Cell Culture Media for Natural Killer (NK) Cells

Abstract

The disclosed invention provides a cell culture media formulation useful for the culture of natural killer (NK) immune cells. Also included are methods of culturing, cryopreserving, and transducing NK cells that include the culture media formulation. TIFF2024529565000013.tif128163

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC § 119(e) from U.S. Provisional Patent Application No. 63 / 230,189, filed August 6, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention The ex vivo and / or in vitro maintenance, study, or expansion of cellular therapeutics requires liquid media specifically formulated for each category or type of cell, as an adequate source of energy and compounds, for maintaining cell health, cell function and phenotype, as well as for cell cycle control.

[0003] Existing media for cell culture of NK cells are limited in terms of supporting the activation and cytotoxicity of NK cells cultured for maintenance and / or expansion, especially in the absence of supplemental cytokines and / or feeder cells. Specifically, the reliance of these media on certain components (e.g., cytokines such as interleukin 15 (IL-15) and others) results in "exhaustion" of cultured NK cells, characterized by reduced production of effector molecules (e.g., interferon-gamma (IFNγ)) and impaired cytotoxic activity against target cells, such as cancer cells. Furthermore, these media often require significant amounts of animal serum (e.g., fetal bovine serum, human serum, etc.), typically in the range of 10-20% volume / volume (v / v), which in turn contributes to increased batch-to-batch variability, as well as increased cost and contamination risk of NK cell immunotherapeutics. Furthermore, the co-culture of antigen-presenting cells (APCs, i.e., feeder cells) to achieve adequate expansion of NK cells introduces undesirable cost, complexity, and quality challenges to NK cell therapeutics. Cryopreservation of NK cells is also difficult due to a decrease in NK cell viability, post-thaw cytotoxic activity, and other functions. NK cell metabolism, transduction, subpopulation / subset content, and differentiation are also suboptimal.

[0004] Thus, there is a need in the art for media formulations and techniques for NK cell culture that overcome the challenges and problems associated with current in vitro and ex vivo culture systems for the generation of more effective NK cell immunotherapeutics.The present invention addresses this need. Summary of the Invention

[0005] The present invention relates to media formulations useful for the ex vivo culture or genetic modification or preservation of Natural Killer (NK) immune cells. In particular, aspects of the invention relate to research and development (R&D) related to NK cell immunotherapeutics, as well as biomanufacturing (e.g., expansion in bioreactors) and clinical use of cellular immunotherapeutics using Natural Killer (NK) cells.

[0006] Thus, in one aspect, the present invention provides a two-part media formulation for culturing natural killer (NK) cells, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and (b) a second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania Somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella Japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L wherein the NK cells are cultured in a first media formulation and then in a second media formulation.

[0007] In another aspect, the invention includes a one-part media formulation for culturing natural killer (NK) cells, comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) Antioxidant supplement at concentrations ranging from 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalogue no. A1345 or equivalent).

[0008] In another aspect, the invention includes a one-part media formulation for culturing natural killer (NK) cells, comprising at least nine of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), (Liii) antioxidant supplement at concentrations between 0.01x and 1000x (antioxidant supplement is Sigma-Aldrich catalogue no. A1345 or equivalent); (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract at a concentration of 1.06E-01 mg / L to 1.06E+04 mg / L.

[0009] In certain embodiments, the formulation is added as a supplement to a basal cell culture medium.

[0010] In another aspect, the present invention provides a method for culturing natural killer (NK) cells or precursor cells thereof, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and (b) a second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L The method further comprises culturing the NK cells or their precursor cells in a cell culture medium formulation comprising:

[0011] In certain embodiments, the NK cells or progenitors thereof are cultured in a first media formulation.

[0012] In certain embodiments, the NK cells or progenitors thereof are cultured in a second media formulation.

[0013] In certain embodiments, the NK cells or progenitors thereof are cultured in a first media formulation and then in a second formulation.

[0014] In certain embodiments, the NK cells or their precursors are cultured ex vivo.

[0015] In certain embodiments, the NK cells or their precursors are cultured in vitro.

[0016] In certain embodiments, the cytotoxic activity of NK cells against target cells is enhanced.

[0017] In certain embodiments, the target cell is a cancer cell.

[0018] In certain embodiments, the activation of NK cells is enhanced.

[0019] In certain embodiments, the dysfunction of NK cells is reduced.

[0020] In certain embodiments, the impairment is fatigue.

[0021] In certain embodiments, the metabolic function of NK cells is enhanced.

[0022] In certain embodiments, high viability and cell expansion of NK cells is achieved.

[0023] In certain embodiments, the use of serum is eliminated or reduced.

[0024] In certain embodiments, the use of cytokine supplements is eliminated or reduced.

[0025] In certain embodiments, the use of antigen presenting cells (APCs) or feeder cells is eliminated or reduced.

[0026] In certain embodiments, the culture of the NK precursor cells induces their differentiation.

[0027] In certain embodiments, the culture of NK cells or progenitor cells modulates the distribution and relative populations of NK cell subsets.

[0028] In certain embodiments, the NK cell subset is memory NK cells.

[0029] In certain embodiments, NK cell subsets are defined by the relative expression of activating and inhibitory receptors.

[0030] In another aspect, the present invention provides a method for cryopreservation of natural killer (NK) cells, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and (b) a second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L The method further comprises the step of: freezing the NK cells in a cryopreservation medium comprising:

[0031] In certain embodiments, the cryopreservation medium comprises a first medium formulation.

[0032] In certain embodiments, the cryopreservation medium comprises a second medium formulation.

[0033] In another aspect, the present invention provides a method for transduction of natural killer (NK) immune cells, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and A second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L a first cell culture step comprising culturing NK cells in a cell culture medium comprising: (b) contacting the NK cell with a viral vector; and (c) a second cell culture step comprising culturing the transduced NK cells in a cell culture medium comprising the cell culture medium formulation of step (a). The method includes the method comprising:

[0034] In certain embodiments, the first and second cell culture steps are performed in a first cell culture preparation.

[0035] In certain embodiments, the first and second cell culture steps are performed in a second cell culture preparation.

[0036] In certain embodiments, the first cell culture step is performed in a first cell culture preparation and the second cell culture step is performed in a second cell culture preparation.

[0037] In certain embodiments, the vector encodes a chimeric antigen receptor (CAR).

[0038] In another aspect, the present invention provides a method for differentiating natural killer (NK) cells from progenitor cells, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and (b) a second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L The method further comprises culturing the progenitor cells in a cell culture medium comprising:

[0039] In certain embodiments, the culture medium comprises a first medium formulation.

[0040] In certain embodiments, the culture medium comprises a second medium formulation.

[0041] In certain embodiments, the culture medium comprises a first media formulation, followed by a second media formulation.

[0042] In certain embodiments, the culture of natural killer (NK) cells modulates the distribution and relative populations of NK cell subsets.

[0043] In certain embodiments, the NK cell subset is memory NK cells.

[0044] In certain embodiments, NK cell subsets are defined by the relative expression of activating and inhibitory receptors. [Brief description of the drawings]

[0045] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, exemplary embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and equipment of the embodiments shown in the drawings.

