Double-target spot embedded antigen receptor, encoding gene thereof, plasmid containing gene, immune T effect cell containing cell and application of cell for HIV-1

A chimeric antigen receptor, dual-target technology, applied in the direction of receptor/cell surface antigen/cell surface determinant, cells modified by introducing foreign genetic material, immunoglobulin, etc., can solve the problem of unsatisfactory effect, CD4 -ζCAR-T cells are susceptible to HIV-1 infection and have not been widely used

Active Publication Date: 2018-05-04
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after CD4-ζCAR-T cells bind to gp120 antigen, although they can activate T cells to kill the combined HIV-1 infected cells, it is difficult for the activated T cells to proliferate further and can only exert a transient effect, and CD4-ζCAR- T cells are susceptible to HIV-1 infection and become new hosts
The effect of this method in late clinical trials is not satisfactory, so far, it has not been widely used

Method used

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  • Double-target spot embedded antigen receptor, encoding gene thereof, plasmid containing gene, immune T effect cell containing cell and application of cell for HIV-1
  • Double-target spot embedded antigen receptor, encoding gene thereof, plasmid containing gene, immune T effect cell containing cell and application of cell for HIV-1
  • Double-target spot embedded antigen receptor, encoding gene thereof, plasmid containing gene, immune T effect cell containing cell and application of cell for HIV-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] A dual-target chimeric antigen receptor for HIV-1, said chimeric antigen receptor comprising CD4 antigen region (comprising CD4Leader region), Linker region, 17b single-chain antibody region (17bscFv), hinge region, transmembrane domain, co-stimulatory domain, signaling domain and suicide gene system region.

[0051] Wherein, the CD4 antigen region is 1-182 amino acids of CD4; the Linker region is selected from connecting peptides of different lengths (G4S) 2, (G4S) 4, (G4S) 6, (G4S) 8 and (G4S) 10, the present invention preferably (G4S)8, a connecting peptide composed of 40 amino acids; the 17b single-chain antibody region is the single-chain antibody 17bscFv directed against the HIV-1 17b epitope; the hinge region is the CD8α hinge region; the transmembrane domain is the CD8α transmembrane region; The co-stimulatory domain is the functional domain of CD137; the signaling domain includes the functional domain of CD3ζ.

Embodiment 2

[0053] The gene encoding the chimeric antigen receptor is CD4-17bscFv-CD8α-CD137-CD3ζ (also known as CD4-17bscFv-CAR DNA fragment).

[0054] Construction of CD4-17bscFv-CD8α-CD137-CD3ζ

[0055] Insert the fusion gene fragment CD4-17bscFv-CD8α-CD137-CD3ζ into the lentiviral expression vector pLent-C-GFP.

[0056] CD4-17bscFv-CD8α-CD137-CD3ζ Module Indication figure 1 (See appendix SEQ ID NO.1 for the complete nucleic acid sequence).

[0057] Module sequences of CD4-17bscFv-CD8α-CD137-CD3ζ

[0058] (1) CD4 antigen nucleic acid artificial sequence (SEQ ID NO.2)

[0059] (2) Linker nucleic acid artificial sequence (SEQ ID NO.3)

[0060] (3) 17b single-chain antibody nucleic acid artificial sequence (SEQ ID NO.4)

[0061] (4) CD8α hinge region nucleic acid artificial sequence (SEQ ID NO.5)

[0062] (5) Artificial nucleic acid sequence of CD8α transmembrane region (SEQ ID NO.6)

[0063] (6) CD137 co-stimulatory region nucleic acid artificial sequence (SEQ ID NO.7)

[0064] (...

Embodiment 3

[0067] Table recombinant plasmid, said recombinant plasmid comprises the above-mentioned gene. The recombinant plasmid is the pLent-CD4-17bscFv-CAR plasmid obtained by inserting the CD4-17bscFv-CD8α-CD137-CD3ζ gene fragment into the linearized pLent-C-GFP DNA fragment.

[0068] The following is the preparation method of the table recombinant plasmid:

[0069] According to artificial nucleic acid sequence of CD4 antigen, artificial nucleic acid sequence of Linker, artificial nucleic acid sequence of 17b single-chain antibody, artificial nucleic acid sequence of CD8 hinge region, artificial nucleic acid sequence of CD8 transmembrane region, artificial sequence of nucleic acid of CD137 co-stimulatory region, artificial nucleic acid sequence of CD3ζ signal transduction region The sequence commissioned Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize its entire expression cassette and insert it into the standard vector pUC57, so it was named pUC-CD4-17bscFv-CAR, and pUC-CD4...

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Abstract

The invention discloses a double-target spot embedded antigen receptor, an encoding gene thereof, a plasmid containing gene, an immune T effect cell containing the gene and application of the gene. The embedded antigen receptor contains a CD4 antigen region (including a Leader region), a Linker region, a 17b single chain antibody region (17bscFv), a hinge region, a membrane spanning structural domain, a co-stimulation structural domain and a signal conduction structural domain. A binding region of gp120 is simulated by virtue of CD4-17bscFv-CAR, and the validity and safety of CD4-17bscFv-CAR are far higher than the validity and safety of a previously reported single-target spot CD4-zetaCAR.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a dual-target chimeric antigen receptor for HIV-1, its coding gene, a plasmid containing the gene, an expression vector, immune T effector cells and its application in HIV-1. Background technique [0002] Human immunodeficiency virus (human immunodeficiency virus Type 1, HIV-1), a type of retrovirus, invades CD4+ T cells, severely damages the body's acquired immune function, and eventually leads to AIDS (Acquired immunodeficiency syndrome). In August 2017, the Chinese Center for Disease Control and Prevention announced the national AIDS epidemic. The number of HIV-1 (human immunodeficiency virus) infections in China reached 718,270, of which 299,169 were suffering from AIDS, and the number of deaths due to AIDS has reached 221,628. . In addition, the number of new HIV-1 infected persons / AIDS patients has reached more than 140,000 each year. These figures show that the situa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/85A61K35/17A61P31/18
CPCA61K35/17C07K14/7051C07K14/70514C07K14/70517C07K14/70596C07K16/1045C07K2317/53C07K2317/622C07K2319/00C07K2319/03
Inventor 刘明录韩国英冯建海王立新金海锋万磊卢永灿韩庆梅刘敏马洪华强邦明张传鹏
Owner SHANDONG XINRUI BIOTECH CO LTD
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