Frozen preparations containing apoptotic cells

Abstract

The present disclosure provides a frozen preparation comprising a population of apoptotic mononuclear enriched cells. The present disclosure also provides a method for producing the frozen preparation of the present disclosure, and a method for thawing and using the frozen preparation of the present disclosure. The frozen preparation of the present disclosure further comprises an isotonic crystalloid solution and a cryoprotectant. Upon thawing, the frozen preparation of the present disclosure maintains the biological activity observed in fresh apoptotic mononuclear enriched cells before freezing.

Description

[Technical field]

[0001] The present disclosure relates to frozen preparations of apoptotic cells and methods for making frozen preparations of apoptotic cells, where the population of apoptotic cells is maintained and stored as a frozen preparation and thawed for use in treating a subject (patient) in need of treatment. [Background technology]

[0002] Cryopreservation is the process of preserving cells by cooling to below freezing. At such low temperatures, all biological activity is effectively stopped, including biochemical reactions that cause cell death. However, cryopreservation is known to cause damage to frozen cells, thereby affecting cell survival. The viability of cells during cryopreservation was measured by the recovery rate and viability of cells after thawing. The viability of cells during cryopreservation is highly influenced by process variables such as the cryopreservation formulation, the freezing cycle and procedure, and the thawing procedure. Phenomena that cause damage to cells during freezing include, but are not limited to, solution effect, extracellular ice formation, dehydration, and intracellular ice formation. In addition, apoptosis has been found to be the main cause of poor recovery of cryopreserved cell populations.

[0003] Apoptotic cells represent one pathway of physiological cell death, most commonly caused by apoptosis, which triggers a series of molecular homeostasis mechanisms, including recognition, immune responses, and elimination processes. Interestingly, apoptotic cells are immunoregulatory cells that can directly or indirectly induce immune tolerance to dendritic cells and macrophages. Apoptotic cells have been found to regulate dendritic cells and macrophages, making them tolerogenic and inhibiting inflammatory activities, such as the secretion of inflammatory cytokines and the expression of costimulatory molecules.

[0004] Populations of apoptotic cells can be produced for therapeutic purposes. As recently summarized, apoptotic cells can show beneficial effects against abnormal immune responses by downregulating both anti-inflammatory and pro-inflammatory cytokines derived from PAMPs and DAMPs in both animal and in vitro models (Trahtemberg and Mevorach (2017) Apoptotic Cells Induced Signaling for Immune Homeostasis in Macrophages and Dendritic Cells. Frontiers in Immunology, vol. 8, 1356). Furthermore, administration of apoptotic cells has been found to be effective in cancer models, independent of other treatments. Moreover, when administration of apoptotic cell populations is performed in combination with other cancer therapies, remarkable synergistic effects can be observed, further delaying disease onset and progression or improving survival (see, for example, International Publication No. WO2018 / 225072).

[0005] Thus, apoptotic cells provide a therapeutic approach to treat a variety of diseases and conditions, including, but not limited to, cytokine storm, cytokine release syndrome, autoimmune diseases, viral infections, osteoarthritis, GVHD, sepsis, pulmonary dysfunction such as ARD and fibrosis, COVID, and cancer.

[0006] It is important that cryopreserved formulations for apoptotic cell populations developed as vehicles for administration of apoptotic cell formulations maintain high cell viability and are able to maintain the integrity and characteristics of the apoptotic cells during thawing. The cryopreserved formulation must also be safe for administration to a recipient. Furthermore, the cryopreserved formulation must maintain its stability over time.

[0007] The composition of the freezing solution also affects cell survival. Therefore, appropriate formulations are necessary to enhance the stability of cell-based preparations, ensure the maintenance of the properties and quality characteristics of the preparation, and prevent damage caused by harmful factors. Several commercially available serum- and protein-free cryopreservation solutions have been developed and are widely used in cell therapy. However, there remains an unmet need for cryopreservation formulations for apoptotic cells and methods for their use.

[0008] The disclosed cryopreserved formulations and methods of use thereof are intended to reduce the known side effects that can be caused by high levels of dimethyl sulfoxide (DMSO) transfusions and to maintain the characteristics and therapeutic efficacy of apoptotic cell populations following thawing of frozen preparations of apoptotic cells. Summary of the Invention [Means for solving the problem]

[0009] In one aspect, the present disclosure provides a frozen formulation comprising a population of apoptotic mononuclear enriched cells, an isotonic crystalloid solution, and a cryoprotectant, the frozen formulation having a pH in the range of about 6.5 to 8.0.

[0010] In a related embodiment, the apoptotic mononuclear enriched cells are about 17-150×10 6 The concentration is 10 cells / ml.

[0011] In another related aspect, the apoptotic mononuclear enriched cells comprise at least one cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells. In yet another related aspect, the at least one cell type comprises a single cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

[0012] In another related embodiment, the isotonic crystalloid solution comprises sodium chloride, potassium chloride, magnesium chloride, sodium acetate, sodium gluconate, or any combination thereof. The isotonic crystalloid solution comprises 140 mmol / L sodium, 5 mmol / L potassium, 1.5 mmol / L magnesium, 98 mmol / L chloride, 27 mmol / L acetate, or 23 mmol / L gluconate.

[0013] In another related embodiment, the cryoprotectant comprises DMSO. In yet another related embodiment, DMSO is included in the frozen formulation of the present disclosure at a concentration of about 2.5%.

[0014] In another related embodiment, the frozen formulation of the present disclosure has a pH of about 7.4.

[0015] In one aspect, the present disclosure provides a method of producing a frozen apoptotic cell preparation, comprising: (a) a resuspension step in which a population of fresh apoptotic mononuclear enriched cells is resuspended in an isotonic crystalloid solution having a pH of about 6.5-8.0; (b) a mixing step of mixing the population of apoptotic mononuclear enriched cells resuspended in the resuspension step (a) with a cryoprotectant; (c) a freezing step, gradually freezing the mixture produced in the mixing step (b) to a temperature of about -196°C.

[0016] In a related aspect, the population of apoptotic mononuclear enriched cells resuspended in the resuspension step (a) is between about 25 and 300×10 6 In yet another related aspect, the apoptotic mononuclear enriched cells comprise at least one cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells. In yet another related aspect, the at least one cell type comprises a single cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

[0017] In another related embodiment, the isotonic crystalloid solution comprises sodium chloride, potassium chloride, magnesium chloride, sodium acetate, or sodium gluconate. In yet another related embodiment, the isotonic crystalloid solution comprises 140 mmol / L sodium, 5 mmol / L potassium, 1.5 mmol / L magnesium, 98 mmol / L chloride, 27 mmol / L acetate, or 23 mmol / L gluconate. In yet another related embodiment, the isotonic crystalloid solution has a pH of about 7.4.

[0018] In another related embodiment, in the mixing step (b), the population of apoptotic mononuclear enriched cells and the cryoprotectant are mixed in a 1:1 (volume / volume) mixing ratio.

[0019] In yet another related embodiment, the cryoprotectant comprises DMSO. In yet another related embodiment, the final concentration of DMSO in the frozen apoptotic cell preparation is about 2.5%.

[0020] In another related aspect, the method of the present disclosure further comprises an irradiation step after the resuspending step (a) and before the mixing step (b), of irradiating the population of apoptotic mononuclear enriched cells.

[0021] In another related embodiment, the concentration of the population of apoptotic mononuclear enriched cells in the frozen apoptotic cell preparation is about 20-100×10 6 cells / ml.

[0022] In one aspect, the present disclosure provides a method of treating a subject using the frozen formulation of the present disclosure described above, comprising: (a) a thawing step of thawing a frozen formulation; (b) administering the thawed frozen formulation, or a portion thereof, to a subject, thereby treating the subject.

[0023] In a related aspect, the method of the disclosure further comprises an analysis step of analyzing the thawed frozen preparation for Annexin V staining, and in the administration step (b), an apoptotic cell population comprising at least 35% Annexin V+ cells is administered. [Brief description of the drawings]

[0024] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0025] The presently disclosed frozen preparations of apoptotic cells and methods for their manufacture are particularly pointed out and distinctly claimed in the concluding portion of this specification, however, the presently disclosed frozen preparations of apoptotic cells and methods for their manufacture, both as to their organization and operation, and also as to their objects, features, and advantages, will best be understood by reading the following detailed description when read in conjunction with the accompanying drawings.

[0026] [Figure 1] FIG. 1 shows the analysis of Allocetra-OTS cells after AnV and PI staining. Frozen preparations of Allocetra-OTS produced using the methods described herein were thawed and stained with Annexin V (AnV; X-axis) and propidium iodide (PI; Y-axis) to assess the prevalence and status of apoptotic cells. The graph shows that 60% of the total number of thawed apoptotic cell populations (including mature and early apoptotic cell populations) met the criteria of AnV 35% or more. The late apoptotic cell population has the highest PI fluorescence intensity (Y-axis). The apoptotic cell population and the late apoptotic cell population are clearly distinguished. The late apoptotic cell population accounted for 22.2% of the total number of cells produced, and was AnV 35% or more. [Diagram 2]2 shows a schematic diagram of one embodiment of the optimized cryopreservation method for apoptotic cells of the present disclosure. The process shown on the left side of the schematic diagram shows the steps in the production of fresh apoptotic cell preparations, and the process shown on the right side of the schematic diagram shows the steps in the production of frozen apoptotic cell preparations. Italic text indicates the differences between both methods. [Diagram 3] FIG. 3 shows a schematic diagram of the main conventional manufacturing process for formulations containing apoptotic cells. [Figure 4A] 4A-4B show the efficacy of CryoStor®-based cryopreservation medium on frozen Allocetra (apoptotic mononuclear cell-enriched cell population). Figure 4A shows the percentage of late apoptotic cells. Figure 4B shows the percentage of apoptotic cells. [Figure 4B] The grey dashed line represents a 7.5% CryoStor® CS-based cryopreservation medium produced by centrifugation after completion of the standard Allocetra manufacturing steps (final DMSO concentration 7.5%). The black line represents a 5% CryoStor® CS-based cryopreservation medium formulation produced by diluting the Allocetra cell suspension 1:1 with commercially available CryoStor® CS-10 medium (final DMSO concentration 7.5%). The grey solid line represents a 2.5% CryoStor® CS-based cryopreservation medium produced by diluting the Allocetra cell suspension 1:1 with commercially available CryoStor® CS-5 medium (final DMSO concentration 2.5%). The dotted line represents the shipping specification for the Allocetra-OTS apoptosis and late apoptosis assays, where apoptotic cells include at least 35% AnV+ cells and at least 35% apoptotic cells (early and mature) based on IPDIM. [Figure 5A] 5A-5B show the efficacy of homemade cryopreservation medium on frozen Allocetra (FIG. 5A) late apoptosis levels, and (FIG. 5B) apoptosis levels over time after thawing of the frozen preparations. [Figure 5B]The grey dashed line indicates the 7.5% DMSO homemade cryopreservation medium, the black line indicates the 5% DMSO homemade cryopreservation medium formulation, and the grey continuous line indicates the 2.5% DMSO homemade cryopreservation medium. The horizontal black dotted line indicates the shipping specification for the Allocetra-OTS apoptosis and late apoptosis assays, where >30% late apoptosis is not acceptable and >35% apoptosis (early and mature) is required. [Figure 6A] 6A-6D show the efficacy of cryopreservation media on late and apoptotic Allocetra populations. [Figure 6B] Figure 6A shows the effect of cryopreservation media on frozen Allocetra late apoptosis levels over time from thawing, as analyzed on AttuneNxT (Figure 6A) and Calibur (Figure 6B) FACS instruments. The grey line represents the homemade cryopreservation media with 2.5% DMSO, and the black line represents the lead cryopreservation formulation in 2.5% CS media. [Figure 6C] The effect of cryopreservation medium on apoptosis levels in frozen Allocetra was also analyzed by AttuneNxT (Figure 6C) and Calibur (Figure 6D) FACS devices. [Figure 6D] The grey line represents the 2.5% DMSO homemade cryopreservation medium and the black line represents the lead cryopreservation formulation in 2.5% CS medium. The horizontal black dotted lines represent the shipping specifications for the Allocetra-OTS apoptosis and late apoptosis assays, where >30% late apoptosis is not acceptable and >35% apoptosis (early and mature) is required. [Figure 7A] Figures 7A-7E show the effect of excipients on cryopreservation medium: Figure 7A shows the effect of 30 mmol / L trehalose on apoptosis and late apoptosis levels of cryopreserved Allocetra after thawing. [Figure 7B] FIG. 7B shows the effect of 0.2% Tween-80 on apoptosis and late apoptosis levels of cryopreserved Allocetra after thawing. [Figure 7C]Figure 7C shows the effect of a combination solution of 1.25% dextran-40 and 0.63% dextrose on apoptosis and late apoptosis levels of cryopreserved Allocetra after thawing. For Figures 7A-C, black bars represent the percentage of apoptosis and grey bars represent late apoptotic cells. [Figure 7D] Figure 7D shows the effect of adding excipients to cryopreservation medium on cell population composition levels: stacked grey bars represent CD3 lymphocyte cell levels, stippled bars with white background represent monocyte / macrophage cell levels, stippled bars with grey background represent granulocyte cell levels, stacked black bars represent B lymphocyte levels, and stacked diagonal striped bars represent NK cell levels. [Figure 7E] FIG. 7E shows the effect of adding excipients to cryopreservation medium on CD45 MNC population levels. [Figure 8A] Figures 8A and 8B show the effect of Allocetra-based media solutions on the levels of late apoptotic (Figure 8A) and apoptotic cells (Figure 8B). [Figure 8B] Black bars indicate results for PBS-based solutions, mesh bars indicate results for saline-based solutions, grey bars indicate results for Hartman-based solutions, and diagonal striped bars indicate results for PlasmaLyte-based solutions. The horizontal black dotted line indicates the shipping specifications for the Allocetra-OTS apoptosis and late apoptosis assays: at least 35% AnV+ and less than 30% PI of all cells in the population. [Figure 9A] 9A and 9B show the effect of the identity of the cryopreservation base solution in homemade media on the levels of Allocetra late apoptotic cells (FIG. 9A) and apoptotic cells (FIG. 9B). [Figure 9B] The black line shows the results of formulating cells in Ringer's lactate solution using LOVO, the grey line shows the results of formulating cells in PlasmaLyte 148 solution, and the dashed black line shows the shipping specification for the Allocetra-OTS apoptosis and late apoptosis assays. [Figure 10A] 10A-10B show the effect of the identity of the cryopreservation base solution in homemade media on the levels of Allocetra late apoptotic cells (FIG. 10A) and apoptotic cells (FIG. 10B). [Figure 10B] Cells were formulated into CryoStor Base Solution (CSB) using LOVO and then either added an equal volume of CS-5 solution to a final concentration of 2.5% (shown as solid black line), or diluted 1:1 with an equal volume mix of commercial CS-10 solution and PlasmaLyte 148 to produce a 2.5% DMSO formulation (shown as dashed grey line), or added WAK's Cryo-Sure DMSO solution to a final concentration of 2.5% (shown as solid black line) to compare with the average standard frozen Allocetra formulation (shown as dashed black line). Horizontal dotted black lines indicate the shipping specifications for the Allocetra-OTS apoptosis and late apoptosis assays. [Figure 11A] Figures 11A and 11B show the effect of liquid nitrogen storage time on the stability of Allocetra cell populations after cryopreservation: (Figure 11A) late apoptotic population (AnV+, PI high+) and (Figure 11B) apoptosis (early apoptotic population (AnV+, PI negative) and mature apoptotic population (AnV+, PI dim)). [Figure 11B] The dashed lines indicate the shipping specifications for the Allocetra-OTS apoptosis and late apoptosis assays, where thawed frozen populations with a percentage of late apoptotic cells less than the dashed line and a percentage of apoptotic cells greater than the dashed line meet the shipping specifications and are acceptable for use. [Figure 12] Figure 12 shows flow cytometry analysis of cell distribution in fresh and frozen preparations. Little change in cell type distribution was observed between fresh and frozen preparations. Markers used for cell identification were as follows: T cells: CD3+ cells, monocytes: CD14+ cells, granulocytes: CD15+ cells, B cells: CD19+ cells, and natural killer (NK) cells: CD56+ cells. [Figure 13A]Figures 13A and 13B are bar graphs showing the measurement of apoptotic cell (e.g., Allocetra) activity as percent inhibition of TNF-α expression by intracellular staining and flow cytometry analysis. Figure 13A shows the activity of fresh formulations at T=0 hours and frozen formulations 1 hour after thawing. [Figure 13B] Figure 13B shows the activity of a fresh formulation stored at 4-8°C for 96 hours (T=96) and a frozen formulation 1 hour after thawing. Results are shown under conditions of high (0.05 ng / mL) and low (0.025 ng / mL) LPS concentrations. [Figure 14] 14 is a bar graph showing the measured activity of apoptotic cells (e.g., Allocetra) from fresh and frozen formulations. Activity was measured as the percentage of inhibition of TNF-α secretion based on assessment of cytokine secretion by mouse TNF-α ELISA. [Figure 15] Figure 15 shows the beneficial effect of Allocetra-OTS on survival of CLP mice. Kaplan-Meier survival curves of CLP mice treated with ertapenem+Allocetra-OTS in fresh or frozen-thawed formulations show that Allocetra provided as either a fresh cell population or a thawed population from the frozen formulation of the present disclosure provided a significant increase in survival compared to mice that did not receive Allocetra. Number of mice in each group (N=12), Representative Results. [Figure 16] Figure 16 shows that CLP mice treated with frozen-thawed and fresh Allocetra-OTS showed higher survival rates than the ertapenem (SOC) group (SOC data not shown). The graph shows improved survival 120 hours after CLP induction compared to the standard of care group. Values ​​on the graph represent the average of four separate experiments, and bars show SD. [Figure 17A]Figures 17A and 17B show the results of analysis of necrotic peripheral blood mononuclear cells (PBMCs). Figure 17A shows the results of analysis of necrotic peripheral blood mononuclear cells (PBMCs) after AnV and PI staining. The graph shows that after necrosis induction, more than 80% of all PBMCs were induced into the necrotic phase and appeared in the AnV+, PI+ necrotic gate, the same gate region as cells in late apoptosis. [Figure 17B] Figure 17B is a bar graph showing measurements of activity in apoptotic (Allocetra-OTS) and necrotic (Necrotic-Allocetra-OTS) cells. Activity was measured as percent inhibition of TNF-α secretion based on mouse TNF-α ELISA. The graph shows that Allocetra-OTS activity was significantly reduced after induction of necrosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the frozen preparations of apoptotic cells according to the present invention, their methods of manufacture, and their uses. However, it will be understood by one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the description of the present invention.

[0028] Cryopreservation and thawing of frozen cell populations is inversely associated with different degrees of activation of apoptotic pathways in the frozen cells (Matsushita et al., (203) Cell Transplantation, Vol. 12, pp. 109-121; Paasch et al. (2004) Biology of Reproduction 71, 1828-1837). The frozen apoptotic cell preparations of the present disclosure are capable of maintaining apoptotic cell function upon thawing following cryopreservation. This ability is surprising and unexpected.

[0029] The present disclosure provides frozen preparations containing apoptotic cell populations that can provide a therapeutic source of apoptotic cells when needed. The ability to freeze apoptotic cells for long-term storage and subsequently provide a therapeutic frozen preparation is beneficial in that upon thawing, the apoptotic cells maintain functional characteristics and activity useful for the treatment of diseases and conditions.

[0030] In some embodiments, one characteristic of a thawed frozen apoptotic cell preparation is that the thawed apoptotic cell population is characterized by at least 35% annexin V+ (AnV) cells with low or no propidium iodide (PI) staining (AnV+PI-; mature apoptotic cells AnV+PI). DIM ), and AnV+PI of 30% or less Hi The aim of the present invention is to provide a method for detecting apoptotic cells comprising the steps of: (a) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (b) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (c) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (d) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (e) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (f) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (g) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (g) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (h) detecting apoptotic cells in a cell population enriched for mononuclear apoptotic cells (late apoptotic cells); (i ...

[0031] In some embodiments, the frozen formulations of the present disclosure contain a population of apoptotic mononuclear enriched cells, an isotonic crystalloid solution, and a cryoprotectant, and have a pH in the range of about 6.5 to 8.0. The mononuclear enriched cells used as a source for producing the apoptotic cell population can include at least one cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

[0032] In some embodiments, the frozen formulation of the present disclosure has a cell density of about 20-150×10 after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 24-150×10 cells / ml after freezing. 6 In some embodiments, the frozen formulation of the present disclosure has a concentration of about 20-100×10 cells / ml after freezing.6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 24-100×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 24-80×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 24-60×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 24-40×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 25-150×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 25-100×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 25-80×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 25-60×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 25-40×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 20-80×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 20-60×10 cells / ml after freezing. 6 In some embodiments, the frozen formulations of the present disclosure have a concentration of about 20-40×10 cells / ml after freezing. 6 The concentration is cells / ml.

[0033] In some embodiments, the frozen formulations of the present disclosure contain cells, and at the time of freezing, at least 35% of the apoptotic cell population are early apoptotic cells or mature apoptotic cells, and less than 30% are late apoptotic cells. In some embodiments, the frozen formulations of the present disclosure contain apoptotic cells, and at the time of freezing, at least 35% of the apoptotic cell population are Annexin V+ (AnV+), and less than 30% are AnV+PI+. High Immediately after thawing the frozen apoptotic cell preparation, the percentage of apoptotic cells shows a gradual increase, but remains within the shipping specification of less than 30% late apoptotic cells. In some embodiments, the thawed apoptotic cells have biological activity useful in the treatment of diseases and conditions. In some embodiments, the thawed apoptotic preparation comprises at least 35% early apoptotic cells and mature apoptotic cells.

[0034] In some embodiments, the frozen formulation of the present disclosure further comprises an isotonic crystalloid solution and a cryoprotectant. In some embodiments, the cryoprotectant comprises dimethylsulfoxide (DMSO). In some embodiments, the percentage of DMSO used in the frozen formulation of the present disclosure is less than that normally used, and may not provide the necessary protection of reducing ice formation in apoptotic cells.

[0035] In some embodiments, the present disclosure provides a method of treating a subject (patient), comprising thawing a frozen apoptotic cell preparation of the present disclosure and administering the thawed apoptotic cell population to the subject.

[0036] Apoptotic Cells

[0037] Apoptosis can be induced in a population of mononuclear enriched cells. In some embodiments, mononuclear enriched cells can be obtained directly from a human subject, for example, but not limited to, using techniques such as leukapheresis. In some embodiments, mononuclear enriched cells can be obtained indirectly, for example, but not limited to, in the case of peripheral blood mononuclear cells (PBMC), from a blood bank. Methods for inducing apoptosis are well known in the art, including, but not limited to, serum deprivation, incubation with corticosteroids (for example, but not limited to, incubation with dexamethasone), activation of cell surface Fas or TNF receptors (by their respective ligands or by crosslinking their receptors with agonistic antibodies), or irradiation (for example, but not limited to, ultraviolet B irradiation or gamma (γ) irradiation).For example, "Roberts KM, et al., (2004) Methods for Inducing Apoptosis. In: Perl A. (eds) Autoimmunity. Methods in Molecular Medicine (TM), vol 102. Humana Press", "Nijhuis et al., Induction of apoptosis by heat and γ-radiation in a human lymphoid cell line; role of mitochondrial changes and caspase activation Int. J. Hyperthermia, December 2006; 22(8): 687-698", "Corinne Petit-Frere, et al., Apoptosis and cytokine release induced by ionizing or ultraviolet B radiation in primary and immortalized human keratinocytes, Carcinogenesis, Volume 21, Issue 6, June 2000, Pages 1087-1095", International Publication No. WO2016 / 170541, International Publication No. WO2019 / 038758, International Publication No. WO2020 / 105034, International Publication No. WO2014 / 087408, International Publication No. WO2018 / 225072 (the above technical or patent documents are incorporated herein by reference in their entirety).

[0038] In some embodiments, induction of apoptosis in mononuclear enriched cell populations (e.g., but not limited to, primary lymphocytes) is achieved by treatment with a corticosteroid, e.g., by treating primary lymphocytes with dexamethasone. In another embodiment, induction of apoptosis in mononuclear enriched cells (e.g., but not limited to, primary lymphocytes) is achieved by irradiation, e.g., by treating primary lymphocytes with gamma irradiation.

[0039] In some embodiments, the apoptotic mononuclear enriched cell population comprises a fresh apoptotic cell population. In some embodiments, the fresh apoptotic mononuclear enriched cell population is maintained on ice or at about 2-8°C. In some embodiments, maintaining the apoptotic mononuclear enriched population on ice is performed for about 24 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population on ice is performed for about 48 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population on ice is performed for about 72 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population on ice is performed for about 96 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population on ice is performed for about 24-96 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population at about 2-8°C is performed for about 24 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population at about 2-8°C is performed for about 48 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population at about 2-8°C is performed for about 72 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population at about 2-8° C. is performed for about 96 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population at about 2-8° C. is performed for about 24-96 hours. In some embodiments, maintaining the apoptotic mononuclear enriched population is performed on ice for about 24-96 hours.

[0040] In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for up to 1 hour. In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for at least 1 hour. In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for more than 1 hour. In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for 1-3 hours. In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for about 1 hour, about 2 hours, or about 3 hours. In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for at least 2 hours. In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for at least 3 hours. In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for more than 2 hours. In some embodiments, the apoptotic mononuclear enriched population is stable at room temperature for more than 3 hours.

[0041] In some embodiments, the fresh apoptotic mononuclear enriched cell population is stored as a frozen formulation containing it, as described herein. In some embodiments, the fresh apoptotic mononuclear enriched cell population is first maintained on ice for up to 96 hours, e.g., up to 24, 48, 72, or 96 hours, and then stored as a frozen formulation containing it, as described herein. In some embodiments, the fresh apoptotic mononuclear enriched cell population is first maintained at about 2-8°C for up to 96 hours, e.g., up to 24, 48, 72, or 96 hours, and then stored as a frozen formulation containing it, as described herein.

[0042] The status of the apoptotic cell population is an important quality attribute of the preparation. To determine the apoptotic status, in some embodiments, annexin V (AnV) staining and propidium iodide (PI) staining were performed as is well known in the art (Trahtemberg, U., Atallah, M., Krispin, A. et al. Calcium, leukocyte cell death and the use of annexin V: fatal encounters. Apoptosis 12, 1769-1780 (2007)). During the early stages of apoptosis, phosphatidylserine (PtdSer) bound to the cell membrane moves from the inner cytoplasmic leaflet of the cell membrane to the outer extracellular leaflet of the cell membrane, exposing PtdSer to the direct environment and making it accessible for AnV binding. After exposure of PtdSer, the apoptotic process proceeds and further caspase activation continues.

[0043] Two subpopulations of apoptotic cells emerge from this dynamic process: AnV+PI - Early apoptotic cells characterized by staining and AnV+ and low PI staining (PI Dim ) are mature apoptotic cells (a dim PI fluorescent population due to the progression of the membrane permeabilization process resulting in a gradual loss of membrane integrity). Apoptotic cells are eventually engulfed by phagocytes. If cells are not phagocytosed, the cell membrane will gradually lose integrity. Loss of cell membrane integrity can be detected by non-selective and massive uptake of fluorescent dyes such as PI, which increases the intensity of PI staining on the cells. PI enters the nucleus and inserts into DNA duplexes, serving as a biomarker for late apoptotic or necrotic cells. In some embodiments, the late apoptotic cell population is characterized by AnV+PI+ High In some embodiments, late apoptotic cells include cells that are not efficiently cleared by phagocytes, and therefore undergo necrosis (secondary necrosis).

[0044] As used herein, the terms "late apoptotic cells", "necrotic cells" and "secondary necrotic cells" are used interchangeably and all have the same properties and meanings.

[0045] In some embodiments, the apoptotic cell population comprises an early apoptotic cell population and a mature apoptotic cell population. In some embodiments, the late apoptotic cell population comprises a necrotic (secondary necrotic) cell population. In some embodiments, the fresh (non-frozen) apoptotic cell population comprises at least 35% apoptotic cells (early apoptotic cells and mature apoptotic cells) and less than 30% late apoptotic cells. In some embodiments, the fresh (non-frozen) apoptotic cell population comprises at least 35% apoptotic cells (early apoptotic cells and mature apoptotic cells) and less than 30% late apoptotic cells. In some embodiments, the fresh (non-frozen) apoptotic cell population comprises at least 35% AnV+ cells (early apoptotic cells and mature apoptotic cells) and less than 30% AnV+IP+ High In some embodiments, the thawed apoptotic cell population comprises at least 35% AnV+ cells (early apoptotic cells and mature apoptotic cells).

[0046] Those skilled in the art will understand that the terms "apoptotic cells", "apoptotic cell population", "Allocetra", and "Allocetra-OTS" are used interchangeably and all have the same nature and meaning, including greater than 35% apoptotic cells (early apoptotic cells and mature apoptotic cells) and less than 30% late apoptotic cells.

[0047] As shown in Figure 1 (see Example 1), differentiation between various apoptotic subpopulations (early, mature, late) was achieved after optimization of the analytical method and improvement of flow cytometry sensitivity. The optimized method with a more precise gating strategy allowed for a more accurate measurement of the apoptotic population. This led to an update of the shipping specifications. Further characterization of late apoptotic cells is provided in Example 2.

[0048] In some embodiments, the fresh frozen apoptotic cell population is frozen after induction of apoptosis. In some embodiments, the fresh frozen apoptotic cell population is frozen after the steps of inducing apoptosis and irradiating the cell population (FIG. 2). As used herein, the term "fresh frozen" encompasses apoptotic cells that have been produced as an apoptotic cell population and have not been previously frozen. In some embodiments, the fresh frozen apoptotic cells are considered to be cryopreserved.

[0049] As used herein, "frozen apoptotic cells", "frozen apoptotic cell population", "frozen Allocetra population", "frozen apoptotic population", "frozen preparation of apoptotic cells", "frozen apoptotic preparation", "cryopreserved apoptotic cells", "cryopreserved Allocetra", "cryopreserved apoptotic population", "cryopreserved apoptotic cell population" and the like are used interchangeably and all have the same nature and meaning. Frozen preparations of apoptotic cells are made from apoptotic cell populations that contain at least 35% apoptotic cells (early apoptotic cells and mature apoptotic cells) and less than 30% late apoptotic cells.

[0050] In some embodiments, the apoptotic mononuclear enriched cell population comprises a cryopreserved apoptotic cell population. When needed for treatment, an appropriate number of cryobags containing the frozen preparation of apoptotic cells can be thawed.

[0051] In some embodiments, thawing comprises rapidly thawing the bag containing the cells in a preheated 37° C. water bath. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells within about 1-10 minutes. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells within about 1-5 minutes. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells within about 2-5 minutes. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells for 2 minutes. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells for 3 minutes. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells for 4 minutes. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells for 5 minutes. In some embodiments, rapidly thawing the cells comprises thawing the bag containing the cells for 6 minutes. In some embodiments, the cells are thawed until no ice crystals remain on their backs and then stored at ambient temperature until use. In some embodiments, the cryopreserved apoptotic cells are thawed using a dry-thaw technique and then stored at ambient temperature until use. As used herein, in some embodiments, the terms "ambient temperature" and "room temperature" are used interchangeably and all have the same properties and meanings.

[0052] In some embodiments, the cryopreserved apoptotic mononuclear enriched cell population is administered to a subject (patient) immediately after thawing. In some embodiments, the cryopreserved apoptotic mononuclear enriched cell population is maintained at ambient temperature ... for about 3 hours after thawing. In some embodiments, the thawed cryopreserved apoptotic population comprises at least 35% apoptotic cells (early apoptotic cells and mature apoptotic cells). In some embodiments, the thawed cryopreserved apoptotic population comprises at least 35% apoptotic cells, including AnV+ and PI+. - or P.I. DIM In some embodiments, the thawed cryopreserved apoptotic population comprises at least 35% apoptotic cells, AnV+ and PI+. DIM Includes:

[0053] One of skill in the art will understand that freezing mononuclear enriched cells for storage prior to induction of apoptosis is not considered to provide previously frozen apoptotic cells. One of skill in the art will understand that freezing mononuclear enriched cells as part of the process of inducing apoptosis is not considered to provide previously frozen apoptotic cells.

[0054] In some embodiments, the apoptotic mononuclear enriched cells are irradiated to reduce proliferation and / or activation of remaining viable cells in the apoptotic cell population. In some embodiments, the apoptotic mononuclear enriched cells are irradiated to reduce the percentage of viable non-apoptotic cells in the population. In some embodiments, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 50% of the cell population. In some embodiments, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 40% of the cell population. In some embodiments, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 30% of the cell population. In some embodiments, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 20% of the cell population. In some embodiments, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 10% of the cell population. In some embodiments, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to 0% of the cell population.

[0055] In another embodiment, the irradiated apoptotic mononuclear enriched cells preserve all of their apoptotic, immunomodulatory and stability properties. In another embodiment, the irradiating step uses UV radiation. In another embodiment, the irradiating step uses gamma radiation. In another embodiment, the apoptotic cells include a reduction in the percentage of viable non-apoptotic cells, an inhibition of cellular activation of viable non-apoptotic cells in the apoptotic cell preparation, a reduction in proliferation of viable non-apoptotic cells in the apoptotic mononuclear enriched cell preparation, or any combination thereof.

[0056] In some embodiments, irradiated apoptotic cells do not have an increased population of dead cells (secondary necrosis; PI+) compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not have an increased population of dead cells (secondary necrosis; PI+) by more than about 1% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not have an increased population of dead cells (secondary necrosis; PI+) by more than about 2% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not have an increased population of dead cells (secondary necrosis; PI+) by more than about 3% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not have an increased population of dead cells (secondary necrosis; PI+) by more than about 4% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not have an increased population of dead cells (secondary necrosis; PI+) by more than about 5% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 6% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 7% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 8% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 9% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 10% compared to non-irradiated apoptotic cells. In some embodiments, irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 15% compared to non-irradiated apoptotic cells.In some embodiments, irradiated apoptotic cells do not exhibit an increase in the population of dead cells (secondary necrosis; PI+) of more than about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to non-irradiated apoptotic cells.

[0057] In some embodiments, the cell populations comprising a reduced or absent fraction of viable non-apoptotic cells provide, in one embodiment, a mononuclear apoptotic cell population that has no viable cells. In some embodiments, the cell populations comprising a reduced or absent fraction of viable non-apoptotic cells provide, in one embodiment, a mononuclear apoptotic cell population that does not induce GVHD in the recipient.

[0058] In some embodiments, apoptotic cells are identified by May-Giemsa stained cell preparations (cytopreps). In some embodiments, cell viability is assessed by trypan blue exclusion. In some embodiments, the apoptotic and necrotic state of cells is identified by Annexin V / propidium iodide staining detected by FACS. In some embodiments, the apoptotic and / or necrotic state of cells is identified by comparison to known necrotic cell populations.

[0059] In some embodiments, the apoptotic cells of the present disclosure do not include necrotic cells. In some embodiments, the apoptotic cells of the present disclosure include less than 30% necrotic cells. In some embodiments, the apoptotic cells of the present disclosure include less than 25% necrotic cells. In some embodiments, the apoptotic cells of the present disclosure include less than 20% necrotic cells. In some embodiments, the apoptotic cells of the present disclosure include less than 15% necrotic cells. In some embodiments, the apoptotic cells of the present disclosure include less than 10% necrotic cells. In some embodiments, the apoptotic cells of the present disclosure include less than 5% necrotic cells.

[0060] Those skilled in the art will understand that in some embodiments, the terms "apoptotic cells", "apoptotic mononuclear enriched cells", "apoptotic cell population", "apoptotic cells", "Allocetra", "ALC" and "Allocetra-OTS" and grammatical variations thereof are used interchangeably, all have the same nature and meaning, and include a population of at least 35% apoptotic cells (early apoptotic cells and mature apoptotic cells) and less than 30% late apoptotic cells. In some embodiments, the population of cells includes at least 35% (AnV+PI) apoptotic cells. - (Early) and AnV+PI+ DIM (mature) are in the early and mature states of apoptosis. In some embodiments, the apoptotic cell population comprises a fresh apoptotic cell population. In some embodiments, the apoptotic cell population comprises a frozen preparation of an apoptotic cell population. In some embodiments, the apoptotic cell population comprises an apoptotic cell population thawed from a frozen preparation of apoptotic cells as described herein.

