WO2024051463A1 - 能够特异性结合ror1的抗体、其偶联药物及其制备方法和应用 - Google Patents

能够特异性结合ror1的抗体、其偶联药物及其制备方法和应用 Download PDF

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WO2024051463A1
WO2024051463A1 PCT/CN2023/113487 CN2023113487W WO2024051463A1 WO 2024051463 A1 WO2024051463 A1 WO 2024051463A1 CN 2023113487 W CN2023113487 W CN 2023113487W WO 2024051463 A1 WO2024051463 A1 WO 2024051463A1
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seq
chain variable
variable region
antibody
ror1
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刘青松
刘静
金锐
王文超
胡晨
齐紫平
王傲莉
王黎
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中国科学院合肥物质科学研究院
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Definitions

  • the present invention relates to the field of medicine, and more specifically, to antibodies that can specifically bind to ROR1, their conjugated drugs, and their preparation methods and applications.
  • ROR1 Receptor tyrosine kinase-like orphan receptor 1
  • ROR1 is a type I transmembrane protein expressed during embryonic development and contributes to polarized migration and organogenesis. Structurally, ROR1 contains a unique extracellular region, a transmembrane domain, and a cytoplasmic tyrosine kinase-like domain composed of an immunoglobulin-like (Ig-like) domain, a Frizzled (Fz) domain, and a kringle (Kr) domain.
  • Ig-like immunoglobulin-like
  • Frizzled Frizzled
  • Kr kringle
  • ROR1 is expressed at high levels during early embryonic development. As the fetus develops, the expression of ROR1 gradually decreases. In normal children and adult tissues, except for rare B lymphocyte precursor cells, ROR1 is lowly or even not expressed. When malignant changes occur in the tissue, ROR1 will initiate a transcription process similar to embryonic development and express high levels of ROR1.
  • ROR1 has been confirmed to be highly activated and expressed in a variety of malignant tumors, such as hematological cancers, including B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL) and Myeloid blood cancers, etc., and solid tumors, including triple negative breast cancer (TNBC), colon cancer, lung cancer, pancreatic cancer, ovarian cancer and other cancers, and are closely related to the poor prognosis of tumor patients (Blood 2016,128( 25):2931-40; Sci Rep 2014,4:5811).
  • CLL B-cell chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • NHL non-Hodgkin lymphoma
  • TNBC triple negative breast cancer
  • colon cancer lung cancer
  • pancreatic cancer pancreatic cancer
  • ovarian cancer and other cancers
  • ROR1 overexpression is involved in the occurrence of resistance to tumor chemotherapy and targeted therapy (Cells 2021,10:142), including resistance to paclitaxel breast cancer treatment (Proc Natl Acad Sci U S A 2019,116(4):1370- 7), resistance to the anti-BCL-2 inhibitor venetoclax in the treatment of chronic lymphocytic leukemia (CLL) (Leukemia 2022, 36(6):1609-18) and resistance to T-DM1 in the treatment of HER2+ breast cancer (EBioMedicine 2019, 43: 211-24) etc.
  • CLL chronic lymphocytic leukemia
  • ROR1 is highly expressed in a variety of solid and hematological malignancies, while its expression is very low in healthy tissues, ROR1 is expected to become a new drug target with broad-spectrum anti-cancer potential like PD-1.
  • ROR1 is expected to become a new drug target with broad-spectrum anti-cancer potential like PD-1.
  • PD-1 broad-spectrum anti-cancer potential
  • ROR1-targeting antibodies and antibody-drug conjugates targeting different binding epitopes. Therefore, the field still needs to develop new ROR1-targeting antibodies and antibody-drug conjugates.
  • an antibody capable of specifically binding ROR1 comprising a heavy chain variable region and a light chain variable region:
  • the heavy chain complementarity determining region of the heavy chain variable region is selected from one or more groups of the heavy chain complementarity determining regions or derivative fragments thereof shown in i) to iii) below:
  • VH-CDR1 shown in SEQ ID NO.11
  • VH-CDR2 shown in SEQ ID NO.12
  • VH-CDR3 shown in SEQ ID NO.13
  • VH-CDR1 shown in SEQ ID NO.19 VH-CDR2 shown in SEQ ID NO.20, and VH-CDR3 shown in SEQ ID NO.21;
  • the light chain complementarity determining region of the light chain variable region is selected from one or more groups of the light chain complementarity determining regions shown in i') to iii') or derivative fragments thereof:
  • VL-CDR1 shown in SEQ ID NO.4 VL-CDR2 shown in SEQ ID NO.5, and VL-CDR3 shown in SEQ ID NO.6;
  • VL-CDR1 shown in SEQ ID NO.14
  • VL-CDR2 shown in SEQ ID NO.:15
  • VL-CDR2 shown in SEQ ID NO.16 VL-CDR3
  • VL-CDR1 shown in SEQ ID NO.22 VL-CDR2 shown in SEQ ID NO.23, and VL-CDR3 shown in SEQ ID NO.24;
  • the derived fragment and its corresponding complementarity determining region have no more than 6 amino acid substitutions and can retain the EC 50 of ROR1-ECD of 0.009nM-0.05nM and the EC 50 of ROR1 of tumor cells of 0.15nM-1.1nM.
  • the heavy chain complementarity determining region of the heavy chain variable region is selected from one or more groups of the heavy chain complementarity determining regions shown in i) to iii) below:
  • VH-CDR1 is D-Y-N-Xa1-H
  • the general sequence formula of VH-CDR2 is Y-I-N-P-N-Xa2-Xa3-Xa4-T-Xa5-Y-N-Q-K-F-Xa6-G
  • the general sequence formula of VH-CDR3 is R-Xa7-Xa8-Xa9-Xa10-Xa11-Xa12-Xa13-D-Xa14; where Xa1 is I, M or L, Xa2 is N or H, Xa3 is D or G, Xa4 is A, N or G, Xa5 is S, N or T, Xa6 is Q, K or E, Xa7 is V or G, Xa8 is R or Y, Xa9 is T or G, Xa10 is S or G, Xa11 is S, T or G, Xa12 is G or Y, X
  • VH-CDR1 is shown in SEQ ID NO.11
  • VH-CDR2 is shown in SEQ ID NO.12
  • VH-CDR3 is shown in SEQ ID NO.13;
  • VH-CDR1 is as shown in SEQ ID NO.19
  • the general sequence formula of VH-CDR2 is T-I-S-D-Xb1-G-S-Y-T-Y-Y-P-D-Xb2-Xb3-K-G
  • VH-CDR3 is as shown in SEQ ID NO.21; among them, Xb1 is A or G, Xb2 is S or N, Xb3 is V or E;
  • the light chain complementarity determining region of the light chain variable region is selected from one or more groups of the light chain complementarity determining regions shown in i') to iii') or derivative fragments thereof:
  • VL-CDR1 The general sequence formula of VL-CDR1 is Xa1'-S-S-Xa2'-Xa3'-I-Xa4'-H-T-N-Xa5'-N-T-Y-L-E, and the general sequence formula of VL-CDR2 is K-V-Xa6'-N-R-F-S, VL -The general sequence formula of CDR3 is F-Q-G-S-Xa7'-Xa8'-P-Y-T; where Xa1' is R, K or T, Xa2' is H or Q, Xa3' is I, N or S, and Xa4' is V or L , Xa5' is A or G, Xa6' is F or S, Xa7' is R or L, Xa8' is F or V;
  • VL-CDR1 is shown in SEQ ID NO.14
  • VL-CDR2 is shown in SEQ ID NO.15
  • VL-CDR3 is shown in SEQ ID NO.16;
  • VL-CDR1 K-A-S-Q-S-V-S-F-Xb1'-G-T-S-L-M-H
  • VL-CDR2 is as shown in SEQ ID NO.23
  • VL-CDR3 is as shown in SEQ ID NO.24; among them, Xb1' is A or P .
  • Xa1 is L
  • Xa2 is H
  • Xa3 is D
  • Xa4 is A or G
  • Xa5 is S or T
  • Xa6 is Q
  • Xa7 is V
  • Xa8 is R
  • Xa9 is T
  • Xa10 is G
  • Xa11 is T
  • Xa12 is G
  • Xa13 is L or F
  • Xa14 is D or Y;
  • Xa1 is I or M
  • Xa2 is N
  • Xa3 is G
  • Xa4 is N or G
  • Xa5 is S
  • N or T is S
  • Xa6 is K or E
  • Xa7 is G
  • Xa8 is Y
  • Xa9 is G
  • Xa10 is S
  • Xa11 is S or G
  • Xa12 is Y
  • Xa13 is AM
  • Xa14 is F or Y.
  • Xa1' is R or K
  • Xa2' is H
  • Xa3' is N
  • Xa4' is V or L
  • Xa5' is A or G
  • Xa6' is S
  • Xa7' is R
  • Xa8' is F
  • Xa1' is R or T
  • Xa2' is H or Q
  • Xa3' is I or S
  • Xa4' is V
  • Xa5' is G
  • Xa6' is F or S
  • Xa7' is R or L
  • Xa8' is F or V.
  • the antibody has one or more of the following combinations of heavy chain complementarity determining regions and light chain complementarity determining regions:
  • Combination 1 is: the heavy chain complementarity determining region or its derivative fragment shown in i), the light chain complementarity determining region or its derivative fragment shown in i');
  • Combination 2 is: ii) the heavy chain complementarity determining region or its derivative fragment shown, ii') the light chain complementarity determining region or its derivative fragment shown;
  • Combination 3 is: iii) the heavy chain complementarity determining region or its derivative fragment shown, iii) the light chain complementation determining region or its derivative fragment.
  • the combination 1 is selected from one or more of the following combinations:
  • Combination 1-1 is: VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO.2, VH-CDR3 shown in SEQ ID NO.3, VH-CDR3 shown in SEQ ID NO.4 VL-CDR1, VL-CDR2 shown in SEQ ID NO.5 and VL-CDR3 shown in SEQ ID NO.6;
  • Combination 1-2 is: VH-CDR1 shown in SEQ ID NO.35, VH-CDR2 shown in SEQ ID NO.36, VH-CDR3 shown in SEQ ID NO.37, and VH-CDR3 shown in SEQ ID NO.38 VL-CDR1, VL-CDR2 shown in SEQ ID NO.39 and VL-CDR3 shown in SEQ ID NO.40;
  • Combinations 1-3 are: VH-CDR1 shown in SEQ ID NO.51, VH-CDR2 shown in SEQ ID NO.52, VH-CDR3 shown in SEQ ID NO.53, and VH-CDR3 shown in SEQ ID NO.54 VL-CDR1, VL-CDR2 shown in SEQ ID NO.55 and VL-CDR3 shown in SEQ ID NO.56;
  • Combinations 1-4 are: VH-CDR1 shown in SEQ ID NO.59, VH-CDR2 shown in SEQ ID NO.60, VH-CDR3 shown in SEQ ID NO.61, and VH-CDR3 shown in SEQ ID NO.62 VL-CDR1, VL-CDR2 shown in SEQ ID NO.63 and VL-CDR3 shown in SEQ ID NO.64;
  • Combinations 1-5 are: VH-CDR1 shown in SEQ ID NO.67, VH-CDR2 shown in SEQ ID NO.68, VH-CDR3 shown in SEQ ID NO.69, and VH-CDR3 shown in SEQ ID NO.70 VL-CDR1, VL-CDR2 shown in SEQ ID NO.71 and VL-CDR3 shown in SEQ ID NO.72;
  • Combinations 1-6 are: VH-CDR1 shown in SEQ ID NO.75, VH-CDR2 shown in SEQ ID NO.76, VH-CDR3 shown in SEQ ID NO.77, and VH-CDR3 shown in SEQ ID NO.78 VL-CDR1, VL-CDR2 shown in SEQ ID NO.79 and VL-CDR3 shown in SEQ ID NO.80;
  • Combinations 1-7 are: VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO.83, VH-CDR3 shown in SEQ ID NO.3, and VH-CDR3 shown in SEQ ID NO.84 VL-CDR1, VL-CDR2 shown in SEQ ID NO.5 and VL-CDR3 shown in SEQ ID NO.6.
  • the combination 3 is selected from one or more of the following combinations:
  • Combination 3-1 is: VH-CDR1 shown in SEQ ID NO.43, VH-CDR2 shown in SEQ ID NO.44, VH-CDR3 shown in SEQ ID NO.45, and VH-CDR3 shown in SEQ ID NO.46 VL-CDR1, VL-CDR2 shown in SEQ ID NO.47 and VL-CDR3 shown in SEQ ID NO.48;
  • Combination 3-2 is: VH-CDR1 shown in SEQ ID NO.19, VH-CDR2 shown in SEQ ID NO.20, VH-CDR3 shown in SEQ ID NO.21, and VH-CDR3 shown in SEQ ID NO.22 VL-CDR1, VL-CDR2 shown in SEQ ID NO.23 and VL-CDR3 shown in SEQ ID NO.24;
  • Combination 3-3 is: VH-CDR1 shown in SEQ ID NO.19, VH-CDR2 shown in SEQ ID NO.85, VH-CDR3 shown in SEQ ID NO.21, and VH-CDR3 shown in SEQ ID NO.22 VL-CDR1, VL-CDR2 shown in SEQ ID NO.23 and VL-CDR3 shown in SEQ ID NO.24.
  • the antibody has one or more of the following technical characteristics:
  • the species source of the heavy chain constant region and light chain constant region of the antibody is independently selected from human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck or goose; and,
  • the constant region of the antibody or antigen-binding fragment thereof is selected from the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and
  • the constant region of the antibody is selected from the sequence of any one of the constant regions of the kappa light chain constant region and the lambda light chain constant region.
  • the antibody has one selected from the group consisting of a heavy chain variable region and a light chain variable region as shown below:
  • a recombinant protein which includes:
  • One or more of the heavy chain complementarity determining region and the light chain complementarity determining region defined in the first aspect are selected from the group consisting of:
  • a tag fragment that assists in the expression and/or purification of the heavy chain complementarity determining region and the light chain complementarity determining region is provided.
  • the recombinant protein further includes a heavy chain constant region, a light chain constant region, or a combination thereof as defined in the first aspect.
  • a CAR construct is provided, the scFV domain of the CAR construct having the heavy chain variable region and the light chain variable region defined in the first aspect.
  • a nucleic acid is provided encoding the antibody described in the first aspect, the recombinant protein described in the second aspect, or the CAR construct described in the third aspect.
  • a vector is provided, the vector containing the nucleic acid described in the fourth aspect.
  • the vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or combinations thereof.
  • a host cell comprising the nucleic acid described in the fourth aspect or the vector described in the fifth aspect.
  • the host cell is a CHO cell, COS cell, NSO cell, HeLa cell, BHK cell or HEK293 cell.
  • a method for preparing the antibody described in the first aspect, the recombinant protein described in the second aspect, or the CAR construct described in the third aspect is provided, and the preparation method includes the following steps:
  • the host cells described in the sixth aspect are cultured, and the antibodies, recombinant proteins or CAR constructs are isolated from the resulting culture.
  • An eighth aspect of the present application provides a recombinant immune cell that expresses the exogenous CAR construct described in the third aspect.
  • the immune cells are selected from one of NK cells and T cells.
  • the ninth aspect of the present application provides the antibody described in the first aspect, the recombinant protein described in the second aspect, the CAR construct described in the third aspect, or the recombinant immune cell described in the eighth aspect. Application in the preparation of drugs for the treatment of ROR1-related diseases or diagnostic products for ROR1-related diseases.
  • the ROR1-related disease is a tumor, an autoimmune disease, a metabolism-related disease or an infectious disease.
  • the tumor is breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, esophageal cancer, kidney cancer, brain glioma, bladder cancer, prostate cancer cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, bone marrow cancer, or angiosarcoma;
  • the inflammation is rheumatoid arthritis, osteoarthritis, gout, Reiter syndrome, psoriatic arthropathy, tuberculous arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis , acute lung injury, chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis;
  • the autoimmune diseases are rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, Sjogren's syndrome or systemic vasculitis, ulcerative colitis, type I diabetes, psoriasis, and multiple sclerosis;
  • the metabolism-related disease is diabetes, food-borne obesity or fat inflammation;
  • the infectious disease is a disease caused by bacterial infection or a disease caused by viral infection.
  • the diagnostic product for ROR1-related diseases is a diagnostic reagent, test strip, test plate or kit.
  • an antibody-conjugated drug is provided, and the antibody-conjugated drug includes:
  • An antibody selected from the antibodies defined in the first aspect is selected from the antibodies defined in the first aspect.
  • the antibody-drug conjugate has one or more of the following technical features:
  • the conjugate is selected from the group consisting of detectable markers, cytotoxic drugs, cytokines, radionuclides, enzymes, or combinations thereof; and,
  • the antibody and the conjugate are coupled through chemical bonds or linkers.
  • the antibody-drug conjugate has one or more of the following technical features:
  • the antibody-conjugated drug has a structure represented by the following molecular formula:
  • D is a cytotoxic drug
  • the linker comprises a sulfhydryl-specific reactive group
  • the cytotoxic drugs are microtubule-targeting drugs, DNA-targeting drugs or topoisomerase inhibitors.
  • a pharmaceutical composition which pharmaceutical composition includes:
  • a diagnostic product for ROR1-related diseases which is characterized by:
  • the diagnostic product is a detection plate, and the detection plate is coated with the antibody or antigen-binding fragment thereof described in the first aspect, the recombinant protein described in the second aspect, and the CAR construct described in the third aspect. Or the recombinant immune cells or combination thereof described in the eighth aspect;
  • the diagnostic product is a detection kit
  • the detection kit includes a primary antibody and a secondary antibody for detecting ROR1, and the primary antibody is the antibody defined in the first aspect.
  • a detection method for ROR1 based on non-diagnostic purposes includes the following steps:
  • the sample to be tested and the antibody described in the first aspect are mixed and reacted, the reaction product is detected, and whether ROR1 is present in the sample to be tested is determined based on the detection results.
  • judging whether ROR1 is present in the sample to be tested according to the reaction results includes:
  • Figure 1 shows the discovery of anti-human ROR1 antibodies in Example 1 of the present application
  • Figure A in Figure 1 shows the enzyme-linked immunosorbent assay (ELISA) detection of a series of originally discovered anti-human ROR1 monoclonal antibodies (original hybridoma) on the culture medium The binding activity of the purified human ROR1 protein extracellular segment (ROR1-ECD);
  • Figure B in Figure 1 shows the flow cytometry fluorescence sorting (FACS) detection of the originally discovered anti-human ROR1 monoclonal antibody (original hybridoma) culture The binding activity of the supernatant to human ROR1-high expression MDA-MB-231 (ROR1-P) and ROR1-low expression MDA-MB-453 (ROR1-N) breast cancer cells;
  • Figure 1 C is 9 The numbers of the monoclonal antibodies (mAb001, mAb002, mAb003, mAb004, mAb005, mAb006, mAb007, mAb008, mAb
  • Figure 2 shows the 9 human-mouse chimeric antibodies (chimeric antibodies) mAb001c, mAb002c, mAb003c, purified in Example 3 of the present application. SDS-PAGE patterns of mAb004c, mAb005c, mAb006c, mAb007c, mAb008c and mAb009c;
  • Figure 3 is an ELISA measurement of the binding affinity (Binding affinity EC 50 ) of human-mouse chimeric antibodies mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c and mAb009c to ROR1-ECD in Example 4 of the present application;
  • Figure 4 shows the ELISA in Example 4 of the present application to detect the binding affinity (Binding affinity EC 50 ) of mAb001c and mAb005c-point mutants to ROR1-ECD;
  • Figure 5 is an analysis and comparison of the expression levels of ROR1 mRNA (compared with ⁇ -actin) in multiple tumor cell lines (lung cancer, breast cancer, colon cancer, hematological tumors, liver cancer, gastric cancer) and 30 kinds of human normal tissues in Example 5 of the present application. ratio);
  • FIG. 6 shows the analysis of Cancer Cell Line Encyclopedia (CCLE) database ROR1 mRNA in the highly invasive and highly metastatic basal-type/triple-negative (Basal-type/TNBC) versus luminal-type (Luminal-type) breasts in Example 5 of the present application. Expression levels in cancer cell lines;
  • Figure 7 shows the Western blot test (Western blot) in Example 6 of the present application to detect the expression of ROR1 protein in different lung cancer cell lines;
  • Figure 8 shows the Western blot test (Western blot) in Example 6 of the present application to detect the expression of ROR1 protein in basal/triple-negative and luminal breast cancer cell lines;
  • Figure 9 shows mAb001c (5 ⁇ g/mL) in Example 7 of the present application against ROR1-high expression (NCI-H446, HT-29, MDA-MB-231, HCC1187, HCC827, NCI-H226) or low expression (MDA- MB-453, MCF-7) binding level of ROR1 on tumor cell surface;
  • Figure 10 is the detection result of the binding affinity (Binding affinity EC 50) of the chimeric antibodies mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c and mAb009c to ROR1 on the surface of MDA-MB-231 cells in Example 8 of the present application. ; This test uses 2 ⁇ 10 5 cells mixed with the antibody in the indicated concentration gradient, and detects MFI after incubation for 1 hour;
  • Figure 11 is the detection result of the binding affinity (Binding affinity EC 50 ) of the chimeric antibodies mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c and mAb009c to NCI-H226 cell surface ROR1 in Example 8 of the present application; this The test uses 2 ⁇ 10 5 cells mixed with antibodies in the indicated concentration gradient, and then incubates for 1 hour to detect MFI;
  • Figure 12 is the FACS detection of the binding affinity (Binding affinity EC 50 ) of mAb001c and mAb005c-point mutants to MDA-MB-231 in Example 8 of the present application;
  • Figure 13 shows the binding affinity (Binding affinity EC 50 ) detection results of the chimeric antibodies mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, mAb009c in Example 9 of the present application for ROR1 transfected on the surface of CHO cells;
  • 2 ⁇ 10 5 cells were mixed with the human-mouse chimeric antibody in the indicated concentration gradient, and MFI was detected after incubation for 1 hour;
  • Figure 14 shows the binding affinity (Binding) of the chimeric antibodies mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, mAb009c in Example 9 of the present application to ROR1 (removed Ig-like domain) transfected on the surface of CHO cells affinity EC 50 ) detection results; this test uses 2 ⁇ 10 5 cells mixed with the antibody in the indicated concentration gradient, and detects MFI after incubation for 1 hour;
  • Figure 15 shows the binding affinity of the chimeric antibodies mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, mAb009c in Example 9 of the present application to ROR1 (Frizzled domain removed) transfected on the surface of CHO cells EC 50 ) detection results; this test uses 2 ⁇ 10 5 cells mixed with the antibody in the indicated concentration gradient, and detects MFI after incubation for 1 hour;
  • Figure 16 shows the binding affinity of the chimeric antibodies mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, mAb009c in Example 9 of the present application to ROR1 (Kringle domain removed) transfected on the surface of CHO cells EC 50 ) detection results; this test uses 2 ⁇ 10 5 cells mixed with the antibody in the indicated concentration gradient, and detects MFI after incubation for 1 hour;
  • Figure 17 is an in vivo anti-tumor activity test of the ROR1 chimeric antibody in Example 10 of the present application.
  • ROR1-high-expressing NCI-H226 lung cancer cells were mixed with 50 ⁇ g of antibody and then inoculated subcutaneously on the back of nude mice. They were observed 2-3 times a week to measure tumor volume and mouse weight;
  • Figure 18 shows the binding of mAb001c, mAb002c, mAb004c, and mAb005c to MDA-MB-231 cells in Example 11 of the present application, resulting in internalization to intracellular lysosomes; the antibodies (5 ⁇ g/mL) were incubated with the cells for 4 °C for 1 hour, or 37°C for 4 hours, then place it in a laser confocal microscope to observe the results;
  • Figure 19 is a hydrophobic interaction chromatography (HIC)-HPLC spectrum of the antibody mAb001c and the antibody drug conjugate mAb001c-vcMMAE in Example 12 of the present application;
  • HIC hydrophobic interaction chromatography
  • Figure 20 is a size exclusion (SEC)-HPLC spectrum of the antibody mAb001c and the antibody drug conjugate mAb001c-vcMMAE in Example 12 of the present application;
  • Figure 21 shows the effects of mAb001c-vcMMAE in Example 13 of the present application on ROR1 high-expression cell lines (MDA-MB-231, HCC1187, HT-29, NCI-H446, HCC827, NCI-N87) and ROR1 low-expression cell lines (MCF -7) Test results of in vitro anti-proliferative activity (IC 50 );
  • Figure 22 shows the in vivo anti-tumor efficacy of mAb001c-vcMMAE (5 mg/kg) in the MDA-MB-231 breast cancer tumor model in Example 14 of the present application;
  • Figure 23 shows the in vivo anti-tumor efficacy of mAb001c-vcMMAE (5 mg/kg, 2.5 mg/kg) in the HT-29 colon cancer tumor model in Example 14 of the present application;
  • Figure 24 shows the binding affinity (Binding EC 50) of the humanized antibody series Hu001-2, 3, 5, 6, 8, 11, 12, 14, and 15 of mAb001c to ROR1-ECD using ELISA in Example 17 of the present application. );
  • Figure 25 shows the binding affinity (Binding affinity EC 50 ) of the humanized antibody series Hu005-35, 40, 41, 42, 43, 44, 45, and 46 of mAb005c to ROR1-ECD using ELISA in Example 17 of the present application;
  • Figure 26 is the ELISA in Example 18 of the present application to detect the binding affinity (Binding affinity EC 50 ) of the humanized antibody series Hu001-2 and Hu005-46 to human ROR1-ECD and human ROR2-ECD;
  • Figure 27 shows the FACS detection of the humanized antibody series Hu001-2, 3, 5, 6, 8, 11, 12, 14, 15 of mAb001c and the humanized antibody series Hu005-35, mAb005c in Example 19 of the present application.
  • Figure 28 shows the FACS detection of the humanized antibody series Hu001-2, 3, 5, 6, 8, 11, 12, 14, 15 of mAb001c and the humanized antibody series Hu005-35, mAb005c in Example 19 of the present application.
  • Figure 29 shows that the humanized antibodies Hu001-02, Hu001-03, Hu005-44, and Hu005-46 in Example 20 of the present application bind to MDA-MB-231 cells, resulting in internalization (Internalization) into intracellular lysosomes;
  • the antibody (5 ⁇ g/mL) was incubated with the cells at 4°C for 1 hour, or at 37°C for 4 hours and then placed under a laser confocal microscope to observe the results;
  • Figure 30 shows the endocytosis of humanized antibodies Hu001-02, Hu001-03, Hu005-44, and Hu005-46 in Example 21 of the present application when they bind to MDA-MB-231 cells at 0, 30 min, 60 min, 120 min, and 240 min. curve;
  • Figure 31 is the endocytosis curve when the humanized antibodies Hu001-02 and Hu005-46 in Example 21 of the present application bind to NCI-N87 cells at 0, 30min, 60min, 120min, and 240min;
  • Figure 32 is the endocytosis curve when the humanized antibodies Hu001-02 and Hu005-46 in Example 21 of the present application bind to NCI-H446 cells at 0, 30min, 60min, 120min, and 240min;
  • Figure 33 is a hydrophobic interaction chromatography (HIC)-HPLC spectrum of the humanized antibody Hu001-2 and the antibody drug conjugate Hu001-2-MMAE in Example 22 of the present application;
  • HIC hydrophobic interaction chromatography
  • Figure 34 is a size exclusion (SEC)-HPLC spectrum of the humanized antibody Hu001-2 and the antibody drug conjugate Hu001-2-MMAE in Example 22 of the present application;
  • Figure 35 is a hydrophobic interaction chromatography (HIC)-HPLC spectrum of the humanized antibody Hu001-3 and the antibody drug conjugate Hu001-3-MMAE in Example 22 of the present application;
  • HIC hydrophobic interaction chromatography
  • Figure 36 is a size exclusion (SEC)-HPLC spectrum of the humanized antibody Hu001-3 and the antibody drug conjugate Hu001-3-MMAE in Example 22 of the present application;
  • Figure 37 is a hydrophobic interaction chromatography (HIC)-HPLC spectrum of the humanized antibody Hu005-44 and the antibody drug conjugate Hu005-44-MMAE in Example 22 of the present application;
  • HIC hydrophobic interaction chromatography
  • Figure 38 is a size exclusion (SEC)-HPLC spectrum of the humanized antibody Hu005-44 and the antibody drug conjugate Hu005-44-MMAE in Example 22 of the present application;
  • Figure 39 is a hydrophobic interaction chromatography (HIC)-HPLC spectrum of the humanized antibody Hu005-46 and the antibody drug conjugate Hu005-46-MMAE in Example 22 of the present application;
  • HIC hydrophobic interaction chromatography
  • Figure 40 is a size exclusion (SEC)-HPLC spectrum of the humanized antibody Hu005-46 and the antibody drug conjugate Hu005-46-MMAE in Example 22 of the present application;
  • Figure 41 is a hydrophobic interaction chromatography (HIC)-HPLC spectrum of the positive reference antibody UC961 and the antibody drug conjugate UC961-MMAE in Example 22 of the present application;
  • HIC hydrophobic interaction chromatography
  • Figure 42 is a size exclusion (SEC)-HPLC spectrum of the positive reference antibody UC961 and the antibody drug conjugate UC961-MMAE in Example 22 of the present application;
  • Figure 43 shows the ELISA detection of humanized antibodies Hu001-2, Hu001-3, Hu005-44, Hu005-46 and antibody conjugates Hu001-2-MMAE, Hu001-3-MMAE, Hu005- in Example 23 of the present application.
  • the binding affinity of 44-MMAE and Hu005-46-MMAE to ROR1-ECD (Binding affinity EC 50 );
  • Figure 44 shows the FACS detection of humanized antibodies Hu001-2, Hu001-3, Hu005-44, Hu005-46 and antibody conjugates Hu001-2-MMAE, Hu001-3-MMAE, Hu005- in Example 24 of the present application.
  • the binding affinity of 44-MMAE and Hu005-46-MMAE to MDA-MB-231 Binding affinity EC 50 );
  • Figure 45 shows the FACS detection of humanized antibodies Hu001-2, Hu001-3, Hu005-44, Hu005-46 and antibody conjugates Hu001-2-MMAE, Hu001-3-MMAE, Hu005- in Example 24 of the present application.
  • the binding affinity of 44-MMAE and Hu005-46-MMAE to NCI-H226 (Binding affinity EC 50 );
  • Figure 46 shows Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, in Example 25 of the present application.
  • Figure 47 shows the in vitro anti-proliferative activity (IC 50 ) test results
  • Figure 48 shows the in vitro anti-proliferative activity of Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, Hu005-46-MMAE, and UC961-MMAE on MDA-MB-231 cells in Example 25 of the present application ( IC 50 ) test results;
  • Figure 49 shows the in vitro anti-proliferative activity (IC 50 ) of Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, Hu005-46-MMAE, and UC961-MMAE on HCC1187 cells in Example 25 of the present application. Test results;
  • Figure 50 shows the in vitro anti-proliferative activity (IC 50 ) test results
  • Figure 51 shows the in vitro anti-proliferative activity of Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, Hu005-46-MMAE, and UC961-MMAE on SK-CO-1 cells in Example 25 of the present application ( IC 50 ) test results;
  • Figure 52 shows the in vitro anti-proliferative activity (IC 50 ) test results
  • Figure 53 is the in vitro anti-proliferative activity (IC 50 ) of Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, Hu005-46-MMAE, and UC961-MMAE in Example 25 of the present application on HCC827 cells. Test results;
  • Figure 54 shows the in vitro anti-proliferative activity (IC 50 ) test results
  • Figure 55 shows the in vitro anti-proliferative activity (IC 50 ) test results
  • Figure 56 shows the in vitro anti-proliferative activity (IC 50 ) test results
  • Figure 57 shows the cytotoxicity (IC 50 value) of Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, Hu005-46-MMAE, and UC961-MMAE in Example 25 of the present application and the cytotoxicity of test cells ROR1 expression levels are directly related to target-specific cytotoxicity;
  • Figure 58 shows the concentration of Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, and Hu005-46-MMAE (5 mg/kg, 2.5 mg/kg) in Example 26 of the present application in MDA-MB-231 In vivo antitumor efficacy in negative breast cancer tumor models;
  • Figure 59 shows the in vivo anti-tumor efficacy of Hu001-2-MMAE, Hu005-46-MMAE, and UC961-MMAE (5 mg/kg, 2.5 mg/kg) in the PA-1 ovarian cancer tumor model in Example 26 of the present application. ;
  • Figure 60 shows the effects of Hu001-2-MMAE, Hu005-44-MMAE, Hu005-46-MMAE, and UC961-MMAE (5 mg/kg, 2.5 mg/kg) in the NCI-N87 gastric cancer tumor model in Example 26 of the present application. Anti-tumor efficacy in vivo;
  • Figure 61 shows the in vivo anti-tumor efficacy of Hu001-2-MMAE and UC961-MMAE (5 mg/kg, 2.5 mg/kg) in the HCC1187 triple-negative breast cancer tumor model in Example 26 of the present application.
  • the technical method of "A, and/or, B, and/or, C, and/or, D" Solution including any one of A, B, C, and D (that is, technical solutions all connected by "logical OR"), including any and all combinations of A, B, C, and D, that is, including A , the combination of any two or any three of B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, a technical solution that is all connected by "logical AND").
  • first”, “second”, “third” and “fourth” etc. are for descriptive purposes only and shall not be understood as indicating or implying relative importance or quantity, nor shall they be understood as implicitly indicating the importance or quantity of indicated technical features.
  • first”, “second”, “third”, “fourth”, etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation of quantity.
  • the technical features described in open format include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
  • the temperature parameters in this application are allowed to be treated at a constant temperature, or to vary within a certain temperature range. It should be understood that the thermostatic treatment described allows the temperature to fluctuate within the accuracy of the instrument control. It is allowed to fluctuate within the range of ⁇ 5°C, ⁇ 4°C, ⁇ 3°C, ⁇ 2°C and ⁇ 1°C.
  • % (w/w) and wt% both represent weight percentage
  • % (v/v) refers to volume percentage
  • % (w/v) refers to mass volume percentage
  • Fragments of the DNA molecules of the antibodies or antigen-binding fragments thereof of the present application can be obtained using conventional techniques, such as PCR amplification or genome library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form single-chain antibodies.
  • recombinant methods can be used to obtain relevant sequence fragments in large quantities. This is usually done by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence fragments from the propagated host cells through conventional methods.
  • artificial synthesis methods can also be used to synthesize relevant sequence fragments, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the DNA encoding the antibody (or fragment thereof, or derivative thereof) of the present application can be obtained completely through chemical synthesis. sequence.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present application through chemical synthesis.
