Methods and compositions for freezing and thawing mammalian cells

Abstract

The present disclosure provides, inter alia, methods for cryopreservation and thawing of cells that result in thawed cells with high cell viability and post-thaw functionality. In some embodiments, a method for cryopreservation of cells on a large scale is provided, the method comprising: (a) contacting the cells with a cryopreservation medium; (b) cooling the cells to -80°C at a controlled rate to minimize the latent heat of thawing; and (c) storing the cells in liquid nitrogen vapor, thereby cryopreserving immune cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 147,737, filed February 9, 2021, and PCT / US2022 / 015627, filed February 8, 2022, the contents of which are incorporated by reference in their entireties for all purposes. [Background technology]

[0002] For example, cryopreservation of biological materials such as cells, tissues, organs, blood products, embryos, sperm, stem cells, fish eggs, etc., involves freezing the biological material to a temperature low enough to stop chemical processes that may damage the material, thereby preserving the material.

[0003] The field of cryopreservation often aims not only to freeze biological material, but also to preserve its viability, i.e., its ability to regain normal biological functions after thawing. When biological material is frozen, the internal fluid generally undergoes a phase transition during which ice crystals may form. The formation of ice crystals may damage the biological material, so that it may not be viable after thawing.

[0004] Therefore, it is desirable to optimize cryopreservation conditions, especially when cryopreserving cells for therapeutic use, to ensure the viability of cells that may need to be transported for use in a variety of applications, such as cell therapy, regenerative medicine, tissue engineering, and many other biomedical applications.

[0005] Suboptimal cryopreservation can lead to batch-to-batch variability in cell viability and function, reduced cell yields, and even the potential selection of subpopulations with different genetic or epigenetic characteristics than the original cells. Regulatory conditions also impact cryopreservation, as they demand robust and reproducible approaches to product freezing, storage, and thawing. Summary of the Invention

[0006] The present application is based, at least in part, on methods and compositions for effective freezing and thawing of mammalian cells. The present invention is based, in part, on the development of a large-scale cryopreservation / thawing method generally applicable to mammalian cells, such as immune cells, particularly modified immune cells suitable for cell therapy. The present application discloses freezing methods that include various cooling, heating, and holding steps that allow for cryopreservation of cells with high viability and function after thawing. Furthermore, the freeze / thawing methods described herein, as described herein, allow for consistent freezing of large-scale (e.g., greater than 10 mL in volume) cell-containing samples in about 60 minutes or less, and also allow for the subsequent administration of the thawed cell samples directly to a subject in need.

[0007] For example, as described in more detail below, the methods described herein can result in immune cell function in vitro and in vivo that is at least equivalent to that of freshly isolated cells. Additionally, the methods and compositions provided herein can be used to preserve large quantities of immune cells, particularly allogeneic modified immune cells suitable for cell therapy, for storage and transportation, for example, to a cell bank or hospital, where the cells can be used for further culture and analysis, or can be directly injected into patients in need thereof. Thus, the present application provides freezing and thawing methods that can be highly effective in preserving large quantities of mammalian cells, particularly modified immune cells suitable for cell therapy.

[0008] In some embodiments, a method for large scale cryopreservation of immune cells is provided, the method comprising: (a) providing a container containing a sample comprising immune cells suspended in a cryopreservation medium, the volume of the sample being at least 5% less than the total volume of the container, the volume of the sample being at least 10 ml; (b) cooling the container in a multi-step process at a controlled rate to a temperature above the freezing temperature of the sample to a temperature of about -80°C or less to minimize the latent heat of thawing; and (c) storing the cells in liquid nitrogen vapor, thereby cryopreserving the immune cells.

[0009] In some embodiments, the controlled rate to minimize the latent heat of fusion includes two or more steps of decreasing the temperature at a rate of 0.75°C / min to 30°C / min to a final temperature of -80°C or less.

[0010] In some embodiments, the total time to achieve cryopreservation of immune cells is less than 120 minutes, less than 110 minutes, less than 100 minutes, less than 90 minutes, less than 80 minutes, less than 70 minutes, or less than 60 minutes. Thus, in some embodiments, the total time to achieve cryopreservation of immune cells is less than 120 minutes. In some embodiments, the total time to achieve cryopreservation of immune cells is less than 110 minutes. In some embodiments, the total time to achieve cryopreservation of immune cells is less than 100 minutes. In some embodiments, the total time to achieve cryopreservation of immune cells is less than 90 minutes. In some embodiments, the total time to achieve cryopreservation of immune cells is less than 80 minutes. In some embodiments, the total time to achieve cryopreservation of immune cells is less than 70 minutes. In some embodiments, the total time to achieve cryopreservation of immune cells is less than 60 minutes.

[0011] In some embodiments, the immune cells are freshly isolated or frozen and thawed at least once.

[0012] In some embodiments, the immune cells are natural or engineered natural killer (NK) cells, αβ T cells, γδ T cells, regulatory T cells (Tregs), induced pluripotent stem cells (iPSCs), iPSC-derived T cells or NK cells, hematopoietic stem cells (HSCs), mesenchymal stromal cells (MSCs), dendritic cells, macrophages, or B cells. Thus, in some embodiments, the immune cells are natural or engineered NK cells. In some embodiments, the immune cells are engineered NK cells. In some embodiments, the immune cells are αβ T cells. In some embodiments, the immune cells are γδ T cells. In some embodiments, the immune cells are Tregs. In some embodiments, the immune cells are iPSCs. In some embodiments, the immune cells are iPSC-derived T cells. In some embodiments, the immune cells are iPSC-derived NK cells. In some embodiments, the immune cells are HSCs. In some embodiments, the immune cells are MSCs. In some embodiments, the immune cells are dendritic cells. In some embodiments, the immune cells are macrophages. In some embodiments, the immune cell is a B cell.

[0013] In some embodiments, the immune cells are cord blood-derived NK cells modified with a chimeric antigen receptor (CAR).

[0014] In some embodiments, the NK cells comprise a chimeric antigen receptor (CAR). The NK cells may comprise any CAR, including, for example, one or more of a CD19 CAR, a B-cell maturation antigen (BCMA) CAR, a glypican-3 (GPC3) CAR, a CD22 CAR, a mesothelin CAR, a MUC1 CAR, an epithelial cell adhesion molecule (EpCAM) CAR, an epidermal growth factor receptor (EGFR) CAR, a CD123 CAR, a CD20 CAR, a HER2 CAR, a GD2 CAR, a CD133 CAR, an EphA2 CAR, and a prostate-specific membrane antigen (PSMA) CAR. Thus, in some embodiments, the NK cells comprise a CD19 CAR. In some embodiments, the NK cells comprise a BCMA CAR. In some embodiments, the NK cells comprise a GPC3 CAR. In some embodiments, the NK cells comprise a CD22 CAR. In some embodiments, the NK cells comprise a mesothelin CAR. In some embodiments, the NK cells comprise a MUC1 CAR. In some embodiments, the NK cells comprise an EpCAM CAR. In some embodiments, the NK cells comprise an EGFR CAR. In some embodiments, the NK cells comprise a CD123 CAR. In some embodiments, the NK cells comprise a CD20 CAR. In some embodiments, the NK cells comprise a HER2 CAR. In some embodiments, the NK cells comprise a GD2 CAR. In some embodiments, the NK cells comprise a CD133 CAR. In some embodiments, the NK cells comprise an EphA2 CAR. In some embodiments, the NK cells comprise a PSMA CAR.

[0015] In some embodiments, the NK cells are modified to express one or more cytokines. In some embodiments, the NK cells are modified to express one or more of IL-15, a complex of IL-15 and IL-15Rα, IL-18, IL-12, IL-7, CCL19. Thus, in some embodiments, the NK cells are modified to express IL-15. In some embodiments, the NK cells are modified to express a complex of IL-15 and IL-15Rα. In some embodiments, the NK cells are modified to express IL-18. In some embodiments, the NK cells are modified to express IL-12. In some embodiments, the NK cells are modified to express IL-7. In some embodiments, the NK cells are modified to express CCL19.

[0016] In some embodiments, the NK cells are modified to express one or more suicide genes. For example, in some instances, the NK cells are modified to express one or more of iCaspase9, non-secretory TNFα, herpes simplex virus thymidine kinase (HSV-TK), uracil phosphoribosyltransferase (UPRTase), and cytosine deaminase (CD). Thus, in some embodiments, the NK cells are modified to express one or more iCaspase9. In some embodiments, the NK cells are modified to express non-secretory TNFα. In some embodiments, the NK cells are modified to express herpes simplex virus thymidine kinase (HSV-TK). In some embodiments, the NK cells are modified to express uracil phosphoribosyltransferase (UPRTase). In some embodiments, the NK cells are modified to express cytosine deaminase (CD).

[0017] In some embodiments, the NK cells are engineered to express CD19 CAR, IL-15, and iCaspase9.

[0018] In some embodiments, the NK cells are genetically modified cord blood NK cells comprising an anti-CD19 binding domain, a transmembrane domain, e.g., the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and an intracellular signaling domain, e.g., a CD19-CAR comprising intracellular signaling domains FcRγ, FcRβ, CD3γ, CD3δ, CD3ζ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. The CD-19 binding domain can be a single chain antibody or single chain antibody fragment, such as an scFv. In one embodiment, the anti-CD19 binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and / or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2. In another embodiment, a CD19 CAR may comprise an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is shown in SEQ ID NO:3), a CD3z signaling domain (an exemplary CD3z sequence is shown in SEQ ID NO:4), and may further comprise a suicide switch such as iCaspase9 and / or IL-15.

[0019] In one embodiment, the genetically engineered cord blood NK cells comprise a nucleic acid molecule encoding a heavy chain variable region of an anti-CD19 binding domain and / or a nucleic acid molecule encoding a light chain variable region of an anti-CD19 binding domain.

[0020] In some embodiments, the total volume of the container is about 50 ml.

[0021] In some embodiments, the total volume of the vessel is about 50 ml and the sample volume is less than 40 ml.

[0022] In some embodiments, the sample volume is about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 31 ml, about 32 ml, about 33 ml, about 34 ml, about 35 ml, about 36 ml, about 37 ml, about 38 ml, about 39 ml, about 40 ml, about 41 ml, about 42 ml, about 43 ml, about 44 ml, or about 45 ml. Thus, in some embodiments, the sample volume is about 10 mL. In some embodiments, the sample volume is about 15 mL. In some embodiments, the sample volume is about 20 mL. In some embodiments, the sample volume is about 15 mL. In some embodiments, the sample volume is about 25 mL. In some embodiments, the sample volume is about 30 mL. In some embodiments, the sample volume is about 35 mL. In some embodiments, the sample volume is about 40 mL. In some embodiments, the sample volume is about 45 mL.

[0023] In some embodiments, the container is a cryovial or a cryobag. Thus, in some embodiments, the container is a cryovial. In some embodiments, the container is a cryobag.

[0024] In some embodiments, the cryovial has an inner dimension between 10 mm and 18 mm. In some embodiments, the cryovial has an outer dimension between 15 mm and 40 mm.

[0025] In some embodiments, the cryovial has an internal dimension of about 13.5 mm.

[0026] In some embodiments, the cryovial has a height of about 40 mm to 50 mm. In some embodiments, the cryovial has a height of about 30 mm to 90 mm.

[0027] In some embodiments, the cryovial has a height of about 48.3 mm.

[0028] In some embodiments, the container is resistant to DMSO.

[0029] In some embodiments, the immune cells are present at a concentration of about 6 to 120 million cells per mL. In some embodiments, the immune cells are present at a concentration of about 6 to 25 million cells per mL.

[0030] In some embodiments, the cryopreservation medium comprises a cryoprotectant, albumin, a disaccharide, and a non-pyrogenic and isotonic crystalloid solution.

[0031] In some embodiments, the cryopreservation medium comprises human serum albumin (HSA), sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride, magnesium chloride, dimethyl sulfoxide (DMSO), and trehalose.

[0032] In some aspects, a method for thawing cryopreserved immune cells on a large scale is provided, the method comprising: (a) heating a water bath to a temperature ranging from 37° C. to 70° C.; (b) transferring a container containing the cryopreserved immune cells to the preheated water bath; and (c) agitating the container at a speed of about 100 to about 250 RPM for an appropriate period of time, thereby obtaining thawed immune cells. In some embodiments, the temperature of the water bath is 55° C. to 65° C. In some embodiments, agitation of the container is performed at a speed of about 100 to 150 RPM.

[0033] In some embodiments, a suitable period of time is between 5 and 15 minutes.

[0034] In some embodiments, a suitable period of time is about 10 minutes.

[0035] In some embodiments, the agitation is performed in an orbital shaker water bath.

[0036] In some embodiments, agitation of the cells in an orbital shaker water bath is at a speed of about 120-150 RPM.

[0037] In one embodiment, the cells are agitated in a 60° C. water bath at 125 RPM for approximately 10 minutes.

[0038] In some embodiments, the temperature of the orbital shaker water bath is about 50°C, 55°C, 60°C, 65°C, 70°C, or 75°C. Thus, in some embodiments, the temperature of the orbital shaker water bath is about 50°C. In some embodiments, the temperature of the orbital shaker water bath is about 55°C. In some embodiments, the temperature of the orbital shaker water bath is about 60°C. In some embodiments, the temperature of the orbital shaker water bath is about 65°C. In some embodiments, the temperature of the orbital shaker water bath is about 70°C. In some embodiments, the temperature of the orbital shaker water bath is about 75°C.

[0039] In some embodiments, the vessel has a total volume of about 50 mL and a sample volume of about 8 mL to 45 mL.

[0040] In some embodiments, the thawed immune cells have a post-thaw viability of 90%, 95%, 97% or more. Thus, in some embodiments, the thawed immune cells have a post-thaw viability of 90%. In some embodiments, the thawed immune cells have a post-thaw viability of 95%. In some embodiments, the thawed immune cells have a post-thaw viability of 97%. In some embodiments, the thawed immune cells have a post-thaw viability of greater than 97%.

[0041] In some embodiments, thawed immune cells retain similar in vitro and / or in vivo functions as freshly isolated immune cells.

[0042] In some embodiments, the method further comprises administering the thawed immune cells to a subject in need thereof.

[0043] In some embodiments, a method is provided for changing the temperature of a sample comprising immune cells from a first temperature above the freezing temperature of the sample to a final temperature of -80°C or less, thereby cryopreserving the sample at a final temperature, the method comprising the steps of: (a) placing the sample at a first temperature above the freezing temperature of the sample, (b) decreasing the first temperature at a first controlled rate to a second temperature, the second temperature being at least 2°C lower than the first temperature, (c) decreasing the second temperature at a second controlled rate to a third temperature, the third temperature being at least 40°C lower than the second temperature, (d) increasing the third temperature at a third controlled rate to a fourth temperature, the fourth temperature being at least 20°C higher than the third temperature, (e) decreasing the fourth temperature at a fourth controlled rate to a fifth temperature, the fifth temperature being at least 10°C lower than the fourth temperature, and (f) decreasing the fifth temperature at a fifth controlled rate to a final temperature, the final temperature being -80°C or less.

[0044] In some embodiments, the first temperature is between about 4° C. and 1° C. In some embodiments, the first temperature is about 4° C. In some embodiments, the first temperature is about 3° C. In some embodiments, the first temperature is about 2° C. In some embodiments, the first temperature is about 1° C.

