Granted Patent B1

US 12,297,451 · App. 17/077,134 · Granted May 13, 2025

Inventors: Ta-Chun Hang (Morristown, NJ); John Zhao (North Arlington, NJ)

Assignee: Regeneron Pharmaceuticals, Inc.

C12N5/0018C12N2500/05C12N2500/14C12N2500/32C12N2500/50C12N2500/60C12N2501/148C12N2501/15C12N2501/22C12N2501/2302C12N2501/24C12N2501/39

Abstract

Claims (40)

1. A method of manufacturing a population of immune effector cells or hematopoietic stem or progenitor cells (HSPCs), comprising culturing the immune effector cells or HSPCs in a culture medium comprising:

a) a base medium for culturing immune effector cells or hematopoietic stem or progenitor cells (HSPCs),

b) a buffer that maintains the pH of the culture medium in the range of about 6.5 to about 7.5,

c) a serum or serum replacement,

d) one or more mono- and di-valent salts selected from the group consisting of CaCl 2 , NaCl, and KCl,

e) a recombinant growth factor; and

f) about 0.75 to about 3.0 mM L-ornithine;

wherein the culture medium has an osmolality of about 275 mOsm/kg to about 320 mOsm/kg, and

wherein cell proliferation is increased compared to immune effector cells or HSPCs grown in a culture medium without L-ornithine and/or an osmolality of about 275 mOsm/kg to about 320 mOsm/kg.

2. The method of claim 1 , wherein cell viability is increased compared to cells grown in a culture medium without L-ornithine and/or an osmolality of about 275 mOsm/kg to about 320 mOsm/kg.

3. The method of claim 1 , wherein CD62L+ expression is increased in the effector cells compared to immune effector cells grown in a culture medium without L-ornithine and/or an osmolality of about 275 mOsm/kg to about 320 mOsm/kg.

4. The method of claim 1 , wherein the cells are genetically modified to express a therapeutic protein, and wherein the therapeutic protein expression is increased compared to genetically modified cells grown in a culture medium without L-ornithine and/or an osmolality of about 275 mOsm/kg to about 320 mOsm/kg.

5. The method of claim 1 , wherein the cells are cultured at about 38° C. to about 39.5° C.

6. The method of claim 1 , wherein the culture medium has an osmolality of about 310 mOsm/kg.

7. The method of claim 1 , wherein the recombinant growth factor increases ornithine decarboxylase (ODC) expression and/or ODC activity in the immune effector cells or HSPCs as compared to immune effector cells or HSPCs grown in a culture media without L-ornithine.

8. The method of claim 7 , wherein the recombinant growth factor is IL-2.

9. The method of claim 1 , wherein:

a) the one or more mono- and di-valent salts comprise NaCl and KCl, and wherein the culture medium comprises a final ratio of NaCl to KCl of about 20:1 to about 30:1;

b) the culture medium comprises about 0.5 mM to about 3 mM CaCl 2 ); and/or

c) the culture medium comprises about 0.40 g/kg to about 0.80 g/kg NaHCO 3 .

10. The method of claim 1 , wherein the culture medium comprises about 0.5 g/kg to about 1.5 g/kg poloxamer 188.

11. The method of claim 1 , wherein the culture medium comprises about 5 mM to about 25 mM HEPES.

12. The method of claim 1 , wherein the culture medium comprises about 40 g/kg to about 60 g/kg heat inactivated (HI) AB serum or gamma irradiated (GI) AB serum.

13. The method of claim 1 , wherein the culture medium further comprises at least one of:

a) at least one of recombinant growth factor or cytokine selected from the group consisting of GM-CSF, G-CSF, IFN-γ, TGFβ, and TNFα;

b) human serum albumin (HSA);

c) cholesterol; and

d) vitamin E.

14. The method of claim 1 , wherein the population of immune effector cells comprises cytotoxic T lymphocytes (CTLs), helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, regulatory T cells, or dendritic cells.

15. The method of claim 1 , wherein the population of hematopoietic stem or progenitor cells (HSPCs) comprise at least one of CD44+, CD34+, CD90+ or CD133+ cells.

16. The method of claim 1 , wherein the one or more mono- and di-valent salts comprises NaCl and KCl, and

wherein the final molar ratio of NaCl to KCl in the culture medium is about 20:1 to about 30:1.

17. The method of claim 1 , wherein

b) the buffer that maintains the pH of the culture medium is 10 mM HEPES buffer;

c) the serum or serum replacement is about 50 mL/kg HI Human AB Serum or GI AB serum;

d) the one or more mono- and di-valent salts are CaCl 2 ), NaCl, and KCl, wherein the medium comprises 1.89 mM CaCl 2 ), about 0.15 g/kg NaCl, and about 0.03 g/kg KC, wherein the final molar ratio of NaCl to KCl in the culture medium is about 28:1;

e) wherein the recombinant growth factor is recombinant human IL-2 growth factor, and wherein the culture medium comprises about 250 IU/mL recombinant human IL-2 growth factor;

f) wherein the medium comprises about 0.25 g/kg L-ornithine HCl;

and wherein the culture medium further comprises about 0.60 g/kg NaHCO 3 , about 1 g/kg poloxamer 188, and about 2 mM L-alanine-L-Glutamine dipeptide; and

wherein the cell culture medium has an osmolality of about 310 mOsm/kg.

Assignments (3)

Continuity (1)

References Cited (400)