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析等，可以将亚克隆细胞分泌的单抗从细胞培养液、腹水或血清中分离出来。
还可以通过基因工程重组技术获得单克隆抗体。利用特异性结合单抗重链和轻链基因的核酸引物进行PCR扩增，可以从杂交瘤细胞中分离得到编码单抗重链和轻链基因的DNA分子。将所得的DNA分子插入表达载体内，然后转染宿主细胞(如E.coli细胞、COS细胞、CHO细胞、或其它不产生免疫球蛋白的骨髓瘤细胞)，并在合适的条件下进行培养，可以获得重组表达的目标抗体。
如本文中所使用的，术语“嵌合抗体”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类 或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567to Cabilly等人；Morrison等人,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的框架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones等人,Nature,321:522 525(1986)；Reichmann等人,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；和Clark,Immunol.Today 21:397 402(2000)。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“多特异性抗体”是指对相同抗原或不同抗原上的至少两个不同表位具有结合特异性的抗体。多特异性抗体可以是全长抗体或所述抗体的片段。术语“双特异性抗体”是指对两种不同的抗原或可识别一个抗原的两种不同表位具有结合特异性的抗体。
如本文中所使用的，术语“人抗体”或“完全人抗体”包括具有源自人种系免疫球蛋白序列的可变区和恒定区(如果存在)的抗体。人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，术语“人抗体”不包括源自另一哺乳动物物种种系(例如小鼠)的CDR序列被嫁接到人框架序列的抗体(即人源化抗体)。完全人抗体或人抗体可以源自携带人抗体基因的转基因小鼠或源自人细胞。
如本文中使用的，术语“特异性结合”、“特异性”、或“对…特异性”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用 于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体(例如，本公开的单克隆抗体ch517,ch704,hu517)以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原(例如，受体酪氨酸激酶样孤儿受体1(ROR1))，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本公开中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“核酸分子”旨在包括DNA分子和RNA分子。核酸分子可以是单链或双链的，并且可以是cDNA。
如本文中所使用的，术语“多肽”是指包含至少两个连续连接的氨基酸残基的链，对链的长度没有上限。多肽中的一个或多个氨基酸残基可含有修饰，例如但不限于任选取代的聚肌氨酸残基修饰、任选取代的糖基化聚乙二醇修饰、糖基化修饰，磷酸化修饰或二硫键修饰。“蛋白质”可以包含一种或多种多肽。
如本文所用，术语“抗ROR1抗体”、“抗ROR1”、“ROR1抗体”、“ROR1单克隆抗体”、“结合ROR1的抗体”和“靶向ROR1的抗体”具有相同的含义，是指能够以足够亲和力结合ROR1蛋白或其片段的抗体，使得该抗体可用作靶向ROR1的诊断剂和/或治疗剂。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化、转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒； 人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行 的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。药学上可接受的载体和/或赋形剂包括但不限于：pH调节剂，表面活性剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂，稳定剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病有效量是指，足以预防、阻止、或延迟疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度，患者自己的免疫系统的总体状态，患者的一般情况例如年龄、体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤或感染)在受试者体内的发生或如果它发生作用减到最小而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。有益或所需的临床结果包括但不限于，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后，无论是可检测或是不可检测的。有效缓解任何特定疾病症状的治疗剂的量可以根据诸如患者的疾病状态，年龄和体重以及药物在受试者中引起期望的反应的能力等因素而变化。疾病症状是否得到缓解可以通过任何临床测量来评估，这些测量通常由医生或其他熟练的医疗保健提供者用于评估该症状的严重程度或进展状态。
如本文中所使用的，术语“患者”和“受试者”、“个体”、“对象”是指接受预防性或治疗性治疗的任何人类或非人类动物，尤其是人。例如，本文所述的抗体、方法和组合物可用于治疗患有癌症的受试者。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物，绵羊，狗，牛，马，猫，鸡，小鼠，大鼠，两栖动物，爬行动物等。
如本文中所使用的，术语“IC50(half maximal inhibitory concentration)”是指被测量的拮抗剂的半抑制浓度。它能指示某一药物或者物质(抑制剂)在抑制某些生物程序(或者是包含在此程序中的某些物质，比如酶，细胞受体或是微生物)的半量。IC50可以衡量抗体灵敏度，IC50越低，说明抗体的灵敏度越高。
如本文中所使用的，术语“EC50(concentration for 50％of maximal effect)”是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体偶联药物(ADC)”，也被称为抗体药物偶联物、抗体-药物偶联物、抗体药物缀合物。抗体偶联药物是由靶向特异性抗原的单克隆抗体与小分子细胞毒性药物通过连接子连接而成，兼具传统小分子化疗的强大杀伤效应及抗体药物的肿瘤靶向性。ADC由三个主要部分组成：负责选择性识别癌细胞表面抗原的抗体，负责杀死癌细胞的药物有效载荷，以及连接抗体和有效载荷的连接子。
有益技术效果
实验表明，本公开的主要优点在于以下一个或多个方面：
1)本公开获得的mAb517和mAb704较临床抗体UC-961具有更优的与人ROR1的亲和力，基于mAb517和mAb704获得的ch517、ch704和Hu517，偶联同等药物的情况下可获得更优的药效；并且本公开获得的mAb517和mAb704可特异性结合人ROR1，基于mAb517和mAb704获得的ch517、ch704和Hu517，也可特异性结合人ROR1。
2)本公开所获得的抗体偶联药物，特别是包含ch517及其人源化的Hu517的抗体偶 联药物，在多种肿瘤模型中均比对照药物有更优的抑瘤效果；并且本公开所获得的抗体偶联药物，特别是包含Hu517的抗体偶联药物，具有显著的旁杀效应。
3)本公开所获得的抗体偶联药物，具有良好的均一性与稳定性，特别是包含人源化的Hu517的抗体偶联药物，体内稳定性高，具有较好的药代动力学性质，包括较高的AUC和较长的半衰期。
附图说明
附图用来提供对本公开技术方案的理解，并且构成说明书的一部分，与本公开的实施例一起用于解释本公开的技术方案，并不构成对本公开技术方案的限制。
图1显示了mAb517,mAb704和UC-961抗体(阳性对照抗体)结合人ROR1的代表性结合曲线；
图2显示了mAb517和mAb704抗体结合人ROR1和人ROR2以及空白对照质粒转染细胞的结果；
图3显示了本公开实施例2制备的裸抗ch517的体积排阻(SEC)色谱图；
图4显示了本公开实施例5制备的抗体-药物偶联物ch517-T1000-exatecan的体积排阻(SEC)色谱图；
图5显示了本公开实施例5制备的抗体-药物偶联物ch517-T1000-exatecan的疏水层析(HIC)色谱图；
图6显示了本公开实施例2制备的裸抗ch704的体积排阻(SEC)色谱图；
图7显示了本公开实施例2制备的裸抗ch704的疏水层析(HIC)色谱图；
图8显示了本公开实施例5制备的抗体-药物偶联物ch704-T1000-exatecan的体积排阻(SEC)色谱图；
图9显示了本公开实施例5制备的抗体-药物偶联物ch704-T1000-exatecan的疏水层析(HIC)色谱图；
图10显示了本公开实施例2制备的裸抗Hu517的体积排阻(SEC)色谱图；
图11显示了本公开实施例2制备的裸抗Hu517的疏水层析(HIC)色谱图；
图12显示了本公开实施例5制备的抗体-药物偶联物Hu517-T1000-exatecan的体积排阻(SEC)色谱图；
图13显示了本公开实施例5制备的抗体-药物偶联物Hu517-T1000-exatecan的疏水层析(HIC)色谱图；
图14显示了本公开所制备的裸抗UC-961的体积排阻(SEC)色谱图；
图15显示了本公开所制备的裸抗UC-961的疏水层析(HIC)色谱图；
图16显示了本公开实施例5制备的抗体-药物偶联物UC-961-T1000-exatecan的体积排阻(SEC)色谱图；
图17显示了本公开实施例5制备的抗体-药物偶联物UC-961-T1000-exatecan的疏水层析(HIC)色谱图；
图18显示了本公开实施例5制备的抗体-药物偶联物UC-961-vc-MMAE的体积排阻(SEC)色谱图；
图19显示了本公开实施例5制备的抗体-药物偶联物UC-961-vc-MMAE的疏水层析(HIC)色谱图；
图20显示了本公开实施例5制备的抗体-药物偶联物HuIgG-T1000-exatecan的体积排阻(SEC)色谱图；
图21显示了本公开实施例5制备的抗体-药物偶联物HuIgG-T1000-exatecan的疏水层析(HIC)色谱图；
图22显示了本公开实施例5制备的抗体-药物偶联物HuIgG-vc-MMAE的体积排阻(SEC)色谱图；
图23显示了本公开实施例5制备的抗体-药物偶联物HuIgG-vc-MMAE的疏水层析(HIC)色谱图；
图24显示了本公开中抗体-药物偶联物Hu517-T1000-exatecan的血药浓度-时间变化曲线图；
图25显示了混合细胞系中的存活细胞数量柱状图；
图26显示了本公开抗体-药物偶联物在Fadu小鼠模型的体内抗肿瘤活性；
图27显示了本公开抗体-药物偶联物针对人胰腺癌BXPC-3的体内抗肿瘤活性；
图28显示了本公开抗体-药物偶联物针对人肺腺癌NCI-H1975的体内抗肿瘤活性；
图29显示了本公开抗体-药物偶联物针对三阴乳腺癌MDA-MB-231的体内抗肿瘤活性；