[0045] In some embodiments, the first control rate is between about 0.75° C. and 1.25° C. per minute.

[0046] In some embodiments, the second temperature is about -2°C.

[0047] In some embodiments, the second control rate is about 20° C. to 30° C. per minute.

[0048] In some embodiments, the third temperature is about -60°C.

[0049] In some embodiments, the third control rate is between about 5° C. and 15° C. per minute.

[0050] In some embodiments, the fourth temperature is about -25°C.

[0051] In some embodiments, the fourth control rate is between 0.75° C. and 1.25° C. per minute.

[0052] In some embodiments, the fifth temperature is about -40°C.

[0053] In some embodiments, the fifth control rate is between 7° C. and 15° C. per minute.

[0054] In some embodiments, the final temperature is -80°C or lower.

[0055] In some embodiments, a method is provided that includes cryopreserving modified immune cells suitable for cell therapy, the method including: (1) providing a container containing a sample comprising immune cells suspended in a cryopreservation medium, wherein the volume of the sample is at least 5% less than the total volume of the container, and the volume of the sample is at least 10 mL; and (2) stepwise freezing a population of modified immune cells at a controlled rate to minimize effects of latent heat of fusion, the stepwise freezing including cooling the cells at a rate of 0.75° C. per minute to 30° C. per minute to a final temperature of −80° C. or less, whereby the cells are cryopreserved. [Brief description of the drawings]

[0056] [Figure 1] FIG. 1A shows an exemplary orbital shaker water bath with adjustable rotation speed and temperature, and FIG. 1B shows an exemplary sample chamber of an orbital shaker with a holder for holding a freezing container or vial for rapid thawing of cryopreserved cells. [Diagram 2] FIG. 1 is a graph showing the freezing profile of 45 ml of cell-free placebo formulation solution using a large volume freezing protocol as described in Example 1. [Diagram 3] Graph showing the freezing profile of 36 ml of CAR-NK cells using the 60 minute freezing protocol described herein, specifically Example 2. CAR-NK cells were frozen in a controlled rate freezer. [Figure 4]FIG. 1 is a graph showing the freezing characteristics of five 50 mL AT vials containing 16 mL, 30 mL, and 45 mL of iCART cells (sometimes referred to herein as iCART). The diagram in the lower left corner shows the arrangement of the 50 ml vials within the freezer. The USB designation refers to the arrangement of the vials within the freezer. [Diagram 5] A is a graph showing the thawing profile of 45 ml and 30 ml frozen iCARTs at a concentration of 80 million cells per ml in an orbital shaker water bath at 60° C., with the orbital shaker rotation speed set at 150 rpm. B is a graph showing the thawing profile of 45 ml, 30 ml, and 16 ml frozen iCARTs at a concentration of 120 million cells per ml in an orbital shaker water bath at 60° C., with the orbital shaker rotation speed set at 150 rpm. [Figure 6] 1 is a graph showing the viability of iCART cells frozen using the freezing and thawing procedure described herein. [Figure 7A] Graph showing equivalent in vitro killing efficacy as a function of E:T (effector:target cell) ratio of frozen CAR-NK cells in 50 mL AT vials at different loading volumes of both 80 and 120 million per mL by the freeze and thaw procedure described herein compared to using fresh cells, and 2 mL cryovials and 2 mL AT vials. [Figure 7B] 1 is a table showing the phenotype of frozen and subsequently thawed CAR-NK cells. [Figure 8A] 1 shows a series of graphs showing that CAR-NK cells at 6 million and 80 million cells per mL using a freeze and thaw procedure have comparable in vitro killing efficacy as a function of E:T ratio to frozen cells in 2 mL cryovials as reference cells. [Figure 8B] Data are compiled showing that the larger 50 mL vials provide high (>=96%) and comparable viability, killing efficiency, and phenotypic results to reference cells in 2 mL cryovials. [Figure 8C]Data are compiled showing that the larger 50 mL vials provide high (>=96%) and comparable viability, killing efficiency, and phenotypic results to reference cells in 2 mL cryovials. [Figure 9A]

[0023] Figure 1 is a series of graphs showing in vivo survival of tumor mouse models administered frozen and thawed CAR-NK cells. The graphs show survival of tumor mouse model animals after administration of frozen and thawed CAR-NK cells (cell suspensions #1-#3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells. Animal survival after administration of CAR-NK cells ("cell suspension #1") is shown. [Figure 9B] A series of graphs showing in vivo survival rates of tumor mouse models administered frozen and thawed CAR-NK cells. The graphs show survival rates of tumor mouse model animals after administration of frozen and thawed CAR-NK cells (cell suspensions #1-#3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells. Animal survival rates after administration of CAR-NK cells to tumor mouse models are shown. [Figure 9C] A series of graphs showing in vivo survival rates of tumor mouse models administered frozen and thawed CAR-NK cells. The graphs show survival rates of tumor mouse model animals after administration of frozen and thawed CAR-NK cells (cell suspensions #1-#3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells. Animal survival rates after administration of CAR-NK cells to tumor mouse models are shown. [Figure 9D] FIG. 1 is a series of graphs showing the in vivo total flux of luciferase fluorescence in tumor mouse models injected with frozen and then thawed CAR-NK cells (cell suspensions #1-3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells. [Figure 9E]FIG. 1 is a series of graphs showing the in vivo total flux of luciferase fluorescence in tumor mouse models injected with frozen and then thawed CAR-NK cells (cell suspensions #1-3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells. [Figure 9F] FIG. 1 is a series of graphs showing the in vivo total flux of luciferase fluorescence in tumor mouse models injected with frozen and then thawed CAR-NK cells (cell suspensions #1-3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells. [Figure 9G] FIG. 11 is a series of images showing luciferase activity in mice injected with frozen and then thawed NK cells (cell suspensions #1-3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells. [Figure 9H] FIG. 11 is a series of images showing luciferase activity in mice injected with frozen and then thawed NK cells (cell suspensions #1-3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells. [Figure 9I] FIG. 11 is a series of images showing luciferase activity in mice injected with frozen and then thawed NK cells (cell suspensions #1-3) compared to vehicle (freezing medium without cells) and freshly rescued CAR-NK cells.

[0057] definition Allogeneic: As used herein, allogeneic refers to any material that is derived from an animal of the same species as the individual into which the material is introduced, but different from that individual.Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical.In some embodiments, allogeneic materials from individuals of the same species can be genetically sufficiently different to interact antigenically.

[0058] Approximately or about: As used herein, the term "approximately" or "about" when applied to one or more target values ​​refers not only to the stated target value, but also to values ​​similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls 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 (above or below) of the stated target value and the stated reference value (except where such number exceeds 100% of possible values), unless otherwise stated or otherwise clear from the context.

[0059] Biological Sample: As used herein, the term "biological sample" includes cells (both eukaryotic and prokaryotic), organs and tissues composed of cells, viruses, all of which may be naturally occurring or genetically or otherwise modified, and may be biologically active molecules, e.g., macromolecules such as cells, nucleic acids, proteins, glycoproteins, lipids, lipoproteins, etc. The present invention has particular application to the cryopreservation of human and other mammalian immune cells.

[0060] Fresh cells or rescued fresh cells: As used herein, the terms "fresh," "fresh cells," or "rescued fresh cells" refer to mammalian cells that have never been frozen and / or that have been frozen but subsequently restimulated and cultured in culture medium and then recovered as fresh cells.

[0061] Container: As used herein, the term "container" shall be given its ordinary meaning and includes carriers, holders, enclosures, and conduits for containing, holding, administering, delivering, or transporting materials such as cryopreserved cells and related compounds. Thus, in one embodiment, the container does not react with DMSO. In another embodiment, the containers (e.g., sterile cryovials) used herein are DEHP-free and DMSO-resistant. Exemplary suitable containers include cryovials, freezer bags, and the like. Various types of cryovials are known, including, for example, AT® cryovials, Nunc™ vials, glass vials, and the like.

[0062] Controlled cooling or controlled rate cooling: As used herein, the term "controlled cooling" or "controlled rate cooling" and similar terms is the process of applying an external cooling regime that results in a temperature reduction of a biological sample that is cooled at a rate of, for example, 0.1°C / min to 50°C / min. In some embodiments, controlled cooling can be achieved using commercially available freezers, such as controlled rate freezers. Various examples of controlled rate freezers include, but are not limited to, CryoMed™ Model 5474, Strex CytoSensei SB02-0920, Custom BioGenic Systems Model 2101, for example.

[0063] Cryoprotectant: As used herein, the term "cryoprotectant" refers to a substance used to protect biological tissue from freezing damage. Exemplary cryoprotectants include, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propanediol.

[0064] Cryopreservation: As used herein, the term "cryopreservation" or "freezing" generally refers to a method of freezing cells such that the viability of the cells is maintained. Cryopreserved cells maintain their viability for extended periods of time in a frozen state, such as one year, five years, ten years, or more in a frozen state. Once thawed, cryopreserved cells can be expanded for both in vitro and in vivo applications.

[0065] Immune cells: As used herein, the term "immune cells" refers to lymphocytes with helper, cytolytic, or regulatory properties, such as, for example, T cells, B cells, NK cells, macrophages, neutrophils, eosinophils, basophils, CD4+ T cells, CD8+ T cells, CD4+CD8+ T cells, CD4+CD8dim T cells, CD4+ regulatory T cells, CD56+CD8+ and CD56-CD57+CD8+ NKT, CAR-T cells, and CD16+CD56+ NK cells. However, as used herein, the term "immune cells" does not only refer to immune cells maintained and expanded in vitro in culture, but also to immune cell populations taken from healthy blood donors, patients, or animals, and to immune cells purified, respectively. In some embodiments, "immune cells" can be used to define T cells modified by expressing a chimeric antigen receptor (CAR). In some embodiments, "immune cells" can be used to define NK cells modified by expressing a CAR. In some embodiments, "immune cells" are HSC cells. In some embodiments, the "immune cells" are MSC cells. In some embodiments, the "immune cells" are cord blood derived immune cells. In some embodiments, the "immune cells" are iPS cell derived immune cells. In some embodiments, the "immune cells" are regulatory T cells. In some embodiments, the "immune cells" are γδ T cells. In some embodiments, the NK cells comprise an anti-CD19 chimeric antigen receptor (CAR), IL-15, and iCaspase9. Thus, in some embodiments, the NK cells are engineered (e.g., using viral transduction or non-viral methods) to express a suicide gene, an anti-CD19 CAR gene, and an IL-15 gene. Exemplary CAR-NK cells comprising CD19 IL-15 and iCaspase9 are described in Leukemia 32 (2018) 520-531, which is incorporated by reference in its entirety.

[0066] In certain embodiments, an exemplary CAR-NK cell comprises a CD19-CAR comprising an anti-CD19 binding domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least 95% identity thereto, and / or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2, or a sequence having at least 95% identity thereto. In some embodiments, the genetically modified cord blood NK cell comprises a nucleic acid molecule encoding the heavy chain variable region of the anti-CD19 binding domain and / or a nucleic acid molecule encoding the light chain variable region of the anti-CD19 binding domain.

[0067] Latent heat or heat of fusion: The term "heat of fusion" or "latent heat", when used in the broadest sense, refers to any substance or phenomenon where, in the process of melting a substance, when heat is added to the substance at a substantially uniform rate, a point is reached where the temperature of the substance temporarily stops increasing while heat is absorbed due to the transformation of the molecular structure and internal energy of the substance. During freezing, the release of latent heat through the phase change from the liquid state to the solid state increases the ambient temperature and vitrification stops. In some embodiments, the latent heat of fusion can cause the ice to melt. In some embodiments, the melting of the ice increases the concentration of sugars, salts, and cryoprotectants (e.g., DMSO or glycerol), which in turn rapidly increases the osmotic pressure of the unfrozen fraction. In some embodiments, the increasing strength of the osmotic pressure causes water to flow out of the cells. In some embodiments, as cooling continues, these processes continue until the viscosity of the unfrozen fraction becomes too high to crystallize any further.

[0068] Minimizing the effect of latent heat of fusion: The term "minimizing the effect of latent heat of fusion" or "minimizing the effect of latent heat of fusion" refers to a process involving the induction of ice crystal formation or ice nucleation outside of cells in a biological sample. In some embodiments, the effect of the latent heat of fusion is minimized by a continuous smooth temperature drop by controlled cooling, allowing ice to form gradually outside the cells while intracellular water is transferred by osmosis. In some embodiments, ice nucleation supports the gradual growth of ice outside the cells, limiting supercooling. In some embodiments, the increase in extracellular ice leads to the dehydration of water from the cells. In some embodiments, the amount of ice crystals formed depends on the initial composition of the solution. In some embodiments, the cryoprotectant retards freezing inside the cells by lowering the freezing point. In some embodiments, the cryoprotectant can penetrate the cells to retard freezing inside the cells.

[0069] Ice nucleation: The term "ice nucleation" refers to the process that occurs in forming ice crystals from a solution, where more ice crystals are thermodynamically favored to form from the water present in the solution. Nucleation is a stochastic process, occurring at specific sites on surfaces within the system. Nucleation can be induced by lowering the temperature or concentrating the water to conditions where the liquid or solution is significantly less thermodynamically stable than the crystals. Nucleation can be further induced by introducing pre-existing ice crystals at a preferred temperature. In some embodiments, ice nucleation can be induced by introducing ice crystals into the container using a copper wire. In some embodiments, ice nucleation can be induced by lowering the temperature.

[0070] Orbital shaker water bath: As used herein, the term "orbital shaker water bath" refers to a water bath apparatus that reliably produces orbital or cyclic motion at a set speed for a predetermined period of time, with a movable tray for supporting items to be agitated, such as in a clinical assay, such as a beaker, flask, test tube, etc.

[0071] Mammal: As used herein, the term "mammal" refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, e.g., chimpanzees, as well as other ape and monkey species, livestock animals such as cows, sheep, pigs, goats, and horses, domestic mammals such as dogs and cats, and laboratory animals including rodents such as mice, rats, and guinea pigs. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, as well as fetuses, are intended to be included within the scope of this term.

[0072] Storage temperature: As used herein, the term "storage temperature" refers to the temperature at which cells are stored. In some embodiments, the cells are stored in liquid nitrogen vapor phase. In some embodiments, the cells are stored at a temperature below -60°C. In another embodiment, the cells are stored at a temperature ranging from -60°C to -140°C. In another embodiment, the cells are stored at a temperature ranging from -60°C to -196°C. In some embodiments, the cells are stored at a temperature below -140°C. In some embodiments, the cells are stored at a temperature below -196°C.

[0073] Transport temperature: As used herein, the term "transport temperature" refers to the temperature at which cells are transported or conveyed, for example, from a first location where the cells may be manufactured and / or cryopreserved to a second location where the cells may be thawed and then administered to a subject in need thereof. In some embodiments, the cells are transported in liquid nitrogen vapor phase. In some embodiments, the cells are transported at or below -140°C. In some embodiments, the cells are stored and / or transported at or below -140°C.

[0074] Vitrification: The term vitrification is defined as the process of rapid freezing of a sample, preferably a biological sample. In some embodiments, vitrification prevents the formation of ice. In some embodiments, the vitrification process requires the presence of a cryoprotectant. In some embodiments, the vitrification process requires a means to rapidly cool the temperature.