[0061] One of skill in the art will appreciate that the formulations and methods of the present disclosure include, in some embodiments, apoptotic cells and uses thereof. In some embodiments, the apoptotic cells are HLA-matched to the recipient (subject in need of the composition comprising the apoptotic cells) as described herein. In some embodiments, the apoptotic cells are mismatched to the recipient (subject in need of the composition comprising the apoptotic cells) as described herein. In some embodiments, the apoptotic cells are mismatched to the foreign donor. In some embodiments, the apoptotic cells that are mismatched to the recipient (subject in need of the composition comprising the apoptotic cells) are irradiated as described in detail herein. In some embodiments, the irradiated mismatched cells are referred to as "Allocetra-OTS" or "ALC-OTS".

[0062] In some embodiments, the fresh apoptotic cells are stable. One of skill in the art will appreciate that in some embodiments, stability includes maintaining apoptotic cell characteristics over time, e.g., maintaining apoptotic cell characteristics when stored at about 2-8°C. In some embodiments, stability includes maintaining apoptotic cell characteristics when stored at freezing temperatures, e.g., temperatures below 0°C. In some embodiments, stability includes maintaining apoptotic cell characteristics when stored at freezing temperatures, e.g., temperatures below about -196°C.

[0063] In some embodiments, the apoptotic population cryopreserved in a frozen state is stored in liquid nitrogen (-196°C) for at least 12 months. In some embodiments, the apoptotic population cryopreserved in a frozen state is stored in liquid nitrogen (-196°C) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the apoptotic population cryopreserved in a frozen state is stored in liquid nitrogen (-196°C) for at least 1 year. In some embodiments, when the cryopreserved apoptotic population is thawed, at least 35% of the thawed population comprises apoptotic cells (early and mature apoptotic cells).

[0064] After thawing, the stability of the apoptotic cell population was confirmed by comparing the concentration of apoptotic cells with the shipping standard (at least 35% early apoptotic cells (AnV+PI-) and mature apoptotic cells (AnV+PI-)). DIM), and 30% or less of late apoptotic cells). In some embodiments, the thawed apoptotic cell population is maintained for at least 3 hours. In some embodiments, the stability of the apoptotic cell population characterized by comprising at least 35% apoptotic cells (early and mature apoptotic cells) after thawing is maintained for at least 1 hour. In some embodiments, the stability of the apoptotic cell population characterized by comprising at least 35% apoptotic cells (early and mature apoptotic cells) after thawing is maintained for at least 2 hours. In some embodiments, the stability of the apoptotic cell population characterized by comprising at least 35% apoptotic cells (early and mature apoptotic cells) after thawing is maintained for at least 3 hours.

[0065] In some embodiments, the thawed apoptotic cell population is maintained on ice. In some embodiments, the thawed apoptotic cell population is maintained in an ice-water slurry. In some embodiments, the thawed apoptotic cell population is maintained at about 4°C. In some embodiments, the thawed apoptotic cell population is maintained in a range of about 0°C to 4°C. In some embodiments, the thawed apoptotic cell population is maintained in a range of about 0°C to 8°C. In some embodiments, the thawed apoptotic cell population is maintained in a range of about 4°C to 8°C.

[0066] In some embodiments, thawing the cryopreserved apoptotic cells increases the percentage of late apoptosis in the population by about 15%, while the early apoptosis level decreases slightly, all within the range of factory specifications. In some embodiments, thawing the cryopreserved apoptotic cells increases the percentage of late apoptosis in the population by 1-20%, while the early apoptosis level decreases slightly, all within the range of factory specifications. In some embodiments, thawing the cryopreserved apoptotic cells increases the percentage of late apoptosis in the population by 1-15%. In some embodiments, thawing the cryopreserved apoptotic cells increases the percentage of late apoptosis in the population by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% percent. In some embodiments, thawing the cryopreserved apoptotic cells increases the percentage of late apoptosis in the population by less than 15%. In some embodiments, thawing the cryopreserved apoptotic cells increases the percentage of late apoptosis in the population by less than 30% of the total cell population.

[0067] In some embodiments, after thawing, the stability of the apoptotic cell population is maintained within shipping specifications for at least 1 hour, 2 hours, or 3 hours. In some embodiments, after thawing, the stability of the apoptotic cell population is maintained within shipping specifications for 1 hour, 2 hours, or 3 hours or more. In some embodiments, after thawing, the stability of the apoptotic cell population is maintained within shipping specifications for 1 hour, 2 hours, or 3 hours.

[0068] In certain embodiments, the apoptotic cells comprise a population of mononuclear apoptotic cells and include a reduction in the proportion of viable non-apoptotic cells in the apoptotic cell preparation, an inhibition of cellular activation of viable non-apoptotic cells in the apoptotic cell preparation, a reduction in proliferation of viable non-apoptotic cells in the apoptotic mononuclear enriched cell preparation, or any combination thereof.

[0069] The present disclosure provides, in some embodiments, a pooled mononuclear enriched apoptotic cell population comprising apoptotic mononuclear cells, wherein the pooled mononuclear enriched apoptotic cell population comprises pooled individual mononuclear cell populations. In some embodiments, the frozen preparation of apoptotic cells of the present disclosure comprises a pooled mononuclear enriched apoptotic cell population, wherein the pooled mononuclear apoptotic cell preparation comprises a reduced percentage of viable non-apoptotic cells in the apoptotic cell preparation, an inhibition of cellular activation of viable non-apoptotic cells in the apoptotic cell preparation, a reduction in proliferation of viable non-apoptotic cells in the apoptotic mononuclear enriched cell preparation, or any combination thereof. In some embodiments, the pooled mononuclear apoptotic cells are irradiated after induction of apoptosis and prior to freezing. In another embodiment, the present disclosure provides, in some embodiments, a pooled mononuclear apoptotic cell preparation uses a white blood cell fraction (WBC) obtained from donated blood. Often, this white blood cell fraction is discarded in blood banks or is intended for use in research.

[0070] In some embodiments, pooled blood comprises third party blood from sources that are HLA-matched or HLA-mismatched with respect to the recipient.

[0071] In some embodiments, the apoptotic cell population to be frozen is inactivated. In another aspect, the inactivation comprises irradiation. In another embodiment, the inactivation comprises T cell receptor inactivation. In another embodiment, the inactivation comprises T cell receptor editing. In another embodiment, the inactivation comprises suppressing or eliminating immune responses in the apoptotic cell preparation. In another embodiment, the inactivation comprises suppressing or eliminating cross-reactivity between multiple individual populations contained in the apoptotic cell preparation. In other embodiments, the inactivation comprises reducing or eliminating T cell receptor activity between multiple individual populations contained in the apoptotic cell preparation. In another embodiment, the inactivated cell preparation comprises reducing the percentage of viable non-apoptotic cells in the apoptotic cell preparation, suppressing cellular activation of viable non-apoptotic cells in the apoptotic cell preparation, reducing proliferation of viable non-apoptotic cells in the apoptotic mononuclear enriched cell preparation, or any combination thereof.

[0072] Those skilled in the art will understand that the terms "apoptotic preparation" and "apoptotic population", and grammatical variations thereof, are used interchangeably and all have the same nature and meaning.

[0073] In another embodiment, the inactivated cell population comprises a reduced number of non-quiescent, non-apoptotic cells compared to non-irradiated cell preparations. In some embodiments, the inactivated cell population comprises 50% viable, non-apoptotic cells. In some embodiments, the inactivated cell population comprises 40% viable, non-apoptotic cells. In some embodiments, the inactivated cell population comprises 30% viable, non-apoptotic cells. In some embodiments, the inactivated cell population comprises 20% viable, non-apoptotic cells. In some embodiments, the inactivated cell population comprises 100% viable, non-apoptotic cells. In some embodiments, the inactivated cell population comprises 0% viable, non-apoptotic cells.

[0074] In some embodiments, the enriched population of apoptotic mononuclear cells comprises apoptotic cells that have been irradiated after induction of apoptosis. In another embodiment, the irradiation comprises gamma irradiation or UV irradiation. In yet another embodiment, the irradiated cell preparation comprises a reduced number of non-quiescent, non-apoptotic cells compared to a non-irradiated cell preparation.

[0075] In some embodiments, the apoptotic cells used in the frozen formulations and methods of the present disclosure are produced by any method known in the art. In another embodiment, the apoptotic cells included in the frozen formulations of the present disclosure are autologous to the subject to be treated. In another embodiment, the apoptotic cells included in the frozen formulations of the present disclosure are allogeneic to the subject to be treated. In another embodiment, the apoptotic cells included in the frozen formulations of the present disclosure include early apoptotic cells known in the art.

[0076] Those of skill in the art will appreciate that the term "autologous" encompasses tissues, cells, nucleic acid molecules, or polypeptides where the donor and recipient are the same individual.

[0077] One of skill in the art will understand that the term "allogeneic" encompasses tissues, cells, nucleic acid molecules, or polypeptides derived from separate individuals of the same species. In some embodiments, the allogeneic donor cells are genetically distinct from the recipient.

[0078] In some embodiments, the step of obtaining a mononuclear enriched cell composition in the disclosed method is accomplished by leukapheresis (white blood cell removal). Those skilled in the art will appreciate that the term "leukapheresis" encompasses apheresis procedures that separate white blood cells from a donor's blood. In some embodiments, the donor's blood is subjected to leukapheresis, whereby a mononuclear enriched cell composition is obtained by the disclosed manufacturing method. It should be noted that the use of at least one anticoagulant is required during leukapheresis, as known in the art, to prevent clotting of the obtained cells.

[0079] In some embodiments, the leukapheresis procedure is configured to allow for the acquisition of a mononuclear enriched cell composition. In some embodiments, the mononuclear enriched cell harvest obtained by leukapheresis comprises at least 65% mononuclear cells. In other embodiments, the mononuclear enriched cell harvest obtained by leukapheresis comprises at least 70%, or at least 80% mononuclear cells. In some embodiments, plasma from the cell donor is acquired in parallel with the step of acquiring the mononuclear enriched cell composition. In some embodiments, the manufacturing method of the present disclosure acquires about 300-600 ml of plasma from the cell donor in parallel with the step of acquiring the mononuclear enriched cell composition.

[0080] In some embodiments, the apoptotic cells contained in the frozen formulation of the present disclosure comprise at least 85% mononuclear cells. In further embodiments, the apoptotic cells contained in the frozen formulation of the present disclosure comprise at least 85% mononuclear cells, 90% mononuclear cells, or more than 90% mononuclear cells. In some embodiments, the apoptotic cells contained in the frozen formulation of the present disclosure comprise at least 90% mononuclear cells. In some embodiments, the apoptotic cells contained in the frozen formulation of the present disclosure comprise at least 95% mononuclear cells.

[0081] It should be noted that in some embodiments, the mononuclear enriched cell preparation at the time of cell harvesting comprises at least 65%, preferably at least 70%, and most preferably at least 80% mononuclear cells, whereas the final pharmaceutical population obtained by the method for producing apoptotic cells used in the methods of the present disclosure comprises at least 85%, preferably at least 90%, and most preferably at least 95% mononuclear cells.

[0082] Those skilled in the art will appreciate that the term "mononuclear cells" encompasses white blood cells having a single lobed nucleus.

[0083] In some embodiments, the apoptotic cell preparation comprises no more than 15% CD15high In some embodiments, the frozen apoptotic cell preparation comprises cells expressing 15% or less CD15 high expressing cells (see Example 1, FIG. 12).

[0084] In some embodiments, the mononuclear enriched cell population comprises at least one cell type selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells. In some embodiments, the mononuclear enriched cell population comprises at least two cell types selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells. In some embodiments, the mononuclear enriched cell population comprises at least three cell types selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells.

[0085] In some embodiments, the mononuclear enriched cell population comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the mononuclear enriched cell population comprises a cell fraction isolated from PBMCs. In some embodiments, the mononuclear enriched cell population comprises one cell fraction isolated from PBMCs. In some embodiments, the mononuclear enriched cell population comprises two cell fractions isolated from PBMCs. In some embodiments, the mononuclear enriched cell population comprises at least one cell fraction isolated from PBMCs. In some embodiments, the mononuclear enriched cell population comprises at least two cell fractions isolated from PBMCs.

[0086] In some embodiments, the mononuclear enriched cell population comprises a single cell type selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells. In some embodiments, the mononuclear enriched cell population comprises two cell types selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells. In some embodiments, the mononuclear enriched cell population comprises three cell types selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells.

[0087] In some embodiments, the mononuclear enriched cell population comprises lymphocytes. In some embodiments, the mononuclear enriched cell population comprises monocytes. In some embodiments, the mononuclear enriched cell population comprises T cells. In some embodiments, the mononuclear enriched cell population comprises B cells. In some embodiments, the mononuclear enriched cell population comprises natural killer cells.

[0088] In some embodiments, the mononuclear enriched cell population comprises lymphocytes and monocytes.

[0089] In another embodiment, the mononuclear cell enriched cell population comprises polymorphonuclear leukocytes, also known as granulocytes (i.e., neutrophils, basophils, eosinophils), in an amount of 15% or less, alternatively 10% or less, typically 5% or less. In another embodiment, the pooled mononuclear cell population is devoid or essentially devoid of granulocytes. In another embodiment, the pooled apoptotic cell preparation disclosed herein comprises less than 5% polymorphonuclear leukocytes.

[0090] Functional analysis of the apoptotic cell population, in some embodiments, includes assaying the ability of the apoptotic cell population to reduce production of cytokines associated with a cytokine storm, including, but not limited to, any one or any combination of IL-6, tumor necrosis factor alpha (TNF-α), IL-1β, and interferon-γ (IFN-γ).

[0091] In some embodiments, the activity of the apoptotic cell population of the present disclosure includes, but is not limited to, inhibition of proinflammatory cytokine expression. In some embodiments, the activity of the apoptotic cell population of the present disclosure includes, but is not limited to, reduction of cytokine storm. In some embodiments, the activity of the apoptotic cell population of the present disclosure includes, but is not limited to, reduction of cytokine release syndrome. In some embodiments, the activity of the apoptotic cell population of the present disclosure includes, but is not limited to, reduction of cytokine storm, reduction of cytokine release syndrome, inhibition of expression of at least one proinflammatory cytokine, or any combination thereof.

[0092] In one embodiment, the apoptotic cells affect cytokine expression levels in macrophages. In another embodiment, the apoptotic cells reduce cytokine expression levels in macrophages. In one embodiment, the apoptotic cells suppress cytokine expression levels in macrophages. In one embodiment, the apoptotic cells inhibit cytokine expression levels in macrophages. In one embodiment, the apoptotic cells affect cytokine expression levels in dendritic cells. In another embodiment, the apoptotic cells reduce cytokine expression levels in dendritic cells. In one embodiment, the apoptotic cells suppress cytokine expression levels in dendritic cells. In one embodiment, the apoptotic cells inhibit cytokine expression levels in dendritic cells.

[0093] In some embodiments, the apoptotic cell population of the present disclosure inhibits IL-6 expression in macrophages. In some embodiments, the fresh apoptotic cell population exhibits inhibition of IL-6 expression in macrophages. In some embodiments, the fresh apoptotic cell population maintained at 4-8°C for up to 96 hours exhibits inhibition of IL-6 expression in macrophages. In some embodiments, the thawed apoptotic cell population exhibits inhibition of IL-6 expression in macrophages. In some embodiments, the thawed apoptotic cell population of the present disclosure thawed after freezing at about -196°C exhibits inhibition of IL-6 expression in macrophages.

[0094] In some embodiments, the apoptotic cell population of the present disclosure inhibits IL-6 expression in dendritic cells (DCs). In some embodiments, fresh apoptotic cell populations exhibit inhibition of IL-6 expression in DCs. In some embodiments, fresh apoptotic cell populations maintained at 4-8°C for up to 96 hours exhibit inhibition of IL-6 expression in DCs. In some embodiments, thawed apoptotic cell populations exhibit inhibition of IL-6 expression in DCs. In some embodiments, thawed apoptotic cell populations of the present disclosure thawed after freezing at about -196°C exhibit inhibition of L-6 expression in DCs.

[0095] In some embodiments, the apoptotic cell population of the present disclosure inhibits TNF-α expression in macrophages. In some embodiments, the fresh apoptotic cell population exhibits inhibition of TNF-α expression in macrophages. In some embodiments, the fresh apoptotic cell population maintained at 4-8° C. for up to 96 hours exhibits inhibition of TNF-α expression in macrophages. In some embodiments, the thawed apoptotic cell population exhibits inhibition of TNF-α expression in macrophages. In some embodiments, the thawed apoptotic cell population of the present disclosure thawed after freezing at about −196° C. exhibits inhibition of TNF-α expression in macrophages.

[0096] In some embodiments, the apoptotic cell population of the present disclosure inhibits TNF-α expression in dendritic cells (DCs). In some embodiments, the fresh apoptotic cell population exhibits inhibition of TNF-α expression in DCs. In some embodiments, the fresh apoptotic cell population maintained at 4-8° C. for up to 96 hours exhibits inhibition of TNF-α expression in DCs. In some embodiments, the thawed apoptotic cell population of the present disclosure exhibits inhibition of TNF-α expression in DCs. In some embodiments, the thawed apoptotic cell population of the present disclosure thawed after freezing at about −196° C. exhibits inhibition of TNF-α expression in DCs.

[0097] In some embodiments, the apoptotic cell population of the present disclosure inhibits INF-γ expression in macrophages. In some embodiments, the fresh apoptotic cell population exhibits inhibition of INF-γ expression in macrophages. In some embodiments, the fresh apoptotic cell population maintained at 4-8° C. for up to 96 hours exhibits inhibition of INF-γ expression in macrophages. In some embodiments, the thawed apoptotic cell population of the present disclosure exhibits inhibition of INF-γ expression in macrophages. In some embodiments, the thawed apoptotic cell population of the present disclosure thawed after freezing at about −196° C. exhibits inhibition of INF-γ expression in macrophages.

[0098] In some embodiments, the apoptotic cell population of the present disclosure inhibits INF-γ expression in dendritic cells (DCs). In some embodiments, the fresh apoptotic cell population exhibits inhibition of INF-γ expression in DCs. In some embodiments, the fresh apoptotic cell population maintained at 4-8° C. for up to 96 hours exhibits inhibition of INF-γ expression in DCs. In some embodiments, the thawed apoptotic cell population of the present disclosure exhibits inhibition of INF-γ expression in DCs. In some embodiments, the thawed apoptotic cell population of the present disclosure thawed after freezing at about −196° C. exhibits inhibition of INF-γ expression in DCs.