图30显示了本公开抗体-药物偶联物针对人肺鳞癌NCI-H226的体内抗肿瘤活性；
图31显示了本公开抗体-药物偶联物针对人非小细胞肺癌PDX LD1-0025-200662的体内抗肿瘤活性；
图32显示了本公开抗体-药物偶联物针对人非小细胞肺癌PDX LD1-0025-200730的体内抗肿瘤活性；
图33显示了本公开抗体-药物偶联物针对人三阴乳腺癌PDX LD1-2009-362263的体内抗肿瘤活性。
序列信息
本公开所涉及的部分序列的信息如下面的表1所示。
表1.部分序列的信息


以上表1中的CDR区序列分别采用Chothia、Kabat和IMGT编号系统定义，也可以采用任何其他的本领域公知的CDR区序列确定方法来鉴定可变区内CDR区的氨基酸残基。
具体实施方式
实施例1毒素连接子的合成
一)化合物L-D-6的合成
1.中间体LD6-C的合成
将中间体LD6-A(Mc-Val-Ala-OH，购自上海皓元化学，2.4g,6.29mmol)与LD6-B(3.18g,6.29mmol)溶解于二氯甲烷与甲醇(v:v＝2:1，90毫升)的混合溶剂中。室温下加入对EEDQ(2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline，1.86g,7.55mmol)。反应液室温搅拌24小时，反应液减压蒸干，经柱层析获得LD6-C(3.9g，收率71％)。LC-MS(ESI,m/z)理论值:867.46，实测值:868.49(M+H)。
2.中间体LD6-D的合成
将中间体LD6-C(2g,2.3mmol)溶解于50毫升无水四氢呋喃中，氩气氛围下冰浴冷 却，加入氢氟酸吡啶复合物(4.6g,46mmol)，反应液在0摄氏度下搅拌2小时，加入水淬灭反应，二氯甲烷萃取，分离出有机层，无水硫酸钠干燥，经柱层析获得中间体LD6-D(1.1g，收率76％)。LC-MS(ESI,m/z)理论值:629.34，实测值:630.31(M+H)。
3.中间体LD6-E的合成
将中间体LD6-D(700mg,1.11mmol)溶解于4毫升无水DMF中，室温下分别加入DIPEA(0.39ml,2.23mmol)与双(4-硝基苯基)碳酸酯(406mg,1.33mmol)。氩气下室温搅拌过夜，减压蒸去溶剂，后加入甲基叔丁基醚搅拌至固体析出，过滤，固体用乙醚淋洗数次，固体干燥后获得中间体LD6-E，直接用于下一步反应。LC-MS(ESI,m/z)理论值:794.35，实测值:795.41(M+H)。
4.中间体LD6-F的合成
将中间体LD6-E(300mg,0.44mmol)溶解于400μL无水DMF中，加入100μL无水吡啶，后再加入依沙替康(exatecan)甲磺酸盐(购自上海皓元，234mg,0.44mmol)与HOBt(60mg,0.44mmol)。氩气下室温搅拌过夜，反应液经反相HPLC制备纯化得中间体LD6-F(230mg,收率48％)。LC-MS(ESI,m/z)理论值:1090.48，实测值:1091.53(M+H)。
5.中间体LD6-G的合成
将中间体LD6-F(200mg,0.183mmol)溶解于1mL无水二氯甲烷中，冰浴下加入300μL三氟乙酸后，恢复至室温搅拌30分钟，减压除去溶剂得终产物得中间体LD6-G的三氟乙酸盐，未经进一步纯化直接用于下一步反应。LC-MS(ESI,m/z)理论值:990.43，实测值:991.47(M+H)。
6.化合物(L-D-6)的合成
将化合物LD6-G(120mg,0.109mmol)溶解于1毫升无水DMF中，分别加入乙酰化-10聚肌氨酸(Ac-Sar10-COOH，84mg,0.109mmol)，HATU(50mg,0.130mmol)和DIPEA(38μL,0.22mmol)后，室温搅拌过夜，减压除去溶剂得终产物后，经反相HPLC制备纯化得化合物(L-D-6)(74mg,收率38％)。LC-MS(ESI,m/z)理论值:1742.81，实测值:1743.85(M+H)。
二)化合物L-D-9的合成
化合物L-D-9的合成与化合物L-D-6的合成步骤与条件相同，只是将最后一步中的原料乙酰化-10聚肌氨酸替换为GPEG-1(参照文献CN107109405A，CN110214141A中相关方法进行合成)。LC-MS(ESI,m/z)理论值:1903.90，实测值:1904.73(M+H)。
四)化合物L-D-3、L-D-4、L-D-5、L-D-7、L-D-8、L-D-10和L-D-11的合成
参照化合物L-D-6的合成方法，合成化合物L-D-3、L-D-4、L-D-5、L-D-7、L-D-8、L-D-10和L-D-11，它们的结构式如下表2所示，制备这些化合物的相关中间体可以商业化购买获得。
表2


实施例2抗体的制备
ROR1单克隆抗体制备
使用人ROR1蛋白胞外区片段抗原(NCBI参考序列:NP_005003.2，氨基酸序列30-403位)免疫Balb/c小鼠(8-12周龄)，并监测其血清效价以决定免疫次数。初次免疫后经过3到4次的加强免疫，取小鼠血清按照本领域常规方法检测滴度。滴度检测合格的小鼠冲击免疫一次后，取全脾和1/2的淋巴结，与骨髓瘤SP2/0细胞系进行PEG融合。融合细胞进行铺板培养，收集所有孔的上清液，使用ELISA方法筛选抗体，将镜检有细胞的阳性孔转到96孔板继续培养。生长7天后，收集所有孔的上清液并使用ELISA方法检测与抗原的反应。阳性孔进一步检测不同稀释度的抗原结合，以进行亲和力排序。免疫原亲和力最高的20个亲代克隆进入亚克隆。通过有限稀释法和ELISA筛选进行亚克隆，得到两株杂交瘤细胞株。对两株杂交瘤细胞株进行小鼠腹水制备，收集纯化后获得两个鼠源单克隆抗体(简称“mAb517”和“mAb704”)。
鼠源ROR1单克隆抗体(mAb517)的序列信息如下：
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:1-3所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:4-6所示，或者
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:7-9所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:10-12所示，或者
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:13-15所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:16-18所示。
鼠源ROR1单克隆抗体(mAb704)的序列信息如下：
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:19-21所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:22-24所示，或者
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:25-27所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:28-30所示，或者
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:31-33所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:34-36所示。以鼠源ROR1单克隆抗体的CDR为基础，对 杂交瘤序列进行人源化修饰，得到嵌合ROR1抗体(简称“ch517”和“ch704”)以及人源化ROR1抗体(简称“Hu517”)，序列如表3所示。
表3.嵌合ROR1抗体以及人源化ROR1抗体的重、轻链可变区氨基酸序列
人源化ROR1抗体(Hu517)的序列信息如下：
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:1、45、3所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:46、5、6所示，或者
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:7-9所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:47、11、12所示，或者
重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:13-15所示；轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:16-18所示。
人-鼠嵌合抗体的制备
通过基因重组技术将ch517重链和轻链可变区序列(参见SEQ ID NO:37、SEQ ID NO:38)，以及ch704重链和轻链可变区序列(参见SEQ ID NO:39、SEQ ID NO:40)克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经测序无误后，采用转染技术和哺乳动物表达系统(FreeStyleTM293T细胞)将构建的嵌合型抗体进行表达，然后纯化，将所获得的人-鼠嵌合型抗体命名为ch517与ch704，其重链及轻链可变区的序列见表1。用同样方法制备阳性对照抗体UC-961，其抗体序列参见专利WO2021159029 A1中SEQ ID NO.2和SEQ ID NO.4，应用UC-961的重链及轻链全长序列(WO2018237335A1中Ab1light chain,Ab1 heavy chain描述的全长序列)分别构建pCDNA3.1表达载体，抗体通过293F悬浮细胞培养表达获得，并通过proteinA/G重力柱对抗体进行纯化。
人源化抗体的制备
在Germline数据库中检索并选取与ch517非CDR区匹配最好的人源化模板，然后将抗体的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，再与IgG1恒定区重组，同时以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变。
通过基因重组技术将所设计的人源化可变区序列克隆入含有人IgG1重链恒定区和 Kappa链恒定区的载体，经测序无误后，利用转染技术和哺乳动物表达系统(FreeStyleTM293细胞)将构建的人源化抗体进行表达，然后纯化，最终获得了1个人源化抗体Hu517，其重链及轻链可变区的序列见表1。
实施例3抗ROR1抗体在人ROR1重组蛋白ELISA中的亲和力检测
将人ROR1蛋白(目录号：RO1-H522y,Acrobiosystems公司)4℃孵育过夜固定到96孔板上。然后将所述板通过在37℃与于PBS中1％的BSA一起孵育1小时来封闭。在封闭之后，将所述板用PBST(含有0.05％Tween20的PBS)洗涤3次。在结合缓冲液(含有0.05％Tween20和0.5％BSA的PBS)中制备连续稀释的抗ROR1抗体并且在37℃将其与固定的蛋白质一起孵育1小时。在结合之后，将所述板用PBST洗涤3次，在37℃与在结合缓冲液中1/10,000稀释的过氧化物酶标记的羊抗鼠IgG或羊抗人IgG(杰克逊免疫研究公司(JacksonImmuno Research))一起孵育1小时，再次洗涤，用TMB显色并且使用1M H2SO4终止。测定450nm-620nm处的吸光度。
mAb517,mAb704和UC-961抗体(阳性对照抗体)结合人ROR1的代表性结合曲线如图1所示，EC50如表4所示。结果表明3个抗体均特异性地结合人ROR1蛋白，而mAb517和mAb704抗体的亲和力显著优于UC-961(阳性对照抗体)。