[0075] The recitation herein of numerical ranges by endpoints includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4, and 5). It is also understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0076] Various aspects of the invention are described in detail in the following sections. The use of the sections is not intended to limit the invention. Each section may be applicable to any aspect of the invention. 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] The process of cryoprotection can be fatal to cell survival. When the medium surrounding the cells is cooled slowly to preserve them, for example immune cells, freezing of the cells rarely occurs at the freezing point of the medium. Samples can be supercooled to temperatures of -21 °C. Excessive supercooling can be detrimental to the survival of some biological samples if they are left to continue cooling in the same container after ice formation.

[0078] Without wishing to be bound by theory, the reason for this adverse effect (lower survival rate) is believed to be as follows: Upon ice formation, the latent heat of melting raises the sample temperature close to the melting point of the medium. At the same time, the temperature of the bath (or other environment) continues to drop at a constant rate, therefore, the greater the degree of supercooling, the greater the temperature difference that exists between the cooling vessel and the sample immediately after ice formation. This increases the cooling rate of the sample beyond the usual rate that is optimal for the survival of the sample (especially in the case of immune cells) until thermal equilibrium with the surroundings is re-established.

[0079] The challenge for cells during cryopreservation is not the ability of the cells to survive storage at ultra-low temperatures (below -120°C), but rather the lethality of the cool-freeze and warm-thaw processes. When cells are cooled to approximately -5°C, both the cells and the surrounding medium are unfrozen and supercooled. Between -5°C and approximately -15°C, ice forms in the external medium (either spontaneously or as a result of artificially introducing ice crystals, i.e., seeding the solution), but the contents of the cell remain unfrozen and supercooled, presumably because the plasma membrane blocks growth of ice crystals into the cytoplasm. The supercooled water within the cell, by definition, has a greater chemical potential than the water in the partially frozen extracellular solution, and therefore water osmotically flows out of the cell and freezes outside.

[0080] Subsequent physical events within the cell depend on the cooling rate. If the cell is cooled too quickly, the intracellular water does not move out of the cell fast enough to maintain equilibrium, the cell becomes increasingly supercooled, intracellular ice formation (IIF) occurs, and the cell dies. However, if the cell is cooled too slowly, severe dehydration with volume shrinkage occurs before the eutectic temperature (the point at which all components in the solution freeze) is reached, resulting in prolonged exposure to high concentrations of solutes (mainly consisting of electrolytes). Both cell shrinkage and prolonged exposure to high concentrations of electrolytes can cause cell damage (so-called "osmotic injury"). As a result, the cell becomes dehydrated and does not freeze intracellularly. Thus, both high and low cooling rates can damage cells, although the mechanisms underlying cell damage are different.

[0081] Cellular function is maintained only if cooling is controlled at an optimal rate that allows cells to lose water fast enough to concentrate intracellular solutes sufficiently to eliminate supercooling while preventing severe cellular dehydration. The optimal cooling rate to maintain cellular function needs to be slow enough to avoid IIF, but fast enough to minimize severe cellular dehydration.

[0082] Moreover, even if cells survive cooling-freezing to low temperatures, they still face a major challenge during the warming-thawing process due to lethal ice recrystallization (LIR), i.e., the growth of small intracellular ice crystals into harmful larger ice crystals during the warming-thawing process. An optimal rapid warming rate or program combined with a cooling rate / program is absolutely necessary to prevent LIR for cellular cryosurvival.

[0083] Controlling the cooling and rapid warming rates for small samples (<5 mL) is easy to achieve in research laboratories. However, modern large-scale industrial manufacturing of therapeutic cells faces a major existing challenge: how to achieve and control optimal cooling and rapid warming rates for cryopreserving large samples (>25 mL), which is becoming a significant technical bottleneck in cryopreservation and commercialization of large-scale production of therapeutic cell products, as well as their shipping and clinical use.

[0084] The methods disclosed herein include: (1) a combination of optimal cooling (with a specific ice nucleation seeding program to prevent overcooling) and rapid warming rate conditions and programs for cryopreservation of mammalian cells, including immune cells (e.g., innate immune cells and developed CAR-T and CAR-NK cells); (2) a method and program for achieving the above optimal cooling rate for large vial samples (e.g., large volume cell suspensions in 10 mL or larger containers) using a liquid nitrogen controlled freezer; and (3) a method and program for achieving the above optimal rapid warming rate for large vial samples (e.g., large volume cell suspensions in 50 mL AT sealed vials) using an orbital shaker water bath at a temperature above 50° C. for cell suspensions with a fill volume in the vial of 8-45 mL and a cell concentration of 6-120 million cells per mL. In some embodiments, the cell concentration is about 6-25 million cells per mL.

[0085] Cryopreservation method It is generally known that when liquid water is cooled, a phase transition from liquid to solid occurs at a critical temperature. This phase transition is a first order transition, which means that the water absorbs or releases an amount of energy per volume (known as latent heat). During the phase transition, the temperature of the water remains constant while heat is added or removed, during which the water is in a mixed state, partly in a liquid state and partly in a solid state. The temperature at which the phase transition occurs can be called the critical temperature of the phase transition. When water is cooled, the temperature of the water decreases until the critical temperature is reached. During continued cooling, the temperature of the water remains constant until the latent heat is removed from the water, after which the temperature of the water, which is in a solid state, decreases again. This means that the time during which the latent heat is removed from the water continues. The time during which the latent heat is removed in the freezing process is the time during which ice crystals can form, which is undesirable when cryopreserving samples containing biological materials such as cells.

[0086] The cryopreservation media described herein, used in the cryopreservation methods described herein, minimize the effects of latent heat (i.e., the effects of ice formation) during cryopreservation, thereby increasing the viability of the cell sample being frozen.

[0087] The cryopreservation methods described herein are performed on a cell suspension of mammalian immune cells. In some embodiments, the stepwise deep freezing procedure is scalable up to 30 or more vials, each having a cell sample volume of 10-40 mL or more. In some embodiments, the freezing procedure is scalable up to 30 vials, 50 vials, or 75 vials. In some embodiments, the current cryopreservation method is performed on a mammalian immune cell suspension in a cryovial, cryobag, AT® vial, or Nunc™ vial, or glass vial. In some embodiments, the current cryopreservation method is performed on a mammalian immune cell suspension in a cryovial, or AT® vial, or any other suitable container. In some embodiments, the suitable container is one that does not react with DMSO. In some embodiments, the cryovials, cryobags, AT® vials, or Nunc™ vials, glass vials, and other containers used for cryopreservation are compatible for use with DMSO. In some embodiments, cryovials, cryobags, AT® vials, or Nunc™ vials, glass vials, and other containers used for cryopreservation are both DEHP-free and DMSO-resistant.

[0088] Various containers that can be used in the cryopreservation method include, for example, cryovials or cryobags. Exemplary cryovials include, for example, AT® vials or Nunc™ vials or glass vials. In some embodiments, the cryopreservation method is performed on a mammalian immune cell suspension in a cryovial or AT® vial or any other suitable container. In some embodiments, a suitable container is one that is resistant to DMSO. In some embodiments, the cryovials, cryobags, AT® vials or Nunc™ vials, glass vials, and other containers used in cryopreservation are compatible for use with DMSO. In some embodiments, the cryovials, cryobags, AT® vials or Nunc™ vials, glass vials, and other containers used in cryopreservation are not chemically reactive with DMSO. In some embodiments, the containers used in cryopreservation are both DEHP-free and DMSO-resistant (e.g., AT® vials). In various embodiments, the containers used herein (e.g., AT® vials) facilitate the direct, sterile transfer of cells to a subject in need thereof via a vial adapter.

[0089] The containers used herein can have a variety of dimensions, including those described herein. In some embodiments, dimensions suitable for use with cryovials described herein are also suitable for use with other containers, such as AT® vials or Nunc™ vials.

[0090] In some embodiments, the freeze / thaw methods described herein allow for consistent freezing and thawing of large cell-containing samples, such as volumes of 10 mL or more. In some embodiments, the freeze / thaw methods described herein can be used on samples having volumes of 10 mL or more. In some embodiments, the freeze / thaw methods described herein can be used on samples having diameters of greater than 225 mm / inch. In some embodiments, the freeze / thaw methods described herein can be used on samples having solution heights of greater than 225 mm / inch. In some embodiments, the freeze / thaw methods described herein can be used on samples having solution thicknesses of greater than 225 mm / inch.

[0091] In some embodiments, the cryovial has dimensions between about 5 mm outer diameter and 100 mm height. In some embodiments, the cryovial has dimensions between about 10 mm outer diameter and 75 mm height. In some embodiments, the cryovial has dimensions between 10 mm outer diameter and 50 mm height.

[0092] In some embodiments, the cryovial has a dimension of 10 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has a dimension of 10.5 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has a dimension of 11.0 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has a dimension between 11.5 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has a dimension of 12.0 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has a dimension of 12.5 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has a dimension of 13.0 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has a dimension of 13.5 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has a dimension of 14.0 mm outer diameter and 48.3 mm height. In some embodiments, the cryovial has an outer diameter of 14.5 mm and a height of 48.3 mm. In some embodiments, the cryovial has an outer diameter of 15.0 mm and a height of 48.3 mm.

[0093] In some embodiments, the cryovial has a height of about 30 mm to about 85 mm. In some embodiments, the cryovial has an outer diameter of about 15 mm to about 40 mm. In some embodiments, the cryovial has a maximum capacity of about 1 mL to 55 mL. A wide variety of cryovials are suitable for the compositions and methods described herein. Exemplary cryovials, including a description of cryovial dimensions, can be found at http: / / www.aseptictech.com / sites / default / files / brochure_vialslines_v3.0.pdf, the contents of which are incorporated herein by reference in their entirety.

[0094] In some embodiments, the cryovial has a height of about 45 mm to 100 mm. In some embodiments, the cryovial has a height of 45.3 mm. In some embodiments, the cryovial has a height of 45.6 mm. In some embodiments, the cryovial has a height of 46 mm. In some embodiments, the cryovial has a height of 46.3 mm. In some embodiments, the cryovial has a height of 46.6 mm. In some embodiments, the cryovial has a height of 47 mm. In some embodiments, the cryovial has a height of 47.3 mm. In some embodiments, the cryovial has a height of 47.6 mm. In some embodiments, the cryovial has a height of 48 mm. In some embodiments, the cryovial has a height of 48.3 mm. In some embodiments, the cryovial has a height of 48.6 mm. In some embodiments, the cryovial has a height of 49 mm. In some embodiments, the cryovial has a height of 49.3 mm. In some embodiments, the cryovial has a height of 50 mm. In some embodiments, the cryovial has a height of 50 mm. In some embodiments, the cryovial has a height of 50 mm. In some embodiments, the cryovial has a height of 55 mm. In some embodiments, the cryovial has a height of 60 mm. In some embodiments, the cryovial has a height of 55 mm. In some embodiments, the cryovial has a height of 65 mm. In some embodiments, the cryovial has a height of 55 mm. In some embodiments, the cryovial has a height of 70 mm. In some embodiments, the cryovial has a height of 75 mm. In some embodiments, the cryovial has a height of 80 mm. In some embodiments, the cryovial has a height of 85 mm. In some embodiments, the cryovial has a height of 90 mm. In some embodiments, the cryovial has a height of 95 mm.In some embodiments, the cryovial has a height of 100 mm.

[0095] In some embodiments, the cryovial has an outer diameter of 10 mm. In some embodiments, the cryovial has an outer diameter of 10.5 mm. In some embodiments, the cryovial has an outer diameter of 11 mm. In some embodiments, the cryovial has an outer diameter of 11.5 mm. In some embodiments, the cryovial has an outer diameter of 12 mm. In some embodiments, the cryovial has an outer diameter of 12.5 mm. In some embodiments, the cryovial has an outer diameter of 13 mm. In some embodiments, the cryovial has an outer diameter of 13.5 mm. In some embodiments, the cryovial has an outer diameter of 14 mm. In some embodiments, the cryovial has an outer diameter of 14.5 mm. In some embodiments, the cryovial has an outer diameter of 15 mm.

[0096] In some embodiments, the cryobag has a width of 11 cm. In some embodiments, the cryobag has a width of 11.3 cm. In some embodiments, the cryobag has a width of 11.5 cm. In some embodiments, the cryobag has a width of 11.7 cm. In some embodiments, the cryobag has a width of 11.9 cm. In some embodiments, the cryobag has a width of 12.1 cm. In some embodiments, the cryobag has a width of 12.3 cm. In some embodiments, the cryobag has a width of 12.5 cm. In some embodiments, the cryobag has a width of 12.7 cm. In some embodiments, the cryobag has a width of 12.9 cm. In some embodiments, the cryobag has a width of 13.1 cm. In some embodiments, the cryobag has a width of 13.3 cm. In some embodiments, the cryobag has a width of 13.5 cm. In some embodiments, the cryobag has a width of 13.7 cm.

[0097] In some embodiments, the cryobag has a length of 14.1 cm. In some embodiments, the cryobag has a length of 14.3 cm. In some embodiments, the cryobag has a length of 14.5 cm. In some embodiments, the cryobag has a length of 14.7 cm. In some embodiments, the cryobag has a length of 14.9 cm. In some embodiments, the cryobag has a length of 15.1 cm. In some embodiments, the cryobag has a length of 15.3 cm. In some embodiments, the cryobag has a length of 15.5 cm. In some embodiments, the cryobag has a length of 15.7 cm. In some embodiments, the cryobag has a length of 15.9 cm. In some embodiments, the cryobag has a length of 16.1 cm. In some embodiments, the cryobag has a length of 16.3 cm. In some embodiments, the cryobag has a length of 16.5 cm. In some embodiments, the cryobag has a length of 16.7 cm.

[0098] In some embodiments, the volume (i.e., maximum capacity) of the cryovial is between 2 ml and 50 ml, e.g., 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28 ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 41 ml, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml, 49 ml, 50 ml, 51 ml, 52 ml, 53 ml, 54 ml, 55 ml, 56 ml, 57 ml, 58 ml, 59 ml, 60 ml, 61 ml, 62 ml, 63 ml, 64 ml, 65 ml, 66 ml, 67 ml, 68 ml, 69 ml, 70 ml, 71 ml, 72 ml, 73 ml, 74 ml, 75 ml, 76 ml, 77 ml, 78 ml, 79 ml, 80 ml, 81 ml, 82 ml, 83 ml, 84 ml, 85 ml, 86 ml, 87 ml, 88 ml, 89 ml, 90 ml, 91 ml, 92 ml, 93 ml, 94 ml, 95 ml, 96 ml, 97 ml, 98 It can be 8ml, 49ml, 50ml, 51ml, 52ml, 53ml, 54ml, 55ml, 56ml, 57ml, 58ml, 59ml, 60ml, 61ml, 62ml, 63ml, 64ml, 65ml, 66ml, 67ml, 68ml, 69ml, 70ml, 71ml, 72ml, 73ml, 74ml, 75ml, 76ml, 77ml, 78ml, 79ml, 80ml, 81ml, 82ml, 83ml, 84ml, 85ml, 86ml, 87ml, 88ml, 89ml, 90ml, 91ml, 92ml, 93ml, 94ml, 95ml, 96ml, 97ml, 98ml, 99ml, or 100ml.

