Serum-free culture medium formulation for cell culture and method of use thereof

Abstract

To provide improved methods for culturing and producing immune cells, e.g., T cells, genetically engineered with recombinant receptors such as chimeric antigen receptors.SOLUTION: Provided herein is a serum-free media for culturing, e.g. cultivating, preparing and / or producing cells, such as immune cells, such as genetically engineered cells. Also provided is a liquid basal media and frozen supplements that can be used to produce serum-free media. The provided embodiments include methods for producing serum-free media and methods for culturing cells, e.g. activating, transducing, cultivating or expanding cells, in the presence of serum-free media.SELECTED DRAWING: Figure 1A

Claims

1. 0.5 mM to 5 mM L-glutamine in dipeptide form; 0.5 mM to 5 mM L-glutamine; IL-2, and one or both of IL-7 and IL-15; and Human serum albumin A serum-free medium containing, The serum-free medium does not contain serum. Serum-free culture medium.

2. The serum-free medium according to claim 1, wherein the dipeptide form of L-glutamine is L-alanyl-L-glutamine.

3. The serum-free medium according to claim 1 or 2, wherein the concentration of L-glutamine in dipeptide form is 1.0 mM to 3.0 mM.

4. The serum-free medium according to any one of claims 1 to 3, wherein the concentration of L-glutamine in the serum-free medium is 1.0 mM to 3.0 mM.

5. A serum-free medium according to any one of claims 1 to 4, wherein the concentration of L-glutamine in dipeptide form is 1.5 mM to 2.5 mM.

6. The serum-free medium according to any one of claims 1 to 5, wherein the concentration of L-glutamine in the serum-free medium is 1.5 mM to 2.5 mM.

7. A serum-free medium according to any one of claims 1 to 6, wherein the medium comprises Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle Basal Medium (BME), F-10, F-12, RPMI 1640, Glasgow Minimum Essential Medium (GMEM), Alpha Minimum Essential Medium (AlphaMEM), Iskov Modified Dulbecco's Medium, M199, or OpTmizer® CTS® T Cell Expansion and Proliferation Basal Medium.

8. The serum-free medium according to any one of claims 1 to 7, wherein the serum-free medium does not contain phenol red.

9. The serum-free medium according to any one of claims 1 to 8, wherein the serum-free medium comprises IL-2 and IL-7.

10. The serum-free medium according to any one of claims 1 to 8, wherein the serum-free medium comprises IL-2 and IL-15.

11. The serum-free medium according to any one of claims 1 to 10, wherein the serum-free medium contains IL-2, which is human recombinant IL-2.

12. serum-free medium, IL-2 at 100 IU / ml to 200 IU / ml, and / or IL-7 at 500 IU / ml to 1400 IU / ml A serum-free medium according to any one of claims 1 to 9, comprising:

13. serum-free medium, IL-2 at 100 IU / ml to 200 IU / ml, and / or IL-15 at 100 IU / ml to 200 IU / ml A serum-free medium according to any one of claims 1 to 8 and 10, comprising:

14. serum-free medium, IL-2 from 100 IU / ml to 200 IU / ml, IL-7 at 500 IU / ml to 1400 IU / ml, and IL-15 at 100 IU / ml to 200 IU / ml A serum-free medium according to any one of claims 1 to 8 and 11, comprising:

15. A method for preparing serum-free culture medium, wherein the method is (a) A basal medium containing the dipeptide form of L-glutamine, wherein the serum-free basal medium is a liquid formulation; (b) A first supplement containing L-glutamine; (c) IL-2, and one or both of IL-7 and IL-15; and (d) A second supplement containing human serum albumin A method that includes the step of combining [something].

16. The first supplement and / or the second supplement are frozen before the combining process, and / or The method includes a step of thawing the first supplement and / or the second supplement before the combining step, The method according to claim 15.

17. The method according to claim 15 or 16, wherein the concentration of the dipeptide form of L-glutamine in the basal culture medium is 0.5 mM to 5 mM.

18. The method according to any one of claims 15 to 17, wherein the dipeptide form of L-glutamine is L-alanyl-L-glutamine.

19. The method according to any one of claims 15 to 18, wherein the concentration of L-glutamine in the first supplement is 20 mM to 100 mM.

20. The method according to any one of claims 15 to 19, wherein the serum-free medium comprises 90% to 97.5% (v / v) of basal medium and 1.25% to 5% (v / v) of a first supplement.

21. The method according to any one of claims 15 to 20, wherein the serum-free medium comprises a second supplement in an amount of 1.25% to 5% (v / v).

22. The method according to any one of claims 15 to 21, wherein the serum-free medium comprises IL-2 and IL-7.

23. The method according to any one of claims 15 to 21, wherein the serum-free medium comprises IL-2 and IL-15.

24. The method according to any one of claims 15 to 23, wherein the serum-free medium contains IL-2, which is human recombinant IL-2.

25. serum-free medium, IL-2 at 100 IU / ml to 200 IU / ml, and / or IL-7 at 500 IU / ml to 1400 IU / ml The method according to any one of claims 15 to 22 and 24, including

26. serum-free medium, IL-2 at 100 IU / ml to 200 IU / ml, and / or IL-15 at 100 IU / ml to 200 IU / ml The method according to any one of claims 15 to 21, 23, and 24, including

27. serum-free medium, IL-2 from 100 IU / ml to 200 IU / ml, IL-7 at 500 IU / ml to 1400 IU / ml, and IL-15 at 100 IU / ml to 200 IU / ml The method according to any one of claims 15 to 21 and 24, including

28. A method for culturing cells, comprising the step of incubating a composition containing T cells in a serum-free medium according to any one of claims 1 to 14.

29. The aforementioned culture, Activation of T cells in the presence of a stimulating agent; Introduction of an active substance containing nucleic acid molecules encoding a heterologous protein into T cells; and / or T cell culture or expansion The method according to claim 28, relating to the present invention.

30. A method for producing genetically modified T cells, wherein the method is (a) A step of bringing a population of T cells, including cells, into contact with an active substance, including a nucleic acid molecule encoding a heterologous protein, under conditions that introduce the nucleic acid encoding the heterologous protein into the T cells of the population; and (b) A step of incubating the T cells in the presence of a stimulating agent before, during, and / or after the contact step, wherein the stimulating agent induces a primary signal that sends a signal for stimulation, activation, and / or expansion of the T cells. Includes, Herein, the method is carried out in a serum-free medium according to any one of claims 1 to 14.

31. The method according to claim 29 or 30, wherein the nucleic acid molecule encoding the heterologous protein encodes a recombinant receptor.

32. The method according to claim 30 or 31, wherein the active substance, which includes a nucleic acid molecule encoding a heterologous protein, is a viral vector encoding a heterologous protein.

33. The method according to claim 32, wherein the viral vector is a lentiviral vector or a gamma retroviral vector.

34. The method according to any one of claims 30 to 33, wherein the heterologous protein is a recombinant receptor.

35. The method according to claim 34, wherein the recombinant receptor is a chimeric antigen receptor.

36. The method according to any one of claims 29 to 35, wherein the stimulating reagent comprises a primary activator that specifically binds to members of the TCR complex and a secondary activator that specifically binds to T cell costimulatory molecules.

37. The primary active substance specifically binds to CD3, and / or The co-stimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. The method according to claim 36.

38. The method according to claim 36 or 37, wherein the primary activator is an anti-CD3 antibody or its antigen-binding fragment, and the secondary activator is an anti-CD28 antibody or its antigen-binding fragment.

39. The method according to any one of claims 28 to 38, wherein the T cells are CD3+ T cells or CD4+ T cells and CD8+ T cells.

40. A serum-free medium according to any one of claims 1 to 14, Instructions for preparing or using the serum-free medium and Manufactured goods, including those mentioned above.

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