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Costimulatory signal domain of chimeric antigen receptor and application thereof

A chimeric antigen receptor and co-stimulatory signal technology, applied in the field of genetic engineering and biological immunotherapy, can solve the problems of increased cytokine secretion, uneven curative effect, limited anti-tumor activity, etc., and achieve the effect of high lethality

Pending Publication Date: 2021-08-13
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the anti-tumor activity of the first-generation CAR-modified T cells is limited in vivo, which will eventually lead to the apoptosis of T cells due to the reduction of T cell proliferation.
The second-generation CAR adds a new co-stimulatory signal CD28 or 4-1BB (CD137) in the cell. Compared with the first-generation CAR, the antigen specificity remains unchanged, but T cell proliferation and cytokine secretion increase, and anti-cell Increased secretion of apoptotic proteins, delayed cell death
On this basis, the third and fourth generation CAR T cells have been developed to enhance the anti-tumor effect by adding two co-stimulatory molecules or adding other multi-targets, but the curative effect is uneven, especially in the treatment of solid tumors. Sensitivity is generally low in

Method used

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  • Costimulatory signal domain of chimeric antigen receptor and application thereof
  • Costimulatory signal domain of chimeric antigen receptor and application thereof
  • Costimulatory signal domain of chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Obtaining the CAR gene whose full-length antigen is HER2 by PCR

[0052] (1) The first step, gene synthesis: synthesize the full-length RD-1(TM+cytoplasmic)-CD3ζ fragment.

[0053] (2) In the second step, primers were synthesized, and the (anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3ζ) fusion fragment was obtained by overlapping PCR method, which specifically included the following two steps:

[0054] (2.1) Use primers F1 and R1 to amplify (anti-HER2 scFV)-(CD8αhinge); use F2 and R2 to amplify (RD-1TM+Cytoplasmic)-(CD3ζ).

[0055] (2.2) Using (anti-HER2 scFV)-(CD8αhinge) and (RD-1TM+Cytoplasmic)-(CD3ζ) as templates and F1 and R2 as primers, the full-length CAR gene was obtained by PCR.

[0056] The amino acid sequence of the synthesized (anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3ζ) is SEQ NO 5.

Embodiment 2

[0057] Example 2 Construction of recombinant plasmid vector of CAR whose antigen is HER2

[0058] (1) The first step, gene synthesis: synthesize the full-length RD-1(TM+cytoplasmic)-CD3ζ fragment.

[0059] (2) The second step is to design primers and use the overlapping PCR method to obtain (anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3ζ) fusion fragment, which specifically includes the following two steps:

[0060] (2.1) Use primers F1 and R1 to amplify (anti-HER2 scFV)-(CD8αhinge); use F2 and R2 to amplify (RD-1TM+Cytoplasmic)-(CD3ζ);

[0061] (2.2) Using (anti-HER2 scFV)-(CD8αhinge) and (RD-1TM+Cytoplasmic)-(CD3ζ) as templates and F1 and R2 as primers, the full-length CAR gene was obtained by PCR.

[0062] (3) Using Mlu I and Spe I as restriction sites, connect the CAR gene synthesized through the above steps and the PCLK vector to obtain the CAR-PCLK plasmid vector.

Embodiment 3

[0063] Example 3 Viral transfection of T lymphocytes and detection of target gene expression

[0064] (1) The new second-generation CAR gene (anti-HER2 scFV)-(CD8αhinge)-(RD-1TM+Cytoplasmic)-(CD3ζ) with chimeric antigen receptor gene (schematic diagram as shown in figure 1 ), traditional second-generation CAR gene (anti-HER2 scFV)-(CD8αhinge+TM)-(4-1BB)-(CD3ζ) (schematic diagram as figure 1 ) into the lentiviral vector PCLK, and co-transfected T cells with two helper vectors psPAX2 and pMD2.G, respectively packaged to obtain virus particles, and obtained high-concentration lentiviral vectors after centrifugation and concentration.

[0065] (2) Lymphocytes were separated by density gradient centrifugation, and lymphocytes were stimulated with CD3 antibody (1ug / ml) and IL-2 (100IU / ml). One day later, the lymphocytes were collected for virus transfection, the lymphocytes were cultured for 48 hours, and the transfected lymphocytes were collected.

[0066] (3) The collected lymph...

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Abstract

The invention belongs to the technical field of genetic engineering and biological immunotherapy, and constructs a novel chimeric antigen receptor (CAR), and a costimulatory signal domain of the novel chimeric antigen receptor comprises a novel costimulatory molecule, namely a reverse Dectin-1 (RD-1) intracellular domain. CAR-T formed by combining the newly constructed chimeric antigen receptor with T cells can secrete cell factors at a higher level and has better anti-tumor activity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and biological immunotherapy, and constructs a new chimeric antigen receptor, which includes a transmembrane region and an intracellular signal transduction domain of a new co-stimulatory molecule RD-1. Background technique [0002] Chimeric Antigen Receptor (CAR) T cell immunotherapy has undergone a series of evolutionary processes. The first-generation CAR-mediated T cell activation is accomplished through the tyrosine activation motif on CD3ζ. CD3ζ is capable of activating T cells, lysing target cells, regulating IL-2 secretion, and signaling required for antitumor activity in vivo. However, the anti-tumor activity of the first-generation CAR-modified T cells is limited in vivo, which will eventually lead to the apoptosis of T cells due to the reduction of T cell proliferation. The second-generation CAR adds a new co-stimulatory signal CD28 or 4-1BB (CD137) in the cell. Compared w...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K14/7051C12N5/0636C12N15/86A61P35/00C07K2319/03C12N2510/00C12N2740/15043A61K2239/22A61K2239/38A61K39/464412A61K2239/31A61K39/464406A61K39/4631A61K2239/59A61K39/4611C12N15/867C07K19/00C12N5/10A61K48/00A61P37/02C07K14/435
Inventor 王玮魏于全
Owner SICHUAN UNIV
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