Preparation and application of Trop-2 specific chimeric antigen receptor cell
A chimeric antigen receptor, specific technology, applied to receptor/cell surface antigen/cell surface determinant, for targeting specific cell fusion, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin Equal direction
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Embodiment 1
[0045] This example provides a method for constructing the above-mentioned CAR molecule into the lentiviral vector pCDH
[0046] Construction of Trop2 antibody scFv: Using anti-Trop-2 Fab and Linker (Gly4Ser) 3 as templates, Vκ, V were designed according to the principle of In-FusionPCR H Amplification primers; amplify Vκ, vH fragments and link them with Linker (Gly4Ser) 3 to form Vκ-Linker (Gly4Ser) 3-VH gene fragments (anti-Trop-2 IgG); use restriction endonuclease Nco I Retroviral vector (SFG.CH2CH3-CD28-4-1BB-CD3ζ) containing human CH2CH3 and intracellular signal transduction region CD28, 4-1BB, CD3ζ gene sequence with Xho I double digestion;
[0047] HRS-28bbZ vector construction: The anti-Trop-2 scFv was connected with the linearized retroviral vector by In-Fusion PCR (Table 1) to construct the third-generation Trop-2 CAR retroviral expression plasmid (Trop-2 CAR retroviral expression plasmid (Trop -2 CAR plasmid), carry out enzyme digestion identification and sequenc...
Embodiment 2
[0051] This example is the preparation of Trop-2 CAR-T cells
[0052] Packaging of CAR lentivirus; pCDH Trop-2 CAR-GFP plasmid and lentivirus auxiliary packaging plasmids pMD2.G and pSPAX2 were extracted by QIAGEN endotoxin-free plasmid large extraction kit. 24h before transfection, 2.5×10 7 Spread 293 to T75 cell culture flasks, replace 293T medium with 10ml serum-free medium 1 hour before transfection, use 293fectin TM TransfectionReage transfected 3 plasmids, and 24 hours after the transformation, the medium was replaced with 15ml of complete medium. After 48 hours, the cell supernatant virus liquid was collected. After the virus supernatant was concentrated in a 0.45μm 100kD ultrafiltration tube, it was stored at -80℃ for later use. . The virus titer was detected by qPCR method, and the virus content after the virus supernatant was concentrated 10 times by ultrafiltration was more than 1.5×10 9 copies / ml.
[0053] T cell transduction: CD4+ / CD8+ T cells were separated...
Embodiment 3
[0055] This example is the killing effect of Trop-2 CAR-T cells on ovarian cancer cells in vitro.
[0056] Ovarian cancer cells OVCAR-3, HO8910, SKOV3, A2780, breast cancer cell MCF-7, and melanoma cell A375 were detected by flow cytometry; the expression levels of Trop-2 mRNA in the above cells were detected by RT-PCR (Table 2). The results of flow cytometry and RT-PCR showed that ovarian cancer cells OVCAR-3, H08910 and breast cancer cells MCF-7 highly expressed Trop-2 antigen, ovarian cancer cells SKOV3 lowly expressed Trop-2 antigen, ovarian cancer cells A2780, Melanoma cell A375 does not express Trop-2 antigen, such as Figure 5A and 5B shown.
[0057] Table 2 Amplification primers for Trop2 and GAPDH fragments
[0058]
[0059] Collect the tumor cells HO8910 and A2780 in the logarithmic growth phase, and set 5 density 5×10 5 Cells / ml, 2×10 5 Cells / ml, 1×10 5 Cells / ml, 5×10 4 Cells / ml were added to 96-well cell culture plate at 100 μL / well; after the cells in ...
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