[0046] [Figure 1] FIG. 1 is a diagrammatic representation of a two-part media formulation, with each part having a specific list of ingredients and their concentration ranges according to Table 01, according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a diagrammatic representation of a two-part media formulation, in accordance with a further embodiment of the present invention, where each part is made by the addition of supplements to a ready-made media, and the supplements are formulated with a specific list of ingredients and their concentration ranges according to Table 01. [Diagram 3] FIG. 3 is a process flow diagram illustrating steps representing an example of the use of a two-part media formulation from FIG. 01 or FIG. 02, where one or both parts of the two-part media formulation are used. [Figure 4] Figures 4A-4B illustrate the cytotoxicity of primary NK cells and cell line NK cells grown in the media formulation of the invention. Figure 4A corresponds to the cytotoxicity observed in target K562 leukemia cells after overnight incubation with effector KHYG1 cells at an effector:target (E:T) ratio of 5:1. Figure 4B corresponds to the cytotoxicity observed in target K562 leukemia cells after overnight incubation with effector primary NK cells (derived from a healthy adult donor) at an effector:target (E:T) ratio of 5:1. In both cases, the control medium is DMEM-F12 and the cytotoxicity measured is natural cytotoxicity only (no ADCC (antibody-dependent cellular cytotoxicity)). The notation used for statistical significance is as follows (unpaired t-test): ns is not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Diagram 5]Figures 5A-5B illustrate that the cytotoxicity of primary human NK cells expanded in Enable-NK™ medium consistently exceeded that of NK cells from the same donor expanded in control medium, regardless of whether the comparison was made in the absence (Figure 5A) or presence (Figure 5B) of anti-GD2 antibodies (for ADCC (antibody-dependent cellular cytotoxicity)). Target cells were CHLA-20-GFP (a human pediatric neuroblastoma cell line) at an effector:target (E:T) ratio of 5:1 (NK:CHLA-20) [co-cultured in DMEM medium]. Target cell death was determined by loss of GFP signal over time. Error bars indicate standard error of the mean (SEM). [Figure 6] Figures 6A-6B illustrate that the cytotoxicity of primary human NK cells expanded in Enable-NK™ medium consistently exceeded that of NK cells from the same donor expanded in control medium, regardless of whether the comparison was made in the absence (Figure 6A) or presence (Figure 6B) of anti-GD2 antibodies (for ADCC (antibody-dependent cellular cytotoxicity)). Target cells were M21-GFP (a human melanoma cell line) at an effector:target (E:T) ratio of 5:1 (NK:M21) [co-cultured in DMEM medium]. Target cell death was determined by loss of GFP signal over time. All error bars indicate standard error of the mean (SEM). [Figure 7]Figures 7A-7B illustrate a comparison of levels of interferon-gamma (IFNγ) measured in supernatant samples of control and Enable-NK media after incubation of effector NK cells cultured alone or co-cultured with target K562 cells. NK cells secrete IFNγ to recruit other immune cells against disease. Figure 7A corresponds to IFNγ levels measured in KHYG1 NK cells after overnight incubation at an effector:target (E:T) ratio of 5:1, and Figure 7B corresponds to IFNγ levels measured in primary NK cells derived from a healthy adult donor. Bars indicate standard error of the mean (SEM). The notation used for statistical significance is as follows (unpaired t-test): ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 8] Figures 8A-8D illustrate flow cytometry quantification of cell surface biomarkers of primary NK cells corresponding to NK cell activation: [Figure 8A] NKG2D, [Figure 8B] NKp46, [Figure 8C] CD69, [Figure 8D] CD16. Graphs represent cell number (y-axis) and fluorescence (x-axis). The notation used for statistical significance is as follows (unpaired t-test): ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 9] Figure 9 illustrates the percentage of target cells (K562-GFP) (y-axis) and the percentage of dead target cells (K562-GFP) (y-axis) both as a function of time (x-axis) after setting up a co-culture of KHYG-1 effector cells and K562-GFP target cells at an effector:target ratio of 1:1 on day 0. Bars indicate standard error of the mean. Notation used for statistical significance (unpaired t-test): ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 10]Figure 10 illustrates a comparison of the mean fluorescence intensity of biomarker levels of KHYG-1 NK cells at various time points of the experiment (day 02, day 10 in both experimental conditions of monoculture and coculture). The notation used for statistical significance is as follows (unpaired t-test): ns is not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 11] Figure 11 illustrates the percentage of target cells (K562-GFP) (y-axis) and the percentage of dead target cells (K562-GFP) (y-axis) both as a function of time (x-axis) after setting up a co-culture of KHYG-1 effector cells and K562-GFP target cells at an effector:target ratio of 1:1 on day 0. Bars indicate standard error of the mean. Notation used for statistical significance (paired t-test): ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 12] Figure 12 illustrates a comparison of the mean fluorescence intensity of biomarker levels of primary NK cells on day 03 in both monoculture and coculture experimental conditions. Data presented are for three healthy adult donors of primary NK cells paired donor-wise between the two media conditions. The notation used for statistical significance is as follows (paired t-test): ns (p-value) is not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 13] Figure 13 illustrates a comparison of CD56dimCD16+CD57+ subpopulations (paired donor-wise data) between the two media conditions on day 03 assessed in primary NK cells (CD3-CD56+) derived from three healthy adult donors. The notation used for statistical significance (paired t-test) is as follows: ns (p-value) is not significant, * p<0.05, ** p<0.01. [Figure 14]Figure 14 illustrates a comparison of CD56dimCD57+NKG2A- subpopulations (paired donor-wise data) between the two media conditions on day 03 assessed in primary NK cells (CD3-CD56+) derived from three healthy adult donors. The notation used for statistical significance (paired t-test) is as follows: ns (p-value) is not significant, * p<0.05, ** p<0.01. [Figure 15] Figure 15 illustrates a comparison of CD56dimCD16+ subpopulations (paired donor-wise data) between the two media conditions on day 03 assessed in primary NK cells (CD3-CD56+) derived from three healthy adult donors. The notation used for statistical significance (paired t-test) is as follows: ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 16] Figure 16 illustrates a comparison of CD56dim subpopulations (paired donor-wise data) between the two media conditions on day 03 assessed in primary NK cells (CD3-CD56+) derived from three healthy adult donors. The notation used for statistical significance (paired t-test) is as follows: ns (p-value) is not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 17] Figure 17 illustrates that normalized NAD values ​​were more than 3-fold higher for KHYG1 NK cells in Enable-NK medium condition. Bars indicate standard deviation. The notation used for statistical significance (unpaired t-test) is as follows: * p<0.05, ** p<0.01. [Figure 18] Figure 18 illustrates a comparison of the efficiency of transduction of NK-92 cells in α-MEM medium (21.0%) compared to Enable-NK part II medium (62.8%) as confirmed by double staining for both NKp-44 and FLAG. In contrast, parental cells showed no significant staining (<1%). [Figure 19]Figure 19 illustrates the number of CHLA-20-GFP target cancer cells per well of co-culture with NK cells as a function of time based on microscopic observation from a Sartorius Incucyte Instrument. Greater cytotoxicity of CHLA-20-GFP target cells was observed for Enable-NK medium (both with and without anti-GD2 Ab) compared to control medium. Error bars are standard error of the mean. [Figure 20] Figure 20 illustrates the fold expansion achieved by Enable-NK medium (various parts) when NK cells were cultured ex vivo in the absence of feeder cells and at reduced serum levels compared to control medium: [A] NK cell line NK3.3, [B] NK cell line KHYG1, and [C] primary human NK cells (derived from a healthy adult donor). Error bars indicate standard error of the mean (SEM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology is used. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting. The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, "element" means one element or more than one element.

[0048] "About," as used herein, when referring to a measurable value, e.g., an amount, length of time, etc., is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for performing the disclosed methods.

[0049] "Basal medium", as used herein, is interchangeable with "prepared medium", "starting medium", and "basal medium", and examples include, but are not limited to, DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12), RPMI-1640 (RPMI: Roswell Park Memorial Institute), MEM (Minimum Essential Medium), and IMDM (Iscove's Modified Dulbecco's Medium).

[0050] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0051] The term "cell culture" or "culturing," as used herein, refers to the maintenance of cells (e.g., NK cells) under controlled conditions, generally outside their native environment. Cells can be cultured to expand and maintain a population of cells for subsequent therapeutic, industrial, or scientific use. Cell culture can be a part of cryopreservation and other processes involving living cells, including, but not limited to, modification or other manipulation of cellular function by inserting exogenous nucleic acid, for example, through transduction or transfection.

[0052] The term "exhaustion" or "immunoexhaustion" as used herein refers to a functional state of immune cells (e.g., NK cells) characterized by impaired or reduced proliferation activity, cytokine production, and / or cytotoxic activity. Exhaustion can be caused by repeated stimulation, signaling through inhibitory signaling receptors, depletion of certain nutrients in the immediate microenvironment, or a combination of all of these factors. The occurrence of exhaustion limits the clinical effectiveness of therapeutic immune cells, e.g., NK cells, due to impaired function.

[0053] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to inform of the usefulness of the compositions and methods of the invention. The instructional material of the kits of the invention may, for example, be attached to a container containing the nucleic acid, peptide, and / or composition of the invention, or may be shipped together with the container containing the nucleic acid, peptide, and / or composition. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the compound are used cooperatively by the recipient.

[0054] In the context of the present invention, the following abbreviations for commonly occurring nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns to the extent that a nucleotide sequence encoding a protein may, in some versions, contain introns.

[0055] As used herein, "Enable-NK medium" refers to the medium of the present invention as defined by the claims.

[0056] The term "polynucleotide", as used herein, is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide are interchangeable as used herein. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning technology and PCR™, etc., and synthetic means.

[0057] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0058] Ranges: Throughout this disclosure, various aspects of the invention may 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 invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0059] explanation The invention provided by this disclosure includes unique media formulations for ex vivo or in vitro culture or genetic modification or storage of natural killer (NK) cells that allow for the generation of more potent and more efficient NK cell therapeutics. The new media formulations incorporate novel combinations of components in specific concentration ranges to create single-part or multi-part media for ex vivo culture or genetic modification or storage of NK cells. The invention is described in enabling detail in the following examples that may represent multiple aspects of the invention.

[0060] FIG. 1 is a diagrammatic representation of a two-part media formulation, with each part being a specific list of ingredients and their concentration ranges according to Table 01, according to one embodiment of the present invention, which is also a preferred embodiment. Media formulation 100, in this example, is a two-part media for the culture of NK cells. In this example, two-part media formulation 100 has a first part 101 formulated using the ingredient list from the "Part 01 Subformulation" section of Table 01, with each ingredient at a specific concentration range listed in "Part 01 Subformulation" of Table 01. In this example, two-part media formulation 100 has a second part 102 formulated using the ingredient list from the "Part 02 Subformulation" section of Table 01, with each ingredient at a specific concentration range listed in "Part 02 Subformulation" of Table 01.

[0061] 02 is a diagrammatic representation of a two-part media formulation according to a further embodiment of the present invention, where each part is made by the addition of a supplement to a ready-made media, where the supplement is formulated with a specific list of ingredients and their concentration ranges according to Table 01. Media formulation 200, in this example, is a two-part media for the culture of NK cells, where part 201 is a supplement formulated using the list of ingredients from the "Part 01 Subformulation" section of Table 01, with each ingredient at the specified concentration range listed in "Part 01 Subformulation" of Table 01, and part 202 is a supplement formulated using the list of ingredients from the "Part 02 Subformulation" section of Table 01, with each ingredient at the specified concentration range listed in "Part 02 Subformulation" of Table 01. In this example, two-part media formulation 200 has a first part made by the addition of part 201, a supplement, to part 203, a ready-made media. In this example, two-part media formulation 200 has a second part created by the addition of part 202, which is a supplement, to part 204, which is a ready-made media. In this example, "ready-made" media refers to any commercially available or non-commercially available media formulation, such as DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12), RPMI-1640 (RPMI: Roswell Park Memorial Institute), MEM (Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), and others.

