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WO2025104700A2 - Méthodes améliorées de cryoconservation et d'adaptation de cellules tueuses naturelles, et compositions associées - Google Patents

Méthodes améliorées de cryoconservation et d'adaptation de cellules tueuses naturelles, et compositions associées Download PDF

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Publication number
WO2025104700A2
WO2025104700A2 PCT/IB2024/061426 IB2024061426W WO2025104700A2 WO 2025104700 A2 WO2025104700 A2 WO 2025104700A2 IB 2024061426 W IB2024061426 W IB 2024061426W WO 2025104700 A2 WO2025104700 A2 WO 2025104700A2
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cells
concentration
medium
cell
car
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WO2025104700A3 (fr
Inventor
Peter Anthony Jones
Nadejda ANDREEV
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Takeda Pharmaceutical Co Ltd
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Takeda Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

  • NK cells are cytotoxic lymphocytes of the immune system. NK cells are cytotoxic against cancerous, pathogen-infected and otherwise damaged cells.
  • NK cells are innate lymphoid cells (ILCs), specifically large granular cytotoxic lymphocytes that bridge the innate and the adaptive arms of the immune response. They make up 10-15% of circulating lymphocytes in the peripheral blood and they also exist in umbilical cord blood. NK cells also exhibit the highest level of cytotoxic activity within the immune system, for example, targeting tumor cells. [0003] NK cells lack specific cell surface antigen receptors. Because of this, NK cells may kill cancerous and pathogen-infected cells without prior sensitization, making them part of the innate immune response. They also have a role in tumor immunosurveillance by directly influencing the adaptive immune response. These features and others make NK cells a particularly attractive cell type for use in adoptive cell therapies.
  • ILCs innate lymphoid cells
  • NK cells from a patient or donor by leukapheresis has limitations as the maximal NK-cell dose is limited and high NK-cell doses may only be obtained for patients with a low body weight. Further, the total number and activity of NK cells may substantially decrease in viral infection and/or cancer, making immunotherapy based on the activation of endogenous NK cells ineffective. Refractory relapses are a major complication in cell transfusions, and repeated infusions of lymphocyte populations are often required.
  • Adoptive cell treatment with allogeneic NK cells for example, derived from fresh cord blood has similar disadvantages, such as timely ex vivo expansion as well as the activation of clinical grade NK cells and in vivo persistence, cytotoxicity, etc. after infusion for therapeutic use.
  • Attorney Docket No. MIL-027WO1 SUMMARY [0006] The present disclosure provides, among other things, improved methods for effectively and efficiently expanding NK cell populations while maintaining, and even enhancing their functionality in-vivo (e.g., killing ability, trafficking, localization, persistence and proliferation), and more efficient and robust methods for cryopreserving NK cells, including CAR NK cells, at high yield such that cytotoxicity and other functionalities are retained to a high degree post-thaw.
  • the present disclosure provides, among other things, improved methods of cryopreserving NK cells, for example, cord-blood derived NK cells, and compositions thereof.
  • This disclosure is based, at least in part, on the discovery of efficient and robust production of NK cells by the fitness-enhanced (FE) methods described herein.
  • the fitness- enhanced process of the present invention improved both cell intrinsic parameters (e.g., cell fitness, armoring, post thaw cell viability and function) as well as cell extrinsic parameters (e.g., persistence, resistance and impact in tumor microenvironment (TME)).
  • methods of the present disclosure specifically result in improved post-thaw viability (e.g., by about 2.4-fold), survival (e.g., by about 2.6-fold), improved cytotoxicity and persistence of CAR-NK cells.
  • frozen cells manufactured in the FE process of the present disclosure maintain a larger percentage of a CD56+ CD16+ NK cell population post-thaw, comparable to fresh cells, increasing anti-tumor efficacy in vivo and for example, decreasing tumor growth.
  • CAR NK cells manufactured by the FE process herein demonstrated greater percent polyfunctionality (the frequency of cells secreting multiple cytokines) and polyfunctional strength index (PSI, which condenses the multi-dimensional secretions into an overall quality metric), which correlates to improved clinical outcomes in cancer patients receiving cell therapies.
  • PSI polyfunctional strength index
  • the methods described herein have numerous benefits over existing methods of producing NK cells representing an improved efficient and robust fitness-enhanced method of producing NK cells for NK cell-based therapies.
  • a method of cryopreserving natural killer (NK) cells comprising: (a) providing NK cells cultured in a culture medium; (b) washing the Attorney Docket No.
  • a method of cryopreserving natural killer (NK) cells comprising a step of culturing NK cells in a medium comprising MEM.
  • step (c) comprises resuspending the washed NK cells in the wash medium from step (b) such that the cells will be at twice their final concentration, and then adding an equal volume of CryoStor CS10 to bring the cells to their final concentration.
  • step (c) comprises resuspending the washed NK cells in a 1:1 mixture of MEM: wash medium further comprising CS10.
  • step (c) comprises resuspending the washed NK cells in a cryoformulation medium comprising CryoStor CS5, but lacking MEM and human serum albumin.
  • a concentration of albumin in the wash medium is between 0% to 20%.
  • the concentration of albumin in the wash medium is 5%.
  • the cryoformulation medium comprises MEM, albumin and one or more cryoprotective agents.
  • the concentration of albumin in the cryoformulation medium is between 0.1% to 10%.
  • the concentration of albumin in the cryoformulation medium is 2.5%.
  • the albumin is human serum albumin.
  • the human serum albumin is obtained from Flexbumin- 25 or Albuminar-25.
  • the cryoprotective agent comprises dimethyl sulfoxide (DMSO).
  • the DMSO is obtained from CryoStor CS10 or CryoStor CS5.
  • a concentration of DMSO in the cryoformulation medium is between 1% to 20%.
  • a concentration of DMSO in the cryoformulation medium is 5%.
  • Attorney Docket No. MIL-027WO1 [0027] In some embodiments, a concentration of DMSO in the cryoformulation medium is 5% and a concentration of albumin is 2.5%.
  • the NK cells are CD56+/CD3- cells. [0029] In some embodiments, the cells are incubated overnight prior to harvest at between 25°C to 32°C. [0030] In some embodiments, the NK cells are obtained from cord blood. [0031] In some embodiments, the NK cells are obtained from peripheral blood.
  • the post-thaw viability of cryopreserved NK cells is at least 50% at 24 hours after thawing.
  • the total amount of NK cells obtained by thawing the cryopreserved cells is between 70% to 100% of the initial cell number prior to cryopreservation.
  • the post-thaw viability of cryopreserved NK cells at 24 hours after thawing is 1.2 to 6.5-fold greater as compared to a control method.
  • cytotoxicity of the post-thawed cells is increased greater than 1.2-fold relative to a control method.
  • a composition comprising NK cells cryopreserved by the method of the present disclosure.
  • a composition comprising NK cells in a cryoformulation medium, wherein the cryoformulation medium comprises Minimal Essential Medium (MEM), albumin and one or more cryoprotective agent.
  • MEM Minimal Essential Medium
  • the cells are cryopreserved at a density of 1 x 10 5 to 2 x 10 8 cells/mL.
  • the cells are cryopreserved at a density of 2 x 10 7 to 1 x 10 8 cells/mL.
  • a concentration of albumin in the cryoformulation is between 0.1% to 10%. [0041] In some embodiments, the concentration of albumin in the cryoformulation is 2.5%. [0042] In some embodiments, the albumin is human serum albumin. [0043] In some embodiments, the human serum albumin is obtained from Flexbumin- 25 or Albuminar-25. [0044] In some embodiments, the cryoprotective agent comprises DMSO. Attorney Docket No. MIL-027WO1 [0045] In some embodiments, the composition comprises a concentration of DMSO between 1% to 20%. [0046] In some embodiments, the concentration of DMSO is 5%.
  • DMSO is obtained from CryoStor CS10 or CryoStor CS5.
  • the NK cells are CD56+/CD3-.
  • the NK cells are obtained from cord blood.
  • the NK cells are obtained from peripheral blood.
  • the post-thaw viability of cryopreserved NK cells is at least 50% at 24 hours after thawing.
  • post-thaw viability of cryopreserved NK cells at 24 hours after thawing is 1.2 to 6.5-fold greater as compared to a control method.
  • total amount of NK cells obtained by thawing the cryopreserved cells is 70% to 100% of the initial cell number prior to cryopreservation.
  • cytotoxicity of the post-thawed product is increased greater than 1.2-fold relative to a control.
  • NK cells are transduced with a chimeric antigen receptor.
  • a method of culturing immune cells comprising (i) a step of culturing the immune cells in a culture medium comprising (a) an AMPK activator, (b) a precursor of nicotinamide adenine dinucleotide (NAD+), (c) arginine supplement and/or (d) a lipid supplement, and/or (ii) a step of culturing the immune cells in between 1-21 % oxygen.
  • the immune cells are NK cells.
  • the immune cells are T cells.
  • the nicotinamide adenine dinucleotide is nicotinamide.
  • the step of culturing the immune cells is in 5% oxygen.
  • the AMPK activator is A-769662 or a derivative thereof.
  • A-769662 or the derivative thereof is at a concentration of 1 ⁇ M to 20 ⁇ M.
  • A-769662 or the derivative thereof is at a concentration of 10 ⁇ M.
  • the AMPK activator is metformin or a derivative thereof.
  • Attorney Docket No. MIL-027WO1 [0062] In some embodiments, metformin or the derivative thereof is at a concentration of 1 ⁇ M to 200 ⁇ M.
  • metformin or the derivative thereof is at a concentration of 100 ⁇ M.
  • the precursor of NAD+ is nicotinamide (NAM), nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN).
  • the nicotinamide (NAM) is at a concentration of between 0.1 mM to 10 mM.
  • the nicotinamide (NAM) is at a concentration of between 5 mM to 7 mM.
  • the nicotinamide mononucleotide is at a concentration of between 0.1 mM to 10 mM.
  • the nicotinamide riboside (NR) is at a concentration of between 0.1 mM to 10 mM.
  • the cells are conditioned in between 5% to 10% oxygen.
  • the cells are conditioned in 5% oxygen.
  • the cells are conditioned in 10% oxygen.
  • the cells are conditioned in 21% oxygen.
  • the culture medium comprises an arginine supplement.
  • the arginine supplement is at a concentration of between 1 mM to 5 mM. [0075] In some embodiments, the arginine supplement is at a concentration of 3 mM. [0076] In some embodiments, the culture medium comprises a lipid supplement. [0077] In some embodiments, a lipid supplement comprises cholesterol or cyclodextrin-complexed cholesterol. [0078] In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.1 mM to 20 mM. [0079] In some embodiments, the lipid supplement is HyClone LS1000.
  • the HyClone LS1000 is at a concentration of between 0.1X and 5X.
  • the culture medium comprises SCGM (Stem Cell Growth Medium), CTS NK-Xpander, NK MACS, AIM V, X-VIVO 10, X-VIVO 15, X- Attorney Docket No. MIL-027WO1 VIVO 20, RPMI-1640, TexMACS, ImmunoCult-XF T Cell Expansion Medium, CTL-Test Medium, KO-DMEM, StemPro-34 SFM, and/or OpTmizer T-cell expansion SFM.
  • the culture medium further comprises one or more of fetal bovine serum (FBS), human serum, pooled human plasma, or human plasma lysate.
  • FBS fetal bovine serum
  • the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of between 2% and 20%.
  • the culture medium further comprises one or more of GlutaMAX (L-alanyl-L-glutamine dipeptide), L-glutamine, MEM amino acids solution, MEM non-essential amino acids solution, MEM vitamin solution, insulin-transferrin- selenium and ascorbic acid.
  • the culture medium comprises at least one cytokine.
  • the at least one cytokine is IL-2, IL-7, IL-12, IL-21, IL- 15 or IL-18.
  • the cytokine is IL-2.
  • the IL-2 is at a concentration of between 10 IU/mL to 750 IU/mL.
  • the IL-2 is at a concentration of between 200 IU/mL to 300 IU/mL.
  • the cytokine is IL-15.
  • the IL-15 is at a concentration of between 0.04 ng/mL to 200 ng/mL.
  • the IL-15 is at a concentration of between 1 IU/mL and 5000 IU/mL. [0093] In some embodiments, the IL-15 is at a concentration of 2.5 ng/mL. [0094] In some embodiments, the cytokines are IL-2 and IL-15. [0095] In some embodiments, the cytokines are IL-2 and IL-21, or IL-2, IL-15 and IL-21. [0096] In some embodiments, the IL-21 is at a concentration of between 0.1 IU/mL to 100 IU/mL, or 0.1 ng/mL to 100 ng/mL.
  • the IL-21 is at a concentration of between 1 IU/mL to 5 IU/mL.
  • a method of treating a disease or disorder comprising administering an effective amount of the composition of the present disclosure.
  • Attorney Docket No. MIL-027WO1 [0099]
  • the subject has cancer.
  • the NK cells show improved cytotoxicity, CAR%, tumor control and/or longer persistence in vivo.
  • the CAR% is increased by 40% relative to a control without increasing the titer, the MOI or the amount of the viral vector.
  • FIG.1A is a graph showing in vivo anti-tumor efficacy at low dose (0.3E6 CAR+ cells/mouse) in donor 1.
  • FIG.1B is a graph showing in vivo anti-tumor efficacy at low dose (0.3E6 CAR+ cells/mouse) in donor 2.
  • FIG.1C is a graph showing in vivo anti- tumor efficacy at high dose (1E6 CAR+ cells/mouse) in donor 1.
  • FIG.1D is a graph showing in vivo anti-tumor efficacy at high dose (1E6 CAR+ cells/mouse) in donor 2.
  • FIG.1E is a graph showing baseline inhibition at 3E5 FE cultured cells as compared to 1E6 baseline CAR+ cells/mouse in donor 1.
  • FIG.1F is a graph showing baseline inhibition at 3E5 FE cultured cells as compared to 1E6 baseline CAR+ cells/mouse in donor 2.
  • FIG.1G is a graph showing CAR-NK persistence in donor 1 after day 6.
  • FIG.1H is a graph showing CAR-NK persistence in donor 2 after day 6. (GRI: growth rate inhibition).
  • FIG.2A is a graph showing viability of 9 donors. Showing average of 2.4X improvement compared to baseline in viability.
  • FIG.2B is a graph showing survival of 9 donors. Showing average of 2.6X improvement compared to baseline in survival.
  • FIG.2C is a bar graph showing cytotoxicity with FE treated cells (F) compared to Control (C) when administered at E:T of 10:1 in BCMA+ CAR NK cells in a donor subset.
  • FIG.2D is a bar graph showing cytotoxicity with FE treated cells (F) compared to Control (C) when administered at E:T of 10:1 in BCMA+ CAR NK cells in a donor subset with 1 donor.
  • FIG. 2E is a bar graph showing cytotoxicity with FE treated cells (F) compared to Control (C) when administered at E:T of 3:1 in BCMA+ cells in a 3 donor subset.
  • FIG.2F is a bar graph showing cytotoxicity with FE treated cells (F) compared to Control (C) when administered at E:T of 0.3:1 in BCMA+ CAR NK cells in BCMA+ cells in another 3 donor subset.
  • FIG.2G is a bar graph showing cytotoxicity with FE treated cells (F) compared to Control (C) when administered at E:T of 1:1 in BCMA+ CAR NK cells in BCMA+ cells in a 2 donor subset.
  • FIG.2H is a bar graph showing cytotoxicity with JJN3 BCMA- CAR NK cells in FE treated cells (F) compared to Control (C) when administered at E:T of 10:1 in a donor subset.