  • the present application also relates to vectors comprising appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present application. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art.
  • the antibody of the present application can be purified by conventional separation and purification means that are well known to persons.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the antibodies of the present application can be expressed within cells, on cell membranes, or secreted outside cells.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, infiltration sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • ADCs Antibody drug conjugates
  • the antibody-conjugated drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and preferably is chemically coupled.
  • the effector molecule is preferably a drug with therapeutic activity.
  • the effector molecule may be one or more of toxic proteins, chemotherapeutic drugs, small molecule drugs or radionuclides.
  • the antibody and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde, etc.
  • the coupling agent utilizing carboxyl groups may be any one or more of aconitic anhydride coupling agents (such as aconitic anhydride) and acyl hydrazone coupling agents (the coupling site is an acyl hydrazone).
  • antibodies are used to connect to a variety of functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (e.g. glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorescent groups
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers e.g. glycol polymers
  • therapeutic agents e.g. glycol polymers
  • Antibodies can be coupled to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be linked to the antibody directly or indirectly through a linker.
  • Typical coupling methods suitable for this application include K-Lock and C-Lock coupling methods.
  • K-Lock coupling method the drug molecule is coupled to the lysine (K) residue in the antibody sequence.
  • C-Lock coupling method the drug molecule is coupled to the cysteine (C) residue in the antibody sequence.
  • Residues the drug molecule is coupled to the cysteine (C) residue in the antibody sequence.
  • Antibodies can be conjugated with drugs to form antibody drug conjugates (ADCs).
  • ADCs contain a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers.
  • Degradable linkers are typically susceptible to degradation in the intracellular environment, such as at the target site, allowing the drug to be released from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases, such as lysosomal or endosomal proteases, or sugar linkers, such as those that can be degraded by glucuronides. Enzymatic degradation of glucuronide-containing linkers.
  • Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH-sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Nondegradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.
  • the linker Before being connected to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and the connection is achieved through the active reactive group.
  • Thiol-specific reactive groups are preferred and include, for example, maleimides, halogenated amides (e.g., iodine, bromo, or chlorinated); halogenated esters (e.g., iodine, bromo, or chlorinated). ); Halogenated methyl ketones (e.g. iodine, bromo or chlorinated), benzyl halides (e.g.
  • Linkers may include, for example, maleimides linked to the antibody via thiosuccinimide.
  • the drug can be any cytotoxic, cytostatic, or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has reactive groups before being attached to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Catharanthus roseus alkaloids, etc.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins.
  • PBDs pyrrolo[1,4]benzodiazepines
  • indolinobenzodiazepines indolinobenzodiazepines
  • oxazolidinobenzodiazepines vinca alkaloids
  • 7-ethyl-10-hydroxycamptothecin SN38
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive part of the linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug, forming an ADC.
  • linker typically, functional groups on the linker are selected to facilitate specific reaction with appropriate reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety.
  • the drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides), isocyanates and isothiocyanates (suitable for reaction with amines) and activated esters, such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols).
  • ketones and aldehydes suitable for reaction with hydrazides and alkoxyamines
  • phosphines suitable for reaction with azides
  • isocyanates and isothiocyanates suitable for reaction with amines
  • activated esters such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols).
  • drug refers broadly to any compound that has a desired biological activity and possesses reactive functional groups for the preparation of conjugates described herein. Desired biological activities include diagnosis, cure, mitigation, treatment, and prevention of disease in humans or other animals.
  • drug refers to compounds that include drugs recognized in the Official National Pharmacopoeia, as well as, for example, the Official Allopathic Pharmacopoeia of the United States, the Official National Formulary, or any supplement thereof, so long as they have the requisite reactive functional groups. Typical drugs are listed in the Physician's Desk Drug Reference (PDR) and the U.S. Food and Drug Administration's (FDA) Orange Book. It should be understood that as new drugs are continuously discovered and developed, these drugs should also be included in the "drugs" of the conjugate drugs described in this application.
  • PDR Physician's Desk Drug Reference
  • FDA U.S. Food and Drug Administration's
  • the present application provides an antibody capable of specifically binding to ROR1, the antibody comprising a heavy chain variable region and a light chain variable region:
  • the heavy chain complementarity determining region of the heavy chain variable region is selected from one or more groups of the heavy chain complementarity determining regions or derivative fragments thereof shown in i) to iii) below:
  • VH-CDR1 shown in SEQ ID NO.11
  • VH-CDR2 shown in SEQ ID NO.12
  • VH-CDR3 shown in SEQ ID NO.13
  • VH-CDR1 shown in SEQ ID NO.19 VH-CDR2 shown in SEQ ID NO.20, and VH-CDR3 shown in SEQ ID NO.21;
  • the light chain complementarity determining region of the light chain variable region is selected from one or more groups of the light chain complementarity determining regions shown in i') to iii') or derivative fragments thereof:
  • VL-CDR1 shown in SEQ ID NO.4 VL-CDR2 shown in SEQ ID NO.5, and VL-CDR3 shown in SEQ ID NO.6;
  • VL-CDR1 shown in SEQ ID NO.14 VL-CDR2 shown in SEQ ID NO.:15
  • VL-CDR3 shown in SEQ ID NO.16;
  • VL-CDR1 shown in SEQ ID NO.22 VL-CDR2 shown in SEQ ID NO.23, and VL-CDR3 shown in SEQ ID NO.24;
  • the derived fragment and its corresponding complementarity determining region have no more than 6 amino acid substitutions and can retain the EC 50 of ROR1-ECD of 0.009nM-0.05nM and the EC 50 of ROR1 of tumor cells of 0.15nM-1.1nM.
  • the derived fragment is formed by amino acid substitution of no more than 6 positions relative to its corresponding complementarity-determining region, and retains the same biological activity as its corresponding complementation-determining region.
  • the derivative fragment may be substituted at 1, 2, 3, 4, 5 or 6 positions of its corresponding complementarity determining region, which may be one amino acid replaced by another amino acid, or one amino acid may be replaced by multiple ( For example 2) amino acid substitutions.
  • derivative fragments refer to those having 1, 2, or 3 amino acids compared with the amino acid sequence of the antibody of this application. Acids are replaced by amino acids with similar or similar properties to form polypeptides. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table-1.
  • the affinity EC 50 of the derived fragment to ROR1 (such as human ROR1 protein extracellular domain, ROR1-ECD) is 0.02nM-0.05nM, optionally 0.02-0.046nM, optionally 0.02nM-0.028nM.
  • the EC 50 of the affinity of the derived fragment to ROR1 on the surface of tumor cells is 0.06nM-0.08nM, optionally 0.06nM-0.073nM, optionally 0.06nM-0.067nM.
  • Antibody as used herein, the term “antibody” or “immunoglobulin” is a heterotetrameric protein of about 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical Composed of heavy chain (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains.
  • VH variable domain
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. .
  • Special amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence and contribute to the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the variable regions of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of natural heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial ⁇ -sheet structure can be formed.
  • the CDRs in each chain are held closely together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)). Constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant region. There are 5 main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • variable regions which are separated into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a cyclic structure, and the ⁇ -sheets formed by the FRs between them are close to each other in spatial structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. You can determine which amino acids constitute the FR or CDR region by comparing the amino acid sequences of antibodies of the same type.
  • This application includes not only complete antibodies, but also fragments of antibodies with immunological activity or recombinant proteins formed by antibodies and other sequences. Therefore, the present application also includes fragments, derivatives and analogs of said antibodies.
  • antibodies include those of animal origin (e.g., mouse, rabbit, sheep, cow, horse, etc.) prepared using techniques well known to those skilled in the art. pig, dog, cat, camel, donkey, deer, mink, chicken, duck or goose), chimeric, humanized or fully human antibodies.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard recombinant DNA techniques and are useful antibodies.
  • a chimeric antibody is a molecule in which the different parts are derived from different animal species, such as a chimeric antibody having a variable region from a mouse monoclonal antibody, and a constant region from a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent 4,816,397, which is incorporated herein by reference in its entirety).
  • Humanized antibodies refer to antibody molecules derived from non-human species, having one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see U.S. Patent 5,585,089, This article is incorporated by reference in its entirety).
  • CDRs complementarity determining regions
  • These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • the antibodies in this application may be chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies targeting human ROR1.
  • the antibody may be monospecific, bispecific, trispecific, or more multiple specificities, and at least one of its antigen-binding fragments includes the heavy chain complementarity determining region and the light chain complementarity defined in this application. decision zone.
  • the heavy chain complementarity determining region of the heavy chain variable region is selected from one or more groups of the heavy chain complementarity determining regions shown in i) to iii) below:
  • VH-CDR1 is D-Y-N-Xa1-H
  • the general sequence formula of VH-CDR2 is Y-I-N-P-N-Xa2-Xa3-Xa4-T-Xa5-Y-N-Q-K-F-Xa6-G
  • the general sequence formula of VH-CDR3 is R-Xa7-Xa8-Xa9-Xa10-Xa11-Xa12-Xa13-D-Xa14; where Xa1 is I, M or L, Xa2 is N or H, Xa3 is D or G, Xa4 is A, N or G, Xa5 is S, N or T, Xa6 is Q, K or E, Xa7 is V or G, Xa8 is R or Y, Xa9 is T or G, Xa10 is S or G, Xa11 is S, T or G, Xa12 is G or Y, X
  • VH-CDR1 is shown in SEQ ID NO.11
  • VH-CDR2 is shown in SEQ ID NO.12
  • VH-CDR3 is shown in SEQ ID NO.13;
  • VH-CDR1 is as shown in SEQ ID NO.19
  • the general sequence formula of VH-CDR2 is T-I-S-D-Xb1-G-S-Y-T-Y-Y-P-D-Xb2-Xb3-K-G
  • VH-CDR3 is as shown in SEQ ID NO.21; among them, Xb1 is A or G, Xb2 is S or N, Xb3 is V or E;
  • the light chain complementarity determining region of the light chain variable region is selected from one or more groups of the light chain complementarity determining regions shown in i') to iii') or derivative fragments thereof:
  • VL-CDR1 The general sequence formula of VL-CDR1 is Xa1'-S-S-Xa2'-Xa3'-I-Xa4'-H-T-N-Xa5'-N-T-Y-L-E, and the general sequence formula of VL-CDR2 is K-V-Xa6'-N-R-F-S, VL -The general sequence formula of CDR3 is F-Q-G-S-Xa7'-Xa8'-P-Y-T; where Xa1' is R, K or T, Xa2' is H or Q, Xa3' is I, N or S, and Xa4' is V or L , Xa5' is A or G, Xa6' is F or S, Xa7' is R or L, Xa8' is F or V;
  • VL-CDR1 is shown in SEQ ID NO.14
  • VL-CDR2 is shown in SEQ ID NO.15
  • VL-CDR3 is shown in SEQ ID NO.16;
  • VL-CDR1 K-A-S-Q-S-V-S-F-Xb1'-G-T-S-L-M-H
  • VL-CDR2 is as shown in SEQ ID NO.23
  • VL-CDR3 is as shown in SEQ ID NO.24; among them, Xb1' is A or P .
  • Xa1 is L
  • Xa2 is H
  • Xa3 is D
  • Xa4 is A or G
  • Xa5 is S or T
  • Xa6 is Q
  • Xa7 is V
  • Xa8 is R
  • Xa9 is T
  • Xa10 is G
  • Xa11 is T
  • Xa14 is D or Y;
  • Xa1 is I or M
  • Xa2 is N
  • Xa3 is G
  • Xa4 is N or G
  • Xa5 is S
  • N or T is S
  • Xa6 is K or E
  • Xa7 is G
  • Xa8 is Y
  • Xa9 is G
  • Xa10 is S
  • Xa11 is S or G
  • Xa12 is Y
  • Xa13 is AM
  • Xa14 is F or Y.
  • Xa1’ is R or K
  • Xa2’ is H
  • Xa3’ is N
  • Xa4’ is V or L
  • Xa5’ is A or G
  • Xa6’ is S
  • Xa7’ is R
  • Xa8’ is F;
  • Xa1' is R or T
  • Xa2' is H or Q
  • Xa3' is I or S
  • Xa4' is V
  • Xa5' is G
  • Xa6' is F or S
  • Xa7' is R or L
  • Xa8' is F or V.
  • the antibody has one or more of the following combinations of heavy chain complementarity determining regions and light chain complementarity determining regions:
  • Combination 1 is: the heavy chain complementarity determining region or its derivative fragment shown in i), the light chain complementarity determining region or its derivative fragment shown in i');
  • Combination 2 is: ii) the heavy chain complementarity determining region or its derivative fragment shown, ii') the light chain complementarity determining region or its derivative fragment shown;
  • Combination 3 is: iii) the heavy chain complementarity determining region or its derivative fragment shown, iii) the light chain complementation determining region or its derivative fragment.
  • the antibody has one or more of the following technical features:
  • the combination 1 is selected from one or more of the following combinations:
  • Combination 1-1 is: VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO.2, VH-CDR3 shown in SEQ ID NO.3, VH-CDR3 shown in SEQ ID NO.4 VL-CDR1, VL-CDR2 shown in SEQ ID NO.5 and VL-CDR3 shown in SEQ ID NO.6;
  • Combinations 1-2 are: VH-CDR1 shown in SEQ ID NO.35, VH-CDR2 shown in SEQ ID NO.36, VH-CDR3 shown in SEQ ID NO.37, and VH-CDR3 shown in SEQ ID NO.38 VL-CDR1, VL-CDR2 shown in SEQ ID NO.39 and VL-CDR3 shown in SEQ ID NO.40;
  • Combinations 1-3 are: VH-CDR1 shown in SEQ ID NO.51, VH-CDR2 shown in SEQ ID NO.52, VH-CDR3 shown in SEQ ID NO.53, and VH-CDR3 shown in SEQ ID NO.54 VL-CDR1, VL-CDR2 shown in SEQ ID NO.55 and VL-CDR3 shown in SEQ ID NO.56;
  • Combinations 1-4 are: VH-CDR1 shown in SEQ ID NO.59, VH-CDR2 shown in SEQ ID NO.60, VH-CDR3 shown in SEQ ID NO.61, and VH-CDR3 shown in SEQ ID NO.62 VL-CDR1, VL-CDR2 shown in SEQ ID NO.63 and VL-CDR3 shown in SEQ ID NO.64;
  • Combinations 1-5 are: VH-CDR1 shown in SEQ ID NO.67, VH-CDR2 shown in SEQ ID NO.68, VH-CDR3 shown in SEQ ID NO.69, and VH-CDR3 shown in SEQ ID NO.70 VL-CDR1, VL-CDR2 shown in SEQ ID NO.71 and VL-CDR3 shown in SEQ ID NO.72;
  • Combinations 1-6 are: VH-CDR1 shown in SEQ ID NO.75, VH-CDR2 shown in SEQ ID NO.76, VH-CDR3 shown in SEQ ID NO.77, and VH-CDR3 shown in SEQ ID NO.78 VL-CDR1, VL-CDR2 shown in SEQ ID NO.79 and VL-CDR3 shown in SEQ ID NO.80;
  • Combinations 1-7 are: VH-CDR1 shown in SEQ ID NO.1, VH-CDR2 shown in SEQ ID NO.83, VH-CDR3 shown in SEQ ID NO.3, and VH-CDR3 shown in SEQ ID NO.84 VL-CDR1, VL-CDR2 shown in SEQ ID NO.5 and VL-CDR3 shown in SEQ ID NO.6;
  • the combination 3 is selected from one or more of the following combinations:
  • Combination 3-1 is: VH-CDR1 shown in SEQ ID NO.43, VH-CDR2 shown in SEQ ID NO.44, VH-CDR3 shown in SEQ ID NO.45, and VH-CDR3 shown in SEQ ID NO.46 VL-CDR1, VL-CDR2 shown in SEQ ID NO.47 and VL-CDR3 shown in SEQ ID NO.48;
  • Combination 3-2 is: VH-CDR1 shown in SEQ ID NO.19, VH-CDR2 shown in SEQ ID NO.20, VH-CDR3 shown in SEQ ID NO.21, and VH-CDR3 shown in SEQ ID NO.22 VL-CDR1, VL-CDR2 shown in SEQ ID NO.23 and VL-CDR3 shown in SEQ ID NO.24;
  • Combination 3-3 is: VH-CDR1 shown in SEQ ID NO.19, VH-CDR2 shown in SEQ ID NO.85, VH-CDR3 shown in SEQ ID NO.21, and VH-CDR3 shown in SEQ ID NO.22 VL-CDR1, VL-CDR2 shown in SEQ ID NO.23 and VL-CDR3 shown in SEQ ID NO.24.
  • the antibody has one or more of the following technical features:
  • the species source of the heavy chain constant region and light chain constant region of the antibody or antigen-binding fragment thereof is independently selected from the group consisting of human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, and donkey. , deer, mink, chicken, duck or goose; and,
  • the constant region of the antibody is selected from the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.
  • the constant region of the antibody is selected from the sequence of any one of the constant regions of the kappa light chain constant region and the lambda light chain constant region.
  • the antibody has one or more groups selected from the following combinations of heavy chain variable regions and light chain variable regions:
  • the antibodies are original murine antibodies mAb001, mAb002, mAb003, mAb004, mAb005, mAb006, mAb007, mAb008, and mAb009.
  • the antibody is a human-mouse chimeric antibody mAb001c, mAb001c_v1, mAb002c, mAb003c, mAb004c, mAb005c, mAb005c_v1, mAb006c, mAb007c, mAb008c, mAb009c.
  • the antibody is humanized antibody Hu001-2, Hu001-3, Hu001-5, Hu001-6, Hu001-8, Hu001-11, Hu001-12, Hu001-14, Hu001-14 , Hu001-15, Hu005-35, Hu005-40, Hu005-41, Hu005-42, Hu005-43, Hu005-44, Hu005-45, Hu005-46.
  • the antibody provided in the first aspect of the application has one or more properties selected from the group consisting of:
  • This application provides three major categories of highly specific and high-affinity antibodies targeting ROR1, which can be used in combination to construct CAR constructs, recombinant immune cells containing CAR constructs, antibody-conjugated drugs, etc., and can also be used for (a) preparing detection reagents, detection plates or kits; and/or (b) preparing drugs for preventing and/or treating ROR1-related diseases.
  • This application provides a recombinant protein, which includes:
  • One or more of the heavy chain complementarity determining region and the light chain complementarity determining region defined in the first aspect are selected from the group consisting of:
  • a tag fragment that assists in the expression and/or purification of the heavy chain complementarity determining region and the light chain complementarity determining region is provided.
  • the recombinant protein further includes a heavy chain constant region, a light chain constant region, or a combination thereof as defined in the first aspect.
  • the tag sequence includes but is not limited to 6His tag.
  • the recombinant protein includes but is not limited to fusion protein.
  • the recombinant protein may be a monomer, dimer, or multimer.
  • the present application provides a CAR construct whose scFV domain has the heavy chain variable region and the light chain variable region defined in the first aspect.
  • scFV domain is the specific binding region of ROR1.
  • the present application provides a nucleic acid encoding the antibody or antigen-binding fragment thereof described in the first aspect, the recombinant protein described in the second aspect, or the CAR construct described in the third aspect.
  • the present application provides a vector, which contains the nucleic acid described in the fourth aspect.
  • the vector includes but is not limited to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or combinations thereof.
  • the present application provides a host cell comprising the nucleic acid described in the fourth aspect or the vector described in the fifth aspect.
  • the nucleic acid can be integrated into the genome of the host cell.
  • the host cells include but are not limited to CHO cells, COS cells, NSO cells, HeLa cells, BHK cells and HEK293 cells.
  • the present application provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect, the recombinant protein described in the second aspect, or the CAR construct described in the third aspect.
  • the preparation method includes the following steps:
  • the host cells described in the sixth aspect are cultured, and the antibodies or antigen-binding fragments thereof, recombinant proteins or CAR constructs are isolated from the resulting culture.
  • culture is performed under appropriate culture conditions, including but not limited to: appropriate culture medium, appropriate temperature, appropriate time, etc.
  • the antibody of the present application may be a chimeric antibody or a humanized antibody.
  • the preparation method of the chimeric antibody includes the steps:
  • the chimeric antibody is expressed by transfecting animal cells.
  • the preparation method of humanized antibodies includes the steps:
  • the nucleic acid described in the fourth aspect of the present application is implanted into a template containing the human antibody FR region, and then cloned into an expression vector containing the human antibody constant region, and then the humanized antibody is expressed by transfecting animal cells.
  • the present application provides a recombinant immune cell that expresses the exogenous CAR construct described in the third aspect.
  • the immune cells may be selected from the group consisting of, but not limited to: NK cells and T cells.
  • the source of the immune cells can be humans or other mammals (such as mice).
  • the present application provides the antibody or antigen-binding fragment thereof described in the first aspect, the recombinant protein described in the second aspect, the CAR construct described in the third aspect, or the recombinant immune cell described in the eighth aspect.
  • treatment includes prevention, control, auxiliary treatment, etc.
  • the ROR1-related disease is a ROR1-high-expressing tumor, an autoimmune disease, a metabolism-related disease, or an infectious disease.
  • the tumor may be a solid tumor or a blood cancer.
  • the tumor with high expression of ROR1 refers to the ratio of ROR1 transcript and/or protein level L1 in tumor tissue to the transcript and/or protein level L0 in normal tissue, L1/L0 ⁇ 2, preferably Land ⁇ 3.
  • the tumor is breast cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, esophageal cancer, kidney cancer, glioma, bladder cancer, prostate cancer cancer, endometrial cancer, cervical cancer, leukemia, lymphoma, bone marrow cancer, or angiosarcoma;
  • the inflammation is rheumatoid arthritis, osteoarthritis, gout, Reiter syndrome, psoriatic arthropathy, tuberculous arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis , acute lung injury, chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis;
  • the autoimmune disease is rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, Sjogren's syndrome or systemic vascular disease inflammation, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis;
  • the metabolism-related disease is diabetes, food-borne obesity or fat inflammation;
  • the infectious disease is a disease caused by bacterial infection or a disease caused by viral infection.
  • the drug may be in the form of an antibody-conjugated drug or a pharmaceutical composition, which is used to treat tumors with high ROR1 expression, tumor migration, or tumor resistance.
  • a pharmaceutical composition which is used to treat tumors with high ROR1 expression, tumor migration, or tumor resistance.
  • it can be used to:
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • the tumor resistance includes: resistance to tumor immunotherapy drugs, resistance to tumor targeted therapy drugs, resistance to conventional tumor chemotherapy, and insensitivity to radiotherapy.
  • the diagnostic product for ROR1-related diseases is a diagnostic reagent, test strip, test plate or kit.
  • the detection reagents, detection plates or kits can be used for:
  • the antibody-conjugated drug provided in the tenth aspect and the pharmaceutical composition provided in the eleventh aspect of this application can target a special cell population and bind to a specific protein (antigen) on the cell surface, thereby endocytosis or Drug penetration causes the drug to be released into cells in an active form. Therefore, the antibody-conjugated drugs and pharmaceutical compositions of the present application can be used to treat target diseases and can be administered to subjects in a therapeutically effective amount through appropriate routes.
  • a subject in need of treatment may be a patient at risk for, or suspected of having, a condition related to the activity or expression of a particular antigen. Such patients can be identified through routine physical examination.
  • parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • This application provides an antibody-conjugated drug, which includes:
  • An antibody selected from the antibodies defined in the first aspect is selected from the antibodies defined in the first aspect.
  • the conjugate may be selected from the group consisting of, but not limited to: detectable markers, cytotoxic drugs, cytokines, radionuclides and enzymes.
  • Cytotoxic drug refers to a substance that inhibits or prevents cell expression activity, cell function and/or causes cell damage.
  • the term includes radioactive isotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • aristatins e.g., aristatin E, aristatin F, MMAE and MMAF
  • chlortetracycline maytansetol, ricin, ricin A-chain, combretastatin, polymethacrylate Carmicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine , vinblastine, colchicine, dihydroxyanthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, capsula root Toxin A chain, alpha-sarcina, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin
  • aristatins
  • the cytotoxic drug is a compound with high cytotoxicity, preferably monomethyl auristatin, calicheamicin, maytansinoids, or a combination thereof; more preferably, it is selected from: Monomethylaristatin-E (MMAE), monomethylaristatin-D (MMAD), monomethylaristatin-F (MMAF), or combinations thereof.
  • MMAE Monomethylaristatin-E
  • MMAD monomethylaristatin-D
  • MMAF monomethylaristatin-F
  • the cytotoxic drug is a cytotoxic drug used for cancer treatment, or a protein or polypeptide with desired biological activity, such as a toxin, such as abrin toxin, ricin A, Pseudomonas exotoxin , and diphtheria toxin; other suitable proteins include tumor necrosis factor, alpha-interferon, beta-interferon, neurogenic growth factor, platelet-derived growth factor, tissue plasminogen growth factor, and biological response modifiers, e.g.
  • Lymphokines interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), Granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF Granulocyte macrophage colony-stimulating factor
  • granulocyte colony-stimulating factor or other growth factors.
  • cytotoxic drug is maytansinoids or maytansinoids.
  • Maytansine compounds inhibit cell proliferation by inhibiting microtubule formation by tubulin.
  • Maytansinoids are derivatives of maytansine. Both maytansinoids and maytansinoids have high cytotoxicity, but their clinical application in cancer treatment has great limitations, mainly due to the low selectivity of these molecules for tumors. However, this high cytotoxicity has prompted them to become the drug moiety of choice for antibody-drug conjugates.
  • the structure of desacetylmaytansine is listed below.
  • Otostatin peptide drugs are analogs of Dolastatin 10, which is a biologically active polypeptide isolated from the marine mollusk Aplysia. Aplysiatoxin 10 inhibits tubulin polymerization by binding to tubulin (the same binding region as vincristine).
  • Aplysia toxin 10, otostatin peptide PE, and otostatin peptide E are all linear polypeptides containing four amino acids (three of which are unique to Aplysia toxin compounds) and a C-terminal amide group.
  • Two representative otostatin peptide compounds, monomethyl otostatin peptide E (MMAE) and monomethyl otostatin peptide F (MMAF) are the drugs of choice for antibody-drug conjugates.
  • PBD pyrrolo[2,1-c][1,4]benzodi-azepines
  • PBD dimers PBD is a type of natural product produced by Streptomyces. Its unique property is its ability to form non-twisted covalent adducts at the minor groove of DNA, specifically at the purine-guanine-purine sequence.
  • the application of PBD as part of a small molecule strategy to target DNA sequences and as a novel anticancer and antibacterial drug has attracted increasing interest.
  • a flexible carbon chain is used to connect the C8/C8' hydroxyl groups of two PBD units, and the resulting dimer has enhanced biological activity.
  • PBD dimers are thought to produce sequence-selective DNA damage, such as inverted 5'-Pu-GATC-Py-3' interstrand cross-links, resulting in their biological activity. These compounds have been shown to be highly potent cytotoxic drugs and may serve as candidates for antibody-drug conjugates.
  • One cytotoxic drug is a derivative of PNU-159682, the main active metabolite of Nemorubicin in human liver microsomes, which is 3000 times more active than MMDX and doxorubicin.
  • Cytotoxic drugs are not limited to the categories mentioned above but include all drugs that can be used in antibody-drug conjugates. and especially those that are capable of coordination through an amide bond with a linker, such as by having a basic amine group (primary or secondary amine).
  • cytotoxic drugs can be divided into: microtubule-targeting drugs, DNA-targeting drugs or topoisomerase inhibitors.
  • the antibody and the conjugate can be coupled through, but are not limited to, chemical bonds or linkers.
  • the linker contains a thiol-specific reactive group, which can be selected from the group including but not limited to: 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinate.
  • the linker is selected from the group consisting of 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-val-cit-PAB), 6-maleimido Caproyl-alanine-phenylalanine-p-aminobenzyloxycarbonyl (MC-ala-phe-PAB), maleimidopropionyl-valine-citrulline-p-aminobenzyloxycarbonyl (MP -val-citPAB), maleimidopropionyl-alanine-phenylalanine-p-aminobenzyloxycarbonyl (MP-ala-phe-PAB), N-succinimidyl 4-(2-pyridine thio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 4-(2-pyridyl) N-hydroxys
  • the antibody-conjugated drug may have a structure represented by the following molecular formula:
  • Ab is an antibody
  • D is a cytotoxic drug.
  • p is selected from 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In some of these embodiments, p is a value of 1-8.
  • the embodiments of the present application also relate to a method for preparing an ADC, which may further include: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • methods of the present application include conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate.
  • the methods of the present application further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
  • the antibody-conjugated drug of the present application can be delivered and administered by conventional methods in this field. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • composition which includes:
  • modes of administration of pharmaceutical compositions include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection, and topical administration, inhalation.
  • the pharmaceutical composition may be administered orally, enema, or parenterally.
  • the administration period of the pharmaceutical composition may be intermittent administration, periodic administration, continuous administration or long-term administration.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders and granules.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) Humectants, For example, glycerol; (d) disintegrating agent, such as agar, Calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) retarder, such as paraffin; (f) absorption accelerator, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl mono
  • the dosage form may also contain buffering agents.
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules may be prepared using coatings and shell materials such as enteric casings and other materials well known in the art. They may contain opacifying agents and the release of the active ingredients or compounds in such compositions may be in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxy substances. If necessary, the active ingredient can also be in the form of microcapsules with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, specifically, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.
  • Liquid dosage forms for parenteral injection may contain physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include vegetable oil, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, water, ethanol, Polyols and suitable mixtures thereof.
  • the water-soluble antibody can be administered by drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is administered by infusion.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients.
  • a sterile preparation of the antibody in a suitable soluble salt form can be dissolved and administered in a pharmaceutically acceptable excipient such as water for injection, 0.9% saline, or 5% dextrose solution.
