Natural killer (NK) cells cryopreservation

Abstract

The present invention provides, among other things, compositions and methods for protecting cell therapy products from cryoinjury, in particular immune cells (e.g., NK cell products), during cryopreservation. The cryoformulation compositions and methods of the present invention reduce cryoinjury during cryopreserving process, thereby increasing cell therapy product recovery, viability and function after cryopreservation.

Description

Attorney Docket No. MIL-050WO1 NATURAL KILLER (NK) CELLS CRYOPRESERVATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No.63 / 683,525 filed on August 15, 2024, the entire contents of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] The cryopreservation of cell therapy products is essential to allow for storage and off-the-shelf administration to patients, especially for allogeneic products designed to treat many patients from a single manufacturing batch.

[0003] In general, the cryopreservation of cell therapy products, is challenging due to damage to cellular structures such as membranes, proteins, organelles, etc. from ice crystal formation and osmotic stress, among other factors. DMSO-based and glycerol-based cryoformulations alone are insufficient to completely preserve pre-cryopreservation function of cell therapy products, even when they also contain various other cryoprotective agents such as human serum albumin or culture medium. Additional cryoprotective agents and supplements may be required to further reduce cryoinjury and improve cell therapy product recovery and function.

[0004] With respect to NK cell products, due to their unique biology (e.g., their physical characteristics and cytotoxic granules), NK cells are particularly difficult to cryopreserve while retaining their proper function. In particular, NK cells include preformed cytotoxic granules that can rupture during cryopreservation due to ice crystal formation, causing the release of granzymes, perforin, and granulysin into the cytoplasm, inducing cell apoptosis.

[0005] There is a need, then, for formulations and strategies that can overcome these problems during the cryopreservation process of NK cells. The present invention provides several strategies to preserve NK cell function and recovery after cryopreservation. SUMMARY

[0006] The present invention provides, among other things, compositions and methods for protecting cell therapy products from cryoinjury, in particular NK cell products, during cryopreservation. In some aspects, the present invention provides compositions and methods that reduce cryoinjury during cryopreserving process, thereby increasing cell therapy product recovery, viability and function after cryopreservation.Attorney Docket No. MIL-050WO1

[0007] In one aspect, the present invention provides a method for cryopreserving natural killer (NK) cells; the method comprises pretreating the NK cells with ectoine prior to cryopreserving the pretreated NK cells.

[0008] In some embodiments, the method for cryopreserving natural killer (NK) cells comprises i) treating the NK cells with ectoine, ii) harvesting the treated NK cells, and iii) cryopreserving the treated NK cells. In other embodiments, the method may further comprise culturing NK cells in a cell culture medium to expand NK cells prior to treating the cultured NK cells with ectoine.

[0009] In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 1 mM to 200 mM.

[0010] In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 1 mM to 20 mM.

[0011] In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 5 mM to 100 mM.

[0012] In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 10 mM to 50 mM.

[0013] In some embodiments, the NK cells are treated with ectoine at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM.

[0014] In some embodiments, the NK cells are treated with ectoine at a concentration of 5 mM.

[0015] In some embodiments, the NK cells are treated with ectoine at a concentration of 10 mM.

[0016] In some embodiments, the NK cells are treated with ectoine at a concentration of 20 mM.

[0017] In some embodiments, the NK cells are treated with ectoine for 1 to 48 hours prior to cryopreserving the NK cells. In some embodiments, the NK cells are treated with ectoine for 1 to 24 hours prior to cryopreserving the NK cells. In some embodiments, the NK cells are treated with ectoine for 1 to 4 hours prior to cryopreserving the NK cells. In some embodiments, the NK cells are treated with ectoine for 4 to 12 hours prior to cryopreserving the NK cells. In some embodiments, the NK cells are treated with ectoine for 8 to 16 hours prior to cryopreserving the NK cells.Attorney Docket No. MIL-050WO1

[0018] In some embodiments, the NK cells are treated with ectoine for 1 hour, 2 hours, 3 hours, 3 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours or more prior to cryopreserving the pretreated NK cells.

[0019] In some embodiments, ectoine is added to the cell culture medium for treatment.

[0020] In some embodiments, ectoine is added to a wash buffer and / or preformulation buffer. In accordance, the method for cryopreserving natural killer (NK) cells comprises processing the NK cells in a preformulation and / or wash buffer comprising ectoine prior to cryopreserving the processed NK cells. The preformulation buffer and / or wash buffer comprises ectoine at a concentration ranging from 1 mM to 200 mM, or 5 mM to 100 mM, or 10 mM to 100 mM. As a non-limiting example, the preformulation buffer and / or wash buffer comprises ectoine at a concentration of 50 mM.

[0021] In another aspect, the present invention provides a method of cryopreserving NK cells; the method comprises i) culturing NK cells in a cell culture medium, ii) treating the NK cells with IL-18 at a pre-determined concentration at a pre-determined time point, iii) harvesting the treated NK cells, iv) washing the pretreated NK cells to remove most or all residual IL-18, and v) cryopreserving the pretreated NK cells.

[0022] In some embodiments, the method of cryopreserving NK cells comprises pretreating NK cells with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL prior to cryopreserving the pretreated NK cells.

[0023] In some embodiments, the method of cryopreserving NK cells comprises pretreating NK cells with IL-18 at a concentration ranging from 50 U / mL to 2000 U / mL prior to cryopreserving the pretreated NK cells.

[0024] In some embodiments, the method of cryopreserving NK cells comprises pretreating NK cells with IL-18 at a concentration ranging from 150 U / mL to 3000 U / mL prior to cryopreserving the pretreated NK cells.

[0025] In some embodiments, the method of cryopreserving NK cells comprises pretreating NK cells with IL-18 at a concentration ranging from 350 U / mL to 2000 U / mL prior to cryopreserving the pretreated NK cells.

[0026] In some embodiments, the method of cryopreserving NK cells comprises pretreating NK cells with IL-18 at a concentration of 50 U / mL, 100 U / mL, 150 U / mL, 200 U / mL, 250 U / mL, 300 U / mL, 350 U / mL, 400 U / mL, 450 U / mL, 500 U / mL, 550 U / mL, 600 U / mL, 650 U / mL, 700 U / mL, 750 U / mL, 800 U / mL, 900 U / mL, 1000 U / mL, 1500 U / mL, 2000 U / mL, 2500 U / mL, 3000 U / mL, 3500 U / mL, 4000 U / mL, 4500 U / mL, or 5000 U / mLAttorney Docket No. MIL-050WO1 prior to cryopreserving the pretreated NK cells. As a non-limiting example, the NK cells are treated with IL-18 at a concentration of 750 U / mL. In another non-limiting example, the NK cells are treated with IL-18 at a concentration of 1500 U / mL. In yet another non-limiting example, the NK cells are treated with IL-18 at a concentration of 2000 U / mL.

[0027] In some embodiments, the method of cryopreserving NK cells comprises pretreating NK cells with IL-18 for 1 to 48 hours before harvesting the NK cells for cryopreservation.

[0028] In some embodiments, the NK cells are pretreated with IL-18 for 1 to 24 hours before harvesting the NK cells for cryopreservation.

[0029] In some embodiments, the NK cells are pretreated with IL-18 for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, or 48 hours before harvesting the NK cells for cryopreservation. In one example, the NK cells are pretreated with IL-18 for 24 hours before harvesting the NK cells for cryopreservation.

[0030] In some embodiments, IL-18 is added to the cell culture medium for pretreatment.

[0031] In another aspect, the present invention provides a method of cryopreserving NK cells; the method comprises i) culturing NK cells in a cell culture medium, ii) treating the NK cells with IL-18 and IL-15 at pre-determined concentrations of IL-18 and IL-15 at a pre- determined time point, iii) harvesting the treated NK cells, and iv) cryopreserving the pretreated NK cells.

[0032] In some embodiments, the NK cells are pretreated with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL and IL-15 at a concentration ranging from 10 IU / mL to 3000 IU / mL. In some examples, the concentration of IL-15 ranges from 50 IU / mL to 2000 IU / mL, or 100 IU / mL to 2000 IU / mL, or 150 IU / mL to 750 IU / mL. As non-limiting examples, the concentration of IL-15 is 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, 750 IU / mL, 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL, 1000 IU / mL, 1500 IU / mL, 2000 IU / mL, 2500 IU / mL, or 3000 IU / mL.

[0033] In some embodiments, the NK cells are pretreated with IL-18 and IL-15 for 1 to 48 hours before harvesting the NK cells for cryopreservation. In some embodiments, the NK cells are pretreated with IL-18 and IL-15 for 1 to 24 hours, or 4 to 24 hours, or 12 to 24 hours, or 24 to 48 hours before harvesting the NK cells for cryopreservation. As non-limiting examples, the NK cells are pretreated with IL-18 and IL-15 for 1 hour, 2 hours, 3 hours, 4Attorney Docket No. MIL-050WO1 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, or 48 hours before harvesting the NK cells for cryopreservation.

[0034] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises pretreating NK cells with ectoine and IL-18 as described herein. In other embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises pretreating NK cells with ectoine and IL-18 and IL-15 as described herein. A skilled person in the art would understand any combination of pretreatments described herein, e.g., different concentration of ectoine, IL-18 and IL-15, and various pretreatment times, can be employed.

[0035] In some embodiments, the pretreated NK cells described herein are harvested for cryopreservation using a cryoformulation medium. In some embodiments, the cryoformulation medium comprises ectoine. In some embodiments, the cryoformulation medium comprises trehalose. In other embodiments, the cryoformulation medium comprises ectoine and trehalose.

[0036] In some embodiments, the cryoformulation medium comprises ectoine at a concentration ranging from 1 mM to 200 mM, or 5 mM to 200 mM, or 5 mM to 100 mM, or 10 mM to 100 mM, or 10 mM to 50 mM. As non-limiting examples, the cryoformulation medium comprises ectoine at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. As a non-limiting example, the concentration of ectoine in the cryoformulation medium is 25 mM.

[0037] In some embodiments, the cryoformulation medium comprises trehalose at a concentration from 10 mM to 1000 mM, or 10 mM to 500 mM, or 100 mM to 1000 mM, or 100 mM to 500 mM, or 50 mM to 500 mM. As non-limiting examples, the cryoformulation medium comprises trehalose at a concentration of 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, or 1000 mM.

[0038] In some embodiments, the cryoformulation medium further comprises dimethyl sulfoxide (DMSO) and / or glycerol.Attorney Docket No. MIL-050WO1

[0039] In accordance, the present invention provides cryopreserved NK cells using methods described herein, post-thawing NK cells and compositions thereof. In some embodiments, the composition comprising NK cells in a cryoformulation medium comprising ectoine at a concentration from 1 mM to 200 mM. In some embodiments, the composition comprising NK cells in a cryoformulation medium comprising ectoine at a concentration from 5 mM to 200 mM. In some embodiments, the composition comprising NK cells in a cryoformulation medium comprising ectoine at a concentration from 5 mM to 100 mM. In some embodiments, the composition comprising NK cells in a cryoformulation medium comprising ectoine at a concentration from 10 mM to 100 mM. In some embodiments, the composition comprising NK cells in a cryoformulation medium comprising ectoine at a concentration from 10 mM to 50 mM.

[0040] In some embodiments, the composition comprising NK cells in a cryoformulation medium comprises trehalose. The cryoformulation medium comprises trehalose at a concentration ranging from 10 mM to 1000 mM.

[0041] In some embodiments, the composition comprising NK cells in a cryoformulation medium comprises ectoine and trehalose.

[0042] In some embodiments, the composition comprising NK cells in a cryoformulation medium comprises ectoine, trehalose, and DMSO.

[0043] In some embodiments, the composition comprising NK cells in a cryoformulation medium comprises ectoine, trehalose, glycerol and DMSO.

[0044] In some embodiments, the NK cells described herein are obtained from blood, for example, cord blood and peripheral blood. As non-limiting examples, the NK cells are from a healthy donor. In some embodiment, the NK cells described herein are from a cell culture. In some embodiments, the NK cells described herein are primary cells or derived from stem cells (e.g., iPS cells). The primary NK cells in some examples, are further expanded in vitro. In some embodiments, the NK cells described herein are modified, for example, engineered to express one or more therapeutics (e.g., a chimeric antigen receptor (CAR)). In some embodiments, the NK cells are CD56+ / CD3- cells.

[0045] In accordance with the present invention, post-thaw viability of cryopreserved NK cells described herein is at least 35% at 24 hours after thawing. In some embodiments, post- thaw viability of cryopreserved NK cells described herein is 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or more at 24 hours after thawing. In some examples, post-thaw viability is measured utilizing acridine orange and propidium iodide (AO / PI) staining.Attorney Docket No. MIL-050WO1

[0046] In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2 to 2.0-fold, or 1.5 to 5.0-fold greater as compared to cryopreserved NK cells in medium without ectoine. In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2 to 2.0-fold, or 1.5 to 5.0-fold greater as compared to cryopreserved NK cells in medium without trehalose. In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2 to 2.0-fold, or 1.5 to 5.0-fold greater as compared to cryopreserved NK cells without pretreatment of IL-18. In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2 to 2.0-fold, or 1.5 to 5.0-fold greater as compared to cryopreserved NK cells without pretreatment of IL- 18 and IL-15. In some examples, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0-fold greater as compared to cryopreserved NK cells in medium without ectoine. In some examples, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0-fold greater as compared to cryopreserved NK cells in medium without trehalose. In some examples, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0-fold greater as compared to cryopreserved NK cells without pretreatment of IL-18, or IL-18 and IL-15.

