Immune cell co-expressing IL-21 and hrCD16 chimeric receptor and application thereof
A technology of IL-21 and immune cells, applied in the direction of blood/immune system cells, receptors/cell surface antigens/cell surface determinants, genetically modified cells, etc., can solve the problem of CD16 chimeric receptor without detailed research, etc. problem, achieve the effect of enhancing ADCC function and avoiding functional impairment
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Embodiment 1
[0092] The construction of embodiment 1 lentiviral expression plasmid
[0093] CD16.BBz (SEQ ID NO: 9) hrCD16.BBz (SEQ ID NO: 10) and hrCD16.BBz-P2A-IL-21 genes (SEQ ID NO: 8) were synthesized by Shanghai Jierui Bioengineering Co., Ltd. and cloned into Blank lentiviral expression plasmid (pXW-EF1α-MCS-P2A-EGFP) obtained pXW-EF1α-CD16.BBz (abbreviated as pXW-CD16.BBz), pXW-EF1α-hrCD16.BBz (abbreviated as pXW-hrCD16.BBz) and pXW-EF1α-hrCD16.BBz-P2A-IL-21 (abbreviated as pXW-hrCD16.BBz-IL-21) recombinant lentiviral expression plasmid, the plasmid map is as follows figure 1 shown.
Embodiment 2
[0094] Packaging, concentration and titer determination of embodiment 2 lentivirus
[0095] 1.1 Packaging of lentivirus
[0096] Treatment of HEK293T cells: 24 hours before transfection, HEK293T cells in logarithmic growth phase were collected and seeded in 10 cm cell culture dishes (6-8×10 6 cells), the cells were grown in 10 mL of complete DMEM medium at 37 °C, 5% CO 2 Culture in a cell culture box for 18-24 hours, and transfection can be carried out when the cell density reaches above 70-90%.
[0097] HEK293T cell transfection: Add 1 mL of basic DMEM medium into a 15 mL centrifuge tube, and make the lentiviral expression plasmid (pXW-CD16.BBz, pXW-hrCD16.BBz or pXW-hrCD16.BBz-IL- 21): Packaging plasmid psPAX2: envelope plasmid PMD2.G = 1:3:1 Prepare a transfection mixture, the total amount of plasmids is 15 μg / dish. Add 30 μL of TurboFect transfection reagent at a ratio of plasmid amount (μg): transfection reagent (μL) = 1:2, incubate at room temperature for 15-20 min,...
Embodiment 3
[0102] Example 3 Infection and Expansion of T Cells
[0103] Add the lentiviral vector (LV-XW-hrCD16.BBz or LV-XW-hrCD16.BBz-IL-21) (MOI=5-10) obtained by packaging and concentrating in Example 2 to 1×10 6 In a 48-well flat-bottomed plate of pre-activated peripheral blood mononuclear cells, add the pro-infection reagent protamine sulfate and adjust the working concentration to 10 μg / mL, centrifuge at 1000×g for 90 min at 32°C, and replace with a fresh one after culturing overnight. The T cell growth medium continued to culture. Add fresh T cell growth medium every 2-3 days, and adjust the cell density to 0.5-2×10 6 cells. From 6 to 7 days after infection, the immunomagnetic beads of activated T cells were removed, and the T cells modified with hrCD16 chimeric receptor and / or IL-21 were continued to be cultured, and subsequent functional experiments could be carried out after the cells rested.
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