METHODS FOR GENERATING GAMMA DELTA T-CELLS AND RELATED COMPOSITIONS
Provided herein, inter alia, are compositions and methods including T-cell cultures enriched for gdT cells, the gdT cells expressing a CAR, and related methods for generating said cells. In an aspect provided herein is a method for generating a T-cell culture enriched for gamma delta T-cells (gd T-cells or T cells). In another aspect, a method for generating a gdT-cell expressing a Chimeric Antigen Receptor (CAR) is provided. The method includes introducing a nuclei acid encoding a CAR to a gdT-cell obtained as provided herein, including embodiments thereof.
1 . A method for generating a T-cell culture enriched for gamma delta T-cells (gdT-cells) comprising:
(a) contacting a population of immune cells with a first media composition thereby forming an initial cell culture, wherein said population of immune cells comprises alpha beta T-cells (abT-cells), gdT-cells, and monocytes, wherein the ratio of abT-cells to gdT-cells in said population of immune cells is at least about 16:1, wherein the ratio of monocytes to gdT-cells is at least about 2:1, and wherein said first media composition comprises a bisphosphonate, interleukin-7 (IL-7) and interleukin-2 (IL-2);
(b) incubating said initial cell culture for a first time period of about one to about three days, thereby forming a monocyte-depleted immune cell culture;
(c) after said first time period, contacting said monocyte-depleted cell culture with interleukin-7 (IL-7) and interleukin-2 (IL-2), but not a bisphosphonate, thereby forming a monocyte-depleted cell culture in contact with a second media composition;
(d) incubating said monocyte-depleted cell culture in contact with said second media composition for a second time period of about one to about three days;
thereby forming an expanded gdT cell culture, wherein the ratio of abT-cells to gdT-cells in said expanded gdT cell culture is less than about 10:1;
(e) removing abT-cells from said expanded gdT cell culture thereby forming an abT-cell-depleted culture;
(f) replacing said second media composition with a third media composition comprising interleukin-7 (IL-7) and interleukin-2 (IL-2), wherein said third media composition does not comprise a bisphosphonate; and,
(g) incubating said abT-cell-depleted culture for at least one day in contact with said third media composition, thereby forming a T-cell culture enriched for gdT-cells, wherein the ratio of abT-cells to gdT-cells in said T-cell culture enriched for gdT-cells is less than about 1:2.
2 . The method of claim 1 , wherein said population of immune cells is obtained from peripheral blood.
3 . The method of claim 1 , wherein said population of immune cells is obtained by fresh leukapheresis.
4 . The method of claim 1 , wherein said bisphosphonate is zoledronate (zoledronic acid), clodronate, etidronate, alendronate, pamidronate, or neridronate.
5 . The method of claim 1 , wherein abT-cells are not removed prior to step (e).
6 . The method of claim 1 , wherein the concentration of IL-7 is about 250 U/mL.
7 . The method of claim 1 , wherein the ratio of abT-cells to gdT-cells of said expanded gdT cell culture is less than about 5:1.
8 . The method of claim 1 , wherein the ratio of abT-cells to gdT-cells of said expanded gdT cell culture is less than about 1:1.
9 . The method of claim 1 , wherein the ratio of abT-cells to gdT-cells of said T-cell culture enriched for gdT-cells is less than about 1:5.
10 . The method of claim 1 , wherein the ratio of abT-cells to gdT-cells of said T-cell culture enriched for gdT-cells is less than about 1:10.
11 . The method of claim 1 , wherein the ratio of abT-cells to gdT-cells of said T-cell culture enriched for gdT-cells is less than about 1:50.
12 . The method of claim 1 , wherein the ratio of abT-cells to gdT-cells of said T-cell culture enriched for gdT-cells is less than about 1:100.
13 . The method of claim 1 , wherein the ratio of abT-cells to gdT-cells of said T-cell culture enriched for gdT-cells is less than about 1:500.
14 . The method of claim 1 , wherein the ratio of abT-cells to gdT-cells of said T-cell culture enriched for gdT-cells is less than about 1:1000.
15 . The method of claim 1 , wherein the percentage of abT-cells of total cells in said T-cell culture enriched for gdT-cells is about 1% or less.
16 . The method of any of claims 1 to 15 , said method further comprising incubating said abT-cell-depleted culture 2, 3, 4, 5, or 6 days.
17 . The method of any of claims 1 to 16 , said method further comprising: (h) cryopreserving said T-cell culture enriched for gdT-cells.
18 . The method of claim 17 , wherein said cryopreserving comprises suspending said T-cell culture enriched for gdT-cells in a cryopreservation medium.
19 . The method of claim 17 or 18 , wherein said cryopreservation medium comprises between about 5% and about 10% dimethylsulfoxide (DMSO).
20 . A method of generating a gdT-cell expressing a Chimeric Antigen Receptor (CAR), said method comprising introducing a nucleic acid encoding a CAR to a gdT-cell obtained by any of the methods of claims 1 to 19 .
21 . A T-cell culture enriched for gdT cells obtained by any of the methods of claims 1 to 19 .
22 . A T-cell population expressing a Chimeric Antigen Receptor (CAR), comprising the T-cell culture enriched for gdT cells of claim 21 , wherein the gdT cells comprise a nucleic acid encoding said CAR.
23 . The T-cell population of claim 22 , wherein said CAR comprises an anti-IsoMSLN binding molecule.
24 . The T-cell population of claim 22 , wherein said CAR comprises a chimeric PD1 binding molecule.