  • Dosage forms for topical administration include ointments, powders, patches, sprays, and inhalants.
  • the pharmaceutical composition of the present application consists of the active ingredient mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the “active ingredients” in the above-mentioned pharmaceutical preparations in this aspect refer to the ingredients in the pharmaceutical composition that can play the role of "medicine”. It can be understood that the drugs in the embodiments of the present application can be prepared into appropriate clinical dosage forms by adding different pharmaceutically acceptable carriers. These clinical dosage forms include but are not limited to the dosage forms mentioned above.
  • the present application provides a diagnostic product for ROR1-related diseases.
  • the diagnostic product is a detection plate, and the detection plate is coated with the antibody or antigen-binding fragment thereof described in the first aspect, and the recombinant protein described in the second aspect.
  • the diagnostic product is a detection kit
  • the detection kit includes a primary antibody and a secondary antibody for detecting ROR1
  • the primary antibody is the antibody defined in the first aspect.
  • the detection plate can be, but is not limited to, an immune chromatography plate, or can also be a detection test paper.
  • the detection plate can include a substrate (substrate, base pad or support plate) and chromatography paper.
  • the detection kit may include at least two containers, one for placing the antibody described in the first aspect; the other for placing the secondary antibody against the antibody.
  • the detection kit may also include instructions for use, buffers, etc.
  • This application provides a detection method for ROR1.
  • the detection method includes the following steps:
  • the sample to be tested and the antibody described in the first aspect are mixed and reacted, the reaction product is detected, and whether ROR1 is present in the sample to be tested is determined based on the detection results.
  • judging whether ROR1 is present in the sample to be tested according to the reaction results includes:
  • the detection method may be based on diagnostic purposes or may be based on non-diagnostic purposes.
  • samples (samples) to be tested include cells, tissue samples and biopsy specimens.
  • biopsy shall include all types of biopsies known to those skilled in the art. Biopsy as used in this application may thus include, for example, resection samples of tumors, tissue samples prepared by endoscopic methods, or puncture or needle biopsy of organs.
  • Samples to be tested as used in this application include fixed or preserved cell or tissue samples.
  • This application provides a method for treating ROR1-related diseases, which method includes the following steps:
  • ROR1-related diseases refers to the ninth aspect.
  • the method further includes: administering other drugs or treatments to the subject for combined treatment.
  • the other drugs or treatment methods include but are not limited to: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.
  • the anti-tumor immunotherapy drugs include but are not limited to: PD-1 and PD-L1 monoclonal antibodies.
  • modes of administration include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) injection, and topical administration, and inhalation.
  • a "subject” is an animal, which may be a human or other non-human mammal. Subjects include, but are not limited to, users of the drug and patients with diseases, disorders and/or symptoms.
  • the term "mammal” in this application mainly refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats (such as rats, mice), Pigs, cattle, sheep, horses, humans, etc., primates are preferred, and humans are more preferred.
  • the present application provides a method for inhibiting the growth and migration of tumor cells.
  • the method includes the following steps: administering to a subject an effective amount of the antibody described in the first aspect of the present application and the recombinant immune cell described in the eighth aspect. , the antibody conjugated drug described in the tenth aspect, the pharmaceutical composition described in the eleventh aspect, or a combination thereof.
  • the method further includes: administering other drugs or treatments to the subject for combined treatment.
  • the present application provides a method for inhibiting tumor growth in a model animal.
  • the method includes the following steps: administering to the model animal an effective amount of the antibody described in the first aspect of the present application and the recombinant immune cell described in the eighth aspect. , the antibody conjugated drug described in the tenth aspect, the pharmaceutical composition described in the eleventh aspect, or a combination thereof.
  • the method further includes: administering other drugs or treatment methods to the model animal for combined treatment.
  • an effective amount refers to the component corresponding to this term that achieves treatment, prevention, reduction and/or alleviation of specific
  • the dosage of diseases, disorders and/or symptoms in this application, unless otherwise specified, refers to the dosage that achieves treatment, prevention, alleviation and/or alleviation of diseases, disorders and/or symptoms associated with high expression of ROR1.
  • a safe and effective amount is applied to the subjects.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the effective dose may vary depending on the mode of administration and the severity of the disease to be treated.
  • the selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the bifunctional antibody conjugate drug such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, and the patient's immunity. conditions, route of administration, etc.
  • the antibody conjugate drug of the present application is administered at a dose of about 0.0001 mg-50 mg/kg animal body weight (preferably 0.001 mg-10 mg/kg animal body weight) every day, satisfactory effects can be obtained.
  • several divided doses may be administered daily, or the dosage may be proportionally reduced as dictated by the exigencies of the treatment situation.
  • the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
  • the chimeric antibodies described above can bind to ROR1-ECD antigen with high specificity, and their EC50 values measured by ELISA are all ⁇ 0.02nM;
  • the chimeric antibody has extremely high binding affinity against multiple strains of tumor cells with high expression of ROR1. Its EC50 is 0.15nM-1.1nM as measured by FACS, based on the similarity of the antibody CDR region sequence and the antibody's antigen-binding activity. and the uniqueness of the binding epitope, we prefer mAb001c and mAb005c for subsequent development;
  • a series of humanized antibodies designed based on mAb001c have higher ROR1 protein binding affinity and cell binding affinity; their EC50 is 0.010nM-0.015nM measured by ELISA; their EC50 is 0.19nM-0.62nM measured by FACS;
  • the chimeric antibody has significant anti-tumor effects in vivo and has no obvious toxic side effects on mammals;
  • a series of humanized antibodies designed based on mAb005c have higher ROR1 protein binding affinity and cell binding affinity; their EC50 is 0.009nM-0.02nM measured by ELISA; their EC50 is 0.12nM-0.22nM measured by FACS.
  • the chimeric antibodies and humanized antibodies have excellent endocytic properties
  • Tumor cells with high expression of ROR1 have extremely high killing effect, and the killing activity is related to the expression level of ROR1; at the same time, the chimeric antibody ADC and humanized antibody ADC drugs have excellent effects on tumor models with different expression levels of ROR1 in vivo. Antitumor activity.
  • ROR1-ECD extracellular domain of human ROR1 protein
  • NCBI NCBI: NP_005003.2 amino acid positions 30 to 403
  • gene cloning technology and mammalian vector expression system were used to obtain the C-terminus polyhistidine-tagged antigen.
  • the specific amino acid sequence is as follows ( SEQ ID NO.86):
  • the human ROR1 extracellular domain protein expressed and prepared in HEK293T cells was used to immunize SJL mice at a dosage of 50 ⁇ g/mouse to prepare immune splenocytes; mouse myeloma cells (SP2/0) and feeder cells were prepared in a timely manner to Prepare for integration needs.
  • the immune spleen cells and SP2/0 cells are fused through an electrofusion instrument (BEX, model LF301), resuspended in HAT complete medium containing feeder cells, and inoculated into a 96-well plate for culture. Positive well screening was performed by ELISA/FACS method.
  • the cells in the positive wells are cloned and cultured through the limiting dilution method, and cells with high potency, good shape, and monoclonal growth are screened through ELSIA or FASCS to continue subclonal screening until the positive clone rate is 100%.
  • the cell line can be expanded and cultured and a library can be established.
  • Step 3 Determination of biological activity and specificity of mouse monoclonal antibodies targeting human ROR1:
  • Panel A the enzyme-linked immunosorbent assay (ELISA) was used to detect the monoclonal cell culture supernatant, and all 9 monoclones were able to specifically bind to the purified human ROR1-ECD protein.
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry fluorescence sorting
  • panel C purified monoclonal antibody samples were used for subtype detection.
  • mAb001, mAb004 ⁇ mAb009 were all identified as IgG2b/k, and mAb002 and mAb003 were identified as IgG2c/k.
  • mAb001, mAb002, mAb003, mAb004, mAb005, mAb006, mAb007, mAb008, and mAb009 are prioritized for antibody sequencing identification.
  • 5'RACE and conventional PCR techniques were used to amplify heavy chain (VH) and light chain (VL) variable region fragments, cloned into vectors, conventional sequencing and analysis through the Kabat database (http://www.bioinf.org.uk ) to obtain the following heavy chain variable region (VH), light chain variable region (VL) amino acid sequence, and complementarity determining region (CDR) information.
  • the underlined "_" indicates the CDR-1/2/3 amino acid sequence.
  • 9 groups of expressed variable region fragments see SEQ ID NO.7, SEQ ID NO.41, SEQ ID NO.49, SEQ ID NO.17, SEQ ID NO.25, SEQ ID NO.57, SEQ ID NO.65, SEQ ID NO.73, SEQ ID NO.81, SEQ ID NO.9, SEQ ID NO.42, SEQ ID NO.50, SEQ ID NO.18, SEQ ID NO.27, SEQ ID
  • the nucleic acids of NO.58, SEQ ID NO.66, SEQ ID NO.74, SEQ ID NO.82 were cloned into the vector containing the human IgG1 heavy chain constant region and the Kappa chain constant region.
  • mammalian HEK was used.
  • -293T cell expression system expresses and purifies the constructed chimeric antibodies (see Figure 2).
  • the obtained human-mouse chimeric antibodies are numbered mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, and mAb008c. , mAb009c.
  • the UC-961 disclosed in the invention patent application US20150232569A1 was prepared using the same method as a control.
  • variable region sequence of the antibody contains several unfavorable amino acids, which have been modified by point mutations.
  • amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) after point mutations are listed below ("_" indicates the CDR amino acid sequence).
  • the point mutation (PTM) was obtained by matching the above point mutation template and cloned into hIgG1, and the corresponding chimeric antibody mutant of the point mutation was obtained from the ⁇ vector.
  • the numbering of the human-mouse chimeric antibody and the corresponding human-mouse chimeric antibody mutant, and the sequence numbering of the heavy chain variable region and light chain variable region of the human-mouse chimeric antibody are as follows: -4 summarized list.
  • mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, and mAb009c have strong affinity for human ROR1-ECD, with EC 50 of 0.012nM, 0.013nM, 0.013nM, respectively.
  • the test results are shown in Figure 4.
  • the human-mouse chimeric antibody mutants of mAb001c and mAb005c also have strong affinity for human ROR1-ECD, with EC 50 of 0.028nM and 0.046nM respectively.
  • tumor cell line groups such as lung cancer, breast cancer, colon cancer, blood tumors, liver cancer, gastric cancer
  • This example also analyzes and compares the expression levels of ROR1 mRNA in breast cancers of different molecular types (eg, luminal type versus basal type).
  • Example 6 ROR1 protein is highly expressed in various tumor cells
  • Lung cancer cells with high ROR1 expression (NCI-H226, HCC827, NCI-H446), triple-negative breast cancer cells (MDA-MB-231, HCC1187), colon cancer HT-29 cells and breast cancer cells with low ROR1 expression (MDA -MB-453, MCF-7) as target cells to determine the binding of human-mouse chimeric antibody mAb001c to ROR1 on the cell surface (mAb001c is taken as an example, other human-mouse chimeric antibodies are not listed).
  • the test results are shown in Figure 9.
  • the human-mouse chimeric antibody mAb001c can specifically bind to tumor cells with high ROR1 expression.
  • the order of binding rate and fluorescence intensity is HCC1187, NCI-H226, HCC827, HT-29, and MDA-MB-231. , NCI-H446, while showing weak binding fluorescence intensity to tumor cells MDA-MB-453 and MCF-7 with low ROR1 expression.
  • Example 8 Determination of binding affinity of human-mouse chimeric antibodies to ROR1 on tumor cell surface
  • Triple-negative breast cancer cells MDA-MB-231 and non-small cell lung cancer NCI-H226 with high ROR1 expression were used as target cells, and 100 ⁇ L of the test human-mouse chimeric antibody was diluted according to a 5-fold gradient from 33.3nM to 0.002nM.
  • the primary antibody was mixed with 2 ⁇ 10 5 MDA-MB-231 or NCI-H226 suspended in 100 ⁇ LRPMI-1640 serum-free medium, and then incubated at 4°C for 1 h.
  • the cells were washed twice with PBS to remove unbound First antibody, then incubate the target cells with 200 ⁇ L, 2.5 ⁇ g/mL, PE-labeled secondary antibody for 30 min at 4°C.
  • mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, and mAb009c have excellent binding affinity to MDA-MB-231, with EC 50 of 0.15nM, 0.20nM, 1.1nM, respectively.
  • mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, and mAb009c also have excellent binding affinities for NCI-H226, with EC 50s of 0.11nM, 0.14nM, 0.76nM, and 0.46 respectively.
  • mAb001c is better than that of UC-961 (EC 50 is 0.13nM), and the binding activity of mAb002c and mAb005c is equivalent to that of UC-961;
  • the test results are shown in Figure 12.
  • the mAb001c and mAb005c mutants also have excellent binding affinity to MDA-MB-231.
  • the EC 50 value of mAb001c_v1 is 0.073nM; the EC 50 value of mAb005c_v1 is 0.067nM.
  • Example 9 Determination of the binding activity of human-mouse chimeric antibodies to each domain of the extracellular segment of ROR1
  • CHO-ROR1 extracellular segment
  • CHO-ROR1 Ig-like domain removed
  • CHO-ROR1 Frizzled domain removed
  • 100 ⁇ L of the test human-mouse chimeric antibody diluted according to a 5-fold gradient from 33.3nM to 0.002nM or a 5-fold gradient from 200nM to 0.013nM was used as the primary antibody, respectively.
  • mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c and mAb009c have excellent binding affinity to CHO-ROR1 (extracellular segment), with EC 50 of 0.32nM, 0.60nM, 1.47nM, 0.32nM, 0.57nM, 0.71nM, 0.54nM, 0.7nM, and 0.79nM respectively, among which The binding activity of mAb001c, mAb004c, mAb005c, and mAb007c is significantly better than that of UC-961 (EC 50 is 0.74nM);
  • mAb001c, mAb002c, mAb004c, mAb006c, mAb007c, mAb008c, and mAb009c have excellent binding affinity to CHO-ROR1 (Ig-like domain removal), with EC 50 of 0.36nM, 0.35nM, and 0.35 respectively.
  • mAb003c and mAb005c are similar to UC-961 and basically do not bind to CHO-ROR1 (Iglike domain removed);
  • mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, and mAb009c have excellent binding affinity to CHO-ROR1 (Frizzled domain removed), with EC 50 of 0.85nM and 0.86nM respectively. , 1.78nM, 1.37nM, 1.02nM, 1.64nM, 1.25nM, 1.67nM, 1.68nM, among which the binding activity of mAb001c, mAb002c, and mAb005c is significantly better than that of UC-961 (EC 50 is 1.11nM);
  • mAb001c, mAb002c, mAb003c, mAb004c, mAb005c, mAb006c, mAb007c, mAb008c, and mAb009c have excellent binding affinity to CHO-ROR1 (Kringle domain removed), with EC 50 of 0.38nM and 0.66nM respectively. , 1.74nM, 0.84nM, 0.91nM, 1.59nM, 1.03nM, 1.50nM, 1.62nM; among them, the binding activity of mAb001c and mAb002c is significantly better than that of UC-961 (EC 50 is 0.79nM).
  • the above results show that the chimeric antibodies mAb001c-mAb009c in this example have excellent binding affinity (EC 50 ⁇ 2nM) for ROR1 transferred on the cell surface; more importantly, mAb001c, mAb002c, mAb004c, mAb006c, mAb007c, mAb008c, mAb009c
  • the binding region for ROR1 is a non-Ig-like domain (Frizzled or Kringle domain), which is obviously different from UC-961 (see US20150232569A1 for binding to Ig-like domains, which is consistent with the results of this example), and both mAb003c and mAb005c bind In the ROR1-Ig-like region, and the binding activity of mAb001c and mAb005c against ROR1 is significantly better than that of UC-961. Therefore, the antibody of this embodiment has significant differential advantages over UC-961.
  • the results are shown in Figure 17.
  • the human-mouse chimeric antibodies mAb001c, mAb004c, and mAb005c can significantly inhibit the growth of NCI-H226 tumors in nude mice (vs. hIgG group, P ⁇ 0.05); the control drug UC-961 has a negative effect on NCI-H226 tumors in nude mice.
  • H226 tumors have basically no obvious inhibitory activity.
  • Example 11 ROR1 human-mouse chimeric antibody binds to tumor cells and results in endocytosis into intracellular lysosomes
  • MDA-MB-231 cells with high ROR1 expression at 60% density were spread in a laser confocal culture dish and cultured overnight.
  • 5 ⁇ g/mL of ROR1 human-mouse chimeric antibodies mAb001c, mAb002c, mAb004c, and mAb005c were added and incubated at 37°C.
  • Incubate for 4 hours and 1 hour at 4°C wash three times with PBS to remove unbound human-mouse chimeric antibodies, add 4% paraformaldehyde, and fix at room temperature for 30 minutes. Wash three times with PBS, and permeabilize the cells with 0.4% TritonX-100 for 10 min.
  • rabbit anti-human LAMP-2 antibody was added and incubated at 37°C for 1 h to mark the location of cell lysosomes.
  • Use PBS to wash away unbound antibodies add R-PE-labeled goat anti-human and Alexa Fluor 488-labeled donkey anti-rabbit secondary antibodies and incubate at 37°C for 30 minutes. Unbound secondary antibodies were washed away, stained with DAPI for 10 min to mark the location of the cell nucleus, and the endocytosis of the antibody was observed using a laser confocal microscope (20 ⁇ ).
  • DMSO solution of vcMMAE (purchased from Shanghai Haoyuan Chemical) according to the final molar ratio of drug to human-mouse chimeric antibody 8:1, add DMSO according to 10% of the total volume of the reaction solution, shake and mix, and place in a refrigerated constant temperature mixer. Reaction on homogenizer, 4°C, 1h. Use an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore) to replace the sample storage buffer. First use 30mM His-Hac containing 10% DMSO, pH5.5 buffer to ultrafiltrate 3 times, and then use 30mM His-Hac without DMSO. Ultrafiltrate at pH 5.5 for 6 times, sterilize through a filtration device with a pore size of 0.22 microns, and store at -80°C. The resulting antibody conjugate is named mAb001c-vcMMAE.
  • Example 13 In vitro antiproliferative activity of ROR1 human-mouse chimeric antibody drug conjugate against tumor cells with high ROR1 expression
  • the cell lines used in this example were purchased from Nanjing Kebai Biotechnology and Shanghai Yuchi Biotechnology, and were cultured according to the corresponding instructions, including: MCF-7, MDA-MB-231, NCI-N87, NCI-H446, HT-29 , HCC827, HCC1187.
  • the above cells in the logarithmic growth phase were seeded into a 96-well cell culture plate at a density of 250-1500 cells per well (depending on the growth rate of different cells), 150 ⁇ L/well, 37°C, 5% CO 2. After culturing for about 3-5 hours, add different concentrations of mAb001c-vcMMAE. Set 2-4 duplicate wells for each drug concentration, as well as corresponding vehicle control and blank control wells.
  • mAb001C-vcMMAE has no obvious inhibitory effect on the proliferation of MCF-7 cells with low ROR1 expression (IC 50 >100nM), but it has no significant inhibitory effect on MDA-MB-231, NCI-N87, and NCI-H446 with high ROR1 expression.
  • HT-29, HCC827, and HCC1187 all showed strong inhibitory effects on cell proliferation (IC 50 ⁇ 10nM).
  • the cytotoxicity (IC 50 value) of the ROR1-antibody drug conjugate showed that the cytotoxic activity of the ROR1-antibody drug conjugate was directly related to the ROR1 expression level of the tested cells, and therefore was judged to be a ROR1 target-specific cell toxicity.
  • tumor volume 200 ⁇ L of cell matrix gel suspension containing 5 ⁇ 10 6 MDA-MB-231 and HT-29 were inoculated subcutaneously into the back of immunodeficient mice (NCG or Balb/c-nude).
  • NCG immunodeficient mice
  • VH UC-961-Heavy chain variable region
  • VL UC-961-Light chain variable region
  • Example 8 Refer to the measurement methods in Example 4, Example 8, and Example 9 to evaluate the ELISA affinity of mAb001c, mAb005c, and UC-961 against ROR1-ECD protein, the affinity of cell surface ROR1, and the activity of the antigen-binding region (epitope). Carry out testing, and the comparison results are summarized in Table-5;
  • Example 10 Refer to the detection method in Example 10 to detect the in vivo anti-tumor effects of mAb001c, mAb005c and UC-961 against the NCI-H226 transplanted tumor model with high ROR1 expression.
  • the comparison results are summarized in Table-6;
  • the preferred antibody has excellent biological activity and specificity. Specifically, the preferred antibody has a high affinity for ROR1-ECD (the EC 50 value measured by ELISA is 0.009nM-0.05nM). In addition, the preferred antibody has good binding affinity to ROR1 on the surface of tumor cells (its EC 50 value is 0.15nM-1.1nM as determined by FACS), and can be used as a therapeutic antibody targeting ROR1.
  • the antibody-drug conjugate (ADC) has specific ROR1-dependent anti-tumor activity; the preferred antibody-drug conjugate (ADC) has no obvious toxic side effects on cells with low ROR1-expression, but has no obvious toxic side effects on cells with low ROR1-expression.
  • Tumor cells with high expression of ROR1 have extremely high killing activity, and their IC 50 value is 1nM-9nM as measured by cell proliferation inhibition test.
  • Both the antibody and the ADC have significant anti-tumor activity in vivo and have no visible toxic side effects on mammals such as model mice.
  • humanization of mAb001c resulted in five variable regions of humanized heavy chains (SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, and SEQ ID NO.32). , and the variable regions of 2 humanized light chains (SEQ ID NO. 33, SEQ ID NO. 34).
  • humanization of mAb005c resulted in eight variable regions of the humanized heavy chain (SEQ ID NO.93, SEQ ID NO.94, SEQ ID NO.95, SEQ ID NO.96, SEQ ID NO.97 , SEQ ID NO.98, SEQ ID NO.99, SEQ ID NO.100), and the variable region of a humanized light chain (SEQ ID NO.101).
  • the designed humanized variable region was cloned into a vector containing human IgG1 heavy chain constant region and Kappa chain constant region through genetic recombination technology. After sequencing, the transfection technology and mammalian expression system (Expi293F cells) were used to The constructed humanized antibody expression vector was used for combined expression of heavy chain and light chain. Finally, the mAb001c group obtained 9 humanized antibodies, and the mAb005c group obtained 8 humanized antibodies. The corresponding heavy chain and light chain combinations of each antibody are as follows As shown in Table-7.
  • the 17 humanized antibodies in Table 7 were diluted in gradients, and their affinity to the ROR1-ECD protein was measured using the ELISA method.
  • the experimental method was as described in Example 4.
  • the experimental results are shown in Figure 24.
  • the humanized antibodies Hu001-02, 03, 05, 06, 08, 11, 12, 14, and 15 all have strong binding affinity to the ROR1-ECD protein, with an EC 50 value of 0.010nM-0.015nM.
  • the experimental results are shown in Figure 25.
  • the humanized antibodies Hu005-35, 40, 41, 42, 43, 44, 45, and 46 all have strong binding affinity to the ROR1-ECD protein, with an EC 50 value of 0.009nM. -0.02nM.
  • the humanized antibodies Hu001-02 and Hu005-46 in Table 7 were gradient diluted, and their affinities for human ROR1-ECD and human ROR2-ECD proteins were measured using the ELISA method. Refer to Example 4 for the experimental method.
  • the experimental results are shown in Figure 26.
  • the humanized antibodies Hu001-02 and Hu005-46 bind to the human ROR1-ECD protein specifically and with high affinity, but have basically no obvious binding activity to the human ROR2-ECD protein, indicating that the Humanized antibodies have binding specificity for ROR1 protein.
  • the 17 humanized antibodies in Table 7 were serially diluted, and their affinity to ROR1 on the surface of MDA-MB-231 and NCI-H226 cells was measured by flow cytometry.
  • the experimental method was as described in Example 8.
  • the experimental results shown in Figure 27 show that the humanized antibody has high binding affinity activity to ROR1 on the surface of MDA-MB-231 cells, Hu001-02, 03, 05, 06, 08, 11, 12, 14, 15
  • the binding EC 50 value is 0.19nM-0.47nM; the binding EC 50 value of Hu005-35, 40, 41, 42, 43, 44, 45, 46 is 0.16nM-0.22nM.
  • the experimental results are shown in Figure 28.
  • the humanized antibody has a high binding affinity activity to ROR1 on the surface of NCI-H226 cells, and the EC 50 value of Hu001-02, 03, 05, 06, 08, and 11 is 0.27nM. -0.62nM; the EC 50 value of Hu005-35, 40, 41, 42, 43, 44, 45, 46 binding is 0.12nM-0.18nM.
  • MDA-MB-231 cells with a density of 60% were spread in a laser confocal culture dish, cultured at 37°C overnight, and then 5 ⁇ g/mL of ROR1 humanized antibodies Hu001-02, Hu001-03, Hu005-44, and Hu005-46 were added. , incubate at 37°C for 4h and 4°C for 1h respectively, wash three times with PBS to remove antibodies that are not bound to the cells, and fix with 4% paraformaldehyde at room temperature for 30min. Wash three times with PBS and permeabilize with 0.4% Triton X-100 for 10 minutes.
  • Lamp-2 (rabbit anti-human) antibody was incubated at 37°C for 1 h to mark the location of cellular lysosomes.
  • Use PBS to wash away unbound antibodies, and incubate R-PE-labeled goat anti-human and Alexa Fluor 488-labeled donkey anti-rabbit secondary antibodies at 37°C for 30 minutes. Unbound secondary antibodies were washed away, stained with DAPI for 10 min to mark the location of the cell nucleus, and then the endocytosis of the antibody was observed using a laser confocal microscope (20 ⁇ ).
  • Hu001-2 or Hu001-3 or Hu005-44 or Hu005-46) were incubated on ice for 30 minutes, washed twice with pre-cooled 1 ⁇ PBS, centrifuged to collect the cells, and resuspended in 1.8 mL of 1640 medium (containing 2% FBS), take 300 ⁇ L cell suspension and incubate at 37°C for 0.5h, 1h, 2h, and 4h (take 300 ⁇ L cell suspension respectively as T0 and background reference). After the incubation is completed at each time point, immediately place it on ice to terminate endocytosis.
  • Hu001-2, Hu001-3, Hu005-44, and Hu005-46 can mediate target internalization into cells to a large extent in 0.5h-1h.
  • the antibody to be tested can basically achieve an endocytosis rate of 50%-70%, among which Hu001-2(3) has better endocytosis rate and degree than Hu005-44(46).
  • humanized antibodies Hu001-2, Hu001-3, Hu005-44 and Hu005-46 are coupled with vcMMAE to prepare corresponding antibody-conjugated drugs.
  • vcMMAE For specific coupling methods, refer to Example 12.
  • the obtained antibody-conjugated drugs are named respectively.
  • Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, Hu005-46-MMAE, UC961-MMAE was also prepared as a positive reference substance.
  • the Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, and Hu005-46-MMAE prepared in Example 22 were gradient diluted, and their affinity to the ROR1-ECD protein was determined by ELISA.
  • Hu001-3, Hu005-44, and Hu005-46 are used as parent antibody references, and the experimental methods are as described in Example 4.
  • the results are shown in Figure 43.
  • the humanized antibody-conjugates can all maintain the high binding affinity activity of the parent antibody against the ROR1-ECD protein, and the binding EC 50 value is 0.012nM-0.024nM.
  • Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, Hu005-46-MMAE prepared in Example 22 Gradient dilution, its affinity to ROR1 on the surface of MDA-MB-231 and NCI-H226 cells was measured by flow cytometry, and Hu001-2, Hu001-3, Hu005-44, and Hu005-46 were used as parent antibody references. Experimental methods Refer to Example 8.
  • the cell lines used in this example were purchased from Nanjing Kebai Biotech and Shanghai Yuchi Biotech, and were cultured according to the corresponding instructions, including: breast cancer cell lines: MCF-7, SK-BR-3, MDA-MB-231, HCC1187; intestinal cancer cell lines: HT-29, SK-CO-1, COLO-678, lung cancer cell lines: HCC827, H446; ovarian cancer cell line: PA-1; gastric cancer cell line: NCI-N87.
  • the CCK8 method was used to detect the in vitro killing activity of Hu001-2-MMAE, Hu001-3-MMAE, Hu005-44-MMAE, and Hu005-46-MMAE against various cancer cells.
  • UC961-MMAE was used as a positive reference. For implementation methods, refer to Example 13. .
  • the humanized antibody-conjugate has no obvious inhibitory effect on the proliferation of SK-BR-3 and MCF-7 cells with low ROR1 expression (IC 50 >100nM) (Figure 46, Figure 47), while MDA-MB-231, HCC1187, HT-29, SK-CO-1, COLO-678, NCI-H446, HCC827, PA-1, and NCI-N87 all showed strong resistance to ROR1 expression.
  • Proliferation inhibitory effect Figure 48, Figure 49, Figure 50, Figure 51, Figure 52, Figure 53, Figure 54, Figure 55, Figure 56.
  • cytotoxicity (IC 50 value) of the ROR1 humanized antibody-conjugate showed that the cytotoxic activity of the ROR1-antibody drug conjugate was directly related to the ROR1 expression level of the test cells ( Figure 57), so it was judged For ROR1 target-specific cytotoxicity.
  • Table-8 summarizes the IC 50 values for inhibition of cell proliferation tested.
  • Table-8 ROR1 humanized antibody-conjugate in vitro anti-tumor activity
  • Example 26 In vivo anti-tumor activity of humanized antibody-conjugated drug needles
  • tumor volume 200 ⁇ L of cell matrix gel suspension containing 5 ⁇ 10 6 MDA-MB-231, 1 ⁇ 10 7 HCC1187, 1 ⁇ 10 7 PA-1, and 5 ⁇ 10 6 NCI-N87 were respectively inoculated into immunodeficient mice (NCG or Balb/c-nude) subcutaneously on the back.
  • NCG immunodeficient mice
  • Tumor volume and body weight of nude mice were measured 2-3 times a week and recorded to draw tumor growth curves.
  • the four ROR1 humanized antibody-conjugates showed dose-related therapeutic effects when administered at 5 mg/kg and 2.5 mg/kg. And at a dose of 5 mg/kg, it can completely inhibit the growth of MDA-MB-231 tumors with high ROR1 expression (P ⁇ 0.0001). No obvious tumor growth was seen after 2 weeks of drug withdrawal.
  • the Hu001-2-MMAE and Hu005-46-MMAE showed dose-related therapeutic effects when administered at 5 mg/kg and 2.5 mg/kg. And at the dose of 5 mg/kg, it can completely inhibit the growth of PA-1 tumors with high ROR1 expression (P ⁇ 0.0001;P ⁇ 0.001), and the tumors will recede. No significant recovery of tumor growth was seen after 2 weeks of drug withdrawal, and at 2.5 mg/kg The anti-tumor activity at all doses was significantly better than that of Positive reference UC961-MMAE.
  • the Hu001-2-MMAE, Hu005-44-MMAE, and Hu005-46-MMAE showed dose-related therapeutic effects when administered at 5 mg/kg and 2.5 mg/kg. . And at a dose of 5 mg/kg, it significantly inhibited the growth of NCI-N87 tumors with medium to low expression of ROR1 (P ⁇ 0.0001), and the anti-tumor activities were significantly better than the positive reference UC961-MMAE.
  • the Hu001-2-MMAE showed dose-related therapeutic effects when administered at 5 mg/kg and 2.5 mg/kg. And at a dose of 5 mg/kg, it can completely inhibit the growth of HCC1187 tumors with high ROR1 expression (P ⁇ 0.001), and the anti-tumor activity is significantly better than the positive reference UC961-MMAE.
  • ROR1 humanized antibody-conjugates have significant anti-tumor activity against tumors with high and medium and low expression of ROR1.
  • Table-9 to Table 12 summarize the in vivo tumor inhibition rate (%) of the humanized antibody-conjugate.
  • Table-9 ROR1 humanized antibody-conjugate tumor inhibition rate (%) in MDA-MB-231 tumor model
  • Table-12 ROR1 humanized antibody-conjugate tumor inhibition rate (%) in HCC1187 tumor model
  • the humanized antibody has excellent antigen-binding ability, and the EC 50 value of binding to ROR1-ECD protein is ⁇ 0.03nM; the EC 50 value of binding to cell surface ROR1 is ⁇ 1nM;
  • the humanized antibody has excellent endocytosis properties and can be used as a target for ADC drug development;
  • the humanized antibody-conjugate maintains the high antigen-binding affinity of the parent antibody against ROR1-ECD protein and cell surface ROR1;
  • the humanized antibody-conjugate has significant in vitro anti-proliferative activity against multiple cancer cells, and the killing activity (IC 50 value) is related to the ROR1 expression level;
  • the humanized antibody-conjugate has excellent in vivo anti-tumor activity against CDX models with different expression levels of ROR1, and the therapeutic effect is significantly better than the positive reference UC961-MMAE.

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Abstract

本申请涉及特异性结合ROR1的抗体、其偶联药物及其制备方法和应用。本申请的发明人开发针对ROR1的抗体，该抗体及抗体偶联药物能够高特异性地结合纯化的人ROR1蛋白和多种肿瘤细胞表面的ROR1，具有独特的抗原结合表位，具备显著的抗肿瘤活性，且具有很高的亲和力及很低的免疫原性。

Description

能够特异性结合ROR1的抗体、其偶联药物及其制备方法和应用
相关申请
本申请要求于2022年09月07日递交中国专利局、申请号为2022110989077、发明名称为“能够特异性结合ROR1的抗体、其偶联药物及其制备方法和应用”的中国专利申请的优先权，以及，于2023年07月07日递交中国专利局、申请号为2023108321758、发明名称为“能够特异性结合ROR1的抗体、其偶联药物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及药物领域，更具体地，涉及能够特异性结合ROR1的抗体、其偶联药物及其制备方法和应用。
背景技术
受体酪氨酸激酶样孤儿受体1(ROR1)是一种在胚胎发育过程中表达的I型跨膜蛋白，有助于极化迁移和器官形成。结构上，ROR1含有一个免疫球蛋白样(Ig样)结构域、一个卷曲(Fz)结构域和一个kringle(Kr)结构域组成的独特胞外区、跨膜结构域及细胞质酪氨酸激酶样结构域(J Biol Chem 1992,267:26181-90)，它通过介导非经典Wnt信号通路的传导，参与调控多种生理功能的发挥，其中包括调节细胞分裂、增殖及迁移等。
研究表明ROR1在早期胚胎发育过程中高水平表达，随着胎儿发育，ROR1的表达逐渐下降，正常儿童及成人组织中除了罕见的B淋巴细胞前体细胞外，ROR1低表达甚至不表达。当组织发生肿瘤恶性改变时，ROR1则会启动类似胚胎发育的转录过程，表达高水平的ROR1。目前，ROR1已被证实在多种恶性肿瘤中高度激活表达，如血液系统癌症，包括B细胞慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、非霍奇金淋巴瘤(NHL)和髓系血液癌症等，再如实体瘤，包括三阴性乳腺癌(TNBC)、结肠癌、肺癌、胰腺癌、卵巢癌等多种癌症，且与肿瘤患者的不良预后密切相关(Blood 2016,128(25):2931-40；Sci Rep 2014,4:5811)。此外，ROR1过表达参与肿瘤化疗及靶向治疗耐药的发生(Cells 2021,10:142)，包括紫杉醇乳腺癌治疗的耐药(Proc Natl Acad Sci U S A 2019,116(4):1370-7)，抗BCL-2抑制剂venetoclax治疗慢性淋巴白血病(CLL)的耐药(Leukemia 2022,36(6):1609-18)以及T-DM1治疗HER2+乳腺癌的耐药(EBioMedicine 2019，43：211-24)等。
鉴于ROR1在多种实体和血液恶性肿瘤中都有高表达，而在健康组织中表达量很低，ROR1有望成为像PD-1一样具有广谱抗癌潜力的新药靶点。然而，目前尚缺乏高特异性且针对不同结合表位的ROR1抗体以及抗体偶联药物。因此，本领域仍需要开发新型的靶向ROR1的抗体及抗体偶联药物。
有鉴于此，特提出本申请。
发明内容
本申请的各种实施例，提供一种能够特异性结合ROR1的抗体、其偶联药物及其制备方法和应用，技术方案为：
在本申请的第一方面，提供一种能够特异性结合ROR1的抗体，所述抗体包括重链可变区和轻链可变区：
所述重链可变区的重链互补决定区选自如下i)至iii)所示的重链互补决定区或其衍生片段中的一组或者多组：
i)SEQ ID NO.1所示的VH-CDR1，SEQ ID NO.2所示的VH-CDR2，和SEQ ID NO.3所示的VH-CDR3；
ii)SEQ ID NO.11所示的VH-CDR1，SEQ ID NO.12所示的VH-CDR2，和SEQ ID NO.13所示的VH-CDR3；和
iii)SEQ ID NO.19的VH-CDR1，SEQ ID NO.20所示的VH-CDR2，和SEQ ID NO.21所示的VH-CDR3；
和/或，
所述轻链可变区的轻链互补决定区选自i’)至iii’)所示的轻链互补决定区或其衍生片段中的一组或者多组：
i’)SEQ ID NO.4所示的VL-CDR1，SEQ ID NO.5所示的VL-CDR2，和SEQ ID NO.6所示的VL-CDR3；
ii’)SEQ ID NO.14所示的VL-CDR1，SEQ ID NO.:15所示的VL-CDR2，和SEQ ID NO.16所示的 VL-CDR3；和
iii’)SEQ ID NO.22所示的VL-CDR1，SEQ ID NO.23所示的VL-CDR2，和SEQ ID NO.24所示的VL-CDR3；
所述衍生片段与其对应的互补决定区具有不超过6个位点的氨基酸替换且能够保留与ROR1-ECD EC50为0.009nM-0.05nM以及与肿瘤细胞ROR1EC50为0.15nM-1.1nM。
在本申请的一些实施方式中，所述重链可变区的重链互补决定区选自如下i)至iii)所示的重链互补决定区中的一组或者多组：
i)VH-CDR1的序列通式为D-Y-N-Xa1-H，VH-CDR2的序列通式为Y-I-N-P-N-Xa2-Xa3-Xa4-T-Xa5-Y-N-Q-K-F-Xa6-G，VH-CDR3的序列通式为R-Xa7-Xa8-Xa9-Xa10-Xa11-Xa12-Xa13-D-Xa14；其中，Xa1为I、M或者L，Xa2为N或者H，Xa3为D或者G，Xa4为A、N或者G，Xa5为S、N或者T，Xa6为Q、K或者E，Xa7为V或者G，Xa8为R或者Y，Xa9为T或者G，Xa10为S或者G，Xa11为S、T或者G，Xa12为G或者Y，Xa13为L、F或者AM，Xa14为D、F或者Y；
ii)VH-CDR1如SEQ ID NO.11所示，VH-CDR2如SEQ ID NO.12所示，VH-CDR3如SEQ ID NO.13所示；
iii)VH-CDR1如SEQ ID NO.19所示，VH-CDR2的序列通式为T-I-S-D-Xb1-G-S-Y-T-Y-Y-P-D-Xb2-Xb3-K-G，VH-CDR3如SEQ ID NO.21所示；其中，Xb1为A或者G，Xb2为S或者N，Xb3为V或者E；
或/和，
所述轻链可变区的轻链互补决定区选自i’)至iii’)所示的轻链互补决定区或其衍生片段中的一组或者多组：
i’)VL-CDR1的序列通式为Xa1’-S-S-Xa2’-Xa3’-I-Xa4’-H-T-N-Xa5’-N-T-Y-L-E，VL-CDR2的序列通式为K-V-Xa6’-N-R-F-S，VL-CDR3的序列通式为F-Q-G-S-Xa7’-Xa8’-P-Y-T；其中，Xa1’为R、K或者T，Xa2’为H或者Q，Xa3’为I、N或者S，Xa4’为V或者L，Xa5’为A或者G，Xa6’为F或者S，Xa7’为R或者L，Xa8’位F或者V；
ii’)VL-CDR1如SEQ ID NO.14所示，VL-CDR2如SEQ ID NO.15所示，VL-CDR3如SEQ ID NO.16所示；
iii’)VL-CDR1的序列通式为K-A-S-Q-S-V-S-F-Xb1’-G-T-S-L-M-H，VL-CDR2如SEQ ID NO.23所示，VL-CDR3如SEQ ID NO.24所示；其中，Xb1’为A或者P。
在本申请的一些实施方式中，i)所示的重链互补决定区中：Xa1为L，Xa2为H，Xa3为D，Xa4为A或者G，Xa5为S或者T，Xa6为Q，Xa7为V，Xa8为R，Xa9为T，Xa10为G，Xa11为T，Xa12为G，Xa13为L或者F，Xa14为D或者Y；或者，
Xa1为I或者M，Xa2为N，Xa3为G，Xa4为N或者G，Xa5为S、N或者T，Xa6为K或者E，Xa7为G，Xa8为Y，Xa9为G，Xa10为S，Xa11为S或者G，Xa12为Y，Xa13为AM，Xa14为F或者Y。
在本申请的一些实施方式中，i’)所示的轻链互补决定区中Xa1’为R或者K，Xa2’为H，Xa3’为N，Xa4’为V或者L，Xa5’为A或者G，Xa6’为S，Xa7’为R，Xa8’为F；或者，
Xa1’为R或者T，Xa2’为H或者Q，Xa3’为I或者S，Xa4’为V，Xa5’为G，Xa6’为F或者S，Xa7’为R或者L，Xa8’为F或者V。
在本申请的一些实施方式中，所述抗体具有如下所示的重链互补决定区和轻链互补决定区的组合中的一组或者多组：
组合1为：i)所示的重链互补决定区或其衍生片段，i’)所示的轻链互补决定区或其衍生片段；
组合2为：ii)所示的重链互补决定区或其衍生片段，ii’)所示的轻链互补决定区或其衍生片段；
组合3为：iii)所示的重链互补决定区或其衍生片段，iii)所示的轻链互补决定区或其衍生片段。
在本申请的一些实施例中，所述组合1选自如下组合中的一组或者多组：
组合1-1为：SEQ ID NO.1所示的VH-CDR1、SEQ ID NO.2所示的VH-CDR2、SEQ ID NO.3所示的VH-CDR3、SEQ ID NO.4所示的VL-CDR1、SEQ ID NO.5所示的VL-CDR2以及SEQ ID NO.6所示的VL-CDR3；
组合1-2为：SEQ ID NO.35所示的VH-CDR1、SEQ ID NO.36所示的VH-CDR2、SEQ ID NO.37所示的VH-CDR3、SEQ ID NO.38所示的VL-CDR1、SEQ ID NO.39所示的VL-CDR2以及SEQ ID NO.40所示的VL-CDR3；
组合1-3为：SEQ ID NO.51所示的VH-CDR1、SEQ ID NO.52所示的VH-CDR2、SEQ ID NO.53所示的VH-CDR3、SEQ ID NO.54所示的VL-CDR1、SEQ ID NO.55所示的VL-CDR2以及SEQ ID NO.56所示的VL-CDR3；
组合1-4为：SEQ ID NO.59所示的VH-CDR1、SEQ ID NO.60所示的VH-CDR2、SEQ ID NO.61所示的VH-CDR3、SEQ ID NO.62所示的VL-CDR1、SEQ ID NO.63所示的VL-CDR2以及SEQ ID NO.64所示的VL-CDR3；
组合1-5为：SEQ ID NO.67所示的VH-CDR1、SEQ ID NO.68所示的VH-CDR2、SEQ ID NO.69所示的VH-CDR3、SEQ ID NO.70所示的VL-CDR1、SEQ ID NO.71所示的VL-CDR2以及SEQ ID NO.72所示的VL-CDR3；
组合1-6为：SEQ ID NO.75所示的VH-CDR1、SEQ ID NO.76所示的VH-CDR2、SEQ ID NO.77所示的VH-CDR3、SEQ ID NO.78所示的VL-CDR1、SEQ ID NO.79所示的VL-CDR2以及SEQ ID NO.80所示的VL-CDR3；
组合1-7为：SEQ ID NO.1所示的VH-CDR1、SEQ ID NO.83所示的VH-CDR2、SEQ ID NO.3所示的VH-CDR3、SEQ ID NO.84所示的VL-CDR1、SEQ ID NO.5所示的VL-CDR2以及SEQ ID NO.6所示的VL-CDR3。
在本申请的一些实施例中，所述组合3选自如下组合中的一组或者多组：
组合3-1为：SEQ ID NO.43所示的VH-CDR1、SEQ ID NO.44所示的VH-CDR2、SEQ ID NO.45所示的VH-CDR3、SEQ ID NO.46所示的VL-CDR1、SEQ ID NO.47所示的VL-CDR2以及SEQ ID NO.48所示的VL-CDR3；
组合3-2为：SEQ ID NO.19所示的VH-CDR1、SEQ ID NO.20所示的VH-CDR2、SEQ ID NO.21所示的VH-CDR3、SEQ ID NO.22所示的VL-CDR1、SEQ ID NO.23所示的VL-CDR2以及SEQ ID NO.24所示的VL-CDR3；
组合3-3为：SEQ ID NO.19所示的VH-CDR1、SEQ ID NO.85所示的VH-CDR2、SEQ ID NO.21所示的VH-CDR3、SEQ ID NO.22所示的VL-CDR1、SEQ ID NO.23所示的VL-CDR2以及SEQ ID NO.24所示的VL-CDR3。
在本申请的一些实施方式中，所述抗体具有如下技术特征中的一个或者多个：
(1)所述抗体的重链恒定区和轻链恒定区的种属来源独立地选自人、鼠、兔、羊、牛、马、猪、狗、猫、骆驼、驴、鹿、貂、鸡、鸭或者鹅；和，
(2)所述抗体或其抗原结合片段的恒定区选自选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE及IgD任何其中之一恒定区的序列；和
(3)所述抗体的恒定区选自κ轻链恒定区和λ轻链恒定区任何其中之一恒定区的序列。
在本申请的一些实施方式中，所述抗体具有选自如下所示的重链可变区和轻链可变区的组合中的一组：
(1)SEQ ID NO.7所示的重链可变区和SEQ ID NO.9所示的轻链可变区；
(2)SEQ ID NO.41所示的重链可变区和SEQ ID NO.42所示的轻链可变区；
(3)SEQ ID NO.49所示的重链可变区和SEQ ID NO.50所示的轻链可变区；
(4)SEQ ID NO.17所示的重链可变区和SEQ ID NO.18所示的轻链可变区；
(5)SEQ ID NO.25所示的重链可变区和SEQ ID NO.27所示的轻链可变区；
(6)SEQ ID NO.57所示的重链可变区和SEQ ID NO.58所示的轻链可变区；
(7)SEQ ID NO.65所示的重链可变区和SEQ ID NO.66所示的轻链可变区；
(8)SEQ ID NO.73所示的重链可变区和SEQ ID NO.74所示的轻链可变区；
(9)SEQ ID NO.81所示的重链可变区和SEQ ID NO.82所示的轻链可变区；
(10)SEQ ID NO.8所示的重链可变区和SEQ ID NO.10所示的轻链可变区；
(11)SEQ ID NO.26所示的重链可变区和SEQ ID NO.27所示的轻链可变区；
(12)SEQ ID NO.28所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(13)SEQ ID NO.28所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
(14)SEQ ID NO.29所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(15)SEQ ID NO.29所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
(16)SEQ ID NO.30所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(17)SEQ ID NO.31所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(18)SEQ ID NO.31所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
(19)SEQ ID NO.32所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(20)SEQ ID NO.32所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
(21)SEQ ID NO.93所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(22)SEQ ID NO.94所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(23)SEQ ID NO.95所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(24)SEQ ID NO.96所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(25)SEQ ID NO.97所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(26)SEQ ID NO.98所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(27)SEQ ID NO.99所示的重链可变区和SEQ ID NO.101所示的轻链可变区；和，
(28)SEQ ID NO.100所示的重链可变区和SEQ ID NO.101所示的轻链可变区。
在本申请的第二方面，提供一种重组蛋白，所述重组蛋白包括：
第一方面中定义的重链互补决定区和轻链互补决定区中的一个或者多个；和，
协助所述的重链互补决定区和轻链互补决定区表达和/或纯化的标签片段。
在本申请的一些实施方式中，所述重组蛋白还包括第一方面中定义的重链恒定区、轻链恒定区或其组合。
在本申请的第三方面，提供一种CAR构建物，所述CAR构建物的scFV结构域具有第一方面中定义的重链可变区和轻链可变区。
在本申请的第四方面，提供一种核酸，所述核酸编码第一方面中所述的抗体、第二方面中所述的重组蛋白或者第三方面中所述的CAR构建物。
在本申请的第五方面，提供一种载体，所述载体含有第四方面所述的核酸。
在本申请的一些实施方式中，所述载体选自细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其组合。
在本申请的第六方面，提供一种宿主细胞，所述宿主细胞包含第四方面中所述的核酸或者第五方面中所述的载体。
在本申请的一些实施方式中，所述宿主细胞为CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞或HEK293细胞。
在本申请的第七方面，提供第一方面中所述的抗体、第二方面中所述的重组蛋白或者第三方面中所述的CAR构建物的制备方法，所述制备方法包括如下步骤：
培养第六方面中所述的宿主细胞，从所得培养物中分离抗体、重组蛋白或者CAR构建物。
本申请的第八方面，提供一种重组的免疫细胞，所述重组的免疫细胞表达外源的第三方面中所述的CAR构建物。
在本申请的一些实施方式中，所述免疫细胞选自NK细胞和T细胞中的一种。
本申请的第九方面，提供第一方面中所述的抗体、第二方面中所述的重组蛋白、第三方面中所述的CAR构建物或者第八方面中所述的重组的免疫细胞在制备治疗ROR1相关疾病的药物或者ROR1相关疾病的诊断产品中的应用。
在本申请的一些实施方式中，所述ROR1相关疾病为肿瘤、自身免疫疾病、代谢相关疾病或者感染疾病。
在本申请的一些实施方式中，所述肿瘤为乳腺癌、肺癌、胰腺癌、卵巢癌、前列腺癌、结直肠癌、胃癌、肝癌、食道癌、肾癌、脑胶质瘤、膀胱癌、前列腺癌、子宫内膜癌、宫颈癌、白血病、淋巴瘤、骨髓癌或者血管肉瘤；
所述炎症为风湿性关节炎、骨关节炎、痛风、莱特尔综合征、牛皮癣性关节病、结核性关节炎、肾小球性肾炎、全身性红斑狼疮、克罗恩病、溃疡性结肠炎、急性肺损伤、慢性阻塞性肺疾病或者特发性肺纤维化；
所述自身免疫疾病为类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、干燥综合征或者系统性血管炎、溃疡性结肠炎、I型糖尿病、银屑病、多发性硬化症；
所述代谢相关疾病为糖尿病、食源性肥胖或者脂肪炎症；
所述感染疾病为细菌感染所致疾病或者病毒感染所致疾病。
在本申请的一些实施方式中，所述ROR1相关疾病的诊断产品为诊断试剂、试纸条、检测板或者试剂盒。
在本申请的第十方面，提供一种抗体偶联药物，所述抗体偶联药物包括：
抗体，所述抗体选自第一方面中定义的抗体；和，
与所述抗体偶联的偶联物。
在本申请的一些实施方式中，所述抗体偶联药物具有如下技术特征中的一个或者多个：
所述偶联物选自可检测标记物、细胞毒性药物、细胞因子、放射性核素、酶或其组合；和，
所述抗体和所述偶联物通过化学键或连接子进行偶联。
在本申请的一些实施方式中，所述抗体偶联药物具有如下技术特征中的一个或者多个：
所述抗体偶联药物具有如下分子式所示的结构：
其中：
Ab为所述抗体，
LU为连接子，
D为细胞毒性药物；
所述连接子包含巯基特异性的活性反应基团；和，
所述细胞毒性药物为微管靶向药物、DNA靶向药物或者拓扑异构酶抑制剂。
在本申请的第十一方面，提供一种药物组合物，所述药物组合物包括：
第一方面中所述的抗体、第二方面中所述的重组蛋白、第三方面中所述的CAR构建物或者第八方面中所述的重组的免疫细胞、第十方面中所述的抗体偶联药物或者其结合；和，
药学上可接受的载体。
在本申请的第十二方面，提供一种ROR1相关疾病的诊断产品，其特征在于，
所述诊断产品为检测板，所述检测板上包被有第一方面中所述的抗体或其抗原结合片段、第二方面中所述的重组蛋白、第三方面中所述的CAR构建物或者第八方面中所述的重组的免疫细胞或者其结合；
或者，所述诊断产品为检测试剂盒，所述检测试剂盒包含检测ROR1的一抗和二抗，所述一抗为第一方面中定义的抗体。
在本申请的第十三方面，一种基于非诊断目的的ROR1的检测方法，所述检测方法包括如下步骤：
提供待测样本，
将所述待测样本和第一方面中所述的抗体进行混合反应，检测反应产物并根据检测结果判断所述待测样本中是否存在ROR1。
在本申请的一些实施方式中，根据反应结果判断所述待测样本中是否存在ROR1包括：
当在所述反应产物中检测到抗原-抗体复合物，则判断所述待测样本中含有ROR1；
当在所述反应产物中未检测到抗原-抗体复合物，则判断所述待测样本中不含ROR1。
本申请的一个或多个实施例细节在下面的描述中提出，本申请的其他特征、目的和优点将从说明书及其权利要求书变得明显。
附图说明
为了更清楚地说明本申请实施例或传统技术中的技术方案，下面将对实施例或传统技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据公开的附图获得其他的附图。
图1为本申请实施例1中的抗人ROR1抗体的发现；图1中A图为酶联免疫吸附实验(ELISA)检测一系列原始发现的抗人ROR1单克隆抗体(original hybridoma)培液上清对纯化的人ROR1蛋白胞外段(ROR1-ECD)的结合活性；图1中B图为流式细胞荧光分选仪(FACS)检测原始发现的抗人ROR1单克隆抗体(original hybridoma)培养上清对人源ROR1-高表达的MDA-MB-231(ROR1-P)、ROR1-低表达的MDA-MB-453(ROR1-N)乳腺癌细胞的结合活性；图1中C图为9个单克隆抗体的编号(mAb001、mAb002、mAb003、mAb004、mAb005、mAb006、mAb007、mAb008、mAb009)，纯化后抗体的亚型鉴定及针对ROR1-ECD蛋白和MDA-MB-231细胞的结合亲和力EC50值；
图2为本申请实施例3中纯化的9个人-鼠嵌合抗体(chimeric antibody)mAb001c、mAb002c、mAb003c、 mAb004c、mAb005c、mAb006c、mAb007c、mAb008c及mAb009c的SDS-PAGE图谱；
图3为本申请实施例4中的ELISA测定人-鼠嵌合抗体mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c及mAb009c对ROR1-ECD的结合亲和力(Binding affinity EC50)；
图4为本申请实施例4中的ELISA检测mAb001c及mAb005c-点突变体对ROR1-ECD的结合亲和力(Binding affinity EC50)；
图5为本申请实施例5中的分析对比多个肿瘤细胞株(肺癌、乳腺癌、结肠癌、血液病肿瘤、肝癌、胃癌)与人30种正常组织中ROR1mRNA的表达水平(与β-actin的比值)；
图6为本申请实施例5中的分析Cancer Cell Line Encyclopedia(CCLE)数据库ROR1mRNA在高侵袭、高转移的基底型/三阴型(Basal-type/TNBC)对比管腔型(Luminal-type)乳腺癌细胞株中的表达水平；
图7为本申请实施例6中的免疫印迹试验(Western blot)检测ROR1蛋白在不同肺癌细胞株中的表达情况；
图8为本申请实施例6中的免疫印迹试验(Western blot)检测ROR1蛋白在基底型/三阴型及管腔型乳腺癌细胞株中的表达情况；
图9为本申请实施例7中的mAb001c(5μg/mL)针对ROR1-高表达(NCI-H446、HT-29、MDA-MB-231、HCC1187、HCC827、NCI-H226)或低表达(MDA-MB-453、MCF-7)肿瘤细胞表面ROR1的结合水平；
图10为本申请实施例8中的嵌合抗体mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c及mAb009c对MDA-MB-231细胞表面ROR1的结合亲和力(Binding affinity EC50)检测结果；本试验采用2×105个细胞与所示浓度梯度的抗体混合，孵育1小时后检测MFI；
图11为本申请实施例8中的嵌合抗体mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c及mAb009c对NCI-H226细胞表面ROR1的结合亲和力(Binding affinity EC50)检测结果；本试验采用2×105个细胞与所示浓度梯度的抗体混合，孵育1小时后检测MFI；
图12为本申请实施例8中的FACS检测mAb001c及mAb005c-点突变体对MDA-MB-231的结合亲和力(Binding affinity EC50)；
图13为本申请实施例9中的嵌合抗体mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对CHO细胞表面转染的ROR1的结合亲和力(Binding affinity EC50)检测结果；本试验采用2×105个细胞与所示浓度梯度的人-鼠嵌合抗体混合，孵育1小时后检测MFI；
图14为本申请实施例9中的嵌合抗体mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对CHO细胞表面转染的ROR1(去除Ig样结构域)的结合亲和力(Binding affinity EC50)检测结果；本试验采用2×105个细胞与所示浓度梯度的抗体混合，孵育1小时后检测MFI；
图15为本申请实施例9中的嵌合抗体mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对CHO细胞表面转染的ROR1(去除Frizzled结构域)的结合亲和力(Binding affinity EC50)检测结果；本试验采用2×105个细胞与所示浓度梯度的抗体混合，孵育1小时后检测MFI；
图16为本申请实施例9中的嵌合抗体mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对CHO细胞表面转染的ROR1(去除Kringle结构域)的结合亲和力(Binding affinity EC50)检测结果；本试验采用2×105个细胞与所示浓度梯度的抗体混合，孵育1小时后检测MFI；
图17为本申请实施例10中的所述ROR1嵌合抗体在体内抗肿瘤活性测试。体内试验采用ROR1-高表达的NCI-H226肺癌细胞与50μg抗体混匀后接种至裸鼠背部皮下，每周观察2-3次，测量肿瘤体积和小鼠体重；
图18为本申请实施例11中的mAb001c、mAb002c、mAb004c、mAb005c与MDA-MB-231细胞结合导致内吞(Internalization)至细胞内溶酶体；所述抗体(5μg/mL)与细胞孵育4℃1小时，或37℃4小时后置于激光共聚焦显微镜观察结果；
图19为本申请实施例12中的抗体mAb001c及抗体药物偶联物mAb001c-vcMMAE的疏水作用层析(HIC)-HPLC图谱；
图20为本申请实施例12中的抗体mAb001c及抗体药物偶联物mAb001c-vcMMAE的分子排阻(SEC)-HPLC图谱；
图21为本申请实施例13中的mAb001c-vcMMAE对ROR1高表达细胞株(MDA-MB-231、HCC1187、HT-29、NCI-H446、HCC827、NCI-N87)及ROR1低表达细胞株(MCF-7)体外抗增殖活性(IC50)的检测结果；
图22为本申请实施例14中的mAb001c-vcMMAE(5mg/kg)在MDA-MB-231乳腺癌肿瘤模型中的体内抗肿瘤药效；
图23为本申请实施例14中的mAb001c-vcMMAE(5mg/kg，2.5mg/kg)在HT-29结肠癌肿瘤模型中的体内抗肿瘤药效；
图24为本申请实施例17中的ELISA检测mAb001c的人源化抗体系列Hu001-2、3、5、6、8、11、12、14、15对ROR1-ECD的结合亲和力(Binding affinity EC50)；
图25为本申请实施例17中的ELISA检测mAb005c的人源化抗体系列Hu005-35、40、41、42、43、44、45、46对ROR1-ECD的结合亲和力(Binding affinity EC50)；
图26为本申请实施例18中的ELISA检测人源化抗体系列Hu001-2、Hu005-46对人ROR1-ECD和人ROR2-ECD的结合亲和力(Binding affinity EC50)；
图27为本申请实施例19中的FACS检测mAb001c的人源化抗体系列Hu001-2、3、5、6、8、11、12、14、15及mAb005c的人源化抗体系列Hu005-35、40、41、42、43、44、45、46对MDA-MB-231的结合亲和力(Binding affinity EC50)；
图28为本申请实施例19中的FACS检测mAb001c的人源化抗体系列Hu001-2、3、5、6、8、11、12、14、15及mAb005c的人源化抗体系列Hu005-35、40、41、42、43、44、45、46对NCI-H226的结合亲和力(Binding affinity EC50)；
图29为本申请实施例20中的人源化抗体Hu001-02、Hu001-03、Hu005-44、Hu005-46与MDA-MB-231细胞结合导致内吞(Internalization)至细胞内溶酶体；所述抗体(5μg/mL)与细胞孵育4℃1小时，或37℃4小时后置于激光共聚焦显微镜观察结果；
图30为本申请实施例21中的人源化抗体Hu001-02、Hu001-03、Hu005-44、Hu005-46与MDA-MB-231细胞结合0，30min，60min，120min，240min时的内吞曲线；
图31为本申请实施例21中的人源化抗体Hu001-02、Hu005-46与NCI-N87细胞结合0，30min，60min，120min，240min时的内吞曲线；
图32为本申请实施例21中的人源化抗体Hu001-02、Hu005-46与NCI-H446细胞结合0，30min，60min，120min，240min时的内吞曲线；
图33为本申请实施例22中的人源化抗体Hu001-2及抗体药物偶联物Hu001-2-MMAE的疏水作用层析(HIC)-HPLC图谱；
图34为本申请实施例22中的人源化抗体Hu001-2及抗体药物偶联物Hu001-2-MMAE的分子排阻(SEC)-HPLC图谱；
图35为本申请实施例22中的人源化抗体Hu001-3及抗体药物偶联物Hu001-3-MMAE的疏水作用层析(HIC)-HPLC图谱；
图36为本申请实施例22中的人源化抗体Hu001-3及抗体药物偶联物Hu001-3-MMAE的分子排阻(SEC)-HPLC图谱；
图37为本申请实施例22中的人源化抗体Hu005-44及抗体药物偶联物Hu005-44-MMAE的疏水作用层析(HIC)-HPLC图谱；
图38为本申请实施例22中的人源化抗体Hu005-44及抗体药物偶联物Hu005-44-MMAE的分子排阻(SEC)-HPLC图谱；
图39为本申请实施例22中的人源化抗体Hu005-46及抗体药物偶联物Hu005-46-MMAE的疏水作用层析(HIC)-HPLC图谱；
图40为本申请实施例22中的人源化抗体Hu005-46及抗体药物偶联物Hu005-46-MMAE的分子排阻(SEC)-HPLC图谱；
图41为本申请实施例22中的阳性参照抗体UC961及抗体药物偶联物UC961-MMAE的疏水作用层析(HIC)-HPLC图谱；
图42为本申请实施例22中的阳性参照抗体UC961及抗体药物偶联物UC961-MMAE的分子排阻(SEC)-HPLC图谱；
图43为本申请实施例23中的ELISA检测人源化抗体Hu001-2、Hu001-3、Hu005-44、Hu005-46及抗体偶联物Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE对ROR1-ECD的结合亲和力(Binding affinity EC50)；
图44为本申请实施例24中的FACS检测人源化抗体Hu001-2、Hu001-3、Hu005-44、Hu005-46及抗体偶联物Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE对MDA-MB-231的结合亲和力(Binding affinity EC50)；
图45为本申请实施例24中的FACS检测人源化抗体Hu001-2、Hu001-3、Hu005-44、Hu005-46及抗体偶联物Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE对NCI-H226的结合亲和力(Binding affinity EC50)；
图46为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、 Hu005-46-MMAE、UC961-MMAE对SK-BR-3细胞的体外抗增殖活性(IC50)的检测结果；
图47为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对MCF-7细胞的体外抗增殖活性(IC50)的检测结果；
图48为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对MDA-MB-231细胞的体外抗增殖活性(IC50)的检测结果；
图49为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对HCC1187细胞的体外抗增殖活性(IC50)的检测结果；
图50为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对HT-29细胞的体外抗增殖活性(IC50)的检测结果；
图51为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对SK-CO-1细胞的体外抗增殖活性(IC50)的检测结果；
图52为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对Colo-678细胞的体外抗增殖活性(IC50)的检测结果；
图53为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对HCC827细胞的体外抗增殖活性(IC50)的检测结果；
图54为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对NCI-H446细胞的体外抗增殖活性(IC50)的检测结果；
图55为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对PA-1细胞的体外抗增殖活性(IC50)的检测结果；
图56为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE对NCI-N87细胞的体外抗增殖活性(IC50)的检测结果；
图57为本申请实施例25中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE的细胞毒性(IC50值)与受试细胞的ROR1表达水平直接相关，表现为靶标特异性细胞毒性；
图58为本申请实施例26中的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE(5mg/kg，2.5mg/kg)在MDA-MB-231三阴性乳腺癌肿瘤模型中的体内抗肿瘤药效；
图59为本申请实施例26中的Hu001-2-MMAE、Hu005-46-MMAE、UC961-MMAE(5mg/kg，2.5mg/kg)在PA-1卵巢癌肿瘤模型中的体内抗肿瘤药效；
图60为本申请实施例26中的Hu001-2-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE(5mg/kg，2.5mg/kg)在NCI-N87胃癌肿瘤模型中的体内抗肿瘤药效；
图61为本申请实施例26中的Hu001-2-MMAE、UC961-MMAE(5mg/kg，2.5mg/kg)在HCC1187三阴性乳腺癌肿瘤模型中的体内抗肿瘤药效。
具体实施方式
下面结合附图、实施方式和实施例，对本申请作进一步详细的说明。应理解，这些实施方式和实施例仅用于说明本申请而不用于限制本申请的范围，提供这些实施方式和实施例的目的是使对本申请公开内容理解更加透彻全面。还应理解，本申请可以以许多不同的形式来实现，并不限于本文所描述的实施方式和实施例，本领域技术人员可以在不违背本申请内涵的情况下作各种改动或修改，得到的等价形式同样落于本申请的保护范围。此外，在下文的描述中，给出了大量具体的细节以便提供对本申请更为充分地理解，应理解，本申请可以无需一个或多个这些细节而得以实施。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述实施方式和实施例的目的，不是旨在于限制本申请。
术语
除非另外说明或存在矛盾之处，本文中使用的术语或短语具有以下含义：
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A及/或B”包括A、B和A+B三种并列方案。又比如，“A，及/或，B，及/或，C，及/或，D”的技术方 案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，也即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。
本申请中涉及“多个”、“多种”、“多次”、“多元”等，如无特别限定，指在数量上大于2或等于2。例如，“一种或多种”表示一种或大于等于两种。
本文中所使用的“其组合”、“其任意组合”、“其任意组合方式”等中包括所列项目中任两个或任两个以上项目的所有合适的组合方式。
本文中，“合适的组合方式”、“合适的方式”、“任意合适的方式”等中所述“合适”，以能够实施本申请的技术方案、解决本申请的技术问题、实现本申请预期的技术效果为准。
本文中，“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本申请保护范围的限制。
本申请中，“进一步”、“更进一步”、“特别”等用于描述目的，表示内容上的差异，但并不应理解为对本申请保护范围的限制。
本申请中，“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“可选”各自独立。
本申请中，“第一方面”、“第二方面”、“第三方面”、“第四方面”等中，术语“第一”、“第二”、“第三”、“第四”等仅用于描述目的，不能理解为指示或暗示相对重要性或数量，也不能理解为隐含指明所指示的技术特征的重要性或数量。而且“第一”、“第二”、“第三”、“第四”等仅起到非穷举式的列举描述目的，应当理解并不构成对数量的封闭式限定。
本申请中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本申请中，涉及到数值区间(也即数值范围)，如无特别说明，可选的数值分布在上述数值区间内视为连续，且包括该数值范围的两个数值端点(即最小值及最大值)，以及这两个数值端点之间的每一个数值。如无特别说明，当数值区间仅仅指向该数值区间内的整数时，包括该数值范围的两个端点整数，以及两个端点之间的每一个整数，在本文中，相当于直接列举了每一个整数，比如t为选自1-10的整数，表示t为选自由1、2、3、4、5、6、7、8、9和10构成的整数组的任一个整数。此外，当提供多个范围描述特征或特性时，可以合并这些范围。换言之，除非另有指明，否则本文中所公开之范围应理解为包括其中所归入的任何及所有的子范围。
本申请中的温度参数，如无特别限定，既允许为恒温处理，也允许在一定温度区间内存在变动。应当理解的是，所述的恒温处理允许温度在仪器控制的精度范围内进行波动。允许在如±5℃、±4℃、±3℃、±2℃、±1℃的范围内波动。
本申请中，％(w/w)与wt％均表示重量百分比，％(v/v)指体积百分比，％(w/v)指质量体积百分数。
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。除非和本申请的申请目的和/或技术方案相冲突，否则，本申请涉及的引用文献以全部内容、全部目的被引用。本申请中涉及引用文献时，相关技术特征、术语、名词、短语等在引用文献中的定义也一并被引用。本申请中涉及引用文献时，被引用的相关技术特征的举例、优选方式也可作为参考纳入本申请中，但以能够实施本申请为限。应当理解，当引用内容与本申请中的描述相冲突时，以本申请为准或者适应性地根据本申请的描述进行修正。
抗体的制备
本申请的抗体或其抗原结合片段的DNA分子的片段可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。一旦获得了有关的序列信息，就可以用重组法来大批量地获得有关序列片段。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列片段。
此外，还可用人工合成的方法来合成有关序列片段，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本申请的抗体(或其片段，或其衍生物)的DNA 序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本申请蛋白序列中。
本申请还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本申请抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本申请的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本申请的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
抗体偶联药物(ADC)
本申请还提供了抗体偶联药物(antibody-drug conjugate，ADC)。典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物、检测试剂、稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
典型的适用于本申请的偶联方式，包括K-Lock和C-Lock两种偶联方式。在K-Lock偶联方式中，药物分子偶联于抗体序列中赖氨酸(K)残基，在C-Lock偶联方式中，药物分子偶联于抗体序列中的半胱氨酸(C)残基。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细 胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)、7-乙基-10-羟基喜树碱(SN38)、依沙替康(Exatecan)及其类似物等。
在本申请中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
药物(Drug)
如本文所用，“药物”泛指任何具有期望的生物活性，并具有反应性官能团以便制备本申请所述偶联物的化合物。期望的生物活性包括，诊断，治愈，缓解，治疗，预防人或其它动物的疾病。因此，只要具有必需的反应性官能团，术语“药物”涉及的化合物包括正式国家药典，以及例如美国正式同种疗法药典，正式全国处方集，或者其任何增补本等确认的药物。典型的药物列于医师案头用药参考(PDR)和美国食品药品监督管理局(FDA)的橙皮书。应理解，随着新型药物不断被发现和发展，这些药物也应纳入本申请所述偶联药物的中的“药物”。
本申请的第一方面
本申请提供一种能够特异性结合ROR1的抗体，所述抗体包括重链可变区和轻链可变区：
所述重链可变区的重链互补决定区选自如下i)至iii)所示的重链互补决定区或其衍生片段中的一组或者多组：
i)SEQ ID NO.1所示的VH-CDR1，SEQ ID NO.2所示的VH-CDR2，和SEQ ID NO.3所示的VH-CDR3；
ii)SEQ ID NO.11所示的VH-CDR1，SEQ ID NO.12所示的VH-CDR2，和SEQ ID NO.13所示的VH-CDR3；和
iii)SEQ ID NO.19的VH-CDR1，SEQ ID NO.20所示的VH-CDR2，和SEQ ID NO.21所示的VH-CDR3；
和/或，
所述轻链可变区的轻链互补决定区选自i’)至iii’)所示的轻链互补决定区或其衍生片段中的一组或者多组：
i’)SEQ ID NO.4所示的VL-CDR1，SEQ ID NO.5所示的VL-CDR2，和SEQ ID NO.6所示的VL-CDR3；
ii’)SEQ ID NO.14所示的VL-CDR1，SEQ ID NO.:15所示的VL-CDR2，和SEQ ID NO.16所示的VL-CDR3；和
iii’)SEQ ID NO.22所示的VL-CDR1，SEQ ID NO.23所示的VL-CDR2，和SEQ ID NO.24所示的VL-CDR3；
所述衍生片段与其对应的互补决定区具有不超过6个位点的氨基酸替换且能够保留与ROR1-ECD EC50为0.009nM-0.05nM以及与肿瘤细胞ROR1EC50为0.15nM-1.1nM。
在其中一个实施例中，所述衍生片段是相对其对应的互补决定区经过不超过6个位点的氨基酸替换形成的，保留有与其对应的互补决定区一致的生物学活性。例如，所述衍生片段相对于其对应的互补决定区1、2、3、4、5或者6个位点发生替换，可以是一个氨基酸被另一个氨基酸替换，也可以是一个氨基酸被多个(例如2个)氨基酸替换。
在本申请中，衍生片段(保守性变异体)，指与本申请抗体的氨基酸序列相比，有1、2、3个氨基 酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表-1进行氨基酸替换而产生。
表-1
在其中一个实施例中，所述衍生片段对ROR1(如人ROR1蛋白胞外区，ROR1-ECD)的亲和力EC50为0.02nM-0.05nM，可选地为0.02-0.046nM，可选地为0.02nM-0.028nM。
在另一优选例中，所述衍生片段对肿瘤细胞表面ROR1的亲和力EC50为0.06nM-0.08nM，可选地为0.06nM-0.073nM，可选地为0.06nM-0.067nM。
抗体，如本申请所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本申请所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本申请不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的重组蛋白。因此，本申请还包括所述抗体的片段、衍生物和类似物。
在本申请中，抗体包括用本领域技术人员熟知技术所制备的动物源的(例如鼠、兔、羊、牛、马、 猪、狗、猫、骆驼、驴、鹿、貂、鸡、鸭或者鹅)、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。本申请中的抗体可以是靶向人ROR1的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本申请中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性，其抗原结合片段中至少一个包含本申请中定义的重链互补决定区和轻链互补决定区。
在其中一个实施例中，所述重链可变区的重链互补决定区选自如下i)至iii)所示的重链互补决定区中的一组或者多组：
i)VH-CDR1的序列通式为D-Y-N-Xa1-H，VH-CDR2的序列通式为Y-I-N-P-N-Xa2-Xa3-Xa4-T-Xa5-Y-N-Q-K-F-Xa6-G，VH-CDR3的序列通式为R-Xa7-Xa8-Xa9-Xa10-Xa11-Xa12-Xa13-D-Xa14；其中，Xa1为I、M或者L，Xa2为N或者H，Xa3为D或者G，Xa4为A、N或者G，Xa5为S、N或者T，Xa6为Q、K或者E，Xa7为V或者G，Xa8为R或者Y，Xa9为T或者G，Xa10为S或者G，Xa11为S、T或者G，Xa12为G或者Y，Xa13为L、F或者AM，Xa14为D、F或者Y；
ii)VH-CDR1如SEQ ID NO.11所示，VH-CDR2如SEQ ID NO.12所示，VH-CDR3如SEQ ID NO.13所示；
iii)VH-CDR1如SEQ ID NO.19所示，VH-CDR2的序列通式为T-I-S-D-Xb1-G-S-Y-T-Y-Y-P-D-Xb2-Xb3-K-G，VH-CDR3如SEQ ID NO.21所示；其中，Xb1为A或者G，Xb2为S或者N，Xb3为V或者E；
或/和，
所述轻链可变区的轻链互补决定区选自i’)至iii’)所示的轻链互补决定区或其衍生片段中的一组或者多组：
i’)VL-CDR1的序列通式为Xa1’-S-S-Xa2’-Xa3’-I-Xa4’-H-T-N-Xa5’-N-T-Y-L-E，VL-CDR2的序列通式为K-V-Xa6’-N-R-F-S，VL-CDR3的序列通式为F-Q-G-S-Xa7’-Xa8’-P-Y-T；其中，Xa1’为R、K或者T，Xa2’为H或者Q，Xa3’为I、N或者S，Xa4’为V或者L，Xa5’为A或者G，Xa6’为F或者S，Xa7’为R或者L，Xa8’位F或者V；
ii’)VL-CDR1如SEQ ID NO.14所示，VL-CDR2如SEQ ID NO.15所示，VL-CDR3如SEQ ID NO.16所示；
iii’)VL-CDR1的序列通式为K-A-S-Q-S-V-S-F-Xb1’-G-T-S-L-M-H，VL-CDR2如SEQ ID NO.23所示，VL-CDR3如SEQ ID NO.24所示；其中，Xb1’为A或者P。
在其中一个实施例中，i)所示的重链互补决定区中：
Xa1为L，Xa2为H，Xa3为D，Xa4为A或者G，Xa5为S或者T，Xa6为Q，Xa7为V，Xa8为R，Xa9为T，Xa10为G，Xa11为T，Xa12为G，Xa13为L或者F，Xa14为D或者Y；
或者，
Xa1为I或者M，Xa2为N，Xa3为G，Xa4为N或者G，Xa5为S、N或者T，Xa6为K或者E，Xa7为G，Xa8为Y，Xa9为G，Xa10为S，Xa11为S或者G，Xa12为Y，Xa13为AM，Xa14为F或者Y。
在其中一个实施例中，i’)所示的轻链互补决定区中：
Xa1’为R或者K，Xa2’为H，Xa3’为N，Xa4’为V或者L，Xa5’为A或者G，Xa6’为S，Xa7’为R，Xa8’为F；
或者，
Xa1’为R或者T，Xa2’为H或者Q，Xa3’为I或者S，Xa4’为V，Xa5’为G，Xa6’为F或者S，Xa7’为R或者L，Xa8’为F或者V。
在其中一个实施例中，所述抗体具有如下所示的重链互补决定区和轻链互补决定区的组合中的一组或者多组：
组合1为：i)所示的重链互补决定区或其衍生片段，i’)所示的轻链互补决定区或其衍生片段；
组合2为：ii)所示的重链互补决定区或其衍生片段，ii’)所示的轻链互补决定区或其衍生片段；
组合3为：iii)所示的重链互补决定区或其衍生片段，iii)所示的轻链互补决定区或其衍生片段。
在其中一个实施例中，所述抗体具有如下技术特征中的一个或者多个：
所述组合1选自如下组合中的一组或者多组：
组合1-1为：SEQ ID NO.1所示的VH-CDR1、SEQ ID NO.2所示的VH-CDR2、SEQ ID NO.3所示的VH-CDR3、SEQ ID NO.4所示的VL-CDR1、SEQ ID NO.5所示的VL-CDR2以及SEQ ID NO.6所示的VL-CDR3；
组合1-2为：SEQ ID NO.35所示的VH-CDR1、SEQ ID NO.36所示的VH-CDR2、SEQ ID NO.37所示的VH-CDR3、SEQ ID NO.38所示的VL-CDR1、SEQ ID NO.39所示的VL-CDR2以及SEQ ID NO.40所示的VL-CDR3；
组合1-3为：SEQ ID NO.51所示的VH-CDR1、SEQ ID NO.52所示的VH-CDR2、SEQ ID NO.53所示的VH-CDR3、SEQ ID NO.54所示的VL-CDR1、SEQ ID NO.55所示的VL-CDR2以及SEQ ID NO.56所示的VL-CDR3；
组合1-4为：SEQ ID NO.59所示的VH-CDR1、SEQ ID NO.60所示的VH-CDR2、SEQ ID NO.61所示的VH-CDR3、SEQ ID NO.62所示的VL-CDR1、SEQ ID NO.63所示的VL-CDR2以及SEQ ID NO.64所示的VL-CDR3；
组合1-5为：SEQ ID NO.67所示的VH-CDR1、SEQ ID NO.68所示的VH-CDR2、SEQ ID NO.69所示的VH-CDR3、SEQ ID NO.70所示的VL-CDR1、SEQ ID NO.71所示的VL-CDR2以及SEQ ID NO.72所示的VL-CDR3；
组合1-6为：SEQ ID NO.75所示的VH-CDR1、SEQ ID NO.76所示的VH-CDR2、SEQ ID NO.77所示的VH-CDR3、SEQ ID NO.78所示的VL-CDR1、SEQ ID NO.79所示的VL-CDR2以及SEQ ID NO.80所示的VL-CDR3；
组合1-7为：SEQ ID NO.1所示的VH-CDR1、SEQ ID NO.83所示的VH-CDR2、SEQ ID NO.3所示的VH-CDR3、SEQ ID NO.84所示的VL-CDR1、SEQ ID NO.5所示的VL-CDR2以及SEQ ID NO.6所示的VL-CDR3；
所述组合3选自如下组合中的一组或者多组：
组合3-1为：SEQ ID NO.43所示的VH-CDR1、SEQ ID NO.44所示的VH-CDR2、SEQ ID NO.45所示的VH-CDR3、SEQ ID NO.46所示的VL-CDR1、SEQ ID NO.47所示的VL-CDR2以及SEQ ID NO.48所示的VL-CDR3；
组合3-2为：SEQ ID NO.19所示的VH-CDR1、SEQ ID NO.20所示的VH-CDR2、SEQ ID NO.21所示的VH-CDR3、SEQ ID NO.22所示的VL-CDR1、SEQ ID NO.23所示的VL-CDR2以及SEQ ID NO.24所示的VL-CDR3；
组合3-3为：SEQ ID NO.19所示的VH-CDR1、SEQ ID NO.85所示的VH-CDR2、SEQ ID NO.21所示的VH-CDR3、SEQ ID NO.22所示的VL-CDR1、SEQ ID NO.23所示的VL-CDR2以及SEQ ID NO.24所示的VL-CDR3。
在其中一个实施例中，所述抗体具有如下技术特征中的一个或者多个：
(1)所述抗体或其抗原结合片段的重链恒定区和轻链恒定区的种属来源独立地选自人、鼠、兔、羊、牛、马、猪、狗、猫、骆驼、驴、鹿、貂、鸡、鸭或者鹅；和，
(2)所述抗体的恒定区选自选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE及IgD任何其中之一恒定区的序列。
(3)所述抗体的恒定区选自κ轻链恒定区和λ轻链恒定区任何其中之一恒定区的序列。
在其中一个实施例中，所述抗体具有选自如下所示的重链可变区和轻链可变区的组合中的一组或者多组：
(1)SEQ ID NO.7所示的重链可变区和SEQ ID NO.9所示的轻链可变区；
(2)SEQ ID NO.41所示的重链可变区和SEQ ID NO.42所示的轻链可变区；
(3)SEQ ID NO.49所示的重链可变区和SEQ ID NO.50所示的轻链可变区；
(4)SEQ ID NO.17所示的重链可变区和SEQ ID NO.18所示的轻链可变区；
(5)SEQ ID NO.25所示的重链可变区和SEQ ID NO.27所示的轻链可变区；
(6)SEQ ID NO.57所示的重链可变区和SEQ ID NO.58所示的轻链可变区；
(7)SEQ ID NO.65所示的重链可变区和SEQ ID NO.66所示的轻链可变区；
(8)SEQ ID NO.73所示的重链可变区和SEQ ID NO.74所示的轻链可变区；
(9)SEQ ID NO.81所示的重链可变区和SEQ ID NO.82所示的轻链可变区；
(10)SEQ ID NO.8所示的重链可变区和SEQ ID NO.10所示的轻链可变区；
(11)SEQ ID NO.26所示的重链可变区和SEQ ID NO.27所示的轻链可变区；
(12)SEQ ID NO.28所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(13)SEQ ID NO.28所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
(14)SEQ ID NO.29所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(15)SEQ ID NO.29所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
(16)SEQ ID NO.30所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(17)SEQ ID NO.31所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(18)SEQ ID NO.31所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
(19)SEQ ID NO.32所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
(20)SEQ ID NO.32所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
(21)SEQ ID NO.93所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(22)SEQ ID NO.94所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(23)SEQ ID NO.95所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(24)SEQ ID NO.96所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(25)SEQ ID NO.97所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(26)SEQ ID NO.98所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
(27)SEQ ID NO.99所示的重链可变区和SEQ ID NO.101所示的轻链可变区；和，
(28)SEQ ID NO.100所示的重链可变区和SEQ ID NO.101所示的轻链可变区。
在其中一个实施例中，所述的抗体为原始的鼠源抗体mAb001、mAb002、mAb003、mAb004、mAb005、mAb006、mAb007、mAb008、mAb009。
在其中一个实施例中，所述的抗体为人-鼠嵌合抗体mAb001c、mAb001c_v1、mAb002c、mAb003c、mAb004c、mAb005c、mAb005c_v1、mAb006c、mAb007c、mAb008c、mAb009c。
在其中一个实施例中，所述的抗体为人源化抗体Hu001-2、Hu001-3、Hu001-5、Hu001-6、Hu001-8、Hu001-11、Hu001-12、Hu001-14、Hu001-14、Hu001-15、Hu005-35、Hu005-40、Hu005-41、Hu005-42、Hu005-43、Hu005-44、Hu005-45、Hu005-46。
本申请第一方面提供的所述抗体具有选自下组的一个或多个特性：
(a)抑制ROR1的生物活性；
(b)特异结合ROR1-ECD蛋白或肿瘤细胞表面的ROR1；
(c)结合肿瘤细胞ROR1能快速介导靶标内吞；
(d)抑制肿瘤细胞迁移或转移；
(f)抑制肿瘤生长，提高联合用药的抗肿瘤疗效；
(g)能够减少抗肿瘤治疗耐药性的出现。
本申请提供3大类靶向ROR1的高特异性和高亲和力的抗体，可以联合应用，用于构建CAR构建物、包含CAR构建物的重组的免疫细胞、抗体偶联药物等，还可以用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备预防和/或治疗ROR1相关疾病的药物。
本申请的第二方面
本申请提供一种重组蛋白，所述重组蛋白包括：
第一方面中定义的重链互补决定区和轻链互补决定区中的一个或者多个；和，
协助所述的重链互补决定区和轻链互补决定区表达和/或纯化的标签片段。
在本申请的一些实施方式中，所述重组蛋白还包括第一方面中定义的重链恒定区、轻链恒定区或其组合。
本申请中，所述标签序列包括但不限于6His标签。
本申请中，所述重组蛋白(或多肽)包括但不限于融合蛋白。
本申请中，所述重组蛋白可以为单体、二聚体、或多聚体。
本申请的第三方面
本申请提供一种CAR构建物，所述CAR构建物的scFV结构域具有第一方面中定义的重链可变区和轻链可变区。
可以理解的是，scFV结构域为ROR1的特异性结合区。
本申请的第四方面
本申请提供一种核酸，所述核酸编码第一方面中所述的抗体或其抗原结合片段、第二方面中所述的重组蛋白或者第三方面中所述的CAR构建物。
本申请的第五方面
本申请提供一种载体，所述载体含有第四方面所述的核酸。
本申请中，所述载体包括但不限于细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其组合。
本申请的第六方面
本申请提供一种宿主细胞，所述宿主细胞包含第四方面中所述的核酸或者第五方面中所述的载体。
本申请中，所述的核酸可以整合到所述宿主细胞的基因组中。
本申请中，所述宿主细胞包括但不限于CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞和HEK293细胞。
本申请的第七方面
本申请提供第一方面中所述的抗体或其抗原结合片段、第二方面中所述的重组蛋白或者第三方面中所述的CAR构建物的制备方法，所述制备方法包括如下步骤：
培养第六方面中所述的宿主细胞，从所得培养物中分离抗体或其抗原结合片段、重组蛋白或者CAR构建物。
可以理解的是，培养是在合适的培养条件下，包括但不限于：合适的培养基、合适的温度、合适的时间等。
参照第一方面中定义的，本申请的抗体可以是嵌合抗体，也可以人源化抗体。
在其中一些实施例中，所述嵌合抗体的制备方法，包括步骤：
将本申请第四方面所述的核苷酸克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达嵌合抗体。
在其中一些实施例中，所述人源化抗体的制备方法，包括步骤：
将本申请第四方面所述核酸植入含人源抗体FR区的模板，再将其克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达人源化抗体。
本申请的第八方面
本申请提供一种重组的免疫细胞，所述重组的免疫细胞表达外源的第三方面中所述的CAR构建物。
本申请中，所述免疫细胞可以选自包括但不限于：NK细胞和T细胞。
本申请中，所述免疫细胞的来源可以人，也可以是其他哺乳动物(例如鼠)。
本申请的第九方面
本申请提供第一方面中所述的抗体或其抗原结合片段、第二方面中所述的重组蛋白、第三方面中所述的CAR构建物或者第八方面中所述的重组的免疫细胞在制备治疗耐药性肿瘤的药物、治疗ROR1相关疾病的药物或者ROR1相关疾病的诊断产品中的应用。
本申请中，治疗，包括预防、控制、辅助治疗等。
在其中一个实施方式中，所述ROR1相关疾病为ROR1高表达肿瘤、自身免疫疾病、代谢相关疾病或者感染疾病。
本申请中，肿瘤可以是实体瘤，也可以是血癌。
本申请中，所述的ROR1高表达的肿瘤指肿瘤组织中ROR1转录本和/或蛋白的水平L1与正常组织中转录本和/或蛋白的水平L0之比，L1/L0≥2，较佳地≥3。
在本申请的一些实施方式中，所述肿瘤为乳腺癌、肺癌、胰腺癌、卵巢癌、前列腺癌、结直肠癌、胃癌、肝癌、食道癌、肾癌、脑胶质瘤、膀胱癌、前列腺癌、子宫内膜癌、宫颈癌、白血病、淋巴瘤、骨髓癌或者血管肉瘤；
所述炎症为风湿性关节炎、骨关节炎、痛风、莱特尔综合征、牛皮癣性关节病、结核性关节炎、肾小球性肾炎、全身性红斑狼疮、克罗恩病、溃疡性结肠炎、急性肺损伤、慢性阻塞性肺疾病或者特发性肺纤维化；
所述自身免疫疾病为类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、干燥综合征或者系统性血管 炎、溃疡性结肠炎、I型糖尿病、银屑病、多发性硬化症；
所述代谢相关疾病为糖尿病、食源性肥胖或者脂肪炎症；
所述感染疾病为细菌感染所致疾病或者病毒感染所致疾病。
本申请中，药物的形式可以是抗体偶联药物，也可以是药物组合物，其用于治疗ROR1高表达的肿瘤、肿瘤迁移或肿瘤耐药。在其中一些实施例中，可以用于：
(a)特异结合肿瘤细胞，和/或肿瘤微环境中的免疫/基质细胞的ROR1；
(b)抑制肿瘤/肿瘤微环境中过度活化的ROR1生物功能；
(c)抑制肿瘤细胞迁移或转移；
(d)抑制肿瘤生长，提高联合用药的抗肿瘤疗效；
(e)抗体依赖的细胞介导的细胞毒性作用(ADCC)。
在其中一些实施例中，所述的肿瘤耐药包括：肿瘤免疫治疗药物的耐药、肿瘤靶向治疗药物的耐药、常规肿瘤化疗的耐药，放射治疗的不敏感。
在其中一些实施例中，所述ROR1相关疾病的诊断产品为诊断试剂、试纸条、检测板或者试剂盒。所述检测试剂、检测板或试剂盒可以用于：
(1)检测样品中的ROR1蛋白；
(2)检测肿瘤细胞中内源性的ROR1蛋白；
(3)检测表达ROR1蛋白的肿瘤细胞。
本申请以下第十方面的提供的抗体偶联药物、第十一方面提供的药物组合物，可以靶向瞄准特殊的细胞群体，与细胞表面特异蛋白(抗原)结合，从而通过结合物内吞或药物渗入使得药物以活性形式释放到细胞内，因此，本申请的抗体偶联药物、药物组合物可以用于治疗目标疾病，可以以治疗有效量，通过合适的途径给予受试者。需要治疗的受试者可以是有风险，或怀疑患有与特定抗原的活性或表达量有关病症的患者。这样的患者可以通过常规体检来鉴定。
常规方法，已知的医学领域的普通技术人员，可以施用抗体偶联药物、药物组合物给受试者，给药的方式取决于疾病的要治疗的类型或疾病的部位。例如，口服，肠胃外给药，通过吸入喷雾，局部，直肠，经鼻，口腔，阴道或通过植入进行给药。本文所用的术语“肠胃外”包括皮下，皮内，静脉内，肌内，关节内，动脉内，滑膜内，胸骨内，鞘内，病灶内和颅内注射或输注技术。
本申请的第十方面
本申请提供一种抗体偶联药物，所述抗体偶联药物包括：
抗体，所述抗体选自第一方面中定义的抗体；和，
与所述抗体偶联的偶联物。
本申请中，所述偶联物可以选自包括但不限于：可检测标记物、细胞毒性药物、细胞因子、放射性核素和酶。
“细胞毒性药物”是指抑制或阻止细胞表达活性、细胞功能和/或造成细胞破坏的物质。该术语包括放射性同位素、化学治疗剂以及毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体。包括但不限于：耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂以及糖皮质激素和其它化学治疗剂，以及放射性同位素，如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212或213、P32和包括Lu177在内的Lu的放射性同位素。抗体也可与能够将前药转化成其活性形式的抗癌前药活化酶偶联。
可选地，所述细胞毒性药物选用具有高细胞毒性的化合物，优选单甲基澳瑞他汀(monomethyl auristatin)、加利车霉素、美登素类、或其组合；更佳地选自：单甲基阿里他汀-E(MMAE)、单甲基阿里他汀-D(MMAD)、单甲基阿里他汀-F(MMAF)、或其组合。
可选地，所述细胞毒性药物选用用于癌症治疗的细胞毒性药物，或具有期望生物活性的蛋白或多肽，例如一种毒素，如相思子毒素，蓖麻毒素A，假单胞菌外毒素，和白喉毒素；其他合适的蛋白包括肿瘤坏死因子，α-干扰素，β-干扰素，神经原生长因子，血小板衍生生长因子，组织型纤酶溶原生长因子，以及生物反应调节制剂，例如淋巴因子，白细胞介素-1(IL-1)，白细胞介素-2(IL-2)，白细胞介素-6(IL-6)， 粒细胞巨噬细胞集落刺激因子(GM-CSF)，粒细胞集落刺激因子，或其它生长因子。
一种细胞毒性药物是美登素或类美登素。美登素化合物通过抑制微管蛋白的微管形成来抑制细胞增殖。类美登素是美登素的衍生物。美登素和类美登素都具有高效的细胞毒性，但是它们在癌症治疗的临床应用上具有很大的局限性，这主要是源于此类分子对肿瘤的低选择性。但是，这种高细胞毒性促使它们成为抗体药物偶联物的首选药物部分。以下列出了去乙酰基美登素的结构。
一种细胞毒性药物是耳抑素肽类药物。耳抑素肽类药物是海兔毒素10(Dolastatin10)的类似物，而后者是从海洋软体动物海兔体内分离出来的具有生物活性的多肽。海兔毒素10通过结合微管蛋白(与长春新碱同样的结合区域)而抑制微管蛋白聚合。海兔毒素10，耳抑素肽PE，耳抑素肽E都是线性多肽，含有四个氨基酸(其中三个氨基酸是海兔毒素类化合物所独有的)和C-端酰胺基团。两个代表性的耳抑素肽类化合物，单甲基耳抑素肽E(MMAE)和单甲基耳抑素肽F(MMAF)，都是抗体药物偶联物的首选药物。
一种细胞毒性药物是吡咯并苯二氮卓类(pyrrolo[2,1-c][1,4]benzodi-azepines，PBDs)或者PBD二聚体类(PBD dimers)。PBD是一类由链霉菌产生的天然产物，其独特特性在于能够在DNA小沟，确切是在嘌呤-鸟嘌呤-嘌呤序列处，形成非扭曲的共价加和物。应用PBD作为部分小分子策略靶向锁定DNA序列以及作为新型的抗癌和抗菌药物引起了越来越多的兴趣。应用一个柔性碳链连接两个PBD单元的C8/C8'的羟基基团，所得的二聚体具有增强的生物活性。PBD二聚体被认为是可以产成序列选择性的DNA损伤，例如倒序的5'-Pu-GATC-Py-3'链间交联，从而导致其生物活性。这些化合物已被证明是高效的细胞毒性药物，可作为抗体药物偶联物的备选药物。
一种细胞毒性药物是PNU-159682衍生物，PNU-159682是Nemorubicin在人肝微粒体中的主要活性代谢产物，与MMDX和阿霉素相比，活性提高3000倍。
细胞毒性药物并不仅仅局限于上述提到的类别，还包括所有可用于抗体药物偶联物的药物。并且尤其是那些能够通过与接头的酰胺键来配位，如通过具有碱性胺基(一级胺或二级胺)来配位的细胞毒素。
本申请中，所述细胞毒性药物可以分为：微管靶向药物、DNA靶向药物或者拓扑异构酶抑制剂。
本申请中，所述抗体和所述偶联物可以通过但不限于化学键或连接子进行偶联。
本申请中，所述连接子包含巯基特异性的活性反应基团，可以选自包括但不限于：4-(N-马来酰亚胺基甲基)环己烷-1-甲酸琥酸亚胺酯、马亚酰亚胺基己酰基、6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧基羰基或者双取代马来酰亚胺类连接子。可选地，所述连接子选自6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-val-cit-PAB)、6-马来酰亚氨基己酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MC-ala-phe-PAB)、马来酰亚氨基丙酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MP-val-citPAB)、马来酰亚氨基丙酰基-丙氨酸-苯丙氨酸-对氨基苄氧羰基(MP-ala-phe-PAB)、N-琥珀酰亚氨基4-(2-吡啶基硫基)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯(SMCC)、4-(2-吡啶基二硫代)丁酸-N-羟基琥珀酰亚胺酯(SPDB)或N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)。在其中一个实施例中，所述连接子为6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-val-cit-PAB)。
本申请中，所述抗体偶联药物可以具有如下分子式所示的结构：
其中：
Ab为抗体，
LU为连接子，
D为细胞毒性药物。
可选地，p是选自1-10，例如为1、2、3、4、5、6、7、8、9、10。在其中一些实施例中，p为1-8的值。
本申请实施例还涉及制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。在某些实施方式中，本申请方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本申请方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
本申请的抗体偶联药物，可以通过本领域常规的方法递送给药。例如，它可以通过使用脂质体、水凝胶、环糊精，生物可降解的纳米胶囊，或生物粘附性微球被引入到细胞中。
本申请的第十一方面
本申请提供一种药物组合物，所述药物组合物包括：
第一方面中所述的抗体、第二方面中所述的重组蛋白、第三方面中所述的CAR构建物或者第八方面中所述的重组的免疫细胞、第十方面中所述的抗体偶联药物或者其结合；和，
药学上可接受的载体。
在一些实施方式中，药物组合物的施用方式包括但不限于：口服、直肠、肠胃外(静脉内、肌肉内或皮下)注射、和局部给药、吸入。
在其中一些实施例中，药物组合物可通过口服、灌肠或肠胃外的形式给药。
在其中一些实施例中，药物组合物的给药周期可为间歇给药、周期性给药、持续给药或长期给药。
用于口服给药的固体剂型可以包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、 碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性成分或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性成分也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，具体例如，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。如悬浮液可包含悬浮剂，具体例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物。
用于肠胃外注射的液体剂型可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，以及用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水或非水载体、稀释剂、溶剂或赋形剂包括植物油、二甲基乙酰胺、二甲基甲酰胺、乳酸乙酯，碳酸乙酯、肉豆蔻酸异丙酯、水、乙醇、多元醇及其适宜的混合物。对于静脉内注射，水溶性抗体可以通过点滴方法，由此含有抗体和生理上可接受的赋形剂的药物制剂输注给药。生理上可接受的赋形剂可以包括，例如，5％葡萄糖，0.9％盐水，林格溶液或其它合适的赋形剂。肌内制剂，例如，抗体的一个合适的可溶盐形式的无菌制剂，可以溶解和施用的药用赋形剂诸如水换注射液，0.9％盐水，或5％葡萄糖溶液。
用于局部给药的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。
本申请的药物组合物由活性成分在无菌条件下与药学上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合而成。
本方面的上述药物制剂中的“活性成分”指药物组合物中能够发挥“药物”作用的成分。可以理解的是，本申请实施例的药物可以添加不同的药学上可接受的载体从而制备成合适的临床剂型，这些临床剂型包括但不限于上文所述剂型。
本申请的第十二方面
本申请提供一种ROR1相关疾病的诊断产品，所述诊断产品为检测板，所述检测板上包被有第一方面中所述的抗体或其抗原结合片段、第二方面中所述的重组蛋白、第三方面中所述的CAR构建物或者第八方面中所述的重组的免疫细胞或者其结合；
或者，所述诊断产品为检测试剂盒，所述检测试剂盒包含检测ROR1的一抗和二抗，所述一抗为第一方面中定义的抗体。
本申请中，所述检测板可以但不限于免疫层析板，也可以成为检测试纸，所述检测板可以包括基片(基板、基垫或者支撑板)以及层析纸等。
本申请中，所述检测试剂盒可以至少包括两个容器，一个用于放置第一方面所述的抗体；另一个用于放置抗所述抗体的二抗。
在其中一些实施例中，检测试剂盒还可以包括使用说明书、缓冲剂等。
本申请的第十三方面
本申请提供一种ROR1的检测方法，所述检测方法包括如下步骤：
提供待测样本，
将所述待测样本和第一方面中所述的抗体进行混合反应，检测反应产物并根据检测结果判断所述待测样本中是否存在ROR1。
在本申请的一些实施方式中，根据反应结果判断所述待测样本中是否存在ROR1包括：
当在所述反应产物中检测到抗原-抗体复合物，则判断所述待测样本中含有ROR1；
当在所述反应产物中未检测到抗原-抗体复合物，则判断所述待测样本中不含ROR1。
本申请中，检测的方法可以是基于诊断目的的，也可以是基于非诊断目的的。
本申请中，所述待测样本(样品)包括细胞、组织样本和活检标本。本申请使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本申请中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本申请中使用的待测样本包括固定的或保存的细胞或组织样本。
本申请的第十四方面
本申请提供一种治疗ROR1相关疾病的方法，所述方法包括如下步骤：
给予受试者有效量的本申请第一方面所述的抗体、第八方面中所述的重组的免疫细胞、第十方面中 所述的抗体偶联药物、第十一方面中所述的药物组合物或其组合。
ROR1相关疾病的定义参照第九方面。
在其中一些实施例中，所述方法还包括：给予受试者施用其他药物或治疗方法进行联合治疗。
在其中一些实施例中，所述的其他药物或治疗方法包括但不限于：抗肿瘤免疫治疗药物、肿瘤靶向药物、肿瘤化疗药物、肿瘤放射治疗。
在其中一些实施例中，所述的抗肿瘤免疫治疗药物包括但不限于：PD-1、PD-L1单抗。
在其中一些实施例中，施用方式包括但不限于：口服、直肠、肠胃外(静脉内、肌肉内或皮下)注射、和局部给药、吸入。
本申请中，“受试者”是动物，可以是人，也可以是非人的其他哺乳动物。受试者包括但不限于药物的食用者和具有疾病、病症和/或症状的患者。本申请中术语“哺乳动物”主要是指温血脊椎类哺乳动物，包括但不限于：如猫、狗、兔、熊、狐狸、狼、猴子、鹿、鼠(如大鼠、小鼠)、猪、牛、羊、马、人等，优选灵长类动物，更优选为人。
本申请的第十五方面
本申请提供了一种抑制肿瘤细胞生长和迁移的方法，所述方法包括如下步骤：给予受试者有效量的本申请第一方面所述的抗体、第八方面中所述的重组的免疫细胞、第十方面中所述的抗体偶联药物、第十一方面中所述的药物组合物或其组合。
在其中一些实施例中，所述方法还包括：给予受试者施用其他药物或治疗方法进行联合治疗。
“其他药物或治疗方法”、“抗肿瘤免疫治疗药物”、“受试者”参照第十四方面中定义。
本申请的第十六方面
本申请提供了一种抑制肿瘤在模型动物体内生长的方法，所述方法包括如下步骤：给予模型动物有效量的本申请第一方面所述的抗体、第八方面中所述的重组的免疫细胞、第十方面中所述的抗体偶联药物、第十一方面中所述的药物组合物或其组合。
在其中一些实施例中，所述方法还包括：给予模型动物施用其他药物或治疗方法进行联合治疗。
“其他药物或治疗方法”、“抗肿瘤免疫治疗药物”参照第十四方面中定义。
本申请的上述第十四、十五、十六方面中，在本申请中，“有效量”是指该术语所对应的组分在受试者中实现治疗、预防、减轻和/或缓解特定疾病、病症和/或症状的剂量，本申请中，如无特别限定，指实现治疗、预防、减轻和/或缓解高表达ROR1相关疾病、病症和/或症状的剂量。给予受试者药物的过程中，是将安全有效量的适用于受试者。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。以本申请所述的抗体偶联药物为例，其有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的双功能抗体偶联药物的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本申请的抗体偶联药物每天以约0.0001mg-50mg/kg动物体重(较佳的0.001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。细胞株为常规的市售产品或购自ATCC，质粒均为市售产品。
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。
以下实施例中，申请人通过广泛而深入的研究，经过大量筛选，意外地获得了9个抗ROR1单克隆抗体，分别命名为mAb001-mAb009并选择人IgG1,κ为骨架构建人-鼠嵌合抗体，将其分别命名为mAb001c-mAb009c。进一步测试上述抗体后获得结果如下：
第一、所述的嵌合抗体均能够高特异性地结合ROR1-ECD抗原，ELISA测定其EC50值均≦0.02nM；
第二、所述的嵌合抗体针对多株ROR1高表达的肿瘤细胞具有极高的结合亲和力，FACS测定其EC50为0.15nM-1.1nM，综合抗体CDR区序列的相似性、抗体针对抗原结合活性及结合表位的独特性，我们优选mAb001c和mAb005c用于后续开发；
第三、基于mAb001c设计的一系列人源化抗体具有更高的ROR1蛋白结合亲和力及细胞结合亲和力；ELISA测定其EC50为0.010nM-0.015nM；FACS测定其EC50为0.19nM-0.62nM；
第四、所述嵌合抗体具备显著的体内抗肿瘤作用，而对于哺乳动物本身没有明显的毒副作用；
第五、基于mAb005c设计的一系列人源化抗体具有更高的ROR1蛋白结合亲和力及细胞结合亲和力；ELISA测定其EC50为0.009nM-0.02nM；FACS测定其EC50为0.12nM-0.22nM。
第六、所述嵌合抗体及人源化抗体均具备优异的内吞特性；
第七、所述嵌合抗体-药物偶联物及人源化抗体-偶联物(ADC)均具有优异的抗增殖活性，即在体外对ROR1-低表达的细胞无明显毒副作用，而对ROR1-高表达的肿瘤细胞具有极高的杀伤作用，杀伤活性与ROR1表达水平相关；同时所述嵌合抗体ADC及人源化抗体ADC药物在体内对ROR1不同表达水平的肿瘤模型均具备优异的抗肿瘤活性。
本申请涉及的各序列的代表含义如下：
表-2

实施例1靶向人ROR1单克隆抗体的发现和制备
步骤①杂交瘤细胞的制备：
首先，制备人ROR1蛋白的胞外区(ROR1-ECD)，作为备用抗原。参照NCBI:NP_005003.2氨基酸的第30位到第403位，采用基因克隆技术和哺乳动物载体表达体系获得碳末端多组氨酸标记(C-terminus polyhistidine-tagged)的抗原，具体氨基酸序列如下(SEQ ID NO.86)：
接着，采用上述在HEK293T细胞表达和制备的人ROR1胞外区蛋白免疫SJL小鼠，用量为50μg/只，以制备免疫脾细胞；适时的制备鼠骨髓瘤细胞(SP2/0)和饲养细胞以备融合之需。待上述三种细胞准备完毕，通过电融合仪(BEX，型号LF301)介导融合免疫脾细胞和SP2/0细胞，用含有饲养细胞的HAT完全培养基重悬，接种到96孔板中培养，通过ELISA/FACS法进行阳性孔筛选。
最后，再对阳性孔的细胞通过有限稀释法进行克隆化培养，通过ELSIA或FASCS筛选效价高、形态好、呈单克隆生长的细胞继续进行亚克隆筛选，直到筛选阳性克隆率全为100％，即可对该细胞株进行扩大培养和建库。
步骤②靶向人ROR1鼠源单克隆抗体的制备和纯化：
将步骤①中筛选出来的杂交瘤细胞在滚瓶中扩大培养14天后，收集细胞培养上清，经0.45μm滤膜过滤后，将所得培养上清恒速加入事先平衡好的Protein A亲和树脂柱(GE，Cat#17-1279-02)中，并用0.1M Tris-HCl(PH＝8.0,含有1.5M NaCl)平衡柱子。
然后采用0.1M柠檬酸钠缓冲液洗脱平衡柱，收集洗脱液并进行脱盐处理，0.22μm滤膜过滤除菌，UV280定量并进行SDS-PAGE电泳、SEC-HPLC及内毒素检测。
分装所得纯化的单克隆抗体，-80℃冻存备用。
步骤③靶向人ROR1鼠源单克隆抗体的生物活性和特异性的确定：
经过反复筛选，对选定的9个杂交瘤单克隆抗体进行生物活性和靶向特异性测定。
如图1中A图所示，采用酶联免疫吸附法(ELISA)检测单克隆细胞培液上清，9个单克隆均能特异性结合纯化的人ROR1-ECD蛋白。
如图1中B图所示，采用流式细胞荧光分选仪(FACS)检测单克隆细胞培液上清，9个单克隆均可以特异性的结合人源ROR1-高表达的MDA-MB-231细胞(ROR1-P)，而对ROR1-低表达的MDA-MB-453细胞(ROR1-N)无明显结合活性。
如图1中C图所示，采用纯化后的单克隆抗体样品进行亚型检测，mAb001，mAb004～mAb009均鉴定为IgG2b/k，mAb002、mAb003为IgG2c/k。
随后对9种纯化单克隆抗体进行梯度稀释，采用ELISA检测对纯化的ROR1-ECD蛋白的结合活性EC50分别为0.049nM、0.028nM、0.023nM、0.038nM、0.043nM、0.059nM、0.066nM、0.053nM、0.062nM。FACS检测对MDA-MB-231细胞ROR1结合活性EC50分别为0.073nM、0.11nM、0.45nM、0.15nM、0.16nM、0.19nM、0.15nM、0.11nM、0.16nM。
实施例2单克隆抗体测序、互补决定区(CDR)的鉴定
基于优异的特异性及亲和力，优先选取mAb001、mAb002、mAb003、mAb004、mAb005、mAb006、mAb007、mAb008、mAb009进行抗体测序鉴定。采用5’RACE和常规PCR技术扩增重链(VH)、轻链(VL)可变区片段，克隆入载体，采用常规测序并通过Kabat数据库分析(http://www.bioinf.org.uk)得到以下重链可变区(VH)、轻链可变区(VL)氨基酸序列、互补决定区(CDR)信息，下划线“_”所示为CDR-1/2/3氨基酸序列。
(1)mAb001