[0047] In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2 to 2.0-fold, or 1.5 to 5.0-fold greater as compared to cryopreserved NK cells without one or more pretreatments described herein. In some examples, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0-fold greater as compared to cryopreserved NK cells without one or more pretreatments described herein.

[0048] In some embodiments, cytotoxicity of the post-thawed NK cells is increased, including short-term cytotoxicity. In some embodiments, anti-tumor function of the post- thawed NK cells retains or improves, as measured by repeat killing capacity.

[0049] In another aspect, the present inventions further provide a method of treating a disease comprising administering to a subject in need thereof an effective amount of the composition comprising NK cells described herein. The NK cells, in some aspects, are used for allogeneic cell therapy.

[0050] In some embodiments, the disease is a cancer, an inflammatory disorder, or an autoimmune disorder.Attorney Docket No. MIL-050WO1

[0051] Also provided by the present invention are frozen and thawed cells produced by the methods of the invention, as well as pharmaceutical compositions incorporating the cells, and methods of treatment using the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The drawings are for illustrative purposes only and are not meant to be limiting.

[0053] FIG.1 shows exemplary NK cell 24-hour post-thaw viability after pre-treatment with IL-18 and / or IL-15, or ectoine after overnight recovery in medium containing 100 IU / mL IL-2 treatment. Exemplary condition 4 yielded the highest post-thaw NK cell viability relative to control.

[0054] FIG.2 shows exemplary NK cell 24-hour post-thaw fold-survival after pre- treatment with IL-18 and / or IL-15, or ectoine after overnight recovery in medium containing 100 IU / mL IL-2 treatment relative to control. Exemplary condition 4 yielded the highest post- thaw NK cell fold survival.

[0055] FIG.3 demonstrates the from-vial recovery of exemplary NK cells after pre- treatment with IL-18 and / or IL-15, or ectoine relative to control. Exemplary condition 4, wherein the IL-18 concentration is the lowest, had the largest improvement in recovery.

[0056] FIG.4A – FIG.4B show the effect that the exemplary pre-treatment conditions have on short-term NK cell cytotoxicity relative to control after overnight rest with 100 IU / mL IL-2. FIG.4A shows the percent cytolysis of cryopreserved NK cells after exposure to A549 cells and the associated area under curve (AUC) quantification at all effector:target (E:T) ratios for all conditions. FIG.4B shows the percent cytolysis of cryopreserved NK cells after exposure to MM.1S tumor cells and the associated AUC quantification at all E:T ratios for all conditions.

[0057] FIG.5A- FIG.5B show the results of an exemplary repeat antigen stimulation assays upon repeat challenge against A549 lung tumor cells relative to the control of pre- treated NK cells. FIG.5A depicts the exemplary killing kinetics upon repeat challenge. FIG. 5B is the associated AUC quantification of the repeated antigen stimulation assays against the A549 lung tumor cells.

[0058] FIG.6A – FIG.6D depict exemplary repeated antigen stimulation assays with multiple challenge against A549 lung tumor cells using cryopreserved NK cells pre-treated with either a combination of IL-18 and IL-15 (FIG.6A – FIG.6B) or ectoine (FIG.6C – FIG.6D) relative to control. Further, these assays were conducted at effector:target (E:T)Attorney Docket No. MIL-050WO1 ratios of either 5:1 (FIG.6A, FIG.6C) or 2.5:1 (FIG.6B, FIG.6D) to assess effective killing.

[0059] FIG.7A – FIG.7B depict exemplary repeated antigen stimulation assays with multiple challenge against MM.1S multiple myeloma cells using cryopreserved NK cells pre- treated with cytokines (IL-18, or IL-18 / IL-15) or ectoine relative to control. FIG.7A shows all exemplary pre-treatment conditions and their effective killing against MM.1S tumor cells. FIG.7B describes the same, but highlights condition 4 at an E:T ratio of 5:1 relative to control.

[0060] FIG.8A – FIG.8E show the expression and extracellular / intracellular staining and of various cytokines (Granzyme Band TNFα) in the exemplary frozen drug product versus freshly harvested NK cells. FIG.8A depicts the percentage of CD56+ CD3- pre- treated cryopreserved NK cells with Granzyme B+ expression FIG.8B depicts the geometric mean fluorescence intensity (gMFI) of Granzyme B in the frozen drug product versus the freshly harvested NK cells. FIG.8C depicts the percentage of CD56+ CD3- pre-treated cryopreserved NK cells with perforin+ expression. FIG.8D shows the same but for TNFα+ expression. FIG.8E depicts the gMFI of perforin in the frozen drug product versus the freshly harvested NK cells.

[0061] FIG.9A – FIG.9E are the quantifications of expression exemplary apoptotic markers on freshly harvested NK cells versus the pre-treated frozen drug product NK cells, including caspase3(high) caspase7+ expression (FIG.9A), caspase3(low) caspase7+ expression (FIG.9B), cleaved PARP(high) expression (FIG.9C), cleaved PARP(low) expression (FIG.9D), and AnnexinV+ expression on live cells (FIG.9E).

[0062] FIG.10A – FIG.10D are quantifications of the expression of exemplary classical NK markers on freshly harvested NK cells and frozen drug product NK cells.

[0063] FIG.11A – FIG.11C are quantifications of the expression and staining of exemplary antiapoptotic markers in freshly harvested NK cells versus the pre-treated frozen drug product NK cells, including BCL-2+ expression (FIG.11A, FIG.11B for BCL-2 intracellular protein staining) and TRAF1+ expression (FIG.11C). DETAILED DESCRIPTIONS

[0064] The present invention relates to compositions and methods for protecting cell therapy products from cryoinjury, in particular NK cell products, during cryopreservation. In many aspects, the present invention provides, among other things, compositions and methods that reduce cryoinjury during cryopreserving process thereby increasing cell therapy productAttorney Docket No. MIL-050WO1 recovery, viability and function after cryopreservation; the reduction of cryoinjury can be achieved by pretreating cell therapy products, for example NK cells, prior to cryopreserving cell therapy products (e.g., NK cells).

[0065] NK cells are difficult to cryopreserve due to their physical characteristics and cytotoxic granules. Freeze / thaw is damaging to cells, particularly to NK cells. In general, much apoptosis after thawing manufactured NK cells, especially before fitness enhancement (FE) process was established, was observed.

[0066] With the hypothesis that inducing pro-survival factors could further boost post- thaw viability, which would improve overall NK health post-thaw and therefore their function in vivo, the inventors of the present invention directly address several mechanisms that cause injury to cells during cryopreservation and provide solutions that efficiently reduce cryoinjury to cell products, particularly to NK cell products (e.g., cord blood derived NK cells).

[0067] One strategy, among other things, relates to protecting NK cells from cryoinjury by degranulating the cells prior to cryopreservation. The degranulation is achieved by pretreating NK cells with IL-18, or IL-18 and IL-15 at a predetermined concentration at a predetermined time point prior to cryopreserving the NK cells during manufacturing. In accordance, the present invention provides, among other things, compositions and methods for cryopreserving NK cells by pretreating the cells with IL-18, or IL-18 in combination with IL-15, prior to cryopreserving the cells.

[0068] Another strategy, among other things, relates to pretreating cells with a novel cryoprotectant, ectoine, to reduce cryoinjuries caused, for example, by ice crystal formation and osmotic stress during cryopreserving process. In another aspect, the present invention provides compositions and methods for protecting NK cells from cryoinjury by pretreatment of NK cells prior to cryopreservation with ectoine.

[0069] One or more cryoprotection solutions in the present disclosure can be combined to maximize protection to cell therapy products (e.g., NK cells).

[0070] The present invention provides, among other things, compositions comprising NK cells cryopreserved in a cryoformulation medium. The NK cells are pretreated to reduce cryoinjury with any combination of ectoine, IL-18 and IL-15. Furthermore, he cryoformulation medium are optimized by including one or more cryoprotectants, such as ectoine, trehalose, DMSO and / or glycerol.

[0071] 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 aspectAttorney Docket No. MIL-050WO1 of the invention. Exemplary embodiments are for illustration only. In this application, the use of “or” means “and / or” unless stated otherwise. As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. A. Definitions

[0072] 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. In certain embodiments, 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.

[0073] 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.

[0074] Allogeneic cell therapy: As used herein, the term “allogeneic cell therapy” refers to a type of cellular therapy that uses cells from a donor (e.g., a healthy person) to treat a patient. Cells from a single donor can be used to treat one or more patients. Donor cells (e.g., NK cells isolated from a donor’s blood) can undergo extensive expansion and screening before establishing a master cell bank, which is the basis for generating allogeneic cell therapies. Cells from the donor can be modified (e.g., expressing a chimeric antigen receptor) or unmodified. The cells can be derived from multiple potential cell sources, including but not limited to donated tissues, umbilical cord blood, placenta, bone marrow, and induced pluripotent and embryonic stem cells. An advantage of allogeneic strategies, among other things, is “off-the-shelf” supply. For example, in urgent medical situations, allogeneic therapies utilize donor’s cells, allowing storage and immediate availability to treat multipleAttorney Docket No. MIL-050WO1 patients. In the context of the present disclosure, the NK cells described herein are used for allogeneic cell therapy.

[0075] Cryoformulation: As used herein, the terms “cryoformulation,” “cryoformulation medium,” “cryoformulation solution,” “ cryoformulation composition,” “cryopreservation composition,” and “cryopreservation medium” refer to a composition or medium or a solution (or a composition when diluted or dissolved) in which a biological material (e.g., a cell therapy product) is immersed before cryopreservation or freezing, or to a composition or medium or solution which can be used to treat the cells or tissues prior to freezing. A cryoformulation medium or a cryopreservation composition contains one or more cryoprotectants. In certain embodiments, a cryoformulation medium or solution may be a freezing solution, a vitrification solution, a lyophilization solution and / or a mixture of such solutions. In certain embodiments, a cryoformulation medium or solution is a cryopreservation solution. In certain embodiments, the cryopreservation composition refers to a composition or medium for storing or freezing a biological material (e.g., a cell therapy product) at a temperature at or below about 8° C., at or below about 4° C., at or below about 0° C., at or below about −20° C., at or below about −70° C., at or below about −135° C., at or below about −196° C, or in liquid nitrogen.

[0076] Cryopreservation: As used herein, the term “cryopreservation” refers to a process including at least one step of lowering the temperature of a biological material (e.g., a cell therapy product) from a temperature that is above the freezing temperature of the biological material (or of a mixture of the biological material and a cryopreservation composition) to a temperature that is below that freezing temperature. Cryopreservation encompasses freezing, vitrification and lyophilization. The term “cryopreservation of cells” means to freeze and preserve cells for the purpose of maintaining the cells over a desired period of time without sub-culturing.

[0077] Cryopreservative agent: As used herein, the term “cryo-preservative agent” or “cryoprotectant” or ‘cryoprotecting agent” means a substance which for example in an appropriate solution is used to protect a sample (e.g., cell therapy product) from freezing damage. Cryopreserve agents cab be divided roughly into two different categories; substances that permeate the cell membrane and impermeable substances. Examples of known cryoprotectants are for example DMSO, polyols etc. In some embodiments, the present disclosure includes but is not limited to, cryopreservative agents of DMSO, glycerol, ectoine and trehalose.Attorney Docket No. MIL-050WO1

[0078] Culture: The term “culture” or “cell culture” or “culturing” refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. In various methods described herein, 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 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.

[0079] Expansion: As used herein, the term “expansion” or “expanding” refers to a method of growing a cell population in tissue culture that increases the number of cells in that population. Cells that have undergone ex vivo expansion are referred to as “expanded”.

[0080] Immune cells: As used herein, the term “immune cell” or “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, neutrophils, mast cells, eosinophils, and any combination thereof. In various embodiments, the immune cells produced using methods described herein are NK cells, characterized as CD56+ / CD3- cells.

[0081] Natural Killer (NK) Cell: As used herein, “natural killer cell” or NK cell is a lymphoid cell defined by its marker expression and function / activity. For example, in humans an NK cell expresses CD56 (CD56+). In a further embodiment, such NK cells may express CD56 and CD16 (CD56+ / CD16+). In another example, such NK cells may express CD56 but not express CD3 (CD56+ / CD3-). NK cells may express variable levels of CD56. For example, NK cells may be “CD56high” 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. As another example, NK cells may be “CD56dim” 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.

[0082] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0083] Preservation: As used herein, the term “preservation” refers to the process of maintaining a biological material (e.g., a cell therapy product) under conditions in which its biological activity is considerably reduced while it nonetheless remains viable and mayAttorney Docket No. MIL-050WO1 resume essentially normal biological activity when taken out of the preservation state. A specific example of preservation is cryopreservation.

[0084] Storage: As used herein, the term “storage” or “storing” refers to the placing of cells under relatively constant and / or ambient conditions for extended periods of time, for example, for at least about one hour, at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about one week, at least about two weeks, at least about four weeks, at least about three months, at least about six months, at least about one year, at least about two years, or even longer in some cases, without making use of their viability, e.g., without rehydrating or otherwise activating the cells. The cells are typically in the dormant, non-physiologically active state during storage (e.g., in a dried state, a frozen state, etc.). Typically, during storage, cells stored under relatively constant and / or ambient conditions are left undisturbed or are only minimally disturbed (e.g., the cells may be moved from one location to another, the temperature may fluctuate around a setpoint, etc.). For example, if the temperature is to be controlled during storage (e.g., at a certain setpoint), the cells may be kept in a liquid nitrogen freezer (e.g., a freezer that is cooled using liquid nitrogen, i.e., at a temperature of about −196° C.), a −80° C. freezer, a conventional freezer (e.g., that maintains an environment of roughly −10° C.), a refrigerator (e.g., about 4° C.), an incubator (e.g., at body temperature, about 37° C.), a (heated) water bath, or the like. Accordingly, A “storage temperature” is the temperature at which the biological material is stored. In certain embodiments, the storage temperature is at or below about 8° C., at or below about 4° C., at or below about 0° C., at or below about −20° C., at or below about −60° C., at or below about −70° C., at or below about −135° C., at or below about −196° C., or in liquid nitrogen.

[0085] Subject: As used herein, the term “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. In many embodiments, 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.

[0086] Trehalose: Trehalose is a disaccharide found at high concentrations in a wide variety of organisms that are capable of surviving almost complete dehydration and has been shown to stabilize certain cells during freezing. Trehalose is thought to maintain thermodynamic stability of membranes by preserving phospholipid head group spacing andAttorney Docket No. MIL-050WO1 inhibiting lipid phase transitions and separation during freezing. Trehalose does not easily penetrate lipid bilayers and must be loaded into cells through endocytosis or other methods that temporarily disrupt the cell membrane.

[0087] Treatment: As used herein, the terms “treatment,” “treat,” “therapy” and synonyms thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a disease, for example cancer. Those in need of such treatment include those already with a disease as well as those prone to getting the disease or those in whom a disease is to be prevented.

[0088] Viability: As used herein the term “viability” or “viable” refers to a cell that is capable of normal growth and development after having been cryopreserved and thawed. One of the most crucial factors in any cryobiological procedure is defining the term “viability”. That definition should include one or more critical functions that accurately reflect how the cell normally carries out its biological function. The ultimate test for viability is the reproduction of the cell through two or more generations after freezing. Any methods known to those skilled in the art, such as alamar blue assays, may also be used for cell viability measurements. B. Cryopreservation of NK Cells

[0089] The cryopreservation of cell therapy products is essential to allow for storage and off-the-shelf administration to patients, especially for allogeneic products designed to treat many patients from a single manufacturing batch.

[0090] During cryopreservation of cell therapy products, two major mechanisms for injury to cells and tissues during freezing have been emphasized. First, there are the obvious mechanical injuries which can occur due to either extra or intracellular ice crystal formation. Second, there is the danger of osmotic dehydration (osmotic stress). Current cryopreservation technology consists of different strategies to maintain a balance between these two forms of injury. Basically, when freezing is performed at a rapid rate, there is a tendency for ice crystals to form both intracellularly and extracellularly. However, when cryopreservation is performed at slower rates, there is a tendency for ice crystal formation to occur first in the extracellular medium. As the extracellular ice forms, the cells are exposed to an increasingly hyperosmotic environment. This is due to water sequestration as the ice crystals grow. The cells shrink due to transport of water out of the cell in response to the osmotic imbalance caused by the increasing extracellular solute concentrations. The net result of combining optimal cooling rates and cryoprotective agents is that less of theAttorney Docket No. MIL-050WO1 freezable intracellular water will be converted to ice and osmotic cellular dehydration is limited.

[0091] Many agents with protective properties have been used for cryoprotection. Permeating cryoprotective agents, such as DMSO (dimethysulfoxide) and glycerol, act by penetrating the cell membrane and reducing the intracellular water concentration, thereby reducing the amount of ice formed at any temperature. A variety of nonpermeating protective agents have also been used including such compounds as polyvinylpyrrolidone, hydroxyethyl starch, monosaccharides, and sugar alcohols. For some cells, combination of these two classes of cryoprotective agents may give optimal viability.

[0092] Different cells face their own challenges for cryopreservation. Natural killer (NK) cell therapy products, because of their unique biological features, are particularly difficult to cryopreserve while retaining their proper function. In particular, in addition to the two general factors, NK cells have preformed cytotoxic granules that can rupture during cryopreservation due to ice crystal formation, causing the release of granzymes, perforin, and granulysin into the cytoplasm, which can induce cell apoptosis.

[0093] There is a need, then, for methods, agents and composition that can overcome the problems of ice crystal formation, toxicity and possible osmotic dehydration, granulation during the cryopreservation process of NK cell products. The embodiments below illustrate several strategies to solve these issues. 1. Pretreatment of cells prior to cryopreservation

[0094] In some aspects, NK cells can be pretreated prior to cryopreservation or during the cryopreserving process. a. Pretreatment with Ectoine

[0095] In one aspect, the present disclosure relates to use of novel cryoprotectants such as ectoine to prevent cryoinjury, thereby protecting a cell therapy product. Ectoine is added as a pretreatment (e.g., in a cell culture medium) prior to cryopreservation and / or in a preformulation buffer and / or wash buffer when harvesting cells for cryopreservation with an optimized concentration and timing.

[0096] Ectoine is a binding zwitterionic amino acid derivative, a natural, low molecular weight (cell-permeating), organic osmolyte that is produced by various halophilic (salt- loving) and xerophilic (drought-tolerant) microorganisms to help them survive in extreme environmental conditions, such as high salinity or desiccation (e.g., Galinski et al., Eur. JAttorney Docket No. MIL-050WO1 Biochem., 1985, 149(1), 135-139). Ectoine belongs to a class of compounds called compatible solutes, which can accumulate in high concentrations within cells without interfering with normal cellular functions. Characterized by a cyclic ring structure containing a carboxyl group and a methyl group, Ectoine structures water around it to create hydration shells that protect cell membranes, proteins and other cellular components under stress conditions (Zaccai et al., Sci. Pep., 2016, 6:31434). Ectoine has been extensively characterized as an osmoprotectant and stabilizer for cells and biomolecules. Ectoine binds to water molecules better than other well-known osmoprotectants / cryoprotectants (e.g., glycerol) and is well tolerated by humans, animals, and various cell cultures. Without wishing to be bound by any theory, it is proposed that ectoine benefits cells during cryopreservation by providing osmotic stress protection through balancing osmotic pressure across cell membrane, and stabilizing proteins, membranes, and nucleic acids. Ectoine can potentially reduce ice crystal formation. Additionally, ectoine is non-toxic, even at high concentrations.

[0097] The inventors of the present invention discovered that pretreating NK cells with ectoine prior to cryopreserving the pretreated cells can increase NK cells recovery, viability and function (e.g., cytotoxicity of NK cells) at least 24 hours after freeze-thaw.

[0098] In one embodiment, the present invention provides a method for cryopreserving natural killer (NK) cells; the method comprises pretreating the NK cells with ectoine prior to cryopreserving the pretreated NK cells.

[0099] In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 1 mM to 200 mM. In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 5 mM to 200 mM. In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 5 mM to 100 mM. In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 10 mM to 100 mM. In some embodiments, the NK cells are treated with ectoine at a concentration ranging from 10 mM to 50 mM.

[0100] In some embodiments, the NK cells are treated with ectoine at a concentration of 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM.

[0101] In some embodiments, the NK cells are pretreated with ectoine at a concentration of 5 mM.

[0102] In some embodiments, the NK cells are pretreated with ectoine at a concentration of 10 mM.Attorney Docket No. MIL-050WO1

[0103] In some embodiments, the NK cells are pretreated with ectoine at a concentration of 20 mM.

[0104] In some embodiments, the NK cells are pretreated with ectoine at a concentration of 25 mM.

[0105] In some embodiments, the NK cells are pretreated with ectoine at a concentration of 50 mM.

[0106] In some embodiments, the NK cells are pretreated with ectoine at a concentration of 100 mM.

[0107] In some embodiments, the NK cells are pretreated with ectoine at a concentration of 200 mM.

[0108] In some embodiments, the NK cells are pretreated with ectoine for 1 to 48 hours prior to cryopreserving the NK cells. In some embodiments, the NK cells are pretreated with ectoine for 1 to 24 hours prior to cryopreserving the NK cells. In some embodiments, the NK cells are treated with ectoine for 1 to 4 hours prior to cryopreserving the NK cells. In some embodiments, the NK cells are treated with ectoine for 4 to 12 hours prior to cryopreserving the NK cells. In some embodiments, the NK cells are treated with ectoine for 8 to16 hours prior to cryopreserving the NK cells.

[0109] In some embodiments, the NK cells are pretreated with ectoine for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or more prior to cryopreserving the NK cells. In some embodiments, the NK cells are pretreated with ectoine for 28 hours, 30 hours, 36 hours or 48 hours.

[0110] In one embodiment, the NK cells are pretreated with ectoine for 1 hour.

[0111] In one embodiment, the NK cells are pretreated with ectoine for 8 hours.

[0112] In one embodiment, the NK cells are pretreated with ectoine for 12 hours.

[0113] In one embodiment, the NK cells are pretreated with ectoine for 16 hours.

[0114] In one embodiment, the NK cells are pretreated with ectoine for 24 hours.

[0115] In some embodiments, ectoine is added to the cell culture medium for pretreatment.

[0116] In some embodiments, ectoine is added to a pre-formulation buffer. The method for cryopreserving NK cells comprises processing the NK cells in a pre-formulation buffer comprising ectoine prior to cryopreserving the processed NK cells. The pre-formulation buffer comprises ectoine at a concentration ranging from 1 mM to 200 mM, or 5 mM to 200Attorney Docket No. MIL-050WO1 mM, or 5 mM to 100 mM, or 10 mM to 100 mM, or 10 mM to 50 mM. For example, the pre- formulation buffer comprises ectoine at a concentration of 10 mM, 25 mM, 50 mM, 100 mM or 200 mM. In one embodiment, the pre-formulation buffer comprises ectoine at a concentration of 50 mM. In one embodiment, the pre-formulation buffer comprises ectoine at a concentration of 100mM.

[0117] Ectoine treatment prior to cryopreservation improves viable cell recovery from the vial immediately post-thaw.

[0118] The present invention has demonstrated that ectoine treatment improved short-term cytotoxicity of cell therapy products (NK cells) (e.g., cytotoxicity on tumor cells), and improved anti-tumor functional persistence in a 14-day repeat antigen challenge assay (RAS) on tumor cells. The ectoine pretreatment also increased perforin, GM-CSF, and TNFa release from the thawed NK cells, in response to tumor cells (e.g., A549 tumor cells). The ectoine pretreatment also reduced caspase 3 / 7 and cleaved PARP signal post-thaw (apoptotic markers).

[0119] Without wishing to be bound by any theory, it is proposed that ectoine can protect membranes and proteins from intracellular and extracellular ice crystal damage. In some aspects, ectoine pretreatment can be used for protecting other cell therapy products for cryopreservation, including but not limited to immune cells (e.g., T cells) and stem cells (e.g., iPS cells). Therefore, the present invention further provides a method of cryopreserving a cell therapy product which comprises pretreating the cell therapy product with ectoine prior to cryopreserving the pretreated cells. b. Pretreatment with IL-18 and IL-15

[0120] In another aspect, the present disclosure relates to pretreating NK cells for degranulation prior to cryopreservation of the cells.

[0121] Due to their unique biology, NK cells are particularly difficult to cryopreserve while retaining their proper function. In particular, NK cells have preformed cytotoxic granules that can rupture during cryopreservation due to ice crystal formation, causing the release of granzymes, perforin, and granulysin into the cytoplasm, which induces cell apoptosis (Berjis et al., Nature Comm., 2024, 162(8): 4511-4520).

[0122] It has been reported that interleukin 18 (IL-18) plays an important role in NK cells survival. IL-18 is a known NK stimulatory cytokine that has potential to improve NK cytotoxicity, cytokine production (especially IFN-g), proliferation, and survival. Hodge et al, demonstrated that IL-18 has ability to protect NK cells by inducing prosurvival factorsAttorney Docket No. MIL-050WO1 (Hodge et al, Journal of Interferon & Cytokine Research, 2006, 706-718), in which it indicates that the addition of IL-18 needs only a short window to have its effect.

[0123] Interleukin 15 (IL-15) can also induce NK cell survival and proliferation, as an essential driver of NK cell development and activation. IL-15 expression correlates with NK cell infiltration in human tumor samples. IL-15, together with IL-12, also indirectly regulates NK cell functions by controlling metabolism via mTORC1 activation, which stimulates nutrient uptake, glycolysis, and OXPHOS, thereby providing the energy for NK cell proliferation, proper functions and enhanced persistence. In comparison to IL-2, IL-15 has a more potent effect on NK cell expansion. Combination of IL-21 and IL-15 has been shown to selectively promote the expansion of cytolytic CD56+CD16+ subtype of NK cells.

[0124] IL-15 and IL-18, among their multiple stimulatory effects on NK cells, can induce degranulation (Fehniger et al, Immunol., 1999, 162(8):4511-4520), which may contribute to their cryoprotective properties. A recent publication by Berjis et al (Nature Comm., 2024, 15: 3937), showed that pretreating NK cells with IL-15 and IL-18 prior to cryopreservation, can increase NK cell recovery post-thaw and maintain their anti-tumor function as non- cryopreserved NK cells. However, the concentrations and timing of pretreatment of NK cells using IL-18 and IL-15 needs to be optimized to achieve maximal protection (e.g., increased degranulation and antiapoptotic genes).

[0125] The present inventors tested different concentrations of IL-18 and IL-15, and demonstrated the addition of a particular combination of cytokines (IL-18 and IL-15) during manufacturing at the right concentration and with the right timing can protect cryopreserved NK cells. Furthermore, the present inventors tested combined treatment of NK cells with ectoine, a cryoprotectant, and cytokines (IL-18 and IL-15); the combination enhance protection of cryopreserved NK cells.

[0126] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises i) culturing NK cells in a cell culture medium, ii) treating the NK cells with IL-18 at a pre-determined concentration at a pre-determined time point, iii) harvesting the treated NK cells, and iv) cryopreserving the pretreated NK cells.

[0127] In some embodiments, the method of cryopreserving NK cells comprises pretreating NK cells with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL prior to cryopreserving the pretreated NK cells. The biologic activity unit (U) of cytokines can be measured using any assays (e.g., a cell proliferation assay) known in the art. For example, the biologic activity unit (U) of IL-18 is measured by its effect on NK cells under standardized condition.Attorney Docket No. MIL-050WO1

[0128] In some embodiments, the NK cells are pretreated with IL-18 at a concentration ranging from 50 U / mL to 4000 U / mL, or 50 U / mL to 3000 U / mL, or 50 U / mL to 2000 U / mL, or 100 U / mL to 4000 U / mL, or 100 U / mL to 3000U / mL, or 100U / mL to 2000 U / mL, or 150 U / mL to 3000 U / mL, or 200 U / mL to 3000 U / mL, or 200 U / mL to 2000 U / mL, or 350 U / mL to 3000 U / mL, or 350U / mL to 2000U / mL prior to cryopreserving the pretreated NK cells.

[0129] In some embodiments, the NK cells are pretreated with IL-18 at a concentration ranging from 150 U / mL to 3000 U / mL prior to cryopreserving the pretreated NK cells.

[0130] In some embodiments, the NK cells are pretreated with IL-18 at a concentration ranging from 350 U / mL to 2000 U / mL prior to cryopreserving the pretreated NK cells.

[0131] In some embodiments, the method of cryopreserving NK cells comprises pretreating NK cells with IL-18 at a concentration of 50 U / mL, 100 U / mL, 150 U / mL, 200 U / mL, 250 U / mL, 300 U / mL, 350 U / mL, 400 U / mL, 450 U / mL, 500 U / mL, 550 U / mL, 600 U / mL, 650 U / mL, 700 U / mL, 750 U / mL, 800 U / mL, 850 U / mL, 900 U / mL, 950 U / mL, 1000 U / mL, 1500 U / mL, 2000 U / mL, 2500 U / mL, 3000 U / mL, 3500 U / mL, 4000 U / mL, 4500 U / mL, or 5000 U / mL prior to cryopreserving the pretreated NK cells.

[0132] In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 750 U / mL.

[0133] In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 1500 U / mL.

[0134] In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 2000 U / mL.

[0135] In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 3000 U / mL.

[0136] In some embodiments, the NK cells are pretreated with IL-18 at a concentration ranging from 25ng / mL to 3000ng / mL prior to cryopreserving the pretreated NK cells. IL-18 is at a concentration ranging from 25ng / mL to 2000ng / mL, or 50ng / mL to 2000ng / mL, or 50ng / mL to 1000ng / mL, or 100ng / mL to 2000ng / mL, or 100ng / mL to 1000ng / mL. In some embodiments, the NK cells are pretreated with IL-18 at a concentration of 25ng / mL, 30ng / mL, 35ng / mL, 40ng / mL, 45ng / mL, 50ng / mL, 55ng / mL, 60ng / mL, 65ng / mL, 70ng / mL, 75ng / mL, 80ng / mL, 85ng / mL, 90ng / mL, 95ng / mL, 100ng / mL, 110ng / mL,, 120ng / mL, 130ng / mL, 140ng / mL, 150ng / mL, 160g / mL, 170ng / mL, 180ng / mL, 190ng / mL, 200ng / mL, 250ng / mL, 300ng / mL, 350ng / mL, 400ng / mL, 450ng / mL, 500ng / mL, 550ng / mL, 600ng / mL,Attorney Docket No. MIL-050WO1 650ng / mL, 700ng / mL, 750ng / mL, 800ng / mL, 850ng / mL, 900ng / mL, 950ng / mL, 1000ng / mL, 1500ng / mL, 2000ng / mL, 2500ng / mL, or 3000ng / mL.

[0137] In some embodiments, the NK cells are pretreated with IL-18 for 1 to 48 hours before harvesting the NK cells for cryopreservation.

[0138] In some embodiments, the NK cells are pretreated with IL-18 for 1 to 24 hours before harvesting the NK cells for cryopreservation. In some embodiments, the NK cells are pretreated with IL-18 for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 20 hours, 24 hours, 26 hours, 30 hours, 36 hours, or 48 hours before harvesting the NK cells for cryopreservation.

[0139] In one example, the NK cells are pretreated with IL-18 for 24 hours before cryopreservation.

[0140] In one example, the NK cells are pretreated with IL-18 for 18 hours before cryopreservation.

[0141] In one example, the NK cells are pretreated with IL-18 for 12 hours before cryopreservation.

[0142] In one example, the NK cells are treated with IL-18 at least one hour before cryopreservation.

[0143] In one embodiment, the NK cells are treated with IL-18 at concentration of 750 U / mL for 24 hours before cryopreservation.

[0144] In one embodiment, the NK cells are treated with IL-18 at concentration of 1500 U / mL for 24 hours before cryopreservation.

[0145] In one embodiment, the NK cells are treated with IL-18 at concentration of 2000 U / mL for 24 hours before cryopreservation.

[0146] In some embodiments, IL-18 is added to the cell culture medium for pretreatment.

[0147] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises i) culturing NK cells in a cell culture medium, ii) treating the NK cells with IL-18 and IL-15 at pre-determined concentrations of IL-18 and IL-15 at a pre- determined time point, iii) harvesting the treated NK cells, and iv) cryopreserving the pretreated NK cells.

[0148] In some embodiments, the NK cells are pretreated with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL and IL-15 at a concentration ranging from 10 IU / mL to 3000 IU / mL.Attorney Docket No. MIL-050WO1

[0149] In some examples, the concentration of IL-15 ranges from 50 IU / mL to 2000 IU / mL, or 100 IU / mL to 2000 IU / mL, or 150 IU / mL to 1000 IU / mL, or 150 IU / mL to 750 IU / mL. As non-limiting examples, the concentration of IL-15 is 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, 750 IU / mL, 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL, 1000 IU / mL, 1500 IU / mL, 2000 IU / mL, 2500 IU / mL, or 3000IU / mL.

[0150] In one embodiment, the concentration of IL-15 is 750 IU / mL.

[0151] In one embodiment, the concentration of IL-15 is 1500 IU / mL.

[0152] The biological activity units, e.g., International Units (IU) of IL-15 are calculated using a validated assay known in the art. For example, IL-15 is tested side-by-side with an internal standard set by the World Health Organization (WHO). The activity of IL-15 is then normalized to the activity level of the standard. The WHO-defined standard can be obtained from the National Institute for Biological Standards and Control (NIBSC).

[0153] In some embodiments, the NK cells are pretreated with IL-18 at a concentration ranging from 25ng / mL to 3000ng / mL and IL-15 at a concentration ranging from 0.5ng / mL to 250ng / mL. In some embodiments, IL-15 is at a concentration ranging from 1ng / mL to 200ng / mL, or 10ng / mL to 100ng / mL, or 20ng / mL to 150ng / mL, or 15ng / mL to 250ng / mL. In some embodiments, IL-15 is at a concentration of 0.5ng / mL, 1ng / mL, 5ng / mL, 10ng / mL, 15ng / mL, 20ng / mL, 25ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 120ng / mL, 150ng / mL, 200ng / mL or 250ng / mL.

[0154] In some embodiments, the NK cells are pretreated with IL-18 and IL-15 for 1 to 48 hours before harvesting the NK cells for cryopreservation. In some embodiments, the NK cells are pretreated with IL-18 and IL-15 for 1 to 24 hours, or 4 to 24 hours, or 12 to 24 hours, or 24 to 48 hours before harvesting the NK cells for cryopreservation. As non-limiting examples, the NK cells are pretreated with IL-18 and IL-15 for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, or 48 hours before harvesting the NK cells for cryopreservation.

[0155] In one example, the NK cells are pretreated with IL-18 and IL-15 for 24 hours before cryopreservation.

[0156] In one example, the NK cells are pretreated with IL-18 and IL-15 for 18 hours before cryopreservation.

[0157] In one example, the NK cells are pretreated with IL-18 and IL-15 for 12 hours before cryopreservation.Attorney Docket No. MIL-050WO1

[0158] In one example, the NK cells are treated with IL-18 and IL-15 at least one hour before cryopreservation.

[0159] In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 750 U / mL and IL-15 at a concentration of 750 IU / mL for 24 hours before cryopreservation. In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 750 U / mL and IL-15 at a concentration of 1500 IU / mL for 24 hours before cryopreservation.

[0160] In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 1500 U / mL and IL-15 at a concentration of 750 IU / mL for 24 hours before cryopreservation. In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 1500 U / mL and IL-15 at a concentration of 1500 IU / mL for 24 hours before cryopreservation.

[0161] In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 2000 U / mL and IL-15 at a concentration of 750 IU / mL for 24 hours before cryopreservation. In one embodiment, the NK cells are pretreated with IL-18 at a concentration of 2000 U / mL and IL-15 at a concentration of 1500 IU / mL for 24 hours before cryopreservation.

[0162] In some embodiments, IL-18 and IL-15 are added to the cell culture medium to pretreat NK cells.

[0163] Pretreating NK cells with IL-18 or IL-18 and IL-15 before cryopreservation improves 24-hour post-thaw viability, 24-hour post-thaw survival and proliferation, and / or viable cell recovery from the vial immediately post-thaw.

[0164] In some embodiments, pretreating NK cells with IL-18 or IL-18 and IL-15 before cryopreservation improves short-term cytotoxicity of the cell therapy product on tumor cells.

[0165] In some embodiments, pretreating NK cells with IL-18 or IL-18 and IL-15 before cryopreservation improves anti-tumor functional persistence in a 14-day repeat tumor challenge assay on tumor cells.

[0166] In some embodiments, pretreating NK cells with IL-18 or IL-18 and IL-15 before cryopreservation reduces caspase 3 / 7 and cleaved PARP signal post-thaw (apoptotic markers), increases TNFα intracellular detection post-thaw, and / or increases TRAF1 expression pre- and post-thaw.

[0167] Without wishing to be bound by any theory, it is proposed that IL-18 and / or IL-15 pre-treatment before cryopreservation induces NK degranulation, including reducing the effects of cryodamage to cytotoxic granules. reduction in intracellular Granzyme B levels via degranulation, and the induction of antiapoptotic genes. c. Combination of pretreatmentAttorney Docket No. MIL-050WO1

[0168] To maximize protection of NK cell products during cryopreservation process, the protection solutions described herein are used in combination.

[0169] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises pretreating NK cells with ectoine and IL-18 concurrently as described herein.

[0170] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises pretreating NK cells with ectoine and IL-18 and IL-15 concurrently as described herein.

[0171] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises i) pretreating NK cells with IL-18, ii) harvesting the pretreated NK cells using a pre-formulation buffer comprising ectoine; and iii) cryopreserving the harvested NK cells in a cryoformulation medium.

[0172] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises i) pretreating NK cells with IL-18 and IL-15, ii) harvesting the pretreated NK cells using a pre-formulation buffer comprising ectoine; and iii) cryopreserving the harvested NK cells in a cryoformulation medium.

[0173] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises i) pretreating NK cells with IL-18 and ectoine, ii) harvesting the pretreated NK cells using a pre-formulation buffer comprising ectoine; and iii) cryopreserving the harvested NK cells in a cryoformulation medium.

[0174] In some embodiments, the present invention provides a method of cryopreserving NK cells; the method comprises i) pretreating NK cells with IL-18, IL-15 and ectoine, ii) harvesting the pretreated NK cells using a pre-formulation buffer comprising ectoine; and iii) cryopreserving the harvested NK cells in a cryoformulation medium.

[0175] A killed person in the art would understand any combination of pretreatments described herein, e.g., different concentration of ectoine, IL-18 and IL-15, and pretreatment times, can be employed.

[0176] In some embodiments, NK cells treated with ectoine, IL-18 and / or IL-15 are preserved in a cryoformulation medium comprising ectoine. In some embodiments, NK cells treated with ectoine, IL-18 and / or IL-15 are preserved in a cryoformulation medium comprising trehalose. In some embodiments, NK cells treated with ectoine, IL-18 and / or IL- 15 are preserved in a cryoformulation medium comprising ectoine and trehalose. In some embodiments, NK cells treated with ectoine, IL-18 and / or IL-15 are preserved in a cryoformulation medium comprising ectoine and trehalose, and glycerol and / or DMSO.Attorney Docket No. MIL-050WO1 2. Cryoformulation Compositions

[0177] As described herein, pretreated NK cells are processed for cryopreservation. In some embodiments, the pretreated NK cells are cryopreserved using a cryoformulation medium.

[0178] A cryoformulation medium typically includes cryoprotective agents to improve viability of cells after freeze-thaw. A variety of substances have been used or investigated as potential additives to enhance viability and function of cells in the freezing process.

[0179] The inventors of the present disclosure have found that in addition to pretreatment of cells as described herein, inclusion of ectoine and / or trehalose in the cryoformulation medium also is surprisingly effective in preserving cell therapy products, e.g., NK cells.

[0180] In some embodiments, the cryoformulation medium for preserving a cell therapy product comprises ectoine.

[0181] In some embodiments, the cryoformulation medium comprises ectoine at a concentration ranging from 1 mM to 200 mM, or 5 mM to 200 mM, or 10 mM to 200 mM, or 10 mM to 100 mM, or 10 mM to 50 mM. As non-limiting examples, the cryoformulation medium comprises ectoine at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM.

[0182] In one embodiment, the cryoformulation medium comprises ectoine at a concentration of 25 mM.

[0183] In some embodiments, the cryoformulation medium comprises trehalose.

[0184] In some embodiments, the cryoformulation medium comprises trehalose at a concentration from 10 mM to 1000 mM, or 10 mM to 500 mM, or 10 mM to 300 mM, or 25 mM to 500 mM, or 25 mM to 350 mM. As non-limiting examples, the cryoformulation comprises trehalose at a concentration of 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, or 1000 mM.

[0185] In other embodiments, the cryoformulation medium comprises ectoine and trehalose. In some embodiments, the cryoformulation medium comprises ectoine at aAttorney Docket No. MIL-050WO1 concentration ranging from 1 mM to 200 mM and trehalose at a concentration from 10 mM to 1000 mM.

[0186] In some embodiments, the cryoformulation medium comprising ectoine and / or trehalose further comprises one or more cryoprotectants that help maintain the viability of cells under cryopreservation and prevent cells from damage that may be caused in cryopreservation.

[0187] Non-limiting examples of cryoprotectants include permeating cryoprotectants such as glycerol, polyethylene glycol, ethylene glycol, propylene glycol (1,2-propanediol, propane-1,2-diol), and DMSO, and non-permeating cryoprotectants such as saccharides (e.g., sucrose, trehalose, maltose), sugars, starches (e.g., hydroxyethyl starch), protein (e.g., albumin such as serum albumin), percoll, ficol, polyethylene glycol, dextran, polyvinyl pyrrolidone, polyvinylalcohol (PVA), serum, plasma and other macromolecules.

[0188] In some embodiments, the cryoformulation medium described herein further comprises dimethyl sulfoxide (DMSO) and / or glycerol. In some embodiments, glycerol and / or DMSO act as the primary cryoprotective agent, combined with other components that serve both cryoprotective and non-cryoprotective functions in the cryoformulation composition.

[0189] In some embodiments, the cryoformulation medium described herein comprises DMSO at a concentration between 1% to 20% (v / v) or any discrete quantity therebetween. As non-limiting examples, the cryoformulation medium described herein comprises DMSO at a concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (v / v). In one embodiment, the cryoformulation medium described herein comprises DMSO at a concentration of 1% (v / v). In one embodiment, the cryoformulation medium described herein comprises DMSO at a concentration of 5% (v / v). In one embodiment, the cryoformulation medium described herein comprises DMSO at a concentration of 10% (v / v).

[0190] In some embodiments, the cryoformulation medium described herein comprises glycerol at a concentration between 1% to 20% (v / v) or any discrete quantity therebetween. As non-limiting examples, the cryoformulation medium described herein comprises glycerol at a concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (v / v).

[0191] In some embodiments, the cryoformulation medium described herein comprises CryoStor CS10. In some embodiments, the cryoformulation medium described herein comprises CryoStor CS5.Attorney Docket No. MIL-050WO1

[0192] In some embodiments, the cryoformulation medium described herein further comprises albumin, for example, serum albumin (e.g., human serum albumin or HAS), plasma albumin (e.g., human plasma albumin), bovine serum albumin, and / or synthetic serum albumin), ovalbumin, plant albumin, or combinations thereof. Albumin may be either of natural origin (e.g., purified from a natural source) or of recombinant origin (recombinant albumin).

[0193] In other embodiments, the cryoformulation medium described herein may further comprise amino acids, cytokines, lipids, growth factors, antibiotics, antimycotics, steroid hormones, protein hormones, serum, proteins, salts, formamide, methoxylated compounds, and / or polymers (e.g., polyvinyl pyrrolidone and polyvinyl alcohol). 3. Cryopreserving Process and Methods

[0194] In some embodiments, the present disclosure provides processes for preserving a cell therapy product. The present processes have particular applicability to the cryopreservation of a NK cell therapy product.

[0195] In general, the cryopreserving process described herein comprises i) preparing a cell sample, ii) pretreating the cell sample as described herein, iii) collecting the pretreated cell sample and iv) cryopreserving the cell sample for storage.

[0196] The starting material for cryopreservation is generally a cell sample, including but not limited to a primary cell sample and a cell culture.

[0197] In terms of preparation for freezing, the cells may be disaggregated to form a suspension by either enzymatic or mechanical means. Similarly, passaging after thawing can be achieved mechanically. The skilled person will understand that there are many standard methods for preparing a cell suspension for the purposes of freezing or further passaging the cells.

[0198] In one aspect, the process for cryopreserving natural killer (NK) cells comprises i) preparing a cell sample comprising NK cells, ii) treating the NK cells with ectoine, iii) harvesting the treated NK cells, and iv) cryopreserving the treated NK cells. The method comprises culturing NK cells in a cell culture medium to expand NK cells for preparing the NK cell sample.

[0199] In some embodiments, the cell culture medium is spiked with ectoine at a concentration ranging from 1 mM to 200 mM a day before harvesting the cells for cryopreservation. In some embodiments, the cell culture medium is spiked with ectoine at a concentration ranging from 1 mM to 20 mM a day before harvesting the cells forAttorney Docket No. MIL-050WO1 cryopreservation. In some embodiments, the cell culture medium is spiked with ectoine at a concentration ranging from 1 mM to 10 mM a day before harvesting the cells for cryopreservation. In some embodiments, the cell culture medium is spiked with ectoine at a concentration of 5 mM a day before harvesting the cells for cryopreservation. In some embodiments, the cell culture medium is spiked with ectoine at a concentration of 10 mM a day before harvesting the cells for cryopreservation.

[0200] In some embodiments, the process comprises harvesting the cells using a pre- formulation buffer (i.e., a wash buffer) comprising ectoine. The buffer may comprise ectoine at a concentration ranging from 10 mM to 200 mM. In some examples, the buffer comprises ectoine at a concentration ranging from 50 mM to 100 mM. In some examples, the buffer comprises ectoine at a concentration of 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80mM, 85 mM, 90mM, 95mM, 100 mM, 110 mM, 120 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160mM, 170 mM, 180 mM, 190 mM, or 200 mM. In some examples, the buffer comprises ectoine at a concentration of 50 mM. In some examples, the buffer comprises ectoine at a concentration of 100 mM.

[0201] In some embodiments, the preformulation buffer and / or wash buffer comprises Minimal Essential Medium (MEM) and HSA.

[0202] In some embodiments, the process comprises cryopreserving the treated NK cells using a cryoformulation medium comprising ectoine as described herein. In some examples, the process comprises diluting the pre-formulation buffer comprising ectoine with a Cryostor CS10 solution at a ratio 1:1 to form a cryoformulation medium. Accordingly, the cryoformulation medium comprising ectoine at a concentration half of its concentration in the pre-formulation buffer.

[0203] A skilled person in the art would understand that the cryopreservation steps described herein can be added or skipped. Exemplary processes described herein are for illustration only. In some examples, the process comprises pretreating cells to be preserved using ectoine, and cryopreserving the cells in a cryoformulation medium comprising ectoine. In other examples, the process comprises harvesting cells to be preserved in a preformulation buffer comprising ectoine, and cryopreserving the cells in a cryoformulation medium comprising ectoine (i.e., no pretreatment with ectoine). In yet other examples, the process comprises pretreating cells to be preserved using ectoine, harvesting the pretreated cells in a preformulation buffer comprising ectoine and cryopreserving the cells in a cryoformulation medium comprising ectoine.Attorney Docket No. MIL-050WO1

[0204] As a non-limiting example, the processing comprises pretreating NK cells with ectoine at a concentration of 10 mM for 24 hours; harvesting the pretreated cells in a preformulation buffer comprising ectoine at a concentration of 50 mM; and preserving the cells in a medium by diluting the preformulation buffer with a Cryostor CS10 solution at a ratio of 1:1, thereby forming a cryoformulation medium comprising ectoine at a concentration of 25 mM.

[0205] In another aspect, the process for cryopreserving natural killer (NK) cells comprises i) preparing a cell sample comprising NK cells, ii) treating the NK cells with IL- 18, or IL-18 and IL-15, iii) harvesting the treated NK cells, and iv) cryopreserving the treated NK cells. The method comprises culturing NK cells in a cell culture medium to expand NK cells for preparing the NK cell sample.

[0206] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL two days before harvesting the cells for cryopreservation.

[0207] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL a day before harvesting the cells for cryopreservation.

[0208] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL about 18 hours before harvesting the cells for cryopreservation.

[0209] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL about 4 hours before harvesting the cells for cryopreservation.

[0210] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL about 1 hour before harvesting the cells for cryopreservation.

[0211] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL and IL-15 at a concentration ranging from 10 IU / mL to 3000 IU / mL two days before harvesting the cells for cryopreservation.

[0212] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL and IL-15 at a concentration ranging from 10 IU / mL to 3000 IU / mL a day before harvesting the cells for cryopreservation.

[0213] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL and IL-15 at a concentration rangingAttorney Docket No. MIL-050WO1 from 10 IU / mL to 3000 IU / mL about 18 hours before harvesting the cells for cryopreservation.

[0214] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL and IL-15 at a concentration ranging from 10 IU / mL to 3000 IU / mL about 4 hours before harvesting the cells for cryopreservation.

[0215] In some embodiments, the cell culture medium is spiked with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL and IL-15 at a concentration ranging from 10 IU / mL to 3000 IU / mL about one hour before harvesting the cells for cryopreservation.

[0216] In one embodiment, the cell culture medium comprising NK cells is spiked with IL-18 at a concentration ranging of 750 U / mL and IL-15 at a concentration ranging from 750 IU / mL 24 hours before harvesting the cells for cryopreservation. In one embodiment, the cell culture medium comprising NK cells is spiked with IL-18 at a concentration ranging of 750 U / mL and IL-15 at a concentration ranging from 1500 IU / mL 24 hours before harvesting the cells for cryopreservation. In one embodiment, the cell culture medium comprising NK cells is spiked with IL-18 at a concentration ranging of 1500 U / mL and IL-15 at a concentration ranging from 750 IU / mL 24 hours before harvesting the cells for cryopreservation. In one embodiment, the cell culture medium comprising NK cells is spiked with IL-18 at a concentration ranging of 1500 U / mL and IL-15 at a concentration ranging from 1500 IU / mL 24 hours before harvesting the cells for cryopreservation. In one embodiment, the cell culture medium comprising NK cells is spiked with IL-18 at a concentration ranging of 2000 U / mL and IL-15 at a concentration ranging from 750 IU / mL 24 hours before harvesting the cells for cryopreservation. In one embodiment, the cell culture medium comprising NK cells is spiked with IL-18 at a concentration ranging of 2000 U / mL and IL-15 at a concentration ranging from 1500 IU / mL 24 hours before harvesting the cells for cryopreservation.

[0217] In some embodiments, the process comprises harvesting the IL-18 pretreated NK cells in a pre-formulation buffer comprising ectoine.

[0218] In some embodiments, the process comprises harvesting the IL-18 and IL-15 pretreated NK cells in a pre-formulation buffer comprising ectoine.

[0219] In some embodiments, the process comprises preserving the IL-18 pretreated NK cells in a cryoformulation medium comprising ectoine.

[0220] In some embodiments, the process comprises preserving the IL-18 and IL-15 pretreated NK cells in a cryoformulation medium comprising ectoine.Attorney Docket No. MIL-050WO1

[0221] In some embodiments, the process comprises harvesting the IL-18 pretreated NK cells in a pre-formulation buffer comprising ectoine and preserving the IL-18 pretreated NK cells in a cryoformulation medium comprising ectoine. In some examples, the process comprises diluting the pre-formulation buffer comprising ectoine with a Cryostor CS10 solution at a ratio 1:1 to form a cryoformulation medium. Accordingly, the cryoformulation medium comprising ectoine at a concentration half of its concentration in the pre-formulation buffer.

[0222] In some embodiments, the process comprises harvesting the IL-18 and IL-15 pretreated NK cells in a pre-formulation buffer comprising ectoine and preserving the IL-18 and IL-15 pretreated NK cells in a cryoformulation medium comprising ectoine. In some examples, the process comprises diluting the pre-formulation buffer comprising ectoine with a Cryostor CS10 solution at a ratio 1:1 to form a cryoformulation medium. Accordingly, the cryoformulation medium comprising ectoine at a concentration half of its concentration in the pre-formulation buffer.

[0223] In some embodiments, the pretreatments described here can be combined to further enhance cell viability and / or maintain function of preserved cells. Exemplary processes described herein are for illustration only.

[0224] In some embodiments, the process comprises spiking the cell culture medium with IL-18 and ectoine concurrently before harvesting the cells for cryopreservation. In some embodiments, the process comprises spiking the cell culture medium with IL-18 and ectoine concurrently and harvesting the pretreated cells using a pre-formulation buffer comprising ectoine. In other embodiments, the process comprises spiking the cell culture medium with IL-18 and ectoine concurrently, harvesting the pretreated cells using a pre-formulation buffer comprising ectoine and cryopreserving the cells using a cryoformulation medium comprising ectoine.

[0225] In some embodiments, the process comprises spiking the cell culture medium with IL-18, IL-15 and ectoine concurrently before harvesting the cells for cryopreservation. In some embodiments, the process comprises spiking the cell culture medium with IL-18, IL-15 and ectoine concurrently and harvesting the pretreated cells using a pre-formulation buffer comprising ectoine. In other embodiments, the process comprises spiking the cell culture medium with IL-18, IL-15 and ectoine concurrently, harvesting the pretreated cells using a pre-formulation buffer comprising ectoine and cryopreserving the cells using a cryoformulation medium comprising ectoine.Attorney Docket No. MIL-050WO1

[0226] In any of the methods described herein, the method may further comprise freezing the NK cells in a plurality of cryovials for storage and / or transfer. A standard freezing process may be employed. For example, a low freezing process which permits fluids to move out of the cells at a rate that results in balanced osmotic pressure between cell and medium by the time the medium freezes is used. A mechanical or a controlled rate freezer may be used to freeze the NK cells in the methods described herein. A controlled rate freezer can be programmed to cool the cells to around −80° C. at a particular rate. A typical freezing rate for cryopreservation of most cells to −80° C. is −1° C. / minute. In some embodiments, the freezing step(s) may be carried out at a rate in the range of about −0.5 to about −10° C. / minute, preferably about −3 to about −5° C. / minute, e.g., around −1, −2, −3, −4, −5 or −10° C. / minute. The final freezing temperature may be between about −70° C. to about −130° C.

[0227] In some embodiments, NK cells described herein are preserved in a vial (e.g., a 50 mL AT vial) in a cryopreservation medium 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 NK 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). C. Compositions

[0228] Also provided by the present disclosure are frozen and thawed cells produced by the methods of the present invention, as well as pharmaceutical compositions incorporating the cells.

[0229] In some embodiments, provided herein is a composition comprising NK cells cryopreserved by the method of the present disclosure.

[0230] In some embodiments, the composition comprises a cell therapy product cryopreserved in a cryoformulation medium comprising ectoine. The cell therapy product is a NK cell therapy product.

[0231] In some embodiments, the composition is frozen.

[0232] In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration from 1 mM to 200 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration from 5 mM to 100 mM. In one embodiment, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 5Attorney Docket No. MIL-050WO1 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM. In one embodiment, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 20 mM. In one embodiment, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 25 mM. In one embodiment, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 50 mM.

[0233] In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising trehalose at a concentration ranging from 10 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising trehalose at a concentration ranging from 10 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising trehalose at a concentration ranging from 100 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising trehalose at a concentration ranging from 100 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising trehalose at a concentration ranging from 50 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising trehalose at a concentration of 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, or 1000 mM.

[0234] In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine and trehalose. In some embodiments, the cryoformulation medium comprises ectoine at a concentration ranging from 1 mM to 200 mM and trehalose at a concentration ranging from 10 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 1 mM to 200 mM and trehalose at a concentration ranging from 10 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 1 mM to 200 mM and trehalose at a concentration ranging from 100 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 1 mM to 200 mM and trehalose at a concentration ranging fromAttorney Docket No. MIL-050WO1 100 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 1 mM to 200 mM and trehalose at a concentration ranging from 50 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 10 mM to 100 mM and trehalose at a concentration ranging from 10 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 10 mM to 100 mM and trehalose at a concentration ranging from 10 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 10 mM to 100 mM and trehalose at a concentration ranging from 100 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 10 mM to 100 mM and trehalose at a concentration ranging from 100 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration ranging from 10 mM to 100 mM and trehalose at a concentration ranging from 50 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 50 mM and trehalose at a concentration ranging from 10 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 50 mM and trehalose at a concentration ranging from 10 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 50 mM and trehalose at a concentration ranging from 100 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 50 mM and trehalose at a concentration ranging from 100 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 50 mM and trehalose at a concentration ranging from 50 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 25 mM and trehalose at a concentration ranging from 10 mM to 1000 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 25 mM and trehalose at a concentration ranging from 10 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 25 mM and trehalose at a concentration ranging from 100 mM to 1000 mM. In some embodiments, theAttorney Docket No. MIL-050WO1 composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 25 mM and trehalose at a concentration ranging from 100 mM to 500 mM. In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine at a concentration of 25 mM and trehalose at a concentration ranging from 50 mM to 500 mM.

[0235] In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine, trehalose, and DMSO.

[0236] In some embodiments, the composition comprises NK cells in a cryoformulation medium comprising ectoine, trehalose, glycerol and DMSO.

[0237] In some embodiments, the NK cell product comprises NK cells obtained from cord blood. In some embodiments, the NK cell product comprises NK cells obtained from peripheral blood.

[0238] In some embodiments, the NK cells are primary cells.

[0239] In some embodiments, the NK cells are differentiated from stem cells (e.g., iPS cells).

[0240] In some embodiments, the NK cells are human cells. As non-limiting examples, the NK cells are from a healthy donor.

[0241] In some embodiment, the NK cells described herein are from a cell culture. The primary NK cells in some examples, are further expanded in vitro.

[0242] In some embodiments, the NK cells are modified cells, for example, engineered to express one or more therapeutics. In some examples, the NK cells are modified to express a chimeric antigen receptor (CAR). The CAR comprises an extracellular antigen binding domain, a hinge region, a transmembrane domain and at least one intracellular signaling domain. The antigen binding domain recognizes and binds to an antigen, for example, a cancer associated antigen.

[0243] In some embodiments, the NK cells are CD56+ / CD3- cells. The percentage of CD56+ / CD3- cells in a NK cell product can be readily determined by one of ordinary skill by known methods in the art, such as flow cytometry. 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%.

[0244] In some embodiments, the composition comprises around 1 million NK cells to around 150 million NK cells, preferably around 30 million NK cells or around 120 million NK cells.Attorney Docket No. MIL-050WO1

[0245] In some embodiments, post-thaw viability of cryopreserved NK cells described herein is at least 35% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells described herein is 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70% or more 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. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 35% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 40% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 45% at 24 hours after thawing. In some embodiments, 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 at least 55% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 60% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 65% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 70% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 75% at 24 hours after thawing. In some embodiments, post- thaw viability of cryopreserved NK cells is at least 80% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 85% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is at least 90% at 24 hours after thawing. In some embodiments, post-thaw viability of cryopreserved NK cells is 95% or more at 24 hours after thawing.

[0246] In some embodiments, total amount of NK cells obtained by thawing the cryopreserved cells is between 50% to 100% of the initial cell number prior to cryopreservation (i.e., recovery of cryopreserved cells). In some embodiments, total amount of NK cells obtained by thawing the cryopreserved cells is between 70% to 100% of the initial cell number prior to cryopreservation. In some embodiments, total amount of NK cells obtained by thawing the cryopreserved cells is 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the initial cell number prior to cryopreservation. In some embodiments, post-thawing recovery of NK cells is 60% of the initial cell number prior to cryopreservation. In some embodiments, post-thawing recovery of NK cells is 70% of the initial cell number prior to cryopreservation. In some embodiments, post-thawing recovery of NK cells is 80% of the initial cell number prior to cryopreservation. In some embodiments, post-thawing recovery of NK cells is 90% of the initial cell number prior to cryopreservation. In someAttorney Docket No. MIL-050WO1 embodiments, post-thawing recovery of NK cells is 100% of the initial cell number prior to cryopreservation.

[0247] In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2 to 2.0-fold, or 1.5 to 5.0-fold greater as compared to cryopreserved NK cells in a cryoformulation medium without ectoine. In some examples, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0-fold greater as compared to cryopreserved NK cells in a cryoformulation medium without ectoine. In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0- fold greater as compared to cryopreserved NK cells in a cryoformulation medium without ectoine.

[0248] In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2 to 2.0-fold, or 1.5 to 5.0-fold greater as compared to cryopreserved NK cells in a cryoformulation medium without trehalose. In some examples, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0-fold greater as compared to cryopreserved NK cells in a cryoformulation medium without trehalose. In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0- fold greater as compared to cryopreserved NK cells in a cryoformulation medium without trehalose.

[0249] In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2 to 2.0-fold, or 1.5 to 5.0-fold greater as compared to cryopreserved NK cells without pretreatment described herein. In some examples, survival of cryopreserved NK cells described herein at 24 hours after thawing is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0-fold greater as compared to cryopreserved NK cells without pretreatment described herein. In some embodiments, survival of cryopreserved NK cells described herein at 24 hours after thawing is 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0-fold greater as compared to cryopreserved NK cells without pretreatment described herein.

[0250] In some embodiments, cytotoxicity of post-thawed NK cells is increased, including short-term cytotoxicity. In some embodiments, thawed NK cells described herein have increased cytolysis of target cells (e.g., tumor cells) at a low E:T ratio as compared to thawed NK cells without pretreatment.Attorney Docket No. MIL-050WO1

[0251] In some embodiments, perforin release of cryopreserved NK cells is increased post-thawing. In some embodiments, granzyme release of cryopreserved NK cells is decreased post-thawing.

[0252] In some embodiments, cryopreserved NK cells described maintain anti-tumor function post-thawing, as measured by repeat killing capacity of NK cells.

[0253] In some embodiments, NK cells described herein are preserved in a vial (e.g., a 50 mL AT vial) in a cryopreservation medium 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 NK 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).

[0254] In some embodiments, NK cells preserved in a cryovial (e.g., a 50 mL container) are between about 1.0×107and 1.0 X 1010cells. The vial containing cryopreserved NK cells is ready for transport, for example, to a clinical site. In some embodiments, cryopreserved cells and compositions described herein are optionally subjected to an intermediate storing temperature for a desired period of time. In some embodiments, the intermediate storing temperature, ranges from between about 0° C. and −100° C., including about −50° C. to about −60° C., about −60° C. to about −70° C., about −70° C. to about −80° C., about −80° C. to about −90° C., about −90° C. to about −100° C. In some embodiments, cryopreserved cells and compositions described herein may be stored at an intermediate storing temperature for a pre-determined period of time before transfer to longer term storage.

[0255] In some embodiments, the present invention provides a pharmaceutical composition comprising NK cells preserved as described herein. Pharmaceutical compositions of the present invention may comprise a 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., aluminum hydroxide); and preservatives.

[0256] In some embodiments, compositions of the present invention are formulated for intravenous administration. D. Methods of UsesAttorney Docket No. MIL-050WO1

[0257] Also provided by the present disclosure are methods of treatment using frozen and thawed cells (e.g., frozen and thawed NK cells) produced by the methods of the present invention, as well as pharmaceutical compositions incorporating the cells. The NK cells produced and cryopreserved by the methods described herein can be used for various applications. For example, NK cells contained within the cryopreservation medium described herein, are suitable for adoptive cell therapy. In some examples, NK cells contained within the cryopreservation medium described herein, are suitable for allogeneic cell therapy.

[0258] In another aspect, the present inventions provides a method of treating a disease comprising administering to a subject in need thereof an effective amount of the composition comprising NK cells described herein.

[0259] In some embodiments, the disease is a cancer, an inflammatory disorder, or an autoimmune disorder.

[0260] In some embodiments, NK cells contained within the cryopreservation medium described herein is useful for the treatment of a cancer or a tumor. In certain embodiments, 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. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is a B-cell malignancy (e.g., diffuse large B-cell lymphoma).

[0261] In some embodiments, the NK cell based adoptive cell therapy is used in combination with one or more additional cancer treatments, such as for example lymphodepleting chemotherapy.

[0262] In some embodiments, NK cells contained within the cryopreservation medium described herein is used for the treatment of an inflammatory disorder.

[0263] In some embodiments, NK cells contained within the cryopreservation medium described herein is used for the treatment of an autoimmune disorder.

[0264] The autoimmune disorders include but are not limited to, lupus, autoimmune hepatitis, juvenile and rheumatoid arthritis, psoriatic arthritis, diabetes type 1, Multiple sclerosis (MS), hashimoto’s thyroiditis, grave’s disease, acquired hemophilia, acromegaly, aplopecia areata, autoimmune addison’s disease, autoimmune hepatitis, autoimmune gastritis, Autoimmune Encephalitis, Autoimmune pancreatitis, Autoimmune Retinopathy, Bullous pemphigoid, Balo Disease, Celiac disease, Crohn’s disease, Cryptogenic organizing pneumonia (COP), Dermatitis herpetiformis, Eczema / Atopic Dermatitis, Endometriosis, Glomerulonephritis, autoimmune thyroiditis, Hypogammaglobulinemia, Inclusion Body Myositis (IBM), Interstitial cystitis, Lyme Disease, Lichen sclerosus, NeuromyelitisAttorney Docket No. MIL-050WO1 Optica / Devic’s disease, Ocular Cicatricial pemphigoid, Primary sclerosing cholangitis, Psoriasis, Psoriatic Arthritis, Pure Red Cell Aplasia, Polymyalgia rheumatica, Reactive arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Sjögren Syndrome, Small fiber sensory neuropathy, Sydenham’s chorea, Systemic Lupus Erythematosus, Testicular Autoimmunity, Ulcerative Colitis, and Vitiligo.

[0265] In some embodiments, NK cells cryopreserved as described herein are subsequently thawed prior to administration to a patient in need thereof.

[0266] NK cells described herein 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.

[0267] NK cells described herein 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.

[0268] In some embodiments, cryopreserved cells are thawed for use (e.g., implantation) by transferring a cryovial containing the cryopreserved cells to a water bath having a temperature around body temperature, for example a temperature of around 37° C., or any other suitable temperature. In other embodiments, a step-up thawing process having a step-up heating rate is used. For example, the cryovial may, in some embodiments, be placed in sequential storage environments with increasing temperatures before being transferred to a temperature that is around body temperature, for example a water bath having a temperature of around 37° C., or any other suitable temperature. In some embodiments, the final incubator is located in the operating room, allowing a surgeon to immediately administer NK cells. In some embodiments, after thawing the NK cells may be transferred to a culture dish and cultured under appropriate conditions for a period of approximately 30 minutes, one hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, one week, two weeks, or more than three weeks prior to being transported to the desired location (e.g., the operating room for delivery to a patient). In some embodiments, the thawed cells are directly administered to a patient.

