Restimulation of cryopreserved tumor infiltrating lymphocytes
The present disclosure provides methods for re-stimulating TIL populations that lead to improved phenotype and increased metabolic health of the TILs and provides methods of assaying for TIL populations to determine suitability for more efficacious infusion after re-stimulation.
1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising:
(i) performing a first expansion by culturing a first population of TILs obtained from a tumor resected from a patient;
in a cell culture medium comprising IL-2 to produce a second population of TILs; and
(ii) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is at least 100-fold greater in number than the second population of TILs, and wherein the second expansion is performed for at least 14 days in order to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs.
2. The method according to claim 1 , wherein the method further comprises:
(iii) performing an additional second expansion by supplementing the cell culture medium of the third population of TILs with additional IL-2, additional OKT-3, and additional APCs, wherein the additional second expansion is performed for at least 14 days to obtain a larger therapeutic population of TILs than obtained in step (ii), wherein the larger therapeutic population of TILs comprises an increased subpopulation of effector T cells and/or central memory T cells relative to the third population of TILs.
3. The method according to claim 2 , wherein after step (ii), the cells are removed from the cell culture and cryopreserved in a storage medium prior to performing step (iii).
4. The method according to claim 3 , wherein the cells are thawed prior to performing step (iii).
5. The method according to claim 2 , wherein step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dosage of the TILs.
6. The method of according to claim 2 , wherein steps (i) through (ii) or (iii) are performed within a period of about 40 days to about 50 days.
7. The method according to claim 2 , wherein steps (i) through (ii) or (iii) are performed within a period of about 42 days to about 48 days.
8. The method according to claim 2 , wherein steps (i) through (ii) or (iii) are performed within a period of about 42 days to about 45 days.
9. The method according to claim 2 , wherein steps (i) through (ii) or (iii) are performed within about 44 days.
10. The method according to claim 2 , wherein the cells from steps (ii) or (iii) express CD4, CD8, and TCR αβ at levels similar to freshly harvested cells.
11. The method according to claim 2 , wherein the effector T cells and/or central memory T cells in the therapeutic population of TILs in step (iii) exhibit one or more characteristics selected from the group consisting of expression of CD27, expression of CD28, longer telomeres, increased CD57 expression, and decreased CD56 expression, relative to effector T cells and/or central memory T cells in the third population of cells.
12. The method according to claim 11 , wherein the effector T cells and/or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
13. The method according to claim 1 , wherein the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).
14. The method according to claim 13 , wherein the PBMCs are added to the cell culture on any of days 9 through 17 in step (ii).
15. The method according to claim 1 , wherein the APCs are artificial APCs (aAPCs).
16. The method according to claim 1 , further comprising the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a high-affinity T cell receptor.
17. The method according to claim 1 , further comprising the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused with at least one endodomain of a T-cell signaling molecule.
18. The method according to claim 1 , wherein the therapeutic population of TILs are formulated for infusion into a patient.
19. The method according to claim 1 , wherein step (ii) further comprises a step of removing the cells from the cell culture medium.
20. The method according to claim 1 , wherein step (ii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dosage of the TILs.
21. The method according to claim 20 , where the number of TILs sufficient for a therapeutically effective dosage is from about 2.3×10 10 to about 13.7×10 10 .
22. A population of expanded TILs made according to the method of claim 1 .
23. A population of expanded TILs made according to the method of claim 1 , wherein the expanded TILs have at least a two-fold increase in basal glycolysis as compared to thawed cryopreserved TILs.
24. A method for assessing the metabolic activity of a TIL cell population made according to the method of claim 1 , comprising measuring the basal glycolysis of the cells, measuring the basal respiration of the cells, measuring the spare respiratory capacity (SRC) of the cells, and/or measuring the glycolytic reserve of the cells.