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, the method comprising:
(a) performing a first expansion by (i) thawing a cryopreserved tumor digest comprising a first population of TILs from a tumor that was resected from the subject, digested after the resection, and cryopreserved after the digestion, and (ii) culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs;
(b) 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, and wherein the second expansion is performed for about 7 to 14 days in order to obtain the third population of TILs;
(c) harvesting the third population of TILs obtained from step (b);
(d) transferring the harvested third population of TILs from step (c) into an infusion bag; and
(e) cryopreserving the infusion bag comprising the harvested TIL population from step (d) using a cryopreservation process.
2. The method according to claim 1 , wherein the tumor digest in step (a) was prepared by incubating a sample of the tumor that was resected from the subject in an enzymatic media.
3. The method according to claim 2 , further comprising disrupting the tumor sample mechanically so as to dissociate the tumor sample.
4. The method according to claim 3 , further comprising purifying the disassociated tumor sample using a density gradient separation.
5. The method according to claim 2 , wherein the enzymatic media comprises DNase.
6. The method according to claim 5 , wherein the enzymatic media comprises 30 units/mL of DNase.
7. The method according to claim 2 , wherein the enzymatic media comprises collagenase.
8. The method according to claim 7 , wherein the enzymatic media comprises 1.0 mg/mL of collagenase.
9. The method according to claim 1 , wherein the cell culture medium is CTS Optimizer.
10. The method according to claim 1 , wherein the third population of TILs harvested in step (c) comprises sufficient TILs for use in administering a therapeutically effective dosage to a subject.
11. The method according to claim 10 , wherein the therapeutically effective dosage of TILs comprises from about 1×10 9 to about 9×10 10 TILs.
12. The method according to claim 1 , wherein the APCs comprise peripheral blood mononuclear cells (PBMCs).
13. The method according to claim 1 , wherein the third population of TILs in step (c) provides for increased efficacy, increased interferon-gamma (IFN-γ) production, increased polyclonality, increased average IP-10, and/or increased average MCP-1 when administered to the subject.
14. The method according to claim 1 , wherein the first expansion is performed within from about 3 to 11 days.
15. The method according to claim 1 , wherein steps (a) through (e) are performed within about 24 days.
16. The method according to claim 1 , wherein step (a) further comprises adding the thawed tumor digest into a closed system prior to culturing the first population of TILs.
17. The method according to claim 16 , wherein the transition from step (a) to step (b), the transition from step (b) to step (c), or the transition from step (c) to step (d) occurs without opening the system.
18. The method according to claim 16 , wherein the transition from step (a) to step (b) occurs without opening the system.
19. The method according to claim 16 , wherein the transition from step (b) to step (c) occurs without opening the system.
20. The method according to claim 16 , wherein the transition from step (c) to step (d) occurs without opening the system.
21. The method according to claim 1 , wherein step (a) further comprises adding the thawed tumor digest into a closed system prior to culturing the first population of TILs, wherein the transition from step (a) to step (b) occurs without opening the system, wherein the transition from step (b) to step (c) occurs without opening the system, and wherein the transition from step (c) to step (d) occurs without opening the system.