Process for generating therapeutic compositions from manipulated cells

Abstract

This invention provides a method for preparing a composition of manipulated cells. [Solution] The method includes (a) a step of incubating an input composition containing CD4+ primary human T cells enriched T cells under stimulating conditions including (i) a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, and (ii) the presence of one or more cytokines, at least one of which is recombinant human IL-2 or contains recombinant human IL-2, thereby generating a stimulated composition; and (b) a step of introducing a recombinant receptor into the stimulated composition, thereby generating an engineered composition containing engineered T cells.

Claims

1. A method for preparing a composition of manipulated cells, comprising the following steps: (a) A step of incubating an input composition containing one or both CD4+ and CD8+ primary human T cells under stimulating conditions, thereby producing a stimulated composition; wherein the incubation step is (i) an anti-CD3 antibody or its CD3-binding fragment, and an anti-CD28 antibody or its CD28-binding fragment, (ii) Recombinant IL-2 and recombinant IL-15, and (iii) N-acetylcysteine It is carried out in the presence of; (b) a step of introducing a recombinant receptor, which is a chimeric antigen receptor (CAR) or a T cell receptor (TCR), into cells of the stimulated composition, thereby producing an engineered composition comprising engineered T cells; wherein the recombinant receptor can bind to a target antigen expressed on cells of disease or pathological condition; and (c) A step of culturing the manipulated composition under conditions that promote the expansion of the manipulated T cells, thereby producing an output composition containing the manipulated T cells; where, (i) The culturing step is carried out in the presence of recombinant IL-2 and recombinant IL-15; (ii) At least part of the culturing process is carried out using mixing and perfusion, wherein the culturing process is started under conditions without perfusion, and then the concentration of the cells is increased to at least 0.2 × 10 6 When a predetermined concentration of live cells / mL is reached, the perfusion is initiated; and (iii) The culturing step is carried out for 2 to 10 days including both of the values, and until the output composition contains at least a threshold number of T cells or viable T cells, where the threshold number of T cells or viable T cells is at least four times the number of T cells or viable T cells of the manipulated cell composition before culturing the manipulated composition.

2. The method according to claim 1, wherein the input composition contains more than 70% CD3+ primary human T cells.

3. The input composition is 200 × 10 6 Cell ~300×10 6 The method according to claim 1 or 2, comprising cells.

4. The method according to any one of claims 1 to 3, wherein the incubation step is carried out in the presence of recombinant IL-2, recombinant IL-15, and recombinant IL-7.

5. The method according to any one of claims 1 to 4, wherein in the incubation step, the concentration of recombinant IL-2 is 10 IU / mL to 200 IU / mL.

6. The method according to any one of claims 1 to 5, wherein the concentration of recombinant IL-15 in the incubation step is 1 IU / mL to 25 IU / mL.

7. The method according to any one of claims 4 to 6, wherein in the incubation step, the concentration of recombinant IL-7 is 100 IU / mL to 1000 IU / mL.

8. The method according to any one of claims 1 to 7, wherein the input composition is a first input composition enriched with CD8+ primary human T cells, and the method further comprises the following: (a) A step of separately incubating a second input composition enriched with CD4+ primary human T cells; here, These CD8+ primary human T cells were isolated from the same biological sample as the CD4+ primary human T cells, and The incubation step of the second input composition is carried out in the presence of (i) an anti-CD3 antibody or its CD3-binding fragment, and an anti-CD28 antibody or its CD28-binding fragment, and (ii) one or more cytokines, thereby generating the second stimulated composition; (b) a step of introducing a recombinant receptor, which is a CAR or TCR, into the cells of the second stimulated composition, thereby generating a second engineered composition containing engineered T cells; and (c) A step of culturing the second manipulated composition under conditions that promote the expansion of the manipulated T cells, thereby producing a second output composition containing the manipulated T cells.

9. The method according to claim 8, wherein the recombinant receptor introduced into the second stimulated composition is the same recombinant receptor introduced into the first stimulated composition.

10. The method according to any one of claims 1 to 9, wherein the anti-CD3 antibody or its CD3-binding fragment, and / or the anti-CD28 antibody or its CD28-binding fragment, are present on the surface of the beads.