[0062] FIG. 03 is a process flow diagram illustrating steps representing an example of the use of a two-part media formulation from FIG. 01 or FIG. 02, where one or both parts of the two-part media formulation are used.

[0063] In one example of the process illustrated in Figure 03, when performing the culture of NK cells, the two parts of the two-part media formulation are used in sequence, with the first part media being used for the expansion of NK cells, with the option of serum reduction or complete serum withdrawal (i.e., serum-free media). The cultured NK cells are then transferred from the first part media to the second part media of the two-part media formulation by centrifugation, dilution of the first part media with the second part media, or other means. The second part media is then used for the culture of the NK cells for activation.

[0064] In another example of the process illustrated in Figure 03, when performing the culture of NK cells, the first part medium of the two-part medium formulation is used without the use of the second part medium. The first part medium is used for the expansion of NK cells with the option of serum reduction or complete serum withdrawal (i.e. serum-free medium). The first part medium is also used for the culture of NK cells for activation.

[0065] In yet another example of the process illustrated in Figure 03, when performing the culture of NK cells, the second part medium of the two-part medium formulation is used without the use of the first part medium. The second part medium is used for the expansion of NK cells with the option of serum reduction or complete serum withdrawal (i.e. serum-free medium). The second part medium is also used for the culture for activation of NK cells.

[0066] The present inventors provide a unique cell culture media formulation for natural killer (NK) immune cells that facilitates more robust NK cell cytotoxic activity while also supporting cell maintenance and cell proliferation at reduced serum levels in the medium. The above-mentioned aspects and examples of the present invention aim to optimally achieve the desired properties of the NK cell therapy (e.g., serum reduction, proliferation, higher cytotoxicity due to activation) through the use of individual versions (i.e., subformulations) of the media formulation optimized for the specific desired properties of the resulting NK cell therapy, although other desirable properties are also supported. As an example, the present inventors provide a unique cell culture media formulation for natural killer (NK) immune cells that facilitates more robust NK cell cytotoxic activity while also supporting cell maintenance and cell proliferation at reduced serum levels in the medium through the use of specific combinations of optimized individual components and specific concentration ranges of optimized individual components in the media formulation, as shown in Table 01 below.

[0067] [Table 1] TIFF2024529565000003.tif224166TIFF2024529565000004.tif224166TIFF2024529565000005.tif70166Table 01: Tabular summary of the ingredients and their concentration ranges for each subformulation, including the Part 01 subformulation, the Part 02 subformulation (which is in addition to the Part 01 subformulation ingredients), and the Part 03 subformulation (which is in addition to the Part 01 and Part 02 subformulation ingredients).

[0068] In Table 01, the following acronyms are used: HSA (human serum albumin), PVA (polyvinyl acetate), 2-ME (2-mercaptoethanol), IGF-1 (insulin-like growth factor 1), 4-1BBL (4-1BB ligand or ligand of CD137), mM (millimolar), mg / L (milligrams per liter). In Table 01, for the plant extracts, their concentrations in preferred embodiments of the invention (summarized in Table 01) were determined based on equivalent dry plant extract in the final volume of medium, expressed in mg / L concentration units. The botanical names of the plant extracts listed in Table 01 are as follows: Ashwagandha (botanical name: Withania somnifera, family: Solanaceae); Kumquat (botanical name: Fortunella japonica or Citrus Japonica, family: Rutaceae); Uncaroabo (botanical name: Pelargonium sidoides, family: Geraniaceae); Echinacea (botanical name: Echinacea purpurea, family: Asteraceae); Ginseng (botanical name: Panax ginseng, family: Araliaceae); Elderberry (botanical name: Sambucus nigra, family: Malvaceae); Reishi (botanical name: Ganoderma Lucidum, family: Ganodermaceae); Mistletoe (botanical name: Viscum Album, family: Santalaceae). In alternative embodiments of the invention, the use of said plant extracts may be replaced by botanical varieties of said plant extracts, or by chemically synthesized phytochemicals, or a combination of phytochemicals, or a subset of the phytochemicals that make up said plant extracts.

[0069] The media formulations represented by the specific component combinations and concentration ranges summarized in Table 01, including Part 01 subformulation, Part 02 subformulation, and Part 03 subformulation, were made by the addition of some of these components to DMEM / F12 medium (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12). The component concentration ranges summarized in Table 01 include the concentrations of some components already contributed by the basal medium, DMEM / F12 medium (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12). Thus, although DMEM / F12 basal medium was used for media formulation development, alternative media, which may be basal or complex media (e.g., RPMI1640, MEM, IMDM, etc.), may be used instead as the basal or starting pre-made medium to which additional components may be added to generate alternative embodiments of the present invention; RPMI1640 (RPMI: Roswell Park Memorial Institute; MEM: Minimum Essential Medium; IMDM: Iscove's Modified Dulbecco's Medium). Regardless of the basal / defined medium used, the basal DMEM / F12 medium is interchangeable with other media as it is possible to ensure that the final concentrations of ingredients, including added components, reach the levels outlined in Table 01.

[0070] Alternative embodiments of the invention include unique combinations of a subset of the ingredients listed in Table 01. Alternative embodiments of the invention also include unique combinations of ingredients listed in Table 01 (either the entire list or a subset of the list) when combined with an ingredient not listed in Table 01. Alternative embodiments of the invention further include multi-part media formulations with more than two parts, e.g., three-part media formulations. An example of a three-part media formulation is one in which a first part is formulated using the ingredient list and concentration ranges from the "Part 01 Subformulation" section of Table 01, a second part is formulated using the ingredient list and concentration ranges from the "Part 02 Subformulation" section of Table 01, and a third part is formulated using the ingredient list and concentration ranges from the "Part 03 Subformulation" section of Table 01. In this example, the first part of the three-part media formulation is optimized for gradual serum reduction or withdrawal while also supporting other desirable properties of the NK cell therapeutic (e.g., proliferation or activation). In this example, the second part of the three-part media formulation is also optimized for NK cell expansion while also supporting other desirable properties of the NK cell therapy (e.g., serum reduction or activation). In this example, the third part of the three-part media formulation is also optimized for NK cell activation while also supporting other desirable properties of the NK cell therapy (e.g., proliferation or serum reduction). In yet other alternative embodiments of the invention, other examples of multi-part media formulations may be made, where each part of the multi-part media formulation is optimized for a particular desirable property of the NK cell therapy while also supporting other desirable properties of the NK cell therapy.

[0071] Table 02 below is a subset of Table 01 and summarizes examples of some unique, non-typical media formulation components that have been combined in specific concentration ranges and added to a basal DMEM / F12 medium as part of the comprehensive media formulation represented in Table 01. Table 01 is a more comprehensive list of components, and therefore includes non-typical / non-obvious components (examples in Table 02) as well as some of the more conventional / typical components that are incorporated into media formulations for culturing cells. The incorporation of unique combinations of both non-typical and conventional components in specific concentration ranges (Table 01), as well as all variations of media formulations that may emerge from this unique combination framework (Table 01), represent alternative aspects of the present invention. For the present invention, the "primary" concentration range of each individual component is broader and includes a "secondary" range, which in turn includes a "tertiary" range.

[0072] [Table 2] Table 02: Tabular summary of some examples of atypical / non-obvious ingredients and their concentration ranges for each subformulation. Listed are five examples of atypical / non-obvious ingredients for the Part 01 subformulation. For the Part 02 subformulation, three additional example ingredients are listed. For the Part 03 subformulation, one additional example of atypical / non-obvious ingredients is listed.

[0073] Possible embodiments of the present invention may be represented by a variety of individual media formulations that can be uniquely created by incorporating specific combinations of ingredients [from Table 01, at specific concentration ranges for the ingredients] that may constitute a stand-alone media product for NK cells, or specific combinations of supplemental ingredients that may be added to any pre-made media.

[0074] To provide further detail regarding alternative embodiments, the present invention covers not only the specific formulation details (combinations and concentration ranges) summarized in Table 01, but also derivative and / or alternative formulations that may be based on Table 01. Examples of alternative embodiments of the present invention include, but are not limited to, the following: Permutations and combinations of subsets of the components listed in Table 01 to create derivative or alternative subformulations of a Part 01 or Part 02 or Part 03 medium, such as, but not limited to, a subset of only molecular components, a subset of only plant extract components, or plant extract variants derived from the same botanical family. Permutations and combinations of a subset of molecular components of plant extracts, whether derived from plants, chemically synthesized, or recombinantly synthesized. Examples include, but are not limited to, iscador peptides derived from molecular components of mistletoe, flavonoid molecules derived from elderberry, etc. Combinations of ingredients listed in Table 01 (and subsets thereof) with other ingredients not listed in Table 01, such as molecular components and botanical extracts not listed in Table 01. Examples of other plant extracts / ingredients include resveratrol, carrageenan (e.g., lambda carrageenan), neem (botanical name: Azadirachta Indica), turmeric (Curcuma Longa), banyan tree (Euphorbia Peplus), Homalanthus Nutans (Euphorbiaceae), Phyllanthus Poilanei, Ascophyllum nodosum, Aphanizomenon flos-aquae, Nigella Sativum, garlic (Allium Sativum), onion (Allium Cepa), and onopordum. Acanthium), as well as certain compounds derived from plant / botanical sources, including but not limited to diallyl disulfide (from garlic), genistein, curcumin, ingenol mebutate, prostratin, phyllanthusmin C, ascophyllan, and fucoidan. Higher or lower concentrations of the ingredients and their concentration ranges listed in Table 01 (the relative ratios of the concentrations of the individual ingredients are maintained). · Combinations of ingredients that are the same as those outlined in Table 01 (or a subset thereof), but at concentration levels different from those summarized in Table 01. · Dried plants / whole herbs or plant extracts made by various extraction methods, e.g. solvent extraction (immersion / Soxhlet) or the use of ultrasound, microwave or supercritical fluids. Use of any one of the subformulations of the described preferred embodiments to achieve all three of the following objectives: serum reduction, proliferation, and enhanced cytotoxicity. Formulation of the components listed in Table 01 as a medium supplement, either premixed and / or with individual components provided separately (e.g., as part of a kit, e.g., in lyophilized form) rather than preformulated with the basal medium.