  • MIL-027WO1 2I is a bar graph showing cytotoxicity with JJN3 BCMA- CAR NK cells in FE treated cells (F) compared to Control (C) when administered at E:T of 3:1 in a 3 donor subset.
  • FIG.2J is a bar graph showing cytotoxicity with JJN3 BCMA- CAR NK cells in FE treated cells (F) compared to Control (C) when administered at E:T of 0.3:1 in a 3 donor subset.
  • FIG.2K is a bar graph showing cytotoxicity with JJN3 BCMA- CAR NK cells in FE treated cells (F) compared to Control (C) when administered at E:T of 1:1 in a 3 donor subset.
  • FIG.2L shows %CAR+ in control (Baseline) and FE treated CAR-NK, in vitro.7 out of 10 donors had increased CAR+%.
  • FIG.2M shows % loss in CAR expression in control (Baseline) and FE treated CAR-NK, comparing the %CAR+ both before and after cryopreservation, in vitro.
  • FIG.2N is a bar graph showing the results of RAS (repeat antigen stimulation) assay, in vitro, when treated with CAR NK cells manufactured with FE (F) or Control (C) process in donor 1.
  • FIG.2O is a bar graph showing elimination of target cells in vitro, when treated with CAR NK cells manufactured with FE (F) or Control (C) process in donor 2.
  • FIG.2P is a bar graph showing elimination of target cells in vitro, when treated with CAR NK cells manufactured with FE (F) or Control (C) process in donor 3.
  • FIG.3A is a bar graph showing amounts of various receptor retention on the harvest day in CAR NK cells manufactured with FE (F) or Control (C).
  • FIG.3B shows tumor burden in mice dosed with CAR NK-cells processed with FE compared to control, which is a measurement of anti-tumor activity.
  • FIG.3C is a depiction of tumor burden in mice treated with CAR NK-cells processed with FE (A3) compared to control (B1).
  • FIG.4A is a bar graph showing expression level (MFI; mean fluorescence intensity) of the CAR protein on CAR-NK cells cultured with various excipients and under various oxygen conditions.
  • the CAR MFI reflects the number of CAR proteins present on the surface of the NK cells.
  • FIG.4B is a bar graph showing viability at 24-hour post-thaw of CAR-NK cultured with various excipients and under various oxygen conditions.
  • FIG.4C shows the percent cytolysis of target tumor cells with CAR-NK cultured under various oxygen concentrations (in a medium comprising 10 ⁇ M A-769662, 7mM nicotinamide, 3mM Arginine and indicated percentage oxygen).
  • FIG.4D shows short-term post thaw viability (24h) of CAR-NK cultured under various oxygen concentrations.
  • FIG.4E shows percent cytolysis of target tumor cells treated with CAR-NK cells cultured in the presence or absence of 2.5 ng/ml IL-15.
  • FIG.4F is a bar graph showing the post-thaw viability of CAR-NK grown in the presence or absence of 2.5 ng/ml IL-15.
  • FIG.4G shows percent cytolysis of CAR NK growing in the presence or absence of LS1000 (100x dilution per product protocol).
  • FIG.4H is a bar graph showing the post-thaw viability at 24 h of CAR-NK cultured in the Attorney Docket No.
  • FIG.4I shows percent cytolysis of CAR-NK grown in the presence of 10 ⁇ M A-769662, 7mM NAM, 3mM L- Arginine in 21% oxygen, and in the presence of 100 ⁇ M metformin (AMPK inhibitor) and 7mM NAM.
  • FIG.4J is a bar graph showing the post-thaw viability at 24 h of CAR-NK cultured in the presence of A-769662 (AMPK inhibitor), NAM, L-Arginine in 21% oxygen, and in the presence of metformin (AMPK inhibitor) and NAM.
  • FIG.4K shows a level of waste product (lactate) at harvest day, produced by CAR-NK produced in the presence or absence of media changes.
  • FIG.4L shows a level of waste product (ammonia) at harvest day, produced by CAR-NK produced in the presence or absence of media changes.
  • FIG.4M shows a level of waste product (LDH) at harvest day, produced by CAR-NK produced in the presence or absence of media changes.
  • FIG.4N shows percent cytolysis by CAR-NK produced in a process where medium is changed or is unchanged.
  • FIG.4O shows percent viability of CAR-NK produced in a process where medium is changed on the shown days or is unchanged.
  • FIG.5A is a bar graph showing polyfunctionality of CAR-NK samples expressing 2, 3, 4 or 5+ cytokines, manufactured with A-769662, nicotinamide, L-arginine, or 5% O2, which all demonstrated remarkably greater polyfunctionality versus the control process.
  • FIG.5B is a bar graph showing polyfunctional strength index (PSI) of CAR-NK samples expressing various cytokines functioning as effectors, stimulatory proteins, chemoattractant, regulatory proteins and inflammatory proteins.
  • FIG.6A is a bar graph showing expansion of CAR-NK cultured under various conditions indicated.
  • FIG.6B is a bar graph showing cytotoxicity of CAR-NK cultured under the following conditions indicated in Table 1.
  • FIG.6C is a bar graph showing viability of NK cells cultured under conditions indicated in Table 1 after 1 day or 4 days in the absence of target cells.
  • FIG.6D is a bar graph showing Granzyme B secretion by CAR-NK cells cultured under indicated conditions.
  • FIG.6E is a bar graph showing interferon gamma secretion by CAR-NK cells cultured under indicated conditions.
  • FIG.6F is a graph showing post-thaw survival of CAR-NK cultured under various conditions.
  • FIG.6G is a graph showing post-thaw viability at 4-days post-thaw of CAR-NK cultured under various conditions.
  • FIG.6H is a graph showing basal oxygen consumption rate in a Seahorse Mito Stress Test assay at 4-days post-thaw of CAR-NK-cells cultured under various conditions. Only the first three points were basal oxygen consumption, after which various compounds were added.
  • FIG.6I is a graph showing basal oxygen consumption rate by NK-cells cultured under various conditions.
  • FIG.7A is a graph showing tumor volume growth with CAR-T cells cultured using various additives indicated in Example 7.
  • FIG.7B is a depiction of tumors in mice treated with CAR-T cells treated with various additives indicated in Example 7.
  • FIG.7C is a graph showing relative body weight in mice treated with CAR-T cells treated with various additive indicated in Example 7.
  • FIG.7D is a bar graph showing polyfunctionality of various cytokines expressed by CAR-T cells cultured using various additives indicated.
  • FIG.7E is a bar graph showing polyfunctional strength index (PSI) of various cytokines functioning as effectors, stimulatory proteins, chemoattractant, regulatory proteins and inflammatory proteins.
  • PSI polyfunctional strength index
  • FIG.8A is bar graph showing percent viability of various blood donors and CAR constructs post thaw, when washed with control medium or MEM/HSA.
  • FIG.8B is bar graph showing percent viability of various blood donors and CAR constructs after 24 h, when washed with control medium or MEM/HSA.
  • FIG.8C is bar graph showing percent viability of various blood donors and CAR constructs after 48 h, when washed with control medium or MEM/HSA.
  • FIG.8D is bar graph showing percent viability of various blood donors and CAR constructs after 72 h, when washed with control medium or MEM/HSA.
  • FIG.8E is bar graph showing percent viability of various blood donors and CAR constructs post thaw, when washed with control medium or MEM/HSA.
  • FIG.8F is bar graph showing percent viability of various blood donors and CAR constructs after 24 h, when washed with control medium or MEM/HSA.
  • FIG.8G is bar graph showing percent viability of various blood donors and CAR constructs after 48 h, when washed with control medium or MEM/HSA.
  • FIG.8H is bar graph showing percent viability of various blood donors and CAR constructs after 72 h, when washed with control medium or MEM/HSA.
  • each graph is data from a different donor.
  • Each bar within each graph is data for a different CAR construct tested within that donor
  • FIG.8I is bar graph showing percent viability of blood donor D511 after 24 h, when washed with control medium or MEM/HSA.
  • FIG.8J is a graph showing % killing in MOLM-13 cells 24 h post thaw, at various E:T ratios of CD123 DAP10 CAR NK or untransfected CAR NK isolated from donor D511 washed with control medium or MEM/HSA.
  • FIG.8K is a graph showing % viability of CAR NK isolated from 2 donors – A and B washed with control medium or MEM/HSA.
  • FIG.8L is a graph showing normalized improvement with MEM/HSA wash + cryo.
  • FIG.8M is a graph showing absolute improvement with MEM/HSA wash + cryo. Attorney Docket No.
  • administering As used herein, the terms “administer,” “administering,” “administration” “introducing” or “introduction” are used interchangeably in the context of delivering therapeutic cells, for example, NK cells (e.g., CD56+/CD3- cells, cord blood derived NK cells, CAR NK cells), into a subject, by a method or route which results in delivery of such cells.
  • NK cells e.g., CD56+/CD3- cells, cord blood derived NK cells, CAR NK cells
  • Various methods are known in the art for administering cells, including for example intravenously, topically, orally, intramuscularly, intraperitoneally, intrathecally, subcutaneously or transdermally. Cells can be administered with or without a carrier.
  • Adoptive Cell Therapy As used herein interchangeably, the terms “adoptive cell therapy” or “adoptive cell transfer” or “cell therapy” or “ACT” refer to the transfer of cells, for example, a population of CD56+/CD3- NK cells generated using methods described herein and administered into a subject or patient in need thereof. In some embodiments, the cell is a CD56+/CD3-immune cell made using a method described herein and additionally expressing a CAR.
  • Animal As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development.
  • animal refers to non-human animals, at any stage of development.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
  • animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms.
  • an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • Antigen-Specific Targeting Domain provides the CAR with the ability to bind to a target antigen of interest.
  • the antigen-specific targeting domain targets an antigen of clinical interest against which it would be desirable to trigger an effector immune response that results in tumor killing.
  • the antigen-specific targeting domain may be any protein or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof).
  • the antigen-specific targeting domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.
  • antigen-specific targeting domains include, for example, antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules/receptors, or receptor binding domains thereof, and tumor binding proteins.
  • the antigen-specific targeting domain is, or is derived from, an antibody.
  • An antibody-derived targeting domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen.
  • Allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.
  • allogeneic material from individuals of the same species may be sufficiently different genetically to interact antigenically.
  • Approximately or about As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). It is understood that when the term “about” or “approximately” is used to modify a stated reference value, the stated reference value itself is covered along with values that are near the stated reference value on either side of the stated reference value.
  • Chimeric Antigen Receptor As used herein, the term “chimeric antigen receptor” or “CAR” engineered receptors which can confer an antigen specificity onto cells (for example immune cells such as NK cells, T cells such as naive T cells, central memory T cells, effector memory T cells or combinations thereof). CARs are also known as artificial T- cell receptors, chimeric T-cell receptors or chimeric immunoreceptors. In some Attorney Docket No. MIL-027WO1 embodiments, the CARs of the invention comprise an antigen-specific targeting domain, an extracellular domain, a transmembrane domain, optionally one or more co-stimulatory domains, and an intracellular signaling domain.
  • a CAR is introduced into NK cells (e.g., CD56+/CD3- cells) made using a method described herein, such that to redirect specificity for a desired cell-surface antigen or MHC-peptide complex.
  • NK cells e.g., CD56+/CD3- cells
  • These synthetic receptors typically contain a target binding domain that is associated with one or more signaling domains via a flexible linker in a single fusion molecule.
  • the target binding domain is used to direct the NK cells to specific targets on the surface of pathologic cells (e.g., a cancer cell) and the signaling domains contain molecular machinery for NK cells activation and proliferation.
  • the flexible linker which usually passes through the NK cell membrane (i.e., forming a transmembrane domain) allows for cell membrane display of the target binding domain of the CAR.
  • CARs have successfully allowed immune cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Gross et al., (1989) Transplant Proc., 21(1 Pt 1): 127-30; Jena et al., (2010) Blood, 116(7):1035-44).
  • a CAR's extracellular binding domain may be composed of a single chain variable fragment (scFv) derived from fusing the variable heavy and light regions of a murine or humanized monoclonal antibody.
  • the extracellular binding domain comprises a single domain antibody.
  • scFvs may be used that are derived from Fab's (instead of from an antibody, e.g., obtained from Fab libraries). In various embodiments, this scFv is fused to a transmembrane domain and then to an intracellular signaling domain.
  • the first generation CARs comprised target binding domains attached to a signaling domain derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs were shown to successfully redirect immune cells to the selected target, but they failed to provide prolonged expansion and antitumor activity in vivo.
  • Culture refers to the maintenance, growth and/or differentiation of cells in an in vitro environment.
  • cells are cultured in a particular cell culture medium (or “media” in case of a plural) which facilitates or promotes the growth or differentiation of one type of Attorney Docket No. MIL-027WO1 cell into a different type of cell.
  • a cell culture medium acts as a source of nutrients, hormones and/or other factors helpful to propagate and/or sustain the cells.
  • Differentiating The terms “differentiating,” “inducing,” “converting,” “deriving” and the like refer to process in which a cell of one phenotype undergoes a change to a cell of another phenotype.
  • Engineered The term “engineered”, as used herein, describes a polynucleotide, polypeptide or a cell that has been designed or modified and/or whose existence and production require intervention and/or activity. For example, an engineered cell that is intentionally designed to elicit a particular effect and that differs from the effect of naturally occurring cells of the same type.
  • an engineered cell is a CD56+/CD3- cell derived from iPSCs or HPCs using a method described herein and further expresses a chimeric antigen receptor.
  • Enriched means a cell population that has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the specific cell type within the cell population as determined by flow cytometry or other analytical method.
  • Ex vivo means a process in which cells are removed from a living organism and are propagated outside the organism (e.g., in a test tube, in a culture bag, in a bioreactor).
  • Functional equivalent or derivative denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence.
  • a functional derivative or equivalent may be a natural derivative or is prepared synthetically.
  • Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved.
  • the substituting amino acid desirably has chemico-physical properties which are similar to that of the substituted amino acid. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like.
  • Immune cells refers to cells of the immune system, including, but not limited to, T cells, NK cells, T/NK cells, dendritic cells, macrophages, B cells, neutrophils, erythrocytes, monocytes, basophils, Attorney Docket No. MIL-027WO1 neutrophils, mast cells, eosinophils, and any combination thereof.
  • the immune cells produced using methods described herein are NK cells, characterized as CD56+/CD3- cells.
  • iPSC Induced Pluripotent Stem Cell
  • iPSC induced pluripotent stem cell
  • iPSC refers to a pluripotent stem cell artificially derived (e.g., induced) from a non-pluripotent cell, typically an adult somatic cell, for example, by inducing expression of one or more genes (including POU4F1/OCT4 (Gene ID; 5460) in combination with, but not restricted to, SOX2 (Gene ID; 6657), KLF4 (Gene ID; 9314), cMYC (Gene ID; 4609), NANOG (Gene ID; 79923), LIN28/ LIN28A (Gene ID; 79727)).
  • POU4F1/OCT4 Gene ID; 5460
  • SOX2 Gene ID; 6657
  • KLF4 Gene ID; 9314
  • cMYC Gene ID; 4609
  • NANOG Gene ID; 79923
  • LIN28/ LIN28A Gene
  • the stem cells may be genetically modified at any stage with markers or gene so that the markers or genes are carried through to any stage of culturing.
  • the markers may be used to purify or enrich the differentiated or undifferentiated stem cell populations at any stage of culture.
