Potency assay and manufacturing method

Abstract

The present disclosure relates to improved cellular compositions and potency assays for obtaining the same. Such compositions and assays may be suitable for use in the context of Tcell mediated disorders such as graft versus host disease (GvHD).

Description

Abstract: This disclosure relates to improved cell compositions and efficacy assays for obtaining them. Such compositions and assays can be applied to cases of T cell-mediated dysregulation, such as graft-versus-host disease (GvHD).

Claims

CLAIMS:

1. A method of selecting a cell population for use in treatment of graft versus host disease (GvHD), the method comprising: (i) obtaining a population of mesenchymal lineage precursor or stem cells (MLPSCs); (ii) culturing the MLPSC population in a culture medium; (iii) determining under culture conditions the level of IL-2RA inhibition; and, (iv) selecting for use in therapy an MLPSC population that inhibits IL-2RA by at least 56% under culture conditions.

2. The method of claim 1 , which comprises selecting an MLPSC population for use in therapy which inhibits IL-2RA by at least 60% under culture conditions.

3. The method according to claim 1 or claim 2, further comprising cryopreserving the selected cells.

4. A method for determining therapeutic efficacy of a population of culture expanded mesenchymal lineage precursor or stem cells (MLPSCs): (i) obtaining a population of MLPSCs; (ii) culturing the cells in a culture medium; and (iii) determining under culture conditions the level of IL-2RA inhibition, wherein the method determines therapeutic efficacy for graft versus host disease (GvHD) and, wherein at least 56% IL-2RA inhibition under culture conditions is indicative of therapeutic efficacy.

5. The method of claim 4, wherein > 60% IL-2RA inhibition under culture conditions is indicative of therapeutic efficacy.

6. A method of manufacturing drug product which comprises a population of MLPSCs, the method comprising: acquiring a determination of whether a test population of MLPSCs inhibits IL-2RA at a predetermined level under culture conditions, and processing at least a portion of the test population ofMLPSCs as a drug product if the test population of MLPSCs inhibits IL-2RA by at least the predetermined level under culture conditions, thereby manufacturing the drug product; or discarding at least a portion of the test population of MLPSCs if the population of mesenchymal stem cells inhibits IL-2RA by less than the predetermined level under culture conditions, wherein the predetermined level is 56%.

7. The method of claim 6, wherein the predetermined level > 60% under culture conditions.

8. A method of treating a subject with graft versus host disease (GvHD), the method comprising administering to a subject in need thereof a composition comprising a culture expanded population of mesenchymal lineage precursor or stem cells (MLPSCs), wherein the MLPSCs inhibit IL-2RA by at least 56% under culture conditions.

9. The method of claim 8, wherein the culture expanded MLPSCs which comprise the administered composition inhibit IL-2RA by > 60% under culture conditions.

10. The method according to any one of claims 1 to 5, 8 and 9, wherein the GvHD is acute GvHD.

11. The method according to any one of claims 1 to 5, 8 and 9, wherein the GvHD is chronic GvHD.

12. The method according to any one of claims 1 to 5 and 8 to 1 1, wherein the GvHD is pediatric GvHD.

13. The method according to any one of claims 1 to 5 and 8 to 12, wherein the GvHD is refractory to steroid therapy.

14. The method according to any one of claims 1 to 5 and 8 to 13, wherein the GvHD is Grade D GvHD.

15. The method according to any one of claims 8 to 14, wherein treatment increases 100 day survival to greater than 60%, greater than 70%.

16. The method according to any one of claims 1 to 15, wherein the MLPSCs are mesenchymal stem cells.

17. A composition comprising a population of culture expanded MLPSCs, wherein the population of MLPSCs are selected based on a predetermined level of IL-2RA inhibition under culture conditions, wherein the predetermined level of IL-2RA inhibition is at least 56%.

18. The composition of claim 17, wherein the predetermined level of IL-2RA inhibition is > 60%.

19. The composition of claim 17 or 18, wherein the MLPSCs are mesenchymal stem cells.

20. The composition of claim 19, wherein the mesenchymal stem cells are also selected based on expression of one or more or all of CD29, CD54, CD73, CD90, CD102, CD105, CD106, CD166, MHC1.

21. The method according to any one of claims 1 to 16, or the composition according to any one of claims 17 to 20, wherein level of IL-2RA inhibition is > 65%.

22. A method of manufacturing drug product which comprises a population of MLPSCs, the method comprising(i) acquiring a determination of whether a test population of MLPSCs inhibits IL-2RA at a predetermined level under culture conditions, wherein step (i) comprises co-culturing the test population of MLPSCs with CD3 / CD28 activated PBMCs; and determining the level of IL-2RA inhibition relative to a control population of CD3 / CD28 activated PBMCs in the absence of MLPSCs; wherein the predetermined level of IL- 2RA inhibition is at least 56%.

23. The method of claim 22, wherein the control population of CD3 / CD28 activated PBMCs express at least 12,000 pg / ml IL-2RA.

24. The method of claim 22 or 23, wherein CD3 / CD28 activation of PBMCs comprises stimulating PBMCs with an anti- CD3 antibody and an anti-CD28 antibody.

25. The method according to any one of claims 22 to 24, wherein the PBMCs are co-cultured with MLPSCs at a ratio of 5 PBMC:1 MPLSC.

26. The method according to claim 25, wherein IxlO6PBMCs are co-cultured with 2xl05MLPSCs.

27. The method according to any one of claims 22 to 26, wherein the PBMCs and MLPSCs are co-cultured for a period of about 72 hours.

28. The method according to any one of claims 22 to 27, wherein the level of IL- 2RA inhibition is determined by:(i) measuring IL-2RA levels in a test population of MLPSCs co- cultured with CD3 / CD28-activated PBMCs, a positive control population of CD3 / CD28-activated PBMCs in the absence of MLPSCs, and a negative control population of un-activated PBMCs in the absence of MLPSCs;(ii) constructing a standard curve to determine IL-2RA levels in the positive control population, negative control population, and test population; and(iii) calculating % inhibition of IL-2RA levels on the positive control population by the test population of MLPSCs cocultured with CD3 / CD28-activated PBMCs, relative to the positive control CD3 / CD28-activated PBMC population.

29. The method of claim 28, wherein the IL-2RA levels are measured by enzyme-linked immunosorbent assay (ELISA).

30. The method of claim 29 or 29, wherein the standard curve is generated using a four-parameter logistic (4- PL) curve fit.

Images