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Chimeric antigen recipient cell taking ROBO1 as target, preparation and application thereof

A chimeric antigen receptor, antigen technology, applied in receptor/cell surface antigen/cell surface determinant, genetically modified cells, for targeting specific cell fusion, etc., can solve the problem of low tumor cell specificity

Pending Publication Date: 2018-12-11
ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This chimeric receptor T cell can only activate the anti-tumor effect of T cells after 2-3 chimeric antigen receptors recognize the antigen. low specificity

Method used

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  • Chimeric antigen recipient cell taking ROBO1 as target, preparation and application thereof
  • Chimeric antigen recipient cell taking ROBO1 as target, preparation and application thereof
  • Chimeric antigen recipient cell taking ROBO1 as target, preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 Preparation of lentiviral expression vector

[0116] According to "Engineering high-affinity PD-1 variants for optimized immunotherapy and immune-PET imaging" (Maute RL, Gordon SR, Mayer AT, etc. Proceedings of the National Academy of Sciences, 2015, 112(47): E6506-E6514.) The HAC sequence information of the PD-1 mutant molecule, the synthesis of the extracellular domain of the PD-1 protein or its mutant gene sequence, as shown in SEQ ID NO:1; search and synthesize the known human anti-ROBO1 from the GenBank database. FN3scFv (hereinafter referred to as scFv) gene sequence, IRES internal ribosome entry site, human CD8 transmembrane region gene sequence, human 4-1BB intracellular region gene sequence, CD3ζ intracellular region gene sequence, respectively, as SEQ ID NO: 2 -6 shown.

[0117] The sequence of the above-mentioned genes was sequenced according to scFv gene, CD8 gene, human 4-1BB intracellular region gene and CD3ζ intracellular region gene, IRES internal ...

Embodiment 2

[0119] Example 2 Preparation of Lentivirus

[0120] 1. 24 hours before transfection, about 8×10 per dish 6 The 293T cells were seeded into a 15cm petri dish. Ensure that the cells are at about 80% confluence and are evenly distributed in the culture dish during transfection.

[0121] 2. Prepare solution A and solution B

[0122] Solution A: 6.25mL 2×HEPES buffer (5 large dishes are used to pack together, the best effect).

[0123] Solution B: Add the following plasmid mixture: 112.5μg scFv-CD8TM-4-1BB-CD3ζ-IRES- (the extracellular domain of PD-1 protein or its mutant) (target plasmid); 39.5μg pMD2.G( VSV-Genvelop); 73μg pCMVR8.74 (gag, pol, tat, rev); 625μL 2M calcium ion solution. Total volume of solution A: 6.25 mL.

[0124] 3. Mix solution B thoroughly, add solution A dropwise while vortexing solution A gently, and let stand for 5-15 minutes. Gently vortex the above-mentioned mixed solution of A and B, add dropwise to the petri dish containing 293T cells, and gently shake the pet...

Embodiment 3

[0127] Example 3 Preparation of CAR-T cells

[0128] Follow the steps in Example 3 of CN201610226230.9 to prepare CAR-T cells.

[0129] After virus infection, use flow cytometry to detect CAR-T positive rate. The flow cytometry diagram is as follows figure 2 As shown, the results showed that the positive rate was 51.38%, and CAR-T cells were successfully prepared. ( figure 2 : Flow cytometry detects the positive expression rate of CAR-T, figure 2 -A is T cell that has not been infected by virus, as a control; figure 2 -B is T cell after virus infection. The antibody is fluorescently labeled with FITC and is indicated on the abscissa. If the T cell successfully expresses the CAR molecule, the signal value will increase significantly. )

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Abstract

The invention discloses a chimeric antigen recipient cell taking ROBO1 as a target, a preparation and an application thereof and especially discloses a reinforced CAR-T cell and CAR-NK cell taking ROBO1 as the target. The cell is capable of stably expressing CAR element while performing secretory expression on extracellular domain molecules or mutants of PD-1 protein and stopping the interaction of PD-1 / PD-L1 molecules. An animal test proves that the cell has an excellent anti-tumor effect, and the cell, compared with a common CAR modified cell taking ROBO1 as the target, is capable of obviously reducing recurrence rate of tumors and increasing survival rate.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a chimeric antigen receptor cell targeting ROBO1, in particular an enhanced CAR-T cell and CAR-NK cell targeting ROBO1, and preparation and application. Background technique [0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) immunotherapy is an adoptive immunotherapy method that has developed rapidly in recent years, in which CAR is usually composed of antigen binding domains and transmembrane domains. , Co-stimulatory signal transduction area and other components, which through genetic modification technology, fusion expression of tumor-associated antigen antibodies on the surface of autologous T cells, modified T cells therefore have targeted killing power to tumor cells. CAR-T therapy has significant effects in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered to be one of the most promising tumor treatment methods. In addition to hema...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N5/10C12N15/867A61P35/00
CPCC12N5/0636C12N5/0646C07K16/30C07K2319/33C07K2319/03C12N2510/00A61K2239/17A61K39/4644A61K39/4613A61K39/4631A61K2239/55A61K39/4611A61K38/17A61P35/00C12N15/867A61K38/00C07K2317/622C07K16/2803A61K2039/505C12N2501/515C07K14/7051C07K14/70517C07K14/70578C07K14/70521C07K2317/24C07K2317/55C07K2317/76C07K2319/02C07K2319/30
Inventor 李华顺王保垒韩昆昆
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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