[0099] As used herein, "fill volume" refers to the volume of a sample containing cells in a container. In some embodiments, the fill volume is less than the maximum capacity of the container. In some embodiments, the fill volume in a vial can be between 15% and 90% of the maximum capacity of the vial. For example, the fill volume in a vial can be 15% of the maximum capacity, 20% of the maximum capacity, 25% of the maximum capacity, 30% of the maximum capacity, 35% of the maximum capacity, 40% of the maximum capacity, 45% of the maximum capacity, 50% of the maximum capacity, 55% of the maximum capacity, 60% of the maximum capacity, 65% of the maximum capacity, 70% of the maximum capacity, 75% of the maximum capacity, 80% of the maximum capacity, 85% of the maximum capacity, or 90% of the maximum capacity. In some embodiments, a 2 ml cryovial has a fill volume of 1 ml. In some embodiments, a 50 ml cryovial has a fill volume of 8 ml to 45 ml. In some embodiments, a 50 ml cryovial has a fill volume of 8 ml. In some embodiments, the 50 ml cryovial has a fill volume of 10 ml. In some embodiments, the 50 ml cryovial has a fill volume of 12 ml. In some embodiments, the 50 ml cryovial has a fill volume of 14 ml. In some embodiments, the 50 ml cryovial has a fill volume of 16 ml. In some embodiments, the 50 ml cryovial has a fill volume of 18 ml. In some embodiments, the 50 ml cryovial has a fill volume of 20 ml. In some embodiments, the 50 ml cryovial has a fill volume of 22 ml. In some embodiments, the 50 ml cryovial has a fill volume of 24 ml. In some embodiments, the 50 ml cryovial has a fill volume of 26 ml. In some embodiments, the 50 ml cryovial has a fill volume of 28 ml. In some embodiments, the 50 ml cryovial has a fill volume of 30 ml. In some embodiments, the 50 ml cryovial has a fill volume of 32 ml. In some embodiments, the 50 ml cryovial has a fill volume of 34 ml. In some embodiments, the 50 ml cryovial has a fill volume of 36 ml.In some embodiments, the 50 ml cryovial has a fill volume of 38 ml. In some embodiments, the 50 ml AT vial has a fill volume of 40 ml. In some embodiments, the 50 ml cryovial has a fill volume of 42 ml. In some embodiments, the 50 ml cryovial has a fill volume of 44 ml. In some embodiments, the 50 ml cryovial has a fill volume of 45 ml. In some embodiments, the cryocontainer can have a volume of about 50 ml, about 75 ml, about 100 ml, about 250 ml, about 500 ml, about 750 ml, about 1 L, or greater than about 1 L.

[0100] In some embodiments, the mammalian immune cells may be cryopreserved at a concentration between about 6M / ml and about 120M / ml. For example, in some embodiments, the immune cells may be cryopreserved at a concentration of 6M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 10M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 15M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 20M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 25M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 30M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 35M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 40M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 45M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 50M / ml. In some embodiments, the immune cells may be cryopreserved at a concentration of 55M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 60M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 65M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 70M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 75M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 80M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 85M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 90M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 100M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 105M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 110M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 115M / ml. In some embodiments, immune cells may be cryopreserved at a concentration of 120M / ml.

[0101] In some embodiments, the CAR NK cells are administered at a concentration of about 1 x 10 in a volume of about 36 mL in a 50 mL cryovial. 6 ~1×10 7 In some embodiments, the CAR NK cells are provided in a dose of between about 200 x 10 cells in a 50 mL cryovial. 6 ~800×10 6 In some embodiments, the CAR NK cells are suspended in a cryopreservation medium as described herein and subsequently cryopreserved as disclosed herein. Such cryopreserved NK cells can then be stored as described herein. The sample containing the NK cryopreserved NK cells is then thawed as described herein. The thawed cells are then administered to a patient in need thereof. In some embodiments, a volume of about 33 mL, 34 mL, 35 mL, or 36 mL of the thawed cells is administered to a patient in need thereof using a vial adapter for sterile administration. In some embodiments, a volume of about 33 mL of the thawed cells is administered to a patient in need thereof using a vial adapter for sterile administration. In some embodiments, a volume of about 34 mL of the thawed cells is administered to a patient in need thereof using a vial adapter for sterile administration. In some embodiments, a volume of about 35 mL of the thawed cells is administered to a patient in need thereof using a vial adapter for sterile administration. In some embodiments, a volume of about 36 mL of the thawed cells is administered to a patient in need thereof using a vial adapter for sterile administration.

[0102] In some aspects, a medium suitable for cryopreserving cells (i.e., cryopreservation medium) comprises a cryoprotectant, albumin, a disaccharide, and a non-pyrogenic and isotonic crystalloid. In various embodiments, cells once cryopreserved in the described medium can be thawed and subsequently administered to a patient without the need to reconstitute or resuspend the cells in another medium or solution.

[0103] In some embodiments, mammalian immune cells are cryopreserved in a medium containing one or more cryoprotectants.Various cryoprotectants are known in the art, including, for example, dimethylsulfoxide (DMSO), glycerol, and propanediol, among others.In some embodiments, the cryopreservation medium comprises DMSO as a cryoprotectant.

[0104] In some embodiments, cryopreservation of mammalian immune cells includes up to nine stages of deep freezing procedures. In some embodiments, the staged freezing reduces the latent heat of fusion of the biological sample being frozen. In some embodiments, the cells are cooled in a temperature drop stage having a selected temperature drop rate. In some embodiments, the rate of temperature drop in the temperature drop stage is about 10° C. per minute. In some embodiments, other change rates are used, for example, about 1° C. / min, about 2° C. / min, about 5° C. / min, about 7° C. / min, about 12° C. / min, about 15° C. / min, about 17° C. / min, about 20° C. / min, about 25° C. / min, or rates within the above values. In some embodiments, the temperature drop stage may include a rapid freezing (e.g., maximum temperature drop) step. For example, the rapid freezing step may include a rapid freezing temperature change rate with an increased temperature drop rate, which is selected to advantageously improve cell integrity or cell viability. In contrast, some embodiments, contrary to conventional wisdom, do not use a rapid freezing step. Applicants have discovered that in certain embodiments, the maximum rate of temperature reduction is as detrimental as certain slower reduction rates, while certain intermediate rates unexpectedly provide improved cryopreservation quality and / or improved cell viability. In some embodiments, the temperature reduction step may include cooling the cells adhered to the substrate at a rate of approximately 10° C. per 10 seconds, 10° C. per 20 seconds, 10° C. per 30 seconds, 10° C. per 40 seconds, 10° C. per 50 seconds, 10° C. per 60 seconds, 10° C. per 70 seconds, 10° C. per 80 seconds, 10° C. per 90 seconds, 10° C. per 100 seconds, 10° C. per 110 seconds, 10° C. per 120 seconds, 10° C. per 130 seconds, 10° C. per 140 seconds, 10° C. per 150 seconds, 10° C. per 160 seconds, 10° C. per 170 seconds, 10° C. per 180 seconds, 10° C. per 190 seconds, or 10° C. per 200 seconds. In some embodiments, the temperature is maintained at a predetermined temperature for a predetermined time. The cooling rate during the temperature drop-down phase is set to improve cell viability. For example, a particular cooling rate during the temperature drop-down phase improves cell function after cryopreservation. In some embodiments, the cooling rate during the temperature drop-down phase is configured to improve cell integrity or stability.In some embodiments, the cryopreserved cells substantially maintain their therapeutic efficacy after cryopreservation, e.g., the cells are suitable for direct transplantation to a target site in a patient after thawing of the cryopreserved cells, resulting in therapeutic efficacy roughly equivalent (or greater) than non-cryopreserved cells.

[0105] In some embodiments, the cryopreservation method includes one or more holding steps lasting from about 1 minute to about 10 minutes. In some embodiments, the holding step is for about 1 minute. In some embodiments, the holding step is for about 3 minutes. In some embodiments, the holding step is for about 5 minutes. In some embodiments, the holding step is for about 10 minutes. The holding step can be performed at a variety of temperatures. For example, in some embodiments, the holding step is performed at a temperature of about -2.0°C. In some embodiments, the holding step is performed at a temperature of about -25°C. In some embodiments, the holding step is performed at a temperature of about -40°C. In some embodiments, the holding step is performed at a temperature of about -60°C. In some embodiments, the cryopreservation method disclosed herein includes one, two, three, four, five, or more holding steps. In some embodiments, the method includes one holding step. In some embodiments, the method includes two holding steps. In some embodiments, the method includes three holding steps. In some embodiments, the method includes four holding steps. In some embodiments, the method includes five holding steps. In some embodiments, the method includes more than five holding steps.

[0106] In some embodiments, the total time for the cryoprotection process is less than 120 minutes. In some embodiments, the total time for the cryoprotection process is less than 100 minutes. In some embodiments, the total time for the cryoprotection process is less than 90 minutes. In some embodiments, the total time for the cryoprotection process is about 60 minutes.

[0107] In some embodiments, the final temperature is about -80°C. In some embodiments, the final temperature is less than -80°C. In some embodiments, the final temperature is -90°C. In some embodiments, the final temperature is -96°C. In some embodiments, the final temperature is -120°C. In some embodiments, the final temperature is -196°C.

[0108] In some embodiments, the freezing methods disclosed herein include a series of steps that result in a controlled change in temperature. In some embodiments, the method may include a series of steps that include lowering the temperature. In some embodiments, the method includes a series of steps that include increasing the temperature.

[0109] In some embodiments, a method of changing the temperature of a sample containing cells from a first temperature to a final temperature of -80°C or less, thereby cryopreserving the sample at a final temperature, the method comprising the steps of: (a) placing the sample at a first temperature above a freezing temperature of the sample; (b) decreasing the first temperature to a second temperature at a first controlled rate, where the second temperature is at least 2°C lower than the first temperature; (c) decreasing the second temperature to a third temperature at a second controlled rate, where the third temperature is at least 40°C lower than the second temperature; (d) increasing the third temperature at a third controlled rate to a fourth temperature, where the fourth temperature is at least 20°C higher than the third temperature; (e) decreasing the fourth temperature at a fourth controlled rate, where the fifth temperature is at least 10°C lower than the fourth temperature; and (f) decreasing the fifth temperature at a fifth controlled rate to a final temperature, where the final temperature is -80°C or less.

[0110] In some embodiments, the first temperature is between about 4°C and 0°C.

[0111] In some embodiments, the first control rate is between about 0.75° C. and 1.25° C. per minute.

[0112] In some embodiments, the second temperature is about -2°C.

[0113] In some embodiments, the second control rate is between about 20° C. and 30° C. per minute.

[0114] In some embodiments, the third temperature is about -60°C.

[0115] In some embodiments, the third control rate is between about 5° C. and 15° C. per minute.

[0116] In some embodiments, the fourth temperature is about -25°C.

[0117] In some embodiments, the fourth control rate is between 0.5° C. and 1.25° C. per minute.

[0118] In some embodiments, the fifth temperature is about -40°C.

[0119] In some embodiments, the fifth control rate is between 7° C. and 15° C. per minute.

[0120] In some embodiments, the final temperature is -80°C or lower.

[0121] In some embodiments, a method is provided that includes cryopreserving modified immune cells suitable for cell therapy, the method comprising stepwise freezing a population of modified immune cells at a controlled rate to minimize latent heat of thawing, the stepwise freezing comprising cooling the cells at a rate of 0.5° C. per minute to 30° C. per minute to a final temperature of −80° C. or lower, thereby cryopreserving the cells.

[0122] In some embodiments, a method of thawing cryopreserved modified immune cells is provided, the method comprising heating a container containing the cryopreserved modified immune cells to a temperature between 37° C. and 70° C. and agitating the cells at a speed between about 100 and about 250 RPM for a suitable period of time until the cells are thawed.

[0123] In some embodiments, methods are provided that include cryopreserving modified immune cells suitable for cell therapy using a cryopreservation medium as described herein, the methods comprising stepwise freezing a population of modified immune cells at a controlled rate to minimize latent heat of thawing, the stepwise freezing comprising cooling the cells at a rate of between 0.5° C. per minute and 30° C. per minute to a final temperature of −80° C. or lower, thereby cryopreserving the cells.

[0124] In some embodiments, a method of thawing cryopreserved modified immune cells is provided, the method comprising heating a container containing the cryopreserved modified immune cells to a temperature between 37° C. and 70° C. and agitating the cells at a speed between about 100 and about 250 RPM for a suitable period of time until the cells are thawed.

[0125] In some embodiments, the cells are stirred at a speed of about 100 RPM to about 250 RPM. In some embodiments, the cells are stirred at a speed of about 100 RPM to about 150 RPM. In some embodiments, the cells are stirred at a speed of about 100 RPM to about 125 RPM. In some embodiments, the cells are stirred at a speed of about 100 RPM. In some embodiments, the cells are stirred at a speed of about 125 RPM. In some embodiments, the cells are stirred at a speed of about 150 RPM. In some embodiments, the cells are stirred at a speed of about 200 RPM. In some embodiments, the cells are stirred at a speed of about 250 RPM.

[0126] In some embodiments, the total thawing time is between about 5 minutes and 20 minutes. Thus, in some embodiments, the total thawing time is about 5 minutes. In some embodiments, the total thawing time is about 10 minutes. In some embodiments, the total thawing time is about 15 minutes. In some embodiments, the total thawing time is about 20 minutes.

[0127] In certain embodiments, the cryopreserved cells are CAR-NK cells, such as cord blood-derived NK cells modified to express CD-19 CAR, IL-15, and iCaspase9, which are cryopreserved using the methods described herein and then thawed in an orbital shaker water bath at 60° C. and about 125 rpm for about 10 minutes. Once thawed, the cells may be administered to a subject in need.

[0128] In some embodiments, the thawed cells are stable for about 1-6 hours. Thus, in some embodiments, the thawed cells are stable for about 2-4 hours. In some embodiments, the thawed cells are stable for about 1-2 hours. In some embodiments, the thawed cells are stable for about 1 hour. In some embodiments, the thawed cells are stable for about 2 hours. In some embodiments, the thawed cells are stable for about 3 hours. In some embodiments, the thawed cells are stable for about 4 hours. In some embodiments, the thawed cells are stable for about 5 hours. In some embodiments, the thawed cells are stable for more than 5 hours.

[0129] In some embodiments, the thawed cells can be administered to a subject in need thereof during the period during which the thawed cells are stable. For example, in some embodiments, the thawed cells are administered to a patient in need thereof within about 30 minutes to 5 hours after thawing. In some embodiments, the thawed cells are administered to a patient in need thereof within about 30 minutes to 2 hours after thawing. In some embodiments, the thawed cells are administered to a patient in need thereof immediately after thawing.

[0130] In some embodiments, the mammalian immune cells can be frozen for approximately 1-5 hours, 5-12 hours, 12-24 hours, 24-48 hours, 48 ​​hours to 1 week, 1 week to 2 weeks, 2-3 weeks, 3-4 weeks or more, and overlapping ranges thereof, hi some embodiments, the cells are cryopreserved for more than 1 month, more than 1 year, more than 5 years, more than 10 years, or more.

[0131] In some embodiments, the immune cells can be freshly isolated, hi some embodiments, the immune cells are frozen and thawed at least once.