[0075] It will be apparent to one of ordinary skill in the art that the media formulations of the present invention may be provided using some or all of the features and components mentioned without departing from the spirit and scope of the present invention. It will also be apparent to one of ordinary skill in the art that the above embodiments are examples of a single broader invention that may have a broader scope than any of the specific descriptions taught. Many variations can be made to the descriptions without departing from the spirit and scope of the present invention.

[0076] The present invention may be used for cellular immunotherapy applications, resulting in a wide variety of usefulness and potential applications, including but not limited to: [a] research use of NK cells, and [b] clinical use of NK cell therapeutics (e.g., after ex vivo / bioreactor expansion). Primary desired outcomes of the present invention for NK cells include, but are not limited to: [i] Higher cytotoxic activity of NK cells resulting in enhanced killing of diseased cells (e.g., cancer cells or virus-infected or other diseased cells) that may lead to better therapeutic outcomes. Such availability of enhanced cytotoxic activity may be conferred through apoptotic or necrotic mechanisms, with or without enhancement of the natural cytotoxic mechanisms of NK cells or the ADCC (antigen-dependent cellular cytotoxicity) mechanisms of NK cells, or amplification of the overall immune response through signaling of NK cells to other immune cells. [ii] Enhanced NK cell activation resulting in improved NK cell cytotoxicity as well as overall biological function, which may translate into better research and clinical outcomes. [iii] Elimination or reduction of the dependency of NK cells on cytokines or chemokines in cell culture for cytotoxicity or proliferation, which may result in not only reduced cell exhaustion but also reduced costs. [iv] Elimination or reduction of APCs (antigen presenting cells, also called "feeder cells") for proliferation (i.e., ex vivo expansion or growth) or activation of NK cells. [v] Reduction or adaptation of serum dependency of NK cells for cell maintenance and expansion (e.g., complete serum withdrawal or serum exclusion leading to serum-free media) resulting in improved batch-to-batch consistency and lower cost of ex vivo / bioreactor expansion of NK cells. Enhanced cell proliferation at conventional serum levels or enhanced cell viability in various serum conditions are other benefits that may be exploited. [vi] Reduction of cellular dysfunction (e.g., exhaustion or anergy or senescence), resulting in improved in vivo persistence of NK cells, particularly in the tumor microenvironment. [vii] Enhancement of NK cell metabolism resulting in better performance in all aspects of NK cell function, including but not limited to NK cell cytotoxicity, reduced NK cell exhaustion, effective interfacing between the innate and adaptive immune systems, or regulating the overall immune response through immune amplification or immune homeostasis. [viii] Improved cryopreservation of NK cells leading to improved logistics, supply chain, and efficacy of NK cell therapeutics. Associated benefits include easier direct thawing of frozen NK cells into low serum (or serum-free) media conditions. Benefits also include enhanced cell motility or migration of NK cells, which in turn positively impacts cryopreservation of NK cells. [ix] Improved transduction of NK cells, e.g., using viral vectors, for better transduction efficiency, resulting in genetically engineered or modified NK cell therapeutics (e.g., CAR-NK cells; CAR: chimeric antigen receptor), which are more effective or the identification of the highest performing clonal CAR-NK populations is facilitated by the present invention. [x] Improvements in transfection of NK cells, for example to use non-viral methods such as electroporation. [xi] Modulation of NK cell subset distribution and relative populations, for example, based on the effects of the present invention on NK cell surface receptors (e.g., activating and inhibitory receptors) or NK cell memory. This may provide the benefit of influencing NK cell performance in vivo (e.g., in the tumor microenvironment). [xii] Differentiation of stem or progenitor cells into NK cells can be made more effective and efficient by the present invention, resulting in more effective NK cell therapeutics (e.g., iPS cell-derived NK cells with higher biological activity; iPS cells are induced pluripotent stem cells).

[0077] In addition to the above summarized applications of the present invention specifically relating to NK cell immunotherapeutics, additional applications of the present invention may be provided through the following.

[0078] [xiii] Biological functions of other cells of the immune system, i.e., enhanced cellular immunity exhibited by immune cells other than NK cells, such as T cells, monocytes, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils (including subsets and combinations thereof), which can confer cellular immunity. Also included are certain subsets of immune cells, such as natural killer T cells (NK-T cells) and gamma-delta (γδ) T cells. A specific example is the enhanced phagocytosis exhibited by macrophages when cultured in the preferred embodiment of the medium formulation described in the present invention (among several possible examples). Better therapeutic outcomes can be achieved for single agent or combination therapy. [xiv] Enhancement of the effectiveness of humoral immunity, for example through improvement of the biological function of B cells of the immune system that generate antibodies against specific antigens. Better therapeutic outcomes can be achieved for monotherapy or combination therapy. [xv] Use of the invention as a direct therapeutic agent, not for culturing immune cells ex vivo, for example when a formulation resulting from the invention is directly injected in vivo (e.g. into a tumor to induce better immune function against cancer). [xvi] The use of the present invention for in vitro diagnostics using immune cells, whereby the biological function of the immune cells is enhanced and improved sensitivity of the in vitro diagnostics is achieved, for example for the purpose of developing personalized immunotherapeutic regimens. [xvii] Enhancement of biological function of non-immune cells (specific examples include hybridoma cells, CHO cells (CHO: Chinese Hamster Ovary), etc.), which can provide the benefit of improved productivity in the biomanufacturing of polymer biotechnology products.

[0079] Having given a description of the present invention above and particularly summarized with respect to the preferred embodiments corresponding to Table 01, the following next section highlights, by way of exemplary experimental data, some examples of implementation and the illustration of the applicability of the preferred and other embodiments of the present invention. EXAMPLES

[0080] Experimental Example The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention is not limited to these examples, but rather encompasses all variations that become evident as a result of the teachings provided herein.

[0081] Example 1: Enhancement of NK cell cytotoxicity by Enable-NK medium Enable-NK medium enhances the cytotoxicity of natural killer (NK) cells against diseased cells such as cancer cells. The enhancement of NK cell cytotoxicity facilitated by Enable-NK medium is comprehensive and covers all three broad mechanisms of NK cell cytotoxicity: [a] innate / cell-mediated cytotoxicity independent of antigen recognition, [b] ADCC (antibody-dependent cellular cytotoxicity) based on antigen recognition, and [c] signaling to other immune cells for amplification of immune responses. As exemplified by the experimental data disclosed herein, Enable-NK medium enhances the appearance of all three of these cytotoxic mechanisms of NK cells.

[0082] Experimental methodology: To experimentally evaluate the impact of Enable-NK medium on natural killer (NK) cell cytotoxicity, the methodology centered on the in vitro quantification of cancer cell viability when co-cultured in the presence of NK cells. The ratio of effector NK cells to target cancer cells (effector:target ratio, i.e., E:T ratio), and the duration of co-culture are examples of experimental factors that directly affect the outcome of such co-culture assays; the higher the E:T ratio and the longer the duration of co-culture incubation, the higher the cytotoxicity of the target cancer cells.

[0083] With the above experimental framework at its core, experimental determination of the specific impact of Enable-NK medium on NK cell cytotoxicity was based on quantification of relative cytotoxicity between experimental groups, where the experimental groups differed only in the medium composition for the culture / expansion of NK cells before using the cultured / expanded NK cells in the co-culture assay with the cancer cells. As outlined in Figures 4-8, a comparison of medium composition was performed, e.g., comparing DMEM-F12 medium (with higher serum concentration) with DMEM-F12 medium combined with Enable-NK components (with lower serum concentration) by co-culture assay incubations (with NK effector cells and target cancer cells) performed in the respective medium conditions (Figures 4, 7, 8). Another example is a comparison of RPMI-1640 medium and Enable-NK medium for NK cell expansion (both with the same serum levels) by co-culture assay incubation (with NK effector cells and target cancer cells) performed in identical media conditions (DMEM, different from either of the two experimental groups), as illustrated in Figures 5 and 6. The NK cells used in these co-culture assays were the human NK cell line KHYG1 (Figures 4, 7) and primary human NK cells derived from healthy adult donors (Figures 4-8). The target cancer cells used were K562 leukemia cells (Figures 4, 7), CHLA-20 neuroblastoma cells (Figure 5), and M21 melanoma cells (Figure 6).

[0084] Endpoint quantification of target cell viability was investigated by propidium iodide (PI) staining assessed through flow cytometry (Figure 4) and also by biomarker quantification (Figure 8), where carboxyfluorescein succinimidyl ester (CFSE) staining was used to distinguish target cells from effector cells. Target cell viability was also determined by quantification of green fluorescent protein (GFP) in viable target cancer cells through the Sartorius Incucyte® live cell analysis system (Figures 5, 6). Quantification of soluble interferon-gamma (IFNγ) in NK cell supernatant samples (Figure 7) was investigated using a Luminex Magpix instrument.

