CD19 and CD30 dual-target chimeric antigen receptor and its application
A chimeric antigen receptor and single-chain antibody technology, applied in the field of biomedicine, can solve the problems of tumor recurrence and poor effect in patients, and achieve the effect of high-efficiency targeting, high-efficiency killing effect, and avoiding the phenomenon of immune escape.
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Embodiment 1
[0065] Example 1 Construction of CAR Molecular Carrier
[0066] In this example, firstly, the coding gene (SEQ ID NO: 10) of the anti-CD19 and CD30 dual-target chimeric antigen receptor is genetically synthesized, and restriction endonuclease Pme1 digestion sites are added to the C-terminal and N-terminal of the coding gene respectively and its protective base and restriction endonuclease Spe1 restriction site and its protective base;
[0067] The coding gene was double-digested with restriction endonucleases Pme1 and Spe1, and the digested product containing cohesive ends was recovered by agar gel electrophoresis, and then ligated into the linearized pWPXLd-eGFP plasmid (containing Sticky ends), the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent), and a lentiviral vector containing the coding gene of CAR targeting CD19 and CD30 dual targets was obtained.
[0068] In this example, the antigen-binding domains were constructed simulta...
Embodiment 2
[0069] Example 2 lentiviral packaging
[0070] In order to introduce CAR molecules into T cells, 293T cells were used to prepare recombinant lentiviruses, and when 293T cells were spread to 80-90% of the bottom of a 100mm culture dish, the lentiviruses were packaged:
[0071] 2 hours before virus packaging, replace the medium with DMEM containing 1% fetal bovine serum, and add 6mL / 100mm culture dish;
[0072] Prepare the plasmid mixture shown in Table 1, the pWPXLd-expression plasmid includes the lentiviral vector containing the coding gene of CAR targeting CD19 and CD30 dual targets, and the lentivirus containing the coding gene of CAR targeting CD19 single target Vector, a lentiviral vector containing a gene encoding a CAR targeting a CD30 single target, and the pWPXLd-eGFP plasmid is an empty vector that does not contain a gene encoding a CAR;
[0073] Table 1
[0074]
[0075] Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature...
Embodiment 3
[0081] Example 3 T cell activation and lentiviral transfection
[0082] Peripheral blood mononuclear cells (PBMC) were separated from whole blood using Ficoll density gradient centrifugation kit (GE Company), and after red blood cells were removed, T cells were sorted out using MACS Pan-T magnetic beads;
[0083] The sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100 U / mL + streptomycin 0.1 mg / mL) to a cell concentration of 2.5×10 6 pcs / mL for use;
[0084] CD2 / CD3 / CD28 T cell activation expansion kit (Miltenyi Company) was used to activate T cells, that is, the coated magnetic beads were mixed with T cells at a ratio of 1:2, and the final density of T cells was 5×10 6 piece / mL / cm 2 , after mixing, place at 37°C, 5% CO 2 The incubator was stimulated for 48 hours;
[0085] After T cells were activated for 48 hours, demagnetize the beads, centrifuge at 300g for 5 minutes, remove the supernatant, resuspend T cells with fresh medium, add recombinan...
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