表4.mAb517、mAb704和UC-961抗体结合人ROR1的EC50
实施例4使用过表达质粒转染流式测定来确定抗体对人ROR1和人ROR2的结合特异性
培养293T(人胚肾细胞，中国科学院细胞库,目录号：SCSP-502)使细胞密度达到80％，收集细胞，铺6孔板，调整细胞密度至2-5×105/ml。每孔铺3mL。第二天进行质粒转染，分别将3μg的人ROR1质粒和3μg的人ROR2质粒与9μL PEI混合，静置20分钟，期间更换无血清培养基。将静置完毕的质粒加入细胞中，混匀，放回培养箱。6小时后更换完全培养基并培养过夜。第三天取出细胞，用胰酶进行消化，消化细胞经过1000rpm离心5min后用10％山羊血清(购自北京索莱宝科技有限公司，货号SL038)封闭30min。抗ROR1抗体mAb517和mAb704以100nM加入细胞中孵育1h，PBS清洗2遍后加入APC标记的羊抗鼠二抗(购自Jackson ImmunoResarch，稀释倍数1:500)孵育1h，最后PBS清洗2遍后进行流式细胞仪分析结果。
mAb517和mAb704抗体结合人ROR1和人ROR2以及空白对照质粒转染细胞的结果 如图2所示，结果表明mAb517和mAb704均特异性地结合人ROR1转染细胞且不结合人ROR2转染细胞。同时，mAb517和mAb704抗体在空白对照质粒转染细胞中也没有结合反应。
实施例5抗体药物偶联物的制备
一)通用操作方法
所制备的抗体-药物偶联物(式(I))可以按照下面所述的常见操作对其进行浓缩、换液、纯化以及测量抗体浓度、计算每个抗体所携带的药物分子的平均分子数以鉴别抗体-药物偶联物。
操作A：抗体或抗体-药物偶联物的浓缩
取一根超滤管(Amicon Ultra，50000MWCO，密理博公司)，加入需要浓缩的抗体或者抗体-药物偶联物的溶液，离心至需要体积后取出。
操作B：抗体浓度的测量
使用酶标仪(Multiskan GO,赛默飞世尔公司),根据制造商定义的方法测量抗体的吸收，该吸收与抗体在该波长下的吸收系数的比值即为抗体的浓度。在这里，使用了抗体的特征吸收波长280nm，该波长下抗体的吸收系数通常在1.3mL mg-1cm-1到1.8mL mg-1cm-1之间。
操作C：抗体的换液
根据制造商(赛默飞世尔公司)提供的说明书，将zeba脱盐柱(5mL,40K MWCO)事先用含有氯化钠(50mM)和EDTA(2mM)的磷酸盐缓冲液(50mM,pH 7.0)平衡。每个zeba脱盐柱(5mL)上样2mL，离心(1000g，4分钟)，收集流穿部分。该组分可以通过操作A进行浓缩，通过操作B进行抗体浓度的测定，用PBS7.0/EDTA对抗体浓度进行调整。
操作D：抗体-药物偶联物的纯化
根据制造商(赛默飞世尔公司)提供的说明书，将zeba脱盐柱(5mL,40K MWCO)事先用任何合适的存储缓冲液进行平衡。存储缓冲液包括但不限于含有150mM氯化钠的组氨酸-醋酸缓冲液(20mM组氨酸，pH5.5)，含有50mM氯化钠的磷酸缓冲液(50mM,pH 7.0)。将含有抗体-药物偶联物的反应液(大约2mL)加入到5mL规格的zeba脱盐柱上，离心(1000g，4分钟)后，收集流穿部分(大约2mL)。该方法采用了凝胶过滤的纯化工艺，将收集到的组分应用于脱盐柱离心洗脱，重复两次洗脱过程，除去未结合的药物连接子和低分子量的化合物比如：还原剂三(2-羧乙基)膦盐酸盐(TCEP)，N-乙酰-(L)-半胱氨酸(NAC)、二甲亚砜等，得到抗体-药物偶联物。
操作E：测量抗体-药物偶联物中抗体的浓度以及平均每个抗体上连接的药物分子个数(DAR值)
抗体-药物偶联物上偶联的药物浓度可以通过测量抗体-药物偶联物水溶液在280nm和370nm下的紫外吸收值并通过下面的公式计算而得到。
在任何给定的波长下，一个体系的总吸光度等于该系统中存在的所有吸光化学物质的吸光度之和(吸光度的可加和性)。因此，假设抗体与药物在偶联前后，抗体与药物的摩尔吸收系数不变，则抗体-药物偶联物中的抗体浓度和药物浓度可以用如下公式表示。
A280＝AD,280+AA,280＝εD,280CD+εA,280CA    公式(1)
A370＝AD,370+AA,370＝εD,370CD+εA,370CA    公式(2)
在这里，A280代表了抗体-药物偶联物水溶液在280nm下的总吸收值，A370代表了抗体-药物偶联物水溶液在370nm下的总吸收值。AA,280代表了抗体在280nm下的吸收值，AA,370代表了抗体在370nm下的吸收值，AD,280代表了药物分子在280nm下的吸收值，AD,370代表了药物分子在370nm下的吸收值，εA,280代表了抗体在280nm下的摩尔消光系数，εA,370代表了抗体在370nm下的摩尔消光系数，εD,280代表了药物分子在280nm下的摩尔消光系数，εD,370代表了药物分子在370nm下的摩尔消光系数，CA代表了抗体-药物偶联物中抗体的浓度，CD代表了抗体-药物偶联物中药物分子的浓度。
在这种情况下，εA,280，εA,370，εD,280和εD,370都是已知的数值(通过抗体的序列计算得到或者化合物的UV吸收测量得到)。比如，εA,280可以通过抗体的氨基酸序列利用一种已知的方法计算得到(蛋白质科学(Protein Science)，1995年，第4卷，第2411-2423页)。抗体在370nm下通常没有吸收，所以εA,370通常是0。εD,280和εD,370的值可以通过测量药物分子分别在280nm和370nm下吸光度随浓度的变化值，再利用朗伯-比尔定律(吸光度＝摩尔浓度×摩尔消光系数×光程)计算得到。CA和CD可以通过测量抗体-药物偶联物在280nm和370nm下的吸光值A280和A370然后解二元一次方程组公式(1)和(2)得到。另外，通过CD除以CA，可以得到平均每个抗体上连接的药物分子的数量(DAR值)。
操作F：抗体-药物偶联物中聚集体的测量
抗体-药物偶联物中聚集体的测量可以通过在高效液相色谱中使用体积排阻色谱进行。分析方法如下所示：
高效液相色谱系统：Agilent 1260 Infinity II HPLC system
检测器：Ultraviolet absorption spectrometer(检测波长：280nm)
色谱柱型号:TOSOH TSKgel G3000SWXL(7.8×300mm,5μm)
流动相：200mmol/L KHPO4,150mmol/L NaCl,15％(v/v)异丙醇,pH7.0
流速：0.75mL/min
分析时间：18min
柱温：室温
进样量：50μg
数据分析:质控品(QC，Hu517裸抗)的体积排阻色谱图如图10所示。值得注意的是，在这种分析方法下，150kDa的质控品的主峰(单峰)的保留时间在9.5-10.5分钟之间。同时，聚集体的保留时间应该早于上述单体的保留时间。
操作G：抗体-药物偶联物疏水性比较与DAR值测定
抗体-药物偶联物的疏水性可以在高效液相色谱上利用疏水层析色谱柱进行分析。分析方法如下：
高效液相色谱系统：Agilent 1260 Infinity II HPLC system
检测器：Ultraviolet absorption spectrometer(检测波长：280nm)
色谱柱型号：TOSOH TSKgel Butyl-NPR(4.6mm I.D.×3.5cm,2.5μm)
流动相A：mol/L(NH4)2SO4,50mmol/L KHPO4,pH7.0
流动相B：50mmol/L KHPO4,25％(v/v)isopropanol,pH7.0
分析时间：25min
柱温：室温
洗脱程序(B％)：0％-25％(0-1分钟),25％(1-3分钟),25％-80％(3-13分钟),80％(13-17分钟),80％-0％(17-17.10分钟),0％(17.10-25分钟)
进样量：10μL
数据分析：质控品(QC，Hu517裸抗)的疏水层析色谱图如图11所示。相比于未偶联上药物分子的裸抗，抗体-药物偶联物有更高的疏水性，因此其保留时间更长。众所周知，保留时间越短的样品的疏水性越弱。通过计算可获得抗体偶联药物的DAR值。
二)制备抗体-药物偶联物ch517-T1000-exatecan
步骤：抗体-药物偶联物
抗体的还原：通过上述的操作B(抗体在280nm下的消光系数采用1.462mL mg-1cm-1)和操作C将抗体用含有EDTA的PBS7.0缓冲液进行换液，换液后的抗体浓度为8.6mg/mL。在2.3mL的ch517蛋白的水溶液中加入0.184mL的5mM的TCEP溶液(相当于抗体含量的7倍当量)，确保反应液的pH值在7.0±0.1。将混合液置于37摄氏度环境中反应2小时。
抗体与药物连接子之间的偶联：将上述混合液置于4摄氏度上孵育10分钟。紧接着 将溶解在N,N-二甲基乙酰胺(DMA)中的药物连接子L-D-6(T1000-exatecan)加入到混合液中，加入体积为0.66mL(相当于抗体含量的25倍当量)。将混合液放置在22摄氏度继续反应30分钟。
抗体-药物偶联物的纯化：将上述的反应液采用操作D中描述的方法进行纯化，最终获得抗体-药物偶联物ch517-T1000-exatecan。
抗体-药物偶联物的表征：采用上述的操作E(εD,280＝6384和εD,370＝16180)，操作F和操作G对获得的抗体-药物偶联物进行表征。
通过操作E测量计算得到抗体药物偶联物的浓度为7.85mg/mL，通过操作E测量计算得到每个抗体上平均偶联的小分子的个数为8.86，即DAR值为约8.86。通过操作F测量得到裸抗ch517的聚集体含量为2.55％(图3)，抗体-药物偶联物ch517-T1000-exatecan的聚集体含量为6.58％(图4)。通过操作G测量得抗体-药物偶联物ch517-T1000-exatecan的保留时间为6.718min(图5)。
三)制备抗体-药物偶联物ch704-T1000-exatecan
步骤：抗体-药物偶联物
抗体的还原：通过上述的操作B(抗体在280nm下的消光系数采用1.48mL mg-1cm-1)和操作C将抗体用含有EDTA的PBS7.0缓冲液进行换液，换液后的抗体浓度为7.2mg/mL。在2.75mL的ch704蛋白的水溶液中加入0.184mL的5mM的TCEP溶液(相当于抗体含量的7倍当量)，确保反应液的pH值在7.0±0.1。将混合液置于37摄氏度环境中反应2小时。
抗体与药物连接子之间的偶联：将上述混合液置于4摄氏度上孵育10分钟。紧接着将溶解在N,N-二甲基乙酰胺(DMA)中的药物连接子L-D-6(T1000-exatecan)加入到混合液中，加入体积为0.66mL(相当于抗体含量的25倍当量)。将混合液放置在22摄氏度继续反应30分钟。
抗体-药物偶联物的纯化：将上述的反应液采用操作D中描述的方法进行纯化，最终获得抗体-药物偶联物ch704-T1000-exatecan。
抗体-药物偶联物的表征：采用上述的操作E(εD,280＝6384和εD,370＝16180)，操作F和操作G对获得的抗体-药物偶联物进行表征。
通过操作E测量计算得到抗体药物偶联物的浓度为8.01mg/mL，通过操作E测量计算得到每个抗体上平均偶联的小分子的个数为8.68，即DAR值为约8.68。通过操作F测量得到裸抗ch704的聚集体含量为13.21％(图6)，抗体-药物偶联物ch704-T1000-exatecan的聚集体含量为1.54％(图8)。通过操作G测量得裸抗ch704的保留时间为3.527min(图7)，抗体-药物偶联物ch704-T1000-exatecan的保留时间为6.423min(图 9)。
四)制备抗体-药物偶联物Hu517-T1000-exatecan
