CN109790518A

CN109790518A - Modified T cells, methods for their preparation and uses

Info

Publication number: CN109790518A
Application number: CN201880002752.8A
Authority: CN
Inventors: 王皓毅; 张永平; 刘晓娟; 程晨; 张兴颖; 李娜
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2017-05-08
Filing date: 2018-05-08
Publication date: 2019-05-21
Also published as: WO2018205926A1; CN109797171A

Abstract

提供了一种通过基因编辑制备经修饰的T细胞的方法，以及通过该方法制备的经修饰的T细胞及其用途。Provided are a method for preparing modified T cells by gene editing, and the modified T cells prepared by the method and uses thereof.

Description

PCT国内申请，说明书已公开。PCT domestic application, the description has been published.

Claims

A method of modified T cell is prepared, includes the steps that reducing or eliminating repressible protein in T cell and expresses.
The method of claim 1 wherein the T cell is the T cell comprising exogenous T-cell receptor (TCR) or Chimeric antigen receptor (CAR).
The method of claims 1 or 2, wherein the repressible protein being reduced or eliminated of expressing is selected from PD1, LAG-3, CTLA-4, Foxp3, Tim3 and combinations thereof.
The method of any one of claim 1-3, the wherein combination expressed the repressible protein being reduced or eliminated and be selected from PD1 and TIM3, the combination of PD1 and CTLA-4, the combination of PD1 and LAG3, the combination of CTLA-4 and TIM3, the combination of the combination of CTLA-4 and LAG3, TIM3 and LAG3, the combination of PD1, TIM3 and CTLA-4, the combination of PD1, CTLA-4 and LAG3, the combination of CTLA-4, TIM3 and LAG3, the combination of PD1, TIM3 and LAG3 or the combination of PD1, CTLA-4, TIM3 and LAG3.
The method of any one of claim 1-4, wherein by being reduced or eliminated described in antisense RNA, antagomir, siRNA, shRNA, meganuclease, Zinc finger nuclease, activating transcription factor sample effector nuclease or the implementation of CRISPR system.
Method for claim 5, wherein the CRISPR system is CRISPR/Cas9 system.
Method for claim 6, wherein the CRISPR/Cas9 system targets one or more the nucleotide sequence in SEQ ID NO:5,6,13,17,22-26 into the cell.
The method of any one of claim 2-7, wherein the TCR or CAR includes the antigen-binding domains for tumor associated antigen.
Method for claim 8, wherein the tumor associated antigen is selected from CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), carcinomebryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), EGF-R ELISA (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, bifunctional sialyltransferase gangliosides GD2, ductal epithelium mucoprotein, gp36, TAG-72, glycosyl sphingolipid, the relevant antigen of glioma, β-human chorionic gonadotrophin, alpha Fetoprotein (AFP), external source Agglutinin reactivity AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestines Carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme (prostase), prostate enzyme spcificity antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and Telomerase, prostate cancer antigen -1 (PCTA-1), MAGE, ELF2M, Neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, present main group of tumour-specific peptide epitopes Knit histocompatibility complex (MHC) molecule, 5T4, ROR1, Nkp30, NKG2D, tumor stroma antigen, the extra domain A (EDA) and extra domain B (EDB) of fibronectin, the A1 structural domain (TnC A1) of tenascin-C, fibroblast GAP-associated protein GAP (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptor, endothelial factor, major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF17), TNFRSF17 (UNIPRO T Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708) and FCRL5 (UNIPROT Q68SN8).
The method of any one of claim 8-9, wherein the antigen-binding domains are selected from monoclonal antibody, the antibody of synthesis, human antibody, humanized antibody, single domain antibody, single chain antibody variable region and its antigen-binding fragment.
The method of any one of claim 2-10, wherein the CAR includes scFv (P4), CD8 hinge area, CD28 transmembrane domain, CD28 costimulation structural domain and the CD3 ζ signal transduction structural domain for mesothelin.
The method of claim 11, wherein the CAR includes amino acid sequence shown in SEQ ID NO:32.
The modified T cell prepared by the method for any one of claim 1-12.
The T cell of modification, wherein the expression of the repressible protein in the T cell is reduced or eliminated compared with unmodified T cell.
The modified T cell of claim 14, wherein the T cell is the T cell comprising exogenous T-cell receptor (TCR) or Chimeric antigen receptor (CAR).
