Methods for Preparing Genetically Modified Cells
The present disclosure provides methods for improving the preparation efficiency of genetically modified immune cells and improving the quality of immune cells.
1 . A method for preparing genetically modified immune cells, the method comprising:
(a) providing a sample containing immune cells;
(b) sorting the sample to obtain a first immune cell population enriched in immune cells;
(c) activating the first immune cell population to obtain a second immune cell population;
(d) culturing the second immune cell population to obtain a third immune cell population;
(e) genetically modifying the third immune cell population to obtain a fourth immune cell population; and
(f) culturing the fourth immune cell population to obtain genetically modified immune cells.
2 . The method of claim 1 , wherein in step (c) the first immune cell population is activated with microbeads coated with activating agents.
3 . The method of claim 2 , wherein the activating is performed with a microbead-to-cell ratio ranging from about 0.5:1 to about 5:1.
4 . The method of claim 2 , wherein the activating agents are selected from the group consisting of: antibodies or fragments thereof, cytokines, recombinant costimulatory molecules, small drug inhibitors, and combinations thereof.
5 . The method of claim 1 , wherein the activating agents are anti-CD3 and/or anti-CD28 antibodies or fragments thereof.
6 . The method of claim 1 , wherein in step (c) the activating is performed with a density of the first immune cell population ranging from about 0.5×10 6 cells/ml to about 10×10 6 cells/ml.
7 . The method of claim 1 , wherein in step (b) the sorting is performed by mixing the sample with sorting magnetic beads.
8 . The method of claim 7 , wherein the sorting comprises positive sorting and/or negative sorting.
9 . The method of claim 7 , wherein the positive sorting comprises using anti-CD4 and/or anti-CD8 antibodies or fragments thereof.
10 . The method of claim 1 , wherein in step (f), for the culturing, when the fourth immune cell population has a density of less than 2×10 6 cells/ml, no perfusion is carried out; when the fourth immune cell population has a density of greater than or equal to 2×10 6 cells/ml and less than 4×10 6 cells/ml, perfusion is carried out at a rate of 0.5 V/day to 1 V/day; and when the fourth immune cell population has a density of greater than or equal to 4×10 6 cells/ml, perfusion is carried out at a rate of 1 V/day to 2 V/day, wherein V is the volume of a culture system.
11 . The method of claim 1 , wherein step (b) to step (f) are performed in about 4 days to about 5 days.
12 . The method of claim 1 , wherein step (d) is performed for about 1.5 days to about 3 days.
13 . The method of claim 1 , wherein step (e) is performed for about 0.5 days to about 2.5 days.
14 . The method of claim 1 , wherein step (f) is performed for about 1 day to about 3.5 days.
15 . The method of claim 1 , wherein the immune cells are T cells or T cell subsets.
16 . The method of claim 1 , wherein in step (e) the genetically modifying is transducing or transfecting.
17 . The method of claim 1 , wherein in step (e) the genetically modifying comprises introducing into the third immune cell population a polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
18 . The method of claim 1 , wherein in step (e) the genetically modifying comprises transducing the third immune cell population with lentiviral vectors, gamma-retroviral vectors, alpha-retroviral vectors, or adenoviral vectors.
19 . (canceled)
20 . The method of claim 1 , wherein the sample is peripheral blood, cells, fresh apheresis, cryopreserved apheresis, monocyte collections, peripheral blood mononuclear cells (PBMCs), or combinations thereof.
21 . (canceled)
22 . Genetically modified immune cells prepared by the method of claim 1 .
23 - 24 . (canceled)