NKT-cell subset for in vivo persistence and therapeutic activity and propagation of same
Embodiments of the disclosure include methods and compositions for producing NKT cells effective for immunotherapy and also methods and compositions for providing an effective amount of NKT cells to an individual in need of immunotherapy. In specific embodiments, the NKT cells are CD62L+ and have been exposed to one or more costimulatory agents to maintain CD62L expression. The NKT cells may be modified to incorporate a chimeric antigen receptor, in some cases.
1. A method of preparing human Type I natural killer T (NKT) cells for use in immunotherapy, comprising the steps of: isolating human NKT cells from human peripheral blood mononuclear cells (PBMCs), to generate an NKT-selected cell fraction and an NKT-depleted cell fraction, and expanding said NKT-selected cell fraction by culturing in the presence of at least:
(1) IL-21 and second cytokine selected from the group consisting of IL-2, IL-7, IL-15, IL-12, TNF-alpha; and
(2) irradiated NKT-depleted PBMCs loaded with alpha-Galactosylceramide (aGalCer) or irradiated artificial antigen-presenting cells (aAPCs) expressing CD1-d loaded with aGalCer;
wherein the majority of human Type I NKT cells in said expanded NKT-selected cell fraction are CD62L positive human Type I NKT cells.
2. The method according to claim 1 , wherein the NKT-selected cell fraction is expanded in media comprising IL-2.
3. The method according to claim 1 , wherein the NKT-selected cell fraction is expanded in the presence of irradiated aAPCs expressing CD1-d loaded with aGalCer.
4. The method according to claim 1 , wherein a subset of said CD62L positive human Type I NKT cells in said expanded NKT-selected cell fraction are 4-1BB positive.
5. The method according to claim 1 , wherein the NKT-selected cell fraction is expanded by culturing in media comprising IL-2, IL-21, and aGalCer.
6. The method according to claim 2 , wherein the irradiated NKT-depleted PBMCs are autologous to said NKT-selected cell fraction.
7. The method according to claim 1 , wherein the NKT cells are isolated using anti-NKT microbeads.
8. The method according to claim 1 , wherein the irradiated NKT-depleted PBMCs are not autologous to said NKT-selected cell fraction.
9. The method according to claim 3 , wherein the irradiated aAPCs expressing CD1-d are Ramos cells.
10. The method according to claim 1 , wherein the NKT-selected cell fraction is modified to express at least one chimeric antigen receptor (CAR).
11. The method according to claim 1 , wherein said majority of said human Type I NKT cells in said expanded NKT-selected cell fraction are CD62L positive human Type I NKT on day 12 of culture.
12. The method according to claim 1 , wherein said cells in said NKT-selected cell fraction are uncultured human NKT cells.
13. The method according to claim 1 , wherein the NKT-selected cell fraction is cultured in media containing fetal bovine serum.
14. A method of preparing human CD62L-positive enriched human Type I natural killer T (NKT) cells for use in immunotherapy, comprising the steps of:
isolating human NKT cells from uncultured human peripheral blood mononuclear cells (PBMCs), to generate an NKT-selected cell fraction and an NKT-depleted cell fraction, and expanding said NKT-selected cell fraction by culturing in the presence of at least:
(1) IL-21 and second cytokine selected from the group consisting of IL-2, IL-7, IL-15, IL-12, TNF-alpha; and
(2) irradiated NKT-depleted PBMCs loaded with alpha-Galactosylceramide (aGalCer) or irradiated artificial antigen-presenting cells (aAPCs) expressing CD1-d loaded with aGalCer;
wherein the majority of human Type I NKT cells in said expanded NKT-selected cell fraction are CD62L positive human Type I NKT cells.
15. The method according to claim 14 , wherein the NKT cells are isolated using anti-NKT microbeads.
16. The method according to claim 14 , wherein the NKT-selected cell fraction is expanded by culturing in media comprising IL-2 and IL-21.
17. The method according to claim 14 , wherein the irradiated aAPCs are Ramos cells.
18. The method according to claim 14 , wherein the NKT-selected cell fraction is modified to express at least one chimeric antigen receptor (CAR).
19. The method according to claim 14 , wherein the NKT-selected cell fraction is expanded by culturing in media comprising IL-2, IL-21, and aGalCer.
20. The method according to claim 14 , wherein the irradiated NKT-depleted PBMCs are autologous with respect to an individual that receives the immunotherapy.
21. The method according to claim 14 , wherein a subset of said CD62L positive human Type I NKT cells in said expanded NKT-selected cell fraction are 4-1BB positive said human Type I NKT cells.
22. The method according to claim 14 , wherein the NKT-selected cell fraction is cultured in media containing fetal bovine serum.
23. A method of preparing human Type I natural killer T (NKT) cells for use in immunotherapy, comprising the steps of: isolating human NKT cells from uncultured human peripheral blood mononuclear cells (PBMCs), to generate an NKT-selected cell fraction and an NKT-depleted cell fraction, and expanding the NKT-selected cell fraction by culturing in the presence of at least:
(1) IL-21 and second cytokine selected from the group consisting of IL-2, IL-7, IL-15, IL-12, TNF-alpha; and
(2) irradiated NKT-depleted PBMCs loaded with alpha-Galactosylceramide (aGalCer) or irradiated artificial antigen-presenting cells (aAPCs) expressing CD1-d loaded with aGalCer;
wherein said expanded NKT-selected cell fraction comprises a subset of 4-1BB positive human Type I NKT cells.
24. The method according to claim 23 , wherein the NKT cells are isolated using anti-NKT microbeads.
25. The method according to claim 23 , wherein the majority of human Type I NKT cells in said expanded NKT-selected cell fraction are CD62L positive human Type I NKT cells on day 12 of culture.
26. The method according to claim 23 , wherein said second cytokine is IL-2.
27. The method according to claim 23 , wherein the irradiated aAPCs are Ramos cells.
28. The method according to claim 23 , wherein the NKT-selected cell fraction is modified to express at least one chimeric antigen receptor (CAR).
29. The method according to claim 23 , wherein said irradiated NKT-depleted PBMCs are not autologous to said NKT-selected cell fraction.
30. The method according to claim 23 , wherein the irradiated NKT-depleted PBMCs are autologous with respect to an individual that receives the immunotherapy.
31. The method according to claim 23 , wherein the NKT-selected cell fraction is cultured in media containing fetal bovine serum.
32. The method according to claim 23 , wherein the NKT-selected cell fraction is expanded by culturing in media comprising IL-2, IL-21, and aGalCer.
33. The method according to claim 1 , wherein said CD62L positive human Type I NKT cells are modified to express at least one chimeric antigen receptor (CAR).
34. The method according to claim 14 , wherein said CD62L positive human Type I NKT cells are modified to express at least one chimeric antigen receptor (CAR).
35. The method according to claim 23 , wherein said subset of 4-1BB positive human Type I NKT cells is modified to express at least one chimeric antigen receptor (CAR).