Chimeric antigen receptor T cell and application thereof

A chimeric antigen receptor, cell technology, applied in receptor/cell surface antigen/cell surface determinant, application, animal cells, etc., can solve the problem that exogenous IL-7 protein cannot be secreted outside T cells, exogenous IL-7 protein cannot be secreted, and peripheral blood immune cells cannot be recruited into tumors.

Active Publication Date: 2020-01-14
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the exogenous introduction of IL-7 can make T cells transcribe and translate IL-7 protein, the secretion of IL-7 protein to the extracellular space is still strictly controlled by T cells. The research reported by Adachi et al. shows that exogenous IL-7 protein cannot be secreted. to the problem of T cells
[0007] CN109504660A discloses a fourth-generation CAR-T cell and its construction method and application. The CAR includes an extracellular antigen bindin

Method used

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  • Chimeric antigen receptor T cell and application thereof
  • Chimeric antigen receptor T cell and application thereof
  • Chimeric antigen receptor T cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1 Preparation of T cells expressing secretory IL-7 and conditional CCL19

[0128] (1) Optimization strategies for cytokines and chemokines

[0129] T cells do not secrete IL-7 under natural conditions, and exogenously introduced IL-7 cannot be secreted outside T cells after being transcribed and translated. The signal peptides of CAR-T cells are replaced by signal peptides of proteins secreted by T cells, such as signal peptides of IL-2 and IL-4; in order to maximize the recruitment of immune cells into the tumor and improve the anti-tumor effect of CAR-T cells, the invention Humans changed the continuous expression of CCL19 to conditional expression, and used the promoter 6×NFAT-RE-minP regulated by the nuclear factor of activated T cells (NFAT) to regulate the expression of CCL19, so that CAR-T cells recognized tumor antigens and were activated. Only then can the expression of CCL19 be activated, thereby specifically recruiting immune cells into the tumor.

...

Embodiment 2

[0159] Example 2 Expression of CAR molecules, IL-7 and CCL19 by T cells

[0160] Since the lentiviral vector expressing CAR molecules carries the EGFP gene, EGFP can be used to indicate the expression of CAR molecules on T cells. Since the lentiviral vectors expressing IL-7 and CCL19 carry tCD19 (only contains the transmembrane domain and extracellular domain of human CD19), which can indicate the expression of IL-7 and CCL19 on T cells through CD19; when T cells are infected by the virus for 48 hours, EGFP and CD19 on T cells are detected by flow cytometry, and the expression of CAR Molecules, IL-7 and CCL19 T cells; the flow cytometer used in this example is ACEA Novocyte, and the flow cytometry antibody with the clone number HIB19 from Biolegend Company is used to detect CD19.

[0161] The result is as figure 1 Shown are CAR-T cells expressing only CAR (GPC3 CAR-T), CAR-T cells expressing CAR, IL-7 and CCL19 (GPC3-7×19CAR-T), and CAR molecules expressing, secreting IL-7 a...

Embodiment 3

[0162] Example 3 Secretion of IL-7 and CCL19 by CAR-T cells

[0163] Take 2×10 of each of the three CAR-T cells confirmed in Example 2 6 One, cultured with fresh T cell culture medium for 24 hours, collected cell supernatant;6 Each was co-cultured with the same amount of HepG2 tumor cells expressing GPC3 tumor antigen for 24 hours, and the cell supernatant was collected; ELISA kit (R&D system) was used to detect the secretion of IL-7 and CCL19 by each cell.

[0164] Test results such as figure 2 As shown, there is no IL-7 and CCL19 in the culture supernatant of GPC3 CAR-T cells, only CCL19 and no IL-7 in the culture supernatant of GPC3-7×19 CAR-T cells, GPC3-IL2SG-IL7×NFATpro-CCL19 The culture supernatant of CAR-T cells has only IL-7 and no CCL19, and the culture supernatant of GPC3-IL2SG-IL7×NFATpro-CCL19 CAR-T cells co-cultured with HepG2 tumor cells can simultaneously detect IL-7 and CCL19.

[0165] This shows that the modified GPC3-IL2SG-IL7×NFATpro-CCL19 CAR-T cells c...

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Abstract

The invention provides a chimeric antigen receptor T cell and application thereof. The chimeric antigen receptor T cell expresses a chimeric antigen receptor of a specific binding antigen, IL-7 and CCL19; signal peptide of the IL-7 is signal peptide of T cell secretion protein; and a promoter of the CCL19 is an NFAT regulation promoter. According to the chimeric antigen receptor T cell and the application thereof provided by the invention, the IL-7 and the CCL19 are optimized, the signal peptide of the IL-7 is changed, the continuous secretion of the CCL19 is changed to be conditioned secretion, the built CAR-T cell can continuously secrete the IL-7 to the outside of the cell, the secreted CCL19 is expressed after a tumor antigen is recognized, and immune cells are maximally recruited inside a tumor, so that an anti-tumor effect of the CAR-T cell is remarkably improved.

Description

technical field [0001] The invention belongs to the field of tumor cell immunotherapy, relates to a chimeric antigen receptor T cell and its application, in particular to a chimeric antigen receptor T cell stably expressing cytokines and deactivating factors at a high level and its preparation Application in antitumor therapeutic drugs. 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. [0003] At present, CAR-T immunotherapy has made a huge breakthrough in the field of leukemia treatment. The US FDA has approved two CD19-targeting CAR-T cell products for marketing, and many domestic companies have also carried out clinical trials targeting CD19. [0004] Although CAR-T technology has made some breakthroughs in t...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N7/01C07K19/00A61K39/00A61P35/00
CPCC07K14/5418C07K14/521C07K14/7051C07K16/303C12N15/86C12N7/00C12N5/0636A61K39/001142A61K39/00114A61P35/00C07K2319/02C07K2319/03C07K2319/33C07K2317/622C12N2510/00A61K2039/5156
Inventor 李鹏师靖轩秦乐姚瑶林思妙
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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