[0085] Key Results: Media conditions for Figure 4 were as follows: Control media was DMEM / F12 [20% (v / v) fetal bovine serum (FBS) for Figure 4A, 10% human AB serum for Figure 4B]. Enable-NK media for Figure 4A (KHYG1 NK cells) was DMEM / F12 with proprietary Enable-NK components [final FBS concentration of 5% (v / v) for Part I alone, 3.5% (v / v) for Part II media (after Part I incubation)]. Enable-NK media for Figure 4B (primary NK cells) was DMEM / F12 with proprietary Enable-NK components [final human AB serum concentration of 2.5% (v / v)]. Co-culture incubations with K562 cells for the different experimental groups were set up in the respective media at the same E:T ratio (5:1).

[0086] The data summarized in Figure 5 illustrates that the observed primary NK cell cytotoxicity against the target cancer cell line CHLA-20 was greater with Enable-NK medium in direct comparison with control medium (RPMI-1640). This is indicated by the lower y-axis values ​​(number of target cells remaining in the co-culture) for the Enable-NK medium condition when compared with the corresponding control medium condition. The greater NK cell cytotoxicity in response to Enable-NK medium holds true whether or not primary NK cell expansion was investigated in the presence of K562 feeder cells and whether or not anti-GD2 antibodies (ADCC) were used.

[0087] The data summarized in Figure 6 illustrates that the primary NK cell cytotoxicity observed against the target cancer cell line M21 was greater in Enable-NK medium when directly compared to control medium (RPMI-1640). This is indicated by the lower y-axis values ​​(number of target cells remaining in the co-culture) for the Enable-NK medium condition when compared to the corresponding control medium condition. The greater NK cell cytotoxicity in response to Enable-NK medium holds true whether or not primary NK cell expansion was investigated in the presence of K562 feeder cells and whether or not anti-GD2 antibodies (ADCC) were used.

[0088] The media conditions for Figure 7 were as follows: Control media was DMEM / F12 [20% (v / v) fetal bovine serum (FBS) for Figure 7A, 10% human AB serum for Figure 7B]. Enable-NK media for Figure 7A (KHYG1 NK cells) was DMEM / F12 with proprietary Enable-NK components [final FBS concentration of 2% (v / v) in part II media (after serum was gradually reduced from 20% to 2% in part I incubation)]. Enable-NK media for Figure 4B (primary NK cells) was DMEM / F12 with proprietary Enable-NK components [final human AB concentration of 2.5% (v / v) in part II media (after serum was gradually reduced from 10% to 2.5% in part I incubation)]. NK cells were expanded in the respective media for 1 week and then incubated overnight. Corresponding to the primary NK cell data (Figure 7B, monoculture), several cell surface biomarkers associated with NK cell activation were also quantified by flow cytometry and these are summarized below in Figure 8.

[0089] The data summarized in Figure 8 correspond to primary NK cells from the same experiment as for Figure 7, and the experimental conditions for media composition and NK cell incubation in these media conditions were identical. NKG2D, NKp46, and CD69 are activation markers on the surface of NK cells, and CD16 is also responsible for NK cell cytotoxicity and is central to the mechanism of ADCC (antibody-dependent cellular cytotoxicity).

[0090] Discussion, Conclusions, and Implications for NK Cell Therapeutics: As illustrated in Figures 4-8, three distinct but complementary aspects of NK cell cytotoxicity are enhanced by Enable-NK Medium for both the KHYG1 NK cell line and primary NK cells: [i] natural cytotoxicity, [ii] antibody-dependent cellular cytotoxicity (ADCC), and [iii] NK signaling to other immune cells (to recruit other immune cells and amplify the overall immune response against cancer).

[0091] The biological significance of enhanced NK cell cytotoxicity is the enhanced efficacy of natural killer immunotherapeutics against cancer and other diseases (Myers et al., 2021; Shimasaki et al., 2020). Specifically, there are three broad distinct but complementary mechanisms of NK cell cytotoxicity against disease cells: [i] natural / cell-mediated cytotoxicity (Kumar et al., 2022), [ii] ADCC (antibody-dependent cell-mediated cytotoxicity, Ochoa et al., 2017), and [iii] NK cell signaling through secretion of cytokines such as interferon-gamma (IFNγ) and recruitment of other immune cells (e.g., T cells) (Hodgins et al., 2019). When the balance between activating and inhibitory receptors on the NK cell surface is shifted in favor of activating receptors, natural cytotoxicity is enhanced, and the data in this section (Example 1) and the next section (Example 2) exemplify this change in balance, which is facilitated by Enable-NK Media. Modulation of specific subpopulations of NK cells (data from Example 2) also impacts NK cell cytotoxicity. Enhancement of all three of these mechanisms of NK cell cytotoxicity by Enable-NK medium provides an opportunity for synergistic effects and broad clinical applicability of NK cell therapeutics for various diseases, including cancer (Kumar et al., 2022; Nigro et al., 2019).

[0092] In summary, the experimental results summarized in this Example provide evidence that Enable-NK media formulation can support greater efficacy of NK cell therapy by enhancing NK cell cytotoxicity and NK cell signaling to other immune cells.

[0093] References for Example 1: TIFF2024529565000007.tif105164

[0094] Example 2: Reduction of NK cell exhaustion by Enable-NK medium Enable-NK medium reduces the exhaustion of natural killer (NK) cells and also reduces other mechanisms of NK cell dysfunction, namely anergy and senescence. NK cell dysfunction is accompanied by [a] a decrease in the effector function of NK cells against disease cells (e.g., cancer cells), i.e., a decrease in cytotoxic activity, and [b] cell exhaustion and / or anergy and / or senescence, as represented by modification of the expression levels of biomarkers, i.e., an increase in PD-1, TIM-3, LAG-3, TIGIT, NKG2A, and a decrease in CD16 and CD57. As illustrated by the experimental data summarized below, Enable-NK medium reduces the appearance of these outcomes of NK cell dysfunction.

[0095] Experimental methodology: The experimental methodology was based on performing long-term co-cultures of NK cells with target cancer cells in G-Rex® [Gas Permeable Rapid Expansion] plates from Wilson Wolf Manufacturing Corporation (St Paul, MN). The membrane-based G-Rex® platform facilitates gentle medium exchange that does not disturb the cells placed on the membrane, thus allowing the evaluation of the same co-culture samples over a longer period of time.

[0096] In separate experiments, both KHYG-1 NK cells and primary NK cells (derived from three healthy adult donors) were cultured in either control medium (DMEM / F12) or Enable-NK medium (DMEM / F12+Enable-NK components / additives) before co-culture experiments with K562-GFP leukemia cells at an effector:target (E:T) ratio of 1:1 were set up. For experiments with the KHYG-1 cell line, culture in Enable-NK medium was first investigated for 7 days in Enable-NK part I medium (DMEM / F12+Enable-NK part I components / additives) followed by 1 day in Enable-NK part II medium (DMEM / F12+Enable-NK part II components / additives). Then, on day 0, 3 million effector cells and 3 million target cells were added to each well to establish 1:1 co-cultures in separate wells of G-Rex 24-well plates (Wilson Wolf, P / N 80192M). Fetal bovine serum (FBS) content was maintained at 20% (v / v) in control medium and gradually decreased from 20% (v / v) to 5% (v / v) in Enable-NK part I medium, then transitioned to Enable-NK part II medium with 5% FBS content. For experiments with primary NK cells (from three donors), culture in Enable-NK medium was investigated first for 3 days in Enable-NK part I medium, followed by 1 day in Enable-NK part II medium. Then, 1:1 co-cultures were established in separate wells of G-Rex 24-well plates by adding 2 million effector cells and 2 million target cells to each well on day 0. Serum content was maintained at 10% (v / v) human AB serum for both control medium (DMEM / F12) and Enable-NK medium (DMEM / F12+Enable-NK components / additives).

[0097] Target K562-GFP leukemia cells were distinguished from effector NK cells by green fluorescent protein (GFP) fluorescence. K562-GFP cells were obtained from the American Type Culture Collection (ATCC, catalog number CCL-243-GFP). Propidium iodide (PI) staining was used to determine cell viability. Fluorescently labeled antibodies against various cell surface proteins (e.g., PD-1, TIM-3, LAG-3, TIGIT, etc.) were used for detection, and flow cytometry analysis was performed (with appropriate steps for gating and compensation) at the University of Connecticut Health Flow Cytometry Facility.

[0098] Main Results: The main experimental results are summarized in Figures 9 and 10 for the NK cell line KHYG-1 and in Figures 11-16 for the experiments with primary NK cells.

[0099] The two media conditions for Figures 9 and 10 were as follows: Control media was DMEM / F12 with 20% (v / v) fetal bovine serum (FBS). Enable-NK media was DMEM / F12 with a final serum concentration of 5% (v / v) fetal bovine serum (FBS) containing the proprietary Enable-NK additive.

[0100] The two media conditions for Figures 11 and 12 were as follows: Control media was DMEM / F12 with 10% (v / v) human AB serum (HS). Enable-NK media was DMEM / F12 with 10% (v / v) human AB serum (HS) with the proprietary Enable-NK additive.

[0101] The two media conditions for Figures 13 and 14 were as follows: Control media was DMEM / F12 with 10% (v / v) human AB serum (HS). Enable-NK media was DMEM / F12 with 10% (v / v) human AB serum (FBS) with the proprietary Enable-NK additive.