  • Induction medium generally refers to a cell culture media that is used to differentiate a population of cells from a first cell phenotype to a second cell phenotype.
  • the first cell phenotype and the second cell phenotypes comprise a heterogeneous population of cells.
  • the first cell phenotype comprises a heterogeneous population of cells.
  • the second cell phenotype comprises a heterogeneous population of cells.
  • the induction medium is used to differentiate a population of heterogeneous cells to a population of cells that is substantially homogeneous.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell- based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolation step means separating particular cell type from a mixture of cells.
  • a cell type can be defined by one or more markers that are either present or absent on the cell surface of a particular cell type.
  • Various Attorney Docket No. MIL-027WO1 methods of separating particular cell types from a mixture of cells are known in the art, and include, for example fluorescence-activated cell sorting (FACS) and magnetic-bead based sorting strategies such as magnetic-activated cell sorting (MACS).
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-bead based sorting strategies
  • Natural Killer (NK) Cell is a lymphoid cell defined by its marker expression and function/activity.
  • an NK cell expresses CD56 (CD56+).
  • such NK cells may express CD56 and CD16 (CD56+/CD16+).
  • such NK cells may express CD56 but not express CD3 (CD56+/CD3-).
  • NK cells may express variable levels of CD56.
  • NK cells may be “CD56 high ” which means that the NK cells express a high level of CD56 as assessed by methods in the art, for example as assessed by flow cytometry.
  • NK cells may be “CD56 dim ” which means that the NK cells express a low but detectable level of CD56 as assessed by methods in the art, for example as assessed by flow cytometry.
  • NK induction media refers to a cell culture media that is used to generate a population of cells comprising CD56+/CD3- cells.
  • the NK cell induction media comprises one or more of IL-7, IL-2 and a CD3 activator (e.g., anti-CD3 antibody).
  • T cell is a lymphoid cell defined by its marker expression and function/activity.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody that includes a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence.
  • Subject refers to a human or any non- human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term “subject” is used herein interchangeably with “individual” or “patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • Attorney Docket No. MIL-027WO1 Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition.
  • the disease may include cancer, for example, lymphoma and leukemia.
  • therapeutically effective amount of a therapeutic agent (e.g., a cell therapy) means an amount (e.g., a certain number of cells or a cell population enriched with a certain percentage of a particular type or types of cell or cells) that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
  • the CD56+/CD3- cells described herein are modified to express one or more transgenes. In some embodiments, the CD56+/CD3- cells described herein are modified to express a chimeric antigen receptor, such as for example CD19. In some embodiments, between about 100 million and 900 million CD56+/CD3- cells described herein are administered to a subject in need thereof. In some embodiments, between about 100 million and 700 million CD56+/CD3- cells described herein are administered to a subject in need thereof. In some embodiments, between about 100 million and 500 million CD56+/CD3- cells described herein are administered to a subject in need thereof.
  • between about 200 million and 900 million CD56+/CD3- cells described herein are administered to a subject in need thereof. In some embodiments, between about 200 million and 700 million CD56+/CD3- cells described herein are administered to a subject in need thereof. In some embodiments, between about 200 million and 500 million CD56+/CD3- cells described herein are administered to a subject in need thereof.
  • Treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition.
  • Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
  • Attorney Docket No. MIL-027WO1 [0139] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4 and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.” [0140] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention.
  • This disclosure is based, at least in part, on the discovery of efficient and robust production of NK cells from a bulk cell by the fitness-enhanced (FE) methods described herein for the production of NK cells.
  • the fitness-enhanced process of the present invention improved post-thaw viability (e.g., by about 2.4-fold), survival (e.g., by about 2.6-fold), improved cytotoxicity and persistence of CAR-NK cells.
  • a larger percentage of CD56 CD16+ cells were retained post-thaw, to levels comparable to fresh cells, increasing anti-tumor efficacy in vivo and for example, decreasing tumor growth.
  • CAR NK cells manufactured by the FE process herein demonstrated greater percent polyfunctionality (the frequency of cells secreting multiple cytokines) and polyfunctional strength index (PSI, which condenses the multi-dimensional secretions into an overall quality metric), which correlates to improved clinical outcomes in cancer patients receiving cell therapies.
  • PSI polyfunctional strength index
  • the methods described herein have numerous benefits over existing methods of producing NK cells representing an improved efficient and robust fitness-enhanced method of producing NK cells for NK cell-based therapies.
  • Various fitness enhancements are described in greater detail below.
  • a method of cryopreserving natural killer (NK) cells comprising: (a) providing NK cells cultured in a culture medium; (b) washing the NK cells from step (a) in a wash medium comprising Minimal Essential Medium (MEM) and albumin; and (c) resuspending washed NK cells from step (b) in a cryoformulation medium.
  • a composition comprising NK cells in a cryoformulation medium, wherein the cryoformulation medium comprises Minimal Essential Medium (MEM), albumin and one or more cryoprotective agent.
  • a method of culturing natural killer (NK) cells comprising (i) a step of culturing NK cells in a culture medium comprising (a) an AMPK activator, (b) a precursor of nicotinamide adenine dinucleotide (NAD+), (c) arginine supplement and/or (d) a lipid supplement, and/or (ii) a step of culturing NK cells in between 1-21 % oxygen.
  • a culture medium comprising (a) an AMPK activator, (b) a precursor of nicotinamide adenine dinucleotide (NAD+), (c) arginine supplement and/or (d) a lipid supplement, and/or (ii) a step of culturing NK cells in between 1-21 % oxygen.
  • a method of cryopreserving natural killer (NK) cells comprising: (a) providing NK cells cultured in a culture medium; (b) washing the NK cells from step (a) in a wash medium comprising Minimal Essential Medium (MEM) and albumin; and (c) resuspending washed NK cells from step (b) in a cryoformulation medium.
  • MEM Minimal Essential Medium
  • HEM is an art- recognized term that refers to a synthetic cell culture medium originally developed by Harry Eagle.
  • MEM is a modified Basal Medium Eagle media containing higher concentrations of essential nutrients that has been utilized for the cultivation of a variety of cells, e.g., grown in monolayers.
  • MEM is supplemented with non-essential amino acids (NEAA), for example, to formulations that incorporate either Hanks’ or Earle's salts.
  • step (c) comprises resuspending the washed NK cells in the wash medium from step (b) at twice the concentration of cells, and then adding an Attorney Docket No. MIL-027WO1 equal volume of Cryostor CS10.
  • Cryostor CS10 is preformulated freezing media comprising 10% DMSO.
  • step (c) comprises resuspending the washed NK cells in a 1:1 mixture of MEM: wash medium further comprising CS10.
  • step (c) comprises resuspending the washed NK cells in a cryoformulation medium comprising CryoStor CS5, but lacking MEM and human serum albumin.
  • CryoStor CS5 is preformulated freezing media comprising 5% DMSO.
  • Cryoprotectant is a substance used to protect biological tissue from freezing damage (i.e., that due to ice formation).
  • a cryoprotectant is ethylene glycol, dimethyl sulfoxide (DMSO), CryoStor comprising DMSO, glycerol, etc.
  • a concentration of albumin in the wash medium is between 0% to 20%, i.e., 0%, 20% or any discrete quantity therebetween.
  • a concentration of albumin in the wash medium is 0%.
  • the concentration of albumin in the wash medium is between 0% to 10%, i.e., 0%, 10% or any discrete quantity therebetween.
  • a concentration of albumin in the wash medium is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In some embodiments, the concentration of albumin in the wash medium is 1%. In some embodiments, the concentration of albumin in the wash medium is 2%. In some embodiments, the concentration of albumin in the wash medium is 3%. In some embodiments, the concentration of albumin in the wash medium is 4%. In some embodiments, the concentration of albumin in the wash medium is 5%. In some embodiments, the concentration of albumin in the wash medium is 6%. In some embodiments, the concentration of albumin in the wash medium is 7%. In some embodiments, the concentration of albumin in the wash medium is 8%.
  • the concentration of albumin in the wash medium is 9%. In some embodiments, the concentration of albumin in the wash medium is 10%. [0155] In some embodiments, the concentration of albumin in the wash medium is between 11% to 20%, i.e., 11%, 20% or any discrete quantity therebetween. In some embodiments, a concentration of albumin in the wash medium is 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some embodiments, the concentration of albumin in the wash medium is 11%. In some embodiments, the concentration of albumin in the wash medium is 12%. In some embodiments, the concentration of albumin in the wash medium is 13%.
  • the concentration of albumin in the wash medium is 14%. In Attorney Docket No. MIL-027WO1 some embodiments, the concentration of albumin in the wash medium is 15%. In some embodiments, the concentration of albumin in the wash medium is 16%. In some embodiments, the concentration of albumin in the wash medium is 17%. In some embodiments, the concentration of albumin in the wash medium is 18%. In some embodiments, the concentration of albumin in the wash medium is 19%. In some embodiments, the concentration of albumin in the wash medium is 20%. [0156] In some embodiments, the concentration of albumin in the wash medium is 5%. [0157] In some embodiments, the cryoformulation medium comprises MEM, albumin and one or more cryoprotective agents.
  • the concentration of albumin in the cryoformulation medium is between 0.1% to 10%. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.1%, 10% or any discrete quantity including any discrete decimal fraction therebetween. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.1%. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.2%. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.3%. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.4%. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.6%.
  • the concentration of albumin in the cryoformulation medium is 0.7%. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.8%. In some embodiments, the concentration of albumin in the cryoformulation medium is 0.9%. [0159] In some embodiments, the concentration of albumin in the cryoformulation medium is 1%. In some embodiments, the concentration of albumin in the cryoformulation medium is 1.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 2%. In some embodiments, the concentration of albumin in the cryoformulation medium is 2.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 3%.
  • the concentration of albumin in the cryoformulation medium is 3.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 4%. In some embodiments, the concentration of albumin in the cryoformulation medium is 4.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 5.5%. In some embodiments, the concentration of albumin in the cryoformulation Attorney Docket No. MIL-027WO1 medium is 6%. In some embodiments, the concentration of albumin in the cryoformulation medium is 6.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 7%.
  • the concentration of albumin in the cryoformulation medium is 7.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 8%. In some embodiments, the concentration of albumin in the cryoformulation medium is 8.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 9%. In some embodiments, the concentration of albumin in the cryoformulation medium is 9.5%. In some embodiments, the concentration of albumin in the cryoformulation medium is 10%. [0160] In some embodiments, the concentration of albumin in the cryoformulation medium is 2.5%. [0161] In some embodiments, the albumin is human serum albumin.
  • the human serum albumin is obtained from Flexbumin- 25 or Albuminar-25. In some embodiments, the albumin is Flexbumin-25. In some embodiments, the albumin is Albuminar-25.
  • the cryoprotective agent comprises dimethyl sulfoxide (DMSO). [0164] In some embodiments, the DMSO is obtained from CryoStor CS10 or CryoStor CS5. In some embodiments, the cryoprotective agent is CryoStor CS10. In some embodiments, the cryoprotective agent is CryoStor CS5.
  • a concentration of DMSO in the cryoformulation medium is between 1% to 20% or any discrete quantity therebetween. In some embodiments, a concentration of DMSO in the cryoformulation medium is 1%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 2%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 3%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 4%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 5%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 6%.
  • a concentration of DMSO in the cryoformulation medium is 7%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 8%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 9%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 10%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 11%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 12%. In some embodiments, a Attorney Docket No. MIL-027WO1 concentration of DMSO in the cryoformulation medium is 13%.
  • a concentration of DMSO in the cryoformulation medium is 14%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 15%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 16%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 17%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 18%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 19%. In some embodiments, a concentration of DMSO in the cryoformulation medium is 20%. [0166] In some embodiments, a concentration of DMSO in the cryoformulation medium is 5%.
  • a concentration of DMSO in the cryoformulation medium is between 10% to 20% and a concentration of albumin is between 0.1% to 10%.
  • a concentration of DMSO in the cryoformulation medium is 5% and a concentration of albumin is 2.5%.
  • the NK cells are CD56+/CD3- cells. The percentage of CD56+/CD3- cells can be readily determined by one of ordinary skill by known methods in the art, such as flow cytometry. [0170] The percentage of CD56+/CD3- NK cells in the cell population produced is at least about 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95% or more than 95%.
  • the method results in at least about 50% CD56+/CD3- NK cells. In some embodiments, the method results in at least about 55% CD56+/CD3- NK cells. In some embodiments, the method results in at least about 60% CD56+/CD3- NK cells. In some embodiments, the method results in at least about 65% CD56+/CD3- NK cells. In some embodiments, the method results in at least about 70% CD56+/CD3- NK cells. In some embodiments, the method results in at least about 75% CD56+/CD3- NK cells. In some embodiments, the method results in at least about 80% CD56+/CD3- NK cells.
  • the method results in at least about 85% CD56+/CD3- NK cells. In some embodiments, the method results in at least about 90% CD56+/CD3- NK cells. In some embodiments, the method results in at least about 95% CD56+/CD3- NK cells. In some embodiments, the method results in more than 95% CD56+/CD3- NK cells. [0171] In some embodiments, the cells are incubated overnight prior to harvest at between 25°C to 32°C, or any discrete temperature therebetween. In some embodiments, the cells are incubated overnight prior to harvest at 25°C. In some embodiments, the cells are Attorney Docket No. MIL-027WO1 incubated overnight prior to harvest at 27°C.
  • the cells are incubated overnight prior to harvest at 29°C. In some embodiments, the cells are incubated overnight prior to harvest at 30°C. In some embodiments, the cells are incubated overnight prior to harvest at 32°C.
  • the cell phenotype present in each of the steps of the method can be determined.
  • the cell differentiation process can be assessed by various means known in the art. For example, in some embodiments, the cell differentiation of cultured cells can be assessed by obtaining a sample of the cultured cells and subjecting that sample of cultured cells to one or more analytical methods to ascertain the cell phenotype of the cell. Known methods of ascertaining cell phenotype include for example, flow cytometry and immunofluorescence imaging.
  • any suitable sampling and phenotyping assay can be used with cell culture methods described herein to ascertain the progress of the cell differentiation process.
  • the methods described herein include one or more sampling steps to determine cell phenotype at a given time.
  • the NK cells are obtained from cord blood.
  • the NK cells are obtained from peripheral blood.
  • post-thaw viability of cryopreserved NK cells is at least 50% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is between 50%-100% or any discrete quantity therebetween at 24 hours after thawing.
  • post-thaw viability of cryopreserved NK cells is 50% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 55% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 60% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 65% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 70% at 24 hours after thawing.
  • post-thaw viability of cryopreserved NK cells is 75% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 80% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 85% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 90% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 95% at 24 hours after thawing.
  • post-thaw viability of cryopreserved NK cells is 100% at 24 hours after thawing.
  • total amount of NK cells obtained by thawing the cryopreserved cells is between 70% to 100% of the initial cell number prior to Attorney Docket No. MIL-027WO1 cryopreservation.
  • total amount of NK cells obtained by thawing the cryopreserved cells is 70% of the initial cell number prior to cryopreservation.
  • total amount of NK cells obtained by thawing the cryopreserved cells is 75% of the initial cell number prior to cryopreservation.
  • total amount of NK cells obtained by thawing the cryopreserved cells is 80% of the initial cell number prior to cryopreservation. In some embodiments, total amount of NK cells obtained by thawing the cryopreserved cells is 85% of the initial cell number prior to cryopreservation. In some embodiments, total amount of NK cells obtained by thawing the cryopreserved cells is 90% of the initial cell number prior to cryopreservation. In some embodiments, total amount of NK cells obtained by thawing the cryopreserved cells is 95% of the initial cell number prior to cryopreservation.