[0269] When "a therapeutically effective 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). It can generally be stated that a pharmaceutical composition comprising the NK cells described herein may be administeredAttorney Docket No. MIL-050WO1 at a dosage of about 104to 109cells / kg body weight, for example, about 105to 106cells / kg body weight. NK cell compositions may also be administered multiple times at these dosages.

[0270] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the NK cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, 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. 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.

[0271] In one embodiment, the cells can be administered by using infusion techniques that are commonly known in immunotherapy.

[0272] The invention will be further described in connection with the following examples which are set forth for purposes of illustration only.

[0273] In some embodiments, NK cells 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, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizitmab treatment or efaiizumab treatment. In further embodiments, 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.

[0274] In some embodiments, 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. In some embodiments, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g.,Attorney Docket No. MIL-050WO1 Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery. EXAMPLES

[0275] Various aspects of the invention are described in further detail in the following Examples. The following examples describe exemplary modes of making and practicing the present invention. However, these Examples are for illustrative purposes only and are not meant to limit the scope of the invention. Example 1: Pretreatment of NK cells with IL-18 or IL-18 and IL-15

[0276] This example outlines the pre-treatment of NK cells with interleukin-18 (IL-18) alone or in combination with interleukin-15 (IL-15) for the prevention of cryoinjury post freeze-thaw. Further, this example highlights data that indicates great viable cell recovery, cytolytic activity, and effective killing post freeze-thaw.

[0277] The timing of the pre-treatment of the exemplary cryoformulations with IL-18 alone or in combination with IL-15 was carried out as described in Table 1. Herein, the NK cells were pre-treated with IL-18, or IL-18 in combination with IL-15 at various times and concentrations. It is thought that the shorter exposure and low concentrations of IL-18 may prevent cryoinjury due to cellular degranulation into the cell culture. To the contrary, high concentrations of IL-18 in combination with IL-15 may induce stronger degranulation to reduce granzyme-mediated cryoinjury. Table 1. Study Design for IL-18 and IL-15 Pre-Treatment Group Condition Timing of Addition IL-18 IL-15 ID 1 Control (no IL-18) -- -- -- 2 24 hrs before harvest: 150 ~24 hours 150 U / mL -- U / mL IL-18 3 24 hrs before harvest: 300 ~24 hours 300 U / mL - U / mL IL-18 4 24 hrs before harvest : 750 ~24 hours 750 U / mL 750 IU / mL U / mL IL-18 + 150 IU / mL IL- 15Attorney Docket No. MIL-050WO1 5 18 hrs before harvest : 15 ~18 hours 15 U / mL -- U / mL IL-18 6 18hrs before harvest: 50 U / mL ~18 hours 50 U / mL -- IL-18 7 18hrs before harvest: 150 ~18 hours 150 U / mL -- U / mL IL-18 8 one hour of harvest: 750 ~1 hour 750 U / mL 750 IU / mL U / mL IL-18 + 150 IU / mL IL- 15 Post-Thaw Cell Viability

[0278] As shown in FIG.1 conditions laid out in Table 1, after recovery in culture medium containing 100 IU / mL IL-2, yielded 24-hour post-thaw cell viabilities greater than 50% across the board, wherein the best post-thaw viability was in culture pre-treated with high concentrations of IL-18 and IL-15 on the day prior to harvest (condition 4). Further, condition 4 of the same exhibited the highest survival ratio of exemplary NK cells across all conditions screened (see FIG.2). With regards to from vial recovery post-thaw, as seen in FIG.3, condition 5, wherein there was pre-treatment with a low concentration of IL-18 alone, yielded the highest viable recovery of exemplary NK cells relative to the control. Short-Term Cytotoxicity Assays

[0279] The purpose of the cytotoxicity assays as laid out below is to determine the impact of the pre-treatment of the exemplary cytokines, IL-18 and IL-15, on cytotoxicity and cytokine release after an overnight rest period. It should be noted that the cells were also treated with 100 IU / mL IL-2 for these assays.

[0280] Upon exposure to either A549 or MM.1S tumor cells, all conditions exhibited effective cytotoxic (FIG.4A, FIG.4B) activity, wherein NK cells pre-treated using condition 4 (pre-treatment with high concentrations of IL-18 and IL-15 on the day prior to harvest) had the greatest degree of cytolysis relative to the control. Table 2 summarizes the effect that the exemplary pre-treatments and timings had on the type of cytokines released and whether expression of these cytokines were upregulated or downregulated against the different tumor cell lines. NK cells pre-treated using condition 4, which as previously stated had the greatest degree of cytolysis against the A549 and MM.1S tumor cells, showed increased release of perforin upon exposure to both tumor cell lines, and decreased Granzyme B upon exposure to A549 tumor cells, even at low effector:target (E:T) ratios. Table 2. Effect of Pre-Treatment on Cytokine Detection after Tumor Cell Challenge. Treatment Treatment GM-CSF Granzyme B IFNγ Perforin TNFα TimeAttorney Docket No. MIL-050WO1 1 Control MM.1 A549 MM.1S A549 MM.1S A549 MM.1S A549 MM.1S A549 S 2 24 hrs 150 U / mL -- ↑ -- -- -- -- -- -- -- -- prior to IL-18 3 harvest 300 U / mL -- ↑ ↑ -- -- ↑ ↑ -- -- -- IL-18 4 750 U / mL ↑ -- -- ↓ -- -- ↑ ↑ ↑ -- IL-18 + 750 IU / mL IL-15 9 10 mM -- ↑ -- -- -- ↑ ↑ ↑ -- ↑ Ectoine pretreatm ent + 25 mM Ectoine in Cryoform ulation 5 16 hrs 15 U / mL -- ↓ -- ↓ -- ↑ ↑ -- -- ↓ prior to IL-18 6 harvest 50 U / mL -- ↓ -- -- -- -- -- -- -- ↓ IL-18 7 150 U / mL -- ↓ -- ↓ -- ↑ ↑ -- -- ↓ IL-18 8 1 hr prior 750 U / mL -- ↓ ↓ ↓ -- ↑ ↑ -- -- -- to harvest IL-18 + 750 IU / mL IL-15 Repeat Antigen Stimulation Assay

[0281] The purpose of the repeat antigen stimulation assays is to determine the repeat killing capability of the frozen product due to different Day 0 feeder:NK ratios. The exemplary pre-treated cells were further treated with the exemplary cytokines IL-2 and IL-15 at concentrations of 100 IU / mL and 100 pg / mL respectively on restimulation days. The assays took place over the course of 14 days and the target cell lines for the repeat antigen stimulation assays were A549-Luc-NLR and MM.1S-Luc-NLR.The target tumor cells were repeatedly added to rechallenge the NK cells every two to three days.

[0282] As shown in FIG.5A – FIG.5B, all pre-treatment conditions maintained vigorous A549 tumor killing for six challenges relative to the control, wherein exemplary NK cells pre-treated with condition 4 (pre-treatment with high concentrations of IL-18 and IL-15 on 24 hours prior to harvest) exhibited strong killing kinetics immediately post-thaw. Further, exemplary NK cells pre-treated using condition 4 demonstrated strong post-thaw killing kinetics of A549 tumor cells within two days at E:T ratios of 5:1 (FIG.6A) and 2.5:1 (FIG. 6B)Attorney Docket No. MIL-050WO1

[0283] Similarly for post-thaw NK cell challenge against MM.1S cells, all conditions maintained vigorous tumor killing for six challenges (FIG.7A), however all conditions are comparable to the control. Exemplary NK cells pre-treated with condition 4 at an E:T ratio of 5:1 showed great killing kinetics relative to the control (FIG.7B). Phenotyping

[0284] Phenotyping of the exemplary frozen NK cell drug product was performed and compared to fresh NK cell product when pre-treated with exemplary cytokines, IL-18 alone or in combination with IL-15, to assess the expression of cytokines, apoptotic markers, classical NK markers, and antiapoptotic markers by fluorescence activated cell-staining (FACS). The parameters used for FACS-based detection of all markers is summarized in Table 3. Table 3. NK Cryoinjury Panel for FACS Analysis Marker Fluorophore Laser Detector Wavelength Vendor Red 637 nm A 780 / 60750 LP CD16 AF700 Red 637 nm B 730 / 45690 LP Biolegend TRAF1 AF647 Red 637 nm C 670 / 30650 LP BD Cleaved PARP PE / Cy7 Y / G 561 nm A 780 / 60750 LP Biolegend Granzyme B PE / Cy5 Y / G 561 nm B 670 / 30650 LP Biolegend BCL-2 PE-CF594 Y / G 561 nm C 610 / 620600 LP BD CD123 (CAR) PE Y / G 561 nm D 586 / 15 Biolegend Blue 488 nm B 710 / 50690 LP Caspase 7 FITC Blue 488 nm E 500505 LP Novus Perforin BV421 Violet 405 nm I 450 / 50410 LP Biolegend IFNγ BV510 Violet 405 nm G 525 / 50505 LP Biolegend Annexin V BV605 Violet 405 nm E 610 / 20600 LP BD Caspase 3 BV650 Violet 405 nm D 670 / 30635 LP BD TNF-α BV711 Violet 405 nm B 710 / 50690 LP Biolegend CD107a BV785 Violet 405 nm A 780 / 60750 LP Biolegend BUV805 UV 355 nm A 820 / 60770 LP CD56 BUV737 UV 355 nm B 740 / 35690 LP BD BUV661 UV 355 nm C 670 / 30635 LP CD3 BUV615 UV 355 nm D 610 / 20600 LP BUV563 UV 355 nm E 586 / 15550 LP BD Live / Dead BUV496 UV 355 nm F 515 / 30450 LP ThermoFischer

[0285] FIG.8A – FIG.8E showcase the expression and staining and of various cytokines (Granzyme Band TNFα) in the frozen drug product versus freshly harvested NK cells. It is noted that there is a slight increase in the Granzyme B geometric mean fluorescence intensity (gMFI) in condition 8 (pre-treated with high concentration of IL-18 and IL-15 on 24 hours prior to harvest) (see FIG.8B) and increased TNFα expression in the frozen NK cell drug product across all conditions (see FIG.8D).Attorney Docket No. MIL-050WO1

[0286] Further, the pre-treatment condition with the lowest Caspase3(high) Caspase 7+ and Cleaved PARP(high) expression was condition 4 in both fresh and frozen NK cell drug product (FIG.9A – FIG.9B). Other exemplary apoptotic markers were either slightly lower in the frozen NK cell drug product (FIG.9C – FIG.9D) or slightly higher in the case of Annexin V+ (FIG.9E).

[0287] Expression of classical NK markers, namely CD16+ / - and CD56bright / dim, are quantified by the same methodologies as above. The expression of CD16+ / - is relatively the same across all pre-treatment conditions in both fresh and frozen cells (FIG.10A), wherein there is a slight increase in CD16- expression in the frozen NK cell drug product in condition 8 (pre-treated with high concentration of IL-18 and IL-15 on 24 hours prior to harvest) (FIG. 10B). There was no notable difference in expression of CD56bright / dim in either population of NK cells across all pre-treatment conditions (FIG.10C – FIG.10D).

[0288] Relative to antiapoptotic markers, there was no difference in expression of BCL-2 across all conditions in both fresh and frozen NK cells (FIG.11A – FIG.11B), whereas all IL-18 containing conditions have higher TRAF-1 expression than the control in both NK cell populations (FIG.11C). Example 2: Pretreatment of NK cells with Ectoine

[0289] This example outlines the pre-treatment of exemplary NK cells with ectoine for the prevention of cryoinjury post freeze-thaw. Further, this example highlights data that indicates great viable cell recovery, cytolytic activity, and effective killing post freeze-thaw.

[0290] The design for the pre-treatment of the exemplary cryoformulations with ectoine is listed in Table 4. As to the condition 9, NK cells were pre-treated with 10 mM ectoine 24 hours prior to harvest, and washed and diluted with MEM / HSA pre-formulation buffer containing 50 mM ectoine (final concentration of 25 mM ectoine in cryoformulation after 1:1 dilution with CS10). Table 4. Ectoine Pre-Treatment Conditions Study Design ID Condition Timing of IL-18 IL-15 Addition 1 Control -- -- -- (no IL-18) 9 24 hours prior to harvest: 10 mM ectoine -- -- -- pre-treatment Harvest: washed and diluted with 50 mM ectoine in MEM / HSA pre-formulation bufferAttorney Docket No. MIL-050WO1 (final concentration of 25 mM ectoine in cryoformulation after 1:1 dilution with CS10) Post-Thaw Cell Viability

[0291] As shown in FIG.1 conditions laid out in Table 4,after recovery in medium containing 100 IU / mL IL-2, yielded the 24-hour post-thaw cell viability of cultures treated with ectoine (see condition 9) resulted in 54% viability. Further, the cells treated with the same exhibited a survival ratio of exemplary NK cells, also recovered in medium containing 100 IU / mL IL-2, that was around 50% (see FIG.2, condition 9). With regards to from vial recovery post-thaw, as seen in FIG.3 (condition 9), yielded a percent recovery that was comparable to the IL-18 or IL-18 / IL-15 treated NK cells. Short-Term Cytotoxicity Assays

[0292] The purpose of the cytotoxicity assays as laid out below is to determine the impact of the pre-treatment of the exemplary extremolyte, ectoine, on cytotoxicity, cytokine release, and after an overnight rest period. It should be noted that the cells were also treated with 100 IU / mL IL-2 for these assays.