[0102] The two media conditions for Figures 15 and 16 were as follows: Control media was DMEM / F12 with 10% (v / v) human AB serum (HS). Enable-NK media was DMEM / F12 with 10% (v / v) human AB serum (FBS) with the proprietary Enable-NK additive.

[0103] Discussion, Conclusions, and Implications for NK Cell Therapeutics: The primary endpoints indicative of cell exhaustion were [i] attenuation of cytotoxicity and [ii] increased expression levels of all or some of five inhibitory cell surface biomarkers, namely PD-1, TIM-3, LAG-3, TIGIT, and NKG2A. As illustrated in Figures 9-12, both of these endpoint trends were mitigated by culturing NK cells in Enable-NK medium, and this mitigation of the NK cell exhaustion phenotype was observed in both the NK cell line KHYG-1 (Figures 9-10) and primary NK cells (Figures 11-12).

[0104] The biological significance of the reduction in NK cell dysfunction (exhaustion and / or anergy and / or senescence) is an increased likelihood of solid tumor infiltration by NK cells, and a reduction in the dysfunction of NK cells in the tumor microenvironment (Judge et al., 2020 and Wang et al., 2020). Based on the data presented in Figures 9-14, Enable-NK reduces NK cell exhaustion (reduced levels of PD-1, TIM-3, LAG-3, and TIGIT), reduces NK cell anergy (higher levels of CD16 expression), and also reduces NK cell senescence (reduced NKG2A expression and increased CD57 expression). Furthermore, the CD56 expression supported by Enable-NK dim Subpopulations (Figures 10, 12, 16), CD56 dim CD16 + CD57 + Subpopulation (Figure 13), CD56 dim CD57 + NKG2A - subpopulations (Figure 14), and CD56 dim CD16 +The biological significance of the higher levels of subpopulations (Figure 15) is the higher levels of cytotoxicity displayed by these subpopulations of NK cells (Lopez-Verges et al., 2010; Beziat et al., 2010; Haanen et al., 2018).

[0105] In summary, the experimental results summarized in this Example provide evidence that Enable-NK Media Formulation can support greater efficacy of NK cell therapeutics by mitigating NK cell dysfunction manifested by exhaustion and / or anergy and / or senescence.

[0106] References for Example 2: TIFF2024529565000008.tif91163

[0107] Example 3: Enhancement of NK cell metabolism by Enable-NK medium Enable-NK medium enhances the metabolism of natural killer (NK) cells, an effect consistent with the alleviation of NK cell exhaustion (covered in Example 2). The metabolic activity of NK cells is represented by the level of intracellular NAD (nicotinamide adenine dinucleotide). As illustrated by the experimental data summarized below, the metabolic activity of NK cells, represented by NAD levels, is enhanced by Enable-NK medium.

[0108] Experimental methodology: KHYG-1 NK cells were cultured in control medium (DMEM / F12) or Enable-NK medium (DMEM / F12+Enable-NK components / additives). Culture of NK cells in Enable-NK medium was investigated first in Enable-NK part I medium (DMEM / F12+Enable-NK part I components / additives) for 3 days, followed by Enable-NK part II medium (DMEM / F12+Enable-NK part II components / additives) for an additional 4 days (total incubation time of 1 week). For experiments with KHYG-1 NK cells, the fetal bovine serum (FBS) content was maintained at 20% (v / v) in control medium and gradually decreased from 20% (v / v) to 5% (v / v) in Enable-NK part I medium, then transferred to Enable-NK part II medium with 5% FBS content.

[0109] The method for detection of NAD (nicotinamide adenine dinucleotide) was through quantification of the total amount of both oxidized and reduced forms of NAD (NAD+ / NADH) as measured by a bioluminescence-based kit from Promega Corporation (Madison WI, Catalog No. G9071). Total intracellular NAD was normalized to that observed in control medium (NT, i.e., no treatment).

[0110] Results: The intracellular NAD (nicotinamide adenine dinucleotide) levels for the two medium conditions, i.e., NAD values ​​normalized to the control medium, are summarized in FIG. 17 and showed a greater than three-fold difference between the two medium conditions.

[0111] The two media conditions for Figure 17 were as follows: Control medium was DMEM / F12 with 20% (v / v) fetal bovine serum (FBS). Enable-NK medium was DMEM / F12 with a final serum concentration of 5% (v / v) fetal bovine serum (FBS) containing the proprietary Enable-NK supplement.

[0112] Discussion, conclusions, and implications for NK cell therapeutics. In this study, normalized NAD measurements are an indicator of NK cell metabolic robustness. Nicotinamide adenine dinucleotide (NAD) is a coenzyme involved in the regulation of various metabolic pathways, including glycolysis, β-oxidation, and oxidative phosphorylation. NAD also serves as a substrate for poly(ADP-ribose) polymerase (PARP), sirtuins, and NAD glycohydrolase. In addition, NAD controls DNA repair, gene expression, energy metabolism, and stress response (Yaku et al., 2019). In general, nicotinamide adenine dinucleotide (NAD) and its metabolome (NADome) play a critical role in maintaining cellular homeostasis (Braidy et al., 2021). The relevance of NAD with NK cell immune metabolism, for example with respect to energy metabolism in general and mitochondrial metabolism specifically, is well established (O'Brien et al., 201; Choi et al., 2021; and Stein et al., 2012), providing the basis for the therapeutic potential of NAD metabolism (Xie et al., 2020; Takeda et al., 2021).

[0113] Given the negative impact of the tumor microenvironment (TME) on NK cell metabolism, the biological significance of enhancing NK cell metabolism is the enhanced efficacy of natural killer immunotherapeutics against solid tumors (Terren et al., 2019). Activation of NK cells for antitumor effector function appears as enhanced metabolic activity, such as upregulation of glycolysis and nutrient transporters, and thus enhancing NK cell immunometabolism in pathophysiological conditions may enhance the clinical efficacy of NK cell immunotherapeutics (Wang et al., 2020, Terren et al., 2020).

[0114] In summary, the experimental results summarized in this Section C provide evidence that Enable-NK media formulations can support greater efficacy of NK cell therapeutics by enhancing NK cell metabolism.

[0115] References for Example 3: TIFF2024529565000009.tif165164

[0116] Example 4: Enhancement of NK cell viral transduction by Enable-NK medium As illustrated by the experimental data summarized below, Enable-NK enhances the efficiency of viral transduction of natural killer (NK) cells, as exemplified by retroviral transduction of the NK cell line NK-92.

[0117] Experimental methodology: Viral transduction of NKp44-FLAG construct into NK-92 cells was investigated via a bicistronic retroviral vector containing an internal ribosome entry site (IRES) [NKp44 tagged with FLAG (extracellular end) was placed into the pBMN-IRES-GFP vector (with GFP edited out), Phoenix-Ampho viral packaging cell line]. NK-92 cells were cultured in α-MEM medium or Enable-NK part II medium for 2 weeks before transduction and then cultured in the two media for an additional 6 days. Both media conditions had 100 IU / ml IL-2. α-MEM medium had 10% (v / v) heat-inactivated FBS and 10% (v / v) horse serum, while Enable-NK phase II medium had 5% (v / v) heat-inactivated FBS. Cells were then stained for both NKp44 and FLAG and analyzed (flow cytometry) to determine transduction efficiency (percent of cells dually stained for both NKp44 and FLAG out of the total NK cell population).

[0118] Results: Figure 18 shows a comparison of transduction efficiency in NK-92 cells in α-MEM medium (21.0%) compared to Enable-NK part II medium (62.8%) as confirmed by double staining for both NKp-44 and FLAG. In contrast, parental cells showed no significant staining (<1%).

[0119] Discussion, conclusions, and implications for NK cell therapeutics. Significantly higher transduction efficiency (62.8%) was achieved for retroviral transduction of NK-92 cells with Enable-NK part II medium compared to α-MEM medium (21.0%).

[0120] The biological significance of enhanced viral transduction of NK cells is the opportunity to enhance the efficacy of natural killer immunotherapeutics through genetic engineering of NK cells (Childs et al., 2015; Matosevic, 2018). Examples of specific genetic engineering strategies for NK cells include [a] better autocrine cytokine stimulation of NK cells by introduction of genes (e.g., IL-2, IL-15, which may eliminate dependency on exogenous cytokines; [b] better tumor targeting induced by genetic silencing of inhibitory NK cell receptors (e.g., NKG2A); and [c] improved tumor targeting against specific antigens by genetic engineering of CARs (chimeric antigen receptors) on the surface of NK cells (Carlsten et al., 2015; Xie et al., 2020). It is more difficult to achieve viral transduction of natural killer (NK) cells for the purpose of genetic engineering, compared to T cells (Allan et al., 2021; Carlsten et al., 2016). Specific examples include low efficiency of viral transduction and reduced viability of virally transduced NK cells, which are barriers to the generation of next-generation NK cell immunotherapeutics. Thus, improving the efficiency and efficacy of viral transduction of NK cells may lead to a broader range of NK cell therapeutics and improved efficacy of NK cell therapeutics.

[0121] In summary, the experimental results summarized in this Example provide evidence that Enable-NK media formulation can support greater efficacy of NK cell therapeutics by enhancing the efficiency of viral transduction of NK cells.

[0122] References for Example 4: TIFF2024529565000010.tif118165

[0123] Example 5: Improved cryopreservation of NK cells with Enable-NK medium Enable-NK improves the cryopreservation of natural killer (NK) cells, as exemplified by the experimental data summarized below on the cryopreservation performance of primary NK cells.