步骤：抗体-药物偶联物
抗体的还原：通过上述的操作B(抗体在280nm下的消光系数采用1.444mL mg-1cm-1)和操作C将抗体用含有EDTA的PBS7.0缓冲液进行换液，换液后的抗体浓度为29.47mg/mL。在0.87mL的Hu517蛋白的水溶液中加入0.307mL的5mM的TCEP溶液(相当于抗体含量的9倍当量)，确保反应液的pH值在7.0±0.1。将混合液置于37摄氏度环境中反应2小时。
抗体与药物连接子之间的偶联：将上述混合液置于4摄氏度上孵育10分钟。紧接着将溶解在N,N-二甲基乙酰胺(DMA)中的药物连接子L-D-6(T1000-exatecan)加入到混合液中，加入体积为0.24mL(相当于抗体含量的14倍当量)。将混合液放置在22摄氏度继续反应30分钟。
抗体-药物偶联物的纯化：将上述的反应液采用操作D中描述的方法进行纯化，最终获得抗体-药物偶联物Hu517-T1000-exatecan。
抗体-药物偶联物的表征：采用上述的操作E(εD,280＝6384和εD,370＝16180)，操作F和操作G对获得的抗体-药物偶联物进行表征。
通过操作E测量计算得到抗体药物偶联物的浓度为7.21mg/mL，通过操作E测量计算得到每个抗体上平均偶联的小分子的个数为7.91，即DAR值为约7.91。通过操作F测量得到裸抗Hu517的聚集体含量为0.26％(图10)，抗体-药物偶联物Hu517-T1000-exatecan的聚集体含量为1.08％(图12)。通过操作G测量的裸抗Hu517的保留时间为3.707min(图11)，抗体-药物偶联物Hu517-T1000-exatecan的保留时间为6.653min(图13)。
五)制备抗体-药物偶联物UC-961-T1000-exatecan
步骤：抗体-药物偶联物
抗体的还原：通过上述的操作B(抗体在280nm下的消光系数采用1.505mL mg-1cm-1)和操作C将抗体用含有EDTA的PBS7.0缓冲液进行换液，换液后的抗体浓度为6.71mg/mL。在2.98mL的UC-961蛋白的水溶液中加入0.24mL的5mM的TCEP溶液(相当于抗体含量的9倍当量)，确保反应液的pH值在7.0±0.1。将混合液置于37摄氏度环境中反应2小时。
抗体与药物连接子之间的偶联：将上述混合液置于4摄氏度上孵育10分钟。紧接着将溶解在N,N-二甲基乙酰胺(DMA)中的药物连接子L-D-6(T1000-exatecan)加入到混合液中，加入体积为0.186mL(相当于抗体含量的14倍当量)。将混合液放置在22摄 氏度继续反应30分钟。
抗体-药物偶联物的纯化：将上述的反应液采用操作D中描述的方法进行纯化，最终获得抗体-药物偶联物UC-961-T1000-exatecan。
抗体-药物偶联物的表征：采用上述的操作E(εD,280＝6384和εD,370＝16180)，操作F和操作G对获得的抗体-药物偶联物进行表征。
通过操作E测量计算得到抗体药物偶联物的浓度为5.46mg/mL，通过操作E测量计算得到每个抗体上平均偶联的小分子的个数为8.62，即DAR值为约8.62。通过操作F测量得到裸抗UC-961的聚集体含量为7.15％(图14)，抗体-药物偶联物UC-961-T1000-exatecan的聚集体含量为3.01％(图16)。通过操作G测量的裸抗UC-961的保留时间为6.293min(图15)，抗体-药物偶联物UC-961-T1000-exatecan的保留时间为7.534min(图17)。
六)制备抗体-药物偶联物UC-961-vc-MMAE
步骤：抗体-药物偶联物
抗体的还原：通过上述的操作B(抗体在280nm下的消光系数采用1.505mL mg-1cm-1)和操作C将抗体用含有EDTA的PBS7.0缓冲液进行换液，换液后的抗体浓度为7.92mg/mL。在1.83mL的UC-961蛋白的水溶液中加入61.87μL的5mM的TCEP溶液(相当于抗体含量的3.2倍当量)，确保反应液的pH值在7.0±0.1。将混合液置于37摄氏度环境中反应2小时。
抗体与药物连接子之间的偶联：将上述混合液置于4摄氏度上孵育10分钟。紧接着将溶解在N,N-二甲基乙酰胺(DMA)中的药物连接子vc-MMAE(购自上海皓元化学，目录号:HY-15575)加入到混合液中，加入体积为135.33μL(相当于抗体含量的7倍当量)。将混合液放置在22摄氏度继续反应30分钟。
抗体-药物偶联物的纯化：将上述的反应液采用操作D中描述的方法进行纯化，最终获得抗体-药物偶联物UC-961-vc-MMAE。
抗体-药物偶联物的表征：采用上述的操作E，操作F和操作G对获得的抗体-药物偶联物进行表征。
通过操作E测量计算得到抗体药物偶联物的浓度为4.08mg/mL。通过操作F测量得到抗体-药物偶联物UC-961-vc-MMAE的聚集体含量为2.53％(图18)。图19是通过操作G测量抗体-药物偶联物UC-961-vc-MMAE得到的疏水层析图谱，通过计算得到平均每个抗体上连接的药物分子个数为4.49，即DAR值约为4.49(图19)。
七)制备抗体-药物偶联物HuIgG-T1000-exatecan
步骤：抗体-药物偶联物
抗体的还原：通过上述的操作B(抗体在280nm下的消光系数采用1.35mL mg-1cm-1)和操作C将抗体用含有EDTA的PBS7.0缓冲液进行换液，换液后的抗体浓度为15mg/mL。在1.33mL的HuIgG蛋白(北京义翘神州，Cat:HG1K)的水溶液中加入0.27mL的5mM的TCEP溶液(相当于抗体含量的10倍当量)，确保反应液的pH值在7.0±0.1。将混合液置于37摄氏度环境中反应2小时。
抗体与药物连接子之间的偶联：将上述混合液置于4摄氏度上孵育10分钟。紧接着将溶解在N,N-二甲基乙酰胺(DMA)中的药物连接子L-D-6(T1000-exatecan)加入到混合液中，加入体积为0.2mL(相当于抗体含量的15倍当量)。将混合液放置在22摄氏度继续反应30分钟。
抗体-药物偶联物的纯化：将上述的反应液采用操作D中描述的方法进行纯化，最终获得抗体-药物偶联物HuIgG-T1000-exatecan。
抗体-药物偶联物的表征：采用上述的操作E(εD,280＝6384和εD,370＝16180)，操作F和操作G对获得的抗体-药物偶联物进行表征。
通过操作E测量计算得到抗体药物偶联物的浓度为12.09mg/mL，通过操作E测量计算得到每个抗体上平均偶联的小分子的个数为6.71，即DAR值为约6.71。通过操作F测量得到抗体-药物偶联物HuIgG-T1000-exatecan的聚集体含量为4.38％(图20)。通过操作G测量的抗体-药物偶联物HuIgG-T1000-exatecan的保留时间为6.213min(图21)。
八)制备抗体-药物偶联物HuIgG-vc-MMAE
步骤：抗体-药物偶联物
抗体的还原：通过上述的操作B(抗体在280nm下的消光系数采用1.35mL mg-1cm-1)和操作C将抗体用含有EDTA的PBS7.0缓冲液进行换液，换液后的抗体浓度为15mg/mL。在0.8mL的HuIgG蛋白的水溶液中加入40μL的5mM的TCEP溶液(相当于抗体含量的2.5倍当量)，确保反应液的pH值在7.0±0.1。将混合液置于37摄氏度环境中反应2小时。
抗体与药物连接子之间的偶联：将上述混合液置于4摄氏度上孵育10分钟。紧接着将溶解在N,N-二甲基乙酰胺(DMA)中的药物连接子vc-MMAE加入到混合液中，加入体积为112μL(相当于抗体含量的7倍当量)。将混合液放置在22摄氏度继续反应30分钟。
抗体-药物偶联物的纯化：将上述的反应液采用操作D中描述的方法进行纯化，最终获得抗体-药物偶联物HuIgG-vc-MMAE。
抗体-药物偶联物的表征：采用上述的操作E，操作F和操作G对获得的抗体-药物偶联物进行表征。
通过操作E测量计算得到抗体药物偶联物的浓度为9.21mg/mL。通过操作F测量得到抗体-药物偶联物HuIgG-vc-MMAE的聚集体含量为7.67％(图22)。图23是通过操作G测量抗体-药物偶联物HuIgG-vc-MMAE得到的疏水层析图谱。
所制备的抗体-药物偶联物ch517-T1000-exatecan、ch704-T1000-exatecan、Hu517-T1000-exatecan、UC-961-T1000-exatecan、HuIgG-T1000-exatecan的结构式如C-6所示，其中Ab和m分别代表抗体和DAR值。
所制备的抗体-药物偶联物UC-961-vc-MMAE、HuIgG-vc-MMAE的结构式如C-11所示，其中Ab和m分别代表抗体和DAR值。
九)制备其他抗体-药物偶联物
参照通用操作方法制备抗体-药物偶联物C-3、C-4、C-5、C-7、C-8、C-9、C-10、C-11，它们的结构式如下表所示，其中m代表DAR值，为约8或约4；Ab代表抗体，为ch517、ch704或Hu517。


实施例6ROR1抗体-药物偶联物(Hu517-T1000-exatecan)在裸鼠中的药代动力学实验
6周龄的BALB/C nude小鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。按照10mg/kg的给药量静脉注射给予实施例5制备得到的抗体-药物偶联物Hu517-T1000-exatecan，每周1次，共给药1次。给药后在第15分钟、4小时、24小时、2天、4天、7天、14天、21天采集小鼠血液进行ELISA实验评估不同时间点小鼠血液中药物浓度，并计算药代动力学参数。图24显示出抗体-药物偶联物Hu517-T1000-exatecan的血药浓度-时间变化曲线图，表5为其相关药代动力学参数。
表5.Hu517-T1000-exatecan的药代动力学参数
结果显示，药物的ADC浓度和总抗(Total antibody)浓度曲线有较好的重合度，证明该药物的稳定性较高，同时，药代动力学参数显示该药物具有较高的AUC参数和较长的半衰期。
实施例7 ROR1抗体-药物偶联物(Hu517-T1000-exatecan)的体外旁杀效应评估
培养PC9(人肺癌细胞，中国科学院细胞库,目录号：SCSP-5085)和A549(人肺癌细胞，ATCC,目录号：CCL-185)使细胞密度达到80％，收集细胞，以1:3的比例混合PC9(ROR1阳性细胞)和A549(ROR1阴性细胞)铺6孔板，调整混合细胞密度至2-5×104/mL，每孔铺3mL。ADC分子及阴性对照分别以100nM的工作浓度加入混合细胞培养液，静置培养5天。期间定期观察细胞凋亡情况。5天后，取出细胞，用胰酶进行消化，进行细胞计数，分别统计ADC分子和阴性对照孔的活细胞总数。消化细胞经过1000rpm离心5min后用10％山羊血清(购自北京索莱宝科技有限公司，货号SL038)封闭30min。抗ROR1人源化抗体Hu517作为一抗以100nM加入细胞中孵育1h，PBS清洗2遍后1:500加入FITC标记的羊抗人二抗(购自Jackson ImmunoResarch)孵育1h，最后PBS清洗2遍后进行流式细胞仪分析结果。根据检测结果分别计算PC9和A549活细胞个数，绘制细胞存活数量柱状图。
图25为在含有实施例5制备的抗体-药物偶联物Hu517-T1000-exatecan或HuIgG-T1000-exatecan的混合细胞系中的存活细胞数量柱状图。结果表明，对比阴性抗体-药物偶联物HuIgG-T1000-exatecan，Hu517-T1000-exatecan不仅对ROR1阳性细胞PC9产生了较强的杀伤作用，对于ROR1阴性细胞A549也同样产生了杀伤作用，显示出了显著的旁杀效应。
实施例8 ROR1抗体-药物偶联物(ROR1-ADCs)针对人头颈鳞癌CDX Fadu的体内抗肿瘤活性
