WO2023203561A1

WO2023203561A1 - Apoptotic cell - check point inhibitor combination therapy

Info

Publication number: WO2023203561A1
Application number: PCT/IL2023/050401
Authority: WO
Inventors: Dror Mevorach; Shai Novik
Original assignee: Enlivex Therapeutics Rdo Ltd
Priority date: 2022-04-19
Filing date: 2023-04-18
Publication date: 2023-10-26

Abstract

Disclosed herein are combination therapies including apoptotic cells combined with checkpoint inhibitors for the treatment of cancers, including mesothelioma, ovarian cancer, and colon cancer. Effective treatment is observed using apoptotic cells freshly produced or previously frozen.

Description

APOPTOTIC CELL - CHECK POINT INHIBITOR COMBINATION

THERAPY

FIELD OF INTEREST

[001] Disclosed herein are combination therapies comprising administration of an apoptotic mononuclear-enriched cell population and at least one checkpoint inhibitor. Combination therapies disclosed herein may be used for treating, inhibiting the growth of, delaying cancer progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject.

BACKGROUND

[002] Cancer is an abnormal state in which uncontrolled proliferation of one or more cell populations interferes with normal biological functioning. The proliferative changes are usually accompanied by other changes in cellular properties, including reversion to a less differentiated, more developmentally primitive state. The in vitro correlate of cancer is called cellular transformation. Transformed cells generally display several or all of the following properties: spherical morphology, expression of fetal antigens, growth-factor independence, lack of contact inhibition, anchorage-independence, and growth to high density.

[003] The primary cause of lethality in malignant diseases such as lung and skin cancer arise from metastatic spread. In many cases, it is not possible to prevent the onset of metastatic disease since cancers are often metastatic by the time of diagnosis, and even in cases where cancers are diagnosed prior to this stage, complete surgical removal or destruction of primary lesion tissues, which are capable of eventually generating metastases may not be feasible. Metastatic disease may be impossible to diagnose at stages due to the small size of metastatic lesions, and/or the absence of reliable markers in primary lesions upon which to reliably predict their existence. Such lesions may be difficult or impossible to treat via ablative methods due to their being inaccessible, disseminated, and/or poorly localized. Chemotherapy/radiotherapy, the current methods of choice for treatment of certain metastatic malignancies are often ineffective or have suboptimal efficacy, and have the significant disadvantage of being associated with particularly harmful and/or potentially lethal side-effects.

[004] Malignant mesothelioma is an aggressive tumor arising from the cells lining the pleural, peritoneal, and pericardial cavity. This tumor is highly resistance to current conventional therapies.

[005] Checkpoint inhibitors are immunotherapy drugs that specifically allow the patient's immune system to recognize and destroy certain types of cancer. The immune checkpoint pathways targeted in anti-cancer therapy include but are not limited to the cytotoxic T- lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) immune checkpoint pathways, which includes targeting PD-1 and/or PDL-1. CTLA-4 (CD152) is expressed on activated T cells and PD-1 (CD279) receptor is expressed broadly on peripheral CD4+ and CD8+ T cells, B cells, and myeloid cells. The anti-cancer mechanism suggested for checkpoint inhibitors involves ligand-receptor interactions between tumor cells and activated T cells. The interaction between PD-1 receptor on T cells with its ligand, PDL-1, on tumor cells promotes tumor escape. In the presence of the checkpoint inhibitors, for example but not limited to anti-PD-1 or anti-PDL-1 antibodies, T cells become activated and promote elimination of tumor cells.

[006] Apoptotic cells present one pathway of physiological cell death, most commonly occurring via apoptosis, which elicits a series of molecular homeostatic mechanisms comprising recognition, an immune response, and a removal process. Moreover, apoptotic cells are immunomodulatory cells capable of directly and indirectly inducing immune tolerance to dendritic cells and macrophages. Apoptotic cells have been shown to modulate dendritic cells and macrophages and to render them tolerogenic and inhibit proinflammatory activities such as secretion of proinflammatory cytokines and expression of costimulatory molecules.

[007] Combination therapy, a treatment modality that combines two or more therapeutic agents, is a cornerstone of cancer therapy. The amalgamation of anti-cancer drugs enhances efficacy compared to the mono-therapy approach because it targets key pathways, and potentially reduces drug resistance (Mokhtari, Reza Bayat, et al. "Combination therapy in combating cancer." Oncotarget 8.23 (2017): 38022). The combination of chemotherapeutic agents and/or other treatments (e.g., radiation therapy) is often advantageous since the additional agent may have the same or different mechanism of action than the primary therapeutic agents. For example, drug combinations may be employed wherein the two or more drugs being administered act in different manners or in different phases of the cell cycle, and/or where the two or more drugs have nonoverlapping toxicities or side effects, and/or where the drugs being combined each has a demonstrated efficacy in treating the particular disease state manifested by the patient.

[008] There remains an unmet need for combined strategies for treating, preventing, inhibiting the growth of, delaying cancer progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject. Combination therapies comprising a first composition comprising an irradiated, apoptotic mononuclear-enriched cell population and a second composition comprising one or more checkpoint inhibitors, and uses thereof, described herein below, address this need.

SUMMARY

[009] In one aspect, disclosed herein is a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor. In a related aspect, the apoptotic mononuclear-enriched cell population comprises an inactivated apoptotic mononuclear- enriched cell population. In a further related aspect, the inactivated apoptotic mononuclear- enriched cell population comprises an irradiated population, wherein said irradiation is post induction of apoptosis. In certain related aspects, the inactivated apoptotic mononuclear- enriched cell population comprises pooled, apoptotic mononuclear-enriched cell populations. In some related aspects, the pooled, apoptotic mononuclear-enriched cell populations comprise allogeneic cells from HLA matched or HLA unmatched sources, with respect to a recipient subject.

[0010] In another related aspect, the apoptotic mononuclear-enriched cell population comprises a white blood cell (WBC) fraction from a blood donation. In yet another related aspect, the apoptotic 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.

[0011] In a related aspect, the checkpoint inhibitor comprises a CTLA-4, programmed death ligand 1 (PDL-1), PDL-2, programmed cell death protein 1 (PD-1), BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160 or CGEN-inhibitor. In a further related aspect, the checkpoint inhibitor comprises an antibody. In yet another further related aspect, the checkpoint comprises PD-1, said antibody comprises nivolumab, pembrolizumab, cemiplimab, tislelizumab, dostarlimab, retifanlimab, spartalizumab, camrelizumab, or sintilimab. In still a further aspect, the checkpoint comprises PDL-1, and the antibody is selected from atezolizumab, avelumab, durvalumab and cosibelimab. [0012] In one aspect, disclosed herein is a method of treating, inhibiting the growth of, or delaying disease progression, of a cancer or a tumor in a human subject, comprising a step of administering to a subject in need a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor, wherein said method treats, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said human subject. In a related aspect, the method reduces the tumor load or reduces the incidence of the cancer or a tumor in said subject, compared with a subject not administered the combination therapy. In a further related aspect, the method reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, prevents metastasis of said tumor or said cancer, or reduces the rate of metastasis of said tumor or said cancer, or any combination thereof.

[0013] In a related aspect of a method disclosed herein, the apoptotic mononuclear-enriched cell population comprises an inactivated apoptotic mononuclear-enriched cell population. In a further related aspect, the inactivated apoptotic mononuclear-enriched cell population comprises an irradiated population, wherein said irradiation is post induction of apoptosis. In another further related aspect, the inactivated apoptotic mononuclear-enriched cell population comprises pooled, apoptotic mononuclear-enriched cell populations. In certain related aspects, the pooled, apoptotic mononuclear-enriched cell populations comprise allogeneic cells from HLA matched or HLA unmatched sources, with respect to a recipient subject.

[0014] In another related aspect of methods disclosed herein, the apoptotic mononuclear- enriched cell population comprises a white blood cell (WBC) fraction from a blood donation. In a further related aspect, the apoptotic 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.

[0015] In another related aspect of methods disclosed herein, the checkpoint inhibitor comprises a CTLA-4, programmed death ligand 1 (PDL-1), PDL-2, programmed cell death protein 1 (PD-1), BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160 or CGEN-inhibitor. In a further related aspect, the checkpoint inhibitor comprises an antibody. In another further related aspect, the checkpoint comprises PD-1, said antibody comprises nivolumab, pembrolizumab, cemiplimab, tislelizumab, dostarlimab, retifanlimab, spartalizumab, camrelizumab, or sintilimab. In still another further related aspect, when the checkpoint comprises PDL-1, said antibody is selected from atezolizumab, avelumab, durvalumab, and cosibelimab.

[0016] In another related aspect of methods disclosed herein, the cancer or tumor comprises a non-solid cancer or a solid tumor, or comprises a metastasis of a cancer or tumor. In a further related aspect, the non-solid cancer comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non-Hodgkin lymphoma, or plasma cell leukemia, or wherein said solid tumor comprises a sarcoma or a carcinoma, a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilm's tumor, a cervical cancer or tumor, a uterine cancer or tumor, a testicular cancer or tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma.

[0017] In another related aspect of methods disclosed herein, the administration comprises co-administration of said first composition and said second composition in the same or separate compositions. In a further related aspect, the administration comprises administration of said first composition and said second composition at different time points. In yet another further related aspect, the first composition and said second composition are administered at separate sites or at the same sites. In still another further related aspect, the administration comprises Intravenous (IV), Intraperitoneal (IP), subcutaneous (SC), or oral administration, and wherein said first composition and said second composition may be administered by the same or different routes. In yet another further related aspect, following administration of said combination therapy, the subject remains disease free for a time period longer than a subject administered either composition alone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fees.

[0019] The subject matter regarded as the apoptotic cell - check point inhibitor combination therapy and use thereof, is particularly pointed out and distinctly claimed in the concluding portion of the specification. The combination therapy and uses thereof, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0020] Figure 1A-1F. Characteristics of Apoptotic Mononuclear-enriched Cell Population. Figure 1A presents a flow chart presenting embodiments of the steps of a manufacturing process of apoptotic cell populations, wherein anti-coagulants and irradiation following apoptotic induction were included in the process. A skilled artisan would appreciate that apheresis is one type of leukapheresis. Mononuclear cells were also collected by leukopherisis. Figure IB presents an analysis of Allocetra-OTS cells following AnV and PI Staining. A frozen formulation of Allocetra-OTS produced using the methods described herein, was thawed and stained with Annexin V (AnV; X-Axis) and Propidium Iodide (PI; Y-axis) to assess the prevalence and state of the apoptotic cells. The graph shows that 60% of the total thawed apoptotic cell population (comprising the mature and apoptotic cell populations), met the criteria of ≥ 35% AnV. The late apoptotic population was shown to have the highest PI fluorescence intense (Y axis). There is a distinct separation between the apoptotic population and the late apoptotic population. The late apoptotic cell population comprised 22.2% of the total product cell produced and was < 30% AnV. For Figure 1C, a liquid formulation of Allocetra-OTS was stained with Annexin V (AnV; X-Axis) and Propidium Iodide (PI; Y-Axis) to assess the prevalence and state of the apoptotic cells. The graph shows that 42.72% of the total cell population are an apoptotic cell population (comprising the mature and early apoptotic cell populations), which meet the criteria of ≥ 35% AnV. The late apoptotic population was shown to have the highest PI fluorescence intense (Y axis). There is a distinct separation between the apoptotic population and the late apoptotic population. The late apoptotic cell population comprised 6.96% of the total product cell produced and was < 30%. Figure ID shows analysis of necrotic peripheral blood mononuclear cells (PBMCs) following AnV and PI staining. The graph shows that following the necrosis induction, more than 80% of the total PBMCs were induced to necrosis phase and appeared in the AnV+, PI+ necrosis gate, which is the same gated region as cells in late apoptosis. Figure IE presents a bar graph showing measures of the activity of apoptotic cells (Allocetra OTS) and of necrotic cells (“Necrotic Allocetra OTS”), wherein the activity is measured as a percent (%) inhibition of TNF-a secretion based on mouse TNF- α ELISA. The graph shows that following the necrosis induction, Allocetra-OTS activity was significantly impaired. Figure IF presents a schematic of one embodiment of an optimized cryopreservation method for apoptotic cells, as disclosed herein. The stages presented on the left-hand side of the schematic include steps in the preparation of a fresh apoptotic cell formulation, while the stages presented on the right-hand side of the schematic include steps in the preparation of a frozen apoptotic cell formulation. Italicized text provides an indication of difference between the methods.

[0021] Figures 2A-2B. Potency Test. Figures 2A-2B present the results of a potency test that shows the inhibition of maturation of dendritic cells (DCs) following interaction with apoptotic cells, measured by expression of HLA-DR. Figure 2A. HLA-DR mean fluorescence of fresh final product A (tO). Figure 2B. HLA-DR mean fluorescence of final product A, following 24h at 2-8°C.

[0022] Figures 3A-3B. Potency Test. Figures 3A-3B present the results of a potency test that shows the inhibition of maturation of dendritic cells (DCs) following interaction with apoptotic cells, measured by expression of CD86. Figure 3A. CD86 Mean fluorescence of fresh final product A (tO). Figure 3B. CD86 Mean fluorescence of final product A, following 24h at 2-8°C.

[0023] Figure 4. Overview of Apoptotic Cells (Allocetra): Reprograming peritoneal pro-tumor macrophages and Method of Use of Combination Therapy for Treating Mesothelioma.

[0024] The flow schematic shows the establishment of the AB 12 mouse mesothelioma syngeneic model starting at day 0. Once established treatment comprising a combination therapy: Allocetra + an immune checkpoint inhibitor (an anti-CTLA4 antibody) are administered over a series of days, followed by analysis of macrophage reprogramming and survival/histology.