The modified T cell of claims 14 or 15, wherein the repressible protein being reduced or eliminated of expressing is selected from PD1, LAG-3, CTLA-4, Foxp3, Tim3 and combinations thereof.
The modified T cell of any one of claim 14-16, the wherein combination expressed the repressible protein being reduced or eliminated and be selected from PD1 and TIM3, the combination of PD1 and CTLA-4, the combination of PD1 and LAG3, the combination of CTLA-4 and TIM3, the combination of CTLA-4 and LAG3, the combination of TIM3 and LAG3, the combination of PD1, TIM3 and CTLA-4, the combination of PD1, CTLA-4 and LAG3, the combination of CTLA-4, TIM3 and LAG3, the combination of PD1, TIM3 and LAG3 or the combination of PD1, CTLA-4, TIM3 and LAG3.
The modified T cell of any one of claim 14-17, wherein the TCR or CAR includes the antigen-binding domains for tumor associated antigen.
The modified T cell of claim 18, wherein the tumor associated antigen is selected from CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), carcinomebryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), EGF-R ELISA (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, bifunctional sialyltransferase gangliosides GD2, ductal epithelium mucoprotein, gp36, TAG-72, glycosyl sphingolipid, the relevant antigen of glioma, β-human chorionic gonadotrophin, alpha Fetoprotein (A FP), lectin reactivity AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestines Carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme (prostase), prostate enzyme spcificity antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and Telomerase, prostate cancer antigen -1 (PCTA-1), MAGE, ELF2M, Neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, present tumour-specific peptide Major histocompatibility complex (MHC) molecule of epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stroma antigen, the extra domain A (EDA) and extra domain B (EDB) of fibronectin, the A1 structural domain (TnC A1) of tenascin-C, fibroblast GAP-associated protein GAP (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptor, endothelial factor, major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF17), TNFRSF17 ( UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708) and FCRL5 (UNIPROT Q68SN8).
The modified T cell of any one of claim 18-19, wherein the antigen-binding domains are selected from monoclonal antibody, the antibody of synthesis, human antibody, humanized antibody, single domain antibody, single chain antibody variable region and its antigen-binding fragment.
The modified T cell of any one of claim 15-20, wherein the CAR includes scFv (P4), CD8 hinge area, CD28 transmembrane domain, CD28 costimulation structural domain and the CD3 ζ signal transduction structural domain for mesothelin.
The modified T cell of claim 21, wherein the CAR includes amino acid sequence shown in SEQ ID NO:32.
The purposes of the modified T cell of any one of claim 13-22 in the preparation of medicament for cancer treatment.
For the pharmaceutical composition for the treatment of cancer, modified T cell and pharmaceutically acceptable carrier comprising any one of claim 13-23.
The purposes of claim 23 or the pharmaceutical composition of claim 24, wherein the cancer is selected from lung cancer, oophoroma, colon and rectum carcinoma, melanoma, kidney, bladder cancer, breast cancer, liver cancer, lymthoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngocarcinoma, cervical carcinoma, corpus uteri tumor and osteosarcoma.It can include: osteocarcinoma with the example of other cancers of method or medicine composite for curing of the invention, cancer of pancreas, cutaneum carcinoma, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, cancer of the anal region, carcinoma of testis, carcinoma of fallopian tube, carcinoma of endometrium, carcinoma of vagina, vaginal orifice cancer, Hodgkin's disease, non_hodgkin lymphoma, cancer of the esophagus, carcinoma of small intestine, internal system cancer, thyroid cancer, parathyroid carcinoma, adrenal, soft tissue sarcoma, carcinoma of urethra, carcinoma of penis, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumor, lymphocytic lymphoma, bladder cancer, kidney or carcinoma of ureter, carcinoma of renal pelvis, central nervous system (CNS) tumour, primary CNS lymphoma, it is swollen Tumor angiogenesis, tumor of spine, brain stem glioma, pituitary adenoma, Kaposi sarcoma, epidermis shape cancer, squamous cell carcinoma, t cell lymphoma, ambient induced cancer, the combination of cancer and the cancer including Induced by Asbestos.
A kind of kit is used for method of the preparation through modifying T cell of any one of claim 1-12.