[0132] In some embodiments, the cryopreserved cells are thawed for use (e.g., transplantation) by transferring the cryovial containing the cryopreserved cells to a water bath having a temperature near body temperature, e.g., a temperature of about 37° C., or any other suitable temperature. In other embodiments, a "step-up" thawing process with a step-up heating rate is used. For example, in some embodiments, the cryovial may be placed in a continuous storage environment with increasing temperature, e.g., a water bath having a temperature of 50° C. or higher, or any other suitable temperature described herein, prior to transfer to a temperature near body temperature.

[0133] In some embodiments, the cells are thawed at the patient's bedside, allowing the freshly thawed cells to be used immediately. In some embodiments, after thawing, the immune cells may be transferred to a culture dish and cultured under appropriate conditions for a period of about 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 86 hours, 110 hours, 1 week, 2 weeks, or 3 weeks or more before being transported to a desired location (e.g., a surgical room for delivery to a patient). Alternatively, in some embodiments, the substrate seeded with the thawed cells is directly injected into the patient.

[0134] How to unzip Current methods of thawing cryopreserved cells are performed on cell suspensions of mammalian immune cells. In some embodiments, the thawing process is rapid and scalable up to 5 vials. In some embodiments, the thawing process is rapid and scalable up to 10, 15, 20, 25, 30 or more vials. As previously mentioned, slow thawing tends to cause physical damage to the cells due to the formation of ice crystals. Current thawing methods include thawing the immune cells in an orbital shaker water bath or the like. In some embodiments, thawing can be accomplished by a dry thawing device capable of distributing heat evenly throughout the cryopreserved sample to thaw the sample. In some embodiments, thawing is accomplished by adjusting the temperature of the water bath to 40° C. In some embodiments, thawing is accomplished by adjusting the temperature of the water bath to 45° C. In some embodiments, thawing is accomplished by adjusting the temperature of the water bath to 50° C. In some embodiments, thawing is accomplished by adjusting the temperature of the water bath to 55° C. In some embodiments, thawing is accomplished by adjusting the temperature of the water bath to 60° C. In some embodiments, thawing is accomplished by adjusting the temperature of a water bath to 65°C.

[0135] In some embodiments, thawing is accomplished by adjusting the rotation speed of the orbital shaker water bath to between 100 rpm and 250 rpm. In some embodiments, thawing is accomplished by adjusting the rotation speed of the orbital shaker water bath to 120 rpm. In some embodiments, thawing is accomplished by adjusting the rotation speed of the orbital shaker water bath to 125 rpm. In some embodiments, thawing is accomplished by adjusting the rotation speed of the orbital shaker water bath to 130 rpm. In some embodiments, thawing is accomplished by adjusting the rotation speed of the orbital shaker water bath to 135 rpm. In some embodiments, thawing is accomplished by adjusting the rotation speed of the orbital shaker water bath to 140 rpm. In some embodiments, thawing is accomplished by adjusting the rotation speed of the orbital shaker water bath to 145 rpm. In some embodiments, thawing is accomplished by adjusting the rotation speed of the orbital shaker water bath to 150 rpm. In some embodiments, thawing is accomplished by adjusting the rotation speed of an orbital shaker water bath to greater than 150 rpm.

[0136] In some embodiments, the agitation rate does not shear the cells being thawed.

[0137] In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 37° C. and the rotation speed to 100 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 37° C. and the rotation speed to 125 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 37° C. and the rotation speed to 150 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 37° C. and the rotation speed to 175 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 37° C. and the rotation speed to 200 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 45° C. and the rotation speed to 100 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 45° C. and the rotation speed to 125 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 45° C. and the rotation speed to 150 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 45° C. and the rotation speed to 175 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 45° C. and the rotation speed to 200 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 50° C. and the rotation speed to 100 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 50° C. and the rotation speed to 125 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 50° C. and the rotation speed to 150 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the orbital shaker water bath to 50° C. and the rotation speed to 175 rpm. In some embodiments, thawing is accomplished by adjusting the temperature of an orbital shaker water bath to 50° C. and rotation speed to 200 rpm.In some embodiments, thawing is accomplished by adjusting the temperature of the orbital shaker water bath to 60° C. and the rotation speed to 100 rpm. In some embodiments, thawing is accomplished by adjusting the temperature of the orbital shaker water bath to 60° C. and the rotation speed to 125 rpm. In some embodiments, thawing is accomplished by adjusting the temperature of the orbital shaker water bath to 60° C. and the rotation speed to 150 rpm. In some embodiments, thawing is accomplished by adjusting the temperature of the orbital shaker water bath to 60° C. and the rotation speed to 175 rpm. In some embodiments, thawing is accomplished by adjusting the temperature of the orbital shaker water bath to 60° C. and the rotation speed to 200 rpm. An exemplary orbital shaker is shown in FIGS. 1A-1B.

[0138] In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 37° C. and the rotation speed to 100 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 37° C. and the rotation speed to 125 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 37° C. and the rotation speed to 150 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 37° C. and the rotation speed to 175 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 37° C. and the rotation speed to 200 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 45° C. and the rotation speed to 100 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 45° C. and the rotation speed to 125 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 45° C. and the rotation speed to 150 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 45° C. and the rotation speed to 175 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 45° C. and the rotation speed to 200 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 50° C. and the rotation speed to 100 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 50° C. and the rotation speed to 125 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 50° C. and the rotation speed to 150 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 50° C. and the rotation speed to 175 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 50° C. and the rotation speed to 200 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 60° C. and the rotation speed to 100 rpm.In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 60° C. and the rotation speed to 125 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 60° C. and the rotation speed to 150 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 60° C. and the rotation speed to 175 rpm. In some embodiments, thawing is achieved by adjusting the temperature of the dry heating device to 60° C. and the rotation speed to 200 rpm.

[0139] In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 5 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 6 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 7 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 8 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 9 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 10 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 11 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 12 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 13 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 14 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in an orbital shaker water bath for about 15 minutes.

[0140] In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 5 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 6 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 7 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 8 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 9 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 10 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 11 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 12 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 13 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 14 minutes. In some embodiments, thawing is accomplished by incubating the cryopreserved cell suspension in a dry heating apparatus for about 15 minutes.

[0141] For example, thawing cells, such as CAR-NK cells, in this manner allows the thawed cells to retain high viability (e.g., greater than 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%) and functionality similar to non-cryopreserved cells following CAR transduction.

[0142] Determining the effectiveness of freezing and thawing cells In some embodiments, the effectiveness of freeze-thaw is measured by measuring cell viability, for example by staining dead cells to distinguish them from live cells. In some embodiments, the effectiveness of freeze-thaw is measured by measuring metabolic activity, ATP content, or cell proliferation. Any suitable assay for measuring cell viability can be used to assess cell viability. In some embodiments, freeze-thaw is effective when cell viability is greater than 50%. In some embodiments, freeze-thaw is effective when cell viability is greater than 60%. In some embodiments, freeze-thaw is effective when cell viability is greater than 70%. In some embodiments, freeze-thaw is effective when cell viability is greater than 80%. In some embodiments, freeze-thaw is effective when cell viability is greater than 90%.

[0143] In some embodiments, the effectiveness of freeze-thaw is measured by measuring the kill rate of targets at a given effector-to-target (E:T) ratio. The E:T ratio is a given amount of T cells (effector cells) used relative to the amount of target cells. In some embodiments, freeze-thaw is effective when the kill rate of targets is at a particular E:T ratio compared to the E:T ratio of a freshly isolated cell sample.

[0144] How to use cryopreservation In some embodiments, cryopreservation procedures are used in connection with transporting therapeutic cells to their destination of use. In some embodiments, cryopreserved cells are transported in liquid nitrogen dried transport vehicles, for example at temperatures ranging from -140°C to -196°C, to avoid devitrification. Thus, in some embodiments, cryopreserved cells are transported in refrigerated transport containers. In some embodiments, vitrified cells are transported in sterile containers and / or in a sterile environment. In some embodiments, vitrified cells are transported on a large scale to a location, for example, a hospital or cell bank. In some embodiments, the cryopreservation techniques described herein are compatible with 96 hours of vapor phase storage under "transport conditions" and do not adversely affect viability after warming. In some embodiments, cryopreserved cells are thawed at the patient's bedside.

[0145] Cryopreservation medium Provided herein are various cryopreservation media suitable for cryopreserving cells. In some embodiments, the cryopreservation media provided herein are suitable for cryopreserving immune cells. Various immune cells are known, including, for example, NK cells, αβT cells, γδT cells, and T cells, including regulatory T cells, B cells, HSCs, and MSCs. In one embodiment, the immune cells are umbilical cord blood-derived NK cells, iPS cell-derived NK cells, and iPS cell-derived T cells. In one embodiment, the cells are NK cells, particularly allogeneic NK cells expressing CAR (i.e., CAR-NK cells).

[0146] In some aspects, a cryopreservation medium suitable for cryopreservation and subsequent thawing of viable cells comprises a cryoprotectant, albumin, and a non-pyrogenic and isotonic crystalloid solution. In some aspects, a cryopreservation medium suitable for cryopreservation and subsequent thawing of viable cells comprises a cryoprotectant, a disaccharide, albumin, and a non-pyrogenic and isotonic crystalloid solution.

[0147] A variety of cryoprotectants are known in the art, including, for example, dimethyl sulfoxide (DMSO), glycerol, and propanediol, among others. In some embodiments, the cryopreservation medium comprises DMSO as a cryoprotectant. In some embodiments, human serum albumin (HSA) is the albumin in the cryopreservation medium.

[0148] In some embodiments, a suitable cryopreservation medium also comprises a sugar or sugar. In another aspect, a suitable cryopreservation medium described herein comprises HSA, sodium, sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride, magnesium chloride, dimethylsulfoxide (DMSO), and a disaccharide.

[0149] In some embodiments, the saccharide includes a monosaccharide, a disaccharide, a trisaccharide, or a polysaccharide. In some embodiments, the saccharide is a disaccharide. In some embodiments, the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, or chitobiose. In some embodiments, the disaccharide is trehalose.

[0150] In some embodiments, a suitable cryopreservation medium comprises one or more of glucose, xylose, arabinose, fructose, galactose, mannose, mannitol, sorbitol, xylitol, myo-inositol, trehalose, sucrose, lactose, maltose, cellobiose, lactitol, maltitol, methylcellulose, carboxymethylcellulose, dextran, glycogen, amylose, amylopectin, inulin, sodium alginate, ethylcellulose, hydroxyethylcellulose, raffinose, stachyose, xanthan gum, glucosamine, and galactosamine. In some embodiments, a suitable cryopreservation medium comprises trehalose, sucrose, mannitol, and / or dextran. In some embodiments, a suitable cryopreservation medium comprises one or more sugars selected from trehalose, sucrose, and / or mannitol.

[0151] Various concentrations of sugars or sugars can be used in the cryopreservation medium. In some embodiments, the cryopreservation medium comprises between about 0 mM and 500 mM trehalose, sucrose, or mannitol. In some embodiments, the cryopreservation medium comprises between about 0 mM and 200 mM trehalose, sucrose, or mannitol. In some embodiments, the cryopreservation medium comprises between about 0 mM and 100 mM trehalose, sucrose, or mannitol.

[0152] In some embodiments, the cryopreservation medium comprises one or more sugars selected from trehalose, sucrose and / or mannitol at a concentration between about 0-100 mM. In some embodiments, the cryopreservation medium comprises between about 0-100 mM mannitol.

[0153] In some embodiments, the cryopreservation medium comprises between about 10 mM and 100 mM trehalose, hi some embodiments, the cryopreservation medium comprises 30 mM trehalose.

[0154] Thus, in some embodiments, trehalose, sucrose, or mannitol is at about 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, 15 ... 100 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or 200 mM final concentrations in the cryopreservation medium.

[0155] Thus, in some embodiments, trehalose, sucrose, or mannitol is present in the cryopreservation medium at a final concentration of less than 1 mM, less than 10 mM, less than 20 mM, less than 30 mM, less than 40 mM, less than 50 mM, less than 60 mM, less than 70 mM, less than 80 mM, less than 90 mM, less than 100 mM, less than 110 mM, less than 120 mM, less than 130 mM, less than 140 mM, less than 150 mM, less than 160 mM, less than 170 mM, less than 180 mM, less than 190 mM, or less than 200 mM.

[0156] In some embodiments, the cryopreservation medium comprises dextran. In some embodiments, the cryopreservation medium comprises between about 0-20 w / v% dextran. In some embodiments, the cryopreservation medium comprises between about 0-6 w / v% dextran. Thus, in some embodiments, the dextran is at about 0.2 w / v%, 0.4 w / v%, 0.6 w / v%, 0.8 w / v%, 1.0 w / v%, 1.5 w / v%, 2.0 w / v%, 2.5 w / v%, 3.0 w / v%, 3.5 w / v%, 4.0 w / v%, 4.5 w / v%, 5.0 w / v%, 5.5 w / v%, 6.0 w / v%, 6.5 w / v%, 7.0 w / v%, 7.5 w / v%, 8.0 w / v%, 8.5 w / v%, 9.0 w / v%, 9.5 w / v%, 10.

[0033] The cryopreservation medium may be present at a final concentration of 0 w / v%, 10.5 w / v%, 11.0 w / v%, 11.5 w / v%, 12.0 w / v%, 12.5 w / v%, 13.0 w / v%, 13.5 w / v%, 14.0 w / v%, 14.5 w / v%, 15.0 w / v%, 15.5 w / v%, 16.0 w / v%, 16.5 w / v%, 17.0 w / v%, 17.5 w / v%, 18.0 w / v%, 18.5 w / v%, 19.0 w / v%, 19.5 w / v%, or 20.0 w / v%.

[0157] In some embodiments, the cryopreservation medium comprises a non-pyrogenic and isotonic crystalloid liquid. A variety of non-pyrogenic and isotonic crystalloid liquids can be used in the cryopreservation medium described herein. For example, exemplary isotonic crystalloid liquids can be used in the cryopreservation medium described herein, including PLASMA-LYTE A, normal saline, lactate buffer, acetate buffer, acetate and lactate buffer, and dextrose in water solution. In general, non-pyrogenic and isotonic crystalloid liquids comprise one or more of the following: sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride, and magnesium chloride. Thus, in some embodiments, the cryopreservation medium comprises one or more of the following: sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride, and magnesium chloride. In some embodiments, the following are sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride, and magnesium chloride. An example of a commercially available non-pyrogenic and isotonic crystalloid liquid is PLASMA-LYTE A. In some embodiments, the non-pyrogenic and isotonic crystalloid fluid is PLASMA-LYTE A. In some embodiments, the non-pyrogenic and isotonic crystalloid fluid is 0.9% normal saline. In some embodiments, the non-pyrogenic and isotonic crystalloid fluid is a lactate buffer. In some embodiments, the non-pyrogenic and isotonic crystalloid fluid is a dextrose in water solution.

[0158] PLASMA-LYTE A comprises sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride, and magnesium chloride. In some embodiments, the cryopreservation medium comprises about 10% v / v to 75% v / v of Plasma-LYTE A. In some embodiments, the cryopreservation medium comprises about 25% v / v to 50% v / v of PLASMA-LYTE A. In some embodiments, the cryopreservation medium comprises about 40% v / v of PLASMA-LYTE A. Thus, in some embodiments, the cryopreservation medium comprises about 10% v / v, 15% v / v, 20% v / v, 25% v / v, 30% v / v, 35% v / v, 40% v / v, 45% v / v, 50% v / v, 55% v / v, 60% v / v, 65% v / v, 70% v / v, or 75% v / v of PLASMA-LYTE A.

