CD19 and CD20 double-target chimeric antigen receptor and application thereof
A chimeric antigen receptor and dual-target technology, applied in the field of biomedicine, can solve problems such as non-expression of tumor antigens, tumor recurrence, and poor therapeutic effect
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Embodiment 1
[0053] Example 1 Construction of CAR molecular carrier
[0054] In this example, the coding gene of anti-CD19 and CD20 dual-target chimeric antigen receptor (SEQ ID NO: 6) was firstly synthesized, and restriction endonuclease Pme1 restriction sites were added to the C-terminus and N-terminus of the coding gene. Its protective base and restriction endonuclease Spe1 restriction site and its protective base;
[0055] The coding gene was double digested with restriction enzymes Pme1 and Spe1, and the digested product containing sticky ends was recovered by agar gel electrophoresis, and then ligated into the linearized pWPXLd-eGFP plasmid (containing the same double digested by Pme1 and Spe1). Cohesive ends), the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent) to obtain a lentiviral vector containing the genes encoding the CAR targeting CD19 and CD20 dual targets.
[0056] In this example, the antigen-binding domains of the anti-CD19 scF...
Embodiment 2
[0057] Example 2 Lentiviral Packaging
[0058] In order to introduce CAR molecules into T cells, 293T cells were used to prepare recombinant lentiviruses. When 293T cells were spread to 80-90% of the bottom of a 100mm petri dish, lentivirus packaging was performed:
[0059] 2h before virus packaging, the medium was replaced with DMEM containing 1% fetal bovine serum, and the addition amount was 6mL / 100mm culture dish;
[0060] Prepare the plasmid mixture as shown in Table 1. The pWPXLd-expression plasmid includes a lentiviral vector containing the coding gene of CAR targeting CD19 and CD20 dual targets, and lentivirus containing the coding gene of CAR targeting CD19 single target. Vector, lentiviral vector containing the coding gene of CAR targeting CD20 single target, pWPXLd-eGFP plasmid is an empty vector without CAR coding gene;
[0061] Table 1
[0062]
[0063] Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand for 5 min at room temperature;
...
Embodiment 3
[0069] Example 3 T cell activation and lentiviral transfection
[0070] Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using Ficoll density gradient centrifugation kit (GE), and after removal of red blood cells, T cells were sorted by MACS Pan-T magnetic beads;
[0071] The sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100U / mL + streptomycin 0.1 mg / mL) to a cell concentration of 2.5 × 10 6 pcs / mL for use;
[0072] The CD2 / CD3 / CD28 T cell activation and expansion kit (Miltenyi) was used to activate T cells, that is, the coated magnetic beads were mixed with T cells in a ratio of 1:2, and the final density of T cells was 5×10 6 pcs / mL / cm 2 , after mixing, placed at 37°C, 5% CO 2 Incubator stimulation for 48h;
[0073] 48h after T cell activation, demagnetize beads, centrifuge at 300g for 5min, remove supernatant, resuspend T cells in fresh medium, add CAR-expressing recombinant lentivirus or blank control eGFP lentivi...
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