CLTX-NKG2D bispecific chimeric antigen receptor cell as well as preparation method and application thereof
A technology of chimeric antigen receptor and specificity, applied in antibacterial drugs, animal cells, antibody medical components, etc., can solve the problems of low specificity and low killing efficiency of tumor cells
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Embodiment 1
[0127] Example 1 Preparation of expression plasmids specifically targeting chimeric antigen receptors of human CLTX-R and NKG2DL
[0128] The overall design is as follows:
[0129] 1. Determination of the amino acid sequence of chimeric antigen receptors specifically targeting CLTX-R and NKG2DL
[0130] First, the full-length amino acid sequence of human NKG2D (NP_031386.2) was searched from the Genbank database of the National Library of Medicine (NCBI), and the full-length amino acid sequence of CLTX protein was searched from the Uniprot website (Uniprot: P45639).
[0131] Secondly, construct a chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL, that is, determine the amino acid sequence of the chimeric antigen receptor molecule:
[0132] From the amino terminal to the carboxyl terminal, the amino acid sequence of the leader peptide (as shown in SEQ ID No.1), the amino acid sequence of CLTX (such as SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4), human NKG2...
Embodiment 2
[0153] Example 2: Preparation of virus liquid of lentiviral vector
[0154] The recombinant plasmid (KD-618 expression plasmid) obtained in Example 1 specifically targeting human CLTX-R and NKG2DL chimeric antigen receptors and the packaging vectors psPAX2 and VSVG were used according to the ratio of 10:8:5 with Lipofectamine TM 6000 transfection reagents (purchased from Beyontien, product model C0526) were used to co-transfect 293T cells (see the transfection instructions for specific transfection procedures), and replaced with complete medium 6 hours after transfection (purchased from hyclone, The product model is SH30243.01), 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 specific targeting human CLTX-R and The virus liquid of the lentiviral vector of the chimeric antigen receptor of NKG2DL (hereinafter referred to as KD-618...
Embodiment 3
[0155] Example 3: Isolation and culture of T cells
[0156] Fresh peripheral blood from healthy donors was taken, and fresh peripheral blood mononuclear cells were separated by density gradient centrifugation; paramagnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA, product information for Human T-Activator CD3 / CD28) to enrich CD3+ T cells, 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 particles 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 TM T-Cell Expansion SFM), adjusted to a cell concentration of 1×10 6 pcs / ml, finally at 37°C, ...
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