(2) mAb002
(3) mAb003
(4) mAb004

(5) mAb005
(6) mAb006
(7) mAb007
(8) mAb008

(9)mAb009
表-3

实施例3人-鼠嵌合抗体的制备
通过基因重组技术将9组表达可变区片段(参见SEQ ID NO.7、SEQ ID NO.41、SEQ ID NO.49、SEQ ID NO.17、SEQ ID NO.25、SEQ ID NO.57、SEQ ID NO.65、SEQ ID NO.73、SEQ ID NO.81、SEQ ID NO.9、SEQ ID NO.42、SEQ ID NO.50、SEQ ID NO.18、SEQ ID NO.27、SEQ ID NO.58、SEQ ID NO.66、SEQ ID NO.74、SEQ ID NO.82)的核酸克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经测序无误后，采用哺乳动物HEK-293T细胞表达系统将构建的嵌合型抗体表达和纯化(见图2)，所获得的人-鼠嵌合型抗体，分别编号为mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c。用同样方法制备发明专利申请US20150232569A1中公开的UC-961作为对照。
所述抗体的可变区序列含有数个不利氨基酸，对其进行了点突变改造。以下列出经点突变后的重链可变区(VH)、轻链可变区(VL)的氨基酸序列(“_”所示为CDR氨基酸序列)。
以上述点突变模板匹配获得点突变(PTM)克隆到hIgG1,κ载体获得点突变的相应嵌合抗体突变体。
综上所述的人-鼠嵌合抗体和相应的人-鼠嵌合抗体突变体的编号、所述人-鼠嵌合抗体的重链可变区和轻链可变区的序列编号由表-4汇总列出。
表-4：人-鼠嵌合抗体及其突变体

实施例4人-鼠嵌合抗体对人ROR1蛋白亲和力的ELISA测定
用包被液将纯化的人ROR1蛋白胞外区(ROR1-ECD)稀释成1μg/mL，包被ELISA板，100μL/孔，4℃，过夜。洗去多余抗原，用1％BSA于室温封闭2h，然后加入3倍梯度稀释的各人-鼠嵌合抗体，100L/孔，室温孵育2h；洗去未结合的人-鼠抗体，加入合适浓度辣根过氧化物酶标记的抗鼠的二抗，100μL/孔，室温孵育1h。洗去未结合的二抗，加入TMB显色液反应大约10min，加入2N H2SO4，50μL/孔，终止显色反应，然后在450nm处测定其吸光度，并分析数据。
检测结果如图3所示，mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对人ROR1-ECD有很强的亲和性，EC50分别0.012nM、0.013nM、0.013nM、0.012nM、0.009nM、0.009nM、0.012nM、0.02nM、0.017nM，其中mAb001c、mAb004c、mAb005c、mAb006c、mAb007c结合活性略强于UC-961(EC50为0.013nM)；
检测结果如图4所示，mAb001c和mAb005c的人-鼠嵌合抗体突变体同样对人ROR1-ECD有很强的亲和性，EC50分别0.028nM、0.046nM。
实施例5人肿瘤与正常组织基因表达数据库分析
通过下载CCLE(Cancer cell line encyclopedia)数据库、GTEX(人正常组织)数据库的基因表达信息，进行分析ROR1mRNA水平在肿瘤细胞株群组(如肺癌、乳腺癌、结肠癌、血液肿瘤、肝癌、胃癌)相对于人30种正常组织的表达情况。
结果如图5显示，对比CCLE数据库及GTEX数据库，高侵袭性的乳腺癌、肺癌、结肠癌、血液肿瘤、肝癌及胃癌细胞株的平均ROR1mRNA表达水平显著高于正常组织，表明ROR1在肿瘤及正常组织具备卓越的治疗窗口。本申请以ROR1为靶点的抗体在诊断、预防及治疗高侵袭性乳腺癌、肺癌、结肠癌、血液肿瘤、肝癌及胃癌等的应用中将具有显著的治疗效果和广泛的应用前景。
本实施例还分析对比了不同分子分型的乳腺癌(例如：管腔型相对于基底型)中ROR1mRNA的表达水平。
结果如图6显示，相比管腔型(Luminal-type)乳腺癌细胞株，高侵袭、高转移的基底型(Basal-type)乳腺癌细胞株的平均ROR1mRNA表达水平显著高于管腔型，且具有统计学意义。鉴于基底型乳腺癌是临床上“三阴性”乳腺癌的主要来源，本申请以ROR1为靶点的抗体在诊断、预防和治疗三阴性乳腺癌的应用中将具有更为显著的效果。
实施例6 ROR1蛋白在多种肿瘤细胞中呈高表达
针对多种肺癌细胞株(NCI-H1299、A549、HCC78、NCI-H226、NCI-H1975、NCI-H2228、HCC827、NCI-H2009、PC9、Calu-6、Calu-1)及不同分子分型的乳腺癌细胞株(MDA-MB-231、Hs578T、HCC1937、MDA-MB-468、SK-BR-3、MDA-MB-453、BT474、MCF-7)，制备细胞总蛋白，精确定量后，通过免疫印迹试验(Western blot)检测ROR1蛋白的表达水平。结果如图7和图8显示，ROR1蛋白在大多数被检测的肺癌、高侵袭性乳腺癌(基底型/三阴型)细胞株呈异常激活表达。
实施例7人-鼠嵌合抗体对肿瘤细胞表面ROR1的特异性结合
采用ROR1高表达的肺癌细胞(NCI-H226、HCC827、NCI-H446)、三阴性乳腺癌细胞(MDA-MB-231、HCC1187)、结肠癌HT-29细胞和ROR1低表达的乳腺癌细胞(MDA-MB-453、MCF-7)作为靶细胞，测定人-鼠嵌合抗体mAb001c对细胞表面ROR1的结合情况(以mAb001c为例，其余人-鼠嵌合抗体未列出)。采用2×105个肿瘤细胞与人-鼠嵌合抗体混匀(最终浓度5μg/mL)，然后于4℃孵育1小时，PBS洗涤细胞两次以去除未结合的一抗，再加入200μL(2.5μg/mL)PE标记的二抗置4℃孵育30min，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μL PBS中，流式细胞荧光分选仪(FACS)检测结合强度(MFI)。
测试结果如图9所示，人-鼠嵌合抗体mAb001c能特异性结合ROR1高表达的肿瘤细胞，结合率荧光强度顺序依次为HCC1187、NCI-H226、HCC827、HT-29、MDA-MB-231、NCI-H446，而对ROR1低表达的肿瘤细胞MDA-MB-453、MCF-7显示微弱的结合荧光强度。将HCC1187、NCI-H226、HCC827与人-鼠嵌合抗体的结合率(MFI)对比MCF-7与人-鼠嵌合抗体的结合率，可以得出mAb001c的结合率差异分别为17.2倍、16.7与16.6倍。
实施例8人-鼠嵌合抗体对肿瘤细胞表面ROR1结合亲和力的测定
采用ROR1高表达的三阴性乳腺癌细胞MDA-MB-231、非小细胞肺癌NCI-H226作为靶细胞，将100μL按照5倍梯度从33.3nM稀释到0.002nM的受试人-鼠嵌合抗体作为一抗，分别与悬浮于100μLRPMI-1640无血清培养基中的2×105个MDA-MB-231或NCI-H226混匀，然后于4℃孵育1h，PBS洗涤细胞两次以去除未结合的一抗，再将靶细胞与200μL，2.5μg/mL，PE标记的二抗4℃孵育30min，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μLPBS中，通过流式细胞分选仪(FACS)测定受试人-鼠嵌合抗体对细胞表面ROR1的结合亲和力(Binding affinity)。
检测结果如图10所示，mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对MDA-MB-231具有优异的结合亲和力，EC50分别为0.15nM、0.20nM、1.1nM、0.40nM、0.24nM、0.40nM、0.25nM、0.78nM、0.33nM，其中mAb001c结合活性明显优于UC-961(EC50为0.21nM)，mAb002、mAb005c、mAb007c结合活性与UC-961相当；
检测结果如图11所示，mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对NCI-H226同样具有优异的结合亲和力，EC50分别为0.11nM、0.14nM、0.76nM、0.46nM、0.18nM、0.28nM、0.28nM、0.36nM、0.25nM，其中mAb001c结合活性优于UC-961(EC50为0.13nM)，mAb002c、mAb005c结合活性与UC-961相当；
检测结果如图12所示，所述mAb001c和mAb005c的突变体同样对MDA-MB-231同样具有优异的结合亲和力，mAb001c_v1的EC50值为0.073nM；mAb005c_v1的EC50值为0.067nM。
实施例9人-鼠嵌合抗体对ROR1胞外段各结构域结合活性测定
采用基因工程技术构建含跨膜区的人ROR1胞外段(SEQ ID NO.89)、ROR1胞外段去除Ig样结构域(SEQ ID NO.90)、ROR1胞外段去除Frizzled结构域(SEQ ID NO.91)、ROR1胞外段去除Kringle结构域(SEQ ID NO.92)序列分别克隆到pCDNA3.1表达载体中，具体氨基酸序列如下：
SEQ ID NO.89 ROR1胞外段序列(含跨膜)
SEQ ID NO.90 ROR1胞外段去除Ig样结构域
SEQ ID NO.91 ROR1胞外段去除Frizzled结构域
SEQ ID NO.92 ROR1胞外段去除Kringle结构域
分别采用转染有上述各基因表达载体的CHO细胞为靶细胞(分别命名为CHO-ROR1(胞外段)、CHO-ROR1(Ig-like结构域去除)、CHO-ROR1(Frizzled结构域去除)、CHO-ROR1(Kringle结构域去除))，将100μL按照5倍梯度从33.3nM稀释到0.002nM或按照5倍梯度从200nM稀释到0.013nM的受试人-鼠嵌合抗体作为一抗，分别与悬浮于100μLRPMI-1640无血清培养基中的2×105个靶细胞混匀，然后于4℃孵育1h，PBS洗涤细胞两次以去除未结合的一抗，再将靶细胞与200μL，2.5μg/mL，PE标记的二抗4℃孵育30min，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μLPBS中，通过流式细胞分选仪(FACS)测定受试抗体对细胞表面ROR1的结合亲和力(Binding affinity)。
检测结果如图13所示，mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、 mAb008c、mAb009c对CHO-ROR1(胞外段)具有优异的结合亲和力，EC50分别为0.32nM、0.60nM、1.47nM、0.32nM、0.57nM、0.71nM、0.54nM、0.7nM、0.79nM，其中mAb001c、mAb004c、mAb005c、mAb007c结合活性明显优于UC-961(EC50为0.74nM)；
检测结果如图14所示，mAb001c、mAb002c、mAb004c、mAb006c、mAb007c、mAb008c、mAb009c对CHO-ROR1(Ig-like结构域去除)具有优异的结合亲和力，EC50分别为0.36nM、0.35nM、0.35nM、0.37nM、0.36nM、0.37nM、0.45nM；mAb003c和mAb005c与UC-961类似，基本不结合CHO-ROR1(Iglike结构域去除)；
检测结果如图15所示，mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对CHO-ROR1(Frizzled结构域去除)具有优异的结合亲和力，EC50分别为0.85nM、0.86nM、1.78nM、1.37nM、1.02nM、1.64nM、1.25nM、1.67nM、1.68nM，其中mAb001c、mAb002c、mAb005c结合活性明显优于UC-961(EC50为1.11nM)；
检测结果如图16所示，mAb001c、mAb002c、mAb003c、mAb004c、mAb005c、mAb006c、mAb007c、mAb008c、mAb009c对CHO-ROR1(Kringle结构域去除)具有优异的结合亲和力，EC50分别为0.38nM、0.66nM、1.74nM、0.84nM、0.91nM、1.59nM、1.03nM、1.50nM、1.62nM；其中mAb001c、mAb002c结合活性明显优于UC-961(EC50为0.79nM)。
综上结果说明，本实施例嵌合抗体mAb001c-mAb009c针对细胞表面转入的ROR1具有优异结合亲和力(EC50<2nM)；更重要的是，mAb001c、mAb002c、mAb004c、mAb006c、mAb007c、mAb008c、mAb009c针对ROR1结合区域为非Ig-like结构域(Frizzled或Kringle结构域)，与UC-961明显不同(参见US20150232569A1所述结合于Ig样结构域，与本实施例结果一致)，mAb003c和mAb005c均结合于ROR1-Ig-like区域，且mAb001c、mAb005c针对ROR1的结合活性明显优于UC-961，因此，本实施例抗体相对于UC-961具备显著的差异化优势。
实施例10 ROR1人-鼠嵌合抗体在免疫缺陷小鼠移植瘤模型中的抗肿瘤活性
随机将免疫缺陷型裸鼠(Balb/c-nude)分为4组，将50μL含有5×106NCI-H226的细胞悬液与50μL所示人-鼠嵌合抗体混匀(终浓度为50μg/瘤)，取100μL基质胶与上述100μL细胞/抗体混合液混匀，并接种入裸鼠背部皮下(n＝6)。采用hIgG为阴性对照。定期观察人-鼠抗体对皮下瘤生长的抑制作用，每周2-3次测量裸鼠体重及肿瘤大小，绘制肿瘤生长曲线，评定活性。
结果如图17所示，人-鼠嵌合抗体mAb001c、mAb004c、mAb005c均能显著抑制NCI-H226肿瘤在裸鼠体内的生长(vs hIgG组，P<0.05)；对照药UC-961对NCI-H226肿瘤基本无明显抑制活性。
实施例11 ROR1人-鼠嵌合抗体与肿瘤细胞结合导致内吞至细胞内溶酶体
将60％密度的ROR1高表达的MDA-MB-231细胞铺于激光共聚焦培养皿中培养过夜，加入5μg/mL的ROR1人-鼠嵌合抗体mAb001c、mAb002c、mAb004c、mAb005c，分别于37℃孵育4h和4℃孵育1h，采用PBS洗涤三次以去除未结合的人-鼠嵌合抗体，加入4％的多聚甲醛于室温固定30min。PBS洗涤三次，采用0.4％TritonX-100透化细胞10min。PBS洗涤三次后，加入兔抗人LAMP-2抗体于37℃条件下孵育1h，以标记细胞溶酶体的位置。采用PBS洗去未结合的抗体，加入R-PE标记的羊抗人和Alexa Fluor 488标记的驴抗兔二抗于37℃孵育30min。洗去未结合的二抗，用DAPI染色10min以标记细胞核位置，采用激光共聚焦显微镜(20×)观察抗体的抗体内吞情况。
结果如图18所示，mAb001c、mAb002c、mAb004c、mAb005c均能快速并大幅度的被MDA-MB-231细胞内吞至溶酶体。该结果表明，本申请的人-鼠嵌合抗体适合用于制备抗体-药物偶联物(ADC)，提示ROR1ADC将具有良好的ADC药物特性，可用于广谱和高特异性靶向ROR1阳性肿瘤治疗药物的前景。
实施例12 mAb001c-vc-MMAE的偶联制备
将靶向ROR1的人-鼠嵌合抗体mAb001c(7.088mg/ml)置于600ml离心瓶，加入50mM PBS(pH＝7.4)缓冲液稀释人-鼠嵌合抗体浓度至5mg/ml，按照反应液总体积5％加入100mM EDTA水溶液，震荡混匀后加入2mg/ml TCEP水溶液进行人-鼠嵌合抗体还原，TCEP与人-鼠嵌合抗体的摩尔比3∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。按照药物与人-鼠嵌合抗体终浓度摩尔比8∶1加入vcMMAE(购自上海皓元化学)的DMSO溶液，按照反应液总体积10％补充DMSO，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO 30KD，厂家：密理博)置换样品保存buffer，先用含有10％DMSO的30mM His-Hac，pH5.5缓冲液超滤3次，再用无DMSO的30mM His-Hac，pH5.5超滤6次，经0.22微米孔径的过滤装置除菌，-80℃保存，所得抗体偶联物命名为mAb001c-vcMMAE。
结果如图19、图20所示，人-鼠嵌合抗体mAb001c与其抗体偶联物mAb001c-vc-MMAE的HIC-HPLC(图19)和SEC-HPLC(图20)均表明，人-鼠抗体mAb001c经偶联反应后，形成了较高纯度(>98％)的抗体偶联物mAb001c-vc-MMAE，偶联物的分子量与预期值相符，平均DAR值约为3.81。
实施例13 ROR1人-鼠嵌合抗体药物偶联物针对ROR1高表达的肿瘤细胞的体外抗增殖活性
本实例所使用细胞系购自于南京科佰生物和上海誉弛生物，并按照相应的说明进行培养，包括：MCF-7、MDA-MB-231、NCI-N87、NCI-H446、HT-29、HCC827、HCC1187。将上述处于对数生长期的细胞，分别以每孔250-1500个细胞的密度(依不同细胞的生长速率而定)接种至96孔细胞培养板中，150μL/孔，37℃，5％CO2培养约3-5h后，分别加入不同浓度的mAb001c-vcMMAE，每个药物浓度设置2-4个复孔，及相应的溶媒对照和空白对照孔，作用5-6天后(根据细胞生长速度，保证细胞分裂足够次数)，倾去培养液，加入CCK8反应液(购自MCE,cat#HY-K0301)，100μL/孔，于37℃反应至预期颜色深浅，测定每组的细胞活力(OD450nm)，并按照以下公式计算细胞存活率：存活率＝(OD给药-OD空白)/(OD对照-OD空白)×100％。通过GraphPad Prism 5软件分析上述数据，并分别计算上述ROR1抗体-药物偶联物在不同细胞株上的IC50值。
如图21所示，mAb001C-vcMMAE对ROR1低表达的细胞MCF-7的增殖抑制作用不明显(IC50>100nM)，而对ROR1高表达的MDA-MB-231、NCI-N87、NCI-H446、HT-29、HCC827、HCC1187均显示很强的细胞增殖抑制作用(IC50<10nM)。
总体而言，ROR1-抗体药物偶联物的细胞毒性(IC50值)表明ROR1-抗体药物偶联物的细胞毒活性与受试细胞的ROR1表达水平直接相关，因此判断为ROR1靶标特异性细胞毒性。
实施例14 ROR1人-鼠嵌合抗体药物偶联物的体内抗肿瘤活性
分别将200μL含有5×106MDA-MB-231、HT-29的细胞基质胶悬液接种到免疫缺陷小鼠(NCG或Balb/c-nude)背部皮下。待肿瘤体积长至100-200mm3，根据肿瘤体积大小及裸鼠体重随机分组(n＝10)，分别采用5mg/kg、2.5mg/kg剂量，每周尾静脉给药一次，共计给药3周；同时设置PBS作为阴性对照。每周测量2-3次肿瘤体积及裸鼠体重并记录以绘制肿瘤生长曲线。肿瘤体积(V)计算公式为：V＝1/2×a×b2，其中a、b分别表示肿瘤的长、宽。
如图22所示，5mg/kg的mAb001c-vcMMAE能完全抑制ROR1高表达的MDA-MB-231肿瘤生长(P<0.001)，停药2周未见肿瘤明显回复生长；
如图23所示，mAb001c-vcMMAE在给药5mg/kg、2.5mg/kg时显著且呈剂量依赖的抑制HT-29肿瘤的生长(P＝0.0014，P＝0.054)。
实施例15本申请ROR1人-鼠嵌合抗体与传统技术的比较
以发明专利申请号US20150232569A1中公开的UC-961抗体的重链、轻链可变区序列(VH/VL)，人工合成其重链、轻链可变区并克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经测序无误后在Expi-293F细胞体系表达和纯化得到UC-961，将抗体制备的实验条件与实施例3、实施例15保持一致。
UC-961-重链可变区(VH)SEQ ID NO.87
UC-961-轻链可变区(VL)SEQ ID NO.88
对比试验的活性结果总结如下：
1、参照实施例4、实施例8、实施例9中的测定方法，对mAb001c、mAb005c及UC-961针对ROR1-ECD蛋白的ELISA亲和力、细胞表面ROR1的亲和力及抗原结合区域(表位)活性进行检测，对比结果汇总于表-5；
表-5：抗体的测试活性
2、参照实施例10中的检测方法，检测mAb001c、mAb005c及UC-961针对ROR1高表达的NCI-H226移植瘤模型的体内抗肿瘤效果，对比结果汇总于表-6；
表-6：抗体在裸鼠体内的抗肿瘤活性
综上，本申请的人-鼠嵌合抗体：
(1)具有优异生物活性和特异性，具体地，所述的优选抗体具有很高的ROR1-ECD的亲和力(ELISA测定其EC50值为0.009nM-0.05nM)。此外，所述的优选抗体对肿瘤细胞表面的ROR1具有良好的结合亲合力(FACS测定其EC50值为0.15nM-1.1nM)，可用做靶向ROR1的治疗抗体。
(2)针对ROR1具有独特的结合表位，结合活性和结合能力更优。
(3)所述抗体-药物偶联物(ADC)具有特异ROR1-依赖的抗肿瘤活性；所述的优选抗体药物偶联物(ADC)对ROR1-低表达的细胞无明显毒副作用，而对ROR1-高表达的肿瘤细胞具有极高的杀伤活性，细胞增殖抑制试验测定其IC50值为1nM-9nM。
(4)所述抗体和ADC均具有显著的体内抗肿瘤活性，而对于哺乳动物如模式小鼠本身没有可见的毒副作用。
实施例16人源化抗体的制备
在Germline数据库中检索并选取与mAb001c非CDR区(Framework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,κ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。
具体地，mAb001c的人源化实施获得5个人源化重链的可变区(SEQ ID NO.28、SEQ ID NO.29、SEQID NO.30、SEQ ID NO.31、SEQ ID NO.32)，以及2个人源化轻链的可变区(SEQ ID NO.33、SEQ ID NO.34)。
具体地，mAb005c的人源化实施获得8个人源化重链的可变区(SEQ ID NO.93、SEQ ID NO.94、SEQ ID NO.95、SEQ ID NO.96、SEQ ID NO.97、SEQ ID NO.98、SEQ ID NO.99、SEQ ID NO.100)，以及1个人源化轻链的可变区(SEQ ID NO.101)。

通过基因重组技术将所设计的人源化可变区克隆入含有人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(Expi293F细胞)将构建的人源化抗体表达载体进行重链、轻链组合表达，最终mAb001c组获得了了9个人源化抗体，mAb005c组获得了8个人源化抗体，各抗体相应的重链和轻链组合如表-7所示。
表-7：人源化抗体的制备
实施例17人源化抗体对ROR1-ECD的结合亲和力
将表-7中的17个人源化抗体梯度稀释，采用ELISA法测定其对ROR1-ECD蛋白的亲和力，实验方法参照实施例4。
实验结果如图24所示，所述人源化抗体Hu001-02、03、05、06、08、11、12、14、15均对ROR1-ECD蛋白具有很强的结合亲和力，EC50值为0.010nM-0.015nM。
实验结果如图25所示，所述人源化抗体Hu005-35、40、41、42、43、44、45、46均对ROR1-ECD蛋白具有很强的结合亲和力，EC50值为0.009nM-0.02nM。
实施例18人源化抗体对ROR1蛋白的结合特异性
将表-7中的人源化抗体Hu001-02、Hu005-46梯度稀释，采用ELISA法测定其对人ROR1-ECD和人ROR2-ECD蛋白的亲和力，实验方法参照实施例4。
实验结果如图26所示，所述人源化抗体Hu001-02、Hu005-46特异性且高亲和力结合于人ROR1-ECD蛋白，而对人ROR2-ECD蛋白基本无明显结合活性，表明所述人源化抗体针对ROR1蛋白具有结合特异性。
实施例19人源化抗体对肿瘤细胞表面ROR1的结合亲和力
将表-7中的17个人源化抗体梯度稀释，通过流式细胞仪测定其对MDA-MB-231和NCI-H226细胞表面ROR1的亲和力，实验方法参照实施例8。
实验结果图27所示，所述人源化抗体对MDA-MB-231细胞表面ROR1具有很高的结合亲和活性，Hu001-02、03、05、06、08、11、12、14、15结合的EC50值为0.19nM-0.47nM；Hu005-35、40、41、42、43、44、45、46结合的EC50值为0.16nM-0.22nM。
实验结果图28所示，所述人源化抗体对NCI-H226细胞表面ROR1具有很高的结合亲和活性，Hu001-02、03、05、06、08、11结合的EC50值为0.27nM-0.62nM；Hu005-35、40、41、42、43、44、45、46结合的EC50值为0.12nM-0.18nM。
实施例20人源化抗体与肿瘤细胞结合导致内吞至细胞内溶酶体
将60％密度MDA-MB-231细胞铺于激光共聚焦培养皿中，于37℃培养过夜后加入5μg/mL的ROR1人源化抗体Hu001-02、Hu001-03、Hu005-44、Hu005-46，分别于37℃孵育4h和4℃孵育1h，PBS洗涤三次以去除未与细胞结合的抗体，采用4％的多聚甲醛于室温固定30min。PBS洗涤三次，采用0.4％Triton X-100透化10min。PBS洗涤三次后，37℃条件下孵育Lamp-2(兔抗人)抗体1h，以标记细胞溶酶体的位置。采用PBS洗去未结合的抗体，37℃孵育R-PE标记的羊抗人和Alexa Fluor 488标记的驴抗兔二抗30min。洗去未结合的二抗，用DAPI染色10min以标记细胞核位置，之后用激光共聚焦显微镜(20×)观察抗体的抗体内吞情况。
结果如图29所示，Hu001-02、Hu001-03、Hu005-44、Hu005-46能快速并大幅度的被MDA-MB-231细胞内吞至溶酶体。该结果表明，本申请的人源化抗体适合用于制备抗体-药物偶联物(ADC)，用于广谱和高特异性地靶向治疗ROR1高表达肿瘤。
实施例21人源化抗体内吞动力学检测
分别收集对数生长期的MDA-MB-231、NCI-N87及NCI-H446细胞，调整细胞密度为1×107/mL,每组分别取600μL细胞悬液与600μL抗体工作液(60μg/mL Hu001-2或Hu001-3或Hu005-44或Hu005-46)于冰上孵育30min，采用预冷的1×PBS洗两遍，离心收集细胞，并重悬于1.8mL的1640培养基(含2％FBS)，分别取300μL细胞悬液于37℃孵育0.5h，1h，2h，4h(分别取300μL细胞悬液作为T0以及背景参照)，每个时间点孵育完成，立即放在冰上终止内吞，离心并采用预冷的1×PBS洗两遍，加入100μL的R-PE标记的羊抗人二抗于冰上孵育20min，离心并采用预冷的1×PBS洗两遍，加入2％的多聚甲醛10min，通过流式细胞分选仪(FACS)检测荧光强度(MFI)。
结果如图30、图31、图32所示，Hu001-2、Hu001-3、Hu005-44、Hu005-46在0.5h-1h即可较大程度介导靶标内化进入细胞内，在所测时间4h内，待测抗体基本可实现50％-70％的内吞率，其中Hu001-2(3)内吞速率及内吞程度较Hu005-44(46)更优。
实施例22人源化抗体-偶联药物的制备
优选人源化抗体Hu001-2、Hu001-3、Hu005-44及Hu005-46与vcMMAE偶联制备相应的抗体-偶联药物，具体偶联方法参照实施例12，所得抗体-偶联药物分别命名为Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE，同时制备UC961-MMAE作为阳性参照物。
如图33-42所示，人源化抗体Hu001-2、Hu001-3、Hu005-44、Hu005-46、UC961与其抗体偶联物Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE、UC961-MMAE的HIC-HPLC(图33、图35、图37、图39、图41)和SEC-HPLC(图34、图36、图38、图40、图42)均表明，所述抗体经偶联反应后，形成了较高纯度(>97％)的抗体偶联物，偶联物的分子量与预期值相符，平均DAR值分别约为4.15、4.15、4.12、4.07、3.95。
实施例23人源化抗体-偶联药物对人ROR1蛋白亲和力
将实施例22中制备的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE梯度稀释，采用ELISA法测定其对ROR1-ECD蛋白的亲和力，同时采用Hu001-2、Hu001-3、Hu005-44、Hu005-46作为母体抗体参照，实验方法参照实施例4。
结果如图43所示，所述人源化抗体-偶联物均能保持母体抗体针对ROR1-ECD蛋白的高结合亲和活性，结合EC50值为0.012nM-0.024nM。
实施例24人源化抗体-偶联药物对肿瘤细胞表面ROR1的结合亲和力
将实施例22中制备的Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE 梯度稀释，通过流式细胞仪测定其对MDA-MB-231和NCI-H226细胞表面ROR1的亲和力，同时采用Hu001-2、Hu001-3、Hu005-44、Hu005-46作为母体抗体参照，实验方法参照实施例8。
实验结果图44所示，所述人源化抗体-偶联物均能保持母体抗体针对MDA-MB-231细胞表面ROR1的高结合亲和活性，结合EC50值为0.13nM-0.26nM。
实验结果图45所示，所述人源化抗体-偶联物均能保持母体抗体针对NCI-H226细胞表面ROR1的高结合亲和活性，结合EC50值为0.19nM-0.38nM。
实施例25人源化抗体-偶联药物针对ROR1高表达的肿瘤细胞的体外抗增殖活性
本实例所使用细胞系购自于南京科佰生物和上海誉弛生物，并按照相应的说明进行培养，包括：乳腺癌细胞株：MCF-7、SK-BR-3、MDA-MB-231、HCC1187；肠癌细胞株：HT-29、SK-CO-1、COLO-678、肺癌细胞株：HCC827、H446；卵巢癌细胞株：PA-1；胃癌细胞株：NCI-N87。采用CCK8法检测Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE针对各癌种细胞的体外杀伤活性，UC961-MMAE作为阳性参照，实施方法参照实施例13。
如图46-56所示，所述人源化抗体-偶联物对ROR1低表达的SK-BR-3、MCF-7细胞的增殖抑制作用不明显(IC50>100nM)(图46、图47)，而对ROR1高表达的MDA-MB-231、HCC1187、HT-29、SK-CO-1、COLO-678、NCI-H446、HCC827、PA-1、NCI-N87均显示很强的细胞增殖抑制作用(图48、图49、图50、图51、图52、图53、图54、图55、图56。
总体而言，ROR1人源化抗体-偶联物的细胞毒性(IC50值)表明ROR1-抗体药物偶联物的细胞毒活性与受试细胞的ROR1表达水平直接相关(图57)，因此判断为ROR1靶标特异性细胞毒性。表-8汇总了测试细胞增殖抑制的IC50值。
表-8：ROR1人源化抗体-偶联物体外抗肿瘤活性
实施例26人源化抗体-偶联药物针的体内抗肿瘤活性
分别将200μL含有5×106MDA-MB-231、1×107HCC1187、1×107PA-1、5×106NCI-N87的细胞基质胶悬液接种到免疫缺陷小鼠(NCG或Balb/c-nude)背部皮下。待肿瘤体积长至100-300mm3，根据肿瘤体积大小及裸鼠体重随机分组(n＝8-10)，分别采用5mg/kg、2.5mg/kg剂量，每周尾静脉给药一次，共计给药3周；同时设置PBS作为阴性对照。每周测量2-3次肿瘤体积及裸鼠体重并记录以绘制肿瘤生长曲线。肿瘤体积(V)计算公式为：V＝1/2×a×b2，其中a、b分别表示肿瘤的长、宽。
四种优选的ROR1人源化抗体-偶联物：Hu001-2-MMAE、Hu001-3-MMAE、Hu005-44-MMAE、Hu005-46-MMAE及阳性参照UC961-MMAE的体内抗肿瘤结果分别如图57-60所示。
如图58所示，在MDA-MB-231模型中，所述四种ROR1人源化抗体-偶联物在给药5mg/kg、2.5mg/kg时呈剂量相关的治疗效果。并且在5mg/kg的剂量下能完全抑制ROR1高表达的MDA-MB-231肿瘤生长(P<0.0001)，停药2周未见肿瘤明显回复生长。
如图59所示，在PA-1模型中，所述Hu001-2-MMAE、Hu005-46-MMAE在给药5mg/kg、2.5mg/kg时呈剂量相关的治疗效果。并且在5mg/kg的剂量下能完全抑制ROR1高表达的PA-1肿瘤生长(P<0.0001；P<0.001)，肿瘤消退，停药2周未见肿瘤明显回复生长，且在2.5mg/kg剂量下的抗肿瘤活性均明显优于 阳性参照UC961-MMAE。
如图60所示，在NCI-N87模型中，所述Hu001-2-MMAE、Hu005-44-MMAE、Hu005-46-MMAE在给药5mg/kg、2.5mg/kg时呈剂量相关的治疗效果。并且在5mg/kg的剂量显著抑制ROR1中-低表达的NCI-N87肿瘤生长(P<0.0001)，抗肿瘤活性均明显优于阳性参照UC961-MMAE。
如图61所示，在HCC1187模型中，所述Hu001-2-MMAE在给药5mg/kg、2.5mg/kg时呈剂量相关的治疗效果。并且在5mg/kg剂量下能完全抑制ROR1高表达的HCC1187肿瘤的生长(P<0.001)，抗肿瘤活性明显优于阳性参照UC961-MMAE。
总体而言，ROR1人源化抗体-偶联物针对ROR1高表达、中低表达的肿瘤均具备显著的抗肿瘤活性。表-9至表12汇总了所述人源化抗体-偶联物的体内肿瘤抑制率(％)。
表-9：ROR1人源化抗体-偶联物在MDA-MB-231肿瘤模型抑瘤率(％)
表-10：ROR1人源化抗体-偶联物在PA-1肿瘤模型抑瘤率(％)
表-11：ROR1人源化抗体-偶联物在NCI-N87肿瘤模型抑瘤率(％)
表-12：ROR1人源化抗体-偶联物在HCC1187肿瘤模型抑瘤率(％)

综上，ROR1人源化抗体及抗体-偶联物相关实施例的研究结果明确表明：
(1)所述人源化抗体具备优异的抗原结合能力，结合ROR1-ECD蛋白的EC50值<0.03nM；结合细胞表面ROR1的EC50值<1nM；
(2)所述人源化抗体具备优异的内吞特性，可作为ADC药物开发的靶头；
(3)所述人源化抗体-偶联物(偶联vcMMAE)保持母体抗体针对ROR1-ECD蛋白及细胞表面ROR1的高抗原结合亲和力；
(4)所述人源化抗体-偶联物针对多癌种细胞具备显著的体外抗增殖活性，杀伤活性(IC50值)与ROR1表达水平相关；
(5)所述人源化抗体-偶联物针对ROR1不同表达水平的CDX模型均具备优异的体内抗肿瘤活性，治疗效果明显优于阳性参照UC961-MMAE。
以上所述实施方式和实施例的各技术特征可以进行任意合适方式的组合，为使描述简洁，未对上述实施方式和实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为在本说明书记载的范围中。
以上所述实施例仅表达了本申请的几种实施方式，便于具体和详细地理解本申请的技术方案，但并不能因此而理解为对申请专利保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，得到的等价形式同样落于本申请的保护范围。还应当理解，本领域技术人员在本申请提供的技术方案的基础上，通过合乎逻辑的分析、推理或者有限的试验得到的技术方案，均在本申请所附权利要求的保护范围内。因此，本申请专利的保护范围应以所附权利要求的内容为准，说明书及附图可以用于解释权利要求的内容。

Claims (31)

  1. 一种能够特异性结合ROR1的抗体，其特征在于，所述抗体包括重链可变区和轻链可变区，满足如下条件(Ⅰ)和(Ⅱ)中的一个或者多个：
    (Ⅰ)所述重链可变区的重链互补决定区选自如下i)至iii)所示的重链互补决定区或其衍生片段中的一组或者多组：
    i)SEQ ID NO.1所示的VH-CDR1，SEQ ID NO.2所示的VH-CDR2，和SEQ ID NO.3所示的VH-CDR3；
    ii)SEQ ID NO.11所示的VH-CDR1，SEQ ID NO.12所示的VH-CDR2，和SEQ ID NO.13所示的VH-CDR3；和
    iii)SEQ ID NO.19的VH-CDR1，SEQ ID NO.20所示的VH-CDR2，和SEQ ID NO.21所示的VH-CDR3；
    (Ⅱ)所述轻链可变区的轻链互补决定区选自i’)至iii’)所示的轻链互补决定区或其衍生片段中的一组或者多组：
    i’)SEQ ID NO.4所示的VL-CDR1，SEQ ID NO.5所示的VL-CDR2，和SEQ ID NO.6所示的VL-CDR3；
    ii’)SEQ ID NO.14所示的VL-CDR1，SEQ ID NO.:15所示的VL-CDR2，和SEQ ID NO.16所示的VL-CDR3；和
    iii’)SEQ ID NO.22所示的VL-CDR1，SEQ ID NO.23所示的VL-CDR2，和SEQ ID NO.24所示的VL-CDR3；
    所述衍生片段与其对应的互补决定区有不超过约6个位点的氨基酸替换且能够保留与ROR1-ECD EC50为约0.009nM-0.05nM以及与肿瘤细胞ROR1 EC50为约0.15nM-1.1nM。
  2. 根据权利要求1所述的能够特异性结合ROR1的抗体，其特征在于，所述抗体满足如下条件(A)和(B)中的一个或者多个：
    (A)所述重链可变区的重链互补决定区选自如下i)至iii)所示的重链互补决定区中的一组或者多组：
    i)VH-CDR1的序列通式为D-Y-N-Xa1-H，VH-CDR2的序列通式为Y-I-N-P-N-Xa2-Xa3-Xa4-T-Xa5-Y-N-Q-K-F-Xa6-G，VH-CDR3的序列通式为R-Xa7-Xa8-Xa9-Xa10-Xa11-Xa12-Xa13-D-Xa14；其中，Xa1为I、M或者L，Xa2为N或者H，Xa3为D或者G，Xa4为A、N或者G，Xa5为S、N或者T，Xa6为Q、K或者E，Xa7为V或者G，Xa8为R或者Y，Xa9为T或者G，Xa10为S或者G，Xa11为S、T或者G，Xa12为G或者Y，Xa13为L、F或者AM，Xa14为D、F或者Y；
    ii)VH-CDR1如SEQ ID NO.11所示，VH-CDR2如SEQ ID NO.12所示，VH-CDR3如SEQ ID NO.13所示；
    iii)VH-CDR1如SEQ ID NO.19所示，VH-CDR2的序列通式为T-I-S-D-Xb1-G-S-Y-T-Y-Y-P-D-Xb2-Xb3-K-G，VH-CDR3如SEQ ID NO.21所示；其中，Xb1为A或者G，Xb2为S或者N，Xb3为V或者E；
    (B)所述轻链可变区的轻链互补决定区选自i’)至iii’)所示的轻链互补决定区或其衍生片段中的一组或者多组：
    i’)VL-CDR1的序列通式为Xa1’-S-S-Xa2’-Xa3’-I-Xa4’-H-T-N-Xa5’-N-T-Y-L-E，VL-CDR2的序列通式为K-V-Xa6’-N-R-F-S，VL-CDR3的序列通式为F-Q-G-S-Xa7’-Xa8’-P-Y-T；其中，Xa1’为R、K或者T，Xa2’为H或者Q，Xa3’为I、N或者S，Xa4’为V或者L，Xa5’为A或者G，Xa6’为F或者S，Xa7’为R或者L，Xa8’位F或者V；
    ii’)VL-CDR1如SEQ ID NO.14所示，VL-CDR2如SEQ ID NO.15所示，VL-CDR3如SEQ ID NO.16所示；
    iii’)VL-CDR1的序列通式为K-A-S-Q-S-V-S-F-Xb1’-G-T-S-L-M-H，VL-CDR2如SEQ ID NO.23所示，VL-CDR3如SEQ ID NO.24所示；其中，Xb1’为A或者P。
  3. 根据权利要求2所述的能够特异性结合ROR1的抗体，其特征在于，i)所示的重链互补决定区中：
    Xa1为L，Xa2为H，Xa3为D，Xa4为A或者G，Xa5为S或者T，Xa6为Q，Xa7为V，Xa8 为R，Xa9为T，Xa10为G，Xa11为T，Xa12为G，Xa13为L或者F，Xa14为D或者Y；
    或者，
    Xa1为I或者M，Xa2为N，Xa3为G，Xa4为N或者G，Xa5为S、N或者T，Xa6为K或者E，Xa7为G，Xa8为Y，Xa9为G，Xa10为S，Xa11为S或者G，Xa12为Y，Xa13为AM，Xa14为F或者Y。
  4. 根据权利要求2所述的能够特异性结合ROR1的抗体，其特征在于，i’)所示的轻链互补决定区中：
    Xa1’为R或者K，Xa2’为H，Xa3’为N，Xa4’为V或者L，Xa5’为A或者G，Xa6’为S，Xa7’为R，Xa8’为F；
    或者，
    Xa1’为R或者T，Xa2’为H或者Q，Xa3’为I或者S，Xa4’为V，Xa5’为G，Xa6’为F或者S，Xa7’为R或者L，Xa8’为F或者V。
  5. 根据权利要求2所述的能够特异性结合ROR1的抗体，其特征在于，所述抗体具有如下所示的重链互补决定区和轻链互补决定区的组合中的一组或者多组：
    组合1为：i)所示的重链互补决定区或其衍生片段，i’)所示的轻链互补决定区或其衍生片段；
    组合2为：ii)所示的重链互补决定区或其衍生片段，ii’)所示的轻链互补决定区或其衍生片段；
    组合3为：iii)所示的重链互补决定区或其衍生片段，iii)所示的轻链互补决定区或其衍生片段。
  6. 根据权利要求5所述的能够特异性结合ROR1的抗体，其特征在于，所述组合1选自如下组合中的一组或者多组：
    组合1-1为：SEQ ID NO.1所示的VH-CDR1、SEQ ID NO.2所示的VH-CDR2、SEQ ID NO.3所示的VH-CDR3、SEQ ID NO.4所示的VL-CDR1、SEQ ID NO.5所示的VL-CDR2以及SEQ ID NO.6所示的VL-CDR3；
    组合1-2为：SEQ ID NO.35所示的VH-CDR1、SEQ ID NO.36所示的VH-CDR2、SEQ ID NO.37所示的VH-CDR3、SEQ ID NO.38所示的VL-CDR1、SEQ ID NO.39所示的VL-CDR2以及SEQ ID NO.40所示的VL-CDR3；
    组合1-3为：SEQ ID NO.51所示的VH-CDR1、SEQ ID NO.52所示的VH-CDR2、SEQ ID NO.53所示的VH-CDR3、SEQ ID NO.54所示的VL-CDR1、SEQ ID NO.55所示的VL-CDR2以及SEQ ID NO.56所示的VL-CDR3；
    组合1-4为：SEQ ID NO.59所示的VH-CDR1、SEQ ID NO.60所示的VH-CDR2、SEQ ID NO.61所示的VH-CDR3、SEQ ID NO.62所示的VL-CDR1、SEQ ID NO.63所示的VL-CDR2以及SEQ ID NO.64所示的VL-CDR3；
    组合1-5为：SEQ ID NO.67所示的VH-CDR1、SEQ ID NO.68所示的VH-CDR2、SEQ ID NO.69所示的VH-CDR3、SEQ ID NO.70所示的VL-CDR1、SEQ ID NO.71所示的VL-CDR2以及SEQ ID NO.72所示的VL-CDR3；
    组合1-6为：SEQ ID NO.75所示的VH-CDR1、SEQ ID NO.76所示的VH-CDR2、SEQ ID NO.77所示的VH-CDR3、SEQ ID NO.78所示的VL-CDR1、SEQ ID NO.79所示的VL-CDR2以及SEQ ID NO.80所示的VL-CDR3；
    组合1-7为：SEQ ID NO.1所示的VH-CDR1、SEQ ID NO.83所示的VH-CDR2、SEQ ID NO.3所示的VH-CDR3、SEQ ID NO.84所示的VL-CDR1、SEQ ID NO.5所示的VL-CDR2以及SEQ ID NO.6所示的VL-CDR3。
  7. 根据权利要求5所述的能够特异性结合ROR1的抗体，其特征在于，所述组合3选自如下组合中的一组或者多组：
    组合3-1为：SEQ ID NO.43所示的VH-CDR1、SEQ ID NO.44所示的VH-CDR2、SEQ ID NO.45所示的VH-CDR3、SEQ ID NO.46所示的VL-CDR1、SEQ ID NO.47所示的VL-CDR2以及SEQ ID NO.48所示的VL-CDR3；
    组合3-2为：SEQ ID NO.19所示的VH-CDR1、SEQ ID NO.20所示的VH-CDR2、SEQ ID NO.21所示的VH-CDR3、SEQ ID NO.22所示的VL-CDR1、SEQ ID NO.23所示的VL-CDR2以及SEQ ID NO.24所示的VL-CDR3；
    组合3-3为：SEQ ID NO.19所示的VH-CDR1、SEQ ID NO.85所示的VH-CDR2、SEQ ID NO.21所示的VH-CDR3、SEQ ID NO.22所示的VL-CDR1、SEQ ID NO.23所示的VL-CDR2以及SEQ ID NO.24所示的VL-CDR3。
  8. 根据权利要求1至7任一项所述的能够特异性结合ROR1的抗体，其特征在于，所述抗体具有 如下技术特征中的一个或者多个：
    (1)所述抗体的重链恒定区和轻链恒定区的种属来源独立地选自人、鼠、兔、羊、牛、马、猪、狗、猫、骆驼、驴、鹿、貂、鸡、鸭或者鹅；
    (2)所述抗体的恒定区选自选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE及IgD任何其中之一恒定区的序列；和，
    (3)所述抗体的恒定区选自κ轻链恒定区和λ轻链恒定区任何其中之一恒定区的序列。
  9. 根据权利要求8所述的能够特异性结合ROR1的抗体，其特征在于，所述抗体具有选自如下所示的重链可变区和轻链可变区的组合中的一组或者多组：
    (1)SEQ ID NO.7所示的重链可变区和SEQ ID NO.9所示的轻链可变区；
    (2)SEQ ID NO.41所示的重链可变区和SEQ ID NO.42所示的轻链可变区；
    (3)SEQ ID NO.49所示的重链可变区和SEQ ID NO.50所示的轻链可变区；
    (4)SEQ ID NO.17所示的重链可变区和SEQ ID NO.18所示的轻链可变区；
    (5)SEQ ID NO.25所示的重链可变区和SEQ ID NO.27所示的轻链可变区；
    (6)SEQ ID NO.57所示的重链可变区和SEQ ID NO.58所示的轻链可变区；
    (7)SEQ ID NO.65所示的重链可变区和SEQ ID NO.66所示的轻链可变区；
    (8)SEQ ID NO.73所示的重链可变区和SEQ ID NO.74所示的轻链可变区；
    (9)SEQ ID NO.81所示的重链可变区和SEQ ID NO.82所示的轻链可变区；
    (10)SEQ ID NO.8所示的重链可变区和SEQ ID NO.10所示的轻链可变区；
    (11)SEQ ID NO.26所示的重链可变区和SEQ ID NO.27所示的轻链可变区；
    (12)SEQ ID NO.28所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
    (13)SEQ ID NO.28所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
    (14)SEQ ID NO.29所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
    (15)SEQ ID NO.29所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
    (16)SEQ ID NO.30所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
    (17)SEQ ID NO.31所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
    (18)SEQ ID NO.31所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
    (19)SEQ ID NO.32所示的重链可变区和SEQ ID NO.33所示的轻链可变区；
    (20)SEQ ID NO.32所示的重链可变区和SEQ ID NO.34所示的轻链可变区；
    (21)SEQ ID NO.93所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
    (22)SEQ ID NO.94所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
    (23)SEQ ID NO.95所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
    (24)SEQ ID NO.96所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
    (25)SEQ ID NO.97所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
    (26)SEQ ID NO.98所示的重链可变区和SEQ ID NO.101所示的轻链可变区；
    (27)SEQ ID NO.99所示的重链可变区和SEQ ID NO.101所示的轻链可变区；和，
    (28)SEQ ID NO.100所示的重链可变区和SEQ ID NO.101所示的轻链可变区。
  10. 一种重组蛋白，其特征在于，所述重组蛋白包括：
    权利要求1至9任一项中定义的重链互补决定区和轻链互补决定区中的一个或者多个；和，
    协助所述的重链互补决定区和轻链互补决定区中的一个或者多个表达纯化的标签片段。
  11. 根据权利要求10所述的重组蛋白，其特征在于，所述重组蛋白还包括权利要求8中定义的重链恒定区、轻链恒定区或其组合。
  12. 一种CAR构建物，其特征在于，所述CAR构建物的scFV结构域具有权利要求1至9任一项定义的重链可变区和轻链可变区。
  13. 一种核酸，其特征在于，所述核酸编码权利要求1至9任一项所述的抗体、权利要求10或者11所述的重组蛋白或者权利要求12所述的CAR构建物。
  14. 一种载体，其特征在于，所述载体含有权利要求13所述的核酸。
  15. 根据权利要求14所述的载体，所述载体选自细菌质粒、噬菌体、酵母质粒、植物细胞病毒、 哺乳动物细胞病毒如腺病毒、逆转录病毒或其组合。
  16. 一种宿主细胞，其特征在于，所述宿主细胞包含权利要求13所述的核酸、权利要求14或者15所述的载体。
  17. 根据权利要求16所述的宿主细胞，其特征在于，所述宿主细胞为CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞或HEK293细胞。
  18. 权利要求1至9任一项所述的抗体、权利要求10或者11所述的重组蛋白或者权利要求12所述的CAR构建物的制备方法，其特征在于，所述制备方法包括如下步骤：
    培养权利要求16或者17所述的宿主细胞，和，从所得培养物中分离抗体或其抗原结合片段、重组蛋白或者CAR构建物。
  19. 一种重组的免疫细胞，其特征在于，所述重组的免疫细胞表达外源的权利要求10所述的CAR构建物。
  20. 根据权利要求19所述的重组的免疫细胞，其特征在于，所述免疫细胞选自NK细胞和T细胞中的一种。
  21. 权利要求1至9任一项所述的抗体、权利要求10或者11所述的重组蛋白、权利要求12所述的CAR构建物或者权利要求19或者20所述的重组的免疫细胞在制备治疗ROR1相关疾病的药物或者ROR1相关疾病的诊断产品中的应用。
  22. 根据权利要求21所述的应用，其特征在于，所述ROR1相关疾病为肿瘤、自身免疫疾病、炎症、代谢相关疾病或者感染疾病。
  23. 根据权利要求22所述的应用，其特征在于，所述肿瘤为乳腺癌、肺癌、胰腺癌、卵巢癌、前列腺癌、结直肠癌、胃癌、肝癌、食道癌、肾癌、脑胶质瘤、膀胱癌、前列腺癌、子宫内膜癌、宫颈癌、白血病、淋巴瘤、骨髓癌或者血管肉瘤；
    所述炎症为风湿性关节炎、骨关节炎、痛风、莱特尔综合征、牛皮癣性关节病、结核性关节炎、肾小球性肾炎、全身性红斑狼疮、克罗恩病、溃疡性结肠炎、急性肺损伤、慢性阻塞性肺疾病或者特发性肺纤维化；
    所述自身免疫疾病为类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、干燥综合征或者系统性血管炎、溃疡性结肠炎、I型糖尿病、银屑病、多发性硬化症；
    所述代谢相关疾病为糖尿病、食源性肥胖或者脂肪炎症；
    所述感染疾病为细菌感染所致疾病或者病毒感染所致疾病。
  24. 根据权利要求21至23任一项所述的应用，其特征在于，所述ROR1相关疾病的诊断产品为诊断试剂、试纸条、检测板或者试剂盒。
  25. 一种抗体偶联药物，其特征在于，所述抗体偶联药物包括：
    抗体，所述抗体选自权利要求1至9任一项中定义的抗体；和，
    与所述抗体偶联的偶联物。
  26. 根据权利要求25所述的抗体偶联药物，其特征在于，所述抗体偶联药物具有如下技术特征中的一个或者多个：
    所述偶联物选自可检测标记物、细胞毒性药物、细胞因子、放射性核素、酶或其组合；和，
    所述抗体和所述偶联物通过化学键或连接子进行偶联。
  27. 根据权利要求25或者26所述的抗体偶联药物，其特征在于，所述抗体偶联药物具有如下技术特征中的一个或者多个：
    所述抗体偶联药物具有如下分子式所示的结构：
    其中：
    Ab为所述抗体，
    LU为连接子，
    D为细胞毒性药物；
    所述连接子包含巯基特异性的活性反应基团；和，
    所述细胞毒性药物为微管靶向药物、DNA靶向药物或者拓扑异构酶抑制剂。
  28. 一种药物组合物，其特征在于，所述药物组合物包括：
    权利要求1至9任一项所述的抗体、权利要求10或者11所述的重组蛋白、权利要求12所述的CAR构建物或者权利要求19或者20所述的重组的免疫细胞、权利要求25至27中任一项所述的抗体偶联药物或者其结合；和，
    药学上可接受的载体。
  29. 一种ROR1相关疾病的诊断产品，其特征在于，
    所述诊断产品为检测板，所述检测板上包被有权利要求1至9任一项所述的抗体、权利要求10或者11所述的重组蛋白、权利要求12所述的CAR构建物或者权利要求19或者20所述的重组的免疫细胞或者其结合；
    或者，所述诊断产品为检测试剂盒，所述检测试剂盒包含检测ROR1的一抗和二抗，所述一抗为权利要求1至9任一项中定义的抗体。
  30. 一种基于非诊断目的的ROR1的检测方法，其特征在于，所述检测方法包括如下步骤：
    提供待测样本，和，
    将所述待测样本和权利要求1至9任一项所述的抗体进行混合反应，检测反应产物并根据检测结果判断所述待测样本中是否存在ROR1。
  31. 根据权利要求30所述的基于非诊断目的的ROR1的检测方法，其特征在于，根据反应结果判断所述待测样本中是否存在ROR1包括：
    当在所述反应产物中检测到抗原-抗体复合物，则判断所述待测样本中含有ROR1；
    当在所述反应产物中未检测到抗原-抗体复合物，则判断所述待测样本中不含ROR1。
PCT/CN2023/113487 2022-09-07 2023-08-17 能够特异性结合ror1的抗体、其偶联药物及其制备方法和应用 WO2024051463A1 (zh)

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