Targeted-MMSA-1-chimeric-antigen-receptor-modified T lymphocyte as well as preparation method and application thereof
A MMSA-1, chimeric antigen receptor technology, used in genetically modified cells, cells that are targeted for fusion of specific cells, and cells modified by the introduction of foreign genetic material, etc. Good anti-tumor effect
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Embodiment 1
[0032] [Example 1] Construction of MMSA-1 scFv and affinity flow detection
[0033] 1. Preparation of MMSA-1 CAR
[0034] After the wild-type antibody was humanized, MMSA-1 scFv was obtained. The MluI restriction site and CD8 transmembrane signal peptide were inserted before the fragment, and the BamHI restriction site was inserted after the fragment, which was then synthesized by a gene company (Jinweizhi). The pUC57-Amp plasmid containing MluI+CD8a-MMSA-1scFv+BamHI was synthesized by a gene company and was digested with MluI (NEB) and BamHI (NEB). The effect of the enzyme digestion was identified by agarose gel electrophoresis, and the modified gene was obtained by gel recovery. fragment. At the same time, the existing lentiviral backbone plasmid pHR containing the CD8 transmembrane region, 4-1BB co-stimulatory signal region and CD3ZetaTCR activation region was digested with MluI and BamHI, and the long fragments were recovered after identification by agarose gel electropho...
Embodiment 2
[0038] [Example 2] Preparation of MMSA-1 CAR-T cells and detection of infection efficiency
[0039] 1. Preparation of MMSA-1 CAR-T cells
[0040] Take 50 mL of fresh blood, and conduct density gradient centrifugation with lymphocyte separation medium (Tianjin Haoyang) to separate mononuclear cells. Divide mononuclear cells into 1-2 x 10 6 / mL resuspended in CTSTM AIM VTM SFM medium (GIBCO, Cat. No. A3021002). At the same time, 5% ICS (GIBCO, A2596101), CD3 monoclonal antibody (Ebioscience) 50ng / mL and CD28 monoclonal antibody (Ebioscience) 50ng / mL were added to activate T lymphocytes, and cultured at 37°C with 5% CO2 for 48 hours.
[0041] After 2 days of culture, collect the cells and resuspend the cells to 1x10 6 / mL, add the above-mentioned concentrated lentivirus according to MOI=5, add IL-2 (Quangang) and 4ug / mL polybrene (Sigma) at a final concentration of 200U / mL at the same time, mix well, and incubate at 37°C with 5% CO2 for 6- After 8 hours, centrifuge at 300g fo...
Embodiment 3
[0048] [Example 3] Detection of specific killing effect of MMSA-1 CAR-T cells in vitro by flow cytometry
[0049] Flow cytometry was used to detect the expression of MMSA-1 antigen in K562 cell line, RPMI-8226 cell line and primary tumor cells. Take K562 cell line, RPMI-8226 cell line and primary tumor cells 5×10 each 5 Add 1 μL of ZDHHC9 Polyclonal Antibody (Invitrogen) antibody and 1 μL of secondary antibody to each sample, incubate at 4°C in the dark for 15 minutes, centrifuge at 400 g for 5 minutes, wash once with 1 mL of PBS, resuspend the precipitate with 2% paraformaldehyde, and put it on a fascalibur (BD Bioscience) detection.
[0050] Such as image 3 As shown in A, K562 does not express MMSA-1 molecules on the surface, while RPMI-8226 cells and primary MM tumor cells express MMSA-1 molecules on the surface.
[0051] The two kinds of cells were seeded in a 96-well plate at a ratio of 1:1, 1×10 per well 5 For each species, MMSA-1 CAR-T cells and T cells cultured fo...
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