[0159] In some embodiments, the cryopreservation medium comprises about 0.1 mg / mL to about 1 mg / mL of sodium chloride. In some embodiments, the cryopreservation medium comprises about 0.4 mg / mL to about 0.6 mg / mL of sodium chloride. Thus, in some embodiments, the cryopreservation medium comprises about 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, or 1 mg / mL of sodium chloride.

[0160] In some embodiments, the cryopreservation medium comprises about 0.1 mg / mL to about 1 mg / mL of sodium gluconate. In some embodiments, the cryopreservation medium comprises about 0.3 mg / mL to about 0.6 mg / mL of sodium gluconate. Thus, in some embodiments, the cryopreservation medium comprises about 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, or 1 mg / mL of sodium gluconate.

[0161] In some embodiments, the cryopreservation medium comprises about 25% v / v to 75% v / v CS10. In some embodiments, the cryopreservation medium comprises about 40% v / v to 60% v / v CS10. In some embodiments, the cryopreservation medium comprises about 50% v / v CS10. Thus, in some embodiments, CS10 is present in the cryopreservation medium at about 25% v / v, 30% v / v, 35% v / v, 40% v / v, 45% v / v, 50% v / v, 55% v / v, 60% v / v, 65% v / v, 70% v / v, or 75% v / v. In some embodiments, CS10 comprises dimethyl sulfoxide (DMSO).

[0162] In some embodiments, the cryopreservation medium comprises human serum albumin (HSA). In some embodiments, the cryopreservation medium comprises about 0.5% to 25% v / v% HSA. In some embodiments, the cryopreservation medium comprises about 5% to 20% v / v% HSA. In some embodiments, the cryopreservation medium comprises about 10% v / v HSA. In some embodiments, the cryopreservation medium comprises about 1.25% v / v to 5% v / v HSA. In some embodiments, the cryopreservation medium comprises about 2.5% v / v HSA.

[0163] Thus, in some embodiments, the cryopreservation medium comprises about 0.5 v / v%, 1.0 v / v%, 1.5 v / v%, 2.0 v / v%, 2.5 v / v%, 3.0 v / v%, 3.5 v / v%, 4.0 v / v%, 4.5 v / v%, 5.0 v / v%, 6.0 v / v%, 6.5 v / v%, 7.0 v / v%, 7.5 v / v%, 8.0 v / v%, 8.5 v / v%, 9.0 v / v%, 10.0 v / v%, 10.5 v / v%, 11.0 v / v%, 11.5 v / v%, 12.0 v / v%, 12.5 v / v%, 13.0 v / v%, 13.5v / v%, 14.0v / v%, 14.5v / v%, 15.0v / v%, 15.5v / v%, 16.0v / v%, 16.5v / v%, 17.0v / v%, 17.5v / v%, 18.0v / v%, 18.5v / v%, 19.0v / v%, 19.5v Contains HSA of / v%, 20.0v / v%, 20.5v / v%, 21.0v / v%, 21.5v / v%, 22.0v / v%, 22.5v / v%, 23.0v / v%, 23.5v / v%, 24.0v / v%, 24.5v / v%, or 25.0v / v%.

[0164] In some embodiments, the cryopreservation medium comprises HSA, Ca 2+ , Na + , K + , Mg 2+ In another embodiment, the cryopreservation medium comprises one or more of HSA, Na, HEPES, one or more disaccharides, sugar alcohols, dextran, metabolites, and antioxidants. + , K + , Mg 2+ , HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at a concentration of about 0-55 mM, one or more sugars selected from trehalose, sucrose and / or mannitol at a concentration of about 0-100 mM, about 0-6% dextran, adenosine, and glutathione. In some embodiments, the metabolite is adenosine. In some embodiments, the antioxidant is glutathione.

[0165] Thus, in some embodiments, HEPES is present in the cryopreservation medium at about 0 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or 55 mM.

[0166] In some embodiments, the cryopreservation medium comprises human serum albumin (HSA), PLASMA-LYTE A, and CS10.

[0167] In some aspects, the cryopreservation medium is suitable for cryopreserving natural killer (NK) cells. In some embodiments, the NK cells are derived from a primary cell isolate. In some embodiments, the NK cells are cord blood derived NK cells. In some embodiments, the NK cells are derived from a cell line. In some embodiments, the NK cells are fresh cells. In some embodiments, the NK cells have been previously frozen and thawed, e.g., the NK cells are cord blood derived NK cells that have been previously frozen and thawed. In some embodiments, the NK cells comprise a chimeric antigen receptor (CAR), e.g., a CD19 CAR. In some embodiments, the cryopreservation medium comprises NK cells at a concentration of 6 M / mL to 120 M / mL.

[0168] In some embodiments, the CAR-NK cell therapy product is cryopreserved using the formulations described herein. In some embodiments, the CAR-NK cell therapy product is an allogeneic cell therapy product composed of human umbilical cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, CD-19 CAR, and IL-15.

[0169] In some embodiments, a CAR-NK cell therapy product comprises an anti-CD19 binding domain comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and / or a heavy chain variable region set forth in SEQ ID NO: 2. In another embodiment, a CD19 CAR may comprise an anti-CD19 binding domain, a CD28 transmembrane domain (an exemplary CD28 transmembrane sequence is set forth in SEQ ID NO: 3), a CD3z signaling domain (an exemplary CD3z sequence is set forth in SEQ ID NO: 4), and may further comprise a suicide switch such as iCaspase9 and / or IL-15.

[0170] In one embodiment, the CAR-NK cell therapy product includes a nucleic acid molecule encoding a heavy chain variable region of an anti-CD19 binding domain and / or a nucleic acid molecule encoding a light chain variable region of an anti-CD19 binding domain.

[0171] Thus, in some embodiments, the cryopreservation medium comprises NK cells at a concentration of about 1 M / mL, 5 M / mL, 10 M / mL, 15 M / mL, 20 M / mL, 25 M / mL, 30 M / mL, 35 M / mL, 40 M / mL, 45 M / mL, 50 M / mL, 55 M / mL, 60 M / mL, 65 M / mL, 70 M / mL, 75 M / mL, 80 M / mL, 85 M / mL, 90 M / mL, 95 M / mL, 100 M / mL, 105 M / mL, 110 M / mL, 115 M / mL, 120 M / mL, 130 M / mL, 140 M / mL, 150 M / mL, 160 M / mL, 170 M / mL, 180 M / mL, 190 M / mL, 200 M / mL.

[0172] In some embodiments, the genetically modified cord blood NK cells are present at a concentration of 6M / mL to 120M / mL. In some embodiments, the genetically modified cord blood NK cells are present at a concentration of 6M / mL to 200M / mL. In some embodiments, the genetically modified cord blood NK cells are present at a concentration of 6M / mL to 25M / mL. In some embodiments, the genetically modified cord blood NK cells are present at a concentration of 6M / mL to 120M / mL in a medium volume ranging from 30 to 45 ml. In some embodiments, the genetically modified cord blood NK cells are present at a concentration of 6M / mL to 200M / mL in a medium volume ranging from 30 to 45 ml. In some embodiments, the genetically modified cord blood NK cells are present at a concentration of 6M / mL to 25M / mL in a medium volume ranging from 30 to 45 ml.

[0173] In some embodiments, the CAR-NK cells are formulated in the cryopreservation medium provided herein at a concentration ranging from 100 million cells to 900 million cells in a volume of medium ranging from 30 to 45 mL. In a particular embodiment, the CAR-NK cells are present at a concentration of about 200 million cells in a volume of about 36 mL of medium. In another embodiment, the CAR-NK cells are present at a concentration of about 800 million cells in about 36 mL of medium. In some embodiments, the cells in 36 mL of medium are contained in a sterile container (e.g., an AT vial).

[0174] In some embodiments, the NK cells are about 100×10 formulated in the cryopreservation medium described herein. 6 Cells ~ approx. 900×10 6 In some embodiments, the CAR-NK cell therapy product comprises a population of 200×10 cells formulated in a cryopreservation medium described herein. 6 Cells ~ approx. 800×10 6The present invention includes a CAR-NK cell therapy product comprising a cell population ranging from 100 to 2000 cells / mL. In some embodiments, the CAR-NK cell therapy product is an allogeneic cell therapy product comprised of human umbilical cord blood derived NK cells transduced with a retroviral vector expressing iCaspase9, CD19 CAR, and IL-15. In certain embodiments, the CAR-NK cell therapy product is an allogeneic cell therapy product comprised of human umbilical cord blood derived NK cells transduced with a retroviral vector expressing iCaspase9, CD19 CAR, and IL-15, the transduced cells being formulated at a concentration of 6M-120M cells / mL in 36 ml of cryopreservation medium comprising DMSO, trehalose, HSA, and PLASMA-LYTE A. In another embodiment, the CAR-NK cell therapy product is an allogeneic cell therapy product composed of human umbilical cord blood-derived NK cells transduced with a retroviral vector expressing iCaspase9, CD19 CAR, and IL-15, the transduced cells being stored at 800×10 in 36 ml of cryopreservation medium containing DMSO, trehalose, HSA, and PLASMA-LYTE A. 6 The transduced cells are formulated at a concentration of 100 mM KCl, 1 mM Mg ...

[0175] Use of cryopreserved cells The methods and compositions described herein, including CAR-NK cell compositions, are suitable for adoptive cell therapy. Adoptive cell therapy can be used to treat a variety of diseases, including, for example, cancer. In certain embodiments, the frozen and then thawed CAR-NK cell compositions contained within the cryopreservation medium described herein are useful for treating cancer or tumors. In certain embodiments, the cancer includes tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, and liver. In some embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is a B-cell malignancy (e.g., diffuse large B-cell lymphoma).

[0176] In some embodiments, the media described herein are useful for adoptive cell therapy. Thus, in some embodiments, the cells described herein are cryopreserved and thawed according to the instructions provided herein. In some embodiments, the cells are NK cells as described herein. In some embodiments, the NK cells are suspended in a cryopreservation medium as described herein.

[0177] In some embodiments, a CAR-NK cell composition suspended in a cryopreservation medium described herein is used to treat a subject with cancer. In some embodiments, a composition comprising CAR-NK cells in a cryopreservation medium described herein is administered to a subject. In some embodiments, the CAR-NK cells comprise an anti-CD19 CAR gene and an IL-15 gene. In some embodiments, the CAR-NK cells comprise an anti-CD19 CAR gene, an IL-15 gene, and iCaspase9. In some embodiments, the CAR-NK cells are not washed prior to administration to a subject in need thereof. In some embodiments, the CAR-NK cells are washed out of the cryopreservation medium prior to administration to a subject in need thereof.

[0178] In some embodiments, the adoptive cell therapy is combined with one or more additional cancer treatments, such as, for example, lymphocyte-depleting chemotherapy. Thus, in some embodiments, prior to administration of a CAR-NK cell therapy product formulated in a cryopreservation medium as described herein, a subject with cancer undergoes lymphocyte-depleting chemotherapy.

[0179] In some embodiments, the CAR-NK cell therapy product is cryopreserved as described herein and then thawed prior to administration to a patient in need thereof. For example, the CAR-NK cell therapy product described herein is cryopreserved, shipped, thawed, and administered to a patient in need thereof as described herein. Thus, in some embodiments, the CAR-NK cell therapy product is cryopreserved in a formulation described herein and then thawed prior to administration to a patient to treat a B-cell malignancy.

[0180] In some embodiments, the frozen CAR-NK cell therapy product is frozen as described herein and shipped to a patient in need thereof. For example, in some embodiments, a method of shipping a cell therapy product includes (a) contacting the CAR-NK cells with a cryopreservation medium as described herein, (b) cooling the CAR-NK cells to a temperature of -80°C, thereby cryopreserving the mammalian cells, and (c) shipping the cryopreserved mammalian cells to a different location at a temperature of about -20°C to about -190°C or below. In some embodiments, the cells are stored and shipped at about -190°C or below, and in certain embodiments, at or below -140°C. In some embodiments, the shipped cells may be stored in a cryoshipper until administration to a patient.

[0181] In certain embodiments, cells to be used for adoptive cell therapy are cryopreserved in vials (e.g., 50 ml AT vials) at a concentration of 200-800 million cells in a volume of 36 ml of cryopreservation medium. In various embodiments, the vials may be labeled after the cells are frozen and transported labeled in cryoshippers to the treatment site. In some embodiments, the vials may be labeled before the cells are frozen.

[0182] In some embodiments, the cell therapy product is CD19-CAR NK cells, further comprising IL-15 and iCaspase9. In some embodiments, the cell therapy product is cryopreserved in a container at a concentration between about 6 million and 120 million cells per milliliter. In some embodiments, the cell therapy product is cryopreserved in a 50 mL container at a concentration between about 6 million and 120 million cells per milliliter. In some embodiments, the cell therapy product is cryopreserved in a 50 mL container at a concentration between about 3 million and 150 million cells per milliliter. In some embodiments, the cell therapy product is cryopreserved in a 50 mL container at a concentration between about 1 million and 250 million cells per milliliter. In some embodiments, the cell therapy product is cryopreserved in a 50 mL container at a concentration between about 1 million and 350 million cells per milliliter. In some embodiments, the cell therapy product is cryopreserved in a 50 mL container at a concentration between about 1 million and 500 million cells per milliliter.

[0183] In some embodiments, the cell therapy product comprises about 20×10 6 ~100×10 7 In some embodiments, the cell therapy product contains about 100×10 cells in a 50 mL container. 6 ~900×10 6 In some embodiments, the cell therapy product comprises about 50×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 100×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 200×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 100×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 200×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 300×10 cells in a 50 mL container. 6In some embodiments, the cell therapy product contains about 400×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product comprises about 500×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 600×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 700×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 800×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 900×10 cells in a 50 mL container. 6 In some embodiments, the cell therapy product contains about 100×10 cells in a 50 mL container. 7 Contains cells.

[0184] In some embodiments, the cell therapy product is contained in a 50 mL container with a fill volume of between about 20-45 mL. In some embodiments, the cell therapy product is contained in a 50 mL container with a fill volume of about 36 mL. In some embodiments, the cell therapy product is an immune cell, such as a NK cell, a T cell, or a B cell. In some embodiments, the immune cell is engineered to include one or more transgenes (e.g., a chimeric antigen receptor (CAR)). In some embodiments, the cell is a CAR-NK+ cell. In some embodiments, the cell therapy product includes a CD19-CAR, an IL-15 transgene, and iCaspas9. In some embodiments, the cell therapy product is contained in a 50 mL container with a fill volume of about 100×10 6 ~900×10 6 In some embodiments, the amount of cell therapy product present is 100×10 in a 50 ml container. 6 In some embodiments, the amount of cell therapy product present is 200×10 CAR-NK+ cells in a 50 ml container. 6 In some embodiments, the amount of cell therapy product present is 300×10 CAR-NK+ cells in a 50 ml container. 6In some embodiments, the amount of cell therapy product present is 400×10 CAR-NK+ cells in a 50 ml container. 6 In some embodiments, the amount of cell therapy product present is 500×10 CAR-NK+ cells in a 50 ml container. 6 In some embodiments, the amount of cell therapy product present is 600×10 CAR-NK+ cells in a 50 ml container. 6 In some embodiments, the amount of cell therapy product present is 700×10 CAR-NK+ cells in a 50 ml container. 6 In some embodiments, the amount of cell therapy product present is 800×10 CAR-NK+ cells in a 50 ml container. 6 In some embodiments, the amount of cell therapy product present is 900×10 CAR-NK+ cells in a 50 ml container. 6 In some embodiments, the amount of cell therapy product present is 100×10 CAR-NK+ cells in a 50 ml container. 7 CAR-NK+ cells.