11. The method according to claim 10, wherein the bead-to-cell ratio is less than 3:

1.

12. The method according to any one of claims 1 to 11, wherein the concentration of N-acetylcysteine ​​(NAC) is 0.2 mg / mL to 2.0 mg / mL.

13. The method according to any one of claims 1 to 12, wherein the introduction step is performed by a viral vector containing a polynucleotide encoding the recombinant receptor.

14. The aforementioned viral vector (a) Retroviral vectors; (b) lentiviral vector; or (c) Gamma retrovirus vector The method according to claim 13.

15. The method according to any one of claims 1 to 14, wherein the introduction step is carried out in the presence of a transduction adjuvant.

16. The method according to claim 15, wherein the transduction adjuvant is a transduction adjuvant derived from protamine sulfate and / or fibronectin, or comprises the same.

17. The method according to any one of claims 1 to 16, wherein the incubation step is carried out in the presence of recombinant IL-2, recombinant IL-15, and recombinant IL-7.

18. The method according to any one of claims 1 to 17, wherein the concentration of recombinant IL-2 in the culture step is 50 IU / mL to 500 IU / mL.

19. The method according to any one of claims 1 to 18, wherein the concentration of recombinant IL-15 in the culture step is 5 IU / mL to 50 IU / mL.

20. The method according to any one of claims 17 to 19, wherein the concentration of recombinant IL-7 in the culture step is 500 IU / mL to 2000 IU / mL.

21. The method according to any one of claims 1 to 20, comprising the step of removing the anti-CD3 antibody or its CD3-binding fragment, and the anti-CD28 antibody or its CD28-binding fragment from the manipulated composition before the culturing step.

22. The method according to claim 21, wherein the anti-CD3 antibody or its CD3-binding fragment, and the anti-CD28 antibody or its CD28-binding fragment are removed within 7 days from the start of the incubation step.

23. The method according to any one of claims 1 to 22, wherein the culturing step is carried out until the output composition contains at least a threshold number of viable T cells.

24. The method according to claim 23, wherein the culturing step is continued for at least one day after a threshold number of viable T cells has been reached.

25. The method according to claim 23 or claim 24, wherein the threshold number of viable T cells is at least five times the number of viable T cells in the manipulated cell composition before culturing the manipulated composition.

26. The method according to any one of claims 1 to 25, wherein the culturing step is performed until at least 9 days after the start of the incubation step.

27. The method according to any one of claims 1 to 26, wherein the culture step is carried out for 2 to 8 days including both end values, until the output composition contains a threshold number of T cells or a threshold number of viable T cells, wherein the threshold number of T cells or the threshold number of viable T cells are at least four times the number of manipulated cells or viable T cells of the manipulated composition before culture.

28. The threshold number of T cells or viable T cells is 50 × 10 6 Cells or at least 50 × 10 6 The method according to any one of claims 24 to 27, wherein the cell is a cell.

29. The method according to any one of claims 1 to 28, further comprising the step of collecting cells of the output composition after the culturing step.

30. The method according to claim 29, wherein the time between the start of the incubation step of the output composition and the collection of cells is 7 to 15 days.

31. The method according to claim 29 or 30, further comprising the step of formulating collected cells into an output composition for cryopreservation and / or administration to a subject.

32. The method according to claim 31, wherein the cells collected from the output composition are formulated in the presence of pharmaceutically acceptable excipients and / or cryoprotectants.

33. The method according to any one of claims 1 to 32, wherein the input composition comprises primary T cells obtained from a human subject having cancer.

34. The method according to claim 33, wherein the recombinant receptor is capable of binding to a target antigen that is associated with cancer cells or tissues, specific to cancer cells or tissues, and / or expressed on cancer cells or tissues.

35. The method according to any one of claims 1 to 34, wherein the recombinant receptor is a CAR.

36. The method according to any one of claims 1 to 35, wherein the recombinant receptor is an anti-CD19 CAR.

37. The predetermined concentration is at least 0.6 × 10 6 The method according to any one of claims 1 to 36, wherein the amount is live cells / mL.

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