Armed specific chimeric antigen receptor cell targeting cxcr2 ligand and its preparation method and application
A technology of chimeric antigen receptors and armed targets, which can be used to target specific cell fusion, receptors/cell surface antigens/cell surface determinants, polypeptides containing positioning/targeting motifs, etc., and can solve the problem of killing Low efficiency, low specificity of tumor cells, etc., to achieve the effect of high killing rate, simple steps, and good industrial application prospects
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Embodiment 1
[0141] Example 1 Preparation of an expression plasmid (KD-207 lentiviral vector) armed with a chimeric antigen receptor targeting CXCR2 ligand and specifically targeting human NKG2DL
[0142] The overall design is as follows:
[0143] 1. Determination of the amino acid sequence of a chimeric antigen receptor that specifically targets human NKG2DL and is armed with a CXCR2 ligand
[0144] First, the full-length amino acid sequence of human NKG2D (NP_031386.2) and the full-length amino acid sequence of human CXCR2 protein (NP_001548.1) were searched from the Genbank database of the National Library of Medicine (NCBI).
[0145] Secondly, construct a chimeric antigen receptor specifically targeting human NKG2DL that is armed with a CXCR2 ligand, that is, determine the amino acid sequence of the armed chimeric antigen receptor molecule:
[0146] From the amino terminal to the carboxyl terminal, the amino acid sequence of the guide peptide (as shown in SEQ ID No.1), the amino acid ...
Embodiment 2
[0165] Example 2: Preparation of virus liquid (KD-207 virus liquid) of lentiviral vector
[0166] The recombinant plasmid (KD-207 lentiviral vector) obtained in Example 1 expressing chimeric antigen receptors armed with targeting CXCR2 ligands and specifically targeting human NKG2DL, and the packaging vectors psPAX2 and VSVG, according to the ratio of 10:8:5 ratio, with Lipofectamine TM 6000 transfection reagents (purchased from ThermoFisher, product model 11668019) were used to co-transfect 293T cells (see the ThermoFisher transfection manual for specific transfection procedures), and replaced with complete medium 6 hours after transfection (purchased from Life Technologies , the product model is 11995-065), after 48 hours and 72 hours of culture, the cell supernatants rich in lentiviral particles were collected respectively, and the virus supernatants were concentrated by ultracentrifugation to obtain the cells carrying the expression armed targeting CXCR2 ligand A viral fl...
Embodiment 3
[0167] Example 3: Isolation and culture of T cells
[0168] Take fresh peripheral blood from healthy donors, and separate fresh peripheral blood mononuclear cells by density gradient centrifugation; then use paramagnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA, product information To enrich CD3+ T cells for Dynabeads® Human T-Activator CD3 / CD28), specifically, dilute peripheral blood mononuclear cells to a concentration of (10~30)×10 6 single cells / ml, and then mixed the magnetic beads and cells in a culture dish at a ratio of 3:1, incubated at room temperature for 2-3 hours, and used a magnetic particle concentrator (MPC for short, purchased from Invitrogen, USA) company) to enrich CD3+ T cells. Finally, the enriched CD3+ T cells were resuspended in culture medium (purchased from Life Technologies, USA, product information is OpTmizer™ T-Cell Expansion SFM), and the cell solubility was adjusted to 1×10 6 pcs / ml, finally at...
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