Dual-target chimeric antigen receptor and expression vector and application thereof

A technology of chimeric antigen receptors and expression vectors, applied in the fields of application, cancer antigen components, receptors/cell surface antigens/cell surface determinants, etc., can solve the problem of poor anti-tumor effect, tumor heterogeneity and tumor Complex microenvironment and other issues, to achieve good application prospects, strong anti-tumor activity, and strong anti-tumor effect

Active Publication Date: 2020-01-17
华夏源细胞工程集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the application of solid tumors, especially liver cancer, due to the lack of tumor-specific antigens and the heterogeneity of the solid tumor itself and the more complex tumor microenvironment, its anti-tumor effect is not good.

Method used

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  • Dual-target chimeric antigen receptor and expression vector and application thereof
  • Dual-target chimeric antigen receptor and expression vector and application thereof
  • Dual-target chimeric antigen receptor and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The present invention provides a dual-target chimeric antigen receptor, such as figure 1 As shown, the dual-target chimeric antigen receptor includes: sequentially linked signal peptide (CD8α leader), receptor NKG2D extracellular region targeting NKG2DL (NKG2D ECD), targeting GPC3 single-chain antibody (GPC3 scFv), Lengthen the CD8α hinge region (CD8α Hinge), transmembrane region (CD8α transmembrane), co-stimulatory factor and intracellular signal domain (CD3ζ signal), the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 1 in the sequence table, targeting The nucleotide sequence of the NKG2D extracellular region of the receptor of NKG2DL is shown in SEQ ID NO: 2 in the sequence listing, the nucleotide sequence of the GPC3 single-chain antibody is shown in SEQ ID NO: 3 in the sequence listing, and CD8α is lengthened The nucleotide sequence of the hinge region is shown in SEQ ID NO: 4 in the sequence listing, the nucleotide sequence of the transmembrane re...

Embodiment 2

[0032] The present invention provides an expression vector, which includes the vector and the dual-target chimeric antigen receptor provided in Example 1. The vector can be a lentiviral vector.

[0033] The following briefly introduces the preparation method of the expression vector, as follows:

[0034]PCR (Polymerase Chain Reaction, Polymerase Chain Reaction) amplifies and obtains the CAR02 gene sequence, respectively adds restriction site Xba I and restriction site EcoR I to the two ends of the CAR02 gene sequence to obtain the product to be digested, Carry out the double digestion reaction of Xba I and EcoR I with the lentiviral vector plasmid pCDH-EF1-MCS-T2A-copGFP, respectively, to obtain the digestion fragment containing CAR02 and the digestion fragment containing ppCDH-EF1-MCS-T2A-copGFP fragment. The enzyme digestion reaction conditions are as follows: the enzyme digestion temperature is 37° C., and the enzyme digestion time is 30 min. Enzyme digestion system (tot...

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Abstract

The invention discloses a dual-target chimeric antigen receptor and an expression vector and application thereof, and belongs to the field of tumor immunity medicines. The dual-target chimeric antigenreceptor comprises: a signal peptide, a receptor NKG2D extracellular region targeting NKG2DL, a single-chain antibody targeting GPS3, an elongated CD8 alpha hinge region, a transmembrane region, a co-stimulatory factor and an intracellular signal domain sequentially connected, wherein the signal peptide has a nucleotide sequence as shown in SEQ ID NO: 1 in Sequence Listing, the receptor NKG2D extracellular region has a nucleotide sequence as shown in SEQ ID NO: 2, the single-chain antibody has a nucleotide sequence as shown in SEQ ID NO: 3, the elongated CD8 alpha hinge region has a nucleotide sequence as shown in SEQ ID NO: 4, the transmembrane region has a nucleotide sequence as shown in SEQ ID NO: 5, and the intracellular signal domain has a nucleotide sequence as shown in SEQ ID NO: 6. The dual-target chimeric antigen receptor can avoid a problem of escape of tumor cells with low abundant antigen expression, thereby effectively reducing risk of tumor recurrence.

Description

technical field [0001] The present invention relates to the field of tumor immune drugs, in particular to a dual-target chimeric antigen receptor and its expression vector and application. Background technique [0002] Primary liver cancer is one of the common malignant tumors. Most of the primary liver cancers are hepatocellular carcinoma (HCC), which ranks fifth in the incidence of malignant tumors and third in the mortality rate. In my country, the morbidity and mortality of liver cancer ranks second among cancers. [0003] Chimeric Antigen Receptor gene-modified T cell (CAR-T) technology is constructed by introducing the nucleic acid fused with the CAR gene into the genome of autologous or allogeneic T lymphocytes. The application of CAR-T in hematological tumors has shown gratifying therapeutic effects, especially for patients with relapsed and refractory B-lymphoblastic leukemia, and the remission has reached more than 90%. However, in the application of solid tumors...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867A61K39/00A61P35/00
CPCC07K16/303C07K16/2851C07K14/7051C12N15/86A61K39/0011A61P35/00C07K2319/02C07K2319/03C07K2319/33A61K2039/844
Inventor 张坤
Owner 华夏源细胞工程集团股份有限公司
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