[0124] Experimental methodology: Primary NK cells were isolated from healthy adult human donors and expanded in control medium [RPMI-1640 with 1:1 K562 feeder cells (containing membrane-bound IL-15 and 4-1BBL)] or Enable-NK Phase I medium (without feeder cells) with fresh medium added every 2-3 days for 2 weeks (switched from Enable-NK Phase I to Phase II medium on day 12). Both media conditions had 10% v / v human AB serum FBS and 100 IU / ml IL-2 (plus 10 ng / ml IL-15 for Enable-NK medium). Expanded NK cells (magnetically separated after expansion in control medium with feeder cells) were frozen in the respective freezing medium. NK cells expanded in control medium were frozen in a freezing medium recipe of [10% v / v DMSO + 45% v / v FBS + 45% v / v control medium], and NK cells expanded in Enable-NK part I medium were frozen in a freezing medium recipe of [10% v / v DMSO + 45% v / v FBS + 45% v / v Enable part I medium]. NK cells frozen in a freezing medium recipe of [10% v / v DMSO + 45% v / v FBS + 45% v / v control medium] were thawed overnight in control medium (without feeder cells), and NK cells frozen in a freezing medium recipe of [10% v / v DMSO + 45% v / v FBS + 45% v / v Enable part I medium] were thawed overnight in Enable part II medium (without feeder cells). 10 IU / ml IL-2 was added to the overnight cultures. The next day, NK effector cells from both experimental groups were co-cultured with human pediatric neuroblastoma cells CHLA-20-GFP target cells at an effector:target ratio (E:T ratio) of 2.5: 1. These co-culture studies were investigated in identical DMEM media conditions (containing 10% v / v FBS, 100 IU / ml IL-2) with and without anti-disialoganglioside (anti-GD2) antibodies for cytotoxicity analysis via a Sartorius Incucyte® live cell analyzer.

[0125] Results: Data obtained from the Sartorius Incucyte® instrument are summarized in FIG.

[0126] Discussion, conclusions, and implications for NK cell therapeutics. Significantly higher cryopreservation performance was demonstrated by Enable-NK™ medium compared to control medium both in the absence (natural cytotoxicity) and in the presence (natural + antibody-dependent cellular cytotoxicity (ADCC)) of anti-GD2 antibodies. The biological significance of improved cryopreservation of NK cells is an opportunity to make off-the-shelf NK cell therapeutics more performant for clinical use (Fang et al., 2022). Based on in vitro studies (Mark et al., 2020; Damodharan et al., 2020; Min et al., 2018), in vivo studies (Miller et al., 2014), and clinical studies (Szmania et al., 2015), it is well documented in the published literature that cryopreservation (i.e., freezing) and subsequent thawing of NK cells negatively impacts key functions (e.g., cytotoxicity) and viability to a much greater extent than cryopreservation of T cells (Fang et al., 2019). These considerations, together with the much shorter half-life of NK cells, represent substantial limitations to the logistics of clinical use of NK cell therapeutics. There is consensus in the field of NK research that continued improvements in NK cell cryopreservation are critical if NK cell therapeutics are to realize their full clinical potential (Kundu et al., 2021).

[0127] In summary, the experimental results summarized in this Section E provide evidence that Enable-NK media formulations can support greater efficacy of NK cell therapeutics through improved cryopreservation of NK cells.

[0128] References for Example 5: TIFF2024529565000011.tif165164

[0129] Example 6: Reducing serum dependency and feeder cell use for the culture and expansion of NK cells with Enable-NK medium Enable-NK Medium supports the culture and expansion of natural killer (NK) cells such that: [a] animal serum dependency is minimized, and [b] the need for feeder cells is minimized or eliminated. These benefits for the culture and expansion of natural killer (NK) cells are exemplified by the ex vivo expansion of the human NK cell line KHYG1 and primary human NK cells (derived from a healthy adult donor) using Enable-NK Medium, as summarized by the experimental results below.

[0130] Experimental Methodology: NK cell lines NK3.3 and KHYG1, as well as primary NK cells derived from healthy adult donors, were cultured in the media conditions summarized below: NK cells were counted using a hemocytometer or a Countess-II instrument (Thermo Fisher, Waltham MA).

[0131] Results: Some key results of NK cell proliferation are shown in Figure 20. For Figure 20A, control medium was standard medium [with 10% (v / v) human AB serum] and Enable-NK medium was DMEM / F12 with the proprietary Enable-NK additive with reduced serum levels shown in Figure 20A, both containing 50 IU / ml IL-2. For Figures 20B and 20C, control medium was DMEM / F12 [with 20% (v / v) fetal bovine serum (FBS) for Figure 20B and 10% (v / v) human AB serum for Figure 20C] and Enable-NK medium was DMEM / F12 with the proprietary Enable-NK additive with reduced serum levels shown in Figures 20B and 20C, both containing 100 IU / ml IL-2.

[0132] Discussion, Conclusions, and Implications for NK Cell Therapy: Cumulatively, the experimental results for this example demonstrate that NK cells can be cultured and expanded in Enable-NK medium containing significantly reduced serum, in the absence of feeder cells.

[0133] The biological significance of reducing serum dependency in natural killer (NK) cell culture and expansion is the opportunity to avoid challenges presented by serum content, such as batch-to-batch variability, cost, and risk of pathogen contamination (Karnieli et al., 2017). With these considerations, reducing serum dependency is a major process development goal for cellular therapeutics in general (Campbell et al., 2015) and NK cell therapeutics specifically (Moseman et al., 2020). Similarly, for ex vivo culture and expansion of natural killer (NK) cells, the use of feeder cells facilitates the achievement of large fold expansion following NK cell expansion (Gurney et al., 2022), but the use of feeder cells introduces drawbacks such as suboptimal cytotoxic performance of expanded NK cells (Childs et al., 2013) and the potential contamination of live feeder cells into the resulting NK cell therapeutics (Kundu et al., 2021).

[0134] In summary, the experimental results summarized in this Example provide evidence that Enable-NK medium formulations can support greater efficacy of NK cell therapy by culturing and expanding NK cells with reduced or no serum. These results also provide evidence that Enable-NK medium formulations can support greater efficacy of NK cell therapy by culturing and expanding NK cells in the absence or with reduced feeder cells. The opportunity to culture and expand NK cells with reduced / no serum and reduced / no feeder cells, i.e., a combined synergistic benefit, is also provided by Enable-NK medium.

[0135] References for Example 6: TIFF2024529565000012.tif105165

[0136] Enumerated Aspects Enumerated aspects are provided below, the numbering of which should not be construed as indicating importance.

[0137] Aspect 1 is a two-part media formulation for culturing natural killer (NK) cells, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and (b) a second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania Somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella Japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L Including, The NK cells are cultured in a first media formulation and then in a second media formulation; A two-part media formulation is provided.

[0138] Embodiment 2 provides a one-part media formulation for culturing natural killer (NK) cells, comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) Antioxidant supplement at concentrations ranging from 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalogue no. A1345 or equivalent).

[0139]

[0013] Embodiment 3 provides a one-part media formulation for culturing natural killer (NK) cells, comprising at least nine of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), (Liii) antioxidant supplement at concentrations between 0.01x and 1000x (antioxidant supplement is Sigma-Aldrich catalogue no. A1345 or equivalent); (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract at a concentration of 1.06E-01 mg / L to 1.06E+04 mg / L.

[0140] Aspect 4 provides the formulation of any of aspects 1-3, added as a supplement to a basal cell culture medium.

[0141] A fifth aspect of the present invention relates to a method for culturing natural killer (NK) cells or precursor cells thereof, the method comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and (b) a second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L In another aspect, the present invention provides a method for the treatment of NK cells, comprising culturing NK cells or precursor cells thereof in a cell culture medium formulation comprising:

[0142] Embodiment 6 provides the method of embodiment 5, wherein the NK cells or precursor cells thereof are cultured in a first media formulation.

[0143] Embodiment 7 provides the method of embodiment 5, wherein the NK cells or precursor cells thereof are cultured in a second media formulation.

[0144] Embodiment 8 provides the method of embodiment 5, wherein the NK cells, or precursor cells thereof, are cultured in a first media formulation and then in a second formulation.

[0145] Embodiment 9 provides the method of embodiment 5, wherein the NK cells or precursors thereof are cultured ex vivo.

[0146] Embodiment 10 provides the method of embodiment 5, wherein the NK cells or precursors thereof are cultured in vitro.

[0147] Embodiment 11 provides the method of embodiment 5, wherein the cytotoxic activity of the NK cell against the target cell is enhanced.

[0148] Example 12 provides the method of example 11, wherein the target cell is a cancer cell.

[0149] Example 13 provides the method of example 5, wherein activation of NK cells is enhanced.

[0150] Example 14 provides the method of example 5, wherein NK cell dysfunction is reduced.

[0151] Example 15 provides the method of Example 14, wherein the impairment is fatigue.

[0152] Example 16 provides the method of Example 5, wherein the metabolic function of the NK cells is enhanced.

[0153] Embodiment 17 provides the method of embodiment 5, wherein high viability and cell expansion of NK cells is achieved.

[0154] Example 18 provides the method of example 5, wherein the use of serum is eliminated or reduced.

[0155] Example 19 provides the method of example 5, wherein the use of cytokine supplements is eliminated or reduced.

[0156] Embodiment 20 provides the method of embodiment 5, wherein the use of antigen presenting cells (APCs) or feeder cells is eliminated or reduced.

