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Genetically-modified immune cells and preparation method and application thereof

An immune cell and cell technology, applied in the field of immune cells overexpressing HIL-6 and/or L-GP130 and their preparation, can solve the problems of affecting the effect of anti-tumor treatment, affecting the anti-tumor effect of immune cells, etc.

Active Publication Date: 2019-07-26
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recent studies often use methods such as IL-6 receptor monoclonal antibody and IL-6 receptor knockout to block the side effects of IL-6 (PMID: 27076371). However, blocking IL-6 will affect the anti-tumor effect of immune cells. Maximize, affect the effect of anti-tumor therapy

Method used

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  • Genetically-modified immune cells and preparation method and application thereof
  • Genetically-modified immune cells and preparation method and application thereof
  • Genetically-modified immune cells and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Constructing a persistent co-expression system

[0072] Through gene synthesis, the continuous co-expression plasmid vectors pUC57-CAR-2A-HIL-6 and pUC57-CAR-2A-L-GP130 of CAR molecules that recognize tumor antigens and HIL-6 or L-GP130 genes were obtained.

[0073] pUC57-CAR-2A-HIL-6 and pUC57-CAR-2A-L-GP130 were digested to obtain CAR-2A-HIL-6 and CAR-2A-L-GP130 genes, which were connected to the lentiviral vector pWPXLd (containing GFP gene) or PB vector (containing GFP gene), construct pWPXLd-CAR-2A-HIL-6-2A-GFP and pWPXLd-CAR-2A-L-GP130-2A-GFP, or PB-CAR-2A-HIL -6-2A-GFP and PB-CAR-2A-L-GP130-2A-GFP.

[0074] In this embodiment, the amino acid sequence of HIL-6 is SEQ ID NO: 1 or an amino acid sequence having the same or similar activity with more than 80% homology with SEQ ID NO: 1, and the nucleic acid sequence is as shown in SEQ ID NO: 2 The amino acid sequence of L-GP130 is SEQ ID NO: 3 or an amino acid sequence having the same or similar activity w...

Embodiment 2

[0081] Example 2 Constructing a continuous separate co-expression system

[0082] Through molecular cloning, plasmid vectors pUC57-CAR, pUC57-HIL-6, and pUC57-L-GP130, which continuously express CAR molecules that recognize tumor antigens, HIL-6 gene, and L-GP130 gene, were obtained. Digest pUC57-CAR, pUC57-HIL-6 and pUC57-L-GP130 to obtain CAR, HIL-6 and L-GP130 genes, and connect them to lentiviral vector pWPXLd (containing GFP gene) or PB vector (containing GFP gene ), construct pWPXLd-CAR-2A-GFP, pWPXLd-HIL-6-2A-GFP and pWPXLd-L-GP130-2A-GFP, or PB-CAR-2A-GFP, PB-HIL-6-2A-GFP and PB-L-GP130-2A-GFP.

[0083] In this example, the sequence information of the CAR molecule, HIL-6, L-GP130 and vector used in the construction system is the same as that in Example 1.

Embodiment 3

[0084] Example 3 Constructing CAR recognition and activation-induced overexpression system

[0085] Through gene synthesis, the plasmid vector pUC57-CAR expressing the CAR molecule recognizing tumor antigen, NFAT repeat sequence (6 repeat sequences)-IL2 promoter-HIL-6 gene and NFAT repeat sequence-IL2 promoter-L-GP130 gene was obtained , pUC57-NFAT repeat-IL2 promoter-HIL-6 and pUC57-NFAT repeat-IL2 promoter-L-GP130. Digest pUC57-CAR, pUC57-NFAT repeat-IL2 promoter-HIL-6 and pUC57-NFAT repeat-IL2 promoter-L-GP130 to obtain CAR, NFAT repeat-IL2 promoter-HIL-6 and NFAT repeat sequence-IL2 promoter-L-GP130 gene, connected to lentiviral vector pWPXLd (no promoter, containing GFP gene) or PB vector (no promoter, containing GFP gene), construct pWPXLd-CAR-2A- GFP, pWPXLd(no promoter)-NFAT repeat-IL2 promoter-HIL-6-2A-GFP and pWPXLd(no promoter)-NFAT repeat-IL2 promoter-L-GP130-2A-GFP, or PB -CAR-2A-GFP, PB(no promoter)-NFAT repeat-IL2 promoter-HIL-6-2A-GFP and PB(no promoter)-NFAT...

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PUM

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Abstract

The invention provides genetically-modified immune cells and a preparation method and application thereof. The immune cells can overexpress HIL-6 and / or L-GP130. By subjecting HIL-6 or L-GP130 to treatment / overexpression / conditional induced overexpression in the immune cells, the abilities of the immune cells to proliferate, migrate and kill tumors are enhanced significantly, with no influence caused to other immune cells, and without causing or worsening global or local inflammatory responses; the side effects of CAR T (chimeric antigen receptor T-cells) therapy are relieved at the premise ofmaintaining immunological effect and tumor resistance; the genetically-modified immune cells have the potential value in the treatment of malignant tumors and Aids.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a genetically modified immune cell and its preparation method and application, in particular to an immune cell overexpressing HIL-6 and / or L-GP130, its preparation method and application. Background technique [0002] The clinical application of anti-CD19 chimeric antigen receptor (CAR) T cells (tisagenlecleucel and axicabtageneciloleucel) has shown that it has a significant killing effect on CD19-positive B-cell malignancies. However, CAR T cell therapy still has multiple problems. In the treatment of leukemia, CAR T cell therapy has the problem of high recurrence rate, and promoting the maintenance of CAR T cells and the formation of memory T cells may be a technical breakthrough to solve this problem; in addition, the clinical treatment research of anti-CD19 CAR T cells The results show that there is a certain positive correlation between the response rate of patients to CAR T cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K39/00A61P35/00A61P35/02A61P31/18
CPCC12N5/0636C12N15/86A61P35/00A61P35/02A61P31/18C12N2510/00C12N2740/15043C12N2800/107A61K39/4631A61K39/4635A61K39/464412A61K39/464474A61K2239/53A61K2239/38A61K2239/48A61K2239/55A61K39/4611A61K39/46447C07K14/5412C07K14/7155C07K2319/02C07K2319/70C07K14/70503C07K14/7051C07K2319/03C07K2317/622C07K16/2803A61K35/17C07K16/303C07K16/3069C07K16/3092C07K16/32C07K2319/33
Inventor 不公告发明人
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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