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Engineered immune cell targeting CD19 and CD22 and application of engineered immune cell

A targeted, cellular technology, applied in the field of immunotherapy, can solve the problems of high recurrence rate and low safety

Pending Publication Date: 2019-08-23
GRACELL BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are still many shortcomings in the current research on cellular immunotherapy, and there are still problems such as high recurrence rate and low safety.

Method used

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  • Engineered immune cell targeting CD19 and CD22 and application of engineered immune cell
  • Engineered immune cell targeting CD19 and CD22 and application of engineered immune cell
  • Engineered immune cell targeting CD19 and CD22 and application of engineered immune cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0320] Structural design and virus preparation of embodiment 1CAR

[0321] The present invention provides a nucleic acid construct that co-expresses a specific chimeric antigen receptor for the CD19 antigen and a specific chimeric antigen receptor for the CD22 antigen. The co-expression of a CD19-targeting CAR and a CD22-targeting CAR structural component may include a CD19-targeting CAR and a CD22-targeting CAR.

[0322] The structure of each construct is as Figure 1A with 1B shown.

[0323] Construct a lentiviral transfer vector plasmid that has co-expressed CD19 and CD22. The backbone of the plasmid is derived from pCDH; the lentiviral transfer vector plasmid pCDH-EF1α-CD19 / CD22CAR, and the lentiviral envelope plasmid pMD2.G (Addgene, Plasmid#12259 ) and the lentiviral packaging plasmid psPAX2 (Addgene, Plasmid#12260) were simultaneously transferred into 293T using Lipofectamine3000 to prepare the lentiviral expression vector; the virus supernatant was collected on the s...

Embodiment 2

[0324] Example 2 Isolation and expansion of T cells

[0325] Mononuclear cells were isolated from peripheral blood by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich), and mononuclear cells were isolated from peripheral blood and enriched for T cells (EasySep human T cell enrichment kit, Stemcell Technologies) Anti-CD3 / anti-CD28 magnetic beads were used to activate, culture and expand T cells; the culture medium used X-vivo15 (containing 5% FBS, 2mM L-glutamine, 1mM sodium pyruvate, 300IU / ml rhIL2); all cells were Place at 37°C, 5% CO 2 cultured in a constant temperature incubator.

Embodiment 3

[0326] Example 3 Cell Culture

[0327] Cell lines expressing CD19 / CD22: Raji (Burkitt”s lymphoma cell line, ATCC-CCL86) Raji-Luc cell line obtained after screening monoclonal infection of Raji cells with firefly luciferase lentivirus; Hela cell line (human cervical cancer cell line Line, ATCC-CCL2), use CD19, CD22 lentivirus transfection to construct stable transfection strains Hela-CD19 and Hela-CD22, use CD22 to infect Hela-CD19 cell line to obtain double-expressed cell line Hela-CD19 / CD22 cells above all Use RPMI 1640 medium to cultivate; 293T (ATCC-CRL3216) to use DMEM medium to cultivate.All mediums are supplemented with 10% (v / v) bovine serum and 100U / ml of penicillin and streptomycin, 2mM glutamine, 1mM Sodium pyruvate; all cells were kept at 37°C, 5% CO 2 cultured in a constant temperature incubator.

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Abstract

The invention relates to an engineered immune cell targeting CD19 and CD22 and application of the engineered immune cell, and particularly provides a nucleic acid construct having a structure of P1-X-P2-Y (formula I), the definition of each element is as described in the specification. The engineered immune cell can recognize both CD19 and CD22 antigens, and the expression levels of two CARs targeting the CD19 and CD22 are similar in the engineered immune cell, thereby reducing immune escape risks caused by the down-regulation or loss of antigen presentation during recognition of a single-target CAR-T cell in the therapy process, and greatly reducing the difficulty of industrial application of the engineered immune cell.

Description

technical field [0001] The present invention relates to the field of immunotherapy, and more specifically relates to engineered immune cells targeting CD19 and CD22 and applications thereof. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new treatment method for autoimmune anti-cancer. It is a method of using biotechnology and biological agents to culture and expand immune cells collected from patients in vitro, and then reinfuse 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] Chimeric antigen receptors (CARs) consist of an extracellular antigen recognition region, usually scFv (single-chain variable fragment), a transmembrane region, and an intracellular co-stimulatory signal region. The design of CARs has gone through the following process: the first-generation CAR has only one intracel...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C07K19/00A61K35/17A61P35/00
CPCC12N15/86C07K16/2803C07K14/7051C12N5/0636A61K35/17A61P35/00C07K2317/622C12N2740/15043C07K2319/02C07K2319/03C12N2510/00
Inventor 江鹏斐曹卫沈连军朱小彬马安云刘丽萍董琦
Owner GRACELL BIOTECH SHANGHAI CO LTD
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