T lymphocyte modified by double specific chimeric antigen receptors as well as preparation method and application thereof
A chimeric antigen receptor and bispecific technology, applied in the field of bispecific chimeric antigen receptor modified T lymphocytes and its preparation, to achieve the effect of overcoming off-target toxicity, specific recognition and killing
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Embodiment 1E
[0058] Example 1 Determination of EGFRvIII-CD3ζ / PD-L1-4-1BB gene sequence
[0059] 1.1 Obtain human CD8α signal peptide gene (SEQ ID NO.9), Linker region (Gly4-Ser) 3 ( SEQ ID NO.10), human CD8α hinge region (CD8αHinge) (SEQ ID NO.11), human CD8α transmembrane region (CD8αTM) (SEQ IDNO.12), immunoreceptor tyrosine activation motif CD3ζ (SEQ ID NO .13), human 4-1BB hinge region (4-1BB Hinge) (SEQ ID NO.14), human 4-1BB transmembrane region (4-1BB TM) (SEQ ID NO.15) and 4-1BB intracellular Signal region (SEQ ID NO.16) sequence, combined with EGFRvIII antibody light chain variable region (EGFR vIIIscfv-VL) (SEQ ID NO.17), EGFRvIII antibody heavy chain variable region (EGFRvIIIscfv-VH) (SEQ ID NO.18 ), PD-L1 antibody light chain variable region (PD-L1scfv-VL) (SEQ ID NO.19), PD-L1 antibody heavy chain variable region (PD-L1scfv-VH) (SEQ ID NO.20) sequence , combined to form complete EGFRvIII-CD3ζ (SEQ ID NO.5), PD-L1-4-1BB (SEQ ID NO.6), EGFRvIII-CAR (SEQ ID NO.7), PD-L1-CAR (SE...
Embodiment 2
[0060] Example 2 Construction of pCDH-EGFRvIII-CD3ζ, pCDH-PD-L1-4-1BB, pCDH-EGFRvIII-CAR and pCDH-PD-L1-CAR plasmids
[0061] 2.1 Whole gene synthesis:
[0062]Complete EGFRvIII-CD3ζ (SEQ ID NO.5), PD-L1-4-1BB (SEQ ID NO.6), EGFRvIII-CAR (SEQ ID NO.7), PD- L1-CAR (SEQ ID NO.8) sequence, and add Xba I restriction site at its 5' end, and add EcoR I restriction site at its 3' end.
[0063] 2.2 Cloning EGFRvIII-CD3ζ, PD-L1-4-1BB, EGFRvIII-CAR, and PD-L1-CAR into pCDH-EF1α-GFP or pCDH-EF1α-RFP lentiviral expression vectors, respectively. The details are as follows: the pCDH-EF1α-GFP vector, pCDH-EF1α-RFP vector, EGFRvIII-CD3ζ fragment, PD-L1-4-1BB fragment, EGFRvIII-CAR fragment, and PD-L1-CAR fragment were respectively treated with Xba I / EcoR I Digest with enzymes, and recover 8192bp, 8111bp, 1297bp, 1258bp, 1483bp, 1477bp fragments respectively. Use T4 DNA ligase to ligate pCDH-EF1α-GFP vector and EGFRvIII-CD3ζ, EGFRvIII-CAR, PD-L1-CAR, pCDH-EF1α-RFP vector and PD-L1-4-1BB res...
Embodiment 3
[0064] Packaging, concentration and titer determination of embodiment 3 lentivirus
[0065] 3.1 Packaging of lentivirus:
[0066] 3.1.1 Cell treatment: 24 hours before transfection, collect 293T cells of passage 3-10 in logarithmic growth phase, inoculate 293T cells in a 10cm cell culture dish, the inoculum size is 1×10^7, and the cells are contained in 10ml 10 Grow in DMEM medium with %FBS, place in a 5% CO2 cell incubator at 37°C for 18 hours, and transfect when the cell density reaches 60-80%.
[0067] 3.1.2 Co-transfect lentiviral expression vector plasmids (pCDH-EGFRvIII-CD3ζ, pCDH-PD-L1-4-1BB, pCDH-EGFRvIII-CAR, pCDH-PD-L1-CAR) and their packaging plasmids ( pLP1, pLP2, pLP / VSVG);
[0068] 3.1.3 24 hours after transfection, the expression of GFP / RFP fluorescence in 293T cells after transfection was observed under a fluorescent microscope. Collect the 293T culture supernatant at 48 hours and 72 hours after transfection, centrifuge at 3000rpm for 15 minutes, collect the...
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