[0157] Example 21 provides the method of example 5, wherein culturing the NK precursor cells induces their differentiation.

[0158] Embodiment 22 provides the method of embodiment 5, wherein culturing the NK cells or progenitor cells modulates the distribution and relative populations of NK cell subsets.

[0159] Example 23 provides the method of example 22, wherein the NK cell subset is memory NK cells.

[0160] Embodiment 24 provides the method of embodiment 22, wherein the NK cell subsets are defined by the relative expression of activating and inhibitory receptors.

[0161] Embodiment 25 is a method for cryopreservation of natural killer (NK) cells, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and (b) a second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L The method further comprises freezing the NK cells in a cryopreservation medium comprising:

[0162] Example 26 provides the method of example 25, wherein the cryopreservation medium comprises a first medium formulation.

[0163] Example 27 provides the method of example 25, wherein the cryopreservation medium comprises a second medium formulation.

[0164] Embodiment 28 is a method for transduction of natural killer (NK) immune cells, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and A second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L a first cell culture step comprising culturing NK cells in a cell culture medium comprising: (b) contacting the NK cell with a viral vector; and (c) a second cell culture step, comprising culturing the transduced NK cells in a cell culture medium comprising the cell culture medium formulation of step (a). The method includes:

[0165] Example 29 provides the method of example 28, wherein the first and second cell culture steps are carried out in a first cell culture preparation.

[0166] Embodiment 30 provides the method of embodiment 28, wherein the first and second cell culture steps are performed in a second cell culture preparation.

[0167] Example 31 provides the method of example 28, wherein the first cell culture step is performed in a first cell culture preparation and the second cell culture step is performed in a second cell culture preparation.

[0168] Example 32 provides the method of example 28, wherein the vector encodes a chimeric antigen receptor (CAR).

[0169]

[0023] Aspect 33 is a method for differentiating natural killer (NK) cells from progenitor cells, comprising: (a) a first media formulation comprising at least six of the following components: (i) glycine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (ii) L-alanine at a concentration between 1.10E-02 mM and 1.10E+03 mM; (iii) L-asparagine at a concentration between 1.50E-03 mM and 1.50E+02 mM; (iv) L-aspartic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (v) L-glutamic acid at a concentration of 1.50E-03 mM to 1.50E+02 mM; (vi) L-proline at a concentration between 2.50E-03 mM and 2.50E+02 mM; (vii) L-serine at a concentration between 3.50E-03 mM and 3.50E+02 mM; (viii) L-arginine HCl at a concentration between 1.30E-02 mM and 1.30E+03 mM; (ix) L-cystine at a concentration of 2.00E-03 mM to 2.00E+02 mM; (x) L-histidine HCl-H at a concentration of 3.50E-03mM to 3.50E+02mM 2 O; (xi) L-isoleucine at a concentration of 8.20E-03 mM to 8.20E+02 mM; (xii) L-leucine at a concentration between 8.50E-03 mM and 8.50E+02 mM; (xiii) L-lysine hydrochloride at a concentration of 8.96E-03 mM to 8.96E+02 mM; (xiv) L-methionine at a concentration of 2.21E-03 mM to 2.21E+02 mM; (xv) L-phenylalanine at a concentration between 4.20E-03 mM and 4.20E+02 mM; (xvi) L-threonine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xvii) L-tryptophan at a concentration between 9.00E-04 mM and 9.00E+01 mM; (xviii) L-tyrosine at a concentration of 4.09E-03 mM to 4.09E+02 mM; (xix) L-valine at a concentration of 8.50E-03 mM to 8.50E+02 mM; (xx) sodium pyruvate at a concentration between 1.00E-02 mM and 1.00E+03 mM; (xxi) arachidonic acid at a concentration of 3.28E-07 mM to 3.28E-02 mM; (xxii) linoleic acid at a concentration between 3.78E-06 mM and 3.78E-01 mM; (xxiii) linoleic acid at a concentration of 1.80E-06 mM to 1.80E-01 mM; (xxiv) myristic acid at a concentration of 2.19E-06 mM to 2.19E-01 mM; (xxv) oleic acid at a concentration of 1.77E-06 mM to 1.77E-01 mM; (xxvi) palmitic acid at a concentration of 1.95E-06 mM to 1.95E-01 mM; (xxvii) stearic acid at a concentration of 1.76E-06 mM to 1.76E-01 mM; (xxviii) cholesterol at a concentration between 2.84E-05mM and 2.84E+00mM; (xxix) Tween-80 at a concentration of 8.40E-05 mM to 8.40E+00 mM; (xxx) tocopherol acetate at a concentration of 7.40E-06 mM to 7.40E-01 mM; (xxxi) Pluronic F-68 at concentrations between 5.95E-04mM and 5.95E+01mM; (xxxii) insulin having a concentration between 1.72E-05 mM and 1.72E+00 mM; (xxxiii) transferrin at a concentration of 6.88E-07 mM to 6.88E-02 mM; (xxxiv) sodium selenite at a concentration of 3.87E-07 mM to 3.87E-02 mM; (xxxv) myo-inositol at a concentration of 2.00E-03 mM to 2.00E+02 mM; (xxxvi) glucose at a concentration between 2.22E-01 mM and 2.22E+04 mM; (xxxvii) human serum albumin (HSA) at a concentration of 1.50E-05 mM to 1.50E+00 mM; (xxxviii) biotin at a concentration between 8.10E-06 mM and 8.10E-01 mM; (xxxix) folic acid at a concentration of 2.40E-04 mM to 2.40E+01 mM; (xL) Vitamin B12 at a concentration of 1.00E-02mM to 1.00E+03mM; Vitamin C at a concentration of (xLi)2.84E-03mM to 2.84E+02mM; (xLii) Vitamin E at a concentration of 2.40E-03mM to 2.40E+02mM; (xLiii) zinc at a concentration of 1.50E-08mM to 1.50E-03mM; (xLiv) L-ornithine at a concentration of 5.00E-04mM to 5.00E+01mM; (xLv) Creatine Anhydrous at a concentration of 1.00E-02mM to 1.00E+03mM; (xLvi) Polyvinyl alcohol (PVA) with a concentration of 1.16E-01 to 1.16E+04 mM; (xLvii) Ethanolamine at a concentration of 5.00E-04 to 5.00E+01 mM; (xLviii) 2-mercaptoethanol (2-ME) at concentrations between 1.00E-03mM and 1.00E+02mM; (xLix)Androstenedione at a concentration of 1.00E-05 to 1.00E+00 mM; (L) Dextran with a concentration of 1.00E+01 to 1.00E+06 mg / L; (Li) L-alanyl-L-glutamine (Glutamax) at concentrations of 2.50E-02mM to 2.50E+03mM; (Lii) Polyamine supplement at concentrations between 0.01x and 1000x of the 1000x stock (Polyamine supplement is Sigma-Aldrich catalog number P8483 or equivalent), and (Liii) antioxidant supplement at a concentration of 0.01x to 1000x (antioxidant supplement is Sigma-Aldrich catalog number A1345 or equivalent); and (b) a second media formulation comprising the first media formulation and at least three of the following components: (Liv) Nicotinamide at a concentration of 2.50E-02mM to 2.50E+03mM; (Lv) IGF-1 (insulin-like growth factor 1) at a concentration of 2.60E-08mM to 2.60E-03mM; (Lvi) 4-1BBL [4-1BB ligand or ligand for cluster of differentiation 137 (CD137)] at a concentration of 4.20E-09 mM to 4.20E-04 mM; (Lvii) Ashwagandha (botanical name: Withania somnifera) plant extract in a concentration of 6.00E-01mg / L to 6.00E+04mg / L; (Lviii) kumquat (botanical name: Fortunella japonica) plant extract with a concentration of 1.40E-22mg / L to 1.40E-02mg / L; (Lix) Uncaroabo (botanical name: Pelargonium sidoides) plant extract with a concentration of 5.00E-01mg / L~5.00E+05mg / L; (Lx) Echinacea (botanical name: Echinacea purpurea) plant extract with a concentration of 1.01E-01mg / L~1.00E+05mg / L; (Lxi) Panax ginseng (botanical name: Panax ginseng) plant extract with a concentration of 8.43E-02mg / L~8.43E+03mg / L; (Lxii) 2,3-butanediol at a concentration of 9.01E-02 mg / L to 9.01E+03 mg / L; and (Lxiii) Elderberry (botanical name: Sambucus nigra) plant extract with a concentration of 1.06E-01mg / L to 1.06E+04mg / L The method further comprises culturing the progenitor cells in a cell culture medium comprising:

[0170] Example 34 provides the method of example 33, wherein the culture medium comprises a first medium formulation.

[0171] Example 35 provides the method of example 33, wherein the culture medium comprises a second medium formulation.

[0172] Example 36 provides the method of example 35, wherein the culture medium comprises a first medium formulation, followed by a second medium formulation.

[0173] Example 37 provides the method of example 33, wherein culturing natural killer (NK) cells modulates the distribution and relative populations of NK cell subsets.

[0174] Example 38 provides the method of example 33, wherein the NK subset is memory NK cells.

[0175] Embodiment 39 provides the method of embodiment 33, wherein the NK cell subsets are defined by the relative expression of activating and inhibitory receptors.

[0176] Other Aspects The recitation of a list of elements in a definition of a variable herein includes definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with other embodiments or portions thereof.

[0177] The disclosures of each and every patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

[Claim 1] The invention described in the specification of this application.

Images