6周龄的BALB/C nude鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。每只小鼠皮下接种五百万Fadu细胞(中国科学院典型培养物保藏委员会细胞库，货号：TCHu132)。当平均肿瘤体积达到150mm3时，按照10mg/kg的给药量分别静脉注射给予实施例5制备的抗体-药物偶联物ch517-T1000-exatecan和HuIgG-T1000-exatecan；按照5mg/kg的给药剂量静脉注射给予实施例5制备的抗体-药物偶联物UC-961-vc-MMAE和HuIgG-vc-MMAE。每周1次(QW)，共给药2次。给药后使用游标卡尺每周测量两次肿瘤体积，并按照如下公式计算肿瘤体积：TV＝(长*宽^2)/2。
图26显示出Fadu小鼠模型的体内肿瘤体积-时间变化曲线图，结果显示，和阴性对照相比，ch517-T1000-exatecan显著抑制肿瘤生长，UC-961-vc-MMAE没有产生任何肿瘤生长抑制作用。
实施例9 ROR1抗体-药物偶联物(ROR1-ADCs)针对人胰腺癌CDX BXPC-3的体内抗肿瘤活性
6周龄的BALB/C nude小鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。小鼠皮下接种五百万BXPC-3细胞(中国科学院典型培养物保藏委员会细胞库，货号：TCHu 12)。当平均肿瘤体积达到150mm3时，按照10mg/kg的给药量分别静脉注射给予实施例5制备的抗体-药物偶联物ch517-T1000-exatecan，ch704-T1000-exatecan；实施例5制备的抗体-药物偶联物HuIgG-T1000-exatecan并设置阴性对照Vehicle组，静脉注射给与等剂量PBS。每周1次(QW)，共给药3次。给药后使用游标卡尺每周测量两次肿瘤体积，并按照如下公式计算肿瘤体积：TV＝(长*宽^2)/2。
图27显示出胰腺癌小鼠模型的体内肿瘤体积-时间变化曲线图，结果显示，和阴性对照HuIgG-T1000-exatecan和PBS组相比，ch517-T1000-exatecan和ch704-T1000-exatecan均能够部分抑制肿瘤生长，ch517-T1000-exatecan的抑瘤趋势和持续时间更优于ch704-T1000-exatecan。
实施例10 ROR1抗体-药物偶联物(ROR1-ADCs)针对人肺腺癌CDX NCI-H1975的体内抗肿瘤活性
6周龄的BALB/C nude小鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。小鼠皮下接种五百万NCI-H1975细胞(中国科学院典型培养物保藏委员会细胞库，货号：SCSP-597)。当平均肿瘤体积达到150mm3时，按照10mg/kg的给药量分别静脉注射给予实施例5制备的抗体-药物偶联物ch517-T1000-exatecan，ch704-T1000-exatecan；实施例5制备的抗体-药物偶联物HuIgG-T1000-exatecan并设置阴性对照Vehicle组，静脉注射给与等剂量PBS。每周1次，共给药1次。给药后使用游标卡尺每周测量两次肿瘤体积，并按照如下公式计算肿瘤体积：TV＝(长*宽^2)/2。
图28显示出肺腺癌小鼠模型的体内肿瘤体积-时间变化曲线图，结果显示，和阴性对照HuIgG-T1000-exatecan和PBS组相比，ch517-T1000-exatecan和ch704-T1000-exatecan均能够显著抑制肿瘤生长，ch517-T1000-exatecan的抑瘤趋势和持续时间更优于ch704-T1000-exatecan。
实施例11 ROR1抗体-药物偶联物(ROR1-ADCs)针对人三阴乳腺癌CDX MDA-MB-231的体内抗肿瘤活性
6周龄的BALB/C nude小鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。小鼠皮下接种五百万MDA-MB-231细胞(中国科学院典型培养物保藏委员会细胞库，货号：SCSP-5043)。当平均肿瘤体积达到150mm3时，按照5mg/kg的给药量分别静脉注射给予实施例5制备的抗体-药物偶联物Hu517-T1000-exatecan，UC-961-vc- MMAE，UC-961-T1000-exatecan并设置阴性对照Vehicle组，静脉注射给与等剂量PBS。每周1次(QW)，共给药3次，其中UC-961-vc-MMAE组共给药5次。给药后使用游标卡尺每周测量两次肿瘤体积，并按照如下公式计算肿瘤体积：TV＝(长*宽^2)/2。
图29显示出三阴乳腺癌小鼠模型的体内肿瘤体积-时间变化曲线图，结果显示，和阴性对照相比，Hu517-T1000-exatecan，UC-961-T1000-exatecan和UC-961-vc-MMAE均能够显著抑制肿瘤生长，Hu517-T1000-exatecan的抑瘤趋势更优于UC-961-T1000-exatecan，UC-961-vx-MMAE抑瘤程度最弱。
实施例12 ROR1抗体-药物偶联物(ROR1-ADCs)针对人肺鳞癌CDX NCI-H226的体内抗肿瘤活性
6周龄的NU/NU小鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。小鼠皮下接种五百万NCI-H226细胞(中国科学院典型培养物保藏委员会细胞库，货号：TCHu235)。当平均肿瘤体积达到150mm3时，按照10mg/kg和5mg/kg的给药量分别静脉注射给予实施例5制备的抗体-药物偶联物Hu517-T1000-exatecan和UC-961-vc-MMAE；按照10mg/kg的给药剂量静脉注射给予实施例5制备的抗体-药物偶联物HuIgG-T1000-exatecan并设置阴性对照Vehicle组，静脉注射给与等剂量PBS。每周1次，共给药1次。给药后使用游标卡尺每周测量两次肿瘤体积，并按照如下公式计算肿瘤体积：TV＝(长*宽^2)/2。
图30显示出肺鳞癌小鼠模型的体内肿瘤体积-时间变化曲线图，结果显示，和阴性对照相比，Hu517-T1000-exatecan能够显著抑制肿瘤生长，而UC-961-vc-MMAE没有产生任何的肿瘤抑制效果。
实施例13 ROR1抗体-药物偶联物(ROR1-ADCs)针对人非小细胞肺癌PDX LD1-0025-200662的体内抗肿瘤活性
6周龄的NU/NU小鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。小鼠皮下接种非小细胞肺癌瘤块(标本编号为200662，来源于西安立迪生物技术有限公司)。当平均肿瘤体积达到150mm3时，按照10mg/kg的给药量静脉注射给予实施例5制备的抗体-药物偶联物ch517-T1000-exatecan并设置阴性对照Vehicle组，静脉注射给与等剂量PBS。每周1次(QW)，共给药2次。给药后使用游标卡尺每周测量两次肿瘤体积，并按照如下公式计算肿瘤体积：TV＝(长*宽^2)/2。
图31显示出非小细胞肺癌小鼠模型的体内肿瘤体积-时间变化曲线图，结果显示，和阴性对照相比，ch517-T1000-exatecan能够显著抑制肿瘤生长。
实施例14 ROR1抗体-药物偶联物(ROR1-ADCs)针对人非小细胞肺癌PDX LD1-0025-200730的体内抗肿瘤活性
6周龄的NU/NU小鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。小鼠皮下接种结直肠癌瘤块(标本编号为200730，来源于西安立迪生物技术有限公司)。当平均肿瘤体积达到150mm3时，按照2.5mg/kg和5mg/kg的给药量分别静脉注射给予实施例5制备的抗体-药物偶联物Hu517-T1000-exatecan和UC-961-vc-MMAE并设置阴性对照Vehicle组，静脉注射给与等剂量PBS。每周1次(QW)，共给药2次。给药后使用游标卡尺每周测量两次肿瘤体积，并按照如下公式计算肿瘤体积：TV＝(长*宽^2)/2。
图32显示出非小细胞肺癌小鼠模型的体内肿瘤体积-时间变化曲线图，结果显示，和阴性对照相比，Hu517-T1000-exatecan能够显著抑制肿瘤生长，而UC-961-vc-MMAE没有产生任何肿瘤生长抑制作用。
实施例15 ROR1抗体-药物偶联物(ROR1-ADCs)针对人三阴乳腺癌PDX LD1-2009-362263的体内抗肿瘤活性
6周龄的NU/NU小鼠，雌性，体重18-21g，购买于北京维通利华实验动物技术有限公司。小鼠皮下接种三阴乳腺癌瘤块(标本编号为362263，来源于西安立迪生物技术有限公司)。当平均肿瘤体积达到150mm3时，按照10mg/kg和5mg/kg的给药量分别静脉注射给予实施例5制备的抗体-药物偶联物Hu517-T1000-exatecan和UC-961-vc-MMAE并设置阴性对照Vehicle组，静脉注射给与等剂量PBS。每周1次(QW)，共给药2次。给药后使用游标卡尺每周测量两次肿瘤体积，并按照如下公式计算肿瘤体积：TV＝(长*宽^2)/2。
图33显示出三阴乳腺癌小鼠模型的体内肿瘤体积-时间变化曲线图，结果显示，和阴性对照相比，Hu517-T1000-exatecan能够显著抑制肿瘤生长，而UC-961-vc-MMAE仅产生部分的肿瘤生长抑制作用。考虑到两者耐受剂量不同，T1000-exatecan具有更高的耐受剂量，通常情况下是vc-MMAE的5倍以上，因此本发明的Hu517-T1000-exatecan具有更大的治疗窗口。
本公开描述了多个实施例，但是该描述是示例性的，而不是限制性的，并且对于本领域的普通技术人员来说显而易见的是，在本公开所描述的实施例包含的范围内可以有更多的实施例和实现方案。尽管本文示出了许多可能的特征组合，并在具体实施方式中进行了 讨论，但是所公开的特征的许多其它组合方式也是可能的。除非特意加以限制的情况以外，任何实施例的任何特征或要素可以与任何其它实施例中的任何其他特征或要素结合使用，或可以替代任何其它实施例中的任何其他特征或要素。

Claims (27)

  1. 能够特异性结合ROR1的抗体或其抗原结合片段，其包含：
    (1)(a)下述3个重链可变区(VH)互补决定区(CDR)：
    (i)VH CDR1，其具有如SEQ ID NO:37或41所示的VH中含有的CDR1的序列，或者与所述VH中含有的CDR1的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    (ii)VH CDR2，其具有如SEQ ID NO:37或41所示的VH中含有的CDR2的序列，或者与所述VH中含有的CDR2的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；和
    (iii)VH CDR3，其具有如SEQ ID NO:37或41所示的VH中含有的CDR3的序列，或者与所述VH中含有的CDR3的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)下述3个轻链可变区(VL)CDR：
    (iv)VL CDR1，其具有如SEQ ID NO:38或42所示的VL中含有的CDR1的序列，或者与所述VL中含有的CDR1的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    (v)VL CDR2，其具有如SEQ ID NO:38或42所示的VL中含有的CDR2的序列，或者与所述VL中含有的CDR2的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；和
