Construction method and application of a chimeric antigen receptor carrier that enhances migration to tumor sites
A chimeric antigen receptor and carrier technology, applied in the field of tumor cell immunotherapy, can solve problems such as poor treatment effect of solid tumors
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[0233] In a specific embodiment, the present invention provides a method for preparing engineered immune cells, comprising the following steps:
[0234] S1: Using CD3ζ as the intracellular signal segment of CAR, using the CD28 transmembrane region as the transmembrane region, the CD28 intracellular region as a co-stimulatory factor, and the human NKG2D extracellular binding region to recognize NKG2DL, constitute a chimera that specifically recognizes NKG2DL Antigen receptor, and artificially synthesize the corresponding nucleotide sequence: NKG2D-CD8α-CD28-CD3ζ, and connect CAR and CXCR3 nucleotide sequence through the nucleotide sequence of P2A to form a fusion gene: NKG2D-CD8α - CD28-CD3ζ-P2A-CXCR3.
[0235] S2: inserting the fusion gene fragment into the lentiviral expression plasmid to obtain the target plasmid. Packaged as a lentivirus carrying NKG2D-CD8α-CD28-CD3ζ-P2A-CXCR3.
[0236] S3: Adjust the NK-92 cell density to 1 × 10 5 Individual / ml, add virus concentrate by...
Embodiment 1
[0266] Example 1: Synthesis of CAR-NKG2D-P2A-CXCR3 vector
[0267] In this example, the extracellular segment of human NKG2D, the hinge region of human CD8α, the transmembrane region and intracellular region of human CD28, and the intracellular signal region of human CD3ζ were synthesized through the method of whole gene synthesis, which were connected in series with human CXCR3 through P2A The gene fragment constituted, and the two ends were respectively connected with Xba I and BamH I restriction sites.
Embodiment 2
[0268] Example 2: Construction of recombinant CAR-NKG2D-P2A-CXCR3 lentiviral vector
[0269]In this example, the CAR-NKG2D-P2A-CXCR3 (SEQ ID NO: 2) gene fragment obtained in Example 1 was cloned into the pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro lentiviral vector, such as figure 1 shown. Use restriction endonucleases Xba I and BamH I to digest pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro lentiviral vector and CAR-NKG2D-P2A-CXCR3 gene fragment respectively to obtain linearized pCDH -The CMV-MCS-EF1-CopGFP-T2A-Puro lentiviral vector and the digested CAR-NKG2D-P2A-CXCR3 gene fragment were incubated overnight at 16°C using the T4 DNA ligase system. Then the Stbl3 competent cells were transformed, the positive colonies were screened, and the plasmids of the positive colonies were extracted to obtain the CAR-NKG2D-P2A-CXCR3-pCDH expression vector.
[0270] 1. Double digested pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro lentiviral vector
[0271] The double enzyme digestion reaction system is as follows (50μl): ...
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