[0099] In some embodiments, the apoptotic cell population of the present disclosure inhibits IL-1β expression in macrophages. In some embodiments, the fresh apoptotic cell population exhibits inhibition of IL-1β expression in macrophages. In some embodiments, the fresh apoptotic cell population maintained at 4-8° C. for up to 96 hours exhibits inhibition of IL-1β expression in macrophages. In some embodiments, the thawed apoptotic cell population exhibits inhibition of IL-1β expression in macrophages. In some embodiments, the thawed apoptotic cell population of the present disclosure thawed after freezing at about −196° C. exhibits inhibition of IL-1β expression in macrophages.

[0100] In some embodiments, the apoptotic cell population of the present disclosure inhibits IL-1β expression in dendritic cells (DCs). In some embodiments, the fresh apoptotic cell population exhibits inhibition of IL-1β expression in DCs. In some embodiments, the fresh apoptotic cell population maintained at 4-8° C. for up to 96 hours exhibits inhibition of IL-1β expression in DCs. In some embodiments, the thawed apoptotic cell population exhibits inhibition of IL-1β expression in DCs. In some embodiments, the thawed apoptotic cell population of the present disclosure thawed after freezing at about −196° C. exhibits inhibition of IL-1β expression in DCs.

[0101] In some embodiments, the apoptotic cell populations of the present disclosure have activity in decreasing the expression of any of the cytokines / chemokines listed in Tables 1 and 2 below.

[0102] [Table 1]

[0103] In some embodiments, the cytokines Flt-3L, fractalkine, GM-CSF, IFN-γ, IL-1β, IL-2, IL-2Rα, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, and IL-13 of Table 1 are believed to be important in CRS or cytokine storm. In another embodiment, IFN-α, IFN-β, IL-1, and IL-1Rα of Table 1 are believed to be important in CRS or cytokine storm. In another embodiment, M-CSF is of unknown importance. In another embodiment, any of the cytokines listed in Table 1, or any combination thereof, can be used as a marker of apoptotic cell functionality.

[0104] [Table 2]

[0105] In one embodiment, IL-15, IL-17, IL-18, IL-21, IL-22, IP-10, MCP-1, MIP-1α, MIP-1β, and TNF-α from Table 2 are believed to be important in CRS or cytokine storm. In another embodiment, IL-27, MCP-3, PGE2, RANTES, TGF-β, TNF-αR1, and MIG from Table 2 are believed to be important in CRS or cytokine storm. In another embodiment, IL-23 and IL-25 are of unknown importance. In another embodiment, any of the cytokines listed in Table 2, or any combination thereof, can be used as a marker of CRS or cytokine storm and thus apoptotic cell activity. In another embodiment, the murine cytokines IL-10, IL-1β, IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15 / IL-15R, and IL-7 are believed to be important in CRS or cytokine storm.

[0106] Those of skill in the art will understand that the term "cytokine" encompasses cytokines (e.g., interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α), chemokines (e.g., MIP1α, MIP1β, RANTES), and other soluble inflammatory mediators such as reactive oxygen species and nitric oxide.

[0107] The potency of the apoptotic cell populations of the present disclosure, in some embodiments, is analyzed in a mouse cecal ligation and puncture model (CLP).The potency of the apoptotic cell populations of the present disclosure, prior to freezing, in some embodiments, is analyzed in a mouse cecal ligation and puncture model (CLP). The efficacy of the apoptotic cell populations of the present disclosure following thawing of frozen preparations is, in some embodiments, analyzed in a mouse cecal ligation and puncture model (CLP) (see, e.g., Mevorach, D., Zuckerman, T., Reiner, I., Shimoni, A., Samuel, S., Nagler, A., Rowe, JM, and Or, R. (2014). Single infusion of donor mononuclear apoptotic cells as prophylaxis for graft-versus-host disease in myeloablative HLA-matched allogeneic bone marrow transplantation: a phase I / IIa clinical trial. Biol. Blood Marrow Transplant. 20, 58-65; and Trahtemberg and Mevorach, 2017 ibid.).

[0108] In another embodiment, the effect of the apoptotic cells of the present disclosure on cytokine expression levels in macrophages, dendritic cells (DCs), or a combination thereof, results in a reduction in CRS. In another embodiment, the effect of the apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof, results in a reduction in severe CRS. In another embodiment, the effect of the apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof, results in suppression of CRS. In another embodiment, the effect of the apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof, results in suppression of severe CRS. In another embodiment, the effect of the apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof, results in inhibition of CRS. In another embodiment, the effect of the apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof, results in inhibition of severe CRS. In another embodiment, the effect of the apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof, results in prevention of CRS. In another embodiment, the effect of the apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof, results in prevention of severe CRS.

[0109] In some embodiments, administration of the apoptotic cells inhibits one or more proinflammatory cytokines. In some embodiments, the proinflammatory cytokines include IL-1β, IL-6, TNF-α, IFN-γ, or any combination thereof. In some embodiments, inhibition of one or more proinflammatory cytokines includes downregulation of the proinflammatory cytokines, reducing the amount of secretion of one or more proinflammatory cytokines.

[0110] In another embodiment, the apoptotic cells, i.e., fresh and / or thawed cells, of the present disclosure have the activity of inhibiting maturation of dendritic cells following exposure to TLR ligands.In another embodiment, the apoptotic cells, i.e., fresh and / or thawed cells, have the activity of potentially generating tolerogenic dendritic cells, which in some embodiments are capable of migrating, and in some embodiments, the migration is due to CCR7.

[0111] Frozen preparations

[0112] Apoptotic cell populations comprising a population of apoptotic mononuclear enriched cells are described in detail above. Provided herein are embodiments of frozen preparations comprising apoptotic cell populations.

[0113] In some embodiments, the frozen apoptotic cell preparation comprises a population of apoptotic mononuclear enriched cells, an isotonic crystalloid solution, a cryoprotectant, and has a pH in the range of about 6.5-8.0. In some embodiments, the population of apoptotic mononuclear enriched cells to be frozen is freshly prepared prior to freezing. In some embodiments, the population of apoptotic mononuclear enriched cells to be frozen is freshly prepared and stored on ice prior to freezing. In some embodiments, the population of apoptotic mononuclear enriched cells to be frozen is freshly prepared and stored at about 2-8°C prior to freezing.

[0114] It is important to analyze the apoptotic status before freezing the population of apoptotic mononuclear enriched cells. In some embodiments, the apoptotic mononuclear enriched cells before freezing comprise an early apoptotic cell population and a mature apoptotic cell population. In some embodiments, the apoptotic mononuclear enriched cells before freezing comprise at least 35% apoptotic cells and less than 30% late apoptotic cells. In some embodiments, the apoptotic mononuclear enriched cells before freezing comprise at least 35% AnV+ cells and less than 30% AnV+PI+ High In some embodiments, the fresh apoptotic cells comprise a population of enriched mononuclear apoptotic cells generated using methods known in the art.

[0115] The cell density at the time of freezing is important to ensure cell viability. Too high a density may reduce cell viability. Additionally, different cell types may require different densities at the time of freezing, which can be tested prior to freezing. In some embodiments, the concentration of the apoptotic mononuclear enriched cell population at the time of freezing is about 10 × 10 6 ~250×10 6 In some embodiments, the concentration of the apoptotic mononuclear enriched cell population upon freezing is about 20×10 6 ~100×10 6 In some embodiments, the concentration of the apoptotic mononuclear enriched cell population upon freezing is about 20-60×10 cells / ml. 6 In some embodiments, the concentration of the apoptotic mononuclear enriched cell population upon freezing is about 20-250×10 cells / ml. 6 In some embodiments, the concentration of the apoptotic mononuclear enriched cell population upon freezing is about 25-300×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 24-50×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 24-40×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 44-50×10 cells / ml. 6 In some embodiments, the density of the population of apoptotic mononuclear enriched cells upon freezing is about 24-35×10 cells / ml. 6 In some embodiments, the density of the population of apoptotic mononuclear enriched cells upon freezing is about 20-40×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 20-100×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 20-35×10 cells / ml. 6In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 25-35×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 35-45×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 35-40×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 40-50×10 cells / ml. 6 In some embodiments, the density of the apoptotic mononuclear enriched cell population upon freezing is about 40-60×10 cells / ml. 6 Cells / ml.

[0116] As discussed above, the mononuclear enriched cells that produce the apoptotic cells to be frozen, in some embodiments, comprise at least one cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells. In some embodiments, the mononuclear enriched cells that produce the apoptotic cells to be frozen comprise PBMCs. In some embodiments, the mononuclear enriched cells that produce the apoptotic cells to be frozen comprise a fraction of PBMCs that comprises at least one cell type. In some embodiments, the mononuclear enriched cells that produce the apoptotic cells to be frozen comprise a fraction of PBMCs that comprises at least two cell types. In some embodiments, the mononuclear enriched cells that produce the apoptotic cells to be frozen comprise a single cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

[0117] In some embodiments, the frozen preparation of apoptotic mononuclear enriched cells comprises a pooled mononuclear apoptotic cell preparation. In some embodiments, the pooled mononuclear apoptotic cell preparation comprises mononuclear cells in an apoptotic state, the pooled mononuclear apoptotic cells comprising a reduced proportion of viable non-apoptotic cells, an inhibition of cellular activation of viable non-apoptotic cells, a reduction in proliferation of viable non-apoptotic cells, or any combination thereof. In some embodiments, the pooled mononuclear enriched apoptotic cells are irradiated. In some embodiments, the pooled mononuclear enriched apoptotic cell preparation is derived from a white blood cell fraction (WBC) obtained from a blood donation.

[0118] Apoptotic mononuclear enriched cell populations for freezing can be produced by any method known in the art, including, but not limited to, inducing apoptosis in mononuclear cell populations using methylprednisolone, as described in detail in Example 14 of International Publication No. WO2014 / 087408, Example 1 of International Publication No. WO2019 / 038758, and Examples 1, 2, 3, 5, and 6 of International Publication No. WO2016 / 170541 (the above patent documents are incorporated by reference in their entireties).

[0119] Crystalloid fluids, or crystalloid solutions, comprise aqueous solutions of mineral salts and other small water-soluble molecules. Crystalloid solutions, in some embodiments, are isotonic with respect to human plasma. In some embodiments, crystalloid solutions approximate the concentrations of various solutes found in plasma and exert no osmotic effect in vivo. Crystalloid solutions function to increase intravascular volume without perturbing ion concentrations or causing significant fluid shifts between intracellular, intravascular, and interstitial spaces. In some embodiments, frozen preparations of apoptotic cells of the present disclosure comprise isotonic crystalloid solutions.

[0120] In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells is selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2), sodium acetate (NaCH3COO (also abbreviated as NaOAc)), sodium gluconate (NaCH6H6COO), sodium phosphate (NaPH ... 11 In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises sodium chloride (NaCl). In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises potassium chloride (KCl). In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises magnesium chloride (MgCl2). In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises sodium acetate (NaCH3COO (also abbreviated as NaOAc)). In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises sodium gluconate (NaC6H 11 O7).

[0121] In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises 140 mmol / L sodium, 5 mmol / L potassium, 1.5 mmol / L magnesium, 98 mmol / L chloride, 27 mmol / L acetate, or 23 mmol / L gluconate. In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises PLASMA-LYTE 148 (Baxter, USA), also known as Normasol® (Molnlycke, Sweden).

[0122] In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises normal saline (0.9% NaCl solution), lactated Ringer's / Hartmann's solution (lactate buffer solution), acetate buffer solution, acetate and lactate buffer solution, acetate and gluconate buffer, 5% glucose in water, 10% glucose in water, or any combination thereof. In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises normal saline (0.9% NaCl solution). In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises lactated Ringer's / Hartmann's solution (lactate buffer). In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises acetate buffer solution. In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises acetate and lactate buffer solution. In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises acetate and gluconate buffer solution. In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises a 5% dextrose solution in water. In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells comprises a 10% dextrose solution in water.

[0123] In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells is serum-free. In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells is protein-free. In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells is calcium (Ca ++ In some embodiments, the isotonic crystalloid solution present in the frozen preparation of apoptotic mononuclear cells does not contain an antimicrobial agent.

[0124] It is important to ensure the proper acid-base balance of the frozen preparation. In some embodiments, the pH of the frozen preparation affects the quality of the cells when thawed. Maintaining the proper low-temperature dependent ion ratio, controlling pH at low temperature, and preventing the formation of free radicals are important factors during the cryopreservation of cells and affect the quality of the cells when thawed.

[0125] In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 6.5-8.0. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.0-8.0. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 6.5-7.5. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.0-7.5. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.5-8.0. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the frozen preparation comprising apoptotic mononuclear enriched cells has a pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0126] In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 6.5. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 6.6. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 6.7. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 6.8. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 6.9. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.0. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.1. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.2. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.3. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.4. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.5. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.6. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.7. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.8. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 7.9. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of about 8.0.

[0127] In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 6.5. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 6.6. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 6.7. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 6.8. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 6.9. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.0. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.1. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.2. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.3. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.4. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.5. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.6. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.7. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.8. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 7.9. In some embodiments, the frozen formulation comprising apoptotic mononuclear enriched cells has a pH of 8.0.

[0128] Cryoprotectants are used during cryopreservation to prevent cell damage due to freezing, e.g., damage due to ice formation. It is important that the cryoprotectant is non-toxic or has low toxicity, can easily permeate cell membranes, and can bind electrolytes (to increase their concentration during the freezing process) or water molecules (to retard freezing).

[0129] In some embodiments, the cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells comprises glycerol, ethylene glycol, propylene glycol, or dimethyl sulfoxide (DMSO). In some embodiments, the cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells comprises glycerol. In some embodiments, the cryoprotectant present in the frozen preparation of apoptotic mononuclear cells comprises ethylene glycol. In some embodiments, the cryoprotectant present in the frozen preparation of apoptotic mononuclear cells comprises propylene glycol. In some embodiments, the cryoprotectant present in the frozen preparation of apoptotic mononuclear cells comprises dimethyl sulfoxide (DMSO). Commercially available cryoprotectants include, but are not limited to, PentaHibe® (Pharmacosmos, Denmark), Cryostor® CS5 or Cryostor® CD10 (Merck KGaA, Darmstadt, Germany), and mFreSR™ (Stemcell Technologies, USA).

[0130] Typically, a concentration of about 5-15% (vol / vol) of cryoprotectant is required to keep a significant proportion of isolated cells alive after freezing and thawing from liquid nitrogen temperatures (-196°); however, surprisingly, as exemplified in Example 1, a significant proportion of apoptotic mononuclear enriched cells was able to survive and maintain function using concentrations of cryoprotectant less than 5%.

[0131] In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells ranges from about 2.0-10% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells ranges from about 2.5-10% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells ranges from about 2.5-5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells ranges from about 5-10% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells ranges from about 5-15% (volume / volume).

[0132] In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 2.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 2.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 3.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 3.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 4.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 4.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 5.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 5.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 6.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 6.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 7.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 7.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is less than 8.0% (volume / volume).In some embodiments, the concentration (percent (%)) of cryoprotectant present in a frozen preparation of apoptotic mononuclear enriched cells is less than 8.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in a frozen preparation of apoptotic mononuclear enriched cells is less than 9.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in a frozen preparation of apoptotic mononuclear enriched cells is less than 9.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in a frozen preparation of apoptotic mononuclear enriched cells is less than 10% (volume / volume).

[0133] In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 2.0% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 2.5% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 3.0% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 3.5% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 4.0% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 4.5% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 5.0% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 5.5% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 6.0% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 6.5% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 7.0% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 7.5% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 8.0% (volume / volume) or less.In some embodiments, the concentration (percent (%)) of cryoprotectant present in a frozen preparation of apoptotic mononuclear enriched cells is about 8.5% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in a frozen preparation of apoptotic mononuclear enriched cells is about 9.0% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in a frozen preparation of apoptotic mononuclear enriched cells is about 9.5% (volume / volume) or less. In some embodiments, the concentration (percent (%)) of cryoprotectant present in a frozen preparation of apoptotic mononuclear enriched cells is about 10.0% (volume / volume) or less.

[0134] In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 2.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 2.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 3.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 3.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 4.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 4.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 5.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 5.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 6.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 6.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 7.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 7.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 8.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 8.5% (volume / volume).In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 9.0% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 9.5% (volume / volume). In some embodiments, the concentration (percent (%)) of cryoprotectant present in the frozen preparation of apoptotic mononuclear enriched cells is about 10% (volume / volume).

[0135] In some embodiments, the cryoprotectant comprises DMSO, and the DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% (volume / volume). In some embodiments, the cryoprotectant comprises DMSO, and the DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 2.0%. In some embodiments, the DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 2.5%. In some embodiments, the DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 3.0%. In some embodiments, the DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 3.5%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 4.0%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 4.5%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 5.0%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 5.5%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 6.0%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 6.5%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 7.0%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 7.5%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 8.0%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 8.5%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 9.0%.In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 9.5%. In some embodiments, DMSO is included in the frozen preparation of apoptotic mononuclear enriched cells at a concentration of about 10.0% (volume / volume).

[0136] Manufacture of frozen preparations

[0137] The methods of making the frozen preparations of the present disclosure relate to freezing of apoptotic mononuclear enriched cell populations, as described herein.

[0138] In some embodiments, a method for producing a frozen apoptotic cell preparation includes (a) a resuspension step in which a population of fresh apoptotic mononuclear enriched cells is resuspended in an isotonic crystalloid solution having a pH of about 6.5-8.0; (b) a mixing step in which the population of apoptotic mononuclear enriched cells resuspended in the resuspension step (a) is mixed with a cryoprotectant; and (c) a freezing step in which the mixture produced in the mixing step (b) is gradually frozen to a temperature of about -196°C.

[0139] In some embodiments, the apoptotic mononuclear enriched cell population used in the method for producing the frozen preparation comprises an apoptotic mononuclear enriched cell population independent of the method for inducing apoptosis.In some embodiments, the apoptotic mononuclear enriched cell population used in the method for producing the frozen preparation comprises an apoptotic mononuclear enriched cell population produced using methylprednisolone for inducing apoptosis, as described in detail in Example 14 of International Publication No. WO2014 / 087408, Example 1 of International Publication No. WO2019 / 038758, and Examples 1, 2, 3, 5, and 6 of International Publication No. WO2016 / 170541 (the above patent documents are incorporated herein by reference in their entirety).

[0140] In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises at least one cell type selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises at least two cell types selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises at least three cell types selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells.

[0141] In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises a cell fraction isolated from PBMCs. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises one cell fraction isolated from PBMCs. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises two cell fractions isolated from PBMCs. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises at least one cell fraction isolated from PBMCs. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of producing a frozen formulation comprises at least two cell fractions isolated from PBMCs.