[0293] Upon exposure to either A549 or MM.1S tumor cells, NK cells treated with ectoine exhibited effective cytotoxic (FIG.4A, FIG.4B) activity. Repeat Antigen Stimulation Assays

[0294] The purpose of the repeat antigen stimulation assays is to determine the repeat killing capability of the frozen product due to different Day 0 feeder:NK ratios. The pre- treated cells were further treated with the exemplary cytokines IL-2 and IL-15 at concentrations of 100 IU / mL and 100 pg / mL respectively on restimulation days. The assays took place over the course of 14 days and the target cell lines for the repeat antigen stimulation assays were A549-Luc-NLR and MM.1S-Luc-NLR. The target tumor cells were repeatedly added to rechallenge the NK cells every two to three days.

[0295] As shown in FIG.5A – FIG.5B, the ectoine pre-treatment condition maintained vigorous tumor killing for six challenges against A549 tumor cells relative to the control. Similarly for post-thaw NK cell challenge against MM.1S cells, the ectoine pre-treated cells maintained vigorous tumor killing for six challenges (FIG.6A), however this condition was also comparable to the control. Table 2 summarizes the effect that the pre-treatment with ectoine had on cytokine release. Further, exemplary NK cells pre-treated using condition 9 demonstrated enhanced post-thaw killing kinetics of A549 tumor cells within two days at E:TAttorney Docket No. MIL-050WO1 ratios of 5:1 (FIG.6C) and 2.5:1 (FIG.6D). It is noted that there is an increase in perforin release after exposure to A549 and MM.1S cell lines. Phenotyping

[0296] Phenotyping of the exemplary frozen NK cell drug product was performed and compared to fresh NK cell product when pre-treated with the exemplary extremolyte, ectoine, to assess the expression of cytokines, apoptotic markers, classical NK markers, and antiapoptotic markers by fluorescence activated cell-staining (FACS). The parameters used for FACS-based detection of all markers is summarized in Table 3.

[0297] FIG.8A – FIG.8E showcase the expression and staining and of various cytokines (Granzyme B, and TNFα) in the frozen drug product versus freshly harvested NK cells. It is noted that there is a slight increase in the Granzyme B gMFI (see FIG.8B) and increased TNFα expression in the frozen NK cell drug product (see FIG.8D).

[0298] Further, the ectoine pre-treatment condition had the Caspase3(high) Caspase 7+ and Cleaved PARP(high) expression in both fresh and frozen NK cell drug product (FIG.9A – FIG.9B). Expression of other exemplary apoptotic markers were either slightly lower in the frozen NK cell drug product (FIG.9C – FIG.9D) or higher in the case of AnnexinV+ (FIG.9E).

[0299] Expression of classical NK markers, namely CD16+ / - and CD56bright / dim, are quantified by the same methodologies as above in the ectoine pre-treatment condition. The expression of CD16+ is within range of the cytokine pre-treated stocks of FIG.10. However, there is a slight increase in CD16- expression in the frozen drug product NK cells (FIG. 10B). There is no notable difference in expression of CD56bright / dim between the freshly harvested NK cells and frozen drug product NK Cells (FIG.10C – FIG.10D).

[0300] Relative to antiapoptotic markers, there was no difference in expression of BCL-2 across all conditions in both fresh and frozen NK cells (FIG.11A – FIG.11B), whereas all IL-18 containing conditions have higher TRAF-1 expression than the control in both NK cell populations (FIG.11C). Example 3. Optimization of IL-18 and IL-15 pretreatment concentration

[0301] This example outlines the exemplary study design for the pre-treatment of frozen NK cells with high concentrations of IL-18 in combination with high concentrations of IL-15. The study is carried utilizing a range of 750 U / mL to 2000 U / mL of IL-18 in combination with IL-15 at either 750 IU / mL or 1500 IU / mL, wherein the pre-treatment occurs on the dayAttorney Docket No. MIL-050WO1 prior to harvest. The addition timing and pre-treatment dosages are laid out in Table 4. From there, the post-thaw viability, cytotoxicity, and phenotyping will be assessed. Table 5. IL-18 / IL-15 Pre-Treatment Study Design ID Condition Timing of Addition IL-18 (U / mL) IL-15 (IU / mL) 10 Control -- -- -- (no IL-18) 11 Day prior to harvest: Day prior to harvest: 750 750 750 U / mL IL-18 + 750 ~24 hrs IU / mL IL-15 12 Day prior to harvest: Day prior to harvest: 1500 750 1500 U / mL IL-18 + 750 ~24 hrs IU / mL IL-15 13 Day prior to harvest: Day prior to harvest: 2000 750 2000 U / mL IL-18 + 750 ~24 hrs IU / mL IL-15 14 Day prior to harvest: Day prior to harvest: 750 1500 750 U / mL IL-18 + 1500 ~24 hrs IU / mL IL-15 15 Day prior to harvest: Day prior to harvest: 1500 1500 1500 U / mL IL-18 + Morning ~24 hrs 1500 IU / mL IL-15 16 Day prior to harvest: Day prior to harvest: 2000 1500 2000 U / mL IL-18 + ~24 hrs 1500 IU / mL IL-15 Example 4. Optimization of ectoine

[0302] This example outlines the exemplary study design for the pre-treatment of frozen NK cells with various concentrations of ectoine and ectoine spiking conditions pre-harvest. The conditions for the spiking and amounts of ectoine in the cryopreservation buffer are outlined in Table 5. From there, the post-thaw viability, cytotoxicity, and phenotyping will be assessed. Table 6. Ectoine Pre-treatment Study Design Group ID Condition 24-hr Pre-Harvest Pre-Formulation Spike Buffer 17 Control N / A N / A 18 Ectoine: 24-hr 10 mM spike; 50 10 mM Ectoine 50 mM Ectoine mM in pre-formulation buffer 19 Ectoine: 24-hr 5 mM spike; 50 5 mM Ectoine 50 mM Ectoine mM in pre-formulation buffer 20 Ectoine: 50 mM in pre- N / A 50 mM Ectoine formulation buffer 21 Ectoine: 100 mM in pre- N / A 100 mM Ectoine formulation buffer

Claims

Attorney Docket No. MIL-050WO1 CLAIMS 1. A method for cryopreserving natural killer (NK) cells comprising pretreating the NK cells with ectoine prior to cryopreserving the pretreated NK cells.

2. A method for cryopreserving natural killer (NK) cells comprising, culturing NK cells in a cell culture medium, treating the NK cells with ectoine, harvesting the treated NK cells, and cryopreserving the treated NK cells.

3. The method of claim 1 or 2, wherein the NK cells are treated with ectoine at a concentration ranging from 1 mM to 200 mM.

4. The method of claim 3, wherein the NK cells are treated with ectoine at a concentration ranging from 5 mM to 100 mM 5. The method of claim 4, wherein the NK cells are treated with ectoine at a concentration ranging from 10 mM to 50 mM.

6. The method of claim 5, wherein the NK cells are treated with ectoine at a concentration of 10 mM.

7. The method of any one of the preceding claims, wherein the NK cells are treated with ectoine for 1-24 hours prior to cryopreserving the NK cells.

8. The method of any one of the preceding claims, wherein the NK cells are treated with ectoine for 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, or 24 hours prior to cryopreserving the NK cells.

9. The method of any one of the preceding claims, wherein the NK cells are pretreated with IL-18 prior to cryopreserving the NK cells.

10. The method of any one of the preceding claims, wherein the cells are pretreated with IL- 18 and IL-15 prior to cryopreserving the NK cells .Attorney Docket No. MIL-050WO1 11. The method of any one of the preceding claims, wherein the NK cells are cryopreserved using a cryoformulation medium.

12. The method of claim 11, wherein the cryoformulation medium comprises ectoine at a concentration from 1 mM to 200 mM.

13. The method of claim 12, wherein the cryoformulation medium comprises ectoine at a concentration from 5 mM to 200 mM.

14. The method of claim 13, wherein the cryoformulation medium comprises ectoine at a concentration of 25 mM.

15. The method of any one of claims 11-14, wherein the cryoformulation medium comprises trehalose.

16. The method of claim 15, wherein the cryoformulation medium comprises ectoine and trehalose.

17. The method of claim 15 or 16, wherein the cryoformulation medium comprises trehalose at a concentration ranging from 10 mM to 1000 mM.

18. The method of claim 17, wherein the cryoformulation medium comprises trehalose at a concentration ranging from 15 mM to 300 mM.

19. The method of any one of the preceding claims, wherein the NK cells are processed using a pre-formulation buffer prior to cryopreservation, wherein the pre-formulation buffer comprises ectoine.

20. A method for cryopreserving natural killer (NK) cells comprising harvesting the NK cells in a pre-formulation buffer comprising ectoine prior to cryopreserving the harvested NK cells.

21. The method of claim 20, wherein the pre-formulation buffer comprises ectoine at a concentration ranging from 1 mM to 200 mM.

22. The method of claim 20 or 21, wherein the NK cells are cryopreserved using a cryoformulation medium.Attorney Docket No. MIL-050WO1 23. The method of claim 22, wherein the cryoformulation medium comprises ectoine and / or trehalose.

24. The method of claim 23, wherein the cryoformulation medium comprises ectoine at a concentration ranging from 1mM to 200mM, and / or trehalose at a concentration ranging from 10mM to 1000mM.

25. A composition comprising NK cells in a cryoformulation medium comprising ectoine at a concentration from 1 mM to 200 mM.

26. The composition of claim 25, wherein the cryoformulation medium comprises ectoine at a concentration from 5 mM to 100 mM.

27. The composition of claim 25 or 26, wherein the cryoformulation medium further comprises trehalose.

28. The composition of claim 27, wherein the cryoformulation medium comprises trehalose at a concentration ranging from 10mM to 1000mM.

29. The composition of any one of claims 25-28, wherein the NK cells are obtained from cord blood.

30. The composition of any one of claims 25-28, wherein the NK cells are obtained from peripheral blood.

31. The composition of any one of claims 25-28, wherein the NK cells are a cell culture.

32. The composition of any one of claims 25-31, wherein the NK cells are modified.

33. The composition of any one of claims 25-32, wherein the NK cells are modified to express a chimeric antigen receptor (CAR).

34. The composition of any one of any one of claims 25-33, wherein the post-thaw viability of cryopreserved NK cells is 35%, 40%, 50%, or more at 24 hours after thawing.

35. The composition of any one of claims 25-34, wherein survival of cryopreserved NK cells at 24 hours after thawing is at least 1.2 – 2.0 fold greater as compared to NK cells in medium without ectoine.Attorney Docket No. MIL-050WO1 36. The composition of any one of claims 25-35, wherein cytotoxicity of the post-thawed NK cells is increased.

37. A method of treating a disease comprising administering an effective amount of the composition of any one of claims 25-36 to a subject in need thereof.

38. The composition of any one of claims 25-36 for use in a treatment of a disease in a subject in need thereof.

39. The method of claim 37, or the use of claim 38, wherein the disease is a cancer, an inflammatory disorder, or an autoimmune disorder.

40. A method of cryopreserving NK cells comprising culturing NK cells in a cell culture medium, treating the NK cells with IL-18 at a pre-determined concentration at a pre-determined time point, harvesting the treated NK cells, and cryopreserving the pretreated NK cells.

41. A method of cryopreserving NK cells comprising pretreating NK cells with IL-18 at a concentration ranging from 50 U / mL to 5000 U / mL prior to cryopreserving the pretreated NK cells.

42. The method of claim 40 or 41, wherein the NK cells are treated with IL-18 at a concentration ranging from 150 U / mL to 3000 U / mL.

43. The method of any one of claims 40-42, wherein the NK cells are treated with IL-18 at a concentration ranging from 350 U / mL to 2000 U / mL.

44. The method of any one of claims 40-43, wherein the NK cells are treated with IL-18 for 1-48 hours before harvesting the NK cells for cryopreservation.

45. The method of claim 44, wherein the NK cells are treated with IL-18 at least for 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 18 hours, 24 hours, or 48 hours before harvesting the NK cells for cryopreservation.

46. The method of claim 45, wherein the NK cells are treated with IL-18 at least 24 hours before harvesting the NK cells for cryopreservation.Attorney Docket No. MIL-050WO1 47. The method of any one of claims 40-46, wherein the NK cells are treated with IL-18 and IL-15.

48. The method of claim 47, wherein IL-15 is at a concentration ranging from 10 IU / mL to 3000 IU / mL.

49. The method of claim 48, wherein IL-15 is at a concentration ranging from 50 IU / mL to 2000 IU / mL.

50. The method of any one of claims 40-49, wherein the NK cells are treated with ectoine concurrently.

51. The method of any one of claims 40-50, wherein the NK cells are cryopreserved using a cryoformulation medium.

52. The method of claim 51, wherein the cryoformulation medium comprises one or more cryoprotective agents.

53. The method of claim 52, wherein the cryoprotective agents comprises ectoine and / or trehalose.

54. The method of any one of claims 52-53, wherein the cryoprotective agent comprises dimethyl sulfoxide (DMSO).

55. The method of any one of claims 40- 54, wherein the NK cells are cultured and expanded in a culture medium.

56. The method of any one of claims 40-55, wherein the NK cells are obtained from cord blood.

57. The method of any one of claims 40-55, wherein the NK cells are obtained from peripheral blood.

58. The method of any one of claims 40-55, wherein the NK cells are from a cell culture.

59. The method of any one of claims 40-58, wherein the NK cells are modified.

60. The method of claim 59, wherein the NK cells comprises a chimeric antigen receptor (CAR).Attorney Docket No. MIL-050WO1 61. The method of any one of claims 40-60, wherein the NK cells are CD56+ / CD3-.

62. A composition comprising NK cells cryopreserved by the method of any one of the preceding claims.

63. A method for treating cancer in a subject comprising administering to the subject an effective amount of the composition of claim 62.

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