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Double-specific chimeric antigen receptor T cell as well as preparation method and application thereof

A chimeric antigen receptor and bispecific technology, applied to bispecific chimeric antigen receptor T cells, can solve the problems of weak targeting specificity and low killing efficiency

Active Publication Date: 2019-10-15
NANJING KAEDI BIOTHERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In view of the above-mentioned problems and / or other problems in related technologies, the purpose of the present invention is to overcome the weak targeting specificity and low killing efficiency of T cells in the tumor microenvironment to bind and kill tumor cells in the existing tumor clinical technology For the problem of providing bispecific targeting binding polypeptide domains targeting human NKG2DL and human CD47 or functional variants thereof, bispecific chimeric antigen receptors targeting human NKG2DL and human CD47 or functional variants thereof , and its coding nucleotide sequence and its expression vector, engineered immune response cells modified by bispecific chimeric antigen receptors targeting NKG2DL and CD47, and applications thereof

Method used

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  • Double-specific chimeric antigen receptor T cell as well as preparation method and application thereof
  • Double-specific chimeric antigen receptor T cell as well as preparation method and application thereof
  • Double-specific chimeric antigen receptor T cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Example 1. Preparation of bispecific expression plasmids targeting bispecific chimeric antigen receptors that bind to human NKG2DL and CD47

[0165] Step 1. Retrieval of the amino acid sequence of the bispecific chimeric antigen receptor targeting human NKG2DL and CD47

[0166] The receptor of NKG2DL protein -- the full-length amino acid sequence of NKG2D (as shown in SEQ ID No.1) is searched from the Genbank database of the NCBI of the National Library of Medicine of the United States; The full-length amino acid sequence of SIRPα (the receptor of CD47 protein) (as shown in SEQ ID No.2)

[0167] Step 2. Construction of bispecific chimeric antigen receptors targeting NKG2DL and CD47

[0168] Design of the amino acid sequence and coding nucleotide sequence of the bispecific chimeric antigen receptor that binds to NKG2DL and CD47.

[0169] details as follows:

[0170] The amino acid sequence of the bispecific CAR molecule with bispecific targeting binding to human NKG2D...

Embodiment 2

[0199] Example 2. Isolation and culture of T cells

[0200] Fresh peripheral blood from healthy donors was taken, and fresh peripheral blood mononuclear cells were separated by density gradient centrifugation; then, magnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA, product information: Human T-Activator CD3 / CD28, catalog number: 11161D) to enrich CD3 + T cells, specifically, peripheral blood mononuclear cells were diluted to a concentration of (10-30) × 10 6 single cells / ml, then mix the magnetic beads and cells in a culture dish at a ratio of 3:1, and incubate at room temperature for 2 to 3 hours. CD3+ T cells were enriched from Invitrogen (USA). Finally, the enriched CD3+T cells were resuspended in culture medium (purchased from Life Technologies, USA, product information is OpTmizer TM T-Cell Expansion SFM, A1048503), adjusted to cell solubility of 1 × 10 6 cells / ml, and finally cultured in a 37°C, 5% CO2 incubator f...

Embodiment 3

[0201] Embodiment 3. Preparation of virus liquid

[0202] The bispecific CAR expression plasmids obtained in Step 3 of Example 1 respectively targeting human NKG2DL and human CD47, as well as the viral packaging helper plasmids psPAX2 and VSVG, were mixed with polyethyleneimine at a ratio of 10:8:5 Transfection reagent (408727, Sigma) transfection reagent, co-transfect 293T cells (ATCC product, product number: CRL-3216TM); specific packaging plasmid preparation method, see Lenti-X Packaging Single Shots (Takara) instructions; For specific transfection procedures, see the Sigma transfection instructions.

[0203] Replace with complete medium (purchased from Life Technologies, product number: 11995-065) 6 hours after transfection, collect virus supernatant after 48 hours and 72 hours of culture, and centrifuge at 4°C, 3000rpm for 10-15 minutes Afterwards, filter through a 0.45 μm filter membrane, and finally ultracentrifuge at 25,000 rpm for 2 to 3 hours at 4°C to concentrate t...

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Abstract

The invention relates to a double-specific chimeric antigen receptor T cell as well as a preparation method and application thereof. The double-specific chimeric antigen receptor double-specifically targeting human NKG2DL and human CD47 is constructed on the basis of human NKG2D and human SIRPalpha molecules as well as an immune response cell modified with the double-specific chimeric antigen receptor. The novel modified immune response cell can effectively realize targeting attack of multiple tumor cells, particularly positive tumor cells expressing NKG2DL and CD47 and can be used for preparing preparations for treating tumor. The preparation method of the immune response cell modified with the double-specific chimeric antigen receptor double-specifically targeting NKG2DL and CD47 comprises simple steps, the obtained immune response cell modified with the double-specific chimeric antigen receptor double-specifically targeting NKG2DL and CD47 has high specific killing rate for tumor cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine for tumor immunotherapy, and relates to specific chimeric antigen receptor T cells, in particular to a bispecific targeting human NKG2DL and human CD47, comprising human NKG2D and human SIRPα protein or its function A sex variant bispecific chimeric antigen receptor (chimeric antigen receptor, CAR), its modified immune response cells, and its preparation method and application. Background technique [0002] With the rapid development of biotechnology, immune cell therapy has become the fourth largest therapy in the field of cancer treatment. [0003] Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immune checkpoint blockade therapy. Among them, in the field of cell therapy, chimeric antigen receptor-modified immune cells (especially Chimeric Antigen Receptor T-Cell, CAR-T) therapy has undoubtedly become a star that research institutions and ph...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/113C12N7/01C12N5/10A61K39/00A61P35/00A61P35/02A61P31/00A61P37/02A61P29/00A61P37/06A61P39/06
CPCA61K39/0011A61K39/001129A61K2039/5156A61K2039/5158A61P29/00A61P31/00A61P35/00A61P35/02A61P37/02A61P37/06A61P39/06C07K14/70503C07K14/7056C07K2319/00C12N7/00C12N2710/10021C12N2740/10021C12N2740/15021
Inventor 代红久李艳云
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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