[0142] In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen formulation comprises a single cell type selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen formulation comprises two cell types selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen formulation comprises three cell types selected from the group consisting of lymphocytes, monocytes, T cells, B cells, and natural killer cells.

[0143] In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen formulation comprises lymphocytes. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen formulation comprises monocytes. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen formulation comprises T cells. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen formulation comprises B cells. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen formulation comprises natural killer cells.

[0144] In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making the frozen preparation comprises lymphocytes and monocytes.

[0145] In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen preparation comprises polymorphonuclear leukocytes, also known as granulocytes (i.e., neutrophils, basophils, eosinophils), in an amount of 15% or less, alternatively 10% or less, typically 5% or less. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen preparation comprises a pooled mononuclear cell population ... that is devoid or essentially devoid of granulocytes. In some embodiments, the apoptotic mononuclear enriched cell population used in the method of making a frozen preparation comprises a pooled mononuclear cell population that contains less than 5% polymorphonuclear leukocytes.

[0146] In some embodiments of the method of producing a frozen preparation, the mononuclear enriched cells comprise at least one cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

[0147] In some embodiments of producing a frozen preparation, the at least one cell type comprises a single cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

[0148] FIG. 2 is a flow chart comparing the methods of manufacturing fresh and frozen formulations containing apoptotic mononuclear enriched cells. In both manufacturing methods, once apoptosis induction is complete, the cells are resuspended in an isotonic crystalloid solution. In formulations used fresh and those used after storage at about 4-8° C. (up to about 96 hours), the isotonic crystalloid solution used is Ringer's lactate solution. In some embodiments, Plasma-Lyte was used as the isotonic crystalloid solution used with the frozen apoptotic cell population, as shown in FIG.

[0149] In some embodiments for producing frozen preparations of apoptotic mononuclear enriched cell populations, the isotonic crystalloid solution used for resuspension is selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2), sodium acetate (NaCH3COO (also abbreviated as NaOAc)), sodium gluconate (NaCH6H6COO), and sodium phosphate (NaPH6H6COO). 11 In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation comprises sodium chloride (NaCl). In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation comprises potassium chloride (KCl). In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation comprises magnesium chloride (MgCl2). In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation comprises sodium acetate (NaCH3COO (also abbreviated as NaOAc)). In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation comprises sodium gluconate (NaC6H 11 In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation includes sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2), sodium acetate (NaCH3COO (also abbreviated as NaOAc)), and sodium gluconate (NaCH6H6COO). 11 O7) any combination.

[0150] In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation comprises 140 mmol / L sodium, 5 mmol / L potassium, 1.5 mmol / L magnesium, 98 mmol / L chloride, 27 mmol / L acetate, and 23 mmol / L gluconate. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation comprises PLASMA-LYTE 148 (Baxter, USA), also known as Normasol® (Molnlycke, Sweden).

[0151] In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation comprises normal saline (0.9% NaCl solution), lactated Ringer's / Hartmann's solution (lactate buffer solution), acetate buffer solution, acetate and lactate buffer solution, acetate and gluconate buffer, 5% glucose in water, 10% glucose in water, or any combination thereof. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation comprises normal saline (0.9% NaCl solution). In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation comprises lactated Ringer's / Hartmann's solution (lactate buffer solution). In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation comprises an acetate buffer solution. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation comprises an acetate and lactate buffer solution. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation comprises an acetate and gluconate buffer solution. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation comprises a 5% dextrose solution in water. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation comprises a 10% dextrose solution in water.

[0152] In some embodiments, the isotonic crystalloid solution used in the method of producing a frozen preparation of apoptotic mononuclear cells is serum-free. In some embodiments, the isotonic crystalloid solution used in the method of producing a frozen preparation of apoptotic mononuclear cells is protein-free. In some embodiments, the isotonic crystalloid solution used in the method of producing a frozen preparation of apoptotic mononuclear cells is calcium (Ca ++ In some embodiments, the isotonic crystalloid solution used in the method of making a frozen preparation of apoptotic mononuclear cells does not contain an antimicrobial agent.

[0153] In some embodiments, the isotonic crystalloid solution used in the method of producing a frozen preparation of apoptotic mononuclear cells has a pH of about 6.5-8.0. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen preparation has a pH of about 7.0-8.0. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen preparation has a pH of about 6.5-7.5. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen preparation has a pH of about 7.0-7.5. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen preparation has a pH of about 7.5-8.0. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen preparation has a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the frozen preparation comprising apoptotic mononuclear enriched cells has a pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0154] In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 6.5. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 6.6. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 6.7. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 6.8. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 6.9. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 7.0. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 7.1. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 7.2. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of about 7.3. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of about 7.4. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of about 7.5. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of about 7.6. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of about 7.7. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of about 7.8. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of about 7.9. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of about 8.0.

[0155] In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 6.5. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 6.6. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 6.7. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 6.8. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 6.9. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 7.0. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 7.1. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 7.2. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of making a frozen formulation has a pH of 7.3. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of 7.4. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of 7.5. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of 7.6. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of 7.7. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of 7.8. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of 7.9. In some embodiments, the isotonic crystalloid solution used for resuspension in the method of producing a frozen formulation has a pH of 8.0.

[0156] In some embodiments of the method for producing a frozen preparation of apoptotic mononuclear enriched cells, the step of resuspending in an isotonic crystalloid solution includes volume reduction. For example, the volume reduction and medium exchange steps can be performed by the LOVO Cell Processing System (Fresenius Kabi, USA). The LOVO Cell Processing System is an automated, closed-kit cell washing and processing device that pumps the cell solution through a spinning membrane filtration device, allowing for rapid fluid management and fast, efficient cell processing. The processing process performed by the LOVO Cell Processing System includes a series of washing cycles, including volume reduction, followed by cell resuspension in a new selectable solution. This processing process results in a final product that includes the original cells and the new selectable solution. The use of spinning membrane technology results in high-speed continuous flow and non-pelleting cell processing.

[0157] In some embodiments, the resuspension step, including volume reduction, results in approximately 10-150×10 6 A population of apoptotic mononuclear enriched cells is obtained having a cell concentration of about 50-80 million cells / ml. In some embodiments, the resuspension step with volume reduction results in a population of apoptotic mononuclear enriched cells having a cell concentration of about 20-150×10 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 20-100×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 20-100×10 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 25-100×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 25-100×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 25-80×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 25-80×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 25-60×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 25-60×10 cells / ml. 6A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 25-40×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 25-40×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 20-60×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 20-60×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 25-50×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 25-50×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 25-40×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 25-40×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 44-50×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 44-50×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 24-35×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 24-35×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 20-40×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 20-40×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 20-35×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 20-35×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 25-35×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 25-35×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 35-45×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 35-45×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 35-40×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 35-40×10 cells / ml. 6A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 40-50×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 40-50×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of about 40-60×10 cells / ml. In some embodiments, the resuspension step, including volume reduction, results in a population of apoptotic mononuclear enriched cells with a cell concentration of about 40-60×10 cells / ml. 6 A population of apoptotic mononuclear enriched cells is obtained with a cell concentration of cells / ml.

[0158] In some embodiments, the resuspension step, including volume reduction, results in approximately 10×10 9 In some embodiments, a population of apoptotic mononuclear enriched cells with ±20% cells is obtained. In some embodiments, a resuspension step including volume reduction results in a population of 8-12×10 9 In some embodiments, a population of apoptotic mononuclear enriched cells is obtained with ±20% cells. 9 In some embodiments, a population of apoptotic mononuclear enriched cells with ±20% cells is obtained. In some embodiments, a resuspension step including volume reduction results in a population of 8-12×10 9 In some embodiments, a population of apoptotic mononuclear enriched cells is obtained with ±20% cells. In some embodiments, a resuspension step including volume reduction results in a population of apoptotic mononuclear enriched cells with ±8×10 cells at the time of freezing. 9 In some embodiments, a population of apoptotic mononuclear enriched cells is obtained with ±20% cells. In some embodiments, a resuspension step including volume reduction results in a population of apoptotic mononuclear enriched cells with ±9×10 cells at the time of freezing. 9 In some embodiments, a population of apoptotic mononuclear enriched cells is obtained with ±20% cells. 9 In some embodiments, a population of apoptotic mononuclear enriched cells is obtained with ±20% cells. In some embodiments, a resuspension step including volume reduction results in a population of apoptotic mononuclear enriched cells with ±11×10 cells at the time of freezing. 9 In some embodiments, a population of apoptotic mononuclear enriched cells is obtained with ±20% cells. In some embodiments, a resuspension step including volume reduction results in a population of apoptotic mononuclear enriched cells with ±12×10 cells at the time of freezing. 9 A population of apoptotic mononuclear enriched cells with ±20% of the cells is obtained.

[0159] In some embodiments, after resuspension, the resuspended apoptotic mononuclear enriched cells are mixed with a cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is 1:1 (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is such that the cryoprotectant is in the range of about 2.0-15% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is such that the cryoprotectant is in the range of about 2.5-10% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is such that the cryoprotectant is in the range of about 2.5-5% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is such that the cryoprotectant is in the range of about 5-10% (volume / volume). In some embodiments, the mixing ratio of resuspended apoptotic mononuclear enriched cells to cryoprotectant is such that the percentage of cryoprotectant ranges from about 5-15% (volume / volume).

[0160] In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 2.0% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 2.5% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 3.0% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 3.5% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 4.0% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 4.5% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 5.0% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 5.5% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 6.0% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 6.5% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 7.0% (volume / volume). In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is less than 7.5% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio that results in a cryoprotectant percentage of less than 8.0% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio that results in a cryoprotectant percentage of less than 8.5% (volume / volume).In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio of less than 9.0% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio of less than 9.5% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio of less than 10% (volume / volume).

[0161] In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 2.0% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 2.5% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 3.0% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 3.5% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 4.0% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 4.5% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 5.0% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 5.5% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 6.0% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 6.5% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 7.0% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 7.5% (volume / volume) or less of the cryoprotectant. In some embodiments, the mixture ratio of resuspended apoptotic mononuclear enriched cells to cryoprotectant is such that the percentage of cryoprotectant is about 8.0% (volume / volume) or less.In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 8.5% (volume / volume) or less of cryoprotectant. In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 9.0% (volume / volume) or less of cryoprotectant. In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 9.5% (volume / volume) or less of cryoprotectant. In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 10% (volume / volume) or less of cryoprotectant.

[0162] In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 2.0% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 2.5% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 3.0% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 3.5% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 4.0% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 4.5% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 5.0% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 5.5% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 6.0% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 6.5% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 7.0% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with the cryoprotectant at a ratio of about 7.5% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio of about 8.0% (volume / volume) cryoprotectant. In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio of about 8.5% (volume / volume) cryoprotectant.In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio of about 9.0% (volume / volume) cryoprotectant. In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio of about 9.5% (volume / volume) cryoprotectant. In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with cryoprotectant at a ratio of about 10% (volume / volume) cryoprotectant.

[0163] In some embodiments, the cryoprotectant used in the method of producing a frozen preparation of apoptotic mononuclear enriched cells comprises glycerol, ethylene glycol, propylene glycol, or dimethyl sulfoxide (DMSO). In some embodiments, the cryoprotectant used in the method of producing a frozen preparation of apoptotic mononuclear enriched cells comprises glycerol. In some embodiments, the cryoprotectant used in the method of producing a frozen preparation of apoptotic mononuclear enriched cells comprises ethylene glycol. In some embodiments, the cryoprotectant used in the method of producing a frozen preparation of apoptotic mononuclear enriched cells comprises propylene glycol. In some embodiments, the cryoprotectant used in the method of producing a frozen preparation of apoptotic mononuclear enriched cells comprises dimethyl sulfoxide (DMSO). In some embodiments, the cryoprotectant used in the method of producing a frozen preparation of apoptotic mononuclear enriched cells comprises a cryoprotectant contained in a solution.

[0164] In some embodiments, when the cryoprotectant contains DMSO, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 2-10% (volume / volume) of DMSO. In some embodiments, when the cryoprotectant contains DMSO, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 2.5-10% (volume / volume) of DMSO. In some embodiments, when the cryoprotectant contains DMSO, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 2-5% (volume / volume) of DMSO. In some embodiments, when the cryoprotectant contains DMSO, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 2.5-5% (volume / volume) of DMSO. In some embodiments, when the cryoprotectant contains DMSO, the mixing ratio of the resuspended apoptotic mononuclear enriched cells to the cryoprotectant is about 5-10% (volume / volume) of DMSO.

[0165] In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with a cryoprotectant containing DMSO at a ratio where the DMSO content is less than 10% (volume / volume). In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with a cryoprotectant containing DMSO at a ratio where the DMSO content is less than 9.5%, 8.0%, 8.5%, 7.0%, 7.5%, 6.0%, 6.5%, 5.0%, 5.5%, 4.0%, 4.5%, 3.0%, 3.5%, 2.0%, or 2.5% (volume / volume).

[0166] In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with a cryoprotectant containing DMSO at a ratio of 10% (volume / volume) or less of DMSO. In some embodiments, the resuspended apoptotic mononuclear enriched cells are mixed with a cryoprotectant containing DMSO at a ratio of 9.5%, 8.0%, 8.5%, 7.0%, 7.5%, 6.0%, 6.5%, 5.0%, 5.5%, 4.0%, 4.5%, 3.0%, 3.5%, 2.0%, or 2.5% (volume / volume) or less of DMSO.

[0167] In some embodiments, the mixing ratio of resuspended apoptotic mononuclear enriched cells and cryoprotectant containing DMSO is such that the percentage of DMSO is 2.5% (volume / volume) or less than 2.5% (volume / volume). Those skilled in the art will understand that although DMSO is described to show various percentage concentrations of cryoprotectant, other cryoprotectants can be used instead of DMSO to obtain a given percentage concentration of cryoprotectant.

[0168] FIG. 2 shows an embodiment of a method for producing a fresh apoptotic mononuclear enriched cell population and a cryopreserved (cryopreserved) apoptotic mononuclear enriched cell population. In the figure, the apoptotic mononuclear enriched cell population, designated Allocetra-OTS DP (Allocetra Off the shelf Drug Product), can be frozen in multiple bags designed for this purpose. In another embodiment, the frozen Allocetra preparation (apoptotic mononuclear enriched cell population) can be cryopreserved in four bags of 100 ml each (for a total cryopreservation volume of 400 ml). In some embodiments, the frozen apoptotic mononuclear enriched cell preparation can be cryopreserved in a volume of about 10-250×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 20-250×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 20-300×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 25-300×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 25×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 20-30×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 25-40×10 6In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 25-60×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 25-80×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 25-100×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 25-125×10 6 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has a concentration of about 25-150×10 6 With a concentration of ±20% cells / ml.

[0169] In some embodiments, the frozen apoptotic mononuclear enriched cell preparation comprises 10×10 9 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation comprises about 8-12×10 cells. 9 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has about 8×10 9 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has about 9×10 9 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has about 10×10 9 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has about 11×10 9 In some embodiments, the frozen apoptotic mononuclear enriched cell preparation has about 12×10 9 ±20% of the cells.

[0170] In a method for producing a frozen preparation of apoptotic mononuclear enriched cells, the rate of cooling of apoptotic cells resuspended in an isotonic crystalloid solution and containing a cryoprotectant affects the viability and function of the apoptotic cells upon thawing. Additionally, the duration of storage at a final temperature of -196°C also affects the viability and function of the apoptotic cells upon thawing.

[0171] In some embodiments of the method for producing a frozen preparation of apoptotic mononuclear enriched cells, the stepwise freezing step comprises stepwise freezing at gradually decreasing temperatures to reach a temperature of about -196°C. For example, in some embodiments, immediately after the cells are placed in the bag, the bag is placed in a -20°C freezer (placed in a pre-chilled aluminum cassette) for 2 hours, then the bag is placed in a -80°C freezer for 2 hours, after which the bag is transferred to a liquid nitrogen tank for long-term storage. Thus, one of skill in the art will appreciate that gradually freezing an apoptotic preparation comprises a stepwise freezing process starting at -20°C, followed by stepwise decreasing temperatures to -80°C, followed by -196°C.

[0172] In some embodiments, after the apoptosis induction step, viable non-apoptotic cells (apoptosis resistant) may remain (Figure 2). In some embodiments, the presence of these viable non-apoptotic cells is observed before the irradiation step. These viable non-apoptotic cells may be expanded or activated. In some embodiments, a pooled mononuclear apoptotic cell preparation derived from multiple donors and containing viable non-apoptotic cells may be activated against the host, against each other, or against both. Thus, in some embodiments, the cells may be irradiated before the step of adding the cryoprotectant, as shown in Figure 2. Irradiation of cell preparations is considered safe in the art. Currently, irradiation treatment is routinely performed on donated blood to prevent reactions against WBCs.

[0173] The method of producing a frozen preparation of apoptotic mononuclear enriched cells, in some embodiments, comprises the step of irradiating the apoptotic cell population after resuspending the cells in an isotonic crystalloid solution.

[0174] In some embodiments, when the method for producing a frozen preparation of apoptotic mononuclear enriched cells includes irradiating the apoptotic cell population with radiation, the irradiated apoptotic mononuclear enriched cells have reduced cell activation or proliferation compared to a non-irradiated cell preparation. In another embodiment, the irradiation in the irradiation step includes gamma irradiation or UV irradiation. In some embodiments, when the method for producing a frozen preparation of apoptotic mononuclear enriched cells includes irradiating the apoptotic cell population with radiation, the irradiated apoptotic cell population has a reduced number of non-apoptotic cells compared to a non-irradiated cell preparation. In another embodiment, the irradiation step in the method for producing a frozen preparation of the present disclosure includes irradiation at a dose of about 15 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen preparation of the present disclosure includes irradiation at a dose of about 20 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen preparation of the present disclosure includes irradiation at a dose of about 25 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen preparation of the present disclosure includes irradiation at a dose of about 30 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen formulation of the present disclosure includes irradiation at a dose of about 35 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen formulation of the present disclosure includes irradiation at a dose of about 40 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen formulation of the present disclosure includes irradiation at a dose of about 45 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen formulation of the present disclosure includes irradiation at a dose of about 50 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen formulation of the present disclosure includes irradiation at a dose of about 55 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen formulation of the present disclosure includes irradiation at a dose of about 60 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen formulation of the present disclosure includes irradiation at a dose of about 65 Gray (Gy). In another embodiment, the irradiation step in the method for producing a frozen formulation of the present disclosure includes irradiation at a dose of up to 2500 Gray (Gy).In another embodiment, the irradiated pooled apoptotic cell preparation maintains the same or similar apoptotic profile, stability, and potency as the non-irradiated pooled apoptotic cell preparation.

[0175] In some embodiments of the method of producing a frozen preparation of apoptotic cells, irradiating comprises irradiating the apoptotic cell population with radiation to reduce proliferation and / or activation of remaining viable cells in the apoptotic cell population. In some embodiments of the method of producing a frozen preparation of apoptotic cells, the apoptotic mononuclear enriched cells are irradiated to reduce the percentage of viable non-apoptotic cells in the population. In some embodiments of the method of producing a frozen preparation, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 65% of the population. In some embodiments of the method of producing a frozen preparation, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 40% of the population. In some embodiments of the method of producing a frozen preparation, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 30% of the population. In some embodiments of the method of producing a frozen preparation, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 20% of the population. In some embodiments of the method of producing a frozen preparation, the percentage of viable non-apoptotic cells in the inactivated apoptotic cell population is reduced to less than 10% of the population. In some embodiments of the method of producing a frozen preparation, the percentage of viable, non-apoptotic cells in the population of inactivated apoptotic cells is reduced to 0% of the population.

[0176] In another embodiment of the method of producing a frozen population, the irradiated apoptotic mononuclear enriched cells retain all of their apoptotic, immunomodulatory and stability properties. In another embodiment of the method of producing a frozen population, the irradiating step uses UV radiation. In another embodiment, the irradiating step uses gamma radiation. Another embodiment of the method of producing a frozen population includes reducing the percentage of viable non-apoptotic cells in the apoptotic cell preparation, inhibiting cellular activation of viable non-apoptotic cells in the apoptotic cell preparation, reducing proliferation of viable non-apoptotic cells in the apoptotic mononuclear enriched cell preparation, or any combination thereof.

[0177] In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) compared to non-irradiated apoptotic cells. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 1% compared to non-irradiated apoptotic cells. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 2% compared to non-irradiated apoptotic cells. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 3% compared to non-irradiated apoptotic cells. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 4% compared to non-irradiated apoptotic cells. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 5% compared to the apoptotic cells that were not irradiated. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 6% compared to the apoptotic cells that were not irradiated. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 7% compared to the apoptotic cells that were not irradiated. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 8% compared to the apoptotic cells that were not irradiated. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 9% compared to the apoptotic cells that were not irradiated.In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 10% compared to apoptotic cells that were not irradiated. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 15% compared to apoptotic cells that were not irradiated. In some embodiments of the method for producing a frozen preparation, the irradiated apoptotic cells do not increase the population of dead cells (secondary necrosis; PI+) by more than about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to apoptotic cells that were not irradiated.

[0178] In some embodiments, the method of producing a frozen preparation of apoptotic mononuclear enriched cells further comprises irradiating the apoptotic cell population after the resuspension step and prior to the mixing step with the cryoprotectant.

[0179] Treatment using frozen preparations

[0180] The advantage of a frozen preparation containing apoptotic mononuclear enriched cells is that a therapeutic population of apoptotic cells can be made ready for use in treating a subject (patient).

[0181] One of skill in the art will appreciate that the apoptotic state of fresh frozen apoptotic cells may be the same as or may be slightly different from that of frozen and thawed apoptotic cells. Importantly, the frozen populations of apoptotic mononuclear enriched cells of the present disclosure maintain their biological activity (potency), as exemplified in Example 1.

[0182] In some embodiments, a method of treating a subject (patient) using a frozen apoptotic cell preparation of the present disclosure comprises slowly thawing the frozen preparation and then administering the thawed preparation to the subject.

[0183] In some embodiments of the method of treating a subject (patient) using a frozen apoptotic cell preparation of the present disclosure, the step of thawing the frozen apoptotic cell preparation comprises thawing the frozen preparation by placing one or more bags containing the frozen apoptotic cell preparation in a preheated 37° C. water bath and gently agitating until no ice particles remain. In some embodiments of the method of treating a subject using a frozen apoptotic cell preparation of the present disclosure, the step of thawing the frozen preparation comprises thawing the frozen preparation using a dry thawing method until no ice particles remain.

[0184] In some embodiments, the method of treating a subject using a frozen apoptotic cell preparation of the present disclosure comprises resuspending a thawed apoptotic mononuclear cell population in a media formulation for administration. In some embodiments, the thawed apoptotic mononuclear cell preparation is resuspended in an appropriate physiological buffer. In some embodiments, the thawed apoptotic mononuclear cell population is thawed and administered without requiring alteration of the formulation in which the cells were frozen.

[0185] In some embodiments, the method of treating a subject comprises administering to the subject a thawed autologous apoptotic mononuclear enriched cell population. In some embodiments, the method of treating a subject comprises administering to the subject a thawed allogeneic apoptotic cell population.

[0186] In some embodiments, the administration comprises a single injection of the thawed apoptotic cell population, hi some embodiments, the administration comprises multiple injections of the thawed apoptotic cell population.

[0187] In some embodiments, the thawed apoptotic cells are administered as a prophylactic to a subject (patient) previously determined to be at risk for a disease, disorder, or condition. In some embodiments, the thawed apoptotic cells are administered as a prophylactic to a subject who is asymptomatic for a disease, disorder, or condition. In some embodiments, the thawed apoptotic cells are administered to a subject who is exhibiting mild, moderate, severe, or critical symptoms of a disease, disorder, or condition. In some embodiments, the thawed apoptotic cells are administered as a prophylactic to an asymptomatic subject to prevent, reduce the risk of, or delay the onset of mild, moderate, severe, or critical symptoms of a disease, disorder, or condition.

[0188] In some embodiments, the method of treating a subject (patient) in need of treatment comprises treating, preventing, ameliorating, inhibiting, or reducing the incidence in the subject of at least one symptom associated with an immune disease, an autoimmune disease, a cytokine release syndrome (CRS), a cytokine storm, an inflammatory disease, a cancer, osteoarthritis, a vanishing bone disease, an infection, COVID-19, ARDS, a fibrotic lung disease, an infection, or any combination thereof. In some embodiments, the therapeutic method of the present disclosure is for reducing or inhibiting the incidence of CRS or a cytokine storm in a subject.

[0189] In some embodiments, the method of using the thawed apoptotic cells comprises treating an immune disease in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating an autoimmune disease in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating CRS in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating a cytokine storm in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating an inflammatory disease in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating a cancer in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating osteoarthritis in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating an effacing bone disease in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating an infection in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating a viral or bacterial infection in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating COVID-19 in a subject. In some embodiments, the method of using the thawed apoptotic cells comprises treating a subject suffering from ARDS. In some embodiments, the method of using the thawed apoptotic cells comprises treating a subject suffering from a fibrotic lung disease. In some embodiments, the method of using the thawed apoptotic cells comprises treating at least one symptom associated with an infection in a subject, the infection can be viral or bacterial. In other embodiments, the thawed early apoptotic population administered to treat an immune disease, an autoimmune disease, CRS, cytokine storm, an inflammatory disease, cancer, osteoarthritis, effacement bone disease, an infection, or at least one symptom associated with an infection in a subject, or any combination thereof, further comprises reducing the incidence or inhibiting the occurrence of CRS or cytokine storm.

[0190] In some embodiments, the viral, bacterial, or fungal infection causes a cytokine release syndrome or cytokine storm in the subject. In some embodiments, the infection is a SARS-CoV-2 infection. In some embodiments, the infection is an influenza infection. In some embodiments, the influenza infection is H1N1. In other embodiments, the influenza infection is H5N1 avian influenza. In other embodiments, the infection is severe acute respiratory syndrome (SARS). In other embodiments, the subject is suffering from Epstein-Barr virus associated hemophagocytic lymphohistiocytosis (HLH). In other embodiments, the infection is sepsis. In some embodiments, the sepsis is caused by a gram-negative bacteria. In other embodiments, the infection is malaria. In other embodiments, the infection is an Ebola virus infection. In other embodiments, the infection is an Epstein-Barr virus associated infection. In other embodiments, the infection is a variola virus infection. In other embodiments, the infection is a systemic gram-negative bacterial infection. In other embodiments, the infection is Jarisch-Herxheimer syndrome.

[0191] In some embodiments, the cause of cytokine release syndrome or cytokine storm in the subject is hemophagocytic lymphohistiocytosis (HLH). In other embodiments, the HLH is sporadic HLH. In other embodiments, the HLH is macrophage activation syndrome (MAS). In other embodiments, the cause of cytokine release syndrome or cytokine storm in the subject is MAS.

[0192] In some embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is chronic arthritis.In other embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is systemic juvenile idiopathic arthritis (sJIA), also known as Still's disease.

[0193] In some embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is cryopyrin-associated periodic syndrome (CAPS). In other embodiments, the CAPS comprises familial cold autoinflammatory syndrome (FCAS), also known as familial cold urticaria (FCU). In other embodiments, the CAPS comprises Muckle-Wells syndrome (MWS). In other embodiments, the CAPS comprises chronic infantile neurological cutaneous articular (CINCA) syndrome. In yet another embodiment, the CAPS comprises FCAS, FCU, MWS, CINCA syndrome, or any combination thereof. In other embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is FCAS. In other embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is FCU. In other embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is MWS. In other embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is CINCA syndrome. In yet another embodiment, the cause of cytokine release syndrome or cytokine storm in the subject is FCAS, FCU, MWS, CINCA syndrome, or any combination thereof.

[0194] In other embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is cryopyrinopathy, which comprises an inherited or de novo gain-of-function mutation in the NLRP3 gene, also known as the CIASI gene.

[0195] In some embodiments, the cause of the cytokine release syndrome or cytokine storm in the subject is an inherited autoinflammatory disease.

[0196] In some embodiments, the trigger for release of inflammatory cytokines is lipopolysaccharide (LPS), gram-positive toxins, fungal toxins, glycosylphosphatidylinositol (GPI), or modulation of RIG-1 gene expression.

[0197] In other embodiments, the subject suffers from cytokine release syndrome or cytokine storm secondary to treatment.

[0198] In some embodiments, a method of inhibiting or reducing the incidence of cytokine release syndrome (CRS) or cytokine storm in a subject undergoing CART cell cancer therapy comprises administering a thawed apoptotic cell population of the present disclosure.

[0199] In some embodiments, the subject suffers from a disease, disorder, or condition that includes cancer. In some embodiments, the cancer includes bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, or any combination thereof. In other embodiments, the cancer includes carcinoma, sarcoma, leukemia, lymphoma, myeloma, mixed cancer, or any combination thereof. In some embodiments, the lymphoma includes non-Hodgkin's lymphoma. In other embodiments, the cancer includes a solid tumor or solid cancer. In some embodiments, the cancer includes a non-solid tumor. In other embodiments, the cancer includes a non-solid cancer.

[0200] In other embodiments, the subject is suffering from a disease, disorder, or condition involving osteoarthritis, hi other embodiments, the subject is suffering from a disease, disorder, or condition involving a degenerative bone disease.

[0201] In other embodiments, the subject is suffering from a disease, disorder, or condition that includes an infectious disease. In some embodiments, the infectious disease includes chickenpox, common cold, coronavirus, diphtheria, E. coli, giardiasis, HIV / AIDS, infectious mononucleosis, influenza, Lyme disease, malaria, measles, meningitis, mumps, poliovirus infection (polio), pneumonia, Rocky Mountain spotted fever, rubella, Salmonella infection, severe acute respiratory syndrome (SARS), sexually transmitted diseases, shingles, tetanus, toxic shock syndrome, tuberculosis, viral hepatitis, West Nile virus, whooping cough, or any combination thereof.

[0202] In other embodiments, the disease, disorder, or condition comprises a viral or bacterial infection. In some embodiments, the infection is an influenza infection. In some embodiments, the influenza infection is H1N1. In other embodiments, the influenza infection is H5N1 avian influenza. In other embodiments, the infection is severe acute respiratory syndrome (SARS). In other embodiments, the subject is suffering from Epstein-Barr virus associated hemophagocytic lymphohistiocytosis (HLH). In other embodiments, the infection is sepsis. In some embodiments, the sepsis is caused by a gram-negative bacteria. In other embodiments, the infection is malaria. In other embodiments, the infection is an Ebola virus infection. In other embodiments, the infection is a variola virus infection. In other embodiments, the infection is a systemic gram-negative bacterial infection. In other embodiments, the infection is Jarisch-Herxheimer syndrome. In other embodiments, the infection comprises a SARS infection. In other embodiments, the infection comprises a COVID-19 or SARS-CoV-2 infection. In other embodiments, the infection comprises a septate infection. In another embodiment, the infection comprises a malaria infection. In another embodiment, the infection comprises an Ebola virus infection. In another embodiment, the infection comprises an Epstein-Barr virus associated infection.

[0203] In other embodiments, the subject has COVID-19.

[0204] In some embodiments, the disclosed method is a method for treating, preventing, ameliorating, inhibiting, or reducing COVID-19 (or its onset) in a subject. In some embodiments, the disclosed method provides a method for treating COVID-19 in a subject infected with SARS-CoV-2 virus, comprising administering a thawed apoptotic mononuclear enriched population to the subject. In some embodiments, the disclosed method for treating COVID-19 comprises treating, inhibiting, reducing the incidence, ameliorating, or alleviating a symptom of COVID-19. In certain embodiments, the disclosed method for treating COVID-19 comprises preventing the appearance of a symptom of COVID-19. In some embodiments, the disclosed method for treating COVID-19 comprises administering a thawed apoptotic cell population, thereby resulting in a negative PCR result for SARS-CoV-2.

[0205] In some embodiments, the subject is asymptomatic for COVID-19. In other embodiments, the subject has symptoms of COVID-19.

[0206] In some embodiments, the symptoms of COVID-19 include any one or more of organ failure, organ dysfunction, organ injury, cytokine storm, chemokine storm, multisystem inflammatory syndrome, and cytokine release syndrome. In some embodiments, the organ includes the heart, kidney, liver, or any combination thereof. In other embodiments, the symptoms of COVID-19 include one or more hematological abnormalities. In some embodiments, the hematological abnormalities include thrombocytopenia, lymphopenia, neutropenia, neutrophilia, or any combination thereof. In some embodiments, the symptoms of COVID-19 include cardiovascular dysfunction, acute kidney injury, or any combination thereof.

[0207] In some embodiments, treating a subject with COVID-19 includes treating a subject with mild COVID-19. In some embodiments, treating a subject with COVID-19 includes treating a subject with moderate COVID-19. In some embodiments, treating a subject with COVID-19 includes treating a subject with severe COVID-19. In some embodiments, treating a subject with COVID-19 includes treating a subject with severe COVID-19.

[0208] In other embodiments, the subject is suffering from sepsis. In some embodiments, the sepsis comprises severe sepsis. In other embodiments, the sepsis comprises septic shock.

[0209] In other embodiments, the subject suffers from GVHD. In some embodiments, the immune disease is GVHD. In some embodiments, the present disclosure provides a pharmaceutical composition used to treat, prevent, ameliorate, inhibit, or reduce GVHD (or its development) in a subject.

[0210] In some embodiments, a method of treating an immune disorder in a subject comprises administering a thawed apoptotic cell population to a subject receiving a solid organ transplant. In some embodiments, the organ comprises lung, heart, kidney, pancreas, liver, skin, and small intestine. In other embodiments, the solid organ comprises beta cells. In other embodiments, the solid organ is a limb.

[0211] In some embodiments, the subject suffers from a hematopoietic malignancy, has a graft-versus-tumor or graft-versus-leukemia (GVL) effect, has undergone hematopoietic stem cell transplantation (HSCT), has undergone chimeric antigen receptor-expressing T cell (CART cell) cancer therapy, or has undergone a solid organ transplant. In some embodiments, the HSCT comprises an allogeneic HSCT. In some embodiments, administration of the thawed apoptotic cell population occurs up to 24 hours prior to transplantation, simultaneously with transplantation, or up to 15 days after transplantation.

[0212] In some embodiments, the subject is afflicted with a hematopoietic malignancy.

[0213] In some embodiments, the hematopoietic malignancy comprises MDS, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0214] In other embodiments, the subject suffers from acute respiratory distress syndrome (ARDS). ARDS includes, in some embodiments, pulmonary ARDS, and in other embodiments, extrapulmonary ARDS. In other embodiments, the subject suffers from respiratory distress syndrome (RDS), adult respiratory distress syndrome, or shock lung. These are alternative names for ARDS in some embodiments. In some embodiments, symptoms of ARDS include shortness of breath (dyspnea), rapid breathing (tachypnea), bluish skin coloration (cyanosis), or any combination thereof. In some embodiments, ARDS includes diffuse damage to cells that form the barrier of the lung's microsacs, surfactant dysfunction, activation of the immune system, dysfunction of the body's regulation of blood clotting, or any combination thereof.

[0215] In another embodiment, the subject has multisystem inflammatory syndrome in children (MIS-C).In another embodiment, the subject has osteoarthritis.

[0216] Administration

[0217] In some embodiments of the disclosed methods for treating a subject (patient), the method comprises administering to the subject a thawed frozen preparation of apoptotic cells, 6 ~210×10 6 In some embodiments, a dose of about 10-100×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 20×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 30×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 40×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 50×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 60×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 70×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 80×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 90×10 apoptotic cells is administered. 6 In some embodiments, a dose of about 1-15×10 apoptotic cells is administered. 7 In some embodiments, a dose of about 10×10 apoptotic cells is administered. 7 In some embodiments, a dose of about 15×10 apoptotic cells is administered. 7 A dose of apoptotic cells is administered.

[0218] In some embodiments, 10×10 6 In another embodiment, a dose of 10×10 apoptotic cells is administered. 7 In another embodiment, a dose of 10×10 apoptotic cells is administered. 8 In another embodiment, a dose of 10×10 apoptotic cells is administered. 9 In another embodiment, a dose of 10×10 apoptotic cells is administered. 10In another embodiment, a dose of 10×10 11 In another embodiment, a dose of 10×10 apoptotic cells is administered. 12 In another embodiment, a dose of 10×10 apoptotic cells is administered. 5 In another embodiment, a dose of 10×10 apoptotic cells is administered. 4 In another embodiment, a dose of 10×10 apoptotic cells is administered. 3 In another embodiment, a dose of 10×10 apoptotic cells is administered. 2 A dose of apoptotic cells is administered.

