METHODS FOR PREPARATION OF IMMUNE CELLS
Methods for preparation of immune cells in a fully closed system are provided. Specifically, the methods contain the steps of pretreating, cell sorting, activation, transduction and expansion. The present methods greatly improve the preparation efficiency of immune cells, and reduce preparation costs.
1 . A method for culturing genetically modified immune cells, the method comprising:
(a) providing a sample containing immune cells;
(b) optionally, washing the sample to obtain pretreated immune cells;
(c) sorting the pretreated immune cells to obtain enriched immune cells;
(d) activating the enriched immune cells with microbeads coated with activating agents to obtain activated immune cells;
(e) genetically modifying the activated immune cells to obtain genetically modified immune cells; and
(f) expanding the genetically modified immune cells.
2 . The method of claim 1 , wherein the microbeads have a diameter ranging from about 1 μm to about 10 μm.
3 . The method of claim 2 , wherein the microbeads have a diameter ranging from about 2 μm to about 8 μm.
4 . The method of claim 3 , wherein the microbeads have a diameter ranging from about 4 μm to about 5 μm.
5 . The method of claim 1 , wherein in step (d) the activating is performed with a microbead-to-cell ratio ranging from about 0.1 to about 10.
6 . The method of claim 5 , wherein in step (d) the activating is performed with a microbead-to-cell ratio ranging from about 0.5 to about 5.
7 . The method of claim 5 , wherein in step (d) the activating is performed with a microbead-to-cell ratio ranging from about 1 to about 2.
8 . The method of claim 1 , 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.
9 . The method of claim 1 , wherein the activating agents are anti-CD3 and/or anti-CD28 antibodies or fragments thereof.
10 . The method of claim 1 , wherein in step (d) the activating is performed for about 16 hours to about 48 hours.
11 . The method of claim 1 , wherein step (d), step (e) and step (f) are performed in about 2 days to about 5 days.
12 . The method of claim 11 , wherein step (d), step (e) and step (f) are performed in about 3 days to about 4 days.
13 . The method of claim 1 , wherein all steps of the method are performed in about 2 days to about 5 days.
14 . The method of claim 13 , wherein all steps of the method are performed in about 3 days to about 4 days.
15 . The method of claim 1 , wherein steps (c)-(f) are performed in a closed and sterile system.
16 . The method of claim 1 , wherein all steps of the method are performed in a closed and sterile system.
17 . The method of claim 1 , wherein the immune cells are T cells or T cell subsets.
18 . The method of claim 1 , wherein in step (d) the activating is performed with an immune cell density ranging from about 0.5×10 6 cells/ml to about 10×10 6 cells/ml.
19 . The method of claim 18 , wherein in step (d) the activating is performed with an immune cell density ranging from about 2×10 6 cells/ml to about 3×10 6 cells/ml.
20 . The method of claim 1 , wherein in step (e) the genetically modifying is transducing or transfecting.
21 . The method of claim 1 , wherein in step (e) the genetically modifying comprises introducing into the activated immune cells a polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
22 . The method of claim 1 , wherein in step (e) the genetically modifying comprises transducing the activated immune cells with lentiviral vectors, gamma-retroviral vectors, alpha-retroviral vectors, or adenoviral vectors.
23 . The method of claim 1 , wherein in step (e) the genetically modifying comprises transducing the activated immune cells with lentiviral vectors.
24 . The method of claim 1 , wherein in step (b) the washing comprises using a human serum albumin (HSA) solution having a final HSA concentration of 0.1% to 30%.
25 . The method of claim 1 , wherein in step (b) the washing comprises using a human serum albumin (HSA) solution having a final HSA concentration of 0.1% to 10%.
26 . The method of claim 1 , wherein in step (b) the washing comprises centrifuging the sample using a centrifugal force ranging from about 100×g to about 1,000×g.
27 . The method of claim 1 , wherein in step (b) the washing comprises centrifuging the sample for about 100 seconds to about 600 seconds.
28 . The method of claim 1 , wherein in step (b) the washing comprises diluting the sample about 0 to about 5 folds.
29 . The method of claim 1 , wherein in step (b) the washing comprises performing the washing cycle for 1 to 5 times.
30 . The method of claim 1 , wherein step (b) has an output volume ranging from about 5 ml to about 400 ml.
31 . The method of claim 1 , wherein in step (c) the sorting comprises using anti-CD4 and/or anti-CD8 antibodies or fragments thereof.
32 . The method of claim 1 , wherein the sample is peripheral blood, immune cells, monocyte collections, or peripheral blood mononuclear cells (PBMCs).
33 . Genetically modified immune cells prepared by the method of claim 1 .
34 . A cell preparation comprising the genetically modified immune cells of claim 33 .
35 . A pharmaceutical composition comprising the genetically modified immune cells of claim 33 .