Genetically modified immune cell, preparation method therefor, and application
Provided are a genetically modified immune cell, a preparation method therefor, and an application. The immune cell overexpresses HIL-6 and/or L-GP130. HIL-6 or L-GP130 continuous overexpression/conditionally induced overexpression in the immune cell reduces the side effects of CAR-T therapy while maintaining immune and anti-tumour effects, and has potential value in the treatment of malignant tumours and AIDS.
1 . An immune cell overexpressing HIL-6 and L-GP130, wherein the HIL-6 has an amino acid sequence of SEQ ID NO: 1, wherein the L-GP130 has an amino acid sequence of SEQ ID NO: 3, wherein the immune cell is a T cell and wherein the T cell is modified with a CAR molecule.
2 . The immune cell according to claim 1 ;
the T cell is selected from the group consisting of a CD4+ T cell, a CD8+ T cell, a CD4+CD8+ T cell, an NKT cell and a gamma delta (γδ) T cell.
3 . The immune cell according to claim 1 , wherein an antigen recognized by an extracellular scFv sequence of the CAR molecule comprises any one of 5T4, alpha-5 beta-1 (α5β1)-integrin, 707-AP, AFP, ART-4, B7H4, B7-H3, BAGE, beta (β)-integrin/m, Bcr-abl, MN/C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT, hTRT, iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, Mesothelin, myosin/m, MUC1, MUM-1, MUM-2, MUM-3, PSCA, NA88-A, PAP, protease-3, GPC3, p190minor bcr-abl, Pml/RARR, PRAME, PSA, PSM, PSMA, RAGE, RU1, RU2, SAGE, SART-1, SART-3, survivin, TEL/AML1, PD-1, PD-L1, CTLA-4, TIM3, LAG3, TGF3, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, IL-13RIN, CD123, GUCY2C, NY-Eso-1 or NY-Eso-B.
4 . The immune cell according to claim 3 , wherein an scFv of the anti-CD 19 CAR has an amino acid sequence of SEQ ID NO: 5 and a nucleic acid sequence of SEQ ID NO: 6 or an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12;
an scFv of the anti-GPC3 CAR has an amino acid sequence of SEQ ID NO: 13 and a nucleic acid sequence of SEQ ID NO: 14;
an scFv of the anti-MUC1 CAR has an amino acid sequence of SEQ ID NO: 15 and a nucleic acid sequence of SEQ ID NO: 16;
an scFv of the anti-Mesothelin has an amino acid sequence of SEQ ID NO: 17 and a nucleic acid sequence of SEQ ID NO: 18;
an scFv of the anti-PSCA has an amino acid sequence of SEQ ID NO: 19 and a nucleic acid sequence of SEQ ID NO: 20;
an scFv of the anti-HER2 has an amino acid sequence of SEQ ID NO: 21 and a nucleic acid sequence of SEQ ID NO: 22.
5 . The immune cell according to claim 1 , wherein the CAR molecule has an intracellular co-stimulatory signal transduction domain comprising an intracellular domain of any one of molecules of CD28, 4-1BB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, DAP10, CD27, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen 1, CD2, CD7, LIGHT, NKG2C, NKG2D, NKp46, NKp30, NKp44, DNAM1, B7-H3, and CD83, or comprising a combination of intracellular domains of at least two of these molecules.
6 . The immune cell according to claim 5 , wherein the TLR2 has an amino acid sequence of SEQ ID NO: 23 and a nucleic acid sequence of SEQ ID NO: 24;
the 4-1BB has an amino acid sequence of SEQ ID NO: 25 and a nucleic acid sequence of SEQ ID NO: 26;
the CD28 has an amino acid sequence of SEQ ID NO: 27 and a nucleic acid sequence of SEQ ID NO: 28;
the TLR1 has an amino acid sequence of SEQ ID NO: 29 and a nucleic acid sequence of SEQ ID NO: 30;
the DAP10 has an amino acid sequence of SEQ ID NO: 31 and a nucleic acid sequence of SEQ ID NO: 32;
a signal peptide of the CAR molecule has a nucleic acid sequence of SEQ ID NO: 33;
a transmembrane region of the CAR molecule has a nucleic acid sequence of SEQ ID NO: 34;
CD32 of the CAR molecule has a nucleic acid sequence of SEQ ID NO: 35.
7 . A method for preparing the immune cell according to claim 1 , comprising overexpressing HIL-6 and L-GP130 in an immune cell, wherein the HIL-6 has an amino acid sequence of SEQ ID NO: 1, wherein the L-GP130 has an amino acid sequence of SEQ ID NO: 3, wherein the immune cell is a T cell and wherein the T cell is modified with one or two of a CAR molecule.
8 . A pharmaceutical composition, comprising the immune cell according to claim 1 ;
the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, an excipient or a diluent.
9 . A method for the treatment of tumors comprising: administering the immune cell of claim 1 ; wherein the tumor comprises CD19+B-cell acute lymphoblastic leukemia, MUC1+ lung cancer, or GPC3+ liver cancer.
10 . The immune cell according to claim 1 , wherein the T cell has a nucleic acid sequence of SEQ ID NO: 2.
11 . The immune cell according to claim 1 , wherein the T cell has a nucleic acid sequence of SEQ ID NO: 4.