  • total amount of NK cells obtained by thawing the cryopreserved cells is 99% of the initial cell number prior to cryopreservation. In some embodiments, total amount of NK cells obtained by thawing the cryopreserved cells is 100% of the initial cell number prior to cryopreservation.
  • the post-thaw viability of cryopreserved NK cells at 24 hours after thawing is 1.2 to 6.5-fold greater as compared to a control method. In some embodiments, the post-thaw viability of cryopreserved NK cells at 24 hours after thawing is 1.2-fold greater as compared to a control method.
  • the post-thaw viability of cryopreserved NK cells at 24 hours after thawing is 1.4, 1.6, 1.8 to 2-fold greater as compared to a control method. In some embodiments, the post-thaw viability of cryopreserved NK cells at 24 hours after thawing is 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 or 6.5-fold greater as compared to a control method. [0176] In some embodiments, cytotoxicity of the post-thawed cells is increased greater than 1.2-fold relative to a control method. In some embodiments, cytotoxicity of the post-thawed cells is increased 1.2-fold relative to a control method.
  • cytotoxicity of the post-thawed cells is increased 1.4, 1.6, 1.8, or 2.0-fold relative to a control method.
  • a composition comprising NK cells cryopreserved by the method of the present disclosure.
  • a composition comprising NK cells in a cryoformulation medium, wherein the cryoformulation medium comprises Minimal Essential Medium (MEM), albumin and one or more cryoprotective agent.
  • MEM Minimal Essential Medium
  • albumin one or more cryoprotective agent.
  • the cells are cryopreserved at a density of 1 x 10 5 to 2 x 10 8 cells/mL.
  • the cells are at a density of 1 x 10 5 cells/mL. In some embodiments, the cells are at a density of 2 x 10 5 cells/mL. In some embodiments, the cells are at a density of 1 x 10 6 cells/mL. In some embodiments, the cells are at a density of 2 x 10 6 cells/mL. In some embodiments, the cells are at a density of 1 x 10 7 cells/mL. In some embodiments, the cells are at a density of 2 x 10 7 cells/mL. In some embodiments, the cells are at a density of 1 x 10 8 cells/mL. In some embodiments, the cells are at a density of 2 x 10 8 cells/mL.
  • the cells are at a density of 2 x 10 7 to 1 x 10 8 cells/mL.
  • the composition comprises a concentration of albumin between 0.1% to 10%. In some embodiments, the composition comprises a concentration of albumin between 0.1% to 1%. In some embodiments, the composition comprises a concentration of albumin between 1% to 10%. [0181] In some embodiments, the concentration of albumin is 2.5%. [0182] In some embodiments, the albumin is human serum albumin. [0183] In some embodiments, the human serum albumin is obtained from Flexbumin- 25 or Albuminar-25. In some embodiments, the human serum albumin is obtained from Flexbumin-25.
  • the human serum albumin is obtained from Albuminar-25.
  • the cryoprotective agent comprises DMSO.
  • the composition comprises a concentration of DMSO between 1% to 20%. In some embodiments, the composition comprises a concentration of DMSO between 1% to 10%. In some embodiments, the composition comprises a concentration of DMSO between 10% to 20%. [0186] In some embodiments, the concentration of DMSO is 5%.
  • DMSO is obtained from CryoStor CS10 or CryoStor CS5.
  • the culture period is for a time suitable to obtain a population of cells comprising NK cells.
  • the culturing of cells to obtain a population of NK is about 1-6 weeks; 3-5 weeks or around 3 weeks. Accordingly, in some embodiments, the cell culture period is for about 2-6 weeks. In some embodiments, the cell culture period is for about 3-5 weeks. In some embodiments, the cell culture period is for about 3 weeks.
  • the NK cells are obtained from cord blood. [0190] In some embodiments, the NK cells are obtained from peripheral blood. Attorney Docket No. MIL-027WO1 [0191] In some embodiments, provided herein is a composition comprising NK cells cryopreserved by the method described herein.
  • NK cells are CD56+/CD3- cells.
  • the CD56+/CD3- NK cells are produced, for example, by a method comprising the step of culturing a cell population comprising CD4- cells in a medium supplemented with vitamin C.
  • the medium is prepared by adding vitamin C to a basal medium which is used for culture of animal cells.
  • the basal medium examples include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), MEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and Neurobasal Medium (Life Technologies), and mixtures of two or more of these media.
  • IMDM Iscove's Modified Dulbecco's Medium
  • EMEM Eagle's Minimum Essential Medium
  • MEM medium MEM medium
  • Ham's F12 medium RPMI 1640 medium
  • Fischer's medium RPMI 1640 medium
  • Neurobasal Medium Neurobasal Medium
  • the basal medium may also contain one or more of substances such as albumin, human insulin, human transferrin, selenium or sodium selenite, fatty acid, trace elements, 2-mercaptoethanol, thiol glycerol, lipids, amino acids, glutamine, non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and cytokines.
  • the medium used for the production of the CD56 positive NK cells further contains an anti-CD3 antibody (UCHT1) and cytokines.
  • cytokines examples include IL-2 (e.g., 1 U/mL to 1000 U/mL) and IL-7 (e.g., 1 ng/mL to 100 ng/mL).
  • IL-2 e.g., 1 U/mL to 1000 U/mL
  • IL-7 e.g., 1 ng/mL to 100 ng/mL.
  • the CD3 antibody is not limited as long as it specifically recognizes CD3.
  • the concentration of the CD3 antibody in the third set of conditions is about 10 ng/mL to 1000 ng/mL, for example, 10 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, 400 ng/mL, 500 ng/mL, 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, or 1000 ng/mL.
  • vitamin C is used for the production of the CD56 positive K cells under the same conditions as described above.
  • the culture temperature conditions for the culture of a population comprising CD4- cells for production of NK cells is about 37° C.
  • the culture period may be appropriately determined by those skilled in the art by monitoring of the number of CD56 positive NK cells and/or the like.
  • the Attorney Docket No. MIL-027WO1 number of days of the culture is not limited as long as NK cells can be obtained. Examples of the culture period include at least not less than 1 day, not less than 2 days, not less than 3 days, not less than 4 days, not less than 5 days, not less than 6 days, or not less than 7 days.
  • the NK cells obtained may be isolated before further use.
  • the CD56 positive NK cells obtained may be used as a cell population that also contains other cell species, including T cells (NK cell bulk).
  • the resulting cell population obtained by culturing a cell population comprising CD4- cells may include at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 75%, 80%, 85%, 90%, or more than 90% NK cells.
  • the isolation method may be a method well known to those skilled in the art, for example, a method in which the cells are labeled with an anti-CD56 antibody and anti-CD3 antibody, and then isolated using a flow cytometer (fluorescence-activated cell sorting), or a method in which the cells are purified using an affinity column or the like to which a desired antigen is immobilized.
  • the CD56 positive NK cells are CD56+/CD3+.
  • the CD56 positive NK cells are CD56+/CD3-.
  • control NK cells are produced by the above methods.
  • the NK cells are produced by methods further comprising addition of one or more fitness enhancement components as described in greater detail below.
  • Methods and Components of Fitness-Enhanced Process AMPK Activator [0203] In some embodiments, the activator is an AMPK activator.
  • the AMPK activator includes, without limitation, sodium butyrate, trehalose, metformin, phenformin, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), aspirin, A-769662, resveratrol, MT 68-73, PF-06409577, PF-249, 5-(5-hydroxy-isoxazol-3-yl)-furan- 2-phosphonic acid, and combinations thereof.
  • AICAR 5-Aminoimidazole-4-carboxamide ribonucleotide
  • the inhibitor is A- 769662 (4-hydroxy-3-[4-(2-hydroxyphenyl)phenyl]-6-oxo-7H-thieno[2,3-b]pyridine-5- carbonitrile) of formula (I) or a derivative thereof.
  • Attorney Docket No. MIL-027WO1 [0204]
  • the AMPK activity of immune cells is increased by inhibiting a protein.
  • the increasing includes the inhibition of Fyn.
  • increasing of immune cell AMPK activity in accordance with the methods of the present disclosure enhances the longevity of the NK cells of the present disclosure in various manners.
  • Enhanced longevity of immune cells are determined by various factors including, without limitation, enhanced motility, persistent motility, enhanced cellular polarity, enhanced respiratory capacity, an enhanced number of punctate mitochondria, increased mitochondrial mass, reduced conjugation durations, enhanced spare respiratory capacity (SRC), enhanced capacity for oxidative metabolism, and combinations thereof.
  • the methods of the present disclosure can be utilized to increase the AMPK activity of NK cells in vivo in a subject.
  • Methods of increasing the AMPK activity of NK cells in vivo in a subject for example, by introducing a gene (e.g., a gene on an expression vector) to the subject that encodes a protein in the immune cells (e.g., AMPK, Sirtuins, serine/threonine-protein kinase (STK11), CD36, trehalose transporter, derivatives thereof, fusion proteins thereof, subunits thereof, and combinations thereof).
  • a gene e.g., a gene on an expression vector
  • a protein in the immune cells e.g., AMPK, Sirtuins, serine/threonine-protein kinase (STK11), CD36, trehalose transporter, derivatives thereof, fusion proteins thereof, subunits thereof, and combinations thereof.
  • the AMPK activity of immune cells is increased in vivo in a subject by introducing an activator to the subject (e.g., an AMPK activator, an STK11 activator, a Sirtuin activator, a CD36 activator, a Klotho activator, an autophagy activator, doxycycline, Attorney Docket No. MIL-027WO1 and combinations thereof).
  • an activator e.g., an AMPK activator, an STK11 activator, a Sirtuin activator, a CD36 activator, a Klotho activator, an autophagy activator, doxycycline, Attorney Docket No. MIL-027WO1 and combinations thereof.
  • the AMPK activity of immune cells is increased in vivo in a subject by inhibiting a protein in the subject (e.g., Fyn).
  • the AMPK activity of NK cells is increased in vivo in a subject by changing the diet of the subject, for example, by restricting calories, fasting, or diet conditions that mimic fasting.
  • the changing of the diet occurs in a subject that is suffering from cancer.
  • the changing of the diet occurs in a subject that is suffering from cancer and undergoing cancer immunotherapy.
  • the modified NK cells of the present disclosure include an exogenous or endogenous AMPK that is constitutively active.
  • the modified NK cells of the present disclosure include an exogenous or endogenous AMPK that is overexpressed.
  • the AMPK includes, without limitation, the alpha subunit of AMPK, AMPK-ocl, AMPK- oc2, and combinations thereof.
  • the AMPK activator is A-769662.
  • A-769662 is at a concentration of 1 ⁇ M to 20 ⁇ M, or any discrete intervening quantity. In some embodiments, A-769662 is at a concentration of 1 ⁇ M. In some embodiments, A-769662 is at a concentration of 2 ⁇ M. In some embodiments, A-769662 is at a concentration of 3 ⁇ M. In some embodiments, A-769662 is at a concentration of 4 ⁇ M.
  • A-769662 is at a concentration of 5 ⁇ M. In some embodiments, A-769662 is at a concentration of 6 ⁇ M. In some embodiments, A- 769662 is at a concentration of 7 ⁇ M. In some embodiments, A-769662 is at a concentration of 8 ⁇ M. In some embodiments, A-769662 is at a concentration of 9 ⁇ M. In some embodiments, A-769662 is at a concentration of 10 ⁇ M. In some embodiments, A-769662 is at a concentration of 11 ⁇ M. In some embodiments, A-769662 is at a concentration of 12 ⁇ M. In some embodiments, A-769662 is at a concentration of 13 ⁇ M.
  • A-769662 is at a concentration of 14 ⁇ M. In some embodiments, A-769662 is at a concentration of 15 ⁇ M. In some embodiments, A-769662 is at a concentration of 16 ⁇ M. In some embodiments, A-769662 is at a concentration of 17 ⁇ M. In some embodiments, A- 769662 is at a concentration of 18 ⁇ M. In some embodiments, A-769662 is at a concentration of 19 ⁇ M. In some embodiments, A-769662 is at a concentration of 20 ⁇ M. [0212] In some embodiments, A-769662 is at a concentration of 10 ⁇ M.
  • the AMPK activator is metformin.
  • metformin is at a concentration of 1 ⁇ M to 200 ⁇ M, or any discrete intervening quantity. In some embodiments, metformin is at a concentration of 1 ⁇ M to 10 ⁇ M, 10 ⁇ M to 20 ⁇ M, 20 ⁇ M to 30 ⁇ M, 30 ⁇ M to 40 ⁇ M, 40 ⁇ M to 50 ⁇ M, 50 ⁇ M Attorney Docket No. MIL-027WO1 to 60 ⁇ M, 60 ⁇ M to 70 ⁇ M, 70 ⁇ M to 80 ⁇ M, 80 ⁇ M to 90 ⁇ M, 90 ⁇ M to 100 ⁇ M.
  • metformin is at a concentration of 100 ⁇ M to 110 ⁇ M, 110 ⁇ M to 120 ⁇ M, 120 ⁇ M to 130 ⁇ M, 130 ⁇ M to 140 ⁇ M, 140 ⁇ M to 150 ⁇ M, 150 ⁇ M to 160 ⁇ M, 160 ⁇ M to 170 ⁇ M, 170 ⁇ M to 180 ⁇ M, 180 ⁇ M to 190 ⁇ M, 190 ⁇ M to 200 ⁇ M. [0215] In some embodiments, metformin is at a concentration of 100 ⁇ M.
  • Nicotinamide adenine dinucleotide (NAD+) Precursor the cells are cultured in the presence of a precursor of nicotinamide and/or a nicotinamide moiety.
  • nicotinamide moiety refers to nicotinamide as well as to products that are derived from nicotinamide, derivatives, analogs and metabolites thereof, such as, for example, NAD, NADH and NADPH, which effectively and preferentially enhance NK cell proliferation and/or activation.
  • nicotinamide analog refers to any molecule that is known to act similarly to nicotinamide including, without limitation, benzamide, nicotinethioamide (the thiol analog of nicotinamide), nicotinic acid and ⁇ -amino-3- indolepropionic acid.
  • nicotinethioamide the thiol analog of nicotinamide
  • nicotinic acid and ⁇ -amino-3- indolepropionic acid.
  • nicotinamide derivative further refers to any structural derivative of nicotinamide itself or of an analog of nicotinamide.
  • nicotinamide moiety is nicotinamide.
  • an “effective concentration” of nicotinamide and/or other nicotinamide moiety is defined as that concentration of a precursor of nicotinamide and/or a nicotinamide moiety which, when provided to the population of NK cells in culture, for an effective duration of exposure and at an effective time of exposure, results in one or more of increased migration response, improved homing and in-vivo persistence, greater proliferation and increased cytotoxic activity of the NK cells, as compared to control NK cells cultured under identical conditions but with less than 0.1 mM of the precursor of nicotinamide and/or nicotinamide moiety.
  • Concentrations of nicotinamide or nicotinamide moiety precursors suitable for use in some embodiments of the present invention range from about 0.5 to about 10 mM, typically 2.5 or 5.0 mM, based on the effect of these concentrations of nicotinamide on proliferation and NK cell function of about 0.5 mM to about 50 mM, about 1.0 mM to about 25 mM, about 1.0 mM to about 25 mM, about 2.5 mM to about 10 mM, about 5.0 mM to about 10 mM.
