Targeting csf1r chimeric antigen receptor modified nk92mi cells and T cells and their preparation and application
A chimeric antigen receptor and antigen technology, applied in the field of biomedicine
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[0200] See the literature for the preparation of lentivirus 6 .
[0201] Collect NK92MI cell count, take 1×10 6 Put each cell into a 15ml centrifuge tube, add concentrated A2-CAR and A3-CAR virus solution, mix well, MOI=10. After incubation at 37°C for 6 hours, transfer to a cell dish for culture. After 72 hours, NK92MI cells transfected with A2-CAR and A3-CAR were taken, and untransfected NK92MI cells were taken as negative controls, and the expression of A2-CAR and A3-CAR was detected on FACSCalibur (BD Biosciences). A2-CAR-NK92MI and A3 that stably express A2-CAR and A3-CAR were obtained by flow cytometry sorting (FACS Aria III, BD Biosciences) sorting combined with drug screening of penicillin-streptomycin (HyClone). -CAR-NK92MI cells.
[0202] The construction process of CAR-T cells is as follows: PBMCs were extracted from the peripheral blood of healthy people by the Ficoll method, the mononuclear cell layer was absorbed, cultured in a 24-well plate, and about 1×10 ce...
Embodiment 1
[0216] Example 1 Construction and expression of A2-CAR-NK92MI and A3-CAR-NK92MI cell lines
[0217] A2-CAR and A3-CAR are three generations of CAR ( figure 1 A), ScFv includes a heavy chain and a light chain, and contains a signal peptide sequence (MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO.: 4)) in front, followed by an Fc sequence and costimulatory factor sequence, CD3ζ, and then constructs the sequence into pCDH-CMV- On the MCS-EF1-Puro lentiviral vector ( figure 1 A).
[0218] The constructed A2-CAR and A3-CAR were transfected into NK92MI cells in the form of lentiviral transfection, and the stably transfected cells A2-CAR-NK92MI and A3-CAR were obtained by cell flow sorting combined with drug screening -NK92MI( figure 1 B). And the average fluorescence intensity of A2-CAR-NK92MI and A3-CAR-NK92MI cells is very high ( figure 1 C). In order to confirm that CAR was indeed transferred to and expressed in NK92MI cells, Western Blotting was performed to detect the expression of CD...
Embodiment 2293
[0220] Example 2 Construction of 293T-CSF1R and K562-CSF1R cells and detection of CSF1R antigen
[0221] In order to obtain a cell line that highly expresses the CSF1R antigen molecule, the CSF1R molecule (NP_005202.2) was artificially constructed on the pCDH-CMV-MCS-EF1-CopGFP and pCDH-CMV-MCS-EF1-Puro vectors, and then transformed into a lentivirus 293T and K562 cells were transfected, and 293T-CSF1R and K562-CSF1R cells were obtained by flow cytometry sorting and drug screening ( figure 2 ).
[0222] figure 2 : Flow cytometric analysis of the expression of CSF1R antigen molecules in 293T-CSF1R and K562-CSF1R cells. Collect 293T, K562, 293T-CSF1R and K562-CSF1R cells, all of which were stained with antibodies against human CSF1R molecules, wherein 293T and K562 cells were used as controls, and the results of flow cytometry were analyzed as follows: figure 2 shown.
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