[0219] In some embodiments, a high dose of apoptotic cells is administered. In some embodiments, 35×10 6 In another embodiment, a dose of 210×10 apoptotic cells is administered. 6 In another embodiment, a dose of 70×10 apoptotic cells is administered. 6 In another embodiment, a dose of 140×10 apoptotic cells is administered. 6 In another embodiment, a dose of 35-210×10 apoptotic cells is administered. 6 A dose of apoptotic cells is administered.

[0220] In some embodiments, a single dose of apoptotic cells is administered. In some embodiments, multiple doses of apoptotic cells are administered. In some embodiments, two doses of apoptotic cells are administered. In some embodiments, three doses of apoptotic cells are administered. In some embodiments, four doses of apoptotic cells are administered. In some embodiments, five doses of apoptotic cells are administered. In some embodiments, six doses of apoptotic cells are administered. In some embodiments, seven doses of apoptotic cells are administered. In some embodiments, eight doses of apoptotic cells are administered. In some embodiments, nine doses of apoptotic cells are administered. In some embodiments, more than nine doses of apoptotic cells are administered. In some embodiments, multiple doses of apoptotic cells are administered.

[0221] In some embodiments, the apoptotic cells are administered by any method known in the art, including, but not limited to, intravenous, subcutaneous, intranodal, intrathecal, intrapleural, intraperitoneal, parenteral, intraarticular, or intramuscular administration, and direct administration into the thymus.

[0222] In some embodiments, the thawed apoptotic cells are administered therapeutically when cytokine release syndrome occurs. In some embodiments, the thawed apoptotic cells are administered therapeutically when cytokine release syndrome is initiated or when cytokine release is detected that is indicative of the onset of cytokine release syndrome. In some embodiments, the thawed apoptotic cells are administered therapeutically to stop the increase in cytokine levels, reduce cytokine levels, or reduce cytokine levels so that cytokine release syndrome does not occur, such that the subject can avoid sequelae.

[0223] In some embodiments, an effective amount of thawed apoptotic mononuclear enriched cells is administered to a subject. One of ordinary skill in the art will recognize that an "effective amount" (or "therapeutically effective amount") encompasses an amount sufficient to affect a beneficial or desired clinical outcome in a treatment, such as, but not limited to, the treatment of symptoms of a disease, disorder, or condition. An effective amount can be administered to a subject in one or more separate doses. In terms of treatment, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse, or delay the progression of a disease, or to reduce the pathological consequences of a disease or its symptoms, such as, but not limited to, respiratory distress. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one of ordinary skill in the art. In determining the appropriate dosage to achieve an effective amount, several factors are generally considered. Such factors include the age, sex, and weight of the subject (patient), the condition being treated, the severity of the condition, and the form and effective concentration of the apoptotic cell population administered.

[0224] Those skilled in the art can readily determine the number of cells, optional additives, vehicles, and / or carriers in the composition to be administered in the disclosed methods. In general, any additives (in addition to the active cells and / or drug) are present in an amount of 0.001-50% by weight in phosphate buffered saline, and the active ingredient is present in an amount on the order of micrograms to milligrams, e.g., about 0.0001-5% by weight. In other embodiments, it is present in an amount of about 0.0001-1% by weight. In yet other embodiments, it is present in an amount of about 0.0001-0.05% by weight or about 0.001-20% by weight. In further embodiments, it is present in an amount of about 0.01-10% by weight. In other embodiments, it is present in an amount of about 0.05-5% by weight. Of course, for any composition administered to animals or humans, and for any particular method of administration, it is preferable to determine toxicity by determining the lethal dose (LD) and LD50 in an appropriate animal model, e.g., rodents such as mice. It is also preferred to determine the dosage of the composition, the concentration of the components in the composition, and the timing of administering the composition to induce an appropriate response. Such determination does not require undue experimentation from the knowledge of those skilled in the art, this disclosure, and the documents cited herein. The time of sequential administration can be ascertained without undue experimentation.

[0225] One of ordinary skill in the art will understand that the term "about" encompasses deviations of 0.0001-5% from a given numerical value or range. Additionally, the term "about" encompasses deviations of 1-10%, 1-5%, or 1-2.5% from a given numerical value or range. Additionally, the term "about" encompasses deviations of up to 10%, 15%, 20%, or 25% from a given numerical value or range.

[0226] Those of ordinary skill in the art will understand that the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. For example, the term "an agent" or "at least one agent" can include a plurality of agents, including mixtures thereof.

[0227] In some embodiments, "treating" includes therapeutic treatment and "preventing" includes prophylactic treatment, the purpose of which is to prevent or alleviate the targeted pathological condition or disorder as described above. Thus, in some embodiments, treating includes directly affecting symptoms associated with a disease, disorder, or condition, or curing, suppressing, inhibiting, preventing, reducing the severity, delaying onset, or alleviating it. Thus, in some embodiments, "treating," "ameliorating," and "alleviating" refer to, among other things, slowing progression, hastening recovery, increasing the effectiveness of alternative therapies, decreasing resistance to alternative therapies, or any combination thereof. In some embodiments, "preventing" refers to, among other things, delaying the onset of symptoms, preventing recurrence of the disease, decreasing the number or frequency of recurrent episodes, increasing the latency between symptomatic episodes, or any combination thereof. In some embodiments, "suppress" or "inhibit" refers to, among other things, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, increasing patient survival, or any combination thereof.

[0228] Those skilled in the art will appreciate that the term "treatment" encompasses clinical intervention in an attempt to alter the course of disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Benefits of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of direct or indirect pathological consequences of disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can prevent deterioration of the disorder in an affected subject, a subject diagnosed with a disease, or a subject suspected of having a disease. Treatment can also prevent the onset of disease or symptoms of a disorder in a subject at risk of disease or a subject suspected of having a disease. In some embodiments, improved prognosis includes a reduction in hospital stays.

[0229] One of ordinary skill in the art will understand that the term "subject" encompasses vertebrates, and in some embodiments, mammals, and in some embodiments, humans. In some embodiments, a subject includes an adult. In some embodiments, a subject includes a child.

[0230] One of ordinary skill in the art will appreciate that the term "effective amount" encompasses an amount sufficient to obtain a therapeutic effect. In some embodiments, an "effective amount" is an amount sufficient to prevent, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0231] Throughout this application, various embodiments disclosed herein may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, a description in range format should be considered to have specifically disclosed not only each numerical value contained within the range, but also all possible subranges contained within the range. For example, a description of a range of 1 to 6 should be considered to have specifically disclosed the subranges of 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, ..., as well as each numerical value (e.g., 1, 2, 3, 4, 5, and 6) contained within the range. This applies regardless of the breadth of the range.

[0232] Whenever a numerical range is specified herein, it is meant to include all numerical values ​​(fractional or integer) contained within the specified numerical range. The phrases "ranging between" a first specified number and a second specified number, and "ranging from" a first specified number to a second specified number, are used interchangeably herein and are meant to include the first specified number and the second specified number, and all fractional and integer numbers therebetween.

[0233] Working Example

[0234] Example 1. Development of an optimal cryopreservation formulation for cryopreservation of apoptotic cells

[0235] Objective: To develop a cryopreservation formulation for cryopreservation of apoptotic cells, where a high percentage of cells retain apoptotic properties upon thawing.

[0236] method:

[0237] The current manufacturing steps for apoptotic cells (e.g., Allocetra-OTS) consist of two major stages, as shown in Figure 3: (1) collection of apherates (total leukocyte apheresis product) and cryopreservation of key process intermediates (KPIs); (2) thawing of KPIs and production of fresh product (DP; i.e., at least 35% of the cell population contains early and mature apoptotic cells as assessed by AnV and PI staining. Fresh product (DP) is a product that is at least 35% AnV+PI+ DIM staining and <30% AnV+PI+ High The KPI contains the total cell population (consisting of approximately 50% mononuclear cells and 50% red blood cells and platelets) obtained from apheresis that has gone through several steps of washing and centrifugation.

[0238] All manufacturing steps from the starting material to the DP were carried out under Good Manufacturing Practice (GMP) conditions.

[0239] Currently, the standard apoptotic cell population, known as Allocetra-OTS, is formulated as a liquid preparation (fresh) in Hartmann's solution (lactated Ringer's solution) ready for injection into patients in a semi-automated process. See, for example, Example 1 of International Publication No. WO2016 / 170541, which is incorporated herein by reference in its entirety. As the clinical development and use of apoptotic cell populations progresses and the need for functional DPs containing apoptotic cell populations expands to regions farther from the manufacturing source of the DPs, the development of formulations with longer shelf life becomes crucial. Upon thawing, the apoptotic population can be assessed by AnV and PI staining, and the thawed drug product (DP) should have at least 35% AnV+PI+ DIM Shown is staining.

[0240] The first study examined small-scale frozen preparations of apoptotic cells stored in liquid nitrogen (-196°C) for 10 months.

[0241] To mitigate known side effects that can be caused by high concentrations of DMSO infusions, experiments focused on investigating cryopreservation of apoptotic cells (e.g., Allocetra) at DMSO concentrations below 10%.

[0242] result:

[0243] To facilitate the production of cryopreserved preparations, manufacturing changes were introduced to the frozen preparations after the cell suspension incubation step, as shown in Figure 2. These changes were made starting from the volume reduction step. The first difference was that the fresh preparations were resuspended in lactated Ringer's solution (Hartmann's solution) at this stage, whereas the frozen preparations were suspended in Plasma-Lyte (Baxter Healthcare Ltd., Norfolk, UK). No changes were made to the steps up to the volume reduction medium exchange.

[0244] DMSO concentration and comparison of commercial and homemade cryopreservation media

[0245] As a component of freezing media for cell preparations, DMSO is often used at a final concentration of 5-15%. A preliminary study was conducted to investigate the effect of cryopreservation with 7.5% DMSO and the results showed significant damage to key properties of AllocetraDP after thawing. The damage observed included a high concentration of late apoptotic cells, an increase in the late apoptotic cell population, and a decrease in the apoptotic cell population based on the key properties analyzed (% of AnV+PI+ cells) (Figures 4A and 4B).

[0246] In addition, four different cryopreservation solutions were tested: two commercial solutions and two home-made preparations: commercial CryoStor CS5 and CS10 (BioLife Solutions) diluted 1x with Allocetra cell suspension in CS-2.5 and CS-5 media, respectively (2.5% and 5.0% DMSO, respectively).

[0247] The Allocetra cell suspension was prepared as briefly described herein below.

[0248] Peripheral mononuclear cells were collected from a single donor using leukapheresis. The cells were cryopreserved as in the liquid preparations and processed in the same manner as in the liquid preparations up to volume reduction and preparation in LOVO medium. The resuspension for the LOVO and frozen preparations was in PL148 solution instead of Ringer's lactate solution.

[0249] The volume of the LOVO resuspension was also reduced to 220 mL, resulting in a higher cell concentration (50-80 million cells per ml). The cells were then irradiated at 4000 cGy, resulting in a cell concentration that allowed us to have 10 billion cells in 400 mL of fixed-dose formulation after 1:1 dilution with cryopreservation solution.

[0250] Allocetra populations with cell concentrations above 60 million cells / ml after LOVO were diluted with PL148 before mixing them with cryoprotectant.

[0251] After mixing with cryoprotectant, the cells were gently frozen and transferred to liquid nitrogen for long-term storage as described herein.

[0252] The two homemade formulations tested were based on common cryopreservation media containing 5% or 2.5% DMSO (v / v) and 3% (w / w) human serum albumin (HSA) in PlasmaLyte-148 (PL148, Baxter Healthcare) solution. Cells were thawed by placing the bag containing the frozen cell preparation in a preheated 37 °C water bath and gently agitating until no ice particles remained (<5 min). The thawed cells were then kept at ambient temperature (room temperature) for the duration of the study, 3 h.

[0253] The effects of these formulations were tested in several trials immediately after thawing and every hour from 3 to 8 hours after thawing, taking into account the main properties of AllocetraDP. Both the 2.5% DMSO commercial formulation and the homemade formulation showed low concentrations of late apoptosis (more than 30% of the cell product was AnV+PI). HI Allocetra cells were consistently protected from thawing by commercial CS-2.5 medium (CS5 diluted in PL148) in terms of a lower percentage of late apoptotic cells in the overall population (Figures 4A-4B, 5A-5B, and 6A-6D). Therefore, CS-2.5 was selected as the basis for the lead formulation.

[0254] As freezing medium, Cryostor® Cell Cryopreservation Medium CS5 (BioLife Solutions) was chosen. Cryostor® is purchased as a sterile formulation and contains DMSO (5%) and additional proteins / molecules that support cells during freeze / thaw cycles. In the final formulation, Cryostor® was diluted 1:1 with PlasmaLyte cell suspension and DMSO was diluted to 2.5%. The final apoptotic freezing formulation, produced by diluting CS5 1:1 with Plasmalyte and DMSO to 2.5%, contains at least 35% apoptotic cells (early and mature) and no more than 30% late apoptotic cells.

[0255] The effectiveness of this medium was also tested in the presence of various excipients (trehalose, Tween-80, and dextran-dextrose solution) that are said to act as additional stabilizers for the cells or provide physiological osmolarity. However, no significant differences were observed in the percentage of late apoptotic cells levels, apoptotic (early and mature) levels, or cell population markers (Figures 7A-7E), nor between excipient-supplemented medium and control cryopreservation medium. Therefore, the lead cryopreservation recipe was kept without additives.

[0256] Interestingly, the selective freezing medium thus optimized had a lower DMSO concentration (2.5%) than commonly used in the art, which surprisingly resulted in a better protection of apoptotic cells (e.g., Allocetra cells) compared to commercial solutions with higher DMSO concentrations (7.5% or 5%) (see Figures 4A and 4B).

[0257] Formulation of base medium solution for cryopreservation

[0258] The base medium used to prepare the cryoprotectant (CPA) also affects the cryopreservation results. In general, any isotonic electrolyte solution approved for injection can be used as a solvent for preparing the cryopreservation solution. PL148 solution is a common cryopreservation base solution. In the analysis using PL148, the apoptosis and late apoptosis results were comparable to those of PBS solution but slightly higher than those of lactated Ringer's solution (Figures 8A and 8B).

[0259] The identity of the cryopreservation base solution (formulation minus apoptotic cells) was determined early in the formulation development. First, a CPA solution (WAK's Cryo-Sure DMSO) was introduced to the Allocetra cell suspension (consisting of lactated Ringer's solution) after the LOVO process. Plasmalyte-148 solution was tested shortly after as a possible alternative since it serves as a common solvent for cryopreservation and is currently used in the KPI freezing process. Various cryopreservation formulations were tested with a 5% DMSO homemade solution with similar results. After the percentage of DMSO for the cryopreservation formulation was selected, Cryostor® Basal medium (CSB) solution was tested as an alternative to the cryopreservation base medium solution. CryoStor medium is uniquely formulated to address molecular biology aspects of cells during the cryopreservation process with the goal of improving cell viability and function after thawing. Therefore, it was presumed that the complete CryoStor medium would be effective for Allocetra cells. Apoptotic cells (Allocetra) were formulated into CBS solution using the LOVO method. CPA solutions containing a DMSO component were introduced to cells in CSB suspension immediately prior to step-freezing by mixing the cell suspension 1:1 with CS5 solution, mixing the cell suspension 1:1 with a Plasmalyte-148 and CS-10 mixture, or by directly introducing WAK DMSO to the cell suspension to a final concentration of 2.5%. Formulations made by diluting the cell suspension with CS solution performed well, although the lead formulation in which cells were directly formulated in Plasmalyte-148 using the LOVO method performed slightly better (Figures 9A-9B and 10A-10B).

[0260] Production and introduction of freezing medium base solutions

[0261] Since the end-of-life process (LOVO volume reduction and formulation) and the cryopreservation process overlap with each other, a seamless transition between the processes can be achieved, allowing pre-freezing and Allocetra reformulation to be incorporated into a single step of the process flow as part of a process improvement strategy. Therefore, volume reduction and formulation were performed directly on the cryopreservation base medium (PL148 solution). Assuming the retentate volume of LOVO is 220 mL, 40–75 × 10 6 A cell suspension of cells / ml was produced, allowing the production of a fixed dose frozen preparation of 10 billion cells (20% error).

[0262] cell concentration

[0263] The cell concentration per unit volume of cryoprotectant medium affects the viability of the cells. The conventional concentration used for cryopreservation of hematopoietic stem cells in cell therapy is 20–80 × 10 6 A fixed dose frozen apoptotic cell (Allocetra) preparation (FDP) was developed with the goal of producing a frozen preparation of apoptotic cells containing at least 35% apoptotic cells (early apoptotic cells and mature apoptotic cells).

[0264] Dosing of fresh Allocetra formulations was initially based on body size with the goal of reducing variability in drug exposure between subjects (patients). However, due to the benefits of a fixed dose (improved convenience, elimination of waste, improved safety due to reduced medication errors, and improved compliance), the weight-based dosing concept was abandoned after review in favor of a fixed dosing regimen.

[0265] The decision to manufacture a fixed dose of frozen apoptotic cell product was guided by similar considerations. The dose of the frozen apoptotic cell product was selected based on a clinically tested fixed dose of fresh product, 10 billion cells per batch, allowing for administration of 5 or 10 billion fixed dose products to subjects (patients). The FDP production batches were stored frozen in four bags of 100 ml each (for a total frozen storage volume of 400 ml). The estimated concentration of the frozen product was approximately 25 x 10 6 The cell count is ±20% per ml.

[0266] The highest and lowest concentrations of frozen Allocetra were 30×10 6 Cells / ml and 20 x 10 6 cells / ml, both from a fixed dose chosen with a 20% margin of error.

[0267] cryopreservation container

[0268] Origen CryoStore™ cryobags (OriGen Biomedical, USA), specifically designed for cryopreservation of infusion solutions and storage in liquid nitrogen, were selected as the cryopreservation container for the apoptotic cell population. After formulation filling (four bags were filled with 100 mL of frozen formulation each; total filling volume was 400 mL, with a total of approximately 10 billion ± 20% apoptotic cells), the Cryobags were placed in aluminum cassettes to protect the formulation from mechanical damage that could compromise the structure of the container and its sterility. Also, to ensure uniform cell recovery between formulations, it is essential to ensure a consistent and uniform thermal profile across the length and thickness of the Cryobag during freezing.

[0269] Process development studies are currently ongoing to determine optimal loading and stability of the frozen formulation.

[0270] long term storage

[0271] The results of storing frozen preparations of apoptotic cells in liquid nitrogen for at least 10 months are shown in Figures 11A and 11B. Figure 11A shows that there was minimal change in the number of late apoptotic cells in the overall population after 1 week, 1 month, 5 months, and 10 months of storage in liquid nitrogen. Importantly, Figure 11B shows that there was no decrease in the proportion of apoptotic cells (early and mature apoptotic cells) in the overall population after 1 week, 1 month, 5 months, and 10 months of storage in liquid nitrogen.

[0272] Liquid nitrogen storage of fixed-dose formulations is expected to show similar stability in apoptotic and late apoptotic cell percentages following thawing of frozen formulations stored for 10 months or more.

[0273] Drug Product Quality and Biological Properties

[0274] The current process for freezing apoptotic mononuclear cell enriched cell populations is as follows: Briefly, after induction of apoptosis, cells are irradiated at 4000cGy to the desired cell concentration; after 1:1 dilution with cryopreservation solution, 10 billion cells are contained in FDP with a final volume of 400ml; fixed dose preparations with cell concentrations of over 60 million cells / ml after LOVO were diluted with PL148 before mixing with cryoprotectant; after mixing with cryoprotectant, cells were gradually frozen and transferred to liquid nitrogen for long-term storage. Below are the steps taken to compare the quality and biological properties of frozen preparations of apoptotic cells after rapid thawing with freshly generated apoptotic cells.

[0275] After producing the apoptotic mononuclear cell enriched cell population for freezing and selecting the freezing medium formulation, several tests were performed to evaluate the quality and biological properties of the new formulation. Tests were performed on the frozen apoptotic cell preparation immediately after thawing (T=0) and hourly for up to 3 hours to simulate conditions during use (estimated time from thawing to end of administration is approximately 1.5-2 hours for a 60-100 ml bag).

[0276] Basic quality test results: The target population of the apoptotic cell preparation was identified as mononuclear cells (MNC) and assessed by staining for CD45 (a common marker for all MNC subpopulations). The major population of Allocetra-OTS is the MNC population (CD45 positive cells). For an Allocetra population to be considered CD45 positive, CD45 levels must be greater than 85%. This preparation generally has a very high percentage of MNC, greater than 85%. MNC are also characterized by the profile of their subpopulation composition.

[0277] The results shown in Tables 1 and 2 below show that the measurements of the percentage of apoptotic cells (proportions of apoptotic (early and mature) cells and late apoptotic cells) and the CD45+ measurements are in the same range for frozen and fresh preparations. CD45+ is a marker for mononuclear cells (MNC) and is a technical characteristic of frozen apoptotic cells.

[0278] MNCs were characterized based on membrane expression of human CD45, a common marker for all hematopoietic cells (blood cells) except mature erythrocytes (red blood cells) and platelets. Cell preparations (fresh cells and frozen / thawed apoptotic cell batches) were stained with a triple mix containing anti-human CD45 as an MNC marker, anti-human CD42a as a platelet and erythrocyte marker, and anti-human CD235a fluorescent antibodies.

[0279] [Table 3]

[0280] The proportion of apoptotic cells in the population was determined by the ratio of early apoptotic cells (AnV+PI-) to mature apoptotic cells (AnV+PI-). dim Mature apoptotic cells are characterized by being AnV positive and negative for PI staining (low levels of PI staining), in contrast to late apoptotic cells (AnV positive and high PI) and early apoptotic cells (AnV positive and negative PI).

[0281] [Table 4]

[0282] The content of apoptotic cells is an important quality attribute of the preparation. Annexin V (AnV) and propidium iodide (PI) staining was performed to determine the apoptotic status. During the early stage of apoptosis, membrane-bound phosphatidylserine (PtdSer) moves from the inner cytoplasmic leaflet of the cell membrane to the outer extracellular leaflet of the cell membrane, exposing PtdSer to the direct environment and making it accessible for AnV binding. After exposure of PtdSer, the apoptotic process proceeds and further caspase activation continues. Two subpopulations of apoptotic cells are derived from this dynamic process: AnV+PI and AnV+PI. - Early apoptotic cells are characterized by AnV+ and low PI staining, and mature apoptotic cells are characterized by a dim PI fluorescence population due to the progression of the membrane permeabilization process that results in a gradual loss of membrane integrity. Apoptotic cells are eventually engulfed by phagocytes. If cells are not phagocytosed, the plasma membrane gradually loses integrity. Loss of plasma membrane integrity can be detected by nonselective and massive uptake of fluorescent dyes such as PI, which increases the intensity of PI staining on cells. PI enters the nucleus and is inserted into DNA double strands, serving as a biomarker for late apoptotic or necrotic cells.

[0283] Discrimination between two apoptotic subpopulations (early and mature) was achieved after optimization of the analytical method and improvement of the flow cytometry sensitivity. The optimized method with a more precise gating strategy allowed a more accurate measurement of the apoptotic population. This allowed the determination of the apoptotic population with a minimum of 35% early (AnV+PI) apoptotic cells. - ) and mature (AnV+PI Dim ) apoptotic cells and less than 30% late apoptotic cells (AnV+PI+ High This resulted in updates to the shipping standards used throughout, including:

[0284] Measurement of the percentage of apoptotic cell population in the preparation samples was performed using the MEBCYTO Apoptosis Kit (MBL International Corporation, Woburn, MA, USA) containing AnV-FITC conjugation and PI staining. The results showed that apoptotic cells were positive for AnV staining and negative or low for PI staining. All late apoptotic cells were brightly stained with PI and appeared as peaks with very high fluorescence values ​​(high intensity of PI staining) due to the significantly higher absorbance of the dye.

[0285] As shown in Tables 1 and 2 and Figures 13A and 13B, the most basic quality parameters of the frozen preparations remained within the range of the fresh preparations. The only parameter that appeared to be affected in the apoptotic cells of the thawed frozen preparations was the increase in the percentage of late apoptotic cells. During the process of thawing the frozen preparations of Allocetra-OTS cells, an increase in the number of late apoptotic cells was observed. A possible explanation for this phenomenon could be the rapid progression of apoptotic cells to the terminal apoptotic phase due to the freezing and / or thawing process.

[0286] The late apoptotic cell population obtained by the progression of the apoptotic process is referred to in the literature as "secondary necrotic" cells. Such late apoptotic cells are characterized by cytoplasmic swelling and cell membrane damage. As these cells are also engulfed by phagocytosis (in contrast to primary necrotic cells), a high proportion of the secondary necrotic population is not considered to pose a safety concern. Based on evidence from the relevant literature, the late apoptotic population is thought to attenuate inflammatory signals and result in a different immune response compared to classical primary necrotic cells (Sachet, M., Liang, YY, & Oehler, R. (2017). The immune response to secondary necrotic cells. Apoptosis: an international journal on programmed cell death, 22(10), 1189-1204).

[0287] Biological activity - potency test: The biological activity of Allocetra cells was also compared between the two formulations (fresh and frozen) using a potency test. Apoptotic formulation activity is shown as the inhibition of TNF-α expression from activated mouse macrophages. The activity of the frozen formulation was tested 1 hour after thawing and compared to the activity of the fresh formulation at the beginning and end of the storage period (T=0, FIG. 13A; T=96, FIG. 13B).

[0288] The purpose of the analysis of the inhibition rate (%) of TNF-α expression was to determine the ability of apoptotic cells to induce immune regulation by decreasing TNF-α expression in macrophages in the presence of two different concentrations of lipopolysaccharide (LPS) (high LPS: 0.05ng / mL and low LPS: 0.025ng / mL) by flow cytometric analysis. The assay was based on evaluation by intracellular staining and flow cytometric analysis. The interaction of Allocetra cells with a mouse macrophage cell line (RAW264.7 cells) reduced LPS-induced TNF-α upregulation. The reduction of TNF-α positive cells was calculated and the inhibition rate brought about by Allocetra-OTS at two different ratios (RAW:Allocetra 1:5 and 1:20) was shown in a dose-dependent pattern. Basically, macrophages were activated with LPS to secrete TNF-α and then incubated with Allocetra, which inhibits this effect.

[0289] As shown in Figure 13B, the average radioactivity of the frozen batches (1 hour after thawing) was slightly lower than that of the fresh batches at the end of the storage period (T=96) as shown by ICS. However, the range of results for the four frozen batches appears to be within the range of the 20 "fresh" batches. Given the inherent variability of this cell-based assay and the inherent variability between batches, the slight differences observed are unlikely to be related to changes in formulation.

[0290] Further support for the similarity of frozen and fresh formulations was provided by an ELISA-based potency assay, as shown in Figure 14. The assay is based on the assessment of cytokine secretion by mouse TNF-α ELISA. Apoptotic formulation activity is shown as inhibition of TNF-α secretion from activated mouse macrophages. The mean radioactivity of the frozen batches was compared to that of the fresh lots over the storage period (T=0 to T=96 hours).

[0291] The efficacy of the frozen formulation was also evaluated using the murine CLP model, which is considered the gold standard animal model of reproducible sepsis. To evaluate the efficacy of the frozen formulation batches in this model, two different batches were tested in four independent studies. Allocetra-OTS thawed from the frozen formulation was administered up to 1.5 hours after thawing in combination with standard of care (SOC) fluid resuscitation and antibiotic treatment (ertapenem).

[0292] As shown in Figures 15 and 16, the frozen formulation batch showed a significant effect with improved survival compared to the standard treatment group. A similar effect was observed with the fresh formulation batch. Considering the variability of the model, the two formulations showed similarity in terms of biological activity and in vivo efficacy.

[0293] summary:

[0294] These results indicate that the optimized cryopreservation formulation, which contains (1) approximately 10 billion apoptotic cells, (2) an isotonic crystalloid solution containing 140mmol / L sodium, 5mmol / L potassium, 1.5mmol / L magnesium, 98mmol / L chloride, 27mmol / L acetate, and 23mmol / L gluconate, and (3) a final concentration of 2.5% DMSO, can be stored at -196°C for at least 10 months and maintains the biological activity of the apoptotic cells upon thawing. Furthermore, results with the thawed frozen apoptotic cell formulations showed a beneficial effect on viability, as observed with fresh formulation batches (Figures 15 and 16). Overall, in both formulations, Allocetra-OTS-treated mice survived approximately 30% longer than antibiotic-treated CLP mice. These findings further support the biological activity and efficacy of Allocetra-OTS thawed from the frozen formulations, as described herein.

[0295] Conclusion: A significant increase in late apoptotic and mature apoptotic cells was observed after the cryopreservation / thawing process of Allocetra cells in the presence of the formulation containing DMSO (compared to the presence of these populations in the liquid Allocetra formulation). Cryopreservation is known to damage frozen cells and affect cell survival (Matsushita et al. (2003) Cell Transplantation, Vol. 12, pp. 109-121.). Since the biological activity of the formulation is derived mainly from the early apoptotic population, not the mature and late apoptotic population, it was expected that the activity of the formulation would also be affected and reduced after the cryopreservation / thawing process. As shown in Figures 13-16, after thawing of the frozen formulation, despite the increase in mature and late apoptotic cells, Allocetra cells showed almost the same activity as fresh Allocetra cells. The activity of Allocetra from the frozen batch was tested in in vitro models (intracellular staining and ELISA, Figures 13A, 13B, and 14, respectively) and in in vivo models (CLP model, Figures 15-16) and compared to the activity from the fresh batch. As a result, the ability of the disclosed apoptotic cell populations to maintain cellular functionality after freezing and thawing, despite an increase in mature and late apoptotic populations, was surprising and unexpected.

[0296] Example 2: Analysis of late apoptotic cells

[0297] the purpose:

[0298] To compare the appearance of late apoptotic cell populations on AnV / PI flow cytometry histograms with control necrotic cells and to determine overlap gating in flow cytometry.

[0299] method:

[0300] Naïve PBMCs were received from the leukapheresis process and induced into the necrotic process as follows. Cells were incubated overnight at 37°C and 5% CO2 before being transferred to a liquid nitrogen tank for long-term storage before AnV / PI staining. Cells were thawed at 37°C for 2 min. Thawed cells were stained with Annexin V (AnV) and propidium iodide (PI) to assess the prevalence and status of necrotic cells. Necrotic induction promoted all cells into the necrotic state.

[0301] result:

[0302] FIG. 17A shows that after necrosis induction, more than 85% of all PBMCs were induced into the necrotic phase and appeared in the AnV+, PI+ necrotic control gate.

[0303] Control necrotic cells were used as reference cells to characterize the flow cytometry patterns of late apoptotic cells, such as those identified in Figure 1 of Example 1. Results of the analysis showed that late apoptotic cells overlapped with the control necrotic cells generated by at least 80% (Figure 17A and data not shown).

[0304] Figure 17A shows that approximately 90% of the total late apoptotic / necrotic cells colocalized with the defined necrosis control gate. Thus, the late apoptotic cells identified as part of the total manufactured Allocetra / formulation population (see Example 1) overlap with the necrotic cells sorted by flow cytometry. Furthermore, necrotic cells were significantly less active compared to apoptotic cells (Figure 17B).

[0305] summary:

[0306] Comparison by flow cytometry showed that when the AnV / PI staining settings and flow cytometry acquisition and voltage settings were the same, cells localized in the same gate as 85% or more of necrotic cells were defined as Allocetra late apoptotic cells.

[0307] Example 3: Analysis of apoptosis induction time

[0308] the purpose:

[0309] The time to induction of apoptosis is assessed.

[0310] method:

[0311] The standard frozen formulation described in Example 1 was incubated with methylprednisolone for 6 hours. Here, incubations were performed for 1 and 6 hours, and then the percentage of apoptotic cells and the percentage of late apoptotic cells were analyzed. After incubation, the cell suspension was reduced in volume with the LOVO cell processing system and medium-exchanged into PlasmaLyte-148 solution. The cells were then diluted to a fixed volume, irradiated, mixed 1:1 with CS-5 solution, and slowly frozen.

[0312] result:

[0313] Table 3 shows the time course of apoptotic and late apoptotic cell populations after 1 hour of incubation.

[0314] [Table 5]

[0315] A range of apoptosis induction times was examined for the frozen preparations, 1 and 6 hours, and similar findings and characteristics were observed (similar late apoptosis, similar cell recovery, similar activity).

[0316] While certain features of the disclosed frozen preparations of apoptotic cells and methods for producing same have been illustrated and described herein, various modifications, substitutions, changes, and equivalents will occur to those skilled in the art, and it is therefore to be understood that the appended claims are intended to cover all such modifications and alterations that fall within the true spirit and scope of the invention.

Claims

1. 1. A frozen formulation comprising: (a) a population of apoptotic mononuclear condensed cells; (b) an isotonic crystalloid solution; and (c) a cryoprotectant; and a pH in the range of about 6.5 to 8.

0.

2. 2. The frozen formulation of claim 1, The apoptotic mononuclear condensed cells are about 17-150×10 ６ Frozen preparation with a concentration of 100 cells / ml.

3. 2. The frozen formulation of claim 1, A frozen preparation, wherein the apoptotic mononuclear enriched cells comprise at least one cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

4. 4. The frozen formulation of claim 3, The frozen preparation, wherein the at least one cell type comprises a single cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

5. 2. The frozen formulation of claim 1, The frozen formulation, wherein the isotonic crystalloid solution comprises sodium chloride, potassium chloride, magnesium chloride, sodium acetate, sodium gluconate, or any combination thereof.

6. 2. The frozen formulation of claim 1, The isotonic crystalloid solution comprises 140 mmol / L sodium, 5 mmol / L potassium, 1.5 mmol / L magnesium, 98 mmol / L chloride, 27 mmol / L acetate, or 23 mmol / L gluconate.

7. 2. The frozen formulation of claim 1, The cryoprotectant comprises DMSO.

8. 8. The frozen formulation of claim 7, The frozen formulation, wherein the DMSO is present in the formulation at a concentration of about 2.5%.

9. 2. The frozen formulation of claim 1, The frozen formulation has a pH of about 7.

4.

10. 1. A method for producing a frozen apoptotic cell preparation, comprising: (a) a resuspension step in which a population of fresh apoptotic mononuclear enriched cells is resuspended in an isotonic crystalloid solution having a pH of about 6.5-8.0; (b) mixing the population of apoptotic mononuclear enriched cells resuspended in the resuspension step (a) with a cryoprotectant; (c) a freezing step, gradually freezing the mixture produced in said mixing step (b) to a temperature of about -196°C.

11. 11. The method of claim 10, The population of apoptotic mononuclear enriched cells resuspended in the resuspension step (a) is about 25-300×10 ６ The method has a concentration of 10 cells / ml.

12. 11. The method of claim 10, The apoptotic mononuclear enriched cells comprise at least one cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

13. 13. The method of claim 12, The method, wherein the at least one cell type comprises a single cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

14. 11. The method of claim 10, The method, wherein the isotonic crystalloid solution comprises sodium chloride, potassium chloride, magnesium chloride, sodium acetate, or sodium gluconate.

15. 11. The method of claim 10, The isotonic crystalloid solution comprises 140 mmol / L sodium, 5 mmol / L potassium, 1.5 mmol / L magnesium, 98 mmol / L chloride, 27 mmol / L acetate, or 23 mmol / L gluconate.

16. 11. The method of claim 10, The isotonic crystalloid solution has a pH of about 7.

4.

17. 11. The method of claim 10, The method of claim 1, wherein in said mixing step (b), said population of apoptotic mononuclear enriched cells and said cryoprotectant are mixed in a 1:1 (volume / volume) mixing ratio.

18. 11. The method of claim 10, The method, wherein the cryoprotectant comprises DMSO.

19. 20. The method of claim 18, The method of claim 1, wherein the final concentration of DMSO in the frozen apoptotic cell preparation is about 2.5%.

20. 11. The method of claim 10, The method further comprises an irradiation step of irradiating the population of apoptotic mononuclear enriched cells after said resuspending step (a) and before said mixing step (b).

21. 11. The method of claim 10, The concentration of the population of apoptotic mononuclear enriched cells in the frozen apoptotic cell preparation is about 20-100×10 ６ The method according to claim 1, wherein the cell number is 1000 to 10000 cells / ml.

22. 13. A method of treating a subject using the frozen formulation of claim 1, comprising: (a) thawing the frozen preparation; (b) administering the thawed frozen formulation, or a portion thereof, to the subject, thereby treating the subject.

23. 23. The method of claim 22, further comprising an analyzing step of analyzing the thawed frozen preparation for Annexin V staining; The method of claim 1, wherein said administering step (b) comprises administering a population of apoptotic cells comprising at least 35% Annexin V+ cells.

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