    (vi)VL CDR3，其具有如SEQ ID NO:38或42所示的VL中含有的CDR3的序列，或者与所述VL中含有的CDR3的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    或者
    (2)(a)下述3个重链可变区(VH)互补决定区(CDR)：
    (i)VH CDR1，其具有如SEQ ID NO:39所示的VH中含有的CDR1的序列，或者与所述VH中含有的CDR1的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    (ii)VH CDR2，其具有如SEQ ID NO:39所示的VH中含有的CDR2的序列，或者与所述VH中含有的CDR2的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；和
    (iii)VH CDR3，其具有如SEQ ID NO:39所示的VH中含有的CDR3的序列，或者与所述VH中含有的CDR3的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)下述3个轻链可变区(VL)CDR：
    (iv)VL CDR1，其具有如SEQ ID NO:40所示的VL中含有的CDR1的序列，或者与所述VL中含有的CDR1的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    (v)VL CDR2，其具有如SEQ ID NO:40所示的VL中含有的CDR2的序列，或者与所述VL中含有的CDR2的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；和
    (vi)VL CDR3，其具有如SEQ ID NO:40所示的VL中含有的CDR3的序列，或者与所述VL中含有的CDR3的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述重链可变区(VH)中含有的CDR1、CDR2及CDR3，和/或所述轻链可变区(VL)中含有的CDR1、CDR2及CDR3由Kabat、Chothia或IMGT编号系统定义。
  2. 权利要求1所述的抗体或其抗原结合片段，其包含：
    (1)如SEQ ID NO:37或41所示的VH中含有的CDR1、CDR2及CDR3的序列；和/或
    如SEQ ID NO:38或42所示的VL中含有的CDR1、CDR2及CDR3序列；或者
    (2)如SEQ ID NO:39所示的VH中含有的CDR1、CDR2及CDR3的序列；和/或
    如SEQ ID NO:40所示的VL中含有的CDR1、CDR2及CDR3序列；
    优选地，所述重链可变区(VH)中含有的CDR1、CDR2及CDR3，和/或所述轻链可变区(VL)中含有的CDR1、CDR2及CDR3由Kabat、Chothia或IMGT编号系统定义。
  3. 权利要求1所述的抗体或其抗原结合片段，其包含：
    (1)(a)下述3个重链可变区(VH)CDR：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与SEQ ID NO:1相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与SEQ ID NO:2相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，或者其由下述序列组成：SEQ ID NO:45，或与SEQ ID NO:45相比具有一个或几个氨基 酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与SEQ ID NO:3相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)下述3个轻链可变区(VL)CDR：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:4，或与SEQ ID NO:4相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，或者其由下述序列组成：SEQ ID NO:46，或与SEQ ID NO:46相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:5，或与SEQ ID NO:5相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:6，或与SEQ ID NO:6相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:2所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:4所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:45所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:4所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:2所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:46所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:45所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:46所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1； 如SEQ ID NO:2所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:4所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:45所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:4所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:2所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:46所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:1所示的VH CDR1；如SEQ ID NO:45所示的VH CDR2；以及，如SEQ ID NO:3所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:46所示的VL CDR1；如SEQ ID NO:5所示的VL CDR2；以及，如SEQ ID NO:6所示的VL CDR3；
    或者
    (2)(a)下述3个重链可变区(VH)CDR：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:7，或与SEQ ID NO:7相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:8，或与SEQ ID NO:8相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:9，或与SEQ ID NO:9相比具有一个或几个氨基酸的置换、缺失或添加(例如1或2个个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)下述3个轻链可变区(VL)CDR：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:10，或与SEQ ID NO:10相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，或者其由下述序列组成：SEQ ID NO:47，或与SEQ ID NO:47相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:11，或与SEQ ID NO:11相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列， 和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:12，或与SEQ ID NO:12相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:7所示的VH CDR1；如SEQ ID NO:8所示的VH CDR2；以及，如SEQ ID NO:9所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:10所示的VL CDR1；如SEQ ID NO:11所示的VL CDR2；以及，如SEQ ID NO:12所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:7所示的VH CDR1；如SEQ ID NO:8所示的VH CDR2；以及，如SEQ ID NO:9所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:47所示的VL CDR1；如SEQ ID NO:11所示的VL CDR2；以及，如SEQ ID NO:12所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:7所示的VH CDR1；如SEQ ID NO:8所示的VH CDR2；以及，如SEQ ID NO:9所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:10所示的VL CDR1；如SEQ ID NO:11所示的VL CDR2；以及，如SEQ ID NO:12所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:7所示的VH CDR1；如SEQ ID NO:8所示的VH CDR2；以及，如SEQ ID NO:9所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:47所示的VL CDR1；如SEQ ID NO:11所示的VL CDR2；以及，如SEQ ID NO:12所示的VL CDR3；
    或者