[0185] The delivered cell therapy product can be thawed as described herein and then administered to a patient in need thereof. In some embodiments, the cell therapy product is thawed at the patient's bedside. In some embodiments, the cell therapy product is not washed prior to administration to a patient in need thereof. In some embodiments, the thawed cells are administered to a patient in need thereof within about 30 minutes to 2 hours of thawing the cells. In some embodiments, the infusion rate into the subject is about 2 to 3 minutes.

[0186] In some embodiments, the delivered cell therapy product remains frozen for further storage at a different location. In some embodiments, the thawed cells are introduced to a subject in need thereof without separating the cells and the cryopreservation solution. Thus, in some embodiments, the thawed cells are not washed prior to use. The thawed cells and associated cryopreservation solution are preferably warmed to body temperature (i.e., about 37° C.) prior to introduction into the subject. In such circumstances, the dose of cells is based on the number of cells prior to freezing.

[0187] In some embodiments, the thawed cells are further cultured. In some embodiments, culturing includes placing the cells in an incubator, removing the buffer, and replacing the buffer with a culture medium designed for cell growth and / or differentiation. In some embodiments, the cells are incubated in the incubator for about 6-7 hours. In some embodiments, the culture medium designed for cell growth and / or differentiation includes Kubota medium and / or hormone defined medium (HDM) for cell differentiation.

[0188] The viability of thawed cells can be evaluated in vitro and in vivo using various methods known in the art. In some embodiments, in vitro cell viability tests include trypan blue exclusion assay. In some embodiments, other analytical methods, such as gene expression using RT-qPCR, can be used to evaluate the cell viability of thawed cells that have been frozen in different cryopreservation media. A person skilled in the art can select any analytical method that can be applied to evaluate the cell viability of otherwise fresh cells to evaluate the viability of thawed cells.

[0189] In vivo cell viability can generally be evaluated by assessing the in vivo functional characteristics of the administered cells. In some embodiments, in vivo cell viability can be evaluated by assessing the cell number of the cells introduced into a subject in need. Various methods are known in the art for tracking cells and determining the viability of administered cells.

[0190] Cells cryopreserved and thawed using the cryopreservation media described herein allow the cells to be used for any application that primary or freshly isolated cells may have. Cryopreserved and thawed cells retain high viability (e.g., greater than 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95%) and retain physiological properties of their native state, allowing them to be used for a variety of applications, such as genetic manipulation of cells, and for cell therapy purposes, e.g., adoptive cell therapy applications. The sequences disclosed herein: Anti-CD19 light chain variable fragment, VL: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLELKR (SEQ ID NO: 1) Anti-CD19 heavy chain variable fragment, VH: EVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSSQDPA (SEQ ID NO: 2) CD28: FWVLVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 3) CD3ζ: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGP (SEQ ID NO: 4) EXAMPLES

[0191] Other features, objects, and advantages of the present invention will become apparent in the following examples. However, it should be understood that the examples, while showing embodiments of the present invention, are given by way of illustration only, not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the examples.

[0192] The NK cells used in these examples contain an anti-CD19 CAR, IL-15, and iCaspase 9. However, it should be noted that NK cells containing other transgenes can also be used in these methods.

[0193] The CAR-NK cells used in these examples were NK cells derived from umbilical blood units transduced with a gene encoding a tumor targeting CD19-CAR (iC9 / CAR.19 / IL15, Leukemia 32 (2018) 520-531, incorporated herein by reference in its entirety), e.g., NK-CAR cells transduced with a nucleic acid molecule encoding an anti-CD19 binding domain light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1, and / or an anti-CD19 binding domain heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:2.

[0194] Example 1: Scale-up of cryopreservation of mammalian cells in 50 ml vessels. This example demonstrates the freezing characteristics of mammalian cells using a 120 minute freezing procedure.

[0195] First, 45 ml of cryopreservation medium without cells (control) was filled into a 50 ml AT vial containing 50% RPMI, 5% DMSO, 20% 25% human serum albumin, 10% dextran in NaCl. The temperature of the vial was lowered to 4°C in a CryoMed5474 controlled freezer. The amount of heat released from the contents of the AT vial was measured by measuring the solution temperature. To start the process of cryopreservation, the temperature was lowered to -4°C at a rate of 1°C per minute. Then, the temperature was rapidly lowered to -45°C at a rate of 20°C per minute. Next, the temperature was rapidly increased to -10°C at a rate of 10°C per minute. Then, the temperature was rapidly lowered to -20°C at a rate of 0.5°C per minute. Finally, the temperature was slowly lowered to -80°C by lowering the temperature at a rate of 1°C per minute.

[0196] The heat released by the solution contents was observed to remain at -1°C for more than 35 minutes following the freezing procedure, suggesting that ice formation is slow with ample opportunity for ice melting. Furthermore, the temperature of the vial contents differs from the surrounding temperature, increasing the likelihood of non-uniform ice formation. Figure 2 shows the freezing characteristics of cryopreservation media frozen using the above freezing procedure.

[0197] Example 2: Optimized cryopreservation of mammalian cells in 50 ml vessels. This example demonstrates a method for cryopreserving mammalian cells in 50 ml containers, such as 50 ml AT vials, such that the effect of the latent heat of fusion is minimized.

[0198] First, 36 ml of CAR-NK cells were formulated at a concentration of 110 million cells per mL in a medium containing 40% PLASMA-LYTE A + 10% HSA + 50% CS10 and then filled into a 50 ml AT vial. The temperature of the vial was reduced to 4°C in a rate-controlled freezer. A CryoMed 5474 controlled rate freezer was used for these experiments. To initiate the process of cryopreservation, the temperature was reduced to -2°C at a rate of 1°C per minute. The internal temperature of the vial and contents was allowed to equilibrate at -2°C for 3 minutes. The ice nucleation process was induced by rapidly reducing the temperature to -60°C at a rate of 25°C per minute. The interior of the vial and the contents of the vial were allowed to equilibrate at -60°C for 1 minute to absorb the latent heat of melting due to the initiation of ice formation, and the temperature of the cell suspension was maintained below freezing to avoid re-melting of the ice. To avoid promoting extracellular ice formation, which would lead to undesirable intracellular ice formation, the temperature was increased to −30 °C at a rate of 10 °C per minute, allowing a continuous, smooth temperature drop, allowing the intracellular water to continue to move osmotically while gradually forming extracellular ice. The temperature was maintained at −30 °C to allow the cells to freeze uniformly. Finally, the temperature was decreased to −40 °C at a rate of 1 °C per minute. The cell contents were equilibrated at −40 °C for 5 min to maximize extracellular ice formation. The temperature was decreased to a final temperature of −80 °C at a rate of 10 °C per minute to allow the remaining extracellular ice formation. The amount of heat released from the sample, the chamber temperature, and the system temperature were plotted as a function of time. Figure 3 shows the freezing characteristics of CAR-NK cells.

[0199] It was observed that the amount of heat released by the cell contents remained nearly constant throughout the freezing protocol, suggesting a significant reduction in the latent heat of thawing. Moreover, the thawing procedure took less than an hour.

[0200] Example 3: Cryopreservation of mammalian cells in multiple 50 ml vessels. This example shows a method for cryopreserving mammalian cells (e.g., cells suitable for cell therapy) in five 50 ml containers, e.g., 50 ml AT vials, positioned at the four corners and in the center such that the effect of the latent heat of fusion is minimized.

[0201] First, five vessels were prepared with 45 ml of iCAR-T cells in the appropriate cryopreservation suspension medium, containing cells at a concentration of 80 million cells / ml in 50 ml AT vials. The temperature of the vials was lowered to 4°C in a CryoMed5474 controlled freezer. To start the cryopreservation process, the temperature was lowered to -2°C at a rate of 1°C per minute. The temperature of the vial interior and contents was equilibrated at -2°C for 3 minutes. The ice nucleation process was initiated by rapidly lowering the temperature to -60°C at a rate of 25°C per minute. The vial interior and contents were equilibrated at -60°C for 1 minute to absorb the latent heat of melting due to the initiation of ice formation, and the temperature of the cell suspension was maintained below freezing to avoid remelting of the ice. To avoid promoting extracellular ice formation that would cause undesirable intracellular ice formation, the temperature was increased to -30°C at a rate of 10°C per minute to allow a continuous and smooth temperature drop, allowing the intracellular water to continue to move osmotically while allowing the formation of extracellular ice gradually. The temperature was maintained at -30°C to allow the cells to freeze uniformly. Finally, the temperature was ramped down to -40°C at a rate of 1°C per minute. The cell contents were equilibrated at -40°C for 5 minutes to maximize extracellular ice formation. The temperature was ramped down to a final temperature of -80°C at a rate of 10°C per minute to allow for residual extracellular ice formation. The amount of heat released from the samples, the chamber temperature, and the system temperature were plotted as a function of time. Figure 4 shows the freezing characteristics of five 45 ml suspensions of CAR-NK cells.

[0202] It was observed that the amount of heat released from the contents of all five 45 ml cell suspensions remained nearly constant throughout the freezing protocol, suggesting a significant reduction in the influence of the latent heat of melting caused by extracellular ice formation. Furthermore, the use of multiple chambers suggests that the freezing protocol can be scaled up for large volumes of mammalian cell suspensions.

[0203] Example 4: Thawing of cryopreserved mammalian cells. This example demonstrates a method for thawing mammalian cells stored at liquid nitrogen temperatures (-196°C) to reduce lethal ice recrystallization. The viability of thawed cells was measured.

[0204] iCART cells harvested in three different volumes, 16, 30, and 45 mL, in 50 ml AT vials with cell densities of 80 million cells / ml and 120 cells / ml were first cryopreserved using a method to reduce the latent heat of fusion, filling AT vials at 50 mL, freezing, and storing at -196°C. Cells stored at -196°C were thawed by placing the AT vials in an orbital shaker water bath (Benchmark SBL-12) set at 60°C and thawing for up to 600 seconds with a rotation speed of 120 rpm. The temperature of the cell samples was measured. Figures 5A and 5B show the increase in temperature of cell samples over time during thawing of 50 ml AT vials containing 16, 30, and 45 ml of cells.

[0205] The viability of the thawed cells was evaluated. Figure 6 shows the viability of 80 million cells per ml and 120 million cells per ml thawed as described. It was observed that all cells had a viability of over 95%, indicating that the method was successful in preventing the induction of lethal ice recrystallization.

[0206] Example 5: In vitro efficacy of freezing and thawing CAR-NK cells. This example demonstrates the effectiveness of freezing and thawing mammalian CAR-NK cells in 50 mL AT vials compared to 2 mL AT vials or 2 ml cryovials when the mammalian cells are frozen in a manner that minimizes the latent heat of fusion.

[0207] First, 45mL, 30mL, and 10mL of CAR-NK cells were placed in 50mL AT vials at concentrations of 80 and 120 million cells per mL. Next, 1mL of CAR-NK cells was placed in 2mL AT vials at concentrations of 80 and 120 million cells per mL, or in 2mL cryovials at a concentration of 10 million cells per mL as a control. All cells were suspended in cryopreservation suspension medium containing 40% PLASMA-LYTE A, 50% CS10, and 10% human serum albumin.

[0208] CAR-NK cells filled in 50 mL and 2 mL AT vials were frozen as follows: the temperature of the vials was lowered to 4°C in a CryoMed5474 controlled freezer. To start the cryopreservation process, the temperature was lowered to -2°C at a rate of 1°C per minute. The temperature of the vial interior and contents was equilibrated at -2°C for 3 minutes. The ice nucleation process was initiated by rapidly lowering the temperature to -60°C at a rate of 25°C per minute. The vial interior and contents were equilibrated at -60°C for 1 minute to absorb the latent heat of melting due to the initiation of ice formation, and the temperature of the cell suspension was maintained below freezing to avoid remelting of the ice. To avoid promoting extracellular ice formation that would cause undesirable intracellular ice formation, the temperature was increased to -30°C at a rate of 10°C per minute to allow a continuous and smooth temperature drop, allowing the gradual formation of extracellular ice while the intracellular water continues to move osmotically. The temperature was maintained at -30°C to allow the cells to freeze uniformly. Finally, the temperature was decreased to -40 °C at a rate of 1 °C per minute. The cell contents were equilibrated at -40 °C for 5 min to maximize extracellular ice formation. The temperature was decreased to a final temperature of -80 °C at a rate of 10 °C per minute to allow for residual extracellular ice formation. CAR-NK cells in 2 mL cryovials were divided into two groups, one group was frozen using the Benchmark program as a freezing control, and the other group was cultured in medium as a fresh control.

[0209] Frozen cells were stored in the vapor phase of a liquid nitrogen tank (≦-140°C) until thawed. 50 mL and 2 mL AT vials were thawed by placing the AT vials in an orbital shaker water bath (Benchmark SBL-12) set at 60°C and a rotation speed of 150 rpm for up to 600 seconds. Cells frozen in 2 ml cryovials were thawed in an orbital shaker water bath (Benchmark SBL-12) set at 37°C and a rotation speed of 150 rpm for up to 600 seconds.

[0210] The viability of the cells was measured. The functionality of the cells was tested by measuring the killing rate of CAR-NK cells at various E:T ratios. An E:T ratio of 10:1 appeared to be the optimal ratio for comparing the killing function. Table 1 shows the viability and functionality assays of CAR-NK cells. It was observed that more than 93% of the cells were viable as measured by measuring the number of stained dead and live cells. Furthermore, it was observed that the cells in the 50 mL vials using the freeze and thaw procedure described herein showed comparable viability to the cells using the benchmark freeze and thaw method in terms of fresh cells and their killing function (Figure 7A). Furthermore, the CAR-NK cells in the 50 mL vials using the freeze and thaw procedure described herein showed comparable immunophenotype (Table 2) and fresh cells. Figure 7A shows the percentage of killing by CAR-NK cells as a function of E:T ratio. Table 2 shows the preservation of the immunophenotype in frozen and thawed CAR-NK cells.

[0211] [Table 1]

[0212] [Table 2]

[0213] Example 6: In vivo efficacy of freezing and thawing CAR-NK cells. This example demonstrates the in vivo efficacy of freezing and thawing mammalian CAR-NK cells in 50 mL AT vials compared to 2 mL AT vials or 2 ml generic cryovials when the mammalian cells were frozen in a manner that minimized the latent heat of thawing. AT cryovials are proprietary cryovials with specialized container sealing technology.

