Fourth-generation CAR-T cell and application thereof

A cell, the fourth technology, applied in the field of biomedicine, to achieve the effect of enhancing the ability of in vitro proliferation

Pending Publication Date: 2021-01-12
汤朝阳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in the treatment of solid tumors, this technology still has many problems

Method used

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  • Fourth-generation CAR-T cell and application thereof
  • Fourth-generation CAR-T cell and application thereof
  • Fourth-generation CAR-T cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Preparation of CAR-T cells expressing secretory IL-7 and conditional CCL19

[0064] (1) Preparation of lentiviral vector

[0065] The following nucleic acid molecules are synthesized through whole genes:

[0066] ① A CAR molecule formed by tandem nucleic acid sequences of GM-CSF signal peptide, PSCA scFv, CD28 transmembrane domain, CD28 intracellular domain, CD3ζ and 2A peptide;

[0067] ② by IL-7 signal peptide (SEQ ID NO: 11), IL-7 (without IL-7 signal peptide) (SEQ ID NO: 7), 2A peptide (SEQ ID NO: 10) and CCL19 (SEQ ID NO :9) the nucleic acid molecules formed in series by the nucleic acid sequences;

[0068] ③ A secretory IL-7 molecule formed by tandem nucleic acid sequences of IL-10 signal peptide (SEQ ID NO: 6) and IL-7 (without IL-7 signal peptide) (SEQ ID NO: 7);

[0069] ④Conditional CCL19 molecule formed by 3×NFAT-RE-IL2 promoter (SEQ ID NO:8) and CCL19 (SEQ ID NO:9) in series;

[0070] SEQ ID NO: 11:

[0071] atgttccatgtttcttttaggtatatctttggacttc...

Embodiment 2

[0081] Example 2 Secretion of IL-7 and CCL19 by CAR-T cells

[0082] Take 2×10 of each of the three CAR-T cells prepared in Example 1 6 , cultured with fresh T cell medium for 24 hours, and collected the cell supernatant; at the same time, take 2×10 6 A PSCA-secIL7×IL2pro-CCL19 CAR-T was co-cultured with the same amount of PSCA-positive cells K562-PSCA for 24 hours, and the cell supernatant was collected; the ELISA kit (R&D system) was used to detect the expression of each cell on IL-7 and CCL19 Secretion.

[0083] like figure 1 As shown, there is no IL-7 and CCL19 in the culture supernatant of PSCA CAR-T cells, only CCL19 and no IL-7 in the culture supernatant of PSCA-7×19 CAR-T cells, PSCA-secIL7×IL2pro-CCL19 CAR - There was only IL-7 and no CCL19 in the culture supernatant of T cells, and after co-culture with K562-PSCA, PSCA-secIL7×IL2pro-CCL19 CAR-T secreted IL-7 and CCL19.

[0084] It shows that PSCA-secIL7×IL2pro-CCL19 CAR-T cells can use IL-10 signal peptide to suc...

Embodiment 3

[0085] Example 3 In vitro proliferation experiment of CAR-T cells

[0086] Take 2×10 of each of the three CAR-T cells prepared in Example 1 6 One was cultured with fresh T cell medium, counted every two days, and cultured continuously for 7 days to analyze the proliferation of CAR-T cells.

[0087] The results are shown in Table 1. PSCA-7×19 CAR-T has no effect on the proliferation of CAR-T cells because it does not secrete IL-7; the IL-7 expressed by the modified PSCA-secIL7×IL2pro-CCL19 CAR-T can It is successfully secreted into the extracellular space and promotes the proliferation of CAR-T cells.

[0088] Table 1

[0089]

[0090]

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Abstract

The invention provides a fourth-generation CAR-T cell and an application thereof. The fourth-generation CAR-T cell expresses a chimeric antigen receptor specifically binding to an antigen, a secretoryIL-7 and an NFAT regulatory CCL19,wherein the signal peptide of the IL-7 is an IL-10 signal peptide; and a promoter of the CCL19 is an NFAT regulatory promoter. According to the invention, the original signal peptide of the IL-7 is changed into the signal peptide of T cell secretory protein IL-10, and the NFAT regulatory promoter is adopted as the promoter of CCL19, so that the CAR-T cell can secrete the IL-7 to the outside of the cell and conditionally express the CCL19, the proliferation of the CAR-T cell is promoted, immune cells are recruited to the inside of tumors to the greatest extent, and the anti-tumor efficacy of the CAR-T is obviously improved.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a fourth-generation CAR-T cell and its application. Background technique [0002] Chimeric antigen receptor T cell (CAR-T) immunotherapy is to induce T cell activation by expressing a chimeric antigen receptor molecule that specifically recognizes and binds to tumor antigens on the T cell membrane, thereby achieving A technique for specific killing of tumor cells. This technology is currently one of the most promising tumor immunotherapies, and has made great breakthroughs in the field of leukemia treatment. [0003] However, in the treatment of solid tumors, this technology still has many problems. Researchers are trying to improve and optimize CAR-T therapy, and improve the therapeutic effect of CAR-T on solid tumors by improving the affinity of chimeric antigen receptors and knocking out PD-1 of CAR-T cells. [0004] The CAR-T cells constructed by CN109153989A can continu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K19/00C12N15/62C12N15/867C12N7/00A61K39/00A61P35/00
CPCC07K14/7051C07K16/3069C07K14/5418C07K14/521C12N5/0636C12N15/86C12N7/00A61K39/00114A61K39/001142A61P35/00C07K2319/02C07K2319/03C07K2319/33C12N2510/00C12N2740/15021C12N2740/15043
Inventor 汤朝阳秦乐吴迪魏志辉王翠花王艳艳其他发明人请求不公开姓名
Owner 汤朝阳
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