  • an effective concentration of nicotinamide and/or a nicotinamide moiety is a concentration which use thereof in culture "enhances", or results in a net increase of proliferation and/or function of NK cells in culture, compared to "control" cultures having less than 0.1 mM of the nicotinamide and/or nicotinamide moiety and tested from the same cord blood, bone marrow or peripheral blood preparation, in the same assay and under similar culture conditions (duration of exposure to nicotinamide and/or the nicotinamide moiety, time of exposure to nicotinamide and/or the other nicotinamide moiety).
  • the phrase "effective duration of time" of exposure of the NK cells to nicotinamide and/or other nicotinamide moiety is defined as that duration of exposure to nicotinamide during which, when the nicotinamide and/or other nicotinamide is provided to the population of NK cells in culture in an effective concentration and at an effective time of exposure, results in one or more of elevated expression of CD62L, elevated migration response, elevated homing and in-vivo retention, greater proliferation and increased cytotoxic activity of the NK cells, as compared to NK cells cultured under identical conditions without with less than 0.1 mM nicotinamide and/or other nicotinamide moiety.
  • Duration of exposure of the NK cell populations to nicotinamide and/or other nicotinamide moiety suitable for use in some embodiments of the present invention are typically in the range of about 2 hours to about 5 weeks, about 30 hours to about 4 weeks, about 2 days to about 3 weeks, about 1 week, about 2 weeks, about 3 weeks.
  • duration of exposure to nicotinamide and/or other nicotinamide moiety is about 1.0, about, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, about 10.0, about 11.0, about 12.0, about 13.0, about 14.0, about 15.0, about 16.0, about 17.0, about 18.0, about 19.0, about 20.0, about 21.0 days, about 25 days, about 30 days, about 35 Attorney Docket No. MIL-027WO1 days and all effective intermediate durations are contemplated.
  • Effective durations of time of exposure to nicotinamide and/or other nicotinamide moiety can be determined according to any assay of NK proliferation and/or activity. It will be appreciated that exposure of the NK cell populations to nicotinamide and/or other nicotinamide moiety can be initiated with establishment of the cell culture, or at any time during cell culture, even for a short duration just prior to use (e.g., infusion) of NK cells.
  • the phrase "effective exposure time" of the NK cell population to nicotinamide and/or the other nicotinamide moiety is defined as the time at which, during the culture of the NK population, the nicotinamide and/or other nicotinamide moiety is provided to the population of NK cells, in an effective concentration of the nicotinamide and/or other nicotinamide moiety and for an effective duration of time, resulting in one or more of elevated expression of CD62L, elevated migration response, elevated homing and in-vivo retention, greater proliferation and increased cytotoxic activity of the NK cells, as compared to NK cells cultured under identical conditions without with less than 0.1 mM of the nicotinamide and/or other nicotinamide moiety.
  • Time of exposure of the NK cell populations to nicotinamide and/or other nicotinamide moiety suitable for some embodiments of the present invention is typically from seeding of the NK cells to about 5 weeks after culturing, from about 1 hour after seeding to about 3 weeks after culturing, from about 24 hours to about 3 weeks after culturing, and from the time of seeding of the NK cell population in culture.
  • time of exposure of the NK cells to the nicotinamide and/or other nicotinamide moiety is at seeding, about 2 hours after seeding of the cells, about 12 hours after seeding of the cells, about 24 hours after seeding of the cells, about 2 days after seeding of the cells, about 4 days after seeding of the cells, about 7 days after seeding of the cells, about 8.0, about 9.0, about 10.0, about 11.0, about 12.0, about 13.0, about 14.0, about 15.0, about 16.0, about 17.0, about 18.0, about 19.0, about 20.0, about 21.0 days, about 25 days, about 30 days, about 35 days after seeding of the cells and all effective intermediate times are contemplated.
  • culturing NK cell populations with at least one growth factor and effective concentrations of nicotinamide and/or other nicotinamide moiety, provided at an effective exposure time for an effective duration of culture results in at least one of enhanced proliferation and/or enhanced NK cell function of the cultured cells, as compared to NK cells cultured under identical conditions in less than 0.1 mM nicotinamide and/or other nicotinamide moiety.
  • the precursor of NAD+ is nicotinamide (NAM), nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN).
  • the precursor of NAD+ is nicotinamide (NAM).
  • the precursor of NAD+ is nicotinamide riboside (NR).
  • the precursor of NAD+ is nicotinamide mononucleotide (NMN).
  • the nicotinamide (NAM) is at a concentration of between 0.1 mM to 10 mM.
  • the nicotinamide (NAM) is at a concentration of between 0.1 mM to 1 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of between 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, or 1.0 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of between 1 mM to 10 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 1 mM.
  • the nicotinamide (NAM) is at a concentration of 2 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 3 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 4 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 5 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 6 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 7 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 8 mM.
  • the nicotinamide (NAM) is at a concentration of 9 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 10 mM. [0227] In some embodiments, the nicotinamide riboside (NR) is at a concentration of between 0.1 mM to 10 mM, or any discrete intervening quantity. In some embodiments, the nicotinamide riboside (NR) is at a concentration of between 0.1 mM to 1 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 0.1 mM.
  • the nicotinamide riboside (NR) is at a concentration of 0.2 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 0.3 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 0.4 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 0.5 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 0.6 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 0.7 mM.
  • the nicotinamide riboside (NR) is at a concentration of 0.8 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 0.9 mM. [0228] In some embodiments, the nicotinamide riboside (NR) is at a concentration of between 1 mM to 10 mM. In some embodiments, the nicotinamide riboside (NR) is at a Attorney Docket No. MIL-027WO1 concentration of 1 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 2 mM.
  • the nicotinamide riboside (NR) is at a concentration of 3 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 4 mM. [0229] In some embodiments, the nicotinamide (NAM) is at a concentration of between 5 mM to 7 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 5 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 6 mM. In some embodiments, the nicotinamide (NAM) is at a concentration of 7 mM.
  • the nicotinamide riboside (NR) is at a concentration of 8 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 9 mM. In some embodiments, the nicotinamide riboside (NR) is at a concentration of 10 mM.
  • Oxygen conditioning [0231] In some embodiments of the fitness-enhanced methods of the present disclosure, the cells are conditioned in between 5% to 10% oxygen, or any intervening quantity therebetween. In some embodiments, the cells are conditioned in 5% oxygen. In some embodiments, the cells are conditioned in 6% oxygen. In some embodiments, the cells are conditioned in 7% oxygen.
  • the cells are conditioned in 8% oxygen. In some embodiments, the cells are conditioned in 9% oxygen. [0232] In some embodiments, the cells are conditioned in 10% oxygen. In some embodiments, the cells are conditioned in 11% oxygen. In some embodiments, the cells are conditioned in 12% oxygen. In some embodiments, the cells are conditioned in 13% oxygen. In some embodiments, the cells are conditioned in 14% oxygen. In some embodiments, the cells are conditioned in 15% oxygen. In some embodiments, the cells are conditioned in 16% oxygen. In some embodiments, the cells are conditioned in 17% oxygen. In some embodiments, the cells are conditioned in 18% oxygen. In some embodiments, the cells are conditioned in 19% oxygen.
  • the cells are conditioned in 20% oxygen. In some embodiments, the cells are conditioned in 21% oxygen.
  • Arginine supplement [0233] In some embodiments, the culture medium comprises an arginine supplement. [0234] In some embodiments, the arginine supplement is at a concentration of between 1 mM to 5 mM. In some embodiments, the arginine supplement is at a concentration of between 1 mM. In some embodiments, the arginine supplement is at a concentration of between 2 mM. In some embodiments, the arginine supplement is at a concentration of Attorney Docket No. MIL-027WO1 between 3 mM. In some embodiments, the arginine supplement is at a concentration of between 4 mM.
  • the arginine supplement is at a concentration of between 5 mM. [0235] In some embodiments, the arginine supplement is at a concentration of 3 mM.
  • Lipid supplement [0236] In some embodiments, the culture medium comprises a lipid supplement. [0237] In some embodiments, a lipid supplement comprises cholesterol or cyclodextrin-complexed cholesterol. In some embodiments, a lipid supplement comprises cholesterol. In some embodiments, a lipid supplement comprises a cyclodextrin-complexed cholesterol. [0238] In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.1 mM to 20 mM, or any discrete intervening quantity.
  • the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.1 mM to 20 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.1 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.2 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.3 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.4 mM.
  • the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.5 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.6 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.7 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.8 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 0.9 mM.
  • the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 1.0 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 2 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 3 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 4 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 5 mM.
  • the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 6 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 7 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 8 Attorney Docket No. MIL-027WO1 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 9 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 10 mM.
  • the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 11 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 12 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 13 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 14 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 15 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 16 mM.
  • the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 17 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 18 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 19 mM. In some embodiments, the cholesterol or cyclodextrin-complexed cholesterol is at a concentration of 20 mM.
  • the lipid supplement is HyClone LS1000. [0240] In some embodiments, the HyClone LS1000 is at a concentration of between 0.1X and 5X.
  • the HyClone LS1000 is at a concentration of 0.1X. In some embodiments, the HyClone LS1000 is at a concentration of 0.2X. In some embodiments, the HyClone LS1000 is at a concentration of 0.3X. In some embodiments, the HyClone LS1000 is at a concentration of 0.4X. In some embodiments, the HyClone LS1000 is at a concentration of 0.5X. In some embodiments, the HyClone LS1000 is at a concentration of 0.6X. In some embodiments, the HyClone LS1000 is at a concentration of 0.7X. In some embodiments, the HyClone LS1000 is at a concentration of 0.8X.
  • the HyClone LS1000 is at a concentration of 0.9X. In some embodiments, the HyClone LS1000 is at a concentration of 1X. In some embodiments, the HyClone LS1000 is at a concentration of 2X. In some embodiments, the HyClone LS1000 is at a concentration of 3X. In some embodiments, the HyClone LS1000 is at a concentration of 4X. In some embodiments, the HyClone LS1000 is at a concentration of 5X.
  • the culture medium comprises SCGM (Stem Cell Growth Medium), CTS NK-Xpander, NK MACS, AIM V, X-VIVO 10, X-VIVO 15, X- VIVO 20, RPMI-1640, TexMACS, ImmunoCult-XF T Cell Expansion Medium, CTL-Test Medium, KO-DMEM, StemPro-34 SFM, and/or OpTmizer T-cell expansion SFM.
  • SCGM Stem Cell Growth Medium
  • CTS NK-Xpander CTS NK-Xpander
  • NK MACS AIM V
  • X-VIVO 10 X-VIVO 15
  • X- VIVO 20 RPMI-1640
  • TexMACS ImmunoCult-XF T Cell Expansion Medium
  • CTL-Test Medium KO-DMEM
  • StemPro-34 SFM StemPro-34 SFM
  • OpTmizer T-cell expansion SFM OpTmizer T-cell expansion SFM.
  • the culture medium comprises a combination
  • the culture medium comprises one component selected from a group consisting of SCGM (Stem Cell Growth Medium), CTS NK-Xpander, NK MACS, AIM V, X-VIVO 10, X-VIVO 15, X-VIVO 20, RPMI-1640, TexMACS, ImmunoCult-XF T Cell Expansion Medium, CTL-Test Medium, KO-DMEM, StemPro-34 SFM, and OpTmizer T-cell expansion SFM.
  • the culture medium comprises SCGM (Stem Cell Growth Medium).
  • the culture medium comprises CTS NK-Xpander.
  • the culture medium comprises NK MACS.
  • the culture medium comprises AIM V. In some embodiments, the culture medium comprises X- VIVO 10. In some embodiments, the culture medium comprises X-VIVO 15. In some embodiments, the culture medium comprises X-VIVO 20. In some embodiments, the culture medium comprises RPMI-1640. In some embodiments, the culture medium comprises TexMACS. In some embodiments, the culture medium comprises ImmunoCult-XF T Cell Expansion Medium. In some embodiments, the culture medium comprises CTL-Test Medium. In some embodiments, the culture medium comprises KO-DMEM. In some embodiments, the culture medium comprises StemPro-34 SFM. In some embodiments, the culture medium comprises OpTmizer T-cell expansion SFM.
  • the culture medium further comprises one or more of fetal bovine serum (FBS), human serum, pooled human plasma or human plasma lysate. In some embodiments, the culture medium further comprises fetal bovine serum (FBS). In some embodiments, the culture medium further comprises human serum. In some embodiments, the culture medium further comprises pooled human plasma. [0245] In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of between 2% and 20%, or any discrete intervening quantity. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 2%.
  • FBS fetal bovine serum
  • human serum or pooled human plasma is at a concentration of 2%.
  • the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 3%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 4%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 5%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 6%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a Attorney Docket No. MIL-027WO1 concentration of 7%.
  • the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 8%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 9%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 10%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 11%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 12%.
  • the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 13%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 14%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 15%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 16%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 17%.
  • the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 18%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 19%. In some embodiments, the fetal bovine serum (FBS), human serum or pooled human plasma is at a concentration of 20%.
  • the culture medium further comprises one or more of GlutaMAX (L-alanyl-L-glutamine dipeptide), L-glutamine, MEM amino acids, non-essential amino acids, vitamins, insulin-transferrin-selenium and ascorbic acid.
  • the culture medium further comprises GlutaMAX (L-alanyl-L-glutamine dipeptide). In some embodiments, the culture medium further comprises L-glutamine. In some embodiments, the culture medium further comprises MEM amino acids. In some embodiments, the culture medium further comprises non-essential amino acids. In some embodiments, the culture medium further comprises vitamins. In some embodiments, the culture medium further comprises insulin-transferrin-selenium. In some embodiments, the culture medium further comprises ascorbic acid. Cytokines [0247] In some embodiments, the culture medium comprises at least one cytokine.
  • the at least one cytokine is IL-2, IL-7, IL-12, IL-21, IL- 15 or IL-18. In some embodiments, the at least one cytokine is IL-2. In some embodiments, the at least one cytokine is IL-7. In some embodiments, the at least one cytokine is IL-12. In Attorney Docket No. MIL-027WO1 some embodiments, the at least one cytokine is IL-21. In some embodiments, the at least one cytokine is IL-15. In some embodiments, the at least one cytokine is IL-18. [0249] In some embodiments, the cytokine is IL-2.
  • the IL-2 is at a concentration of between 10 IU/mL to 750 IU/mL. In some embodiments, the IL-2 is at a concentration of between 10 IU/mL to 50 IU/mL. In some embodiments, the IL-2 is at a concentration of between 50 IU/mL to 100 IU/mL.
  • the IL-2 is at a concentration of between 100 IU/mL-150 IU/mL, 150 IU/mL-200 IU/mL, 200 IU/mL-250 IU/mL, 250 IU/mL-300 IU/mL, 300 IU/mL-350 IU/mL, 350 IU/mL-400 IU/mL, 400 IU/mL- 450 IU/mL, 450 IU/mL-500 IU/mL, 500 IU/mL-550 IU/mL, 550 IU/mL-600 IU/mL, 600 IU/mL-650 IU/mL, 650 IU/mL-700 IU/mL, 700 IU/mL-750 IU/mL, or any intervening quantity.
  • the IL-2 is at a concentration of between 100 IU/mL, 150 IU/mL, 200 IU/mL, 250 IU/mL, 300 IU/mL, 350 IU/mL, 400 IU/mL, 450 IU/mL, 500 IU/mL, 550 IU/mL, 600 IU/mL, 650 IU/mL, 700 IU/mL, or 750 IU/mL. In some embodiments, the IL-2 is at a concentration of between 200 IU/mL to 300 IU/mL. [0250] In some embodiments, the cytokine is IL-15.
