Enhanced immune cell and application thereof
An immune cell, chimeric antigen receptor technology, applied in the field of biomedicine, can solve problems such as obstacles, tumor cells do not express specific and specific tumor antigens, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0049] Example 1 Construction of CAR Molecular Carrier
[0050] In this example, fragments with NKG2D and 4-1BB and CD3ζ intracellular signal transduction domains were first synthesized, connected to the pWPXLD-EGFP vector, and then the scFv targeting GPC3 was specifically amplified by PCR and cloned into the above expression vector , to obtain a CAR molecule whose antigen binding domain is anti-GPC3 scFv and NKG2D (the amino acid sequence is shown in SEQ ID NO: 3, and the nucleic acid sequence is shown in SEQ ID NO: 4), and the structural diagram is shown in figure 1 shown.
[0051] The constructed plasmid was transformed into Escherichia coli, a single clone with the target plasmid was selected for overnight culture, and the plasmid was extracted using a plasmid extraction kit to obtain a recombinant lentiviral vector.
[0052] In this example, CARs (antiGPC3scFv-CD8α-4-1BB-CD3ζ) and NKG2D CARs (NKG2D-CD8α-4-1BB-CD3ζ) with antigen-binding domains respectively anti-GPC3 scFv...
Embodiment 2
[0053] Example 2 lentiviral packaging
[0054] In this example, the lentiviral vector constructed in Example 1 is packaged with lentivirus, and the steps are as follows:
[0055] (1) Culture 293T cells in a 10cm petri dish, the culture medium is DMEM high glucose medium+10% FBS (fetal bovine serum)+1% double antibody (100×penicillin-streptomycin mixed solution);
[0056] (2) When the 293T cell density in the culture dish reaches 80%, replace the medium with DMEM high glucose medium + 1% FBS + 1% double antibody;
[0057] (3) After replacing the medium and culturing for 2 hours, prepare a transfection reagent, take 500 μL opti-DMEM into a 15 mL centrifuge tube, add 7.2 μL of PEI (linear polyethyleneimine) with a concentration of 10 μg / μL, and mix slightly. Stand still for 5 minutes;
[0058](4) Put 500μL opti-DMEM into a 1.5mL centrifuge tube, take 9μg of recombinant lentiviral vector, 3μg of pMD2.G helper plasmid and 12μg of psPAX, add them to the centrifuge tube, mix well, ...
Embodiment 3
[0065] Example 3 T cell activation and lentiviral transfection
[0066] (1) After sorting Pan T cells from umbilical cord blood, count the cells and adjust the concentration to 1×10 6 cells / mL, then add 10 μL of Miltenyi TransAct T cell reagent to each ml of cell suspension, and replace it with fresh medium (IMDM medium + 5% FBS (fetal bovine serum) + 1% double antibody ( 100×penicillin-streptomycin mixed solution)+IL-2);
[0067] (2) After T cells were activated for 48 h, demagnetize the beads, centrifuge at 300 g for 5 min, remove the supernatant, resuspend T cells with fresh medium, add CAR-expressing recombinant lentivirus or blank control lentivirus (MOI=10), and Add 8μg / mL polybrene and 300IU / mL IL-2, place at 37°C, 5% CO 2 incubator cultivation;
[0068] (3) After 24 hours, centrifuge at 300 g for 5 minutes, remove the supernatant, and resuspend the T cells in fresh medium containing 300 IU / mL IL-2 to obtain CAR-T cells.
[0069] The CAR-T cells constructed in this ...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - 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.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap