Completely humanized EGFRvIII chimeric antigen receptor T cell and preparation method thereof
A fully humanized, cell-based technology, applied in biochemical equipment and methods, receptors/cell surface antigens/cell surface determinants, chemical instruments and methods, etc., can solve problems such as inability to activate human T cells
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Embodiment 1
[0032] Embodiment 1: Pre-Lenti-EF1-EGFRvIII CAR vector construction
[0033] 1. Synthetic ScFV gene: including nucleotides encoding CD8 signal peptide + fully humanized 139 antibody light chain variable region amino acid codon-optimized nucleotides + encoding linker (G4S) 3 Nucleotide + fully humanized 139 antibody heavy chain variable region amino acid codon-optimized nucleotide sequence, the base sequence of which is shown in SEQ ID NO:1. Among them, the sequence of the fully humanized 139 antibody recognizing EGFRvIII can be found in the world patent WO2005010151A2.
[0034] 2. Synthetic 3rd generation CAR gene: including nucleotide fragments encoding CD8 hinge region+CD28 transmembrane region, cytoplasmic region+4-1BB cytoplasmic region+CD3ζ cytoplasmic region, its base sequence is as SEQ ID NO: 2.
[0035] 3. Splicing the synthesized ScFV gene and the synthesized third-generation CAR gene through overlapping PCR reactions to obtain the EGFRvIII CAR gene of the third-gen...
Embodiment 2
[0044] Embodiment 2: cell killing experiment
[0045] 1. Lentivirus packaging:
[0046] (1) Packaging cells 293T cells were cultured at 37°C, 5% CO 2 In the incubator, the medium is DMEM (Hyclone) / 10% FBS (Lanzhou Bailing).
[0047] (2) The day before packaging, digest 293T cells with 0.25% trypsin, 1×10 7 Cells / Dish Seed 10cm Petri dishes.
[0048] (3) When transfecting cells, in addition to the Pre-Lenti-EF1-MCS-EGFRvIII CAR shuttle plasmid, the packaging plasmid psPAX2 and pMD2.0G should also be added for co-transfection, including 5 μg of the Pre-Lenti-EF1-MCS-EGFRvIII CAR plasmid , psPAX2 plasmid 3.75 μg, pMD2.0G plasmid 1.25 μg. For transfection, the mixture of the three plasmids was added to 500 μl MEM medium (Hyclone), and 25 μl Lipofectamine 2000 reagent (Life Technologies) was added to 500 μl MEM medium (Hyclone) in another microcentrifuge tube, and then the diluted transfection Slowly add the staining reagent dropwise to the top of the diluted plasmid, vortex g...
Embodiment 3
[0080] Embodiment 3: animal experiments
[0081] 1. Mouse strain: It is an NSG immunodeficiency mouse strain, and the immune system lacks the functions of T cells, B cells and NK cells.
[0082] 2. Tumor inoculation: BxPC-1 tumor cell line with exogenous high expression of EGFRvIII was used, 3×10 6 Cells / 100ulPBS subcutaneously injected 8 mice, see Figure 5 .
[0083] 3. CART injection: 6 days after tumor cell inoculation, 4 mice were injected into the tail vein of Control, and the other 4 mice were injected into the tail vein of EGFRvIII CART cells (1×10 7 cells / 200ul PBS), see Figure 5 .
[0084] 4. Observe the mice every 2 days and measure their body weight until 22 days after CART cell injection, the subcutaneous tumors of 4 mice treated with EGFRvIII CART disappeared. To plot tumor volume change versus body weight change, see Figure 6 , Figure 7 . Depend on Figure 6 It can be seen that after treatment with CART cells, the tumor volume was measured every 2 da...
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