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PD-1 gene expression silenced engineered immune cell

A technology of immune cells and PD-1, applied in the field of genetic engineering and immune cell therapy, can solve the problems of expensive antibody drugs and difficulties in the development of effective antibodies

Active Publication Date: 2019-04-02
GRACELL BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PD-1 antibody can improve the function of CAR-T cells, but after the antibody is injected, the whole body blocks PD-1, which enhances the activation of autoreactive T cells, interferes with normal T cells in the body, and leads to related toxicity in the body, and the effect of the antibody is only short-lived Yes, the antibody blocking effect will disappear after stopping the drug, it is difficult to develop effective antibodies, and antibody drugs are expensive

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0211] Example 1 Preparation of PD-1 gene knockout T cells

[0212] 1.1 Design sgRNA:

[0213]Design sgRNA (20bp) for the coding region of PD-1 signal peptide, intracellular segment or transmembrane region to knock out PD-1 gene. Synthesis of Oligo1: 5'-CACCG-(sgRNA sequence)-3' and Oligo2: 5'-AAAC-(sgRNA reverse complement sequence)-C-3'; use ultrapure water to dissolve Oligo fragments at a concentration of 100uM, Oligo1 and Oligo2 is mixed according to the following system (total volume 10uL) and then annealed to form double strands: 1uL, Oligo1 (100uM); 1uL, Oligo2 (100uM); 8uL, H 2 O; PCR annealing conditions: 95°C, 5 minutes, slowly cool down to room temperature for 1 hour, dilute 1:200 to anneal double strands.

[0214] 1.2 Plasmid digestion and gel recovery:

[0215] Enzyme digestion system: 2ug, pX330plasmid (Addgene plasmamid #4223); 1uL, BsmBI; 2uL, 10× buffer; add ultrapure water to make up the volume to 20uL. Digestion conditions: 55°C, incubate for 1 hour. Af...

Embodiment 2

[0233] Example 2 CAR and chPD-1 structure and carrier preparation

[0234] The molecular structure of CAR includes: GM-CSF signal peptide, FMC63scFv, CD8 hinge region and transmembrane region, CD137 co-stimulatory domain and CD3ζ intracellular signaling domain (named CD19CAR), the amino acid sequence is shown in SEQ ID NO.:9 Show.

[0235] The molecular structure of chPD-1 includes: CD8 signal peptide, extracellular domain of PD-1, CD8 transmembrane region, CD137 co-stimulatory domain, CD3ζ intracellular signaling domain (named chPD-1-CD137), the amino acid sequence is shown in SEQ ID NO.: 11.

[0236] The gene sequences of CD19CAR and chPD-1-CD137 were synthesized, connected by T2A peptide, and cloned into the backbone of FUW lentiviral vector, and placed under the promoter of EF1α to form Fuw-EF1α-CD19CAR-T2A-chPD-1-CD137 The CD19CAR and chPD-1-CD137 genes can be co-expressed in the cells.

[0237] Three plasmids, Fuw-EF1α-CD19CAR-T2A-chPD-1-CD137, and the lentiviral enve...

Embodiment 3

[0240] Example 3 PD-1(-)chPD-1(+)CD19CAR-T cytokine release detection

[0241] Effector cells PD-1(-)chPD-1(+)CD19CAR-T cells, chPD-1(+)CD19CAR-T cells, CD19CAR-T cells, T cells and target cells (K562 cells, K562-CD19, K562 - CD19PD-L1 cells) were co-cultured at a ratio of 1:1 (cell density was 10^6 / ml), and the supernatant was collected after 24 hours, and the IL-2 after CAR-T stimulation was detected using an ELISA kit (Biolegend) according to the instructions. and IFN-γ release levels.

[0242] The results showed that in the target cell K562-CD19PD-L1 group, PD-1(-)chPD-1(+)CD19CAR-T cells secreted cytokines IFN-γ and IL-2 levels were significantly correlated with those of chPD-1(+)CD19CAR-T cells Similar, higher than CD19CAR-T cells, T cells.

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PUM

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Abstract

The invention relates to a PD-1 gene expression silenced engineered immune cell. In particular, the present invention relates to the PD-1 gene expression silenced engineered immune cell that expressesa CAR or an exogenous TCR that targets a tumor cell marker and a fusion protein that targets PD-L1. The experiments show that the engineered immune cell of the present invention is affected by immunosuppression effect of a PD-1 signal pathway, and synergistically activates immune cells by the fusion protein that targets PD-L1, and synergism provides higher immunization cell activity, the tumor cell killing effect is significantly enhanced, the tumor cells expressing the CAR and / or exogenous TCR-targeted antigen and tumor cells expressing the PD-L1 can be simultaneously killed, tumor cells canbe prevented from immune escape, and the immune cell is difficult to target and relapse.

Description

technical field [0001] The invention belongs to the field of genetic engineering and immune cell therapy, and relates to an engineered immune cell with PD-1 gene expression silence. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture and expand immune cells collected from patients in vitro and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors. [0003] In recent years, chimeric antigen receptor gene-modified T (CAR-T) cells, as "living drugs", have achieved exciting results in the treatment of hematological tumors, and have become a new development direction for tumor treatment. The design of CAR has gone through the following process: the first-generation CAR has only one intra...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85A61P35/00
CPCA61K35/17C12N5/0636C12N5/0646C07K14/7051C07K14/70521C07K16/2827C07K2319/02C07K2319/03C12N2510/00
Inventor 马安云刘丽萍何佳平曹卫
Owner GRACELL BIOTECH SHANGHAI CO LTD
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