  • the IL-15 is at a concentration of between 0.04 ng/mL to 200 ng/mL, or any discrete intervening quantity. In some embodiments, the IL-15 is at a concentration of 0.04 ng/mL to 1 ng/mL. In some embodiments, the IL-15 is at a concentration of 1 ng/mL to 10 ng/mL. In some embodiments, the IL-15 is at a concentration of 10 ng/mL to 100 ng/mL. In some embodiments, the IL-15 is at a concentration of 100 ng/mL to 200 ng/mL. [0251] In some embodiments, the IL-15 is at a concentration of 2.5 ng/mL.
  • NK cells are transduced with CAR.
  • the NK cell are engineered to comprise one or more transgenes.
  • the NK cells can be genetically engineered to express tumor- directed chimeric antigen receptors (CAR), thereby producing antitumor effector cells.
  • the NK cell is engineered to express CAR.
  • cord blood derived NK cell is engineered to express CAR.
  • a cell precursor to the NK cell is engineered to express a CAR.
  • the NK cell is engineered to express a CAR.
  • NK cell precursor cells are engineered to express a CAR.
  • the NK cell is engineered to express a CAR.
  • these transgenic receptors can be directed to tumor- Attorney Docket No. MIL-027WO1 associated antigens that are not protein-derived.
  • NK cells are modified to comprise at least one CAR.
  • a single CAR targets two or more antigens.
  • the NK cells include a receptor that is chimeric, non- natural and engineered.
  • the engineered chimeric antigen receptor has one, two, three, four, or more components, and in some embodiments the one or more components facilitate targeting or binding of the lymphocyte to one or more tumor antigen-comprising cancer cells.
  • the CAR generally comprises at least one transmembrane polypeptide comprising at least one extracellular ligand-biding domain and; one transmembrane polypeptide comprising at least one intracellular signaling domain; such that the polypeptides assemble together to form a Chimeric Antigen Receptor.
  • extracellular ligand-binding domain as used herein is defined as an oligo- or polypeptide that is capable of binding a ligand.
  • the domain will be capable of interacting with a cell surface molecule.
  • the extracellular ligand- binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.
  • the extracellular ligand-binding domain can comprise an antigen binding domain derived from an antibody against an antigen of the target.
  • NK cells can be genetically modified to express one or more chimeric antigen receptors (CAR).
  • the CAR includes an extracellular ligand-binding domain that targets a tumor antigen selected from one or more of the following: CD44, CD19, CD20, CD22, CD23, CD30, CD89, CD123, CS-1, ROR1, mesothelin, c-Met, PSMA, Her2, GD-2, CEA, MAGE A3 TCR, EGFR, HER2/ERBB2/neu, EPCAM, EphA2, CEA, BCMA.
  • the tumor antigen is CD19.
  • the extracellular ligand-binding domain is a single chain antibody fragment (scFv) comprising the light (VL) and the heavy (VH) variable fragment of a target antigen specific monoclonal antibody joined by a flexible linker.
  • the CAR includes a transmembrane domain.
  • transmembrane domain further comprises a stalk region between the extracellular ligand-binding domain and the transmembrane domain.
  • stalk region used herein generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, stalk region Attorney Docket No.
  • a stalk region may comprise up to 300 amino acids, 10 to 100 amino acids, and/or 25 to 50 amino acids.
  • a stalk region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the stalk region may be a synthetic sequence that corresponds to a naturally occurring stalk sequence or may be an entirely synthetic stalk sequence.
  • the CAR contains a signal transducing domain or intracellular signaling domain which contributes to intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the NK cell and immune response.
  • the term "signal transducing domain” refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.
  • NK cells have a CAR that includes a signal transducing domain.
  • the NK cells are genetically modified to express IL- 15R ⁇ /IL-15 complex.
  • transmembrane polypeptides comprise the ability to be expressed at the surface of an immune cell, for example on cord blood derived NK cells, and to interact together for directing cellular response of immune cell against a predefined target cell.
  • the different transmembrane polypeptides of the CAR comprising an extracellular ligand-biding domain and/or a signal transducing domain interact together to take part in signal transduction following the binding with a target ligand and induce an immune response.
  • the transmembrane domain can be derived either from a natural or from a synthetic source.
  • the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • genetic modification of NK cells to express CAR can include steps of: (1) synthesizing a gene corresponding to a specific CAR; (2) preparing a vector containing the gene corresponding to the CAR; and (3) transducing the CD56+ NK cells with the vector containing the CAR gene.
  • Expression vectors that encode the CARs can be introduced as one or more DNA molecules or constructs, where there may be at least one marker that will allow for selection of host cells that contain the construct(s).
  • the constructs can be prepared in conventional ways, where the genes and regulatory regions may be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing, or other convenient means. Particularly, using PCR, individual fragments including all or portions of a functional unit may be isolated, where one or more mutations may be introduced using "primer repair", ligation, in vitro mutagenesis, etc., as appropriate. The construct(s) once completed and demonstrated to have the appropriate sequences may then be introduced into the CTL by any convenient means.
  • the constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors or lentiviral vectors, for infection or transduction into cells.
  • the constructs may include viral sequences for transfection, if desired.
  • the construct may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like.
  • the host cells may be grown and expanded in culture before introduction of the construct(s), followed by the appropriate treatment for introduction of the construct(s) and integration of the construct(s). The cells are then expanded and screened by virtue of a marker present in the construct.
  • Various markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.
  • homologous recombination one may use either OMEGA or O-vectors.
  • the constructs may be introduced as a single DNA molecule encoding at least the CAR and optionally another gene, or different DNA molecules having one or more genes. Other genes include genes that encode therapeutic molecules or suicide genes, for example.
  • the constructs may be introduced simultaneously or consecutively, each with the same or different markers.
  • Vectors containing useful elements such as bacterial or yeast origins of replication, selectable and/or amplifiable markers, promoter/enhancer elements for expression in prokaryotes or eukaryotes, etc. that may be used to prepare stocks of construct DNAs and for carrying out transfections are well known in the art, and many are commercially available. Methods of Use Attorney Docket No.
  • a method of treating a disease or disorder comprising administering an effective amount of the composition of the present disclosure.
  • the subject has cancer.
  • the NK cells show improved cytotoxicity, CAR%, tumor control and/or longer persistence in vivo.
  • the CAR% is increased by 40% relative to a control.
  • the cells according to the invention can be used for treating cancer, viral infections or autoimmune disorders in a patient in need thereof.
  • said isolated cell according to the invention can be used in the manufacture of a medicament for treatment of a cancer, viral infections of autoimmune disorders, in a patient in need thereof.
  • the present invention relies on methods for treating patients in need thereof, said method comprising at least one of the following steps: (a) providing a chimeric antigen receptor cell according to the invention and (b) administrating the cells to said patient.
  • Said treatment can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment.
  • autologous it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor.
  • HLA Human Leucocyte Antigen
  • allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.
  • the described cells are allogeneic. In some embodiments, the described cells are autologous.
  • Treatment can be used to treat patients diagnosed with cancer, viral infection, autoimmune disorders or Graft versus Host Disease (GvHD). Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise nonsolid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.
  • Types of cancers to be treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • Treatment is in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines Attorney Docket No. MIL-027WO1 therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
  • said treatment can be administered into patients undergoing an immunosuppressive treatment.
  • the cell compositions are administered to a patient in combination with one or more additional therapies.
  • the cell compositions are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH.
  • the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded immune cells of the present invention.
  • expanded cells are administered before or following surgery.
  • Said modified cells obtained by any one of the methods described here can be used in a particular aspect of the invention for treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD); therefore in the scope of the present invention is a method of treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating said patient by administering to said patient an effective amount of modified cells comprising inactivated TCR alpha and/or TCR beta genes.
  • the cells can be introduced into a host organism, e.g., a mammal, in a wide variety of ways.
  • the cells may be introduced at the site of the tumor, in specific embodiments, although in alternative embodiments the cells hone to the cancer or are modified to hone to the cancer.
  • the number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, the stability of the recombinant construct, and the like.
  • the cells may be applied as a dispersion, generally being injected at or near the site of interest.
  • the cells may be in a physiologically-acceptable medium.
  • the NK cells of the present invention Attorney Docket No. MIL-027WO1 may express one or more CARs, TCRs, or any other engineered protein or polypeptide domain such as high affinity CD16.
  • the cells are encapsulated to inhibit immune recognition and placed at the site of the tumor.
  • the cells may be administered as desired. Depending upon the response desired, the manner of administration, the life of the cells, the number of cells present, various protocols may be employed. The number of administrations will depend upon the factors described above at least in part.
  • the administration of the cells or population of cells according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally.
  • the cell compositions of the present invention are preferably administered by intravenous injection.
  • Nucleic Acid-Based Expression Systems [0284]
  • the NK cells (e.g., cord blood NK cells) of the invention are engineered to express one or more CARs, TCRs, or any other engineered protein or polypeptide domain such as high affinity CD16 or CD19. Recombinant techniques to generate expression vectors comprising these polypeptides are well known in the art and are described generally below.
  • vectors are used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • expression vector refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed.
  • Attorney Docket No. MIL-027WO1 RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. Promoters and Enhancers [0287]
  • a "promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence.
  • a promoter generally comprises a sequence that functions to position the start site for RNA synthesis.
  • the best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • To bring a coding sequence "under the control of” a promoter one positions the 5' end of the transcription initiation site of the transcriptional reading frame “downstream” of (i.e., 3' of) the chosen promoter.
  • the "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • a promoter may or may not be used in conjunction with an "enhancer,” which refers to a cis- Attorney Docket No. MIL-027WO1 acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5 prime' non-coding sequences located upstream of the coding segment and/or exon.
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
  • promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • promoters that are most commonly used in recombinant DNA construction include the lactamase (penicillinase), lactose and tryptophan (trp) promoter systems.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR.TM., in connection with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • a promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression.
  • promoters for protein expression
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • Attorney Docket No. MIL-027WO1 [0292] Additionally, any promoter/enhancer combination could also be used to drive expression.
  • Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • tissue-specific promoters or elements as well as assays to characterize their activity, is well known to those of skill in the art.
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided.
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available.
  • a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
  • "Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology. [0297] Splicing sites, termination signals, origins of replication, and selectable markers may also be employed. Plasmid Vectors [0298] In certain embodiments, a plasmid vector is contemplated for use to transform a host cell.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are Attorney Docket No. MIL-027WO1 capable of providing phenotypic selection in transformed cells.
  • E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species.
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • the phage lambda GEM.TM.11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.
  • Further useful plasmid vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S transferase (GST) soluble fusion proteins for later purification and separation or cleavage.
  • GST glutathione S transferase
  • Other suitable fusion proteins are those with galactosidase, ubiquitin, and the like.
  • Bacterial host cells for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB.
  • the expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.
  • Viral Vectors [0302] The ability of certain viruses to infect cells or enter cells via receptor mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells).
  • Components of the present invention may be a viral vector that encodes one or more CARs, TCR, or any other engineered protein or polypeptide domain such as high affinity CD16 of the invention.
  • virus vectors that may be used to deliver a nucleic acid of the present invention are described below.
  • Adenoviral Vectors [0303] A particular method for delivery of the nucleic acid involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of Attorney Docket No. MIL-027WO1 gene transfer afforded by these vectors.
  • Adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell specific construct that has been cloned therein.
  • AAV Vectors [0304] The nucleic acid may be introduced into the cell using adenovirus assisted transfection.
  • Adeno associated virus is an attractive vector system for use in the cells of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo.
  • AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988).
  • Retroviral Vectors are useful as delivery vectors because of their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell lines (Miller, 1992).
  • a nucleic acid e.g., one encoding the desired sequence
  • a retroviral vector In order to construct a retroviral vector, a nucleic acid (e.g., one encoding the desired sequence) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983).
  • a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g., by calcium phosphate precipitation for example)
  • the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).
  • the media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer.
  • Retroviral vectors are able to infect a broad variety Attorney Docket No. MIL-027WO1 of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Pat. Nos.6,013,516 and 5,994,136).
  • lentivirus examples include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV.
  • Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.
  • recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
  • a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
  • an "anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell.
  • This process may involve contacting the cancer cells with the expression construct and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single Attorney Docket No. MIL-027WO1 composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s).
  • Tumor cell resistance to chemotherapy and radiotherapy agents represents a major problem in clinical oncology.
  • One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy by combining it with other therapies.
  • cell therapy could be used similarly in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention, as well as pro-apoptotic or cell cycle regulating agents.
  • the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agent and present invention are applied separately to the individual, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and inventive therapy would still be able to exert an advantageously combined effect on the cell.
  • one may contact the cell with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other.
  • cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments.
  • Combination chemotherapies include, for example, abraxane, altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing and also combinations thereof.
  • Radiotherapy For the individual is employed in conjunction with the invention, for example before, during and/or after administration of the invention Radiotherapy [0315]
  • Other factors that cause DNA damage and have been used extensively include what are commonly known as gamma.-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • the terms "contacted" and "exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • Immunotherapies generally rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • Immunotherapy other than the inventive therapy described herein could thus be used as part of a combined therapy, in conjunction with the present cell therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context Attorney Docket No. MIL-027WO1 of the present invention.
  • Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.
  • the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the present invention clinical embodiments.
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • the NK cells as produced and cryopreserved by the methods described herein can be used for various applications.
  • the compositions described herein, including the CAR-NK cell compositions contained within the cryopreservation medium described herein are suitable for adoptive cell therapy.
  • Adoptive cell therapies can be used Attorney Docket No. MIL-027WO1 to treat various disease, including, for example, cancer.
  • the CAR-NK cell compositions contained within the cryopreservation medium described herein is useful for the treatment of a cancer or a tumor.
  • the cancer comprises breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovarian, prostate, brain, pancreatic, skin, bone, bone marrow, blood, thymus, uterine, testicular, and liver tumors.
  • the cancer is a blood cancer.
  • the blood cancer is a B-cell malignancy (e.g., diffuse large B-cell lymphoma).
  • the cryopreservation media described herein is used to suspend cells used for adoptive cell therapy. Accordingly, in some embodiment, CAR-NK cell compositions are suspended in the cryopreservation media described herein.
  • the CAR-NK cell compositions suspended in the cryopreservation media described herein is used to treat a subject who has cancer.
  • the subject is administered a composition comprising CAR-NK cells within the cryopreservation media described herein.
  • the CAR-NK cell comprises an anti-CD19 CAR gene and an IL-15 gene.
  • the CAR-NK cell comprises an anti-CD19 CAR gene, an IL-15 gene, and iCaspase9.
  • the CAR-NK cells are not washed prior to administering to a subject in need thereof.
  • the CAR-NK cells are washed of the cryopreservation media prior to administering to a subject in need thereof.
  • the thawed cells are administered into a patient in need thereof within about 30 minutes and 2 hours from thawing the cells.
  • the rate of intravenous infusion into a subject is between about 2-3 minutes.
  • the adoptive cell therapy is used in combination with one or more additional cancer treatments, such as for example lymphodepleting chemotherapy. Accordingly, in some embodiments, a subject who has cancer receives lymphodepleting chemotherapy before administration of a CAR-NK cell therapy product formulated in a cryopreservation media described herein.
  • a CAR-NK cell therapy product is cryopreserved as described herein and subsequently thawed prior to administration to a patient in need thereof.
  • a CAR-NK cell therapy product as described herein is cryopreserved, transported, thawed, and administered to a patient in need thereof as described herein.
  • the CAR-NK cell therapy product is cryopreserved in a Attorney Docket No. MIL-027WO1 formulation as described herein and subsequently thawed prior to administration to a patient for treatment of B-cell malignancies.
  • the frozen CAR-NK cell therapy product is frozen in a vial (e.g., a 50 ml AT vial) in a cryopreservation media described herein and using a method described herein and transported or shipped in the same vial at a temperature ranging from - 140 °C to -196 °C to a location where a patient is situated, such that the cells can be thawed at the location where the patient is situated and administered aseptically directly to the patient using a syringe connected to the vial with a vial adapter (i.e., vial to vein transfer).
  • a vial e.g., a 50 ml AT vial
  • a method of transporting the cell therapy product comprises: (a) providing the CAR-NK cells in a cryopreservation medium as described herein; (b) cooling the CAR-NK cells to a temperature of -80°C, thereby cryopreserving the mammalian cells; and (c) transporting the cryopreserved mammalian cells to a different location at a temperature of between about -20 °C to about -140 °C or below.
  • the cryopreserved mammalian cells are transported to a different location in a container maintained at a temperature at or below -140°C.
  • the cryopreserved mammalian cells are transported to a different location in a container maintained at a temperature of between -140°C and -196°C. In some embodiments, the cryopreserved mammalian cells are transported to a different location in a cryoshipper. In some embodiments, the transported cells may be stored in a cryoshipper until administration to a patient. In some embodiments, the transported cells are stored at the different location at a temperature at or below -140°C. In some embodiments, the transported cells are stored at the different location at a temperature of between -140°C to -196°C.
  • the transported cells are stored in liquid nitrogen vapor phase from the time of receipt at the different location until a future time of use.
  • the storage thus can be accomplished using clinical site freezers or tanks that maintain temperature at or below -140°C in a vapor phase of liquid nitrogen.
  • the cell therapy product is a population of CD19- CAR-NK cells that further comprise IL-15 and iCaspase9.
  • the cell therapy product is cryopreserved in a container at concentration of between about 6 and 120 million cells per milliliter.
  • the cell therapy product is cryopreserved in a 50 mL container at concentration of between about 6 and 120 million cells per milliliter.
  • the cell therapy product is cryopreserved in a 50 mL container at concentration of between about 3 and 150 million cells per milliliter. In some embodiments, the cell therapy product is cryopreserved in a 50 mL container at concentration of between Attorney Docket No. MIL-027WO1 about 1 and 250 million cells per milliliter. In some embodiments, the cell therapy product is cryopreserved in a 50 mL container at concentration of between about 1 and 350 million cells per milliliter. In some embodiments, the cell therapy product is cryopreserved in a 50 mL container at a concentration of between about 1 and 500 million cells per milliliter.
  • the cell therapy product comprises between about 20 ⁇ 10 6 and 100 X 10 7 cells in 50 mL container. In some embodiments, the cell therapy product comprises between about 100 X 10 6 and 900 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 50 X 10 6 in 50 mL container. In some embodiments, the cell therapy product comprises about 100 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 200 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 200 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 300 X 10 6 cells in 50 mL container.
  • the cell therapy product comprises about 400 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 500 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 600 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 700 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 800 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 900 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 1000 X 10 6 cells in 50 mL container.
  • the cell therapy product comprises about 1500 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 2000 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 2500 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 3000 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 3500 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 4000 X 10 6 cells in 50 mL container. In some embodiments, the cell therapy product comprises about 4500 X 10 6 cells in 50 mL container.
  • the cell therapy product comprises about 5000 X 10 6 cells in 50 mL container. [0330] In some embodiments, the cell therapy product is contained in a 50 mL container at a fill volume of about between 20-45 mL. In some embodiments, the cell therapy product is contained in a 50 mL container at a fill volume of about 36 mL. In some Attorney Docket No. MIL-027WO1 embodiments, the cell therapy product is an immune cell, such as an NK cell, T cell or B cell. In some embodiments, the immune cell is engineered to comprise one or more transgenes, for example a chimeric antigen receptor (CAR). In some embodiments, the cells are CAR-NK+ cells.
  • CAR chimeric antigen receptor
  • the cell therapy product comprises a CD19-CAR, IL-15 transgene and an iCaspase9. In some embodiments, the cell therapy product comprises between about 100 X 10 6 and 900 X 10 6 CAR-NK+ cells in a 50-ml container. In some embodiments, the cell therapy product present is 200 X 10 6 CAR-NK+ cells in a 50-ml container. In some embodiments, the cell therapy product present is 300 X 10 6 CAR-NK+ cells in a 50-ml container. In some embodiments, the cell therapy product present is 400 X 10 6 CAR-NK+ cells in a 50-ml container.
  • the cell therapy product present is 500 X 10 6 CAR-NK+ cells in a 50-ml container. In some embodiments, the cell therapy product present is 600 X 10 6 CAR-NK+ cells in a 50-ml container. In some embodiments, the cell therapy product present is 700 X 10 6 CAR-NK+ cells in a 50-ml container. In some embodiments, the cell therapy product present is 800 X 10 6 CAR-NK+ cells in a 50-ml container. In some embodiments, the cell therapy product present is 900 X 10 6 CAR-NK+ cells in a 50 ml container. [0331] The transported cell therapy product can be thawed as described herein followed by administration to a patient in need thereof.
  • the cell therapy product is thawed at the patient’s bedside. In some embodiments, the cell therapy product is not washed prior to administration into a patient in need thereof. [0332] In some embodiments, the transported cell therapy product remains frozen for further storage at the different location. In some embodiments, the thawed cells is introduced into a subject in need thereof without separating the cells and the cryopreservation solution. Thus, in some embodiments, the thawed cells are not washed prior to use. The thawed cells and accompanying cryopreservation solution is preferably warmed to body temperature (i.e., about 37° C.) prior to introduction into the subject.
  • the dose of the cells is based on the pre-freeze cell count.
  • thawed cells are further cultured.
  • culturing involves placing the cells in an incubator; removing the buffer solution; and replacing the buffer solution with a culture medium designed for the growth and/or differentiation of cells.
  • the cells are incubated in the incubator for between about 6 to 7 hours.
  • the culture medium designed for the Attorney Docket No. MIL-027WO1 growth and/or differentiation of cells comprises Kubota's medium and/or a hormonally defined medium (HDM) for the differentiation of cells.
  • HDM hormonally defined medium
  • Viability of thawed cells can be assessed in vitro as well as in vivo using various methods known in the art.
  • the in vitro cell viability tests includes the Trypan Blue exclusion assay.
  • other analytical methods can be used to assess the cell viability of thawed cells that had been frozen with the different cryopreservation medium, for example, gene expression, through the use of RT-qPCR and the like.
  • a person of ordinary skill in the art can opt for any analytical method to assess the viability of thawed cells that can be applied to assess the cell viability of otherwise fresh cells.
  • Viability of cells in vivo in general, can be assessed by evaluating the functional characteristics of administered cells in vivo.
  • the in vivo viability of cells can be assessed by evaluating the cell number of the cells that have been introduced into a subject in need.
  • Various methods are known in the art for tracing cells and determining viability of administered cells.
  • the cryopreserved and thawed cells using the cryopreservation media described herein allows for using the cells for any purpose that a primary cell or fresh cell isolate can have.
  • the cryopreserved and thawed cells retain high viability (e.g., greater than 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%) and retain physiological characteristics of their native state, which allows the cells to be used for a variety of applications, such as for genetic manipulation of the cells, and for cell therapy purposes such as, for example, in adoptive cell therapy applications.
  • Administration of CAR-NK cells [0337]
  • the CAR-modified NK cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
  • compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., Attorney Docket No. MIL-027WO1 aluminum hydroxide); and preservatives.
  • compositions of the present invention are formulated for intravenous administration.
  • compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • an immunologically effective amount When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • a pharmaceutical composition comprising the NK cells described herein may be administered at a dosage of about 10 4 to 10 9 cells/kg body weight, for example, about 10 5 to 10 6 cells/kg body weight.
  • NK cell compositions may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy.
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the NK cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection.
  • the NK cell compositions of the present invention are preferably administered by i. v. injection.
  • the compositions of NK cells may be injected directly into a tumor, lymph node, or site of infection.
  • cells activated and expanded using the methods described herein are administered to a patient in conjunction with (for example, either before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, Attorney Docket No. MIL-027WO1 cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizitmab treatment or efaiizumab treatment.
  • agents such as antiviral therapy, Attorney Docket No. MIL-027WO1 cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizitmab treatment or efaiizumab treatment.
  • the NK cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fliidaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies
  • immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fliidaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.
  • the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded immune cells of the present invention.
  • expanded cells are administered before or following surgery.
  • the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the scaling of dosages for human administration can be performed according to art-accepted practices.
  • a dose about 1 to about 100 mg is administered to an adult patient.
  • the CAR-NK cell therapy is administered daily for a period between 1 and 30 days.
  • 1 to 10 mg per day is administered daily.
  • about 40 mg per day is administered.
  • NK-cells cultured in control media and FE medium were evaluated with Anti-tumor efficacy.
  • FE fitness-enhanced
  • NK cells were isolated from frozen umbilical cord blood, then cultured with feeder cells. The NK cells were expanded in a humidified incubator set to 5% oxygen (physoxia) and 5% CO 2 , in the presence of culture medium with IL-2, containing 10 ⁇ M A-769662, 7 mM nicotinamide, 3 mM L-arginine, and 2 mM GlutaMAX (L-alanyl-L-glutamine dipeptide).
  • the NK cells were genetically engineered to overexpress a CAR (chimeric antigen receptor) construct targeting a tumor of interest. Subsequently, the NK cells were restimulated with feeder cells and cultured for a period in the same medium at 5% O 2 . Finally, the NK cells were harvested and washed with 5% HSA in phenol-free MEM medium, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, to result in a final cryoformulation containing 2.5% HSA and 5% DMSO. The entire process was performed at- scale according to good manufacturing practices (GMP).
  • GMP good manufacturing practices
  • a control group of CAR-NK cells was also produced from the same initial cord blood unit, however the medium did not contain A-769662, nicotinamide, L-arginine, or GlutaMAX (2 mM L-glutamine was used instead), and the cells were cultured at an incubator setting of 21% O2. In addition, a second cord blood unit was thawed, and the above processes were repeated to generate CAR-NK cells from this second donor. [0346] An in vivo study was performed to assess the anti-tumor efficacy and persistence of the CAR-NK cells. Eight approximately 10- to 12-week-old NSG mice per process group, per donor, and per dose were irradiated with 150 cGy.
  • each mouse was inoculated with luciferized tumor cells.
  • One day prior to CAR-NK injection the mice were bioluminescently imaged and randomized by tumor size.
  • CAR-NK injection was performed 7 days post-tumor inoculation, and the mice were separated by dose level with either 0.3E6 or 1.0E6 CAR-NK cells per mouse.
  • the CAR-NK cells were thawed, washed, and dosed in PBS (vehicle).
  • the tumor volume was monitored via bioluminescent imaging every 7 days until the study endpoint on day 29.
  • Tumor growth rate inhibition (GRI) was calculated as compared to vehicle alone.
  • the persistence of CAR-NK Attorney Docket No.
  • FIG.1A-FIG.1B showed anti-tumor efficacy of CAR-NK cells from two donors at low dose (0.3E6 CAR+ cells/mouse). It was observed that CAR-NK cultured with FE process was more effective compared to control at low dose.
  • FIG.1C-FIG.1D showed anti-tumor efficacy of CAR-NK cells from two donors at high dose (1E6 CAR+ cells/mouse). It was observed that CAR-NK cultured with FE process was significantly more effective compared to control at high dose in at least one of the donors.
  • Table 1 showed the dose of the CAR-NK administered in FIG.1A-1D.
  • Table 1 Dosage of CAR + cells per mouse in FIG.1A-1D for evaluating efficacy of FE vs baseline culture process.
  • Control 1E6 3.0E6 34 Donor 2 FE 0.3E6 1.0E6 29
  • the CAR-NK cells produced in the FE process (5% O 2 physoxic culture with medium containing A-769662, nicotinamide, L-arginine, and GlutaMAX) demonstrated superior anti-tumor control compared to the control process at a matched dose of 0.3E6 CAR- NK cells/mouse, using cells from both cord blood donors.
  • FIG.1E- FIG.1F showed comparison of in vivo anti-tumor efficacy to baseline and vehicle from both donor 1 (FIG.1E) and donor 2 (FIG.1F).
  • FIG.1G and FIG.1H showed the in vivo persistence of CAR-NKs as seen on day 6. It was observed that FE conferred greater persistence on day 6 compared to baseline process. [0351] Overall, the results showed improved tumor control and persistence with the fitness-enhanced process.
  • Attorney Docket No. MIL-027WO1 Example 2. Multi-Donor Pairwise Comparison of FE vs. Baseline Process for In vitro Efficacy in Tumor Killing [0352] This example evaluated the FE culture process with the baseline process in multiple donors. FE versus control process comparisons were repeated in 9 to 10 donors.
  • NK cells were isolated from frozen umbilical cord blood, then cultured with feeder cells. The NK cells were expanded in a humidified incubator set to 5% oxygen (physoxia) and 5% CO 2 , in the presence of culture medium with IL-2, containing 10 ⁇ M A-769662, 7 mM nicotinamide, and 3 mM L-arginine.
  • the NK cells were genetically engineered to overexpress a CAR (chimeric antigen receptor) construct targeting a tumor of interest. Subsequently, the NK cells were restimulated with feeder cells and cultured for a period in the same medium at 5% O 2 . Finally, the NK cells were harvested and washed with 5% HSA in phenol-free MEM medium, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, for a final cryoformulation containing 2.5% HSA and 5% DMSO.
  • CAR chimeric antigen receptor
  • a control group of CAR-NK cells was also produced from the same initial cord blood unit, however the medium did not contain A-769662, nicotinamide, or L-arginine, and the cells were cultured at an incubator setting of 21% O2. In addition, and the FE versus control process comparisons were repeated in 9 or 10 donors. [0354] Prior to cryopreserving the CAR-NK cells, they were assessed by flow cytometry for the %CAR+ on the harvest day. The frozen CAR-NK cells were thawed and assessed for post-thaw viability, survival, short-term cytotoxicity, % CAR+, and long-term killing capability.
  • Post-thaw viability and survival were determined by recovering the CAR- NK cells overnight in culture medium in a humidified incubator, followed by a determination of viability and cell count at 24 hours post-thaw.
  • Short-term cytotoxicity was determined by co-culturing the CAR-NK cells with target tumor cells at several effector-to-target ratios, and then the percent cytolysis of the target cells was determined at approximately 21 hours post- thaw.
  • the percentage of CAR+ cells was determined by flow cytometry immediately post- thaw. A paired t-test or 2-way ANOVA were used to determine significance of comparisons, as appropriate.
  • the cytotoxicity assay comparisons were performed at the effector-to-target cell ratio that allowed for the best discrimination Attorney Docket No. MIL-027WO1 between the FE and control groups.
  • Long-term in vitro killing ability of the CAR-NKs was evaluated in a subset of 3 donors using a repeat antigen stimulation (RAS) assay, wherein target tumor cells were repeatedly added to the CAR-NK cells three times per week over the course of two weeks in the presence of exogenous IL-2, and the number of target tumor cells per well was assessed at regular intervals via flow cytometry.
