Immunomodulatory specific chimeric antigen receptor cells as well as preparation method and application thereof
A chimeric antigen receptor and specific technology, applied in the direction of blood/immune system cells, receptors/cell surface antigens/cell surface determinants, botanical equipment and methods, etc., can solve the problem of low killing efficiency, tumor cell The problem of poor specificity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0133] Example 1 Preparation of expression plasmids for immune checkpoint regulation combined with chimeric antigen receptors specifically targeting human NKG2DL
[0134] The overall design is as follows:
[0135] 1. Determination of the amino acid sequence of a chimeric antigen receptor specifically targeting human NKG2DL through immune checkpoint regulation
[0136] First, the full-length amino acid sequence of human NKG2D (NP_031386.2), the full-length amino acid sequence of PD1 protein (NP_005009.2), and the full-length amino acid sequence of TIGIT protein were searched from the Genbank database of the National Library of Medicine (NCBI). (NP_776160.2).
[0137] Secondly, construct immune checkpoint regulation (PD1 or TIGIT) combined with a chimeric antigen receptor specifically targeting human NKG2DL, that is, determine the amino acid sequence of the chimeric antigen receptor molecule:
[0138]From the amino terminal to the carboxyl terminal, the amino acid sequence of ...
Embodiment 2
[0162] Example 2: Preparation of virus liquid of lentiviral vector
[0163] The recombinant plasmid (KD-593 / 323 expression plasmid) obtained in Example 1 expressing immune checkpoint regulation combined with the chimeric antigen receptor 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 reagent (purchased from ThermoFisher, product model 11668019) co-transfected 293T cells (see ThermoFisher transfection manual for specific transfection operation process), 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 expression of immune checkpoint regulation joint specificity The virus liquid of the len...
Embodiment 3
[0164] Example 3: Isolation and culture of T cells
[0165] 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 6pcs / ml, finally at 37°C, 5...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com