Construction of chimeric antigen receptor-mononuclear/macrophage (CAR-M) and application thereof
A chimeric antigen receptor and macrophage technology, applied to receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, blood/immune system cells, etc. It can solve the problems of impaired phagocytic ability of macrophages and achieve the effect of enhancing phagocytic ability, enhancing anti-tumor immunity, and significant technological innovation characteristics
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Embodiment 1
[0034] This example is the assembly of a chimeric antigen receptor molecule targeting HER2, which includes a leader peptide, an extracellular recognition region, a hinge region, a transmembrane region / intracellular signal region, and the sequence from the N-terminal to the C-terminal is the leader peptide It is the CSF2Rα leader peptide (SEQ ID NO.1), the extracellular recognition region is the anti-HER2 antibody sequence (scFv amino acid sequence, SEQ ID NO.2), the hinge region is the CD28hinge region (SEQ ID NO.3), and the transmembrane region / The intracellular signal region is CD16 (SEQ ID NO.4), CD32 (SEQ ID NO.5), CD64 (SEQ ID NO.6), FCER1G (SEQ ID NO.7) or CD36 (SEQ ID NO.8) . Specific nucleic acid sequence CAR-16 (SEQ ID NO.14), CAR-32 (SEQ ID NO.15) and CAR-64 (SEQ ID NO.16), CAR-R1G (SEQ ID NO.17) and CAR-36 ( SEQ ID NO.18) was artificially synthesized at Sangon Biotechnology to obtain a chimeric antigen receptor sequence. figure 1 A schematic diagram of a chimer...
Embodiment 2
[0037] This example is the preparation of lentivirus with chimeric antigen receptor, and the selected chimeric antigen receptor is CAR-36.
[0038] Experimental materials: OPTI-MEM, Lipofectamine 2000 (lipo2000) were purchased from Invitrogen; HEK293T.
[0039] Experimental steps:
[0040] 1) On the first day, 293T was subcultured, cultured overnight in 10cm*10cm dish with DMEM containing 10% FBS, and its density reached 80%.
[0041] 2) The next day, the cells were transfected, the old medium was sucked off, and 5 mL of preheated OPTI-MEM was quickly added, and then put back into the incubator.
[0042] 3) Prepare the DNA / lipo2000 complex:
[0043] a) Add 1.5mL OPTI-MEM to the EP tube, then add the following plasmids and mix well.
[0044] pLenti-CAR-36 15 μg
[0045] psPAX2 7.5 μg
[0046] pMD2.G 2.5 μg
[0047] b) Add 1.5mL OPTI-MEM to the EP tube, then add 75μL lipo2000, mix well and stand at room temperature for 5min.
[0048] c) Mix a) and b) and leave at room tem...
Embodiment 3
[0058] This example is the construction of chimeric antigen receptor monocyte / macrophage
[0059] Experimental Materials:
[0060] Polybrene was purchased from Sigma; the flow cytometer was BD FACS Aria.
[0061] Experimental steps:
[0062] 1) Spread THP-1 cells in the logarithmic growth phase in a 6-well plate (1 mL), 1×10 6 cell / well.
[0063] 2) Slowly add 1mL virus supernatant and ploybrene (8μg / mL), and shake gently.
[0064] 3) Centrifuge at 1900rpm for 60min (32°C).
[0065] 4) Place in a cell culture incubator for 1 hour.
[0066] 5) Repeat 3) once.
[0067] 6) Change the medium after 24 hours, and then infect once with the lentivirus prepared in Example 2, the operation is the same as above.
[0068] 7) After 48 hours, the expression of GFP in the cells was observed with a fluorescence microscope.
[0069] 8) The above cells were sorted by flow cytometry to obtain GFP positive cells.
[0070] 9) The obtained cells are subjected to expanded culture to obtain ...
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