[0025] Figure 5. Kaplan-Meier survival analysis 136+137

[0026] BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated i.p with 200μg anti-CTLA-4 (BioXcell, BE0164) and/ or Allocetra-OTS cells in 1 hour interval. Mice were monitored daily for clinical score and survival. Mice were sacrificed when reaching score 15. Log-rank p values presented relate to AB 12 cancer control (Group B).

[0027] Figure 6. Kaplan-Meier survival analysis 136+137

[0028] Figure 6 presents a number at risk table for mice survival. Number at risk is the number of subjects at risk immediately before the indicated timepoint. Being "at risk" means that the subject (i.e., mouse) has not had an event (i.e., death) before the indicated timepoint, and is not censored before or at the indicated timepoint.

[0029] Figures 7A and 7B. Clinical Score Presentation - In-vivo 136.

[0030] The clinical scores of mice in the experiment are presented. BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferase-expressing AB12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 200μg anti-CTLA-4 (BioXcell, BE0164) and/or 20x10^{^6} Allocetra-OTS cells at a 1-hour interval, wherein the anti-CTLA- 4 was added 1 hour prior to the addition of Allocetra. Mice were monitored daily for clinical scores and survival and were sacrificed upon reaching a clinical score of 15. Mean +SEM (Figure 7A) and Median +IQR (Figure 7B) are represented. Dead mice received a maximal clinical score of 15, as long as there were live mice in the group. When all mice from a group had died, plotting of the mean and median scores was ended.

[0031] Figures 8A - 8C. Clinical Score Presentation - In-vivo 137.

[0032] BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 200μg anti-CTLA-4 (BioXcell, BE0164) and/or Allocetra-OTS cells after a 1-hour interval, wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. Mice were monitored daily for clinical score and survival and sacrificed upon reaching a score of 15. Mean +SEM (Figure 8A) and median +IQR (Figure 8B) are presented. Dead mice received a maximal clinical score of 15, as long as there were live mice in the group. When all mice from group had died, plotted mean or median were stopped.

[0033] Figure 9. Abdominal Circumference - In-vivo 136

[0034] BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 200μg anti-CTLA-4 (BioXcell, BE0164) and/ or 20x10^{^6} Allocetra-OTS cells ay a 1-hour interval, wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. Abdominal circumference was measured and calculated weekly, starting on day 35 and twice a week from day 54 using two methods: measured circumference using a measuring tape, or measurements of width and height using a caliper and calculation of circumference using the ellipse circumference formula

Values presented as median ± IQR.

[0035] Figures 10A-10B. Abdominal Circumference - In- vivo 137.

[0036] BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 200μg anti-CTLA-4 (BioXcell, BE0164) and/or Allocetra-OTS cells after a 1-hour interval, wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. Abdominal circumference was measured and calculated, starting on day 27 and twice a week from day 33 by two methods: circumference of abdomen by measuring tape (Figure 10A), or measurements of width and height using a caliper and calculating the circumference using the ellipse circumference formula

(Figure 10B).

[0037] Elevation of values at day 39 using caliper measurements (Figure 10B) is due to an improved and adjusted measurement technique to improve the correlation tape measurements, and not a substantial increase in abdominal swelling.

[0038] Figures 11A and 11B. IVIS Imaging - In-vivo 136.

[0039] Figure 11A: BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferaseexpressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 200μg anti-CTLA-4 (BioXcell, BE0164) and/ or 20x10^{^6} Allocetra-OTS cells at a 1-hour interval, wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. IVIS imaging was performed at days 6, 11, 18, 25, 34, 48, 60, and 74 (Perkin- Elmer, Lumina III).

[0040] Figure 11B: BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferase- expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 200μg anti-CTLA-4 (BioXcell, BE0164) and/or 20x10^{^6} Allocetra-OTS cells after a 1-hour interval, wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. IVIS imaging was performed at days 6, 11, 18, 25, 34, 48, 62, and 74. Results show IVIS signal for surviving mice at day 74. The marked mouse had enlarged abdomen and high clinical score, without an IVIS signal.

[0041] Figures 12A-12H. IVIS Imaging - In- vivo 137.

[0042] BALB/c mice (female, 7 weeks) received 0.1x10^6 luciferase- expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated i.p with 200μg anti-CTLA-4 (BioXcell, BE0164) 20x10^6 or 5x10^6 Allocetra-OTS cells in 1 hour interval, wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. Control mice were treated with equivalent amounts of anti-CTLA-4 or Allocetra-OTS in control monotherapies. IVIS imaging was performed at days 6, 11, 18, 25, and 39 (Perkin- Elmer, Lumina III). Figure 12A presents the effect of anti-CTLA-4 and Allocetra-OTS mono- and combination therapies on tumor progression in mouse mesothelioma model (AB 12) at day 6. Figure 12B presents the effect of anti-CTLA-4 and Allocetra-OTS on tumor progression in mouse mesothelioma model (AB 12), at day 11, which is 1 day before treatment, and on day 25, which is 3 days after the 4^th treatment. Figures 12C-12H presents the effect of anti-CTLA-4 and Allocetra-OTS mono- and combination therapies on tumor progression in mouse mesothelioma model (AB 12) at days 18, 25, 39, 53, 67, and 81, respectively. Mice marked with red asterisk had luminescence spillovers from adjacent mice.

[0043] Figure 13A-13C. Graphic Presentation of IVIS Signal.

[0044] Figure 13A Graphic presentation of total flux in the IVIS signal. BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferase-expressing AB 12 tumor cells (AB 12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 200μg anti-CTLA-4 (BioXcell, BE0164) and/or 20x10^{^6} Allocetra-OTS cells after 1 hour interval (marked with orange vertical lines), wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. IVIS imaging was performed at days 6, 11, 18, 25, 34, 48, 60, and 74 (Perkin-Elmer, Lumina III). 7 minutes after luciferin injection, mice were anesthetized by isoflurane in an induction chamber. The mice were placed in a supine position (anterior facing upwards) inside the IVIS chamber. 10 minutes after luciferin injection the mice were imaged (exposure: 10 sec, F-stop: 2, Binning: medium). Analysis was performed by Living Image® software (Perkin Elmer, version 4.5.5). total flux (photon/sec) was evaluated at each time point. Results are presented as group median ± IQR. Black error represents days of injections.

[0045] Figure 13B. Graphic presentation of median radiance of IVIS signal. BALB/c mice (female, 7 weeks) received 0.1x10^{^6} luciferase-expressing AB 12 tumor cells (AB 12- luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 200μg anti-CTLA-4 (BioXcell, BE0164) and/or Allocetra-OTS cells after a 1-hour interval, wherein the anti- CTLA-4 was added 1 hour prior to the addition of Allocetra. IVIS imaging was performed at days 6, 11, 18, 25, 39, 53, 67, and 81 (Perkin-Elmer, Lumina III). 7 minutes after luciferin injection, mice were anesthetized by isoflurane in an induction chamber. The mice were placed in the supine position (anterior facing upwards) inside the IVIS chamber. 10 minutes after luciferin injection, the mice were imaged (exposure: 10 sec, F-stop: 2, Binning: medium). Analysis was performed by Living Image® software (Perkin Elmer, version 4.5.5). Total flux (photon/sec) was evaluated. Results are presented as group median ± IQR. In some groups (i.e., AB12+anti CLTA4+ 5x10^{^6}, some individuals had very low or very high median radiance (See, Figures 12A-12H) leading to a high IQR.

[0046] Figures 14A-14D. Clinical Score as a Predictor of Mortality.

[0047] Untreated, AB 12 tumor-bearing mice, were assigned a clinical score daily, and retrospectively analyzed for mortality outcome (death within 5 days or 10 days) at each timepoint. One-hundred and fourteen (114) data-points of 8 mice were analyzed from In- vivo 136, and total of 218 data-points from 16 mice were analyzed from In-vivo 136+137. The clinical score was evaluated as a predictor of 5 or 10-day mortality using a simple (univariate) logistic regression (Analysis was done with GraphPad Prism 9.3).

[0048] Figure 15. Weight Change post induction of Colon Cancer in a mouse model

[0049] Figure 15 graphically presents ANOVA measures of weight change following induction and treatment modes in mouse colon cancer model. ANOVA revealed no significant changes in body weight.

[0050] Figure 16. Tumor Volume Change post induction of Colon Cancer in a mouse model

[0051] Figure 16 graphically presents ANOVA measures of tumor volume change following induction and treatment modes in a mouse colon cancer model. ANOVA revealed a significant change between the vehicle group to 3 treatment groups in tumor volume. [0052] Figure 17. Tumor Weight Change post induction of Colon Cancer in a mouse model

[0053] The bar graph of Figure 17 shows that one way ANOVA analysis revealed no significant changes in tumor weight.

[0054] Figure 18. Efficacy studies of combination therapy (Allocetra + anti-PD-1 Antibody) in ID8 Luc syngeneic ovarian cancer mouse model

[0055] Figure 18 presents the optical imaging of mice prior to and following induction of ovarian cancer, and mono-therapeutic treatments (anti-PD-1 Ab alone [PD-1 mono- ] and Allocetra alone [Allo mono]) or combination therapies (Allocetra + anti-PDl Ab [Allo + PD-1], Allocetra - low dose + anti-PDl Ab [Allo 1/2 + PD-1], Allocetra - frozen formulation + anti-PDl Ab [Allo F+ PD-1], and Allocetra - intravenous administration + anti-PDl Ab [Allo IV + PD-1]. (See, Table 16 in Example 6 for administration route) [0056] Figure 19. Efficacy studies of combination therapy (Allocetra + anti-PD-1 Antibody) in ID8 Luc syngeneic ovarian cancer mouse model

[0057] Figure 19 graphically portrays the results of mono- and combination therapies in a ovarian cancer mouse model over the first 35 days of treatment. Monotherapies showed effectively the same or very similar results, while the combination therapy demonstrated a synergistic effect using Allocetra and the anti-PDl antibody, wherein the synergism was dramatically enhanced when administration of the anti-PDl antibody was by an intraperitoneal (IP) route. Control vehicle alone - red; anti-PD-1 Ab monotherapy (1.25 mg/kg) monotherapy - green; Allocetra 20M (IP) monotherapy - orange; Allocetra 20M (IP) + Anti-PD-1 Ab (1.25 mg/kg; IP) - dark purple; Allocetra 10M (Low dose IP) + Anti- PD-1 Ab (1.25 mg/kg; IP) - pink; Allocetra 20M (Frozen Formulation; IP) + Anti-PD-1 Ab (1.25 mg/kg; IP) - turquoise; and Allocetra 20M (IV) + Anti-PD-1 Ab (1.25 mg/kg; IP) - blue.

[0058] Figures 20A and 20B. Allocetra efficacy studies in ID8 Luc syngeneic ovarian cancer mouse model

[0059] Figure 20A graphically portrays optical imaging results from the ovarian cancer mouse model from week 1 to week 11, including the results from mouse controls, monotherapies, and various combination therapies. The mean BLI signal (photon flux/sec) is shown in each of the control and drug treatment arms over time. Figure 20B graphically portrays changes in body weight of the ovarian cancer mouse model subjects from week 1 to week 11, including those receiving controls, monotherapies, and various combination therapies. Control vehicle alone - red; anti-PD-1 Ab monotherapy (1.25 mg/kg) monotherapy - green; Allocetra 20M (IP) monotherapy - orange; Allocetra 20M (IP) + Anti- PD-1 Ab (1.25 mg/kg; IP) - dark purple; Allocetra 10M (Low dose IP) + Anti-PD-1 Ab (1.25 mg/kg; IP) - pink; Allocetra 20M (Frozen Formulation; IP) + Anti-PD-1 Ab (1.25 mg/kg; IP) - turquoise; and Allocetra 20M (IV) + Anti-PD-1 Ab (1.25 mg/kg; IP) - blue.

[0060] Figures 21A and 21B. Allocetra efficacy studies in ID8 Luc syngeneic ovarian cancer mouse model

[0061] Figure 21A graphically portrays the optical imaging results for the control (vehicle only) and monotherapies (anti-PD-1 Ab only and Allocetra only 20 x 10^A6 [20M]) for individual mice. Figure 21B graphically portrays the optical imaging results for the different combination therapies for individual mice (anti-PD-1 + Allocetra 20M; anti-PD-1 + Allocetra 10M; anti-PD-1 + Allocetra (F) 20M [Frozen Formulation]; and anti-PD-1 + Allocetra intravenous administration (IV) 20M).

[0062] Figures 22A and 22B. Survival Curves examining Allocetra efficacy studies in ID8 Luc syngeneic ovarian cancer mouse model.

[0063] Figure 22A graphically portrays the survival curve results with a 10M flux Cutoff KM for the mouse subjects of the ovarian cancer mouse model. Specifically examined where: control (vehicle only), monotherapies (anti-PD-1 Ab only and Allocetra only 20 x 10^A6 [20M]), and the different iterations of combination therapies (anti-PD-1 + Allocetra 20M; anti-PD-1 + Allocetra 10M; anti-PD-1 + Allocetra 20M Frozen [Frozen Formulation]; and anti-PD-1 + Allocetra intravenous administration (IV) 20M). Figure 22B graphically portrays the survival curve results with a 5M flux Cutoff KM for the mouse subjects of the ovarian cancer mouse model. Specifically examined where: control (vehicle only), monotherapies (anti-PD-1 Ab only and Allocetra only 20 x 10^A6 [20M]), and the different iterations of combination therapies (anti-PD-1 + Allocetra 20M; anti-PD-1 + Allocetra 10M; anti-PD-1 + Allocetra 20M Frozen [Frozen Formulation]; and anti-PD-1 + Allocetra intravenous administration (IV) 20M). Control vehicle alone - red; anti-PD-1 Ab monotherapy (1.25 mg/kg) monotherapy - green; Allocetra 20M (IP) monotherapy - orange; Allocetra 20M (IP) + Anti-PD-1 Ab (1.25 mg/kg; IP) - dark purple; Allocetra 10M (Low dose IP) + Anti-PD- 1 Ab ( 1.25 mg/kg; IP) - pink; Allocetra 20M (Frozen Formulation; IP) + Anti-PD-1 Ab (1.25 mg/kg; IP) - turquoise; and Allocetra 20M (IV) + Anti-PD-1 Ab (1.25 mg/kg; IP) - blue.

[0064] Figure 23 Follow-up Comparison from Week 1, Week 11, and Week 12.

[0065] Figure 23 shows follow-up comparisons of Bioluminescence from week 1, week 11, and week 12, wherein the mouse in the lower left-hand comer shows significant weight gain and a case of ascites.

[0066] Figure 24 presents a schematic of the AB 12 mesothelioma model and treatment plan. BALB/c mice (female, 7 weeks) received 0.1x10⁶ luciferase-expressing AB12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 125μg or 250μg anti-PD-1 (BioXcell, BE0146) and/or 5x10⁶ or 20x10⁶ Allocetra-OTS cells (Frozen Drug Product; FDP) at a 1-hour interval. On day 12, prior to treatment injections, mice were regrouped for homogeneous spreading of the tumor cells according to the In Vivo Imaging System (IVIS) findings.

[0067] Figure 25 presents follow-up data of weight during the experiment. The weight of mice in the experiment is presented. BALB/c mice (female, 7 weeks) received 0.1x10⁶ luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 125μg or 250μg anti-PD-1 (BioXcell, BE0146) and/or 5x10⁶ or 20x10⁶ Allocetra-OTS cells (FDP) at a 1-hour interval. On day 12, prior to treatment injections, mice were regrouped for homogeneous spreading of the tumor cells according to day 11 IVIS findings. Mice were weighed once a week. Mean +SEM is represented.

[0068] Figures 26A and 26B present the clinical score: Mean (Figure 26A) and Median (Figure 26B). BALB/c mice (female, 7 weeks) received 0.1x10⁶ luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 125μg or 250μg anti-PD-1 (BioXcell, BE0146) and/or 5x10⁶ or 20x10⁶ Allocetra-OTS cells (FDP) at a 1-hour interval. On day 12, prior to treatment injections, mice were regrouped for homogeneous spreading of the tumor cells according to day 11 IVIS findings. Mice were monitored daily for clinical scores and survival and were sacrificed upon reaching a clinical score of 15. Mean +SEM (Figure 26A) and Median +IQR (Figure 26B) are represented. Dead mice received a maximal clinical score of 15, as long as there were live mice in the group. When all mice from a group had died, plotting of the mean and median scores was ended.

[0069] Figure 27 presents IVIS imaging. BALB/c mice (female, 7 weeks) received 0. IxlO⁶ luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 125μg or 250μg anti-PD-1 (BioXcell, BE0146) and/or 5x10⁶ or 20x10⁶ Allocetra-OTS cells (FDP) at a 1-hour interval. IVIS imaging was performed at days 6, 11, 25, 39, 53, 64, and 78 (Perkin-Elmer, Lumina III). Image of 1 day before treatment (day 11) is after re-grouping. D represents an animal that had died.

[0070] Figure 28 presents IVIS imaging at day 78. BALB/c mice (female, 7 weeks) received 0.1x10⁶ luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 125μg or 250μg anti-PD-1 (BioXcell, BE0146) and/or 5x10⁶ or 20x10⁶ Allocetra-OTS cells (FDP) at a 1-hour interval. IVIS imaging was performed at days 6, 11, 25, 39, 53, 64, and 78 (Perkin-Elmer, Lumina III). Image of 1 day before treatment (day 11) is after re-grouping. D represents an animal that had died.

[0071] Figure 29 shows a graphic presentation of total flux of IVIS signal. BALB/c mice (female, 7 weeks) received 0.1x10⁶ luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 125μg or 250μg anti-PD-1 (BioXcell, BE0146) and/or 5x10⁶ or 20x10⁶ Allocetra-OTS cells (FDP) at a 1-hour interval. IVIS imaging was performed at days 6, 11, 25, 39, 53, 64, and 78 (Perkin-Elmer, Lumina III). Seven (7) minutes after luciferin injection, mice were anesthetized by isoflurane in an induction chamber. The mice were placed in a supine position (anterior facing upwards) inside the IVIS chamber. Ten (10) minutes after luciferin injection the mice were imaged (exposure: 10 sec, F-stop: 2, Binning: medium). Analysis was performed by Living Image® software (Perkin Elmer, version 4.5.5). total flux (photon/sec) was evaluated at each time point. Results are presented as group median ± IQR. Black arrow represents days of injections.

[0072] Figures 30A and 30B present Kaplan-Meier survival analysis. BALB/c mice (female, 7 weeks) received 0.1x10⁶ luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated IP with 125μg or 250μg anti-PD-1 (BioXcell, BE0146) and/or 5x10⁶ or 20x10⁶ Allocetra-OTS cells (FDP) at a 1-hour interval. Mice were monitored daily for clinical score and survival and were sacrificed upon reaching a score of 15. Log-rank p values shown here relate to AB 12 cancer control (Group A). Figure 30A shows a graphical representation of the Kaplan-Meier survival analysis. Figure 30B represents the number of mice at risk for survival.

[0073] Number at risk is the number of subjects at risk immediately before the indicated timepoint. Being "at risk" means that the subject (i.e., mouse) has not had an event (i.e., death) before the indicated timepoint, and is not censored before or at the indicated timepoint.

[0074] To calculate the survival probability for each elapsed time, the Kaplan-Meier product limit method is used following the formula:

St = Survival probability at the indicated timepoint.

St-1 = Survival probability at the previous timepoint.

Nt = Number at risk at the indicated timepoint.

Et = The number of events (deaths) at the indicated timepoint.

[0075] The idea behind the number at risk table is that - in order to calculate survival probability using the Kaplan-Meier product limit method - one needs to know how many mice were still accounted for in the study group that had not yet died. Therefore, the number at risk at any specific time point will be equal to the total number of mice remaining in the study group including any mice that died or mice that are censored at this time point. In case that the unit of time is “days”, the number at risk was considered to be those mice who have not yet died or been censored at the beginning of the day (before any death or censoring could occur).

[0076] The number at risk doesn't change unless a mouse dies or is censored. Thus, there is only a need to report a new number at risk at time points where one of these situations occurs. Therefore, the number at risk table, which is displayed under the Kaplan-Meier curve, provides us the information of how many mice are still present (didn't die or censored) at each step of the curve.

[0077] Figure 31 presents the descriptive statistics of average survival.

DETAILED DESCRIPTION

[0078] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the combination therapy and methods of use thereof for treating cancer. However, it will be understood by those skilled in the art that method of treatment using a combination therapy comprising apoptotic cells and checkpoint inhibitors, 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 therapy and methods thereof disclosed herein. [0079] In some embodiments, disclosed herein is a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor. In some embodiments, disclosed herein is a combination therapy comprising a first composition comprising a mononuclear-enriched apoptotic cell population and a second composition comprising one or more checkpoint inhibitors. In some embodiments, disclosed herein is a method of treating, inhibiting the growth of, reducing the incidence of, or any combination thereof, a cancer or a tumor in a subject, comprising a step of administering a combination therapy comprising a first composition comprising a mononuclear-enriched apoptotic cell population and a second composition comprising a checkpoint inhibitor. In some embodiments, disclosed herein is a method of treating, inhibiting the growth of, reducing the incidence of, or any combination thereof, a cancer or a tumor in a subject, comprising a step of administering a combination therapy comprising a first composition comprising a mononuclear-enriched apoptotic cell population and a second composition comprising one or more checkpoint inhibitors. In some embodiments, disclosed herein is a method of increasing survival of a subject suffering from a cancer or a tumor, comprising a step of administering a combination therapy comprising a first composition comprising a mononuclear-enriched apoptotic cell population and a second composition comprising a checkpoint inhibitor. In some embodiments, disclosed herein is a method of increasing survival of a subject suffering from a cancer or a tumor, comprising a step of administering a combination therapy comprising a first composition comprising a mononuclear-enriched apoptotic cell population and a second composition comprising one or more checkpoint inhibitors. In some embodiments, disclosed herein is a method of reducing the size or reducing the growth rate of a cancer or a tumor, or a combination thereof, in a subject, comprising a step of administering a combination therapy comprising a first composition comprising a mononuclear-enriched apoptotic cell population and a second composition comprising a checkpoint inhibitor. In some embodiments, disclosed herein is a method of reducing the size or reducing the growth rate of a cancer or a tumor, or a combination thereof, in a subject, comprising a step of administering a combination therapy comprising a first composition comprising a mononuclear-enriched apoptotic cell population and a second composition comprising one or more checkpoint inhibitors.

[0080] In some embodiments, compositions of apoptotic cells comprise a mononuclear enrich population of apoptotic cells, wherein the apoptotic cells are inactivated, where the population comprises a decreased percent of non-quiescent non-apoptotic viable cells; a suppressed cellular activation of any living non-apoptotic cells; or a reduced proliferation of any living non-apoptotic cells; or any combination thereof. In some embodiments, disclosed herein are populations of apoptotic cells wherein any viable cells are inactivated, resulting in a mononuclear enriched apoptotic cell population comprising a decreased percent of non- quiescent non-apoptotic viable cells; a suppressed cellular activation of any living non- apoptotic cells; or a reduced proliferation of any living non-apoptotic cells; or any combination thereof.

[0081] In some embodiments, a cell population disclosed herein is considered inactivated, when the viable portion of the population has been decreased, or when the cellular activation or proliferation thereof has been suppressed. In some embodiments, inactivation comprises irradiation of the apoptotic cell population, wherein said irradiation is post induction of apoptosis of a mononuclear enriched cell population. In another embodiment, inactivation comprises suppressing or eliminating an immune response in the population. In another embodiment, inactivation comprises suppressing or eliminating cross-reactivity between a apoptotic cell population and any other cell population. In other embodiments, inactivation comprises reducing or eliminating T-cell receptor activity in an apoptotic cell population. In another embodiment, an inactivated cell preparation comprises a decreased percent of living non-apoptotic cells, suppressed cellular activation of any living non-apoptotic cells, or a reduce proliferation of any living non-apoptotic cells, or any combination thereof.

[0082] In another embodiment, an inactivated cell population comprises a reduced number of non-quiescent non-apoptotic cells compared with a non-irradiated cell preparation. In some embodiments, an inactivated cell population comprises less than 50 percent (%) living non-apoptotic cells. In some embodiments, an inactivated cell population comprises less than 40% living non-apoptotic cells. In some embodiments, an inactivated cell population comprises less than 30% living non-apoptotic cells. In some embodiments, an inactivated cell population comprises less than 20% living non-apoptotic cells. In some embodiments, an inactivated cell population comprises less than 0% living non-apoptotic cells.

[0083] In some embodiments, methods disclosed herein treat, inhibit the growth of, or delay disease progression, of a cancer or a tumor in a human subject, wherein the method comprises a step of administering to a subject in need a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor, wherein said method treats, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said human subject. In some embodiments, methods of use of treating, inhibiting the growth of, reducing the incidence of, or any combination thereof, a cancer or a tumor in a subject, comprise a step of administering a combination therapy comprising a first composition comprising an inactivated mononuclear-enriched apoptotic cell population, wherein said population is irradiated post induction of apoptosis, and a second composition comprising one or more checkpoint inhibitors. In some embodiments, methods of use of treating, inhibiting the growth of, reducing the incidence of, or any combination thereof, a cancer or a tumor in a subject, comprise a step of administering a combination therapy comprising a first composition comprising an irradiated mononuclear-enriched apoptotic cell population, wherein said population is irradiated post induction of apoptosis, and a second composition comprising one or more checkpoint inhibitors. In some embodiments, methods of treating a cancer or tumor disclosed herein, comprise an improved method compared with administering either an apoptotic cell population independent of another therapy or a checkpoint inhibitor independent of another therapy.

[0084] In some embodiments, the improvement comprises an improved effectiveness of T cells in attacking a tumor cell. In some embodiments, the improvement comprises an improved effectiveness of T cells in killing a tumor cell. In some embodiments, the improvement comprises an improved effectiveness of natural killer (NK) cells in attacking a tumor cell. In some embodiments, the improvement comprises an improved effectiveness of NK cells in killing a tumor cell. In some embodiments, the improvement comprises an improved cytotoxic effect on a tumor cell. In some embodiments, the improvement comprises a reduction of at least one side effect. In some embodiments, the improvement comprises an increased reduction of tumor size. In some embodiments, the improvement comprises a complete reduction of a tumor. In some embodiments, the improvement comprises a reduction of metastasis. In some embodiments, the improvement comprises the elimination of metastasis. In some embodiments, the improvement comprises enhanced responsiveness, wherein improvements are observed in a shorter time frame, compared with a subject not receiving the combination therapy. In some embodiments, the improvement comprises an increased survival rates compared with a subject not receiving the combination therapy. Mononuclear-enriched Apoptotic Cell Populations

[0085] Apoptosis may be induced in a population of mononuclear-enriched cells. Methods of induction of apoptosis are well known in the art and include but are not limited to serum deprivation; incubation with a corticosteroid, for example by not limited to incubation with dexamethasone; activation of Fas or TNF receptors on the cell surface by their respective ligands, or by cross-linking these receptors with an agonist antibody; or irradiation, for example but not limited to ultraviolet B irradiation or gamma (y) irradiation. (See for example, Roberts K.M., et al., (2004) Methods for Inducing Apoptosis. In: Perl A. (eds) Autoimmunity. Methods in Molecular Medicine™, vol 102. Humana Press; Nijhuis et al., Induction of apoptosis by heat and y-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 Application Publication Numbers WO 2016/170541, WO 2019/038758, WO 2020/105034, WO 2014/087408, and WO 2018/225072, which are herein incorporated in their entirety).

[0086] In some embodiments, mononuclear-enriched cells may be obtained directly, for example but not limited to, from a human subject using techniques such as leukapheresis. In some embodiments, mononuclear-enriched cells may be obtained indirectly, for example but not limited to, from a blood bank when peripheral blood mononuclear cells (PBMC) are used.

[0087] In some embodiments, an apoptotic mononuclear-enriched cell population comprises a white blood cell (WBC) fraction from a blood donation. In some embodiments, an apoptotic 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.

[0088] In some embodiments, inducing apoptosis of a mononuclear enriched cell population, for example but not limited to primary lymphocytes, comprises treatment with a corticosteroid and is affected by treating the primary lymphocytes with dexamethasone. In another embodiment, inducing apoptosis of mononuclear enriched cells, for example but not limited to primary lymphocytes, is via irradiation and is affected by treating the primary lymphocytes with gamma-irradiation. In other embodiments, inducing apoptosis in a mononuclear enriched population comprises use of any method known in the art.

[0089] In some embodiments, an apoptotic mononuclear-enriched cell population comprises a freshly prepared apoptotic cell population. In some embodiments, a freshly prepared apoptotic mononuclear-enriched cell population is maintained on ice or at between about 2-8°C comprises apoptotic cells. In some embodiments, maintenance of an apoptotic mononuclear-enriched population on ice is for about 24 hours. In some embodiments, maintenance of an apoptotic mononuclear-enriched population on ice is for about 48 hours. In some embodiments, maintenance of an apoptotic mononuclear-enriched population on ice is for about 72 hours. In some embodiments, maintenance of an apoptotic mononuclear- enriched population on ice is for about 96 hours. In some embodiments, maintenance of an apoptotic mononuclear-enriched population on ice is for between about 24-96 hours. In some embodiments, maintenance of an apoptotic mononuclear-enriched population at 2-8 °C is for about 24 hours. In some embodiments, maintenance of an apoptotic mononuclear- enriched population at 2-8°C is for about 48 hours. In some embodiments, maintenance of an apoptotic mononuclear-enriched population at 2-8°C is for about 72 hours. In some embodiments, maintenance of an apoptotic mononuclear-enriched population at 2-8°C is for about 96 hours. In some embodiments, maintenance of an apoptotic mononuclear-enriched population at 2-8°C is for between about 24-96 hours. In some embodiments, maintenance of an apoptotic mononuclear-enriched population is on ice for between about 24-96 hours. [0090] In some embodiments, an apoptotic mononuclear-enriched population is stable at room temperature for up to one hour. In some embodiments, an apoptotic mononuclear- enriched population is stable at room temperature for at least one hour. In some embodiments, an apoptotic mononuclear-enriched population is stable at room temperature for more than one hour. In some embodiments, an apoptotic mononuclear-enriched population is stable at room temperature for between about 1 -3 hours. In some embodiments, an apoptotic mononuclear-enriched population is stable at room temperature for about 1, 2, or 3 hours. In some embodiments, an apoptotic mononuclear-enriched population is stable at room temperature for at least 2 hours. In some embodiments, an apoptotic mononuclear- enriched population is stable at room temperature for at least 3 hours. In some embodiments, an apoptotic mononuclear-enriched population is stable at room temperature for more than 2 hours. In some embodiments, an apoptotic mononuclear-enriched population is stable at room temperature for more than 3 hours. [0091] A skilled artisan would appreciate that the terms “stable apoptotic nuclear- enriched populations of cells” or “maintenance of apoptotic nuclear-enriched populations of cells” encompasses apoptotic cells that remain PS-positive (Phosphatidylserinepositive) with only a very small percent of Pl-positive (Propidium iodide-positive) cells. Pl-positive cells provide an indication of membrane stability wherein a Pl-positive cell permits admission into the cells, showing that the membrane is less stable.

[0092] As used herein, a freshly prepared mononuclear-enriched apoptotic cell population is termed a “Fresh Formulation” or a “Liquid Formulation”, wherein these terms may be used interchangeably having all the same meanings and qualities.

[0093] In some embodiments, freshly prepared apoptotic mononuclear-enriched cell populations are stored as a frozen formulation, wherein the frozen formulation comprises an apoptotic cell population. In some embodiments, freshly prepared apoptotic mononuclear- enriched cell populations are first maintained for a time period of up to 96 hours, for example for up to 24, 48, 72, or 96 hours, on ice and are then stored as a frozen formulation, wherein said frozen formulation comprises apoptotic cells. In some embodiments, freshly prepared apoptotic mononuclear-enriched cell populations are first maintained at between about 2- 8°C for up to 96 hours, for example for up to 24, 48, 72, or 96 hours, and are then stored as a frozen formulation wherein said frozen formulation comprises apoptotic cells.

[0094] The state of apoptotic cell populations is an important quality attribute of the product. To determine apoptotic state, Annexin V (AnV) and propidium iodide (PI) staining are 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 initial stage of apoptosis, membrane-bound phosphatidylserine (PtdSer) translocates from the inner, cytoplasmic layer leaflet of the cell membrane to the outer, extracellular layer leaflet of the cell membrane, thus exposing PtdSer to its immediate environment and making it accessible to AnV binding. Following PtdSer exposure, the apoptotic process progresses and continues with activations of additional caspases.

[0095] Two sub-populations of apoptotic cells are derived from this dynamic process: apoptotic cells characterized by AnV+ PI- staining, and mature apoptotic cells characterized by AnV+ and low PI staining (dimly PI fluorescent population due to the progression of the membrane permeabilization process which leads to gradual loss of membrane integrity). [0096] The apoptotic cell is eventually engulfed by phagocytes, if cells are not phagocytosed, the plasma membrane gradually loses its integrity. Loss of plasma membrane integrity can be detected by nonselective and massive uptake of fluorescent dyes such as PI, which leads to high intensity of PI staining on the cells. The PI enters the nucleus and intercalates within the DNA double strand and may serve as a biomarker for late apoptotic or necrotic cells. Late apoptotic cell populations are characterized by AnV+ PI+High staining. In some embodiments, late apoptotic cells comprise cells not efficiently removed by phagocytic cells and therefore the cells become necrotic (secondary necrosis).

[0097] In some embodiments, as used herein, an apoptotic cell population comprises and mature mononuclear-enriched apoptotic cell populations. In certain embodiments, a fresh (non-frozen) apoptotic cell population comprises at least 35% apoptotic cells (and mature apoptosis) and less than 30% late apoptotic cells. In certain embodiments, a fresh (nonfrozen) apoptotic cell population comprises at least 35% AnV+ cells (and mature apoptosis) and less than 30% AnV+ and IP+High cells. In certain embodiments, a thawed frozen formulation of apoptotic cell comprises at least 35% apoptotic cells (and mature apoptosis) and less than 30% late apoptotic cells. In certain embodiments, a thawed frozen formulation of apoptotic cells comprises at least 35% AnV+ cells (and mature apoptosis). In certain embodiments, a thawed frozen formulation of apoptotic cells comprises at least 35% AnV+ cells (and mature apoptosis) and less than 30% AnV+ and IP+High cells.

[0098] A skilled artisan would appreciate that the terms “apoptotic cells”, “apoptotic cell populations”, “Allocetra”, “Allocetra-OTS”, and the like may be used interchangeably having all the same qualities and meanings of a population of mononuclear enriched apoptotic cells comprising wherein at least 35% of the apoptotic cells are in an and mature state of apoptosis (at least 35% AnV+PI- (early) and AnV+PI+^DIM (mature)). In some embodiments, apoptotic cell populations comprise freshly prepared apoptotic cell populations. In some embodiments, apoptotic cell populations comprise frozen formulations of apoptotic cell populations. In some embodiments, apoptotic cell populations comprise apoptotic cell populations that have been thawed from frozen formulations of apoptotic cells, as described herein.

[0099] Embodiments of the apoptotic sub-populations (early, mature, and late) are indicated in Figure IB (See, Example 1).

[00100] In some embodiments, freshly frozen apoptotic cell populations are frozen following induction of apoptosis. In some embodiments, freshly frozen apoptotic cell populations are frozen after induction of apoptosis and a step of irradiating of the apoptotic cell population.

[00101] As used herein, the term “freshly frozen” encompasses apoptotic cells that have been prepared and not previously frozen as an apoptotic cell population. (See, Figure IE) [00102] The skilled artisan would appreciate that freezing of mononuclear enriched cells for storage prior to apoptotic induction would not be considered as providing previously frozen apoptotic cells. The skilled artisan would appreciate that freezing of mononuclear enriched cells as part of the process of apoptotic induction would not be considered as providing previously frozen apoptotic cells.

[00103] In some embodiments, an apoptotic mononuclear-enriched cell population comprises an inactivated apoptotic mononuclear-enriched cell population. In some embodiments, an inactivated apoptotic mononuclear-enriched cell population comprises an irradiated population, wherein said irradiation is post induction of apoptosis.

[00104] In some embodiments, apoptotic mononuclear enriched cells are irradiated in a way that will decrease proliferation and/or activation of residual viable cells within the apoptotic cell population. In some embodiments, apoptotic mononuclear enriched are irradiated in a way that reduces the percentage of viable non-apoptotic cells in a population. In some embodiments, the percent of viable non-apoptotic cells in an inactivated apoptotic cell population is reduced to less than 50% of the population. In some embodiments, the percentage of viable non-apoptotic cells in an inactivated apoptotic cell population is reduced to less than 40% of the population. In some embodiments, the percentage of viable non-apoptotic cells in an inactivated apoptotic cell population is reduced to less than 30% of the population. In some embodiments, the percent of viable non-apoptotic cells in an inactivated apoptotic cell population is reduced to less than 20% of the population. In some embodiments, the percentage of viable non-apoptotic cells in an inactivated apoptotic cell population is reduced to less than 10% of the population. In some embodiments, the percent of viable non-apoptotic cells in an inactivated apoptotic cell population is reduced to 0% of the population.

[00105] In some embodiments, an apoptotic mononuclear-enriched cell population disclosed herein is inactivated. In another embodiment, inactivation comprises irradiation. In another embodiment, inactivation comprises T-cell receptor inactivation. In another embodiment, inactivation comprises T-cell receptor editing. In another embodiment, inactivation comprises suppressing or eliminating an immune response in said preparation. In another embodiment, inactivation comprises suppressing or eliminating cross-reactivity between multiple individual populations comprised in the preparation. In other embodiment, inactivation comprises reducing or eliminating T-cell receptor activity between multiple individual populations comprised in the preparation. In another embodiment, an inactivated cell preparation comprises a decreased percent of living non-apoptotic cells, suppressed cellular activation of any living non-apoptotic cells, or a reduce proliferation of any living non-apoptotic cells, or any combination thereof.

[00106] In another embodiment, an inactivated cell population comprises a reduced number of non-quiescent non-apoptotic cells compared with a non-radiated cell preparation. In some embodiments, an inactivated cell population comprises 50 percent (%) of living non-apoptotic cells. In some embodiments, an inactivated cell population comprises 40% of living non-apoptotic cells. In some embodiments, an inactivated cell population comprises 30% of living non-apoptotic cells. In some embodiments, an inactivated cell population comprises 20% of living non-apoptotic cells. In some embodiments, an inactivated cell population comprises 100% of living non-apoptotic cells. In some embodiments, an inactivated cell population comprises 0% of living non-apoptotic cells.

[00107] In some embodiments, an apoptotic mononuclear-cell-enriched population comprises apoptotic cells irradiated after induction of apoptosis. In another embodiment, the irradiation comprises gamma irradiation or UV irradiation. In yet another embodiment, the irradiated preparation has a reduced number of non-quiescent non-apoptotic cells compared with a non-irradiated cell preparation.

[00108] In another embodiment, the irradiated apoptotic mononuclear enriched cells preserve all their apoptotic-, immune modulation-, stability-properties. In another embodiment, the irradiation step uses UV radiation. In another embodiment, the radiation step uses gamma radiation. In another embodiment, the apoptotic cells comprise a decreased percent of living non-apoptotic cells, comprise a preparation having a suppressed cellular activation of any living non-apoptotic cells present within the apoptotic cell preparation, or comprise a preparation having reduced proliferation of any living non-apoptotic cells present within the apoptotic mononuclear enriched cell preparation, or any combination thereof.

[00109] In some embodiments, an irradiated apoptotic cell preparation as disclosed herein has suppressed cellular activation and reduced proliferation compared with a nonirradiated apoptotic cell preparation. In another embodiment, the irradiation comprises gamma irradiation or UV irradiation. In another embodiment, an irradiated apoptotic cell preparation has a reduced number of non-apoptotic cells compared with a non-irradiated cell preparation. In another embodiment, the irradiation comprises about 15 Grey units (Gy). In another embodiment, the irradiation comprises about 20 Grey units (Gy). In another embodiment, the irradiation comprises about 25 Grey units (Gy). In another embodiment, the irradiation comprises about 30 Grey units (Gy). In another embodiment, the irradiation comprises about 35 Grey units (Gy). In another embodiment, the irradiation comprises about 40 Grey units (Gy). In another embodiment, the irradiation comprises about 45 Grey units (Gy). In another embodiment, the irradiation comprises about 50 Grey units (Gy). In another embodiment, the irradiation comprises about 55 Grey units (Gy). In another embodiment, the irradiation comprises about 60 Grey units (Gy). In another embodiment, the irradiation comprises about 65 Grey units (Gy). In another embodiment, the irradiation comprises up to 2500 Gy. In another embodiment, an irradiated pooled apoptotic cell preparation maintains the same or a similar apoptotic profile, stability and efficacy as a non-irradiated pooled apoptotic cell preparation.

[00110] In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 1% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 2% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 3% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 4% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 5% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 6% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 7% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 8% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 9% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 10% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 15% compared with apoptotic cells not irradiated. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared with apoptotic cells not irradiated.

[00111] In some embodiments, a cell population comprising a reduced or non-existent fraction of living non-apoptotic cells may in one embodiment provide a mononuclear apoptotic cell population that does not have any living / viable cells. In some embodiments, a cell population comprising a reduced or non-existent fraction of living non-apoptotic cells may in one embodiment provide a mononuclear apoptotic cell population that does not elicit GVHD in a recipient.

[00112] In some embodiments, apoptotic cells are verified by May-Giemsa-stained cytopreps. In some embodiments, viability of cells is assessed by trypan blue exclusion. In some embodiments, the apoptotic and necrotic status of the cells are confirmed by annexin V/propidium iodide staining with detection by FACS.

[00113] In some embodiments, apoptotic cells disclosed herein comprise no necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 30 % necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 25% necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 20% necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 15% necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 10% necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 5% necrotic cells.

[00114] The skilled artisan would appreciate that the formulations and methods described herein, in some embodiments comprise apoptotic cells and uses thereof. In some embodiments, as described herein, apoptotic cells are HLA matched to a recipient (a subject in need of a composition comprising the apoptotic cells). In some embodiments, as described herein, apoptotic cells are not matched to a recipient (a subject in need of a composition comprising the apoptotic cells). In some embodiments, apoptotic cells are unmatched from a foreign donor. In some embodiments, the apoptotic cells not matched to a recipient of a composition comprising the apoptotic cells (a subject in need) are irradiated as described herein in detail. In some embodiments, irradiated not matched cells are termed “Allocetra- OTS”, “Allocetra”, “ALC”, or “ALC-OTS”, all having the same meaning and qualities.

[00115] In some embodiments, freshly prepared apoptotic cells are stable. A skilled artisan would appreciate that in some embodiments, stability encompasses maintaining apoptotic cell characteristics over time, for example, maintaining apoptotic cell characteristics upon storage at about 2-8°C. In some embodiments, stability comprises maintaining apoptotic cell characteristic upon storage at freezing temperatures, for example temperatures at or below 0°C. In some embodiments, stability comprises maintaining apoptotic cell characteristics upon storage at freezing temperatures, for example temperatures at about -196 °C.

[00116] In some embodiments, an apoptotic mononuclear-enriched cell population comprises pooled, apoptotic mononuclear-enriched cell populations. In some embodiments, an inactivated apoptotic mononuclear-enriched cell population comprises pooled, apoptotic mononuclear-enriched cell populations. In some embodiments, an irradiated apoptotic mononuclear-enriched cell population comprises pooled, apoptotic mononuclear-enriched cell populations. In some embodiments, a pooled, apoptotic mononuclear-enriched cell populations comprise allogeneic cells from HLA matched or HLA unmatched sources, with respect to a recipient subject.

[00117] This disclosure provides in some embodiments, a pooled mononuclear-enriched apoptotic cell population comprising mononuclear cells in an apoptotic state, wherein said pooled mononuclear-enriched apoptotic cells population comprises pooled individual mononuclear cell populations. In some embodiments, an apoptotic cell population as described herein, comprises a pooled mononuclear-enriched apoptotic cells population and wherein said pooled mononuclear apoptotic cell preparation comprises a decreased percent of living non-apoptotic cells, a suppressed cellular activation of any living non-apoptotic cells, or a reduced proliferation of any living non-apoptotic cells, or any combination thereof. In some embodiments, the pooled mononuclear apoptotic cells have been irradiated after induction of apoptosis. In another embodiment, this disclosure provides a pooled mononuclear apoptotic cell preparation that in some embodiments, uses the white blood cell fraction (WBC) obtained from donated blood. Often this WBC fraction is discarded at blood banks or is targeted for use in research.

[00118] A skilled artisan would appreciate that the term "autologous” may encompass a tissue, cell, nucleic acid molecule or polypeptide in which the donor and recipient is the same person.

[00119] A skilled artisan would appreciate that the term "allogeneic” may encompass a tissue, cell, nucleic acid molecule or polypeptide that is derived from separate individuals of the same species. In some embodiments, allogeneic donor cells are genetically distinct from the recipient.

[00120] In some embodiments, obtaining a mononuclear-enriched cell composition is affected by leukapheresis. A skilled artisan would appreciate that the term “leukapheresis” may encompass an apheresis procedure in which leukocytes are separated from the blood of a donor. In some embodiments, the blood of a donor undergoes leukapheresis and thus a mononuclear-enriched cell composition is obtained according to the production method disclosed herein. It is to be noted, that the use of at least one anticoagulant during leukapheresis is required, as is known in the art, in order to prevent clotting of the collected cells.

[00121] In some embodiments, the leukapheresis procedure is configured to allow collection of mononuclear-enriched cell composition. In some embodiments, cell collections obtained by leukapheresis comprise at least 65% mononuclear cells. In other embodiments, cell collections obtained by leukapheresis comprise at least at least 70%, or at least 80% mononuclear cells. In some embodiments, blood plasma from the cell-donor is collected in parallel to obtaining of the mononuclear-enriched cell composition. In some embodiments, about 300-600ml of blood plasma from the cell-donor are collected in parallel to obtaining the mononuclear-enriched cell composition according to the production method disclosed herein.

[00122] In some embodiments, the apoptotic cells comprised in the frozen formulations disclosed herein comprise at least 85% mononuclear cells. In further embodiments, the apoptotic cells in the frozen formulations disclosed herein contain at least 85% mononuclear cells, 90% mononuclear cells or alternatively over 90% mononuclear cells. In some embodiments, the apoptotic cells in the frozen formulations disclosed herein comprise at least 90% mononuclear cells. In some embodiments, the apoptotic cells in the frozen formulations disclosed herein comprise at least 95% mononuclear cells.

[00123] It is to be noted that, in some embodiments, while the mononuclear-enriched cell preparation at cell collection comprises at least 65%, preferably at least 70%, most preferably at least 80% mononuclear cells, the final pharmaceutical population, following the production method of the apoptotic cells for use in the methods disclosed herein, comprises at least 85%, preferably at least 90%, most preferably at least 95% mononuclear cells.

[00124] A skilled artisan would appreciate that the term “mononuclear cells” may encompass leukocytes having a one lobed nucleus.

[00125] In some embodiments, the apoptotic cell preparation comprises no more than 15% CD15high expressing cells.

[00126] 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.

[00127] In some embodiments, the mononuclear enriched cell population comprises peripheral blood mononuclear cells (PBMC). In some embodiments, the mononuclear enriched cell population comprises a fraction of cells isolated from PBMC. In some embodiments, the mononuclear enriched cell population comprises a fraction of a single type of cells isolated from PBMC. In some embodiments, the mononuclear enriched cell population comprises a fraction of two types of cells isolated from PBMC. In some embodiments, the mononuclear enriched cell population comprises a fraction of at least a single type of cells isolated from PBMC. In some embodiments, the mononuclear enriched cell population comprises a fraction of at least two types of cells isolated from PBMC.

[00128] 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.

[00129] 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.

[00130] In some embodiments, the mononuclear-enriched cell population comprises lymphocytes and monocytes.

[00131] In another embodiment, the mononuclear enriched cell population comprises no more than 15%, alternatively no more than 10%, typically no more than 5% polymorphonuclear leukocytes, also known as granulocytes (i.e., neutrophils, basophils and eosinophils). In another embodiment, a pooled mononuclear cell population is devoid or essentially devoid of granulocytes. In another embodiment, a pooled apoptotic cell preparation as disclosed herein comprises less than 5% polymorphonuclear leukocytes.

[00132] Functional analysis of an apoptotic cell population may in some embodiments comprise assaying the potential of an apoptotic cell population for the ability to reduce production of cytokines associated with the cytokine storm including but not limited to IL- 6, Tumor Necrosis Factor alpha (TNF-a), IL-ip, and interferon-gamma (IFN-y), alone or in combination.

Frozen Formulations

[00133] In some embodiments, a frozen apoptotic cell formulation comprises a population of apoptotic mononuclear enriched cells; an isotonic crystalloid solution; and a cryoprotectant agent; wherein said formulation comprises a pH range of about 6.5-8.0. In some embodiments, the population of apoptotic mononuclear enriched cells frozen was freshly prepared prior to freezing. In some embodiments, the population of apoptotic mononuclear enriched cells frozen was freshly prepared and stored on ice prior to freezing. In some embodiments, the population of apoptotic mononuclear enriched cells frozen was freshly prepared and stored at between about 2-8°C prior to freezing.

[00134] Analysis of apoptotic state is important prior to freezing the population of apoptotic mononuclear enriched cells. In some embodiments, prior to freezing the apoptotic mononuclear enriched cells comprise and mature apoptotic cell populations. In some embodiments, prior to freezing the apoptotic mononuclear enriched cells comprise at least 35% apoptotic cells and less than 30 % late apoptotic cells. In some embodiments, prior to freezing the apoptotic mononuclear enriched cells comprise at least 35% AnV+ cells and less than 30% AnV+ and PI+ ^Hlgh cells. In some embodiments, fresh apoptotic cells comprise a population of mononuclear enriched apoptotic cells prepared from methods known in the art.

[00135] Density of cells at freezing may be critical to ensure the cell viability. Too high density might reduce the cell viability. Further, different cell types may require different densities at freezing, which may be tested prior to freezing. In some embodiments, the concentration of the population of apoptotic mononuclear enriched cells at freezing is about 10 x 10^{^6} - 250 x 10^{^6} cells/ml. In some embodiments, the concentration of the population of apoptotic mononuclear enriched cells at freezing is about 20 x 10^{^6} - 100 x 10^{^6} cells/ml. In some embodiments, the concentration of the population of apoptotic mononuclear enriched cells at freezing is about 20 x 10^{^6} - 60 x 10^{^6} cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 24 x 10⁶ - 50 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 24 x 10⁶ - 40 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 44 x 10⁶ - 50 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 24 x 10⁶ - 35 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 20 x 10⁶ - 40 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 20 x 10⁶ - 35 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 25 x 10⁶ - 35 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 35 x 10⁶ - 45 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 35 x 10⁶ - 40 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 40 x 10⁶ - 50 x 10⁶ cells/ml. In some embodiments, the density of the population of apoptotic mononuclear enriched cells at freezing is about 40 x 10⁶ - 60 x 10⁶ cells/ml.

[00136] As described above, the mononuclear enriched cells from which apoptotic cells to be frozen are produced may in certain 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 from which apoptotic cells to be frozen are produced comprise PBMC. In some embodiments, the mononuclear enriched cells from which apoptotic cells to be frozen are produced comprise a fraction of PBMC comprising at least one cell type. In some embodiments, the mononuclear enriched cells from which apoptotic cells to be frozen are produced comprise a fraction of PBMC comprising at least two cell types. In some embodiments, the mononuclear enriched cells from which apoptotic cells to be frozen are produced comprise a single cell type selected from monocytes, lymphocytes, T cells, B cells, and natural killer cells.

[00137] In some embodiments, a frozen formulation of apoptotic mononuclear enriched cells comprises a pooled mononuclear apoptotic cell preparation. In some embodiments, a pooled mononuclear apoptotic cell preparation comprises mononuclear cells in an apoptotic state, wherein said pooled mononuclear apoptotic cells comprise a decreased percent of living non-apoptotic cells, a suppressed cellular activation of any living non-apoptotic cells, or a reduced proliferation of any living non-apoptotic cells, or any combination thereof. In certain embodiments, the pooled mononuclear enriched apoptotic cells have been irradiated. In some embodiments, disclosed herein is a pooled mononuclear enriched apoptotic cell preparation that originates from the white blood cell fraction (WBC) obtained from donated blood.

[00138] The population of apoptotic mononuclear enriched cells for freezing may be produced by any method known in the art including but not limited to inducing apoptosis in a mononuclear cell population using methylprednisolone as described in detail in International Publication No. WO 2014/087408 at Example 14, International Publication No. WO 2019/038758 at Example 1, and International Publication No. WO 2016/170541 at Examples 1, 2, 3, 5, and 6, which are all hereby incorporated in their entirety. [00139] Crystalloid fluids, i.e., crystalloid solutions, comprise an aqueous solution of mineral salts and other small, water-soluble molecules. Crystalloid solutions may in some embodiments be isotonic to human plasma. In some embodiments, crystalloid solutions approximate concentrations of various solutes found in plasma and do not exert an osmotic effect in vivo. Crystalloid solutions may function to expand intravascular volume without disturbing ion concentration or causing significant fluid shifts between intracellular, intravascular, and interstitial spaces. In some embodiments, a frozen formulation of apoptotic cells disclosed herein comprises an isotonic crystalloid solution.

[00140] In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises sodium chloride (NaCl), potassium chloride (KC1), magnesium chloride (MgCl₂), sodium acetate (NaCH₃COO, also abbreviated NaOAc), or sodium gluconate (NaC₆H₁₁O₇), or any combination thereof. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises sodium chloride (NaCl). In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises potassium chloride (KC1). In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises magnesium chloride (MgCl₂). In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises sodium acetate (NaCH₃COO, also abbreviated NaOAc). In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises or sodium gluconate (NaC₆H₁₁O₇), or any combination thereof.

[00141] In some embodiments, the isotonic crystalloid solution present in a frozen formulation 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, and 23 mmol/L gluconate. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises PLASMA-LYTE 148 (Baxter USA), also known as Normasol® (Molnlycke Sweden).

[00142] In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises a normal saline solution (0.9% NaCl solution), a lactated Ringer' s/Hartman's solution (lactate buffered solution), an acetate buffered solution, an acetate and lactate buffered solution, an acetate and gluconate buffered solution, 5% dextrose in water, or 10% dextrose in water, or any combination thereof. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises a normal saline solution (0.9% NaCl solution). In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises a lactated Ringer' s/Hartman's solution (lactate buffered solution). In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises an acetate buffered solution. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises an acetate and lactate buffered solution. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises an acetate and gluconate buffered solution. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises 5% dextrose in water. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells comprises 10% dextrose in water.

[00143] In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells is serum free. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells is protein free. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells is calcium (Ca⁺⁺) free. In some embodiments, the isotonic crystalloid solution present in a frozen formulation of apoptotic mononuclear cells does not contain antimicrobial agents.

[00144] Ensuring the proper acid-base balance for the frozen formulation is important. In some embodiments, the pH of the freezing formulation affects the quality of the cells at thawing. Maintenance of proper cold-dependent ion ratios, control of pH at lowered temperature, and prevention of the formation of free radicals, are critical elements during cryopreservation of cells and may affect the quality of the cells at thawing.

[00145] In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 6.5-8.0. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.0-8.0. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 6.5-7.5. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.0-7.5. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.5-8.0. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 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 pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 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.

[00146] In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 6.5. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 6.6. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 6.7. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 6.8. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 6.9. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.0. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.1. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.2. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.3. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.4. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.5. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.6. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.7. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.8. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 7.9. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is at about pH 8.0.

[00147] In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 6.5. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 6.6. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 6.7. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 6.8. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 6.9. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.0. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.1. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.2. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.3. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.4. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.5. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.6. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.7. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.8. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 7.9. In some embodiments, the pH of a frozen formulation comprising apoptotic mononuclear enriched cells is pH 8.0.

[00148] Cryoprotectants are used during cry opreservation to prevent cells from damage due to freezing, for example due to ice formation. It is important that cryoprotectants be non- or minimally toxic, able to penetrate cell membranes easily, and able to bind either with electrolytes (to increase concentration in the freezing process) or with water molecules (to delay freezing).

[00149] In some embodiments, the cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises glycerol, ethylene glycol, propylene glycol, or dimethyl sulfoxide (DMSO). In certain embodiments, the cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises glycerol. In certain embodiments, the cryoprotectant agent present in a frozen formulation of apoptotic mononuclear cells comprises ethylene glycol. In certain embodiments, the cryoprotectant agent present in a frozen formulation of apoptotic mononuclear cells comprises propylene glycol. In certain embodiments, the cryoprotectant agent present in a frozen formulation of apoptotic mononuclear cells comprises dimethyl sulfoxide (DMSO). Commercial cryoprotectants available include but are not limited to PentaHibe® (Pharmacosmos, Denmark) Cryostor® CS5 and Cryostor® CD 10 (Merck KGaA, Darmstadt, Germany), and mFreSR™ (Stemcell Technologies, USA).

[00150] While a typical cryoprotectant concentration of about 5% to 15% (vol/vol) is usually required to permit survival of a substantial fraction of isolated cells after freezing and thawing from liquid nitrogen temperature (-196°), as exemplified in Example 1, surprisingly the survival of a substantial fraction of apoptotic mononuclear enriched cells and maintenance of their functionality was obtains using concentrations of cryoprotectant less than 5%.

[00151] In some embodiments, the percent (%) cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises between about 2.0% - 10% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises between about 2.5% - 10% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises between about 2.5% - 5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises between about 5% - 10% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises between about 5% - 15% (vol/vol).

[00152] In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 2.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 2.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 3.0 % (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 3.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 4.0 % (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 4.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 5.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 5.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 6.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 6.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 7.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 7.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 8.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 8.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 9.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 9.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises less than 10% (vol/vol).

[00153] In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 2.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 2.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 3.0 % (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 3.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 4.0 % (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 4.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 5.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 5.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 6.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 6.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 7.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 7.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 8.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 8.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 9.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 9.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises equal to or less than about 10% (vol/vol). [00154] In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 2.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 2.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 3.0 % (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 3.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 4.0 % (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 4.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 5.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 5.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 6.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 6.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 7.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 7.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 8.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 8.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 9.0% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 9.5% (vol/vol). In some embodiments, the % cryoprotectant agent present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 10% (vol/vol).

[00155] In some embodiments, the cryoprotectant agent comprises DMSO and the % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% (vol/vol). In some embodiments, the cryoprotectant agent comprises DMSO and the % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 2.0%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 2.5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 3.0%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 3.5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 4.0%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 4.5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 5.5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 6.0%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 6.5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 7.0%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 7.5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 8.0%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 8.5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 9.0%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 9.5%. In some embodiments, % DMSO present in a frozen formulation of apoptotic mononuclear enriched cells comprises about 10.0% (vol/vol).

Checkpoint Inhibitors

[00156] In some embodiments, the term “checkpoint inhibitor” may encompass any compound or molecule capable of inhibiting the function of a checkpoint protein. In some embodiments, the term “immune checkpoint inhibitor” may encompass any compound or molecule which targets immune checkpoints. An artisan would appreciate that “immune checkpoints” are key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Checkpoint inhibitors can block inhibitory checkpoints, and thereby restore immune system function. In some embodiments, the one or more checkpoint inhibitors comprise immune checkpoint inhibitors.

[00157] A skilled artisan would appreciate that the terms "immune checkpoint inhibitors" (ICIs), "checkpoint inhibitors," and the like may be used interchangeably herein having all the same qualities and meanings, wherein an checkpoint inhibitor encompasses compounds that inhibit the activity of control mechanisms of the immune system. Immune system checkpoints, or immune checkpoints, are inhibitory pathways in the immune system that generally act to maintain self-tolerance or modulate the duration and amplitude of physiological immune responses to minimize collateral tissue damage. Checkpoint inhibitors can inhibit an immune system checkpoint by inhibiting the activity of a protein in the pathway. Immune checkpoint inhibitor proteins include, but are not limited to, CD80, CD28, CD86, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), PDL-1, PDL-2, PD- 1, Ligand of Inducible T-cell co-stimulator (L-ICOS), Inducible T-cell co-stimulator (ICOS), CD276, and V-set domain containing T cell activation inhibitor 1 (VTCN1). As such, ICI inhibitors include antagonists of, for example, Checkpoint inhibitors such as CTLA4, PD-1, or PDL-1. For example, antibodies that bind to CTLA4, PD-1, or PDL-1 and antagonize their function are ICI inhibitors. Moreover, any molecule (e.g., peptide, nucleic acid, small molecule, etc.) that inhibits the inhibitory function of an ICI is an ICI inhibitor. [00158] In some embodiments, checkpoint inhibitors include any agent that blocks or inhibits in a statistically significant manner, the inhibitory pathways of the immune system. Illustrative Checkpoint inhibitors that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL-1, PDL-2, PD-1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γΔ, and memory CD8+ (α,β) T cells, CD 160 (also referred to as BY55), CGEN- 15049, CHK 1 and CHK2 kinases, A2aR and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7.

[00159] In some embodiments, a checkpoint inhibitor comprises an antibody. Checkpoint inhibitors may include antibodies, or antigen binding fragments thereof, other binding proteins, biologic therapeutics or small molecules, which bind to and block or inhibit the activity of one or more of CTLA-4, PDL-1, PDL-2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049.

[00160] Illustrative checkpoint inhibitors include but are not limited to Tremelimumab or Ipilimumab (CTLA-4 blocking antibodies), anti-OX40, PDL-1 monoclonal Antibody (Anti- B7-H1; MEDI4736), MK-3475 (PD-1 blocker), Nivolumab (anti-PD-1 antibody), pembrolizumab (anti-PD-1), CT -Oil (anti-PD-1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL-1 antibody), BMS-936559 (anti-PDL-1 antibody), MPLDL3280A (anti-PDL-1 antibody), MSB0010718C (anti-PDL-1 antibody), atezolizumab (anti-PDL-1 antibody), avelumab (anti-PDL-1 antibody), durvalumab (anti-PDL-1 antibody), cosibelimab (anti-PDL-1 antibody), cemiplimab (anti-PD-1 antibody), tislelizumab (anti- PD-1 antibody), dostarlimab (anti-PD-1 antibody), retifanlimab (anti-PD-1 antibody), spartalizumab (anti-PD-1 antibody), camrelizumab (anti-PD-1 antibody), sintilima b(anti- PD-1 antibody), and Yervoy/ipilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include, but are not limited to PDL-1, PDL-2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

[00161] In some embodiment, checkpoint inhibitors comprise drugs that block the interaction between immune checkpoint receptor programmed cell death protein 1 (PD-1) and its ligand PDL-1. See A. Mullard, "New checkpoint inhibitors ride the immunotherapy tsunami," Nature Reviews: Drug Discovery (2013), 12:489-492. PD-1 is expressed on and regulates the activity of T-cells. Specifically, when PD-1 is unbound to PDL-1, the T-cells can engage and kill target cells. However, when PD-1 is bound to PDL-1 it causes the T- cells to cease engaging and killing target cells. Furthermore, unlike other checkpoints, PD- 1 acts proximately such the PDLs are overexpressed directly on cancer cells which leads to increased binding to the PD-1 expressing T-cells.

[00162] In some embodiments, disclosed herein are checkpoint inhibitors which are antibodies that can act as agonists of PD-1, thereby modulating immune responses regulated by PD-1. In one embodiment, the anti-PD-1 antibodies can be antigen-binding fragments. Anti-PD-1 antibodies disclosed herein are able to bind to human PD-1 and agonize the activity of PD-1, thereby inhibiting the function of immune cells expressing PD-1.

[00163] In some embodiments, the one or more checkpoint inhibitors is selected from CTLA-4, programmed death ligand 1 (PDL-1), PDL-2, programmed cell death protein 1 (PD-1), BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN inhibitors. In some embodiments, the checkpoint inhibitor comprises a CTLA-4 inhibitor. In some embodiments, the checkpoint inhibitor comprises a BTLA inhibitor. In some embodiments, the checkpoint inhibitor comprises a HVEM inhibitor. In some embodiments, the checkpoint inhibitor comprises a TIM3 inhibitor. In some embodiments, the checkpoint inhibitor comprises a GAL9 inhibitor. In some embodiments, the checkpoint inhibitor comprises a LAG3 inhibitor. In some embodiments, the checkpoint inhibitor comprises a VISTA inhibitor. In some embodiments, the checkpoint inhibitor comprises a KIR inhibitor. In some embodiments, the checkpoint inhibitor comprises a 2B4 inhibitor. In some embodiments, the checkpoint inhibitor comprises a CD 160 inhibitor. In some embodiments, the checkpoint inhibitor comprises a CGEN inhibitor.

[00164] In some embodiments, a checkpoint inhibitor comprises one or more checkpoint inhibitors. In some embodiments, the one or more checkpoint inhibitors comprises anti- programmed cell death 1 protein (PD-1, also known as B7 homolog 1 (B7-H1), PDCD1 and CD279). In some embodiments, the one or more checkpoint inhibitors comprises anti-PD-1 ligand 1 (PDL-1 or CD274). PDL-1 on the cell surface binds to PD-1 on an immune cell surface, which inhibits immune cell activity. In some embodiments, antibodies that bind to either PD-1 or PDL-1 and therefore block the interaction may allow T-cells to attack the tumor. In some embodiments, the anti-PD-1 antibody comprises Nivolumab (Opdivo®). In some embodiments, the anti-PD-1 antibody comprises Pembrolizumab (Keytruda®). In some embodiments, the anti-PD-1 antibody comprises Spartalizumab. (anti-PD-1 antibody). In some embodiments, the anti-PD-1 antibody comprises cemiplimab. In some embodiments, the anti-PD-1 antibody comprises tislelizumab. In some embodiments, the anti-PD-1 antibody comprises dostarlimab. In some embodiments, the anti-PD-1 antibody comprises retifanlimab. In some embodiments, the anti-PD-1 antibody comprises spartalizumab. In some embodiments, the anti-PD-1 antibody comprises camrelizumab. In some embodiments, the anti-PD-1 antibody comprises sintilimab. In some embodiments, the anti-PDL-1 antibody comprises Atezolizumab (Tecentriq®). In some embodiments, the anti-PDL-1 antibody comprises Avelumab (Bavencio®). In some embodiments, the anti- PDL-1 antibody comprises Durvalumab (Imfinzi®). In some embodiments, the anti-PD-1 antibody comprises Cemiplimab (Libtayo®). In some embodiments, the anti-PDL-1 antibody comprises cosibelimab.

[00165] In some embodiments, the one or more checkpoint inhibitors comprise a programmed cell death protein 1 (PD-1) inhibitor or programmed death ligand 1 (PDL-1) inhibitor. In some embodiments, when said checkpoint comprises PD-1, said checkpoint inhibitor comprises an antibody and said antibody comprises nivolumab, pembrolizumab, cemiplimab, tislelizumab, dostarlimab, retifanlimab, spartalizumab, camrelizumab, or sintilimab; or when said checkpoint comprises PDL-1 and said checkpoint inhibitor comprises an antibody, said antibody is selected from atezolizumab, avelumab, durvalumab, and cosibelimab.

[00166] In some embodiments, the one or more checkpoint inhibitors comprise a programmed cell death protein 1 (PD-1) inhibitor. In some embodiments, the one or more checkpoint inhibitors comprise a programmed death ligand 1 (PDL-1) inhibitor.

[00167] In some embodiments, the one or more checkpoint inhibitors comprise an antibody. In some embodiments, an antibody or functional fragment thereof comprises a monoclonal antibody, a single chain antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment.

[00168] In some embodiments, disclosed herein are active fragments of any one of the polypeptides or peptide domains disclosed herein, for example but not limited to the active “binding” portion of an antibody. A skilled artisan would appreciate that the term "a fragment" may encompass at least 5, 10, 13, or 15 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids. Fragments disclosed herein can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[00169] The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense and specifically refer to a polyclonal antibody, a monoclonal antibody, or any fragment thereof, which retains the binding activity of the antibody. In certain embodiments, methods disclosed herein comprise use of a chimeric antibody, a humanized antibody, or a human antibody.

[00170] In some embodiments, the term "antibody" refers to intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of specifically interacting with a desired target as described herein, for example, binding to phagocytic cells. In some embodiments, the antibody fragments comprise:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

[00171] Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[00172] In some embodiments, the antibody fragments may be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.

[00173] Antibody fragments can, in some embodiments, be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R., Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[00174] Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al., Proc. Nafl Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or crosslinked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426, 1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

[00175] Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.

[00176] In some embodiments, the antibodies or fragments as described herein may comprise “humanized forms” of antibodies. In some embodiments, the term “humanized forms of antibodies” refers to non-human (e.g., murine) antibodies, which are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'). sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[00177] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[00178] Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al. , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

[00179] In some embodiments, the PD-1 checkpoint inhibitor comprises an antibody. In some embodiments, the PDL-1 checkpoint inhibitor comprises an antibody. In some embodiments, the CTLA-4 checkpoint inhibitor comprises an antibody.

[00180] In some embodiments, the antibody comprises an anti-PDL-1 antibody. In some embodiments, the anti-PDL-1 antibody is selected from atezolizumab, avelumab, durvalumab, and cosibelimab. In some embodiments, the antibody comprises atezolizumab, avelumab, durvalumab, or cosibelimab. In some embodiments, the anti-PDL-1 antibody comprises atezolizumab. In some embodiments, the anti-PDL-1 antibody comprises avelumab. In some embodiments, the checkpoint inhibitor comprises atezolizumab. In some embodiments, the checkpoint inhibitor comprises avelumab. In some embodiments, the checkpoint inhibitor comprises durvalumab. In some embodiments, the checkpoint inhibitor comprises cosibelimab.

[00181] In some embodiments, the antibody comprises an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody comprises nivolumab, pembrolizumab, cemiplimab, tislelizumab, dostarlimab, retifanlimab, spartalizumab, camrelizumab, or sintilimab. In some embodiments, the anti-PD-1 antibody comprises nivolumab. In some embodiments, the anti- PD-1 antibody comprises pembrolizumab. In some embodiments, the anti-PD-1 antibody comprises cemiplimab. In some embodiments, the anti-PD-1 antibody comprises tislelizumab. In some embodiments, the anti-PD-1 antibody comprises dostarlimab. In some embodiments, the anti-PD-1 antibody comprises retifanlimab. In some embodiments, the anti-PD-1 antibody comprises spartalizumab. In some embodiments, the anti-PD-1 antibody comprises camrelizumab. In some embodiments, the anti-PD-1 antibody comprises sintilimab. In some embodiments, the checkpoint inhibitor comprises nivolumab or pembrolizumab. In some embodiments, the checkpoint inhibitor comprises nivolumab. In some embodiments, the checkpoint inhibitor comprises pembrolizumab. In some embodiments, the checkpoint inhibitor comprises cemiplimab. In some embodiments, the checkpoint inhibitor comprises tislelizumab. In some embodiments, the checkpoint inhibitor comprises dostarlimab. In some embodiments, the checkpoint inhibitor comprises retifanlimab. In some embodiments, the checkpoint inhibitor comprises spartalizumab. In some embodiments, the checkpoint inhibitor comprises camrelizumab. In some embodiments, the checkpoint inhibitor comprises sintilimab.

[00182] The PD-1 or PDL-1 checkpoint inhibitors described herein may be used in a combination therapy or use thereof, as described herein.

[00183] In some embodiments, checkpoint inhibitors comprise a specific class of checkpoint inhibitors, which comprise drugs that inhibit CTLA-4. Suitable anti-CTLA4 antagonist agents for use in the combination therapy and methods thereof described herein, include, without limitation, anti-CTLA4 antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, monoclonal anti-CTLA4 antibodies, polyclonal anti-CTLA4 antibodies, chimeric anti-CTLA4 antibodies, MDX-010 (ipilimumab), Tremelimumab, anti-CD28 antibodies, anti-CTLA4 adnectins, anti-CTLA4 domain antibodies, single chain anti- CTLA4 fragments, heavy chain anti-CTLA4 fragments, light chain anti-CTLA4 fragments, inhibitors of CTLA4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No. WO 2001/014424, the antibodies disclosed in PCT Publication No. WO 2004/035607, the antibodies disclosed in U.S. Publication No. 2005/0201994, and the antibodies disclosed in granted European Patent No. EP 1212422 Bl. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO 00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014. Other anti-CTLA-4 antibodies that can be used in a combination therapy or method thereof, and include, for example, those disclosed in: WO 98/42752; U.S. Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145):Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281.

[00184] Additional anti-CTLA4 antagonists include, but are not limited to, the following: any inhibitor that is capable of disrupting the ability of CD28 antigen to bind to its cognate ligand, to inhibit the ability of CTLA4 to bind to its cognate ligand, to augment T cell responses via the co-stimulatory pathway, to disrupt the ability of B7 to bind to CD28 and/or CTLA4, to disrupt the ability of B7 to activate the co-stimulatory pathway, to disrupt the ability of CD80 to bind to CD28 and/or CTLA4, to disrupt the ability of CD80 to activate the co-stimulatory pathway, to disrupt the ability of CD86 to bind to CD28 and/or CTLA4, to disrupt the ability of CD86 to activate the co-stimulatory pathway, and to disrupt the co- stimulatory pathway, in general from being activated. This necessarily includes small molecule inhibitors of CD28, CD80, CD86, CTLA4, among other members of the co- stimulatory pathway; antibodies directed to CD28, CD80, CD86, CTLA4, among other members of the co-stimulatory pathway; antisense molecules directed against CD28, CD80, CD86, CTLA4, among other members of the co-stimulatory pathway; adnectins directed against CD28, CD80, CD86, CTLA4, among other members of the co-stimulatory pathway, RNAi inhibitors (both single and double stranded) of CD28, CD80, CD86, CTLA4, among other members of the co-stimulatory pathway, among other anti-CTLA4 antagonists.

[00185] In some embodiments, checkpoint inhibitors comprise drugs that inhibit TIM-3. Blocking the activation of TIM-3 by a ligand, results in an increase in Thl cell activation. Furthermore, TIM-3 has been identified as an important inhibitory receptor expressed by exhausted CD8+ T cells. TIM-3 has also been reported as a key regulator of nucleic acid mediated antitumor immunity. In one example, TIM-3 has been shown to be upregulated on tumor-associated dendritic cells (TADCs).

[00186] In some embodiments, a combination therapy comprises a second composition comprising one or more checkpoint inhibitors, as described herein.

Pharmaceutical Compositions

[00187] In some embodiments, disclosed herein are compositions for use as part of a combination therapy. In some embodiments, a composition comprises an apoptotic mononuclear-enriched cell population. In some embodiments, a composition comprises a checkpoint inhibitor. In some embodiments, a composition comprises one or more checkpoint inhibitors.

[00188] As used herein, the terms “composition” and pharmaceutical composition” may in some embodiments, be used interchangeably having all the same qualities and meanings. In some embodiments, disclosed herein is a pharmaceutical composition for the treatment of a condition or disease as described herein .

[00189] In some embodiments, disclosed herein are pharmaceutical compositions for use in a combination therapy. In another embodiment, disclosed herein are compositions for use treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject.

[00190] In another embodiment, a pharmaceutical composition comprises an apoptotic cell population. In another embodiment, a pharmaceutical composition comprises an inactivated apoptotic cell population. In another embodiment, a pharmaceutical composition comprises an irradiated, apoptotic cell population. In one embodiment, a pharmaceutical composition as disclosed herein comprises a pooled mononuclear apoptotic cell preparation, wherein said pooled mononuclear apoptotic cell preparation comprises pooled individual mononuclear cell populations, and wherein said pooled mononuclear apoptotic cell preparation comprises a decreased percent of living non-apoptotic cells; a suppressed cellular activation of any living non-apoptotic cells; or a reduced proliferation of any living non-apoptotic cells; or any combination thereof. In another embodiment, a pharmaceutical composition comprises a pooled mononuclear apoptotic cell preparation disclosed herein.

[00191] In another embodiment, in a composition said pooled mononuclear apoptotic cell preparation comprises an inactivation preparation as disclosed herein, for example an irradiated preparation or a preparation wherein said individual cell populations have been irradiated. In another embodiment, a composition further comprises an anti-coagulant.

[00192] Apoptotic cells for use in a composition, a combination therapy, and methods of use thereof are described in detail herein are exemplified in the Examples provided below (See, Examples 1 and 2).

[00193] In some embodiments, apoptotic cells for use in compositions, combination therapies, and methods as disclosed herein are produced in any way that is known in the art. In another embodiment, apoptotic cells for use in compositions, combination therapies, and methods disclosed herein are autologous with a subject undergoing therapy. In another embodiment, apoptotic cells for use in compositions, combination therapies, and methods disclosed herein are allogeneic with a subject undergoing therapy. In another embodiment, a composition comprising apoptotic cells comprises apoptotic cells as disclosed herein or as is known in art.

[00194] In still another embodiment, a pharmaceutical composition for use as a combination therapy, as described herein, comprises an effective amount of an apoptotic cell mononuclear-enriched population, as described herein, in a pharmaceutically acceptable excipient. In yet another embodiment, a pharmaceutical composition for the treatment of cancer, as described herein, comprises an effective amount of an apoptotic cell mononuclear-enriched population, as described herein, in a pharmaceutically acceptable excipient.

[00195] In some embodiments, a composition comprises a checkpoint inhibitor. In some embodiments, a composition comprises a checkpoint inhibitor comprising a PD-1, a PDL-1, a CTLA-4, or a TIM3 checkpoint inhibitor. In some embodiments, a composition comprises a checkpoint inhibitor comprising a PD-1 checkpoint inhibitor. In some embodiments, a composition comprises a checkpoint inhibitor comprising a PDL- 1 checkpoint inhibitor. In some embodiments, a composition comprises a checkpoint inhibitor comprising a CTLA-4 checkpoint inhibitor. In some embodiments, a composition comprises a checkpoint inhibitor comprising a TIM3 checkpoint inhibitor. In some embodiments, a composition comprises any checkpoint inhibitor known in the art.

[00196] In some embodiments, a composition comprises one or more checkpoint inhibitors. In some embodiments, a composition comprising one or more checkpoint inhibitors, comprises multiple compositions, wherein each checkpoint inhibitor is comprised in a separate composition. In some embodiments, a composition comprising one or more checkpoint inhibitors, comprises multiple compositions, wherein checkpoint inhibitors are comprised in multiple composition, together or separate or both. In some embodiments, a composition comprising one or more checkpoint inhibitors, comprises a single composition, wherein each checkpoint inhibitor is comprised in the same composition.

[00197] In still another embodiment, a pharmaceutical composition for use in a combination therapy, as described herein, comprises an effective amount of a checkpoint inhibitor, as described herein, in a pharmaceutically acceptable excipient. In yet another embodiment, a pharmaceutical composition for the treatment of cancer, as described herein, comprises an effective amount of a checkpoint inhibitor, as described herein, in a pharmaceutically acceptable excipient.

[00198] In some embodiments, apoptotic cells comprised in a composition are pooled third party donor cells. In another embodiment, an apoptotic cell supernatant comprised in a composition disclosed herein is collected from apoptotic cells. In another embodiment, an apoptotic cell supernatant comprised in a composition disclosed herein, is collected pooled third-party donor cells.

[00199] In some embodiments, a composition comprises apoptotic cells and a checkpoint inhibitor. In some embodiments, a composition comprises apoptotic cells and checkpoint inhibitor that comprises an antibody or a functional fragment thereof. In some embodiments, apoptotic cells and a checkpoint inhibitor, which may comprise an antibody or a functional fragment thereof, may be comprised in separate compositions. In some embodiments, apoptotic cells and a checkpoint inhibitor, which may comprise an antibody or a functional fragment thereof, may be comprised in the same composition. Whether comprised in the same or different compositions, embodiments of a combination therapy described herein comprises a composition comprising irradiated, apoptotic cells and a composition comprising a checkpoint inhibitor. Thus, in some embodiments, a combination therapy described herein comprises a composition comprising irradiated, apoptotic cells and a checkpoint inhibitor. In other embodiments, a combination therapy described herein comprises a separate composition comprising irradiated, apoptotic cells and a separate composition comprising a checkpoint inhibitor.

[00200] A skilled artisan would appreciate that a "pharmaceutical composition" may encompass a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.

[00201] In some embodiments, disclosed herein is a pharmaceutical composition for a combination therapy for treating, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject.

[00202] A skilled artisan would appreciate that the phrases "physiologically acceptable carrier", "pharmaceutically acceptable carrier", "physiologically acceptable excipient", and "pharmaceutically acceptable excipient", may be used interchangeably may encompass a carrier, excipient, or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered active ingredient .

[00203] A skilled artisan would appreciate that an "excipient" may encompass an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. In some embodiments, excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[00204] Techniques for formulation and administration of drugs are found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[00205] In some embodiments, the composition as disclosed herein comprises a therapeutic composition. In some embodiments, the composition as disclosed herein comprises a therapeutic efficacy.

Combination Therapies

[00206] In some embodiments, the present disclosure provides combination therapies comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor. In some embodiments, the present disclosure provides combination therapies comprising a first composition comprising an inactivated apoptotic mononuclear-enriched cell population, and a second composition comprising a checkpoint inhibitor. In some embodiments, the present disclosure provides combination therapies comprising a first composition comprising an irradiated, apoptotic mononuclear-enriched cell population, wherein said population is irradiated post induction of apoptosis, and a second composition comprising a checkpoint inhibitor.

[00207] In another embodiment, the present disclosure provides a combination therapy comprising an inactivated apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor, wherein said inactivated apoptotic mononuclear-enriched cell population comprises a decreased number of non-quiescent non-apoptotic cells; a suppressed cellular activation of any living non-apoptotic cells; or a reduced proliferation of any living non-apoptotic cells; or any combination thereof compared with a population comprising a non-irradiated cell population.

[00208] In some embodiments, a combination therapy of the present disclosure or use in the methods of the present disclosure comprises compositions comprising one or more checkpoint inhibitors with an apoptotic mononuclear-enriched cell population as described herein.

[00209] In another embodiment, the present disclosure provides a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising one or more checkpoint inhibitors.

[00210] In some embodiments, the combination therapy comprises a first composition comprising an irradiated, apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a PD-1 checkpoint inhibitor. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a PDL-1 checkpoint inhibitor. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a CTLA-4 checkpoint inhibitor. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising a TIM3 checkpoint inhibitor. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising any checkpoint inhibitor known in the art.

[00211] In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising atezolizumab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising avelumab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising durvalumab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising cosibelimab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising cemiplimab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising nivolumab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising pembrolizumab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising cemiplimab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising tislelizumab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising dostarlimab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising retifanlimab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising spartalizumab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising camrelizumab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising sintilimab. In some embodiments, the combination therapy comprises a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising Ipilimumab.

[00212] In some embodiments, checkpoint inhibitors comprised in a composition and useful in the combination therapy and methods of the present disclosure include, but are not limited to, anti-PD-1; anti-CTLA-4; anti- PDL-1; anti-B7-Hl; anti-Programmed cell death

1 ligand 2 (also known as PD-L2, B7-DC); anti-B7-H3; anti-B7-H4; anti-CD137; anti- CD40; anti-CD27; anti-Lymphocyte-activation gene 3 (LAG3); anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM3) (also known as Hepatitis A virus cellular receptor

2 (HAVCR2)); anti-Inducible T-cell co-stimulator (ICOS), and anti-BTLA. In some embodiments, one or more checkpoint inhibitors comprise a CTLA4 inhibitor, which in some embodiments, is ipilimumab (Yervoy®).

[00213] In some embodiments, the second composition comprises one or more checkpoint inhibitors. In some embodiments, the second composition comprises one checkpoint inhibitor. In some embodiments, the second composition comprises two checkpoint inhibitors. In some embodiments, the second composition comprises 3 checkpoint inhibitors. In some embodiments, the second composition comprises 4 checkpoint inhibitors. In some embodiments, the second composition comprises 5 checkpoint inhibitors. In some embodiments, the second composition comprises 6 checkpoint inhibitors. In some embodiments, the second composition comprises more than 5 checkpoint inhibitors. In some embodiments, the second composition comprises at least 2 checkpoint inhibitors.

[00214] The combination therapies of the present disclosure may be administered together with other anti-cancer treatments useful in the treatment of cancer or other proliferative diseases. The disclosure herein further comprises use of the first and second compositions in preparing medicaments for the treatment of cancer or tumors. Methods of Use of a Combination Therapy

[00215] In some embodiments, disclosed herein are methods for treating, inhibiting the growth of, or delaying disease progression, of a cancer or a tumor in a human subject, comprising a step of administering to a subject in need a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor, wherein said method treats, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said human subject. In some embodiments, the method reduces the tumor load or reduces the incidence of the cancer or a tumor in said subject, compared with a subject not administered the combination therapy. In some embodiments, the method reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, prevents metastasis of said tumor or said cancer, or reduces the rate of metastasis of said tumor or said cancer, or any combination thereof.

[00216] In some embodiments, disclosed herein are methods for treating, inhibiting, reducing the incidence of, ameliorating, or alleviating a cancer or a tumor treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, or any combination thereof, comprising the step of administering a combination therapy as disclosed herein.

[00217] In some embodiments, disclosed herein are methods of treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, or any combination thereof, comprising a step of administering to a subject in need a combination therapy comprising a first composition comprising an inactivated, apoptotic mononuclear-enriched cell population, and a second composition comprising one or more checkpoint inhibitors, wherein said inactivated apoptotic mononuclear-enriched cell population comprises

(a) a decreased number of non-quiescent non-apoptotic cells;

(b) a suppressed cellular activation of any living non-apoptotic cells; or

(c) a reduced proliferation of any living non-apoptotic cells;

(d) or any combination thereof compared with a population comprising a non-inactivated cell population.

[00218] wherein said method treats, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said subject. In some embodiments, methods disclosed herein reduce the size and or growth rate of a tumor or cancer. In some embodiments, a method of treating disclosed herein reduces the tumor load or reduces the incidence of the cancer or a tumor in said subject or reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, prevents metastasis of said tumor or said cancer, or reduces the rate of metastasis of said tumor or said cancer, or any combination thereof, in said subject compared with a subject not administered the combination therapy. In some embodiments, methods disclosed herein increase the survival of a subject suffering from a tumor or cancer.

[00219] In some embodiments of the methods disclosed herein, a composition comprising an apoptotic cell population comprises an apoptotic mononuclear-enriched cell population. [00220] In another embodiment, methods as disclosed herein utilize combination therapy with an irradiated apoptotic cell population and one or more checkpoint inhibitors. In some embodiments, a checkpoint inhibitor is any compound or molecule capable of inhibiting the function of a checkpoint protein. In some embodiments, administration of a checkpoint inhibitor, which in some embodiments, is an antibody, produces a net effect of checkpoint inhibition. In another embodiment, compositions and methods as disclosed herein utilize combination therapy comprising an apoptotic cell population and one or more checkpoint inhibitors.

[00221] In some embodiments, combination therapy provides a synergistic effect. In some embodiments, methods of use a combination therapy comprising an of an apoptotic mononuclear-enriched cell population in combination with one or more checkpoint inhibitors improves cancer treatment efficacy in comparison to use of checkpoint inhibitors alone. In some embodiments, methods of use a combination therapy comprising an apoptotic mononuclear-enriched cell population in combination with one or more checkpoint inhibitors improves cancer treatment efficacy in comparison to use of an apoptotic cell population alone. In some embodiments, methods of use a combination therapy comprising an apoptotic cell population in combination with one or more checkpoint inhibitors extends the survival time of a subject suffering from a cancer or tumor in comparison to administering checkpoint inhibitors alone. In some embodiments, methods of use a combination therapy comprising an apoptotic cell population in combination with one or more checkpoint inhibitors extends the survival time of a subject suffering from a cancer or tumor in comparison to administering an apoptotic cell population alone.

[00222] In some embodiments, combination therapy provides an improved therapy compared with using either one or more checkpoint inhibitors independent of another therapy or using an apoptotic cell population independent of another therapy.

[00223] In some embodiments, methods of use of a combination therapy provide an improved effectiveness of T cells in attacking a tumor cell. In some embodiments, the improvement comprises an improved effectiveness of T cells in killing a tumor cell. In some embodiments, the improvement comprises an improved effectiveness of natural killer (NK) cells in attacking a tumor cell. In some embodiments, the improvement comprises an improved effectiveness of NK cells in killing a tumor cell. In some embodiments, the improvement comprises an improved cytotoxic effect on a tumor cell. In some embodiments, the improvement comprises an upregulation of HLA markers on cancer stem cells. In some embodiments, the improvement comprises an upregulation of HLA markers on cancer cells.

[00224] In some embodiments, methods of use of a combination therapy provides a reduction of at least one side effect.

[00225] In some embodiments, methods of use of a combination therapy provide an improved effectiveness resulting in an increased reduction of tumor size. In some embodiments, the improvement comprises a complete reduction of a tumor. In some embodiments, the improvement comprises a reduction of metastasis. In some embodiments, the improvement comprises an elimination of metastasis. In some embodiments, the improvement comprises enhanced responsiveness, wherein improvements are observed in a shorter time frame, compared with a subject not receiving the combination therapy. In some embodiments, the improvement comprises an increased survival rates compared with a subject not receiving the combination therapy.

[00226] In some embodiments, methods of use of a combination therapy comprising the apoptotic cell population in combination with one or more checkpoint inhibitors delays the onset of cancer or the appearance of a tumor, in comparison to use of either an apoptotic cell population or the checkpoint inhibitor alone. In some embodiments, methods of use of a combination therapy comprising the apoptotic cell population in combination with one or more checkpoint inhibitors delay the progression of a cancer, in comparison to use of either therapy alone. In some embodiments, methods of use of a combination therapy comprising the apoptotic cell population in combination with one or more checkpoint inhibitors delay the growth of a tumor, in comparison to use of either therapy alone. In some embodiments, methods of use of a combination therapy comprising the apoptotic cell population in combination with one or more checkpoint inhibitors extends the survival time of a subject suffering from a cancer or tumor in comparison to use of either therapy alone. In some embodiments, methods of use of a combination therapy comprising the apoptotic cell population in combination with one or more checkpoint inhibitors extends the survival time of a subject suffering from a cancer or tumor in comparison to use of either therapy alone. In some embodiments, methods of use of a combination therapy comprising the apoptotic cell population in combination with one or more checkpoint inhibitors extends the survival time of a subject suffering from a solid cancer or tumor, in comparison to use of either therapy alone.

[00227] In some embodiments, the checkpoint inhibitor comprises an anti-PDL-1 antibody. In some embodiments, the anti-PDL-1 antibody is selected from atezolizumab, avelumab, durvalumab, and cosibelimab. In some embodiments, the antibody comprises atezolizumab, avelumab, or durvalumab. In some embodiments, the anti-PDL-1 antibody comprises atezolizumab. In some embodiments, the anti-PDL-1 antibody comprises avelumab. In some embodiments, the anti-PDL-1 antibody comprises durvalumab. In some embodiments, the anti-PDL-1 antibody comprises cosibelimab

[00228] In some embodiments, the checkpoint inhibitor comprises an anti-PD- 1 antibody. In some embodiments, the anti-PD- 1 antibody comprises nivolumab, pembrolizumab, cemiplimab, tislelizumab, dostarlimab, retifanlimab, spartalizumab, camrelizumab, or sintilimab. In some embodiments, the antibody comprises nivolumab, pembrolizumab, cemiplimab, tislelizumab, dostarlimab, retifanlimab, spartalizumab, camrelizumab, or sintilimab. In some embodiments, the anti-PD- 1 antibody comprises nivolumab. In some embodiments, the anti-PD- 1 antibody comprises pembrolizumab. In some embodiments, the anti-PD- 1 antibody comprises cemiplimab. In some embodiments, the anti-PD- 1 antibody comprises tislelizumab. In some embodiments, the anti-PD- 1 antibody comprises dostarlimab. In some embodiments, the anti-PD- 1 antibody comprises retifanlimab, In some embodiments, the anti-PD- 1 antibody comprises spartalizumab. In some embodiments, the anti-PD- 1 antibody comprises camrelizumab. In some embodiments, the anti-PD- 1 antibody comprises sintilimab. [00229] In some embodiments, the checkpoint inhibitor comprises an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody comprises. In some embodiments, the antibody comprises Tremelimumab or Ipilimumab. In some embodiments, the anti-CTLA-4 antibody comprises Tremelimumab. In some embodiments, the anti-CTLA-4 antibody comprises Ipilimumab.

[00230] In some embodiments, methods of use of a combination therapy comprising administration of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PD-1 antibody, delays the onset of cancer or the appearance of a tumor, in comparison to use of either an apoptotic cell population or the antibody alone. In some embodiments, methods of use of a combination therapy comprising an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PD-1 antibody, delays the progression of a cancer, in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PD-1 antibody, delays the growth of a tumor, in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PD-1 antibody, extends the survival time of a subject suffering from a cancer or tumor in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PD- 1 antibody, extends the survival time of a subject suffering from a solid tumor in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PD-1 antibody, extends the survival time of a subject suffering from a non- solid tumor in comparison to use of either the apoptotic cell population or the antibody alone.

[00231] In some embodiments, methods of use comprising administration of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PDL-1 antibody, delays the onset of cancer or the appearance of a tumor, in comