Low dose interleukin-2 pharmaceutical compositions

Abstract

Pharmaceutical compositions include low doses of non-lyophilized liquid interleukin-2 (IL-2). Accordingly, in certain aspects, a low dose liquid interleukin (IL)-2 pharmaceutical composition as presented herein comprises a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 30, wherein the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates.

Description

LOW DOSE INTERLEUKIN-2 PHARMACEUTICAL COMPOSITIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U. S. Provisional Application 63 / 789,496 filed on April 16, 2026. The entire contents of this application are incorporated herein by reference in its entirety.FIELD

[0002] The disclosure relates to new pharmaceutical compositions comprising interleukin-2 substantially devoid of IL-2 / SDS aggregates in solution and their pharmaceutical uses.BACKGROUND

[0003] Interleukin-2 (IL2 or IL-2) is a cytokine that regulates key aspects of the immune system. IL2 has been used in attempts to boost immune responses in patients with cancer, as well as autoimmune and / or inflammatory diseases. IL2 is a potent T cells growth factor that promotes immune responses, including clonal expansion of antigen-activated T cells, drives development of CD4 T-helper (Th)l and Th2 cells, terminally differentiates CD8+cytotoxic T lymphocytes (CTLs), and opposes development of CD4+Th 17 and T-follicular helper (Tfh) cells. IL2 also shapes T cell memory recall responses.SUMMARY

[0004] This disclosure relates to a liquid interleukin (IL)-2 pharmaceutical composition suitable for injection to a patient.

[0005] Accordingly, in certain aspects, a low dose liquid interleukin (IL)-2 pharmaceutical composition as presented herein comprises a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 30, wherein the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, the pharmaceutical composition further comprises a non-ionic isotonizing agent and / or a buffer,

[0006] In certain embodiments, presented herein is a liquid pharmaceutical composition comprising low dose liquid IL-2 and SDS in an SDS to IL-2 mass ratio from about 4 to about 30, a non-isotonizing agent and a buffer, wherein the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In particular embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer.

[0007] In particular embodiments, presented herein is a liquid pharmaceutical composition comprising low dose liquid IL-2 and SDS in an SDS to IL-2 mass ratio from about 8 to about 11, a non-isotonizing agent and a buffer, wherein the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In particular embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer.

[0008] In particular embodiments, presented herein is a liquid pharmaceutical composition comprising low dose liquid IL-2 and SDS in an SDS to IL-2 mass ratio of about 8, about 9, about 10 or about 11, a non-isotonizing agent and a buffer, wherein the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In particular embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer.

[0009] In certain embodiments, the low dose IL, -2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0010] In certain embodiments, the SDS to 11,-2 mass ratio ranges from about 4 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL- 2) is from about 4 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 4 to about 23. In certain embodiments, mass ratio of SDS to interleukin- 2 (IL-2) is from about 4 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (II, -2) is from about 4 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4to about 1 I. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 8. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 7. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 4.

[0011] In certain embodiments, the SDS to IL-2 mass ratio ranges from about 5 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL. -2) is from about 5 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 23. In certain embodiments, mass ratio of SDS to interleukin- 2 (IL-2) is from about 5 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 8. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 7. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 5.

[0012] In certain embodiments, the SDS to IL-2 mass ratio ranges from about 6 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 6 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 8. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 7. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 6.

[0013] In certain embodiments, the SDS to IL-2 mass ratio ranges from about 7 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2)is from about 7 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 7 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 8. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 7.

[0014] In certain embodiments, the SDS to IL-2 mass ratio ranges from about 8 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL- 2) is from about 8 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 19. In certain embodiments, mass ratioof SDS to interleukin-2 (IL-2) is from about 8 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 8.

[0015] In certain embodiments, the SDS to IL. -2 mass ratio ranges from about 9 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL- 2) is from about 9 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 9 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 9.

[0016] In certain embodiments, the SDS to IL-2 mass ratio ranges from about 10 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 10 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 13, In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 10.

[0017] In certain embodiments, the SDS to IL-2 mass ratio ranges from about 11 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 11 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2)is from about 11 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 11 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2). In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 11.

[0018] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 0.0001 mg / ml to about 1 mg / ml, In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 0.001 mg / ml to about 1 mg / ml. In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 0.01 mg / ml to about 1 mg / ml. In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 0.1 mg / ml to about 1 mg / ml. In certain embodiments, the phar aceutical composition comprises a low dose IL-2 of about 0.1 mg / ml.

[0019] In certain embodiments, the pharmaceutical composition comprises one or more unit doses of a low dose IL-2, wherein a unit dose ranges from about 0.0001 mg to about 1 mg. In certain embodiments, a unit dose comprises a low dose IL-2 ranging from about 0.001 mg to about 1 mg. In certain embodiments, a unit dose comprises a low dose IL- 2 ranging from about 0.01 mg to about 1 mg. In certain embodiments, a unit dose comprises a low dose IL-2 ranging from about 0.1 nig to about 1 mg. In certain embodiments, a unit dose comprises a low dose IL-2 of about 0.1 mg.

[0020] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 400 international units (ILT) to about 9xl07ILT / ml. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MILT / mg (MIU = million international units, e.g. 4 MILT = seven million international units) to about 30 MIU / mg.

[0021] In certain embodiments, the specific activity of the low dose IL-2 is between about 5 million international units / mg to about 45 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 40 MILT / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 39 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 38 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 37 MIU / mg. In certain embodiments, the specific activity of the low dose IL, -2 is between about 7 MIU / mg to about 36 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 35 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 34 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MILT / mg to about 33 MILT / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MILT / mg to about 32 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 31 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose 11,-2 is between about 8 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is about 10 MIU / mg.

[0022] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 400 international units (ILT) to about 135 MlU / ml. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 45 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 40 MILT / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 39 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 38 MIU / mg. Incertain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 37 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 36 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 35 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 M IU / mg to about 34 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 33 MI U / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 32 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 31 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 29 MI U / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 28 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 27 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 26 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 4 MIU / mg to about 25 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is about 10 MIU / mg,

[0023] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 500 international units (IU) to about 135 MIU / ml. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 45 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 40 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 39 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 38 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 37 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MILT / mg to about 36 MIU / mg. In certain embodiments, the specific activity of the low dose I L-2 is between about 5 MIU / mg to about 35 MIU / mg. In certainembodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 34 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 33 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 32 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 31 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 29 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 28 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 27 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 26 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 5 MIU / mg to about 25 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is about 10 MIU / mg.

[0024] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 600-international units (IU) to about 135 MIU / nil. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 45 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 40 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 39 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 38 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 37 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 36 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 35 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 34 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 33 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 32 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 31 MIU / mg. InI Icertain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 29 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 28 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 27 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 26 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 6 MIU / mg to about 25 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is about 10 MIU / mg.

[0025] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 700-international units (IU) to about 135 MIU / ml. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 45 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 40 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 39 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 38 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 37 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 36 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 35 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 34 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 33 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 32 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 31 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 28 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 27 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 26 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 7 MIU / mg to about 25 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is about 10 MIU / mg.

[0026] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 800-international units (IU) to about 135 MIU / ml. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 45 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 40 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 39 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 38 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 37 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 36 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 35 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8MIU / mg to about 34 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 33 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 32 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 31 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 29 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 28 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 27 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg to about 26 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 8 MIU / mg toabout 25 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is about 10 MIU / mg.

[0027] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 900-international units (IU) to about 135 MIU / ml. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 45 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 40 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 39 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 38 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 37 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 36 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 35 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 34 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 33 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 32 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 31 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 29 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 28 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 27 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9 MIU / mg to about 26 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 9MIU / mg to about 25 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is about 10 MIU / mg.

[0028] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 1,000-international units (IU) to about 135 MIU / ml. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about45 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 40 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 39 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 38 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 37 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 36 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 35 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 34 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 33 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 32 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 31 MIU / mg. In certain embodiments, the specific activity of the low dose IL -2 is between about 10 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 30 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 29 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 28 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 27 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 26 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is between about 10 MIU / mg to about 25 MIU / mg. In certain embodiments, the specific activity of the low dose IL-2 is about 10 MIU / mg.

[0029] In certain embodiments, the pharmaceutical composition comprises about 0.0001 mg / ml to about 90 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.001 mg / ml to about 10 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.001 mg / ml to about 10 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.01 mg / ml to about 10 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.1 mg / ml to about 10 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.5 mg / mlto about 10 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 1.0 mg / ml to about 10 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.1 mg / ml to about 5 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.5 mg / ml to about 5 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 1.0 mg / ml to about 5 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.1 mg / ml to about 1 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.5 mg / ml to about 1 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.5 mg / ml to about 1.5 mg / ml.

[0030] In certain embodiments, the IL-2 remains stable in the pharmaceutical liquid composition for at least a year as measured by an in vitro biological activity assay. In certain embodiments, the IL-2 remains stable in the pharmaceutical liquid composition for at least a year as measured by an in vivo biological activity assay.

[0031] In certain embodiments, the IL-2 is substantially free SDS / IL-2 microaggregates. In certain embodiments, the IL-2 is substantially free of SDS / IL-2 microaggregates as measured by dynamic light scattering.

[0032] In certain embodiments, the pharmaceutical composition comprises a nonionic isotonizing agent. In certain embodiments, the pharmaceutical composition comprises a non-ionic isotonizing agent, wherein the isotonizing agent is mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the pharmaceutical composition comprises a buffer, e.g., a phosphate buffer. In certain embodiments, the pharmaceutical composition comprises a pH 7-8 phosphate buffer.

[0033] In certain aspects, a method of inducing proliferation of regulatory T cells in vivo, comprises administering to a subject in need thereof, a therapeutically effective amount of a liquid pharmaceutical composition comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 30, a non-ionic isotonizing agent and a buffer, wherein the regulatory T cells increase in the subject, thereby inducing proliferation of regulatory T cells in vivo. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the buffer is a phosphate buffer, e g., a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, asmeasured in an animal model of human cancer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates.

[0034] In another aspect, a method of treating an autoimmune disease or disorder in a subject in need thereof, comprises administering to a subject in need thereof, a pharmaceutical composition comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 20, a non-ionic isotonizing agent and a buffer, wherein the regulatory T cells increase in the subject, thereby inducing proliferation of regulatory T cells in vivo. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL-2 is between about 4 MIU / mg to about 30 MIU / mg. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured in an animal model of human cancer. In certain embodiments, the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured by an in vitro biological activity assay.

[0035] In another aspect, a pharmaceutical composition comprises a therapeutically effective amount of a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 30, a non-ionic isotonizing agent, a buffer and is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 18.In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 4 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 4 to about 8. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 4 to about 7. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 4. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0036] In another aspect, a pharmaceutical composition comprises a therapeutically effective amount of a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 5 to about 30, a non-ionic isotonizing agent, a buffer and is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL- 2) is from about 5 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 toabout 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL- 2) is from about 5 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 8. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 5 to about 7. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 5. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0037] In another aspect, a pharmaceutical composition comprises a therapeutically effective amount of a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 6 to about 30, a non-ionic isotonizing agent, a buffer and is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about6 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 8. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 6 to about 7. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 6. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0038] In another aspect, a pharmaceutical composition comprises a therapeutically effective amount of a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 7 to about 30, a non-ionic isotonizing agent, a buffer and is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL- 2) is from about 7 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 27. In certain embodiments, mass ratio of SDS to interleukin- 2 (IL-2) is from about 7 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is from about 7 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) isfrom about 7 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 7 to about 8. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 7. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0039] In another aspect, a pharmaceutical composition comprises a therapeutically effective amount of a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 8 to about 30, a non-ionic isotonizing agent, a buffer and is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 27. In certain embodiments, mass ratio of SDS to interleukin- 2 (IL-2) is from about 8 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 22, In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 1. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 12, In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 8 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2(IL-2) is from about 8 to about 9. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 8. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0040] In another aspect, a pharmaceutical composition comprises a therapeutically effective amount of a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 9 to about 30, a non-ionic isotonizing agent, a buffer and is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 28. In certain embodiments, mass ratio of SDS to interleukin- 2 (IL-2) is from about 9 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 9 to about 10. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 9. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).2?

[0041] In another aspect, a pharmaceutical composition comprises a therapeutically effective amount of a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 10 to about 30, a non-ionic isotonizing agent, a buffer and is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 28. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 10 to about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL -2) is about 10. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0042] In certain embodiments, the SDS to IL-2 mass ratio ranges from about 11 to about 30. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 29. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 28, In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is fromabout 11 to about 27. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 26. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 25. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 24. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 23. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 22. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 21. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 20. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 19. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 18. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 17. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 16. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 15. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 14. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 13. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is from about 11 to about 12. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2). In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 11. In certain embodiments, mass ratio of SDS to interleukin-2 (IL-2) is about 9. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0043] In another aspect, a method of treating an inflammatory disease or disorder in a subject in need thereof, comprises administering to a subject in need thereof, a pharmaceutical composition comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 30, a non-ionic isotonizing agent and a buffer, wherein the regulatory T cells increase in the subject, thereby inducing proliferation of regulatory T cells in vivo. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL-2 is between about 4 MIU / mg to about 30 MIU / mg. In certain embodiments, the IL -2 has a stable specific activity, at least for a year in the liquid composition, as measured by an invitro biological activity assay. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured in an animal model of human cancer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates.

[0044] In certain embodiments, the subject in need thereof is suffering from an inflammatory disorder. In some embodiments, the inflammatory disorder comprises inflammation, autoimmune disease, atopic diseases arthritis, rheumatoid arthritis (e.g. active),, Sjogren’s syndrome, psoriasis, Behcet's disease, including but not limited to the effects on the skin, alopecia, lupus, Still's disease, Systemic Lupus Erythematosus(SLE) (e.g. active), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis(MS), asthma, COPD, Guillain-Barre disease, Type I diabetes mellitus, thyroiditis (e.g., Graves' disease), graft versus host disease, steroid refractory chronic graft versus host disease, transplantation rejection (e.g. kidney, lung, heart, skin, and the like), kidney damage, and the like.

[0045] In another aspect, a method of treating an inflammatory disease or disorder in a subject in need thereof, comprises administering to a subject in need thereof, a pharmaceutical composition comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 30, a non-ionic isotonizing agent, a buffer and wherein the regulatory T cells increase as compared to a control, inducing proliferation of regulatory T cells in vivo. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL -2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the IL. -2 has a stable specific activity, at least for a year in the liquid composition, as measured in an animal model of human cancer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured by an in vitro biological activity assay.

[0046] In another aspect, a method of treating an immune disorder in a subject in need thereof, comprises administering to a subject in need thereof, a pharmaceutical composition comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratiofrom about 4 to about 30, a non-ionic isotonizing agent, a buffer and wherein the regulatory T cells increase as compared to a control, inducing proliferation of regulatory T cells in vivo. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL-2 is between about 4 MIU / mg to about 30 MIU / mg. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured in an animal model of human cancer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured by an in vitro biological activity assay.

[0047] In certain embodiments, the immune disorder is inflammation, graft versus host disease, transplant rejection, or an autoimmune disorder. In certain embodiments, the immune disorder is graft versus host disease (GVHD). In specific aspects, the GVHD is chronic GVHD. In certain embodiments, the immune disorder is transplant rejection. In certain embodiments, the transplant is an organ transplant, bone marrow or other cell transplant, composite tissue transplant, or a skin graft. In certain embodiments, the immune disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythrematosus, contact hypersensitivity, asthma or Sjogren's syndrome. In certain embodiments, the immune disorder is cancer.

[0048] In certain embodiments, a method of inducing proliferation of regulatory T cells in vivo, comprises administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising IL-2 embodied herein, wherein the regulatory T cells increase in the subject, thereby inducing proliferation of regulatory T cells in vivo.

[0049] In certain embodiments, a method of regulating an immune response associated with a disease or disorder in a subject in need thereof, comprising administering to a subject in need thereof, a pharmaceutical composition comprising a therapeutically effective amount of a IL-2 composition embodied herein, wherein the regulatory’ T cells increase as compared to a control, inducing proliferation of regulatory T cells in vivo. In certain embodiments, the IL-2 has a stable therapeutic activity, at least for a year in the liquid composition. In certain embodiments, the disease or disorder comprises: an autoimmunedisease, cancer, inflammation, a viral infection, a bacterial infection, a neurodegenerative disorder or combinations thereof.

[0050] In certain embodiments, a method of restoring T-cell mediated immune responses in a subject in need thereof comprising administering a therapeutically effective amount comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 40, a non-ionic isotonizing agent and a buffer, wherein the regulatory T cells increase in the subject, thereby inducing proliferation of regulatory T cells in vivo. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the buffer is a phosphate buffer, e g., a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL-2 is between about 4 MIU / mg to about 30 MIU / mg, In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured by an in vitro biological activity assay. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured in an animal model of human cancer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates

[0051] In certain embodiments, a method of treating a subject suffering from an immune deficiency disease comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 30, a non-ionic isotonizing agent, a buffer and wherein the regulatory T cells increase as compared to a control, inducing proliferation of regulatory T cells in vivo. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured in an animal model of human cancer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured by an in vitro biological activity assay.

[0052] In certain embodiments, a method of treating a subject in need of immunotherapy, comprising administering to the subject a pharmaceutical compositioncomprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 10 to about 30, a non-ionic isotonizing agent, a buffer and wherein the regulatory T cells increase as compared to a control, inducing proliferation of regulatory T cells in vivo. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose. In certain embodiments, the buffer is a phosphate buffer, e.g., a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured in an animal model of human cancer. In certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the pharmaceutical composition is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured by an in vitro biological activity assay.

[0053] In certain embodiments, the subject is suffering from cancer, immunodeficiency, immunosuppression, viral infections, an immune disorder or combinations thereof. In certain embodiments, the immune disorder, inflammation, graft versus host disease (GVHD), transplant rejection, or an autoimmune disorder. In certain embodiments, the immune disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythrematosus, contact hypersensitivity, asthma or Sjogren's syndrome. In certain embodiments, the method further comprises administering IL-2.

[0054] The IL-2 molecules embodied herein include IL-2 variants of the present disclosure comprising a nucleic acid sequence or an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the human IL-2 nucleic acid or amino acid sequence (Accession numbers:ENSEMBL: ENSG00000109471, NCBI_Gene:3558, RGD.730858 or amino acid accession number: UniProtKB: P60568), or SEQ ID NO: 2. These include IL-2 variants that comprise an amino acid sequence having an N88R mutation that has at least 60%, at least 61%, at least62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type IL-2 amino acid sequence (e.g. amino acid accession number: UniProtKB: P60568) ), or SEQ ID NO: 2.Embodiments also include IL-2 variants that stimulate Treg cells and comprise an amino acid sequence having N88R and C125S mutations that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% sequence identity to the IL-2 amino acid sequence (amino acid accession number: UniProtKB: P60568) ), or SEQ ID NO: 2. Embodiments also include IL-2 variants that stimulate Treg cells and comprise an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-2 amino acid sequence (amino acid accession number: UniProtKB: P60568) ), or SEQ ID NO: 2.

[0055] Definitions

[0056] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).

[0057] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof areused in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising,”

[0058] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range. Alternatively, particularly with respect to biol ogical systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0059] As used herein, “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastatses. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0060] As used herein, the term “chemokine” refers to soluble factors (e.g., cytokines) that have the ability to selectively induce chemotaxis and activation of leukocytes. They also trigger processes of angiogenesis, inflammation, wound healing, and tumorigenesis. Example chemokines include IL-8, a human homolog of murine keratinocyte chemoattractant (KC).

[0061] The term “chimeric antigen receptor” or “CAR” as used herein refers to an antigen-binding domain that is fused to an intracellular signaling domain capable of activating or stimulating an immune ceil, and in certain embodiments, the CAR also comprises a transmembrane domain. In certain embodiments the CAR's extracellular antigenbinding domain is composed of a single chain variable fragment (scFv) derived from fusing the variable heavy and light regions of a murine or humanized monoclonal antibody.Alternatively, scFvs may be used that are derived from Fab's (instead of from an antibody, e.g., obtained from Fab libraries). In various embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain. “First-generation” CARs include those that solely provide CD3^ signals upon antigen binding, “Second-generation” CARs include those that provide both co-stimulation (e.g., CD28 or CDI37) and activation (CD3Q. “Third-generation” CARs include those that provide multiple co¬ stimulation (e.g. CD28 and CD137) and activation (CD3Q. A fourth generation of CARs have been described, CAR T cells redirected for cytokine killing (TRUCKS) where the vector containing the CAR construct possesses a cytokine cassette. When the CAR is ligated, the CAR T cell deposits a pro-inflammatory cytokine into the tumor lesion. A CAR-T cell is a T cell that expresses a chimeric antigen receptor. The phrase “chimeric antigen receptor (CAR),” as used herein and generally used in the art, refers to a recombinant fusion protein that has an antigen-specific extracellular domain coupled to an intracellular domain that directs the cell to perform a specialized function upon binding of an antigen to the extracellular domain. The terms “artificial T-cell receptor,” “chimeric T-cell receptor,” and “chimeric immunoreceptor” may each be used interchangeably herein with the term “chimeric antigen receptor.”

[0062] As used herein, the term “cell” includes prokaryotic and eukaryotic cells. In one embodiment, a cell of the disclosure is a bacterial cell. In another embodiment, a cell of the disclosure is a fungal cell, such as a yeast cell. In another embodiment, a cell of the disclosure is a vertebrate cell, e.g., an avian or mammalian cell. In a preferred embodiment, a cell of the disclosure is a murine or human cell. As used herein, the term “engineered” (as inan engineered cell) refers to a cell into which a nucleic acid molecule e.g., encoding an IL-2 protein (e.g., a spliced and / or unspliced form of IL-2) or fragments thereof, has been introduced.

[0063] The term ‘‘combination therapy”, as used herein, refers to those situations in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents. When used in combination therapy, two or more different agents may be administered simultaneously or separately. This administration in combination can include simultaneous administration of the two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more agents can be simultaneously administered, wherein the agents are present in separate formulations. In another alternative, a first agent can be administered just followed by one or more additional agents. In the separate administration protocol, two or more agents may be administered a few minutes apart, or a few hours apart, or a few days apart.

[0064] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements— or, as appropriate, equivalents thereof— and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0065] As used herein, the term “cytokine” refers generically to proteins released by one cell population that act on another cell as intercellular mediators or have an autocrine effect on the cells producing the proteins. Examples of such cytokines include lymphokines, monokines; interleukins (“ILs”) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN™ rIL-2; a tumor-necrosis factor such as TNF-a or TNF-p, TGF-pl-3; and other polypeptide factors including leukemia inhibitory factor (“LIF”), ciliary neurotrophic factor (“CNTF”), CNTF-like cytokine (“CLC”), cardiotrophin (“CT”), and kit ligand (“K. L”).

[0066] “Diagnostic” or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The“sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.” The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0067] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, when the transcription product is an mRNA molecule, this is in turn translated into a protein, polypeptide, or peptide.

[0068] As used herein, the terms “freeze-drying” or “lyophilization” refer to a dehydration process comprising freezing a material comprising water and reducing pressure so as to allow frozen water in the material to be removed by sublimation.

[0069] As used herein, the term “immune checkpoint modulator” refers to an agent that interacts directly or indirectly with an immune checkpoint. In some embodiments, an immune checkpoint modulator increases an immune effector response (e.g., cytotoxic T cell response), for example by stimulating a positive signal for T cell activation. In some embodiments, an immune checkpoint modulator increases an immune effector response (e g., cytotoxic T cell response), for example by inhibiting a negative signal for T cell activation (e.g. disinhibition). In some embodiments, an immune checkpoint modulator interferes with a signal for T cell anergy. In some embodiments, an immune checkpoint modulator reduces, removes, or prevents immune tolerance to one or more antigens.

[0070] “Interleukins” are a group of cytokines that play important roles in innate and adaptive immune system function. Some interleukins promote the development and differentiation of B lymphocytes, T lymphocytes, and hematopoietic cells. Many interleukins are produced by helper CD4 T lymphocytes, monocytes, macrophages, and endothelial cells. Interleukins can either enhance or inhibit immune function, depending on the particular interleukin.

[0071] As used herein, the term “human recombinant IL-2 (rhIL-2)” is anun glycosylated protein produced by a microorganism that has been transfected with the human IL-2 codifying DNA sequence or a non-extensive modification of it. In fact, the IL-2 human recombinant version most used in clinical practices is modified version of the naturalhuman IL-2 amino acid sequence, named aldesleukin (PROLEUKIN™) having the following odifications: a) aldesleukin has no the N-terminal alanine present in natural IL-2 b) aldesleukin has serine substituted for cysteine at amino acid position 125. Aldesleukin amino acid sequence is shown in FIG. 1. Construction of the aldesleukin gene is described in US patent 4518584A and related non-US patents. The United States FDA has approved aldesleukin (PROLEUKIN™) for use in the treatment of adults with metastatic renal cell carcinoma and metastatic melanoma. PROLEUKIN™ is supplied as a sterile, white to off- white, lyophilized cake in 20 single-use vials intended for intravenous administration. When reconstituted with 1.2 mL Sterile water for Injection, USP, each mL contains 18 million International Units (1.1 mg) PROLEUKIN™, 50 mg mannitol, and 0.18 mg sodium dodecyl sulfate, buffered with approximately 0,17 mg monobasic and 0.89 mg dibasic sodium phosphate to a pH of 7.5 (range 7.2 to 7.8). Reconstituted or diluted PROLEUKIN™ is stable for up to 48 hours at refrigerated and room temperatures, 2° to 25°C (36° to 77°F) as stated in the Reconstitution and Dilution Directions of the “PROLEUKIN™ (aldesleukin) injection label - FDA” (Reference ID: 3165255).

[0072] As used herein, the term “interleukin 2” (IL-2) denotes a cytokine that regulates the activities of white blood cells (mainly lymphocytes). This cytokine is part of the immune response to microbial infections and exercise its effects by binding to specific receptors present on the surface of lymphocytes. IL-2 binds to IL-2 receptors (e.g., expressed by lymphocytes) and regulates the activity of white blood cells (e.g., leukocytes, lymphocytes), plays important roles in self versus non-self immune recognition, and participates in responses against microbial infectious agents. IL-2 promotes the differentiation of T cells into effector T cells and memory T cells, and also promotes the differentiation of immature T cells into regulatory T cells. In the context of cancer therapy, IL-2 can increase immune cell activation and subsequent immune cell-mediated inhibition of tumor cell growth. Within the context of this disclosure, the term “IL-2” designates any source of IL-2, including mammalian sources such as e.g., human, mouse, rat, primate, and pig, and may be native or obtained by recombinant or synthetic techniques, including recombinant IL-2 polypeptides produced by microbial hosts IL-2 may be or comprise the native polypeptide sequence, or can be an active variant of the native IL-2 polypeptide. In certain embodiments, the IL-2 polypeptide or active variant is derived from a human source, and includes recombinant human IL-2, particularly recombinant human IL-2 produced by microbial hosts.

[0073] Further information about IL-2 can also be found in its “GeneCard” (accession number GC04M123831). Other accession numbers include: HGNC: 6001 Entrez Gene: 3558 Ensembl: ENSG00000109471 OMIM: 147680, NCBI__Gene:3558, RGD:730858 or amino acid accession number: UniProtKB: P60568. IL-2 proteins include native IL-2 proteins, as well as variant IL-2 proteins. A “native” or “wild type” IL-2 sequence, as used herein, refers to a human IL-2 sequence (e.g., Accession No. NP 000577.2), whether purified from natural sources or made using recombinant techniques.

[0074] The natural IL-2 molecule has an alanine amino acid at its N-terminal end. In aldesleukin, this alanine is absent. Human IL-2 sequence (e.g., Accession No. NP 000577.2) (SEQ ID NO:!):

[0075] MYRMQLLSCI LSLALVTNS A PTS S STKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFI ILRPRDLISNIN VI VLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT. Underlined amino acids represent the signal sequence which is absent in the low dose IL-2 formulations embodied herein.

[0076] Aldesleukin sequence: (including Cys modification; i.e., at amino acid position 125, natural IL-2 has a cysteine residue. In aldesleukin, this cysteine is replaced with a serine residue) (SEQ ID NO: 2):

[0077] MPTS S STKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFSQSIIST LT

[0078] The term “immune response” refers to any response that is induced, triggered, or enhanced in a subject by an antigen (e.g., tumor cell antigen). The term includes the development, maturation, differentiation, and activation of immune cells (e.g., B cells and T cells), as well as the production of antibodies against an antigen. The term also includes increasing or decreasing the expression or activity of cytokines that are involved in regulating immune function.

[0079] As used herein, the term “in combination” in the context of the administration of a therapy to a subject refers to the use of more than one therapy for therapeutic benefit. The term “in combination” in the context of the administration can also refer to the prophylactic use of a therapy to a subject when used with at least one additional therapy. The use of the term “n combination” does not restrict the order in which the therapies (e.g., a first and second therapy) are administered to a subject. A therapy can be administered prior to(e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject. The therapies are administered to a subject in a sequence and within a time interval such that the therapies can act together. In a particular embodiment, the therapies are administered to a subject in a sequence and within a time interval such that they provide an increased benefit than if they were administered otherwise. Any additional therapy can be administered in any order with the other additional therapy.

[0080] As used herein, the term “kit” refers to any delivery system for delivering materials. Inclusive of the term “kits” are kits for both research and clinical applications. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery' of reaction reagents (e.g., cytokines, oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to delivery' systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides or liposomes. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery' system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0081] The term “low dose” IL-2 refers an amount of IL-2, for example O. OOOlmg / ml to about Img / ml of IL-2 having a specific activity of at least 7 MIU / mg to about 30 MIU / mg (Remark: MIU means million international units, i.e, 7 MIU::::7 millioninternational units or 7 x 106international units). In certain embodiments, a low dose of IL-2 is about 0.001 mg / ml to about 0,9 mg / ml IL-2 / ml having a specific activity of at least 7 MILT / mg to about 30 MIU / mg. In certain embodiments, a low dose of IL-2 is about 0.001 mg / ml to about 0.9 mg / ml IL-2 / ml having a specific activity of at least 7 MIU / mg to about 30 MIU / mg. In certain embodiments, a low dose of IL-2 is about 0.001 mg / ml to about 0.9 mg / ml IL-2 / nil having a specific activity of at least 7 MIU / mg to about 30 MIU / mg. In certain embodiments, a low dose of IL-2 is about 0.01 mg / ml to about 0.5 mg / ml IL-2 / ml having a specific activity of at least 7 MIU / mg to about 30 MIU / mg. In certain embodiments, a low dose of IL-2 is about 0.1 mg / ml to about 0,5 mg / ml IL-2 / ml having a specifi c activity of at least 7 MILT / mg to about 30 MIU / mg.

[0082] The term “lymphocyte” refers to a subtype of white blood cells that includes T lymphocytes (also called “T cells”), B lymphocytes (also called “B cells”), dendritic cells, and natural killer (NK) cells, T lymphocytes mature in the thymus and play a central role in cell-mediated immunity. T lymphocytes are distinguished from other types of lymphocytes by the presence of T-cell receptors that are present on the cell surface. T lymphocytes are subdivided into several types, including effector T cells, helper T cells, cytotoxic (killer) T cells, memory' cells, regulatory' (suppressor) T cells, and natural killer T cells. Effector T cells refer to a broad category that includes several T lymphocyte types (including helper, killer, and regulatory' cells), and generally are cells that respond to a stimulus. Helper T cells, also known as CD4 T cells, assist other types of white blood cells, playing roles such as assisting with the maturation of B cells into plasma and memory B cells, and activation of cytotoxic T cells and macrophages. Helper T cells are activated by the presentation of an antigen on the surface of an antigen-presenting cell (APC). Cytotoxic (killer) T cells, also known as CD8+T cells, destroy virus-infected cells and tumor cells, and are associated with transplant rejection. Memory T cells recognize foreign invaders such as bacteria and viruses, and also tumor cells. After memory' T cells encounter and respond to their cognate antigen, upon a second encounter, these cells are able to reproduce and mount a stronger and fast immune response. Regulatory (suppressor) T cells (Tregs) are critical for maintaining immunological tolerance; their major role is to downregulate T lymphocyte-mediated immunity near the end of an immune reaction and to inhibit autoreactive T lymphocytes that escaped the process of negative selection in the thymus. Natural killer T cells, which bridge the adaptive and innate immune systems, recognize an antigen presented by CD Id molecules and perform functions such as cytokine production and the release of cytolytic molecules. B lymphocytes secreteantibodies and function in the humoral immunity component of the adaptive immune system. In addition, B lymphocytes can function as antigen-presenting cells and secrete cytokines. B lymphocytes, which mature in the bone marrow, are distinguished from other types of lymphocytes by the presence of B cell receptors on the cell surface. Activation of B lymphocytes can either be T lymphocyte-dependent or T lymphocyte-independent. B lymphocytes are subdivided into several types, including plasmablast cells, plasma cells, lymphoplasmacytoid cells, memory B cells, follicular B cells, marginal zone B cells, B-l cells, B-2 cells, and regulatory B (Breg) cells. NK ceils, which are part of the innate immune system, play major roles in defending against tumor cells and virally infected cells, differentiating these cells from normal cells by recognizing cell surface changes in major histocompatibility complex class I molecules. NK cells are activated by the interferon family of cytokines and once activated release cytotoxic molecules that destroy the altered or infected cells.

[0083] The term “modulator” is used to refer to an entity whose presence in a system in which an activity of interest is observed correlates with a change in level and / or nature of that activity as compared with that observed under otherwise comparable conditions when the modulator is absent. In some embodiments, a modulator is an activator, in that activity is increased in its presence as compared with that observed under otherwise comparable conditions when the modulator is absent. In some embodiments, a modulator is an inhibitor, in that activity is reduced in its presence as compared with otherwise comparable conditions when the modulator is absent. In some embodiments, a modulator interacts directly with a target entity whose activity is of interest. In some embodiments, a modulator interacts indirectly (i.e., directly with an intermediate agent that interacts with the target entity) with a target entity whose activity is of interest. In some embodiments, a modulator affects level of a target entity of interest; alternatively or additionally, in some embodiments, a modulator affects activity of a target entity of interest without affecting level of the target entity. In some embodiments, a modulator affects both level and activity of a target entity of interest, so that an observed difference in activity is not entirely explained by or commensurate with an observed difference in level.

[0084] As used herein a “non-ionic isotonizing agent” is a substance that is used to adjust the osmotic pressure of a solution to match that of bodily fluids (making it isotonic), while not carrying an electrical charge, i.e. “non-ionic.” Common examples of non-ionicisotonizing agents include glycerol, polyethylene glycols (PEGs), sugar alcohols such as sorbitol, mannitol, xylitol, trehalose and the like.

[0085] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0086] The term “peripheral blood mononuclear cell (PBMC)” refers to any peripheral blood having a round nucleus. PBMCs include lymphocytes (e.g., T lymphocytes, B lymphocytes, NK cells) and monocytes. PBMCs can be extracted from whole blood, for example, by using the polysaccharide Ficoll, followed by gradient centrifugation. Following centrifugation, PBMCs are found between the top layer of plasma and the bottom fraction of polymorphonuclear cells and erythrocytes.

[0087] The phrase “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent. Exemplary' earners include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while com starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

[0088] As used herein, “stable” or “highly stable” refers to the activity and / or the integrity of the molecule, e.g. IL-2, in the formulations embodied herein, over extended periods of time, as evaluated by biological activity in vitro, structural studies in vitro and therapeutic activity in vivo. The activity7of the IL-2 can be measured by any standard assay. The activity before storage and after long-term storage, e.g. at least one year, can be compared. Highly stable IL-2 would have the same or slightly less activity as compared to the activity at the time of IL-2 when first formulated.

[0089] The terms “subject”, “patient” or “individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the disclosure find use in experimental animals, in veterinaryapplication, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates. Patients in need of therapy comprise those at risk of developing a certain condition, disease or disorder (e.g. due to genetic, environmental or physical attributes, such as for example, obesity). Patients in need of therapy also include those afflicted with a condition, disease or disorder. The diseases or disorders comprise, for example: autoimmune diseases, cancer, inflammatory' diseases, neurological diseases or disorders, neuroinflammatory diseases or disorders, cardiovascular disease, obesity', diseases or disorders caused by infectious agents such as, for example, viruses, bacteria, fungi, prions, or parasites.

[0090] The term “substantially devoid of microaggregates” refers to a low amount of microaggregates. In order to achieve this goal an SDS / IL-2 mass ratio not below 0.4 was kept throughout the manufacturing process of IL-2 and in the final formulations.

[0091] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.“Treatment” of any disease mentioned herein encompasses an alleviation of at least one symptom of the disease, a reduction in the severity of the disease, or the delay or prevention of disease progression to more serious symptoms that may, in some cases, accompany the disease or to at least one other disease. Treatment need not mean that the disease is totally cured. A useful therapeutic agent needs only to reduce the severity of a disease, reduce the severity of symptom(s) associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition. For example, if the disease is an inflammatory bowel disease, a therapeutic agent may reduce the number of distinct sites of inflammation in the gut, the total extent of the gut affected, reduce pain and / or swelling, reduce symptoms such as diarrhea, constipation, or vomiting, and / or prevent perforation of the gut. A patient's condition can be assessed by standard techniques such as an X-ray performed following a barium enema or enteroclysis, endoscopy, colonoscopy, and / or a biopsy. Suitable procedures vary according to the patient's condition and symptoms.

[0092] “Tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Theterms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

[0093] Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0094] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0095] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0096] FIG. 1 is a series of graphs showing the protein concentration at 2 - 8 °C, 25°C / 60% RH and 40°C / 75 % RH after 0, 1, 2 and 4 weeks.

[0097] FIG. 2 is a series of graphs demonstrating the biological activity in % of nominal biological activity (1 MIU / mL for LI, L2, L3 and SI; 4 MIU / mL for L4) at 2 - 8 °C, 25°C / 60% RH and 40°C / 75 % RH after 0, 1, 2 and 4 weeks.

[0098] FIG.3 is a series of graphs demonstrating the purity (% main peak area) by RP HPLC at 2 - 8CC, 25°C / 60% RH and 40°C / 75 % RH after 0, 1, 2 and 4 weeks.

[0099] FIG. 4 is a series of graphs showing the pre-peaks expressed as % total chromatographic area at 2 - 8 °C, 25°C / 60% RH and 40°C / 75 % RH after 0, 1, 2 and 4 weeks.

[0100] FIG. 5 is a series of graphs showing the pre peaks expressed as % total chromatographic area at 2 - 8 °C, 25°C / 60% RH and 40°C / 75 % RH after 0, 1, 2 and 4 weeks.

[0101] FIG. 6 is a series of graphs demonstrating the purity as % densitometric area at 2- 8 °C, 25°C / 60% RH and 40°C / 75 % RH after 0, 1, 2 and 4 weeks.

[0102] FIG. 7 is a series of graphs demonstrating the purity as % densitometric area at 2 - 8 °C, 25°C / 60% RH and 40°C / 75 % RH after 0, 1, 2 and 4 weeks.

[0103] FIGS. 8A-8 D are a series of gels demonstrating an SDS-PAGE Analysis. SDS-Page 15% rHu IL-2 1 MIU LI LIQ 4 week 2-8 °C. 1- MWM Precision Plus Bio-Rad; 2- Reference IL-2043 DS – Reductor; 3- IL-2 LI 2-8°C DPI - Reductor; 4- IL-2 LI 2-8°C DP2 - Reductor; 5- IL-2 LI 2-8°C DP3 - Reductor; 6- Reference IL-2043 DS - Non Reductor; 7- IL-2 LI 2-8°C DPI -Non Reductor; 8- IL-2 LI 2-8°C DP2 - Non Reductor; 9- IL-2 LI 2-8°C DP3 - Non Reductor. FIG. 8B SDS-Page 15% rHu IL-2 1 MIU LI LIQ-4 week 40 °C. 1-MWM Precision Plus Bio-Rad; 2- Reference IL-2043 DS - Reductor; 3- IL-2 LI 40°C DPI - Reductor; 4- IL-2 LI 40°C DP2 - Reductor; 5- IL-2 LI 40°C DP3 - Reductor; 6- Reference IL-2 043 DS - Non Reductor; 7- IL-2 LI 40°C DPI - Non Reductor; 8- IL-2 LI 40°C DP2 - Non Reductor; 9- IL-2 LI 40°C DP3 - Non Reductor. FIG. 8C: SDS-Page 15% rHu IL-2 1 MIU L3 LIQ-4 week 40 °C. 1- MWM Precision Plus Bio-Rad; 2- Reference IL-2043 DS -Reductor; 3- IL-2 L3 40°C DPI - Reductor; 4- IL-2 L3 40°C DP2 - Reductor; 5- IL-2 L3 40°C DPS - Reductor; 6- Reference IL-2043 DS - Non Reductor; 7- IL-2 L3 40°C DP I - Non Reductor; 8- IL-2 L3 40°C DP2 - Non Reductor; 9- IL-2 L3 40°C DP3 - Non Reductor. FIG.8D: SDS-Page 15% rHu IL-2 1 MIU Lyophilized SI Formulation -4 weeks 40 °C. 1- MWM Precision Plus Bio-Rad; 2- Reference IL-2043 DS - Reductor; 3- IL-2 SI 40°C DPI - Reductor; 4- IL-2 SI 40°C DP2 - Reductor; 5- IL-2 SI 40°C DP3 - Reductor; 6- Reference IL-2 043 DS - Non Reductor; 7- IL-2 SI 40°C DPI - Non Reductor; 8- IL-2 SI 40°C DP2 - Non Reductor; 9- IL-2 SI 40°C DP3 - Non Reductor.

[0104] FIG. 9 is a series of graphs demonstrating the protein concentration (0 = time zero, w = weeks; m = months).

[0105] FIG. 10 is a series of graphs demonstrating the biological Activity in % of nominal biological activity (0 = time zero, w = weeks; m = months.

[0106] FIG. 11 is a series of graphs demonstrating purity. Main peak as % of total RP HPLC area.

[0107] FIG. 12 is a series of graphs demonstrating purity. Pre-peaks as % of total RP HPLC area.

[0108] FIG. 13 is a series of graphs demonstrating purity. Post-peaks as % of total RP HPLC area.

[0109] FIG. 14 is a series of graphs demonstrating purity. Main band in reducing SDS PAGE as % of total densitometric area.

[0110] FIG. 15 is a series of graphs demonstrating purity. Main band in non-reducing SDS PAGE as % of total densitometric area.

[0111] FIG. 16 is a graph demonstrating the SDS:IL-2 relationship as a function of diavolumes, throughout the last downstream step of final concentration and diafiltration, by TFF (Tangential Flow Filtration). Two representative drug substance batches are included. The nominal cutoff of the diafiltration cassette is 5 kDa, and because SDS is above the critical micelle concentration, it is partially retained along with IL-2. This results in a ratio always above 0.4:1 SDS: IL-2, preventing the formation of particles or microaggregates with multiple IL-2 molecules.

[0112] FIG. 17 is a series of plots demonstrating the particle size distribution via DLS (Finished Product). Particle size distribution (Volume and Number profiles) for Batch B0401 (Active) and Batch A0402 (Placebo) measured at room temperature. The Active sample shows two distinct populations: one at < 1 nm (SDS molecules) and one at approximately 2 nm (monomeric rhIL-2). The Placebo sample confirms the absence of the 2 nm peak, showing only the < 1 nm molecular population. Neither sample shows populations in the 8-20 nm range.

[0113] FIG. 18 is a series of linear plots demonstrating the linear fit for NMR DOSY (Diffusion Ordered Spectroscopy) Calculations (Drug Substance). Linear regression analysis for internal standard (Dioxane) and DS Batch IL2040 (rhIL-2 in phosphate buffer) used to determine the experimental Hydrodynamic Radius (RH). The slope values for Dioxane (Slope -2.119 ± 0.007) and the IL-2 protein (Slope -1.622 ± 0.007) yield an RH of 20.41 ± 0.55 A.

[0114] FIG. 19 is a graph and a table demonstrating the far-UV CD spectrum (190- 240 nm) of DS Batch IL2040 demonstrating a characteristic curve for a folded protein. The accompanying data provides deconvolution results indicating 45.2% a-helix content, which aligns with structural predictions (51.5% via Jpred 4 Server) for folded monomeric IL-2. Note: “Other” includes disordered structures and turns. “Nrmsd” stands for normalized rootmean-square deviation, a parameter used to measure how well the theoretical spectrum matches the experimental one.DETAILED DESCRIPTION

[0115] Embodiments of the disclosure are directed to a composition comprising a low dose of highly stable IL-2 in a liquid formulation.

[0116] INTERLEUKIN-2 (IL-2)

[0117] IL-2 is a small 15-kDa cytokine with pleiotropic effects on the immune system. Low doses of IL-2 preferentially bind to the trimeric IL-2 receptor (IL-2R), consisting of IL-2Ra (CD25), IL-2Rβ (CD122), and the common gamma chain (CD 132), which is mainly expressed on immunosuppressive regulatory T (Treg) ceils. Trimeric IL-2Rs are also referred to as high-affinity IL-2Rs because their affinity for IL-2 is about 10-100 times higher than that of dimeric IL~2Rs. Once the limited amounts of trimeric IL-2RS on these cells are saturated, IL-2 also very efficiently associates with and stimulates dimeric IL- 2 Rs, made of CD 122 and CD132, that are principally present on resting antigen-experienced (memory) effector T (Teff) and natural killer (NK) cells. The stimulatory effect of IL-2 on Teff and NK cells motivated trials of high-dose IL-2 for the treatment of cancer, with recombinant human IL-2 (Aldesleukin) becoming the first US Food and Drug Administration approved immunotherapy for the treatment of metastatic renal cell carcinoma (RCC) and metastatic melanoma in 1992 and 1998, respectively. However, the stimulatory effects of high-dose IL-2 on Treg cells, which dampen immune responses against self-antigens including certain tumor antigens, as well as the considerable adverse side effects of IL-2 at high doses due to vascular leak syndrome limited its efficacy in cancer Moreover, the short in vivo half-life of IL-2 in the range of minutes requires frequent applications Thus, only 9.3% of patients with RCC and 4.0% with metastatic melanoma achieved complete responses (CR) on high-dose IL-2 monotherapy. These shortcomings of high-dose IL-2 treatment motivated the development of improved IL-2-based biologic agents with higher selectivity for effector immune cell subsets, reduced toxicity, and prolonged half-life (Raeber, Miro E. et al. A systematic review of interleukin-2-based immunotherapies in clinical trials for cancer and autoimmune diseases. eBioMedicine, Volume 90, 104539).

[0118] Accordingly, a low dose IL-2 is provided herein. In certain embodiments, a low dose interleukin (IL)-2 pharmaceutical composition comprises a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 4 to about 30, a non-ionic isotonizing agent and a buffer, wherein the pharmaceutical composition is a liquid and is substantially devoid of SDS / IL-2 microaggregates. In certain embodiments, the low dose IL-2 present in the liquid pharmaceutical composition has not been subjected to freeze-drying (or lyophilization process).

[0119] In certain embodiments, the formation of SDS / IL-2 microaggregates is avoided by keeping the SDS / IL-2 ratio well above 0.4 mg SDS / mg IL-2 at all steps of the manufacturing process of IL -2. The present disclosure provides a novel manufacturing process for interleukin-2 (IL-2) formulations, particularly suitable for indications requiring dosages below 0.01 mg IL-2 / kg. In certain embodiments, the prevention of sodium dodecyl sulfate (SDS) / IL-2 microaggregate formation by maintaining a controlled SDS / IL-2 mass ratio exceeding 1 mg SDS / mg IL-2 throughout the purification and formulation process. Specifically, following upstream fermentation and intracellular expression of IL-2 as inclusion bodies (IB) in E. coli, the IB are solubilized using a phosphate buffer containing 1% w / v SDS. This step initiates the continuous presence of SDS with soluble IL-2 throughout the downstream process. Subsequent process intermediates maintain an SDS concentration of approximately 0.3% w / v, ensuring IL-2 solubility. Following two chromatographic purification steps, a tangential flow filtration (TFF) concentration step is employed. Due to the SDS concentration exceeding the critical micellar concentration (CMC), SDS is also concentrated during TFF, as it is retained by a 5 kDa nominal molecular weight cutoff (NMWC) diafiltration cassette. A final diafiltration step is performed using a buffer with a minimal SDS concentration, exemplified by 0.044% w / v, which maintains an SDS / IL-2 mass ratio above 0.4:1. This ratio effectively prevents microaggregate formation during TFF. Notably, utilizing this manufacturing process and further increasing the SDS / IL-2 mass ratio above 4 mg SDS / mg IL-2 in the final solution, stable IL-2 formulations were unexpectedly achieved even at concentrations below 1 mg / mL in buffered, isotonic, or nearly isotonic solutions, demonstrating stability for at least one year at 2-8 °C.

[0120] Other methods for producing the non-lyophilized, liquid IL-2 are disclosed by methods described in U. S. Application Nos.: 17 / 116,722 and 18 / 519,193, each of which are incorporated herein by reference in their entireties.

[0121] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 0.0001 mg / ml to about 1 mg / ml. In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.0004 mg / ml to about 30 mg / ml. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose, or trehalose. In certain embodiments the pharmaceutical composition further compri ses a pH 7-8 phosphate buffer. In certain embodiments, the specific activity of IL-2 is between about 7 MILT / mg to about 30 MIU / mg. In certain embodiments, the IL-2 remains stable in the pharmaceutical liquid composition for at least a year as measured by an in vitrobiological activity assay. In certain embodiments, the IL-2 is substantially free and not bound to SDS.

[0122] In certain embodiments, the pharmaceutical composition comprises a low dose of IL-2 plus a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (such as those having less than 10 amino acids), a protein, amino acids, carbohydrates such as glucose, sucrose, or dextrins, chelating agent such as EDTA, glutathione, and / or other stabilizers, excipients, and / or preservatives. The composition may be formulated as a liquid or a lyophilizate. Further examples of components that may be employed in pharmaceutical formulations are presented in Remington's Pharmaceutical Sciences, 23rdEd., Mack Publishing Company, Easton, Pa., (2020) and others as described herein,

[0123] In certain embodiments, the low dose IL-2 compositions are administered to a subject, in need of IL -2 therapy, daily doses up to about 1 mg / mL.

[0124] FORMULATION OF PHARMACEUTICAL COMPOSITIONS

[0125] The compositions may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, intravesicularly or intraperitoneally) administration route. An advantageous method of administration is intravenous infusion. The pharmaceutical therapeutic agent may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed, A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0126] In certain embodiments, the pharmaceutical composition comprises a low dose IL-2 ranging from about 0.0001 mg / ml to about 1 mg / ml, In certain embodiments, the pharmaceutical composition comprises SDS ranging from about 0.0004 mg / ml to about 30 mg / ml. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose, or trehalose. In certain embodiments the pharmaceutical composition further comprises a pH 7-8 phosphate buffer, certain embodiments, the specific activity of IL-2 is between about 7 MIU / mg to about 30 MIU / mg. In certain embodiments, the IL-2 remains stable in the pharmaceutical liquid composition for at least a year as measured by an in vitro biological activity assay. In certain embodiments, the IL-2 is substantially free and not bound to SDS.

[0127] The buffering agent can be of any type with proviso that it is appropriate to formulate interleukin-2 and adapted for injection, in particular subcutaneous injection. The buffering agent refers to acids, salt forms of acids and combination thereof. Buffering agents encompass phosphoric acid, Tris(hydroxymethyl)aminomethane hydrochloride (TRIS-HCl), 4-Morpholinepropanesulfonic acid (MOPS), 4-(2-Hydroxyethyl)piperazine-l -ethanesulfonic acid (HEPES), PIPES, 2,2-Bis(hydroxymethyl)-2,2’,2"-nitrilotriethanol (BIS-TRIS), TRIS- glycine, Bicine, Trici ne, TAPS, TAPSO, MES, citrate, borate, citrate / phosphate, bicarbonate, glutaric acid, succinic acid, salts thereof and combinations thereof The concentration of the buffering agent, in the liquid formulation of the disclosure is typically from 1 mM to 100 mM, preferably from 5 mM to 25 mM, such as from 5 mM to 15 mM. The pH of the liquid composition of the disclosure is preferably from 7.0 to 8 0, for instance pH 7.5. 0.2

[0128] The pharmaceutical compositions may be formulated to release the IL-2 substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with the thymus; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that cancer by using carriers or chemical derivatives to deliver the therapeutic, agent to a particular cell type (e.g., neoplastic cell). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic, level.

[0129] Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in acontrolled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0130] Subject doses of the compounds described herein typically range from about 0.1 pg to 10,000 mg, or from about 1 pg / day to 8000 mg, or from about 10 pg to 100 pg. Stated in terms of subject body weight, typical dosages range from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 10,000 milligram / kg / body weight, etc., can be administered, based on the numbers described above.

[0131] In certain embodiments, exemplary effective doses of the IL-2 molecules embodied herein, include between 0.1 pg / kg and 100 mg / kg body weight, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pg / kg body weight or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg body weight.

[0132] In some cases, the low dose IL-2 pharmaceutical compositions embodied herein, are administered daily, e.g., every 24 hours. Or, the IL-2 is administered continuously or several times per day, e.g., every 1 hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours, every 11 hours, or every 12 hours.

[0133] Exemplary effective daily doses of IL-2 include between 0.1 pg / kg and 100 pg / kg body weight, e.g., 0.1, 0.3, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 pg / kg body weight.

[0134] Alternatively, the IL-2 is administered about once per week, e.g., about once every 7 days. Or, the IL-2 is administered twice per week, three times per week, four timesper week, five times per week, six times per week, or seven times per week. Exemplary effective weekly doses of IL-2 include between 0.0001 mg / kg and 4 mg / kg body weight, e.g., 0.001, 0.003, 0.005, 0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, or 4 mg / kg body weight. For example, an effective weekly dose of IL-2 is between 0.001 pg / kg body weight and 400 ug / kg body weight. Alternatively, IL-2 is administered at a fixed dose or based on body surface area (i.e., per m2).

[0135] In some cases, subjects receive two 6-week cycles consisting of 4 weekly IL-2 intravenous doses followed by a 2-week rest period. Ultimately, the attending physician or veterinarian decides the appropriate amount and dosage regimen.

[0136] The absolute amount will depend upon a variety of factors including the concurrent treatment, the number of doses and the individual patient parameters including age, physical condition, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.

[0137] In certain embodiments, the compositions described herein are administered systemically, intravenously, subcutaneously, intramuscularly, intraperitoneally, intravesically, or by instillation. The inhibitors and the aldesleukin may be administered simultaneously or sequentially.

[0138] Parenteral Compositions

[0139] The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intravesicularly or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

[0140] Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a cancer, the composition mayinclude suitable parenterally acceptable carriers and / or excipients. The IL-2 molecules may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and / or dispersing, agents.

[0141] As indicated above, the pharmaceutical compositions according to the disclosure may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3 -butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w / w of propylene glycol or the like.

[0142] CONTROLLED RELEASE PARENTERAL COMPOSITIONS

[0143] Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the antibody may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.

[0144] Materials for use in the preparation of microspheres and / or microcapsules are, e.g., biodegradable / bioerodible polymers such as polygalactin, poly -(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutam- nine) and, polyflactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).

[0145] IL-2 VARIANTS

[0146] In certain embodiments, the IL-2 is an active variant. Active variants of IL -2 have been disclosed in the literature. Variants of the native IL -2 can be fragments, analogues, and derivatives thereof. By “fragment” is intended a polypeptide comprising only a part of the intact polypeptide sequence. An “analogue” designates a polypeptide comprising thenative polypeptide sequence with one or more amino acid substitutions, insertions, or deletions. Muteins and pseudopeptides are specific examples of analogues. “Derivatives” include any modified native IL-2 polypeptide or fragment or analogue thereof, such as glycosylated, phosphorylated, fused to another polypeptide or molecule, polymerized, etc., or through chemical or enzymatic modification or addition to improve the properties of IL-2 (e.g., stability, specificity, etc.). Active variants of a reference IL-2 polypeptide generally have at least 75%, at least 85%, more preferably at least 90% amino acid sequence identity to the amino acid sequence of the reference IL-2 polypeptide.

[0147] Methods for determining whether a variant IL-2 polypeptide is active are available in the art. An active variant is, for example, a variant that activates Tregs. Examples of IL-2 variants are disclosed, for instance, in EP109748, EP136489, U. S. Pat. No 4,752,585; EP200280, or EP118,617.

[0148] Modifications: In certain embodiments, the IL-2 is modified. For, example, the IL-2 is linked or fused to one or more domains. For example, CD25, CD122 or targeting domains such as for example, an Fc portion of immunoglobulins. In certain embodiments, the IL-2 is modified. For example, pegylated IL-2,

[0149] Numerous strategies have been developed to modify IL-2 therapy and overcome its limitations such as its short half-life, side effects and potential to expand Tregs. These approaches include modifications of IL-2 to increase its half-life to allow for reduced dosing and lower systemic Cmax, such as pegylation, fusion to human serum albumin or monoclonal antibodies and Fc domains. Fc-fusion proteins consist of the Fc region of an IgG antibody and a desired linked protein in which the Fc region can bind to the neonatal Fc receptor (FcRn) rescuing it from degradation. The FcRn belongs to the extensive and functionally divergent family of MHC molecules which are unable to present antigen Instead, its capacity to bind IgG and albumin with high affinity at low7pH, circumvents lysosomal degradation and prolongs the serum half-lives of proteins. Pegylation is a process where inert polyethylene glycol (PEG) moieties are covalently conjugated to proteins, mainly via the amino acids lysine, histidine and cysteine. Pegylation increases the hydrodynamic volume of a protein, leading to a decrease of its rapid renal clearance and increase in its halflife. It further reduces protein aggregation through repulsion between Pegylated surfaces and enhances the thermal stability of proteins.

[0150] Various approaches to limit systemic IL-2 exposure and selectively deliver low dose IL-2 directly to the tumor comprises modifying the IL-2 or linking the IL-2 toanother molecule. These include the fusion of IL-2 to tumor targeting antibodies as well as the fusion to inactivation domains with protease cleavable linkers In certain embodiments, the IL-2 is encapsulated into polymers. For example, the IL-2 can be incorporated into various carriers such as gelatin and chondroitin-6-sulfate, liposomes, bundled carbon nanotubes, IL -2 encapsulating polymeric micelles, chitosan nanoparticles, and cyclodextrin (CD) nanoplexes to list a few.

[0151] In certain embodiments, IL-2 is fused to immune cell targeting antibodies or other ligands such as for example, a receptor. (Rokade S, Damani AM, Oft M, Emmerich J. IL-2 based cancer immunotherapies: an evolving paradigm. Front Immunol. 2024 Jul 24; 15: 1433989. doi: 10.3389 / fimmu.2024.1433989. PMID: 39114660; PMCID:PMC 11303236).

[0152] Pharmaceutical Compositions

[0153] The IL-2 may be formulated as pharmaceutical compositions with pharmaceutically acceptable carriers. The compounds are administered to the subject in an effective amount for treating cancer. An “effective amount”, for instance, is an amount necessary or sufficient to realize a desired biologic effect. An effective amount for treating cancer, for instance, could be that amount necessary to (i) prevent or slow further growth of a cancer and / or (ii) kill existing cancer cells. According to some aspects of the disclosure, an effective amount is that amount of a compound of the disclosure alone or in combination with another medicament, which when combined or co-administered or administered alone, results in a therapeutic response to the disease, either in the prevention or the treatment of the disease. The biological effect may be the amelioration and or absolute elimination of the cancer. In another embodiment, the biological effect is the complete abrogation of the disease, as evidenced for example, by the absence of a symptom of the disease e.g. renal cell carcinoma.

[0154] The effective amount of a compound of the disclosure in the treatment of a disease described herein may vary depending upon the specific compound used, the mode of delivery of the compound, and whether it is used alone or in combination. The effective amount for any particular application can also vary depending on such factors as the disease being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular molecule of the disclosure without necessitating undue experimentation. Combined with the teachings provided herein, by choosing among thevarious active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.

[0155] In certain embodiments, a pharmaceutical composition comprises IL-2 in a range in a range of about 0.0001 mg / ml to about 1 mg / ml, SDS from about 0.0004 mg / ml to 30 mg / ml, a non-ionic isotonizing agent, and / or a pH 7-8 phosphate buffer. In certain embodiments, a pharmaceutical composition consists of IL-2 in a range from about 0.001 mg to 3 mg / mL, SDS in a range of about 0.004 mg / ml to about 90 mg / ml, a non-ionic isotonizing agent and a pH 7-8 phosphate buffer. In certain embodiments, the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose, or trehalose. In certain embodiments the pharmaceutical composition further comprises a pH 7-8 phosphate buffer.

[0156] Subject doses of the compounds described herein typically range from about 0.1 pg to 10,000 mg, or from about 1 pg / day to 8000 mg, or from about 10 pg to 100 pg. Stated in terms of subject body weight, typical dosages range from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 10,000 milligram / kg / body weight, etc., can be administered, based on the numbers described above.

[0157] In certain embodiments, exemplary effective doses of the IL-2 molecules embodied herein include between 0.1 pg / kg and 100 mg / kg body weight, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, or 900 pg / kg body weight or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg body weight.

[0158] In some cases, the IL-2 is administered daily, e.g., every 24 hours. Or, the IL-2 is administered continuously or several times per day, e.g., every 1 hour, ever}' 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every' 7 hours, every 8 hours, every' 9 hours, every 10 hours, every 11 hours, or every 12 hours.

[0159] Exemplary effective daily doses of IL-2 include between 0.1 pg / kg and 100 pg / kg body weight, e.g., 0.1, 0.3, 0.5, 1, 5, 10, 15, 20, 25, 30, 35. 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 pg / kg body weight.

[0160] Alternatively, the IL-2 is administered about once per week, e.g., about once every' 7 days. Or, the IL-2 is administered twice per week, three times per week, four times per week, five times per week, six times per week, or seven times per week. Exemplary' effective weekly doses of IL-2 include between 0.0001 mg / kg and 4 mg / kg body weight, e.g., 0.001, 0.003, 0.005, 0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, or 4 mg / kg body weight. For example, an effective weekly dose of IL-2 is between 0.001 pg / kg body weight and 400 pg / kg body weight. Alternatively, IL-2 is administered at a fixed dose or based on body surface area (i.e., per m2).

[0161] In some cases, subjects receive two 6-week cycles consisting of 4 weekly IL-2 intravenous doses followed by a 2-week rest period. Ultimately, the attending physician or veterinarian decides the appropriate amount and dosage regimen.

[0162] The absolute amount will depend upon a variety of factors including the concurrent treatment, the number of doses and the individual patient parameters including age, physical condition, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.

[0163] In certain embodiments, the compositions described herein are administered systemically, intravenously, subcutaneously, intramuscularly, intraperitoneally, intravesically, or by instillation. The inhibitors and the aldesleukin may be administered simultaneously or sequentially.

[0164] THERAPEUTICS

[0165] It is well established that immunotherapy, including IL-2, is an effective approach for enhancing anti-tumor immunity against certain types of cancer. IL-2 has stimulatory effects on a number of immune cell types including T and B cells, monocytes, macrophages, lymphokine-activated killer cells (LAK) and NK cells (Waldmann, T. A., NatRev Immunol, 6: 595-601, 2006). Based on its ability to provide durable curative antitumor responses, systemic administration of recombinant human IL-2 (PROLEUKIN®) has been approved to treat patients with metastatic melanoma or renal cell carcinoma (Rosenberg, S. A. etal., Ann Surg, 210: 474-484; discussion 484-475, 1989; Fyfe, G. et al., J Clin Oncol, 13: 688-696, 1995; and Atkins, M B. etal., J Clin Oncol, 17: 2105-2116, 1999).Unfortunately, the considerable toxicity associated with this treatment makes it difficult to achieve an effective dose at the site of the tumor and limits the population that can be treated. For example, systemic treatment with IL-2 at tolerated doses induces lymphoid activation in virtually all treated patients, but anti-tumor responses are observed in a minority of these individuals (Rosenberg, S. A. etal., Ann Surg, 210: 474-484; discussion 484-475, 1989). As a result, use of high dose IL-2 is limited to specialized programs with experienced personnel and it is generally offered to patients who are responsive and have excellent organ function (Tarhini, A. A. etal., Curr Opin Investig Drugs, 6: 1234-1239, 2005). Local treatment (intravesical) of superficial bladder cancer patient with IL-2 has been shown to provide tumor regression and prolonged regression free time in a number of clinical studies (Den Otter, W. et al., J Urol, 159: 1183-1186, 1998; and Den Otter, W. etal.. Cancer Immunol Immunother, 57: 931-950, 2008). In a Phase 2 study, systemic IL-2 administration to patients with cisplatin-refractory advance / metastatic urothelial carcinoma (65% of which were bladder cancers) provided a median survival of over 10 months compared to 6-7 months observed using single agent or combination salvage chemotherapy, suggesting further evidence of bladder carcinoma sensitivity to IL-2 therapy (Kim, J. etal., Urol Oncol, 21: 21-26, 2003; and Gallagher, D. J, et al., Cancer, 113: 1284-1293, 2008), However, IL-2 induced toxicities in these patients were significant and limited the treatment regimen (Kim, J. et al., Urol Oncol, 21: 21-26, 2003). Thus, there is a critical need for innovative strategies that enhance the curative effects of IL-2, reduce its toxicity without compromising clinical benefit and expand its utility beyond the currently approved indications.

[0166] Accordingly, the therapeutic methods of the disclosure (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the pharmaceutical compositions embodied herein comprising a pharmaceutical composition having a low dose of IL -2, to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subjector health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).

[0167] In certain embodiments, cells from patients are obtained and cultured with the IL-2 compositions embodied herein, expanded and re-infused to the patient.

[0168] Regulatory T cells (Tregs) are important in the maintenance of immune cell homeostasis as evidenced by the catastrophic consequences of genetic or physical ablation of the Treg population. Specifically, Treg cells maintain order in the immune system by enforcing a dominant negative regulation on other immune cells. Broadly classified into natural or adaptive (induced) Tregs; natural Tregs are CD4 CD25+T-cells which develop and emigrate from the thymus to perform their key role in immune homeostasis. Adaptive Tregs are non-regulatory CD4 T-cells which acquire CD25 (IL-2R alpha) expression outside of the thymus, and are typically induced by inflammation and disease processes, such as autoimmunity and cancer.

[0169] There is increasing evidence that Tregs manifest their function through a myriad of mechanisms that include the secretion of immunosuppressive soluble factors such as IL-9, IL-10 and TGF beta, cell-contact-mediated regulation via the high affinity TCR and other costimulatory molecules such as CTLA-4, GITR, and cytolytic activity. Under the influence of TGF beta, adaptive Treg cells mature in peripheral sites, including mucosa-associated lymphoid tissue (MALT), from CD4+Treg precursors, where they acquire the expression of markers typical of Tregs, including CD25, CTLA4 and GITR / AITR. Upon up-regulation of the transcription factor Foxp3, Treg cells begin their suppressive effect. This includes the secretion of cytokines including IL-10 and TGF beta which may induce cellcycle arrest or apoptosis in effector T cells and blocking co-stimulation and maturation of dendritic cells.

[0170] In some embodiments, the pharmaceutical compositions increase the regulatory T cell (Treg) count (i.e. proliferation) when administered to a subject. For example, the pharmaceutical compositions comprising the IL-2 embodied herein of the present disclosure can increase the regulatory' T cell count in a subject after administration to the subject relative to the subject’s regulatory T cell count before administration. For example, administration of a pharmaceutical composition comprising the IL-2 molecules of the present disclosure to a subject may increase the levels of regulatory T cells in the subject by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold,20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold or 60-fold relative to the subject's regulatory T cell count before administration.

[0171] In some embodiments, the pharmaceutical composition comprising the low dose IL-2 molecules of the present disclosure can increase the regulatory' T cell count in a subject after administration to the subject relative to administration of the IL-2.Administration of a pharmaceutical composition comprising the molecules of the present disclosure to a subject may increase the levels of regulatory T cells in the subject by at least 10%, 20%, 30%, 40%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or 500% relative to an equimolar amount of the low dose IL-2 formulations embodied herein.

[0172] In some embodiments, the low dose IL-2 formulations of the present disclosure can increase the ratio of regulatory T cells (Treg) to conventional T cells (Tconv) in a subject. In humans, daily low dose IL-2 therapy has been used to treat patients with chronic GVHD by augmenting the levels of Tregs (See Koreth J, et al.. Blood. 2016 Jul. 7; 128(1 ): 130-7; and Koreth J. N Engl J Med. 2011 Dec. 1; 365(22):2055-66). In the latter trial, IL-2 (aldesleukin) was administered by daily subcutaneous injection for 12 weeks. Overall, patients in these trials attained a greater than 5 fold increase of Tregs over baseline (Treg levels prior to treatment), a greater than 5 fold increase in their Treg / Tconv ratios, and a 61% clinical response rate. Clinical responses were strongly associated with a Treg / Tconv ratio > 0.2 at the end of the first week of treatment, which was an approximately 2.9 fold increase over the baseline Treg / Tconv ratios. Accordingly, in some embodiments, administration of a pharmaceutical composition comprising the IL-2 molecules of the present disclosure to a subject may increase the Treg / Tconv ratio to at least 0.2, 0.3, 0.4, 0.5, 0.6. 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5. The Treg / Tconv ratio may be maintained at the levels described above over several doses of the IL-2 molecules of the present disclosure, for example over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12,13, 14, 15, 16, 17, 18, 19 or 20 doses.

[0173] The low dose IL-2 formulations embodied herein of the present disclosure may also increase regulatory T cell activity (e.g. immunosuppressive activity). Regulatory T cell function may be determined by measuring the expression of one or more biomarkers, including but not limited to, CD25, FOXP3, CTLA-4, ICOS and CD39. The level of FOXP3, a protein that is necessary for Treg function, is correlated with highly suppressive Tregs (MiyaraM, et al., Immunity. 2009 Jun, 19; 30(6):899-911). The level of CD25 (IL2RA protein) has been associated with increased IL-2 consumption, a major immunosuppressive mechanism of Tregs (Chinen T, etal., Nat Immunol. 2016 November; 17(11):1322-1333).

[0174] Adoptive cell therapy. Adoptive cell therapy (ACT) (including allogeneic and autologous hematopoietic stem cell transplantation (HSCT) and recombinant cell (i.e., CAR-T) therapies) is the treatment of choice for many malignant disorders (for reviews of HSCT and adoptive cell therapy approaches, see, Rager & Porter, Ther Adv Hematol (2011) 2(6) 409-428; Roddie & Peggs, Expert Opin. Biol. Ther. (2011) 11 (4):473-487; Wang et al. Int. J. Cancer. (2015)136, 1751-1768; and Chang, Y J and X. J. Huang, Blood Rev, 2013. 27(1): 55-62). Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including recombinant cells, i.e., CAR-T, CAR-NK). Beyond the necessity for donor- derived cells to reconstitute hematopoiesis after radiation and chemotherapy, immunologic reconstitution from transferred cells is important for the elimination of residual tumor cells. The efficacy of ACT as a curative option for malignancies is influenced by a number of factors including the origin, composition and phenotype (lymphocyte subset, activation status) of the donor cells, the underlying disease, the pre-transplant conditioning regimen and post-transplant immune support (i.e., IL-2 therapy) and the graft-versus-tumor (GV T) effect mediated by donor cells within the graft. Additionally, these factors must be balanced against transplant-related mortality, typically arising from the conditioning regimen and / or excessive immune activity of donor cells within the host (i.e., graft-versus-host disease, cytokine release syndrome, etc.).

[0175] The cells, e.g. CAR-T cells, NK cells, T cells etc., once they have been expanded ex vivo in response to, for example, a tumor or viral antigen, are reinfused into the subject in a therapeutically effective amount. The term “therapeutically effective amount” as used herein means the amount of cells when administered to a mammal, in particular a human, in need of such treatment, is sufficient to treat autoimmune diseases such as rheumatoid arthritis, cancer and the like.

[0176] The precise amount of cells to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of disease and condition of the subject.

[0177] Typically, administration of T cell therapies is defined by number of cells per kilogram of body weight. However, because T cells will replicate and expand after transfer, the administered cell dose will not resemble the final steady-state number of cells.

[0178] In an embodiment, a pharmaceutical composition comprising expanded cells, e.g. CAR-T cells may be administered at a dosage of 104to 109cells / kg body weight. In another embodiment, a pharmaceutical composition comprising the cells may be administered at a dosage of 105to 106cells / kg body weight, including all integer values within those ranges.

[0179] Compositions comprising the cells may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are known in the art (see, for example, Rosenberg et al., 1988, New England Journal of Medicine, 319: 1676). The optimal dosage and treatment regimen for a particular subject can be readily determined by one skilled in the art by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0180] In certain embodiments, administration of any of the compositions embodied herein, e.g. a chimeric antigen receptor (CAR)-T cell, comprising a single chain variable fragment (scFv) which specifically binds to an immune checkpoint protein or receptors thereof, and a chimeric antigen receptor comprising an antigen binding domain; a hinge region; a transmembrane domain; a costimulatory domain; a cytoplasmic signaling domain. T cells for the treatment of cancer or virus infection, can be combined with other cell-based therapies, for example, stem cells, antigen presenting cells, etc.

[0181] Approaches utilizing adoptive NK cell therapy have become of significant interest. In patients receiving autologous HSCT, blood NK cell numbers recover very early after the transplant and the levels of NK cells correlate with a positive outcome (Rueff et al., 2014, Biol. Blood Marrow Transplant. 20, 896-899). Although therapeutic strategies with autologous NK cell transfer have had limited success due to a number of factors, adoptive transfer of ex vivo activated allogeneic (or haplo-identical) NK cells has emerged as a promising immunotherapeutic strategy for cancer (Guillerey et al. 2016. Nature Immunol. 17: 1025-1036). The activity of these cells is less likely to be suppressed by self-MHC molecules compared to autologous NK cells. A number of studies have shown that adoptive therapy with haploidentical NK cells to exploit alloreactivity against tumor cells is safe and can mediate significant clinical activity in AML patients. Taking these findings further, recent studies have focused on optimizing ex vivo activation / expansion methods for NK cells or NK precursors (i.e., stem cells) and pre-transplant conditioning and post-transplant immune support strategies; use of NK cell lines or recombinant tumor-targeting NK cells; evaluationof combination therapies with other agents such as therapeutic antibodies, immunomodulatory' agents (lenalidomide), and anti-KIR and checkpoint antibodies.

[0182] Accordingly, in certain embodiments an NK cell is transfected with a chimeric antigen receptor. In certain embodiments, a CAR-NK cell comprises an antigen binding molecule e.g. an scFv, which specifically binds to an immune checkpoint protein or receptors thereof, and a chimeric antigen receptor comprising an antigen binding domain; a hinge region; a transmembrane domain; a costimulatory domain; a cytoplasmic signaling domain. As indicated herein, ex vivo incubation of CAR-NK cells can be activated, e g. an IL- 15 superagonist.

[0183] NK ceils mediate some of their functions through the secretion of cytokines, such as interferon y (IFN-y), granulocyte-macrophage colony-stimulating factors (GM-CSFs), tumor necrosis factor a (TNF-a), macrophage colony-stimulating factor (M-CSF), interleukin-3 (IL-3), and IL-8. NK cell cytotoxic activity is regulated through a balance of activating and inhibitoiy receptors that enables fine-tuned control of cytotoxic activity, preventing cytotoxicity against healthy cells, while maintaining effective cytotoxic capacity against tumor ceils. Indeed, multiple studies have demonstrated the safety of adoptive NK cell transfer and clinical anti-cancer effects, highlighting the potential for NK cells as an effective cancer immunotherapy ((Parkhurst, M. R, el al. Clin Cancer Res 17, 6287-6297 (2011); Ruggeri, L. et al. Science 295, 2097-2100, (2002); Miller, J. S. et al. Blood 105, 3051-3057, (2005; Bachanova, V. et al. Blood 123, 3855-3863, (2014); Rubnitz, J. E. et al. J Clin Oncol 28, 955-959, (2010)). For example, cytokines including IL-2, IL-12, TNF-a, and IL-1 can induce NK cells to produce cytokines. IFN-a and IL-2 are strong inducers of NK cell cytotoxic activity (G. Trinichieri et al., Journal of Experimental Medicine 160:1147- 1169, 1984; G. Trinichieri andD. Santoli, Journal of Experimental Medicine 147: 1314- 1333, 1977). The presence of IL-2 both stimulates and expands NK cells (K. Oshimi, International Journal of Hematology 63:279-290, 1996). IL-12 has been shown to induce cytokine production from T and NK cells, and augment NK cell-mediated cytotoxicity (M. Kobayashi et al., Journal of Experimental Medicine 170:827-846, 1989).

[0184] NK cells have been expanded from multiple sources, including peripheral blood and umbilical cord blood (CB) ((Denman, C. J. etal. Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells. PLoS One 7, e30264, (2012); Knorr, D. A. et al. Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Transl Med 2, 274-283, (2013); Shah, N, et al.Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity. PLoS One 8, e76781, (2013); Woll, P. S. et al. Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity. Blood 113, 6094-6101, (2009)). Ex vivo NK cell expansion methods have been developed using cytokines in combination with artificial antigen-presenting cells (aAPCs) as feeder cells ((Denman, C. J. etal. PLoS One 7, e30264, (2012); Berg, M. et al. Cytotherapy 11, 341-355, (2009); Gong, W. etal. Tissue Antigens 76, 467-475, (2010); Zhang, H et al., J Immunolher 34, 187-195, (2011)).

[0185] In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered. To achieve this, one would administer to an animal, or human patient, an immunologically effective amount of activated lymphocytes in combination with an adjuvant-incorporated antigenic peptide composition as described herein. The activated lymphocytes will most preferably be the patient's own cells that were earlier isolated from a blood or tumor sample and activated (or “expanded”) in vitro. This form of immunotherapy has produced several cases of regression of melanoma and renal carcinoma, but the percentage of responders were few compared to those who did not respond. More recently, higher response rates have been observed when such adoptive immune cellular therapies have incorporated genetically engineered T cells that express chimeric antigen receptors (CAR) termed CAR T cell therapy. Similarly, natural killer cells both autologous and allogenic have been isolated, expanded and genetically modified to express receptors or ligands to facilitate their binding and killing of tumor cells.

[0186] The compositions of the present disclosure may be prepared in a manner known in the art and are those suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.

[0187] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers and solutes, which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents and the like. It willbe understood that compositions of the disclosure may also include other supplementary physiologically active agents.

[0188] The carrier must be pharmaceutically ‘’acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous and intradermal administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include preparing the carrier for association with the cells. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers.

[0189] In an embodiment, the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.Compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes, which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0190] TREATMENT OF AUTOIMMUNE DISEASES

[0191] The methods of this disclosure can be applied to various autoimmune or immune-related diseases or conditions, for example to treat such diseases or conditions. Autoimmune diseases include diseases that affect organs such as the heart, kidney, liver, lung, reproductive organs, digestive system, or skin. Autoimmune diseases include diseases that affect glands, including the endocrine, adrenal, thyroid, salivary and exocrine glands, and the pancreas. Autoimmune diseases can also be multi-glandular. Autoimmune diseases can target one or more tissues, for example connective tissue, muscle, or blood. Autoimmune diseases can target the nervous system or eyes, ears or vascular system. Autoimmune diseases can also be systemic, affecting multiple organs, tissues and / or systems. In some embodiments, an autoimmune disease or condition is an inflammatory disease or condition.

[0192] Non-limiting examples of autoimmune or immune-related diseases or conditions include inflammation, antiphospholipid syndrome, systemic lupus erythematosus, rheumatoid arthritis, autoimmune vasculitis, celiac disease, autoimmune thyroiditis, posttransfusion immunization, maternal-fetal incompatibility, transfusion reactions, immunological deficiency such IgA deficiency, common variable immunodeficiency, drug-induced lupus, diabetes mellitus, Type I diabetes, Type II diabetes, juvenile onset diabetes, juvenile rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, immunodeficiency, allergies, asthma, psoriasis, atopic dermatitis, allergic contact dermatitis, chronic skin diseases, amyotrophic lateral sclerosis, chemotherapy-induced injury, graft-vs-host diseases, bone marrow transplant rejection, / Xnkylosing spondylitis, atopic eczema, Pemphigus, Behcet's disease, chronic fatigue syndrome fibromyalgia, chemotherapy -induced injury, myasthenia gravis, glomerulonephritis, allergic retinitis, systemic sclerosis, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus including chilblain lupus erythematosus, Sjogren's syndrome, autoimmune nephritis, autoimmune vasculitis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune mediated hematological diseases, Ic-SSc (limited cutaneous form of scleroderma), dc-SSc (diffused cutaneous form of scleroderma), autoimmune thyroiditis (AT), Grave's disease (GD), myasthenia gravis, multiple sclerosis (MS), ankylosing spondylitis, transplant rejection, immune aging, rheumatic / autoimmune diseases, mixed connective tissue disease, spondyloarthropathy, psoriasis, psoriatic arthritis, myositis, scleroderma, dermatomyositis, autoimmune vasculitis, mixed connective tissue disease, idiopathic thrombocytopenic purpura, Crohn's disease, human adjuvant disease, osteoarthritis, juvenile chronic arthritis, a spondyloarthropathy, an idiopathic inflammatory myopathy, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, immune-mediated renal disease, a demyelinating disease of the central or peripheral nervous system, idiopathic demyelinating polyneuropathy, Guillain-Barre syndrome, a chronic inflammatory demyelinating polyneuropathy, a hepatobiliary disease, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, Whipple's disease, an autoimmune or immune-mediated skin disease, a bullous skin disease, erythema multiforme, allergic rhinitis, atopic dermatitis, food hypersensitivity, urticaria, an immunologic disease of the lung, eosinophilic pneumonias, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, a transplantation associated disease, graft rejection or graft-versus-host-disease, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, conditions involving infiltration of T cells and chronic inflammatory responses,atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis, vasculitis (including ANCA), aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRC A), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, central nervous system (CNS) inflammatory' disorders, multiple organ injury' syndrome, mysathenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Stevens-Johnson syndrome, pemphigoid bullous, pemphigus, autoimmune polyendocrinopathies, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), Sheehan's syndrome, autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, Celiac sprue (gluten enteropathy), cryoglobulinemia, and amyotrophic lateral sclerosis (ALS).

[0193] METHODS OF TREATMENT OF C NCER

[0194] In some embodiments, described herein is a method of treating a proliferative disease or condition in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the pharmaceutical compositions comprising the low' dose IL-2 formulations embodied herein.

[0195] In certain embodiments, the low dose IL-2 in the formulations embodied herein, interacts with the IL-2 receptor to stimulate and / or enhance expansion of CD4 helper cells, CD8+effector naive and memory T cells, NK cells, and / or NKT cells. In additional cases, the expansion of Teff cells skews the TeffTreg ratio toward the Teff population which target tumor cells or cells infected with a pathogen, e.g. a virus.

[0196] In some embodiments, the proliferative disease or condition is a cancer. In some cases, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the solid tumor is a metastatic cancer. In some cases, the solid tumor is a relapsed or refractory cancer. In some cases, the solid tumor is castrate-resistant prostate cancer, metastatic castrate-resistant prostate cancer, or metastatic castrate-resistant prostate cancer having DNA damage response (DDR) defects.

[0197] In some embodiments, the pharmaceutical composition comprising the low dose IL-2 formulations embodied herein, is administered to a subject for the treatment of a metastatic cancer. In some instances, the metastatic cancer comprises a metastatic bladder cancer, metastatic bone cancer, metastatic brain cancer, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic eye cancer, metastatic head and neck cancer, metastatic kidney cancer, metastatic lung cancer, metastatic melanoma, metastatic ovarian cancer, metastatic pancreatic cancer, or metastatic prostate cancer. In some cases, the pharmaceutical composition comprising the low dose IL-2 formulations embodied herein, is administered to a subject for the treatment of metastatic bladder cancer, metastatic bone cancer, metastatic brain cancer, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic eye cancer, metastatic head and neck cancer, metastatic kidney cancer, metastatic lung cancer, metastatic melanoma, metastatic ovarian cancer, metastatic pancreatic cancer, or metastatic prostate cancer. In some cases, the pharmaceutical composition comprising the low dose IL-2 formulations embodied herein, is administered to a subject for the treatment of castrate-resistant prostate cancer, metastatic castrate-resistant prostate cancer, or metastatic castrate-resistant prostate cancer having DNA damage response (DDR) defects.

[0018] In some instances, the pharmaceutical composition comprising the low dose IL-2 formulations embodied herein, is administered to a subject for the treatment of a relapsed or refractory cancer. In some instances, the relapsed or refractory cancer comprises arelapsed or refractory bladder cancer, relapsed or refractory bone cancer, relapsed or refractory' brain cancer, relapsed or refractory breast cancer, relapsed or refractory colorectal cancer, relapsed or refractory esophageal cancer, relapsed or refractory eye cancer, relapsed or refractory head and neck cancer, relapsed or refractory kidney cancer, relapsed or refractory lung cancer, relapsed or refractory' melanoma, relapsed or refractory ovarian cancer, relapsed or refractory pancreatic cancer, or relapsed or refractory' prostate cancer. In some cases, the pharmaceutical composition comprising the low dose IL-2 formulations embodied herein, is administered to a subject for the treatment of a relapsed or refractory bladder cancer, relapsed or refractory bone cancer, relapsed or refractory brain cancer, relapsed or refractory breast cancer, relapsed or refractory colorectal cancer, relapsed or refractory' esophageal cancer, relapsed or refractory' eye cancer, relapsed or refractory' head and neck cancer, relapsed or refractory kidney cancer, relapsed or refractory’ lung cancer, relapsed or refractory melanoma, relapsed or refractory ovarian cancer, relapsed or refractory pancreatic cancer, or relapsed or refractory' prostate cancer.

[0199] In some embodiments, the cancer is a treatment-naive cancer. In such cases, the treatment-naive cancer is a cancer that has not been treated by a therapy. In some cases, the treatment-naive cancer is a solid tumor, such as bladder cancer, a bone cancer, a brain cancer, a breast cancer, a colorectal cancer, an esophageal cancer, an eye cancer, a head and neck cancer, a kidney cancer, a lung cancer, a melanoma, an ovarian cancer, a pancreatic cancer, or a prostate cancer. In some embodiments, described herein is a method of treating a treatment-naive solid tumor in a subject in need thereof which comprises administering to the subject the pharmaceutical composition comprising the low dose IL-2 formulations embodied herein.

[0200] In some embodiments, the cancer is a hematologic malignancy. In some instances, the hematologic malignancy comprises a leukemia, a lymphoma, or a myeloma. In some cases, the hematologic malignancy is a T-cell malignancy. In other cases, the hematological malignancy is a B-cell malignancy. Exemplary' hematologic malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary' mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cellprolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0201] IMMUNE DEFICIENCIES

[0202] Immune deficiency diseases are recognized by the World Health Organization. These diseases are characterized by an intrinsic defect in the immune system in which, in some cases, the body is unable to produce any enough, or effective antibodies against infection. In other cases, cellular defenses to fight infection fail to work properly. Typically, primary immune deficiencies are inherited disorders. Patients with inherited immune deficiencies such as adenosine deaminase deficient (ADA)-severe combined immunodeficiency (SCID), X-linked SCID, chronic granulomatous disease (CGD), Wiskott- Aldrich Syndrome, and Fanconi anemia (FA) can benefit from gene therapy, which provides a functioning gene to an affected patient to compensate for the defective one.

[0203] Secondary, or acquired, immune deficiencies are not the result of inherited genetic abnormalities but rather occur in individuals in which the immune system is compromised by factors outside the immune system. Examples include trauma, viruses, chemotherapy, toxins, and pollution. Acquired immunodeficiency syndrome (AIDS) is an example of a secondary immune deficiency disorder caused by a virus, the human immunodeficiency virus (HIV), in which a depletion of T lymphocytes renders the body unable to fight infection. Patients with secondary immune deficiencies can also benefit from genetic therapies.

[0204] The compositions can be used in combination with one or more gene therapies related to primary immune deficiency diseases (PIDDS). These include, for example, Autoimmune Lymphoproliferative Syndrome (ALPS), APS-1 (APEC ED), CARD9, Chronic Granulomatous Disease (CGD), Congenital Neutropenia Syndromes, Common Variable Immunodeficiency (CVID), CTLA4 Deficiency, DOCK8 Deficiency, Glycosylation Disorders with Immunodeficiency, Hyper-Immunoglobulin E Syndromes (HIES), PI3 Kinase Disease, PLAID, Severe Combined Immunodeficiency (SCID), STAT3 Dominant-Negative Disease, WHIM Syndrome, X-Linked Agammaglobulinemia (XLA), X-Linked Lymphoproliferative Disease (XLP), Wiscott-Aldrich syndrome, DiGeorge syndrome, Ataxia-telangectasia, Chronic granulomatous disease, Transient hypogammaglobulinemia of infancy, Agammaglobulinemia, Complement deficiencies, Selective IgA deficiency.

[0205] X-linked severe combined immunodeficiency (SCID-X1) is both a cellular and humoral immune depletion caused by mutations in the common gamma chain gene (yC), which result in the absence of T and natural killer (NK) lymphocytes and the presence of nonfunctional B lymphocytes. SCID-X1 is fatal in the first two years of life unless the immune system is reconstituted, for example, through bone marrow transplant (BMT) or gene therapy. Administration of a therapeutic gene, for example, by an expression vector encoding a therapeutic protein, can be combined with the low dose IL-2 formulations embodied herein. Particular examples of therapeutic genes and / or gene products to treat immune deficiencies associated with severe combined immunodeficiency (SCID) include yC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFXS, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, and SLC46A1;FANC family genes including FancA, FancB, FancC, FancDl (BRCA2), FancD2, FancE, FancF, FancG, Fanci, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3); soluble CD40; CTLA; Fas L; antibodies to CD4, CD5, CD7, CD52; antibodies to IL1, IL2, IL6; an antibody to TCR specifically present on autoreactive T cells; IL4; IL10; IL 12; IL13; ILIRa, sILlRI, sILlRII; sTNFRI; sTNFRII; antibodies to TNF; P53, PTPN22, and DRBl*1501 / DQB 1*0602; globin family genes; WAS; phox; dystrophin; pyruvate kinase; CLN3; ABCD1; arylsulfatase A; SFTPB; SFTPC; NLX2.1; ABCA3; GATAI; ribosomal protein genes; TERT; TERC; DKCI; TINF2; CFTR; LRRK2; PARK2; PARK7; PINK1; SNCA; PSEN1; PSEN2; APP; SOD1; TDP43; FUS; ubiquilin 2; and / or C9ORF72.

[0206] Because most individuals lack a matched donor for BMT or non-autologous gene therapy, haploidentical parental bone marrow depleted of mature T cells is often used; however, complications include graft versus host disease (GVHD), failure to make adequate antibodies hence requiring long-term immunoglobulin replacement, late loss of T cells due to failure to engraft hematopoietic stem and progenitor cells (HSPCs), chronic warts, and lymphocyte dy sregulation.

[0207] Fanconi anemia (FA) is an inherited blood disorder that leads to bone marrow failure. It is characterized, in part, by a deficient DNA-repair mechanism. At least 20% of patients with FA develop cancers such as acute myeloid leukemias, and cancers of the skin, liver, gastrointestinal tract, and gynecological systems. The skin and gastrointestinal tumorsare usually squamous cell carcinomas. The average age of patients who develop cancer is 15 years for leukemia, 16 years for liver tumors, and 23 years for other tumors.

[0208] Cells from FA patients display a characteristic hypersensitivity to agents that produce interstrand DNA crosslinks such as mitomycin C or diepoxybutane. FA genes define a multicomponent pathway involved in cellular responses to DNA cross-links. Five of the FA genes (FANC A, FANCC, FANCE, FANCF and FANCG) have been cloned and the FANCA, FANCC and FANCG proteins have been shown to form a molecular complex with primarily nuclear localization. A number of mutations in the FANCC gene have been identified which are correlated with FA of differing degrees of severity.

[0209] An alternative therapeutic approach to BMT and non-autologous gene therapy in immune and blood disorder failures is ex vivo HSPC gene therapy, where blood or bone marrow derived HSPCs are enriched from patients, transduced with viral vectors to deliver a functional therapeutic gene (e.g., a yC gene for SCID-X1 or a FancA gene for FA), and transplanted back to the patient. The first generation ex vivo gene therapy for SCID-X1 used murine leukemia virus-based gamma retroviral (RV) delivery' and showed significant longterm clinical improvement in treated patients. However, 5 / 20 patients unexpectedly developed T cell leukemia, resulting in the death of one patient. These findings precipitated intense interest in utilization of self-inactivating (SIN) viral vectors and SIN-lentiviral vectors (LV) as alternative vector platforms. While SIN-RV and SIN-LV are currently used in the clinical setting with considerable success, ex vivo gene therapy still faces multiple challenges that include the: 1) extensive ex vivo manipulation of HSPCs required to prepare them for therapeutic use that results in loss of multipotency potential and / or reduced fitness for engraftment following transplantation, 2) various conditioning regimens used to enhance engraftment of gene modified HSPCs add considerable genotoxic risks to patients, and 3) requirement of advanced infrastructures for the collection, culture, transduction, validation, and re-infusion of HSPCs, consequently restricting this form of treatment to a select few institutions worldwide.

[0210] Accordingly, compositions and methods of use include administering to a subject the low dose IL-2 formulations embodied herein. In certain embodiments, the IL-2 therapy is conducted in combination with one or more gene therapies, e.g. expression of a therapeutic agent or a wild-type protein in cases where the expression of that particular protein is the cause of the disease. The method further comprises administration of mobilization factors such as chemokines, G-CSF, granulocyte macrophage colonystimulating factor (GM-CSF), AMD3100 and SCF. G-CSF is a cytokine whose functions in HSPC mobilization can include the promotion of granulocyte expansion and both proteasedependent and independent attenuation of adhesion molecules and disruption of the SDF-1 / CXCR4 axis. Any commercially available form of G-CSF known to one of ordinary' skill in the art can be used in the methods and formulations as disclosed herein, for example, Filgrastim (NEUPOGEN™, Amgen Inc., Thousand Oaks, Calif.) and PEGylated Filgrastim (Pegfilgrastim, NEULASTA™, Amgen Inc., Thousand Oaks, Calif.).

[0211] AMD3100 (MOZOBIL™, PLERIXAFOR™; Sanofi-Aventis, Paris, France), a synthetic organic molecule of the bicyclam class, is a chemokine receptor antagonist and reversibly inhibits SDF-1 binding to CXCR4, promoting HSPC mobilization. AMD3100 is approved to be used in combination with G-CSF for HSPC mobilization in patients with myeloma and lymphoma.

[0212] In certain embodiments, the low dose IL-2 formulations embodied herein can be combined with other cytokines, proteins and the like. See, for example, Colombo, F. et al. Combined IISV-TK / IL-2 gene therapy in patients with recurrent glioblastoma multiforme: biological and clinical results. Cancer Gene Ther 12, 835-848 (2005). doi.org / 10.1038 / sj.cgt.7700851. Robert E. Sobol etal. Interleukin 2 Gene Therapy of Colorectal Carcinoma with Autologous Irradiated Tumor Cells and Genetically Engineered Fibroblasts: A Phase I Study. Clin Cancer Res September 1, 1999 (5) (9) 2359-2365. Philip A. Durost, et al. Human Gene Therapy. Mar 2018.352-365. http: / / doi.org / 10.1089 / hum.2017.072. Kevin S. Goudy etal. Inducible Adeno-Associated Virus-Mediated IL-2 Gene Therapy Prevents Autoimmune Diabetes. The Journal of Immunology’ March 15, 2011, 186 (6) 3779-3786; DOI:10.4049 / jimmunol.1001422. Mahzad Akbarpour et al.. Insulin B chain 9-23 gene transfer to hepatocytes protects from type 1 diabetes by inducing Ag-specific FoxP3;Tregs. Science Translational Medicine. 7, 289ra81 (2015). DOI: 10.1126 / scitranslmed.aaa3032.

[0213] Methods for Isolation of Cells

[0214] Any number of methods known in the art can be used to isolate cells, such as T cells, or any other cell type that may be used in carrying out the treatment of a subject. Thus, also provided are various other genetically engineered cells expressing the chimeric antigen receptors e.g., CARs. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g. myeloid or lymphoid cells, includinglymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+cells, CD8+cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and / or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.

[0215] Among the sub-types and subpopulations of T cells and / or of CD4+and / or of CD8+T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as THI cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T (αβ T) cells, and delta / gamma T (γδ T) cells.

[0216] In certain embodiments, the T cell is a mammalian regulatory T cell (Treg), wherein the Treg cell is CD4+, CD25+, CD127-, FOXP3+and / or Helios+ / -. In other embodiments, the T cell is a mammalian regulatory T cell (Treg), wherein the Treg cell is CD4+, CD25+, CD127-, and / or FOXP3+

[0217] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0218] In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normallynot present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0219] In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for introduction of the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered.

[0220] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0221] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary' samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom.Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0222] In some embodiments, the cells are derived from cell lines, e g,, T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig.

[0223] In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity -based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0224] KITS

[0225] The disclosure provides kits for the treatment or prevention of neoplasia. In one embodiment, the kit includes a therapeutic or prophylactic composition containing a therapeutically effective amount of IL-2, in unit dosage form and one or more therapeutic agents. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0226] If desired the IL-2 and one or more therapeutic agents are provided together with instructions for administering the IL-2 and one or more therapeutic agents to a subject in need of such therapy. The instructions will generally include information about the use of the composition for the treatment or prevention of the particular disease or disorder, e.g. an autoimmune disease. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0227] The following examples further illustrate the invention. These examples are not intended to limit the invention in any manner.EXAMPLES

[0228] EXAMPLE 1: FORMULATION STABILITY ASSESSMENT

[0229] The aim of this study was to assess the stability of recombinant human (rHu) IL-2 in formulations to be used in clinical trials. This study assesses accelerated stability of liquid and lyophilized formulations in vials containing different concentrations of SDS.

[0230] The formulations assessed in this study are:

[0231] Table 1. Liquid formulationsComponent rHu IL-2 and excipients in mg / mL Formulation Formulation Formulation Formulation LI L2 L3L4 rHu IL-2 0.092 (1 MIU) 0.092 (1 MIU) 0.072 (1 MIU) 0.369 (4 MIU) SDS 1.00 0.38 0.100 1.52 Sodium phosphate monobasic di hydrate 0.26 0.26 0.26 0.26Sodium phosphate dibasic anhydrous 1.19 1.19 1.19 1.19Mannitol 50.0 50.0 50.0 50.0 Water for Injection q.s. 1 mL q.s. 1 mL q.s. 1 mL q.s, 1 mL pH 7.0 - 8.0 7.0 - 8.0 7.0 - 8.0 7.0 - 8.0 SDS / rHu IL-2 mass ratio 10.9 4.1 1.4 4.1 Injection volume (dose) 1 mL 1 mL ImL 0.25 mL Current DP formulation was modified by targeting IL-2 protein mass to 0.1 mg / mL (0.085 - 0.115 mg / mL) instead of a formulation obtained by biological units with a target of 1 MIU / mL (and the resulting mass of IL-2 to achieve that potency).

[0232] Table 2. Lyophilized formulation (composition of 1 mL after reconstitution with 1.2 mL of water for injection)Component Formulation SIrHu IL-2 0,092 mgSDS 1.00Sodium phosphate monobasic di hydrate 0.26Sodium phosphate dibasic anhydrous 1.19Mannitol 50.0Water for Injection q.s. 1 mLpH 7.0 - 8.0Injection volume (dose) 1 mL

[0233] MATERIALS AND METHODS

[0234] The materials and methods used for the manufacturing of the development batches are recorded in the development batch records:

[0235] Formulation LI: batch PR1-A1; Formulation L2: batch PR2-A1, Formulation L3: batch PR3-A1, Formulation L4: batch PR4-A1, Formulation SI (555706750): batch 01.

[0236] Stability tests were run in controlled temperature stability chambers at 2 - 8 °C (long term condition), 25 °C / 60 % RH (accelerated condition) and 40 °C / 75 % RH for 4 weeks (stress condition) for formulation selection.

[0237] Two candidate formulations were selected after the 4-week intensive timepoint study: LI and L4. Stability runs continued until 3 months for both formulations at 2 - 8 °C, 25 °C / 60 % and 40 °C / 75 % RH and until 6 months at 2 - 8 °C for L1, which was the most stable of all candidate formulations.

[0238] Analytical methods used are listed below:Time 0 only

[0239] RESULTS

[0240] Selection of the candidate formulation for further stability testing

[0241] The results obtained during the first 4 weeks for each parameter of the five candidate formulations are described below.

[0242] Physical attributes

[0243] Both appearance and osmolality remained within expected values with minor variations in all liquid formulations at all temperatures.

[0244] pH— value

[0245] As expected, the phosphate buffer kept pH well within specifications in all formulations at all temperatures.

[0246] Protein concentration

[0247] Figure 1 summarizes data at all temperatures for the five candidate formulations. L4 has a different protein concentration, because it is meant to be administered in smaller volumes than the rest of them. At 2 - 8 °C and 25 °C / 60 % RH no formulation showed significant change in protein concentration. At 40 °C / 75 % RH all formulations showed a continuous slight decreasing trend in protein concentration.

[0248] SDS concentration

[0249] As expected, SDS concentration remains practically unchanged in all formulations at all testing timepoints.

[0250] Relative potency (in % of nominal biological activity: 1 MIU / mL for L1, L2, L3 and S1; 4 MIU / mL for L4)

[0251] Figure 2 summarizes data at all temperatures for the five candidate formulations. No trend in % relative potency was observed for formulations LI, L2, L4 and SI. The variation observed is most probably attributable to the analytical dispersion of the method and not to storage time. A trend in potency is observed for L3 at 40 °C (ca. 50% drop).

[0252] Purity by RPHPLC

[0253] Figure 3 shows the purity (% main peak area) by RP HPLC at 2-8 °C, 25°C / 60% RH and 40°C / 75 % RH after 0, 1, 2 and 4 weeks.

[0254] Pre-peaks expressed as % total chromatographic area (RP HPLC)

[0255] Figure 4 summarizes data at all temperatures for the five candidate formulations. In liquid formulations % total pre-peak area remains practically unchanged at 2 - 8 °C, increases slightly at 25 °C / 60 % RH and increases significantly at 40 °C / 75 % RH. The most stable formulation is LI followed by L4. These are the formulations with the highest concentration of SDS The lyophilized formulation shows much higher degradation from time zero. In case a lyophilized formulation was pursued in the future, composition or process changes should be made to improve stability during the lyophilization cycle.

[0256] Post-peaks (RP HPLC)

[0257] Figure 5 summarizes data at all temperatures for the five candidate formulations. In liquid formulations the % total post-peak area tends to decrease with time at all storage temperatures. This may be explained because these degradations products are further degraded and appear as pre-peaks (oxidized forms). The lyophilized formulation shows much higher degradation from time zero.

[0258] Non reducing SDS-PAGE with densitometric measurement of band areas

[0259] Figure 6 summarizes the densitometric % area of the main band in SDS PAGE runs. This method measures the degree of oligomerization of rHu-IL2 via the decrease in the % densitometric area of the drug substance (main band). At 2 - 8°C: no decrease in purity was observed for all formulations. At 25°C: no decrease in purity for formulations L1 and L4. Decrease in purity for the other formulations: S1 > L3 > L2. At 40°C: decrease in purity:L3 > S1 > L2 > L4 > L1.

[0260] Reducing SDS — PAGE with densitometric measurement of band areas

[0261] Figure 7 summarizes the densitometric % area of the main band in SDS P AGE runs. This analytical technique may identify whether oligomers already detected by non-reducing SDS PAGE are formed by units of rHu IL-2 linked via disulfide bonds. In this case, bands of these oligomers would co-elute with the parent drug due to reduction of the disulfide linkage with 2-mercaptoethanol pre-treatment. Otherwise, oligomers would remain at the same Rf as in the non-reducing electropherogram.

[0262] As can be observed in Figure 8D, in the case of the lyophilized formulation SI, these oligomers are compatible with dimers and tetramers of IL-2 (apparent molecular weights: monomer ca. 14kDa; dimer ca. 28 kDa and tetramer ca. 56 kDa). Also, in the case of SI, these related impurities seem to be non-reducible (at least under the reducing conditions applied). No significant differences were observed between reducing and non-reducing conditions for impurities after the lyophilization process at 40°C after 4 weeks.

[0263] On the other hand, in the case of the L3 liquid formulation, with the lowest SDS content (Figure 8C), a partially different behavior is observed for the higher molecul r weight impurities after 4 weeks at 40°C. In this case, most of the impurities were not present under reducing conditions. This behavior would be compatible with aggregates linked by disulfide bonds.

[0264] Related impurities of lower molecular weight than IL-2 were only present in liquid formulations (see Figure 8B and 8C for LI and L3 at 40°C after 4 weeks respectively). This degradation pathway might require certain mobility and proximity that would be minimal in the solid structure of the lyophilized formulation. It might be caused by minimal amounts of residual proteolytic enzymes.

[0265] As can be seen, neither higher molecular weight nor lower molecular weight degradation was present after 4 weeks at 2-8°C for LI (Figure 8 A).

[0266] Subvisible particles

[0267] All samples passed the criteria. Subvisible particles may come from the environment during filling, from the primary package or from protein precipitation. The first two sources were present from time zero and may vary broadly from vial to vial. This could explain some differences among the data for all formulations.

[0268] Additional stability data of LI and L4 formulations

[0269] The most stable formulations selected to continue with the assessment study were LI and LA. They were studied for 2 and 3 months in accelerated and stress conditions. LI was finally selected and studied for 3 and 6 months at 2 - 8 °C and L4 was only tested after 3 months at 2 - 8 °C. At these timepoints the analysis included protein concentration, % relative potency, purity by RP HPLC (main band, pre-peaks and post-peaks) and purity by SDS PAGE in reducing and non-reducing conditions. The stability data are summarized in the mentioned order in Figures 9, 10, 11, 12, 13, 14 and 15.

[0270] Table 3: Current Proposed Specifications for IL-2 Drug Product.

[0271] LI remained within these specifications for at least 4 weeks at 25 °C / 60 % RH and 1 week at 40 °C / 75 % RH. Current DP formulation was modified by targeting IL-2 protein mass to 0.1 mg / mL (0.085 - 0.115 mg / mL) instead of a formulation obtained by biological units with a target of 1 MIU / mL (and the resulting mass of IL-2 to achieve that potency).

[0272] Solution LI was considered stable for at least 6 months at 2 - 8 °C with the following specifications (Table 3, stability indicating parameters studied up to 6 months are underlined in gray).

[0273] EXAMPLE 2: FORMULATIONS WITH SDS / IL-2 MASS RATIO OF 10 AND ABOVE ARE STABLE AT LEAST 1 YEAR AT 2 - 8 °C

[0274] The following stability data obtained on another lot of formulation LI confirms that this formulation is suitable for pharmaceutical commercial use because it has at least 1 year stability in refrigerated conditions (2 - 8 °C).

[0275] See Table below. Moreover, excursions up to 25 °C and 40 °C during transport are acceptable, thus rendering the formulation robust for distribution.Table 5: STORAGE: 25 ± 2 °CTable 6: STORAGE: 40 ± 2 °C

[0276] DISCUSSION

[0277] The 4-week data clearly showed that the most stable formulations were LI and L4, particularly considering the pre-peaks in RP HPLC and the non-reducing SDS PAGE results at 40 °C / 75 %RH. The main degradation pathway is the one involving pre-peaks ofRP HPLC, most probably due to oxidation.

[0278] The post-peaks in RP HPLC may be connected to deamidated and acetylated forms already present at time 0. Post-peaks area decreases with time at higher temperatures possibly due to further degradation to oxidized forms.

[0279] Lyophilization in the tested conditions appears to destabilize the protein at this low concentration.

[0280] Data from LI and L4 after longer periods of storage time confirm their stability at 2 - 8 °C.

[0281] EXAMPLE 3: BIOPHYSICAL CHARACTERIZATION AND DIFFERENTIATION OF LIQUID RHIL-2 FORMULATIONS FROM SDS / I L-2 MICROAGGREGATES

[0282] This example demonstrates that the low-dose liquid IL-2 compositions of the present invention are structurally distinct from the microaggregated forms of IL-2 found in existing commercial products, such as PROLEUKIN® (Aldesleukin).

[0283] Commercial rhIL-2 (Aldesleukin) is known to exist as biologically active, non-covalently bound microaggregates with an average size of approximately 27 recombinant IL-2 molecules. These microaggregates typically exhibit a mean hydrodynamic diameter between 8 nm and 20 nm. In contrast, the formulations described herein aim to maintain rhIL-2 in a substantially monomeric state, free from such microaggregates.

[0284] Methods

[0285] Particle size distribution and biophysical properties were compared between a representative rhIL-2 active formulation and a corresponding placebo.

[0286] Active Sample (Batch B0401 / IL2040): rhIL-2 (0.1--mg / mL and 1.95 mg / mL respectively) in 50 mg / mL Mannitol, 1.19 mg / mL Na₂HPO₄, 0.26 mg / mL NaH₂PO₄, and 0.1% sodium dodecyl sulfate (SDS), pH 7,5.

[0287] Placebo Sample (Batch A0402): Identical composition to the active sample but lacking the rhIL-2 protein.

[0288] Drug Product (Batch B0401): rhIL-2 (0.1~mg / mL) in 50 mg / mL Mannitol, 1.19 mg / mL Na₂HPO₄, 0.26 mg / mL NaH₂PO₄, and 0.1% sodium dodecyl sulfate (SDS), pH 7.5.

[0289] Drug Substance (DS) Sample (Batch IL2040): rhIL-2 (1.95 mg / mL) in 50 mg / mL Mannitol, 1.19 mg / mL Na2HPO4, 0.26 mg / mL NaH2PO4, and 0.5% sodium dodecyl sulfate (SDS), pH 7.5. Used for NMR DOSY and Far-UV CD analyses. This sample utilized a different formulation consisting of rhIL-2 at 1.95 mg / mL This sample was diluted in phosphate buffer 1 / 10.

[0290] The SDS:IL-2 ratio of formulations described herein aim to maintain rhIL-2 in a substantially monomeric state, free from such microaggregates.

[0291] Analyses included:

[0292] Dynamic Light Scattering (DLS): Performed using a BetterSize 180 Pro to analyze particle populations by intensity, volume, and number.

[0293] NMR Diffusion Ordered Spectroscopy (DOSY): Performed at 600 MHz using dioxane as an internal standard to determine the Hydrodynamic Radius (RH).

[0294] Circular Dichroism (CD): Far-UV CD (190-240 nm) was used to quantify secondary structure content using the BeStSel server.

[0295] Structural Analysis Results

[0296] Differentiation via DLS

[0297] The DLS analysis revealed clear structural differences between the active formulation and known microaggregated IL-2 products (see figure 17):

[0298] Absence of 10-20 nm Microaggregates: Neither the active nor the placebo samples showed significant populations in the 8 nm to 20 nm range characteristic of SDS / IL-2 microaggregates found m commercial formulations.100299] Monomeric Protein Population: In the active sample (B0401 ), volume and number profiles identified a significant population with a hydrodynamic size of approximately 2 nm. This size is compatible with the hydrodynamic radius of a fully folded 132-residue protein.

[0300] Molecular SDS Population: Both samples exhibited a peak at values less than 1 nm, corresponding to molecular-sized species (e.g., SDS present below its critical micellar concentration).85

[0301] Placebo Verification: The placebo sample (A0402) showed the molecular peak (< 1 nm) but completely lacked the 2 nm peak, confirming that the 2 nm population in the active sample is attributable solely to rhIL-2.

[0302] Validation via NMR DOSY

[0303] To provide independent validation of the monomeric status, the Hydrodynamic Radius (RH) was determined via NMR DOSY (see regression lines in FIG. 18 and explanation in paragraph 00113):

[0304] Measured RH: The experimental value was calculated to be 20.41 ± 0.55 A (approximately 2.04 nm),

[0305] Theoretical Comparison: This value closely matches the theoretical RH of 19.7 A for a fully folded 132-residue protein.

[0306] Secondary Structure via Circular Dichroism

[0307] The Far-UV CD spectrum of the rhIL-2 solution confirmed a properly folded protein (see FIG. 19):

[0308] Secondary Structure Content: Deconvolution of the spectrum indicated α-helix content of 45.2%.

[0309] Folding Status: This value is consistent with the predicted value of 51.5% (Jpred 4 server) and previously reported values for properly folded rhIL-2.

[0310] Conclusion

[0311] The biophysical data confirms that the liquid rhIL-2 compositions of the present invention do not form the 8-20 nm microaggregates typical of current commercial formulations. Instead, the rhIL-2 remains in a folded, compact, monomeric status (ca. 2 nm), distinctly different from the microaggregated preparations described previously.OTHER EMBODIMENTS

[0312] While the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0313] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or86unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.87

Claims

What is claimed:

1. A low dose Interleukin (IL)-2 pharmaceutical composition comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 8 to about 20, a non-ionic isotonizing agent and a buffer, wherein the pharmaceutical composition is a liquid and is substantially devoid of SDS / IL-2 microaggregates.

2. The pharmaceutical composition of claim 1, wherein the SDS to IL-2 mass ratio ranges from about 8 to about 18.

3. The pharmaceutical composition of claim 1, wherein the SDS to IL-2 mass ratio ranges from about 8 to about 11.

4. The pharmaceutical composition of claim 1, wherein the SDS to IL-2 mass ratio ranges from about 10 to about 12.

5. The pharmaceutical composition of claim 1, wherein the SDS to IL-2 mass ratio is about 11.

6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises IL-2 ranging from about 0.0001 mg / ml to about 4 mg / ml.

7. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises SDS ranging from about 0.0004 mg / ml to about 150 mg / ml.

8. The pharmaceutical composition of claim 1, wherein the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose.

9. The pharmaceutical composition of claim 1, wherein the buffer is a phosphate buffer.

10. The pharmaceutical composition of claim 1, wherein the buffer is a pH 7-8 phosphate buffer.

11. The pharmaceutical composition of claim 1, wherein the IL-2 has a specific activity of between about 4 MIU / mg to about 30 MIU / mg.8812. The pharmaceutical composition of claim 1, wherein the IL-2 has a specific activity of between about 7 MIU / mg to about 20 MIU / mg.

13. The pharmaceutical composition of claim 1, wherein the IL-2 remains stable in the pharmaceutical liquid composition for at least a year as measured by an in vitro biological activity assay.

14. The pharmaceutical composition of claim 1, wherein the IL-2 is substantially free and not bound to SDS.

15. A method of inducing proliferation of regulatory T cells in vivo, comprising administering to a subject in need thereof, a therapeutically effective amount of a liquid pharmaceutical composition comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 8 to about 20, a non-ionic isotonizing agent and a buffer, wherein the pharmaceutical composition is a liquid and is substantially devoid of SDS / IL-2 microaggregates, and wherein the regulatory T cells increase as compared to a control, inducing proliferation of regulatory T cells in vivo.

16. The method of claim 15, wherein the non-ionic isotonizing agent comprises mannitol, sorbitol, xylitol, sucrose or trehalose and / or a pH 7-8 phosphate buffer.

17. The method of claim 15, wherein the buffer is a phosphate buffer.

18. The method of claim 15, wherein the buffer is a pH 7-8 phosphate buffer.

19. The method of claim 15, wherein the IL-2 has a specific activity of between about 7 MIU / mg to about 30 MIU / mg.

20. The method of claim 15, wherein the IL-2 has a specific activity of between about 7 MIU / mg to about 20 MIU / mg.

21. The method of claim 15, wherein the IL -2 has a stable specific activity, at least for a year in the liquid composition, as measured in an animal model of human cancer.22, The method of claim 15, wherein the SDS to IL-2 mass ratio ranges from about 10 to 128923. A method of treating an autoimmune disease or disorder in a subject in need thereof, comprising: a therapeutically effective amount of a liquid pharmaceutical composition comprising a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 8 to about 20, a non-ionic isotonizing agent and a buffer, wherein the pharmaceutical composition is a liquid and is substantially devoid of SDS / IL-2 microaggregates and wherein the regulatory T cells increase as compared to a control, inducing proliferation of regulatory T cells in vivo.

24. The method of claim 23, wherein the osmolytes comprise mannitol, sorbitol, xylitol, sucrose, trehalose and / or a pH 7-8 phosphate buffer.

25. The method of claim 23, wherein buffer is a phosphate buffer.

26. The method of claim 23, wherein the buffer is a pH 7-8 phosphate buffer.

27. The method of claim 23, wherein the IL-2 has a specific activity of between about 7 MIU / mg to about 30 MIU / mg.

28. The method of claim 23, wherein the IL-2 has a specific activity of between about 7 MIU / mg to about 20 MIU / mg.

29. The method of claim 23, wherein the IL-2 has a stable specific activity, at least for a year in the liquid composition, as measured by an in vitro biological activity assay.

30. A pharmaceutical composition having a formulation comprising a therapeutically effective amount of a sodium dodecyl sulfate (SDS) to interleukin-2 (IL-2) mass ratio from about 10 to about 20, a non-ionic isotonizing agent and a buffer, wherein the pharmaceutical composition is a liquid and is substantially devoid of SDS / IL-2 microaggregates.

31. The pharmaceutical composition of claim 30, wherein the SDS to IL-2 mass ratio ranges from about 10 to about 12.

32. The pharmaceutical composition of claim 30, wherein the non-ionic isotonizing agent is mannitol.9033. The pharmaceutical composition of claim 30, wherein the buffer is a pH 7-8 phosphate buffer.91

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