[0214] First, 35 ml of CAR-NK cells were frozen in a 50 ml AT vial at a concentration of 80 million cells per mL. The temperature of the vial was lowered to 4°C in a CryoMed5474 controlled freezer. To start the process of cryopreservation, the temperature was lowered to -2°C at a rate of 1°C per minute. The temperature of the vial interior and contents was equilibrated at -2°C for 3 minutes. The ice nucleation process was initiated by rapidly lowering the temperature to -60°C at a rate of 25°C per minute. The interior of the vial and the vial contents were equilibrated at -60°C for 1 minute to absorb the latent heat of melting due to the initiation of ice formation, and the temperature of the cell suspension was maintained below freezing to avoid remelting of the ice. To avoid promoting extracellular ice formation that would cause undesirable intracellular ice formation, the temperature was increased to -30°C at a rate of 10°C per minute to allow a continuous and smooth temperature drop, allowing the intracellular water to continue to move osmotically while allowing the formation of extracellular ice gradually. The temperature was maintained at -30°C to allow cells to freeze uniformly. Finally, the temperature was ramped down to -40°C at a rate of 1°C per minute. The cell contents were equilibrated at -40°C for 5 minutes to maximize extracellular ice formation. The temperature was ramped down to a final temperature of -80°C at a rate of 10°C per minute to allow for residual extracellular ice formation. After freezing was complete, the cryovials were stored in the vapor phase of a liquid nitrogen tank. Table 3 shows the components of the cryopreservation medium.

[0215] [Table 3]

[0216] Cryopreserved cells were thawed by placing the AT vial in an orbital shaker water bath (Benchmark SBL-12) set at 60°C and a rotation speed of 150 rpm for a maximum of 600 s.

[0217] Both in vitro viability, killing rate, and immunophenotype of frozen and thawed CAR-NK were tested. Additionally, CAR-NK cells were injected into female NOD SCID gamma (NSG) mice bearing luciferase-expressing Raji human Burkitt lymphoma (Raji B.luc) xenografts. 12-week-old female NSG mice were obtained from Jackson Laboratory. The in vivo efficacy of the freeze-thaw protocol on CAR-NK cells was measured by assessing their in vivo killing efficacy in NOD / Shi-scid, IL-2Rgamma null immunodeficient mice ("NSG mice"). Natural killing (NK) cell activation is an antigen-dependent process that leads to NK cell proliferation and persistence into effector cells. PBS was used as a negative control.

[0218] The in vivo efficacy of CAR-NK cells was tested in female NOD SCID gamma (NSG) mice bearing luciferase-expressing Raji human Burkitt lymphoma (Raji B.luc) xenografts. One day prior to treatment (D-1), female NSG mice were randomized into groups of five mice per group according to body weight and then administered 1.5 Gray (1.5 Gy) of total body radiation. On day 0, mice were administered 2 × 10 4 Bioluminescent Raji B luc tumor cells were co-administered and treatment was by intravenous injection via the tail vein. In vivo, luciferin was administered to mice and whole-body abdominal images were taken 9 min after substrate injection. Luciferase activity was measured in surviving mice after treatment using an IVIS® Spectrum CT imaging system (PerkinElmer). On the day of imaging, mice were given a luciferin substrate (150 mg / kg total, IP) injection and placed in an anesthesia induction chamber (2.5-3.5% isoflurane in oxygen). After sedation, mice were placed in the imaging chamber for image acquisition 9 min after luciferin substrate injection.

[0219] FIG. 8 shows a graph of in vitro killing rate of cells after thawing in 50 mL AT vials and 2 mL AT vials at different E:T ratios for both 6 and 80 million cells in two cryopreservation media, 40% PLASMA-LYTE A, 10% HSA, 50% CS10, with or without 30 mM trehalose. It was observed that frozen cells in 50 mL AT vials exhibited comparable killing function (FIG. 8). Table 4 summarizes the killing rate at an E:T ratio of 10:1, and the viability and recovery rate of CAR-NK cells frozen and thawed using the sequences described herein. It was observed that CAR-NK cells in 50 ml AT vials exhibited comparable high viability (≧97.0%) as CAR-NK cells frozen in 2 ml AT vials. Table 5 summarizes the phenotypic preservation in CAR-NK cells frozen in 50 ml and 2 ml vials. It was observed that the immunophenotype was similarly preserved in CAR-NK frozen in 50 ml AT vials compared to CAR-NK cells frozen in 2 ml AT vials.

[0220] Figure 9 further illustrates the in vivo efficacy of CAR-NK cells 36 days after administration of CAR-NK cells. The in vivo efficacy of three different CAR-NK cells (e.g., cells taken from different donors) was tested. Two different formulations were used in these studies: 1) 40% PLASMA-LYTE A, 10% HSA, 50% CS10 (T6), and 2) 40% PLASMA-LYTE A, 10% HSA, 50% CS10, 30 mM trehalose (T6T). Negative control mice were injected with media containing cells (vehicle). Mice injected with NK-CAR cells were observed to have a reduced intensity of luciferase expression on days 13 / 14 and 20 / 21 (Figures 9G-9I). The total flux of luciferase fluorescence as a function of time is shown in Figures 9D-9F. Figures 9A-9B show the survival rate of mice as a function of days after treatment. It was observed that mice injected with fresh CAR-NK cells, as well as CAR-NK cells that had been frozen and thawed according to the procedures described herein, exhibited comparable survival rates.

[0221] [Table 4]

[0222] [Table 5]

[0223] Example 7: Treatment of a subject in need with CAR-NK cells - Freezing in vials, shipping, thawing, administration This example describes the freezing, thawing, and exemplary uses of the modified CAR-NK cells described herein. An exemplary use described in this example is the freezing, thawing, and administration of the CAR NK cells to a cancer patient, for example, a patient with diffuse large B-cell lymphoma.

[0224] CAR-NK cells containing CD19, IL-15, and iCaspase9 are suspended in cryopreservation medium containing human serum albumin (HSA), PLASMA-LYTE A, trehalose, and CS10. CAR-NK cells are frozen in 50 mL cryovials at concentrations between 6 M / mL and 25 M / mL with a fill volume of approximately 36 mL of fill sample. Such cryovials may contain 2-4 doses for patients in need. The CAR-NK cells are frozen using the following freezing program: (a) the sample is placed at a first temperature higher than the freezing temperature of the sample; (b) the first temperature is decreased at a first controlled rate to a second temperature, where the second temperature is at least 2° C. lower than the first temperature; (c) the second temperature is decreased at a second controlled rate to a third temperature, where the third temperature is at least 40° C. lower than the second temperature; (d) the third temperature is increased at a third controlled rate to a fourth temperature, where the fourth temperature is at least 20° C. higher than the third temperature; (e) the fourth temperature is decreased at a fourth controlled rate to a fifth temperature, where the fifth temperature is at least 10° C. lower than the fourth temperature; and (f) the fifth temperature is decreased at a fifth controlled rate to a final temperature, where the final temperature is −80° C. or lower. The entire freezing process typically takes less than about an hour.

[0225] Once the cells are frozen, the samples are stored at temperatures below -140°C. Such temperatures can be achieved in a variety of ways, including placing the samples in the liquid nitrogen vapor phase. Frozen samples can remain stored at temperatures below -140°C until use. Cells can be stored for 1 week, 2 weeks, 1 month, 6 months, 1 year, 2 years, 5 years, 10 years, or longer.

[0226] When the cells are needed, for example for use in allogeneic cell therapy, they are transported in a cryoshipper from the frozen storage to a hospital or other location where the patient is waiting for the transplant of the cells. During the transport process, the cells are maintained at a temperature of -140°C or lower until they arrive at the hospital or other location (e.g., a clinic). Upon arrival at that location, the cells are thawed. The cells can be thawed as follows: the container containing the cryopreserved modified immune cells is heated to a temperature of 37°C to 70°C and the cells are agitated at a speed of about 100 to about 250 RPM for an appropriate time until the cells are thawed. Heating can be performed, for example, at a temperature of 60°C to 65°C while agitating the cell sample at a speed of 100 to 125 RPM. Heating can be performed using a water bath or a dry heating device. Typically, the total time to thaw the cells is about 10 minutes overall.

[0227] Thawing of the 50 mL cryovials can be performed at the patient's bedside or other nearby location for easy access to the patient receiving the cells. Once the sample is thawed, the total volume of the sample will be approximately 34-36 mL. Administer up to approximately 34 mL of thawed sample to the patient using a vial adapter for sterile administration. One thawed sample may contain multiple doses.

[0228] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above detailed description, but rather is as set forth in the claims.

Claims

1. 1. A CAR-NK cell therapy product comprising a CAR-NK cell population comprising NK cells genetically modified to express CD19-CAR and IL-15, formulated in a cryopreservation medium comprising PLASMA-LYTE A, trehalose, CS10 and human serum albumin (HSA).

2. The cell therapy product of claim 1, wherein the CD19-CAR has an anti-CD19 binding domain comprising a heavy chain variable region having at least 95% identity to SEQ ID NO:2 and a light chain variable region having at least 95% identity to SEQ ID NO:

1.

3. The cell therapy product of claim 1 or 2, wherein the CD19-CAR comprises a heavy chain variable region having the sequence of SEQ ID NO:2 and / or a light chain variable region having the sequence of SEQ ID NO:

1.

4. A cell therapy product described in any one of claims 1 to 3, wherein the CD19-CAR comprises a transmembrane domain selected from the group consisting of the α chain, β chain or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

5. The cell therapy product of any one of claims 1 to 4, wherein the CD19-CAR comprises an intracellular signaling domain selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ζ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

6. The cell therapy product of any one of claims 1 to 5, wherein the CD19-CAR comprises a CD28 transmembrane domain having at least 95% identity to SEQ ID NO:

3.

7. The cell therapy product of claim 6, wherein the CD19-CAR comprises the CD28 transmembrane domain of SEQ ID NO:

3.

8. The cell therapy product of any one of claims 1 to 7, wherein the CD19-CAR comprises a CD3z signaling domain having at least 95% identity to SEQ ID NO:

4.

9. The cell therapy product of claim 8, wherein the CD19-CAR comprises a CD3z signaling domain of SEQ ID NO:

4.

10. The cell therapy product of any one of claims 1 to 9, wherein the cell therapy product further comprises a suicide switch, the suicide switch being iCaspase9.

11. The cell therapy product of any one of claims 1 to 10, wherein the NK cells are derived from umbilical cord blood, peripheral blood, T cells or iPS cells.

12. The cell therapy product of any one of claims 1 to 11, wherein the cryopreservation medium comprises 25% v / v to 50% v / v PLASMA-LYTE A.

13. The cell therapy product of any one of claims 1 to 12, wherein the trehalose is at a concentration of about 10 mM to 100 mM.

14. 14. The cell therapy product of any one of claims 1 to 13, wherein the 25% w / v HSA is at a concentration of 1.25% v / v to 20% v / v.

15. The cell therapy product of any one of claims 1 to 14, wherein the cryopreservation medium comprises 40% v / v to 60% v / v CS10.

16. 16. The cell therapy product of any one of claims 1 to 15, further comprising a cryoprotectant, wherein the cryoprotectant is DMSO.

17. A composition comprising a cell therapy product according to any one of claims 1 to 16.

18. A cancer therapeutic agent comprising a cell therapy product according to any one of claims 1 to 16 or a composition according to claim 17.

19. Use of a cell therapy product according to any one of claims 1 to 16 or a composition according to claim 17 in the manufacture of a medicament for the treatment of cancer.

20. A cell therapy product comprising a CAR-NK cell population comprising NK cells genetically modified to express CD19-CAR and IL-15, formulated in a cryopreservation medium comprising PLASMA-LYTE A, trehalose, CS10 and HSA, wherein the CD19-CAR comprises an anti-CD19 binding domain comprising a heavy chain variable region having at least 95% identity to SEQ ID NO:2 and a light chain variable region having at least 95% identity to SEQ ID NO:

1.

21. 1. Use of a cell therapy product comprising a CAR-NK cell population comprising NK cells genetically modified to express CD19-CAR and IL-15, formulated in a cryopreservation medium comprising PLASMA-LYTE A, trehalose, CS10 and HSA, wherein the CD19-CAR comprises an anti-CD19 binding domain comprising a heavy chain variable region having the sequence of SEQ ID NO:2 and / or a light chain variable region having the sequence of SEQ ID NO:1, in the manufacture of a medicament for treating cancer.

22. 22. The use according to claim 19 or 21, wherein the cancer is B-cell lymphoma.

23. 1. A method for cryopreservation of immune cells on a large scale, said method comprising: (a) providing a container containing a sample comprising immune cells suspended in a cryopreservation medium, the volume of the sample being at least 5 percent less than a total volume of the container, the volume of the sample being at least 10 ml; (b) cooling the container from a temperature above the freezing point of the sample to a temperature below about −80° C. in a multi-step process at a controlled rate to minimize the latent heat of fusion; (c) storing the cells in liquid nitrogen vapor, thereby cryopreserving the immune cells; The above-mentioned cryopreservation method.

24. 24. The method of claim 23, wherein the cells are stored for 1 week, 2 weeks, 1 month, 6 months, 1 year, 2 years, 5 years or 10 years or more.

25. 25. The method of claim 23 or 24, further comprising thawing the cryopreserved cells, the thawing comprising: (a) heating a water bath to a temperature in the range of 37° C. to 75° C.; (b) transferring the container containing the cryopreserved cells to said pre-heated water bath; (c) agitating said container at a speed of about 100 to about 250 RPM for a suitable period of time, thereby thawing the cells.

26. 26. The method of claim 25, wherein the total viable cells after thawing are about 200 million to about 800 million cells.

27. A cell therapy product comprising a CAR-NK cell population comprising NK cells genetically modified to express a BCMA CAR and IL-15, formulated in a cryopreservation medium comprising PLASMA-LYTE A, trehalose, CS10 and HSA.

28. 30. A cancer treatment comprising the cell therapy product of claim 27.

29. 30. Use of the cell therapy product of claim 27 in the manufacture of a medicament for the treatment of cancer.

30. A cryopreservation medium comprising a cryoprotectant, albumin, a disaccharide and a non-pyrogenic and isotonic crystalloid solution.

31. 31. The cryopreservation medium of claim 30, comprising PLASMA-LYTE A, human serum albumin (HSA), trehalose and a cryoprotectant.

32. 24. The method of claim 23, comprising changing the temperature of a sample containing immune cells from a first temperature above the freezing temperature of the sample to a final temperature of -80°C or lower, thereby cryopreserving the sample at said final temperature, the method comprising: (a) subjecting the sample to a first temperature above a freezing temperature of the sample; (b) decreasing the first temperature to a second temperature at a first controlled rate, wherein the second temperature is at least 2° C. less than the first temperature; (c) decreasing the second temperature at a second controlled rate to a third temperature, wherein the third temperature is at least 40° C. less than the second temperature; (d) increasing the third temperature at a third controlled rate to a fourth temperature, wherein the fourth temperature is at least 20° C. greater than the third temperature; (e) decreasing the fourth temperature at a fourth controlled rate to a fifth temperature, wherein the fifth temperature is at least 10° C. less than the fourth temperature; (f) decreasing the fifth temperature at a fifth controlled rate to a final temperature, wherein the final temperature is −80° C. or lower.

33. 24. The method of claim 23, wherein the multi-step process comprises cooling the cells at a controlled rate of between 0.75° C. / min and 30° C. / min to a final temperature of −80° C. or lower, thereby cryopreserving the cells.

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