  • RAS repeat antigen stimulation
  • the area under the curve (AUC) of target cells during the RAS assay is an indication of how well the CAR-NK cells repeated killed tumor cells over time, with a lower AUC indicating fewer tumor cells and thus better killing by CAR-NKs.
  • AUC area under the curve
  • NK cells were isolated from frozen umbilical cord blood, then cultured with feeder cells. The NK cells were expanded in a humidified incubator set to 5% oxygen (physoxia) and 5% CO 2 , in the presence of a cell culture medium comprising IL-2, containing 10 ⁇ M A-769662, 7 mM nicotinamide, and 3 mM L-arginine.
  • the NK cells were genetically engineered to overexpress a CAR (chimeric antigen receptor) construct targeting a tumor of interest. Subsequently, the NK cells were restimulated with feeder cells and cultured for a Attorney Docket No. MIL-027WO1 period in the same medium at 5% O 2 . Finally, the NK cells were harvested and washed with 5% HSA in phenol-free MEM medium, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, to yield a final cryoformulation containing 2.5% HSA and 5% DMSO.
  • CAR chimeric antigen receptor
  • a control group of CAR-NK cells was also produced from the same initial cord blood unit, however the medium did not contain A-769662, nicotinamide, or L-arginine, and the cells were cultured at an incubator setting of 21% O 2 .
  • NK receptor expression was evaluated using flow cytometry, including CD56 and CD16 expression, both on fresh CAR-NK cells on the harvest day and in the frozen cells immediately post-thaw.
  • Anti-tumor efficacy was evaluated in an in vivo mouse model, as described previously. Five mice per cohort were evaluated, and the tumor volume was evaluated via bioluminescent imaging of luciferized tumor cells. A dose of 5E6 cryopreserved cells was injected per mouse.
  • CAR-NK cells were prepared with variations in the components used in the method to test various fitness enhancement strategies. NK cells were isolated from frozen umbilical cord blood, then cultured with feeder cells at varying oxygen levels, e.g., either 5% O 2 , 10% O 2 , or 21% O 2 (Table 2).
  • Table 2 Summary of Exemplary Formulations to test various Fitness Enhancements CAR-NK MEM/HSA Control BCMA CAR-NK MEM/HSA Control; MEM/HSA in wash buffer and cryoformulation A-769662 + NAM + L-arg + 5% 10 ⁇ M A-769662, 7 mM nicotinamide, 3 mM L- O2 arginine, 5% O2 A-769662 + NAM + L-arg + 10 ⁇ M A-769662, 7 mM nicotinamide, 3 mM L- 10% O2 arginine, 10% O2 A-769662 + NAM + L-arg + 10 ⁇ M A-769662, 7 mM nicotinamide, 3 mM L- 21% O2 arginine, 21% O2 Attorney Docket No.
  • MIL-027WO1 Two physoxic conditions were tested: 5% O 2 mimics venous circulation and 10% O 2 mimics arterial circulation. A standard 21% O 2 (hyperoxic) incubator setting was also tested. All test conditions were manufactured from the same cord blood unit.
  • the NK cells were expanded in a humidified incubator in the presence of culture medium with IL-2.
  • Various medium additives were tested, including 10 ⁇ M A-769662, 100 ⁇ M metformin, 7 mM nicotinamide, 3 mM L-arginine, and 2.5 ng/mL IL-15 (Table 3).
  • Table 3 Summary of Exemplary Formulations to test the effect of IL-15 CAR-NK MEM/HSA Control BCMA CAR-NK MEM/HSA Control; MEM/HSA in wash buffer and cryoformulation IL-152.5 ng/ml Add 2.5 ng/ml IL-15 on the same day as IL- 2 [0364]
  • NK cells were genetically engineered to overexpress a CAR (chimeric antigen receptor) construct targeting a tumor of interest. Subsequently, the NK cells were restimulated with feeder cells and cultured for a period in the same medium and at the same oxygen setting.
  • CAR chimeric antigen receptor
  • NK expansion various medium change schedules were also assessed in the context of 21% O 2 culture without the above additives (Table 4).
  • the NK cells were harvested and washed with 5% HSA in phenol-free MEM medium, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, for a final cryoformulation containing 2.5% HSA and 5% DMSO.
  • a control group of CAR-NK cells was also produced from the same initial cord blood unit, however the medium did not contain A-769662, metformin, nicotinamide, L-arginine, or IL- 15, and the cells were cultured at an incubator setting of 21% O 2 (Table 5).
  • Table 4 Summary of Exemplary Formulations to test the effect of media change Low D9 seed, no media change D9-D21H, D90.25E6 NK/cm 2 , no media change D9- IL-2 every 3 days D21H, 200 IU/mL IL-2 on D9/12/14/15/18.
  • Table 5 Summary of Exemplary Formulations to test the effect of Metformin Attorney Docket No.
  • CAR expression level was assessed by evaluating the median fluorescence intensity (Median FI) by flow cytometry.24h post-thaw viability and short-term cytotoxicity were assessed as described previously. [0368] CAR expression level by fresh CAR-NK cells on harvest day was determined to be greater than control for any conditions containing the combination of A-769662, nicotinamide, and L-arginine, regardless of whether the cells were manufactured at a setting of 5% O2, 10% O2, or 21% O2. The CAR-NK cells manufactured at 10% O2 demonstrated the greatest CAR expression level (FIG.4A).
  • Post-thaw viability was greatly enhanced for all conditions containing the combination of A-769662, nicotinamide, and L-arginine, regardless of whether the cells were manufactured at a setting of 5% O 2 , 10% O 2 , or 21% O 2 .
  • the greatest 24h post-thaw viability was generated for the condition containing the combination of A-769662, nicotinamide, L-arginine, and 5% O 2 culture (FIG.4B). Improvements in post-thaw viability were also obtained by performing additional medium changes throughout the process (FIG.4D).
  • Short-term cytotoxicity was enhanced in conditions containing A-769662, nicotinamide, L-arginine, with the greatest effect observed at 5% and 10% O 2 levels (FIG.4C).
  • LS1000 cyclodextrin-complexed cholesterol
  • AMPK activator and nicotinamide precursor (NAM) were used to assess the polyfunctionality.
  • Various functions including effector function, stimulatory function, chemoattractive function, regulatory function, and inflammatory function were tested.
  • CAR-NK cells were prepared with multiple variations, as detailed below. NK cells were isolated from frozen umbilical cord blood, then cultured with feeder cells. All test conditions were manufactured from the same cord blood unit. The NK cells were expanded in a humidified incubator in the presence of a culture medium comprising IL-2.
  • the NK cells were genetically engineered to overexpress a CAR (chimeric antigen receptor) construct targeting a tumor of interest. Subsequently, the NK cells were restimulated with feeder cells and cultured for a period in the same medium and at the same oxygen setting. During NK expansion, various medium change schedules were assessed in the context of 21% O 2 culture without any additives. In addition, one group was cultured at 5% O 2 (physoxia) and the remaining groups were cultured at 21% O 2 (standard culture conditions, hyperoxia). For the CAR-NK cells produced at an incubator setting of 21%, some were cultured in the presence of various additives, either individually or in combination.
  • CAR chimeric antigen receptor
  • additives were tested individually: 7 mM nicotinamide, 0.5 mM nicotinamide riboside, 3 mM L-arginine, 30 mM L-carnosine, 0.5 mM N-acetylcysteine, 0.85 mM added Ca ++ , 10 nM leptin, 10 ⁇ M A-769662, or 0.6 mM 2-deoxyglucose.
  • additives were also tested.
  • NK cells were harvested and washed with 5% HSA in phenol-free MEM medium, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, for a final cryoformulation containing 2.5% HSA and 5% DMSO.
  • a control group of CAR-NK cells was also produced from the same initial cord blood unit, Attorney Docket No. MIL-027WO1 however the medium did not contain A-769662, metformin, nicotinamide, L-arginine, or IL- 15, and the cells were cultured at an incubator setting of 21% O 2 .
  • Single-cell cytokine secretion was measured on the IsoPlexis IsoLight instrument with the Human Adaptive Immune Kit, which measures 32 cytokines and immunologically relevant molecules produced by each cell within single-cell microchambers by ELISA.
  • a subset of CAR-NK samples was thawed and recovered in a proprietary medium containing IL-2 and IL-15 for one day. All samples were stimulated with PMA and ionomycin (except for one unstimulated negative control) and then loaded onto the IsoLight instrument. Percent polyfunctionality (the frequency of cells secreting multiple cytokines) and polyfunctional strength index (PSI) were generated from the data.
  • the PSI calculation condenses the multi-dimensional secretions into an overall quality metric, combining both sample polyfunctionality and signal intensity for each individual cell. Both polyfunctionality and PSI have been observed to correlate to clinical outcomes in cancer patients receiving cell therapies.
  • CAR-NK samples that were manufactured with A-769662, nicotinamide, L- arginine, or 5% O 2 all demonstrated remarkably greater polyfunctionality and PSI versus the control process, with the increase being most prominent for A-769662 (FIG.5A, FIG.5B).
  • the PSI score indicated increases in the effector, stimulatory, and chemoattractive categories.
  • Example 6 Evaluating Effect of L-Arginine in Exemplary Donor Derived NK cells [0375] This Example evaluated the effect of L-Arginine in the manufacture or culturing of CAR-NK cells and evaluated its effect in addition to A-769662 and nicotinamide. Cell expansion and cytotoxicity were evaluated. [0376] CAR-NK cells were prepared with multiple variations, as detailed below. NK cells were isolated from frozen umbilical cord blood, then cultured with feeder cells. All test conditions were manufactured from the same cord blood unit.
  • the NK cells were expanded in a humidified incubator in the presence of culture medium with IL-2. Several days after feeder cell activation, the NK cells were genetically engineered to overexpress a CAR (chimeric antigen receptor) construct targeting a tumor of interest. Subsequently, the NK cells were restimulated with feeder cells and cultured for a period in the same medium and at the same oxygen setting. During NK expansion, the following additives were tested individually in the presence of 21% O2: 10 ⁇ M A-769662, 7 mM nicotinamide (NAM), 3 mM L-arginine, 25 Attorney Docket No.
  • NK cells were harvested and washed with 5% HSA in Plasma-Lyte, trehalose added, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, for a final cryoformulation containing 30 mM trehalose, 2.5% HSA and 5% DMSO. At cryopreservation, a further test condition was evaluated which contained 0.5% Poloxamer-188 in the cryoformulation.
  • Short-term cytotoxicity increased on both tumor-antigen-positive and tumor-antigen-negative cell lines, for the following combinations: A-769662 + 2-DG + L-arginine; A-769662 + NAM + L- arginine + 5% O2; NAM + L-arginine + 5% O 2 ; 10% O 2 alone; and 2-DG alone. Cytokine release was notably higher for the following combination, which had augmented Granzyme B and IFN ⁇ release: A-769662 + NAM + L-arginine + 5% O 2 (FIG.6D, FIG.6E).
  • Example 7 Benefits of Fitness Enhancement by NAM and AMPK inhibitor are also applicable to iPS-T/iCAR-T Platform [0381] This Example evaluated the effects of nicotinamide and A-769662, as well as 2-deoxyglucose manufactured CAR-T or iPS-T cells in tumor growth in vitro and in vivo. Attorney Docket No.
  • iPSC-derived T cells were differentiated from peripheral blood mononuclear cells (PBMCs). Several days after feeder-free activation, these iPS-T cells were genetically engineered to overexpress a CAR (chimeric antigen receptor) construct targeting a solid tumor of interest, to generate iCAR-T cells. The iCAR-T cells were further expanded in culture medium formulation containing an activating cytokine cocktail. During iCAR-T cell activation and expansion, various individual compounds and combinations of compounds were tested in the medium formulation. A control group was generated using the standard expansion protocol without these compounds.
  • PBMCs peripheral blood mononuclear cells
  • Control (1) 7 mM nicotinamide (2); 3 mM added L-arginine (3); 30 mM L-carnosine plus 30 mM glycyl- sarcosine in combination (4); 0.5 mM N-acetylcysteine (5); 0.6 mM 2-deoxyglucose (6); 0.85 mM added Ca++ (7); 10 ⁇ M A-769662 (8); 25 ⁇ M UK-5099 (9); and 7 mM nicotinamide plus 10 ⁇ M A-769662 (10) in combination.
  • the basal medium contained 1.5 mM Ca++, the addition of 0.85 mM brought the total concentration to 2.35 mM Ca++, mimicking normocalcemia in humans. Since the basal medium contained 0.4 mM L-arginine, the addition of 3 mM brought the total concentration to 3.4 mM L-arginine.
  • the iCAR-T cells were harvested and washed with 5% HSA in phenol-free MEM medium, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, for a final cryoformulation containing 2.5% HSA and 5% DMSO.
  • FIG.7C is graph showing relative body weight in mice treated with CAR-T cells treated with various additives.
  • the polyfunctionality and polyfunctional strength index (PSI) were increased compared to the control in any groups containing nicotinamide, A-769662, L-arginine, 2-deoxyglucose, and added Ca++ (FIG.7D, FIG 7E).
  • the PSI results demonstrated that A-769662 and Ca++ were separately responsible for increases in the chemoattractive strength of the iCAR-T cells.
  • fitness enhancements such as addition of an AMPK activator, nicotinamide, L-arginine, 2-deoxyglucose, and added Ca++ also led to improvement in various properties of iCAR-T cells such as decreased tumor growth, polyfunctionality and chemoattractive strength.
  • Example 8 Multi-Donor Summary of MEM/HSA washed CAR-NK cells on Post-Thaw Viability
  • CAR-NK cells were prepared with multiple variations, as detailed below. NK cells were isolated from frozen umbilical cord blood, then cultured with feeder cells. All test conditions were manufactured from the same cord blood unit. The NK cells were expanded in a humidified incubator in the presence of culture medium with IL-2.
  • the NK cells were genetically engineered to overexpress a CAR (chimeric antigen receptor) construct targeting a tumor of interest. Multiple CAR constructs were tested within multiple cord blood donors. Subsequently, the NK cells were restimulated with feeder cells and cultured for a period in the same medium and at the same oxygen setting. On the final day of the process, the cells were washed and cryopreserved according to two different methods.
  • CAR chimeric antigen receptor
  • NK cells were harvested and washed with 5% HSA in Plasma-Lyte, trehalose added, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, to yield a final cryoformulation containing 30 mM trehalose, 2.5% HSA and 5% DMSO.
  • the second method the NK cells were harvested and washed with 5% HSA in phenol-free MEM medium, and then cryopreserved after adding one volume of CS10 containing 10% DMSO, for a final cryoformulation containing 2.5% HSA and 5% DMSO.

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Abstract

La présente divulgation concerne, entre autres, une méthode de cryoconservation de cellules tueuses naturelles (cellules NK) consistant à : laver des cellules NK cultivées dans un milieu comprenant un milieu essentiel minimum, et les remettre en suspension dans un milieu de cryoformulation comprenant du MEM, de l'albumine et un ou plusieurs agents cryoprotecteurs ; ainsi que des compositions associées. Selon certains aspects, l'invention concerne une méthode de culture de cellules tueuses naturelles (NK) dans un milieu de culture comprenant un activateur d'AMPK, du nicotinamide, un complément d'arginine, éventuellement contenant un complément lipidique, et/ou cultivé dans un milieu contenant entre 1 et 21 % d'oxygène.
PCT/IB2024/061426 2023-11-16 2024-11-15 Méthodes améliorées de cryoconservation et d'adaptation de cellules tueuses naturelles, et compositions associées Pending WO2025104700A2 (fr)

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