A fourth-generation car-t cell fused to express type I il-10 receptor and its preparation method and application
An expression vector, cd19scfv-il10rcar-t technology, applied to CAR-T cells and their application in the field of tumor immunotherapy, can solve the problems of T cell exhaustion, CAR-T cells cannot play an anti-tumor effect for a long time, etc. The effect of alleviating T cell exhaustion and reducing the effect of cytokine storm
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Embodiment 1
[0032] The features and advantages of the present invention can be further understood from the following detailed description in conjunction with the accompanying drawings. The examples provided are merely illustrative of the methods of the present invention, and are not intended to limit the remainder of the present disclosure in any way. [Example 1] Construction of the fourth-generation novel CAR structure (scFv-IL10R-CD28-4-1BB-CD3ζ) lentiviral expression vector
[0033] 1. A lentiviral expression vector expressing human scFv-IL10R-CD28-4-1BB-CD3ζ;
[0034] Result: as figure 1 shown in A
[0035] A is a lentiviral vector inserted to express the fourth-generation novel CAR structure of scFv-IL10R-CD28-4-1BB-CD3ζ; corresponding to figure 2 Identification by restriction enzyme digestion in lanes 1-5.
[0036] The above-obtained CAR complete structural gene was digested with EcoRI and BamHI, and then ligated with the linearized lentiviral expression plasmid pCDH-CMV-mcs-EF...
Embodiment 2
[0054] [Example 2] CD3 + Acquisition of T cells
[0055] human peripheral blood CD3 + Take the acquisition of T cells as an example.
[0056] 1. Preparation of peripheral blood mononuclear cells (PBMC)
[0057] 1. Take 50 mL of human peripheral blood into an anticoagulation tube, and add an equal volume of PBS to dilute;
[0058] 2. Slowly add the diluted blood into a centrifuge tube containing an equal volume of lymphocyte separation solution, not exceeding 2 / 3 of the total volume of the centrifuge tube, centrifuge at 20°C and 400g / min for 15min (speed 9, speed drop 1);
[0059] 3. Absorb the cloudy liquid into a centrifuge tube, 300g, 5min to remove excess liquid;
[0060] 4. Add 3 mL of erythrocyte lysis solution for lysis for 5 minutes;
[0061] 5. Add 5mL PBS 300g, wash 1-2 times in 5min, set aside.
[0062] Remarks: The reagents and materials used above all need to be aseptically processed in advance.
[0063] 2. Magnetic Bead Sorting CD3 + T cells (Pan T CellBio...
Embodiment 3
[0071] [Example 3] CD3 + Culture, activation of T cells and preparation of CD19 scFv-IL10R CAR-T cells 1. CD3 + Culture and activation of T cells
[0072] 1. Culture medium configuration:
[0073]1640 medium: containing 10% FBS, 2mM glutamine (gluamine), 1% MEM non-essential aminoacid solution, 100uM HEPES (hydroxyethylpiperazine ethanethiosulfonic acid), 1mM sodium pyruvate (sodium pyruvate), 1% antibiotic-antimycotic solution (double antibody);
[0074] 2. Each cell culture plate was pre-coated with anti-CD3 at a working concentration of 5 μg / mL, and the isolated cells were diluted to 10 6 cells / ml, 10ml of diluted cells were added to each plate, supplemented with 2μg / ml anti-CD28 and 50U / ml IL-2, 37°C, 5% CO 2 Continue to cultivate 3d;
[0075] 2. Preparation of CD19 scFv-IL10R CAR-T cells
[0076] 1. Take the required virus out of the -80°C refrigerator 60 minutes in advance, and put it in the 2-8°C refrigerator to melt;
[0077] 2. Turn on the UV light of the biosa...
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