    (3)(a)下述3个重链可变区(VH)CDR：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:13，或与SEQ ID NO:13相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:14，或与SEQ ID NO:14相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:15，或与SEQ ID NO:15相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)下述3个轻链可变区(VL)CDR：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:16，或与SEQ ID NO:16相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:17，或与SEQ ID NO:17相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:18，或与SEQ ID NO:18相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:13所示的VH CDR1；如SEQ ID NO:14所示的VH CDR2；以及，如SEQ ID NO:15所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:16所示的VL CDR1；如SEQ ID NO:17所示的VL CDR2；以及，如SEQ ID NO:18所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:13所示的VH CDR1；如SEQ ID NO:14所示的VH CDR2；以及，如SEQ ID NO:15所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:16所示的VL CDR1；如SEQ ID NO:17所示的VL CDR2；以及，如SEQ ID NO:18所示的VL CDR3；
    或者
    (4)(a)下述3个重链可变区(VH)CDR：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:19，或与SEQ ID NO:19相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:20，或与SEQ ID NO:20相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:21，或与SEQ ID NO:21相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)下述3个轻链可变区(VL)CDR：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:22，或与SEQ ID NO:22相比具有 一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:23，或与SEQ ID NO:23相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:24，或与SEQ ID NO:24相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:19所示的VH CDR1；如SEQ ID NO:20所示的VH CDR2；以及，如SEQ ID NO:21所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:22所示的VL CDR1；如SEQ ID NO:23所示的VL CDR2；以及，如SEQ ID NO:24所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:19所示的VH CDR1；如SEQ ID NO:20所示的VH CDR2；以及，如SEQ ID NO:21所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:22所示的VL CDR1；如SEQ ID NO:23所示的VL CDR2；以及，如SEQ ID NO:24所示的VL CDR3；
    或者
    (5)(a)下述3个重链可变区(VH)CDR：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:25，或与SEQ ID NO:25相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:26，或与SEQ ID NO:26相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:27，或与SEQ ID NO:27相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)下述3个轻链可变区(VL)CDR：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:28，或与SEQ ID NO:28相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:29，或与SEQ ID NO:29相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:30，或与SEQ ID NO:30相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:25所示的VH CDR1；如SEQ ID NO:26所示的VH CDR2；以及，如SEQ ID NO:27所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:28所示的VL CDR1；如SEQ ID NO:29所示的VL CDR2；以及，如SEQ ID NO:30所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:25所示的VH CDR1；如SEQ ID NO:26所示的VH CDR2；以及，如SEQ ID NO:27所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:28所示的VL CDR1；如SEQ ID NO:29所示的VL CDR2；以及，如SEQ ID NO:30所示的VL CDR3；
    或者
    (6)(a)下述3个重链可变区(VH)CDR：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:31，或与SEQ ID NO:31相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:32，或与SEQ ID NO:32相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:33，或与SEQ ID NO:33相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    和/或
    (b)下述3个轻链可变区(VL)CDR：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:34，或与SEQ ID NO:34相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:35，或与SEQ ID NO:35相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列， 和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:36，或与SEQ ID NO:36相比具有一个或几个氨基酸的置换、缺失或添加(例如1个或2个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)任一项中所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:31所示的VH CDR1；如SEQ ID NO:32所示的VH CDR2；以及，如SEQ ID NO:33所示的VH CDR3；和/或，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:34所示的VL CDR1；如SEQ ID NO:35所示的VL CDR2；以及，如SEQ ID NO:36所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO:31所示的VH CDR1；如SEQ ID NO:32所示的VH CDR2；以及，如SEQ ID NO:33所示的VH CDR3；并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO:34所示的VL CDR1；如SEQ ID NO:35所示的VL CDR2；以及，如SEQ ID NO:36所示的VL CDR3。
  4. 权利要求1至3任一项所述的抗体或其抗原结合片段，其包含：
    (1)(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO：37所示的序列；
    (ii)与SEQ ID NO：37所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO：37所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO：38所示的序列；
    (v)与SEQ ID NO：38所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO：38所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：
    重链可变区(VH)，其包含具有如SEQ ID NO:37所示的序列或与SEQ ID NO：37所 示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，和轻链可变区(VL)，其包含具有如SEQ ID NO:38所示的序列或与SEQ ID NO：38所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述抗体或其抗原结合片段包含：
    具有如SEQ ID NO:37所示的序列的VH和具有如SEQ ID NO:38所示的序列的VL；
    或者
    (2)(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO：39所示的序列；
    (ii)与SEQ ID NO：39所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO：39所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO：40所示的序列；
    (v)与SEQ ID NO：40所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO：40所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：
    重链可变区(VH)，其包含具有如SEQ ID NO:39所示的序列或与SEQ ID NO：39所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，和轻链可变区(VL)，其包含具有如SEQ ID NO:40所示的序列或与SEQ ID NO：40所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的 序列同一性的序列；
    优选地，所述抗体或其抗原结合片段包含：
    具有如SEQ ID NO:39所示的序列的VH和具有如SEQ ID NO:40所示的序列的VL；
    或者
    (3)(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO：41所示的序列；
    (ii)与SEQ ID NO：41所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO：41所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO：42所示的序列；
    (v)与SEQ ID NO：42所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个、3个、4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO：42所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：
    重链可变区(VH)，其包含具有如SEQ ID NO:41所示的序列或与SEQ ID NO：41所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，和轻链可变区(VL)，其包含具有如SEQ ID NO:42所示的序列或与SEQ ID NO：X所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述抗体或其抗原结合片段包含：
    具有如SEQ ID NO:41所示的序列的VH和具有如SEQ ID NO:42所示的序列的VL。
  5. 权利要求1至4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是人源化的；
    优选地，所述抗体或其抗原结合片段进一步包含人免疫球蛋白的框架区；
    优选地，所述抗体或其抗原结合片段进一步包含人免疫球蛋白的重链框架区(例如，人重链胚系抗体基因所编码的氨基酸序列中所包含的重链框架区)，和/或，人免疫球蛋白的轻链框架区(例如，人轻链胚系抗体基因所编码的氨基酸序列中所包含的轻链框架区)；
    优选地，所述重链框架区和/或轻链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至鼠源残基的回复突变。
  6. 权利要求1至5中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段进一步包含来源于人免疫球蛋白的恒定区；
    优选地，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区；
    优选地，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
  7. 权利要求1至6中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段选自单克隆抗体、小鼠抗体、兔抗体、人源化抗体、全人抗体、嵌合抗体(例如，人鼠嵌合抗体)、双特异性抗体、多特异性抗体、单链抗体、dAb、互补决定区片段、Fv、单链Fv(scFv)、Fd、Fab、Fab'和F(ab')2，
    优选地，所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  8. 根据权利要求1至7中任一项所述的抗体或其抗原结合片段，其以小于或等于1.0×10-8M的KD结合人或猴ROR1。
  9. 分离的核酸分子，其包含编码权利要求1-8中任一项所述的抗体或其抗原结合片段、其重链和/或轻链、或其重链可变区和/或轻链可变区的核酸序列，或者其编码权利要求1-8中任一项所述的抗体或其抗原结合片段、其重链和/或轻链、或其重链可变区和/或轻链可变区。
  10. 载体，其包含权利要求9所述的分离的核酸分子。
  11. 宿主细胞，其包含权利要求9所述的核酸分子或权利要求10所述的载体。
  12. 权利要求1-8中任一项所述的抗体或其抗原结合片段在制备抗体偶联药物中的用途，或在制备用于诊断疾病特别是ROR1介导的疾病或病症的试剂盒中的用途，或在制备用于预防和/或治疗疾病特别是ROR1介导的疾病或病症的药物中的用途。
  13. 如式(I)所示的抗体偶联药物或其药学上可接受的盐，
    其中：
    Q1-L1-L2-L3为连接子，
    Ab为权利要求1-8中任一项所述的抗体或其抗原结合片段，其通过所述的抗体或其抗原结合片段中的S原子偶联至连接子中的Q1，
    T为药物单元或诊断试剂单元，其通过药物单元或诊断试剂单元中的N原子或O原子偶联至连接子中的L3，
    m为1-9之间的数，例如m为2至9之间的数，例如约4，约5，约6，约7，约8，或约9，
    Q1选自：取代的取代的取代的和取代的
    L1为-L1a-C(＝O)-，其中，L1a选自：亚烷基、聚乙二醇基、亚烯基、亚炔基、亚脂环基、亚脂杂环基、亚芳基、亚杂芳基、取代的亚烷基、取代的聚乙二醇基、取代的亚烯基、取代的亚炔基、取代的亚脂环基、取代的亚脂杂环基、取代的亚芳基和取代的亚杂芳基，
    L2为多肽残基或取代的多肽残基，
    L3为或取代的
  14. 权利要求13所述的抗体偶联药物或其药学上可接受的盐，其中L2和/或L3可被CH2NHCH3、聚肌氨酸残基、糖基化聚乙二醇片段、取代的聚肌氨酸残基或取代的糖基化聚乙二醇片段取代。
  15. 权利要求13所述的抗体偶联药物或其药学上可接受的盐，其中所述的多肽残基包含选自以下的氨基酸：苯丙氨酸、异亮氨酸、亮氨酸、色氨酸、缬氨酸、甲硫氨酸、酪氨酸、丙氨酸、苏氨酸、组氨酸、丝氨酸、谷氨酰胺、精氨酸、赖氨酸、天冬酰胺、谷氨酸、脯氨酸、瓜氨酸、天冬氨酸和甘氨酸。
  16. 权利要求13所述的抗体偶联药物或其药学上可接受的盐，其中所述的多肽残基选自：苯丙氨酸-赖氨酸(Phe-Lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-瓜氨酸(Val- Cit)、谷氨酸-缬氨酸-丙氨酸(Glu-Val-Ala)、谷氨酸-缬氨酸-瓜氨酸(Glu-Val-Cit)、缬氨酸-赖氨酸(Val-Lys)、丙氨酸-丙氨酸-丙氨酸(Ala-Ala-Ala)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。
  17. 权利要求13所述的抗体偶联药物或其药学上可接受的盐，其中L1a为-(CH2)n1-，n1为1至10之间的数，例如1至8之间的数，1至6之间的数，1至4之间的数。
  18. 权利要求13或14所述的抗体偶联药物或其药学上可接受的盐，其中L3为被聚肌氨酸残基、糖基化聚乙二醇片段、取代的聚肌氨酸残基或取代的糖基化聚乙二醇片段取代的其中聚肌氨酸残基通过结构单元-X-连接至苯环，
    所述结构单元-X-为或取代的
    所述取代的聚肌氨酸残基为其中n2为4至18之间的数，例如4至12之间的数，R选自：C1-C6烷基、C1-C6环烷基和C1-C6烷氧基，
    所述取代的糖基化聚乙二醇片段为其中n3为4至18之间的数，例如4至12之间的数。
  19. 权利要求13所述的抗体偶联药物或其药学上可接受的盐，其中药物单元选自：
    (i)微管蛋白抑制剂，如单甲基奥瑞他汀E(MMAE)、单甲基奥瑞他汀F(MMAF)、tubulysin或其类似物(例如tubulysin A、tubulysin B、tubulysin D、tubulysin H、tubulysin U、tubulysin V)、艾瑞布林(Eribulin)或其类似物、长春碱或其类似物(例如长春新碱、长春地辛、长春花碱、DAVLBH)、Aur0101；
    (ii)喜树碱类拓扑异构酶抑制剂，如依沙替康(Exatecan，CAS 171335-80-1)、DXd(CAS 1599440-33-1)、7-乙基-10-羟基喜树碱(SN38，CAS 86639-52-3)、倍罗替康(Belotecan，CAS 213819-48-8)、(4-NH2)-Exatecan(AZD'0132，CAS 2495742-21-5)、7-MAD-MDCPT(CAS 765871-81-6)、7-Aminomethyl-10-methyl-11-fluoro camptothecin(CAS 2378616-23-8)；
    (iii)免疫激活剂，如TLR7/8激动剂、STING激动剂。
  20. 权利要求13所述的抗体偶联药物或其药学上可接受的盐，其中Q1-L1-L2-L3-T选 自：

  21. 权利要求13所述的抗体偶联药物或其药学上可接受的盐，其中所述的抗体偶联药物选自：


    其中m、Ab的定义如权利要求13所述。
  22. 制备权利要求13至21任一项所述抗体偶联药物或其药学上可接受的盐的方法，包括：
    还原Ab，得到Ab’；
    使Ab’与式L-D所示的化合物进行偶联反应，得到式(I)所示的抗体偶联药物，
    其中：Ab、Q1、L1、L2、L3、T和m的定义如权利要求13至21任一项中所述。
  23. 药物组合物，其含有权利要求13至21任一项所述的抗体偶联药物或其药学上可接受的盐，以及一种或多种药学上可接受的载体和/或赋形剂。
  24. 权利要求13至21任一项所述的抗体偶联药物或其药学上可接受的盐或权利要求23所述的药物组合物在制备用于预防和/或治疗疾病特别是ROR1介导的疾病或病症的药物中的用途，或在制备用于治疗和/或预防肿瘤的药物中的用途，或在制备用于诊断疾病特别是ROR1介导的疾病或病症的试剂盒中的用途。
  25. 权利要求24所述的用途，其中所述ROR1介导的疾病或病症为ROR1中高表达的癌症，
    优选地，所述ROR1中高表达的癌症选自头颈鳞癌、肺癌(例如非小细胞肺癌，包括肺腺癌和肺鳞癌)、尿道癌、大肠癌、前列腺癌、卵巢癌、胰腺癌、乳腺癌(例如三阴乳腺癌)、膀胱癌、胃癌、胃肠道间质瘤、宫颈癌、食道癌、鳞状细胞癌、腹膜癌、肝癌、肝细胞癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌、子宫癌、唾液腺癌、肾癌、外阴 癌、甲状腺癌、阴茎癌、黑色素瘤、白血病(例如慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL))、恶性淋巴瘤(例如套细胞淋巴瘤(MCL))、浆细胞瘤、骨髓瘤和肉瘤。
  26. 权利要求24所述的用途，其中所述的肿瘤选自头颈鳞癌、肺癌、尿道癌、大肠癌、前列腺癌、卵巢癌、胰腺癌、乳腺癌、膀胱癌、胃癌、胃肠道间质瘤、宫颈癌、食道癌、鳞状细胞癌、腹膜癌、肝癌、肝细胞癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌、子宫癌、唾液腺癌、肾癌、外阴癌、甲状腺癌、阴茎癌、黑色素瘤、白血病、恶性淋巴瘤、浆细胞瘤、骨髓瘤和肉瘤；
    优选地，所述肿瘤选自头颈鳞癌、胰腺癌、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、套细胞淋巴瘤(MCL)、卵巢癌、乳腺癌、前列腺癌、肺癌、黑色素瘤和结肠直肠癌；
    优选地，所述肿瘤选自头颈鳞癌、胰腺癌、非小细胞肺癌和三阴乳腺癌；
    优选地，所述非小细胞肺癌选自肺腺癌和肺鳞癌。
  27. 试剂盒或组合物，其包含权利要求1-8中任一项所述的抗体或其抗原结合片段，权利要求9的分离的核酸分子，权利要求10的载体，权利要求11的宿主细胞，13至21任一项所述的抗体偶联药物或其药学上可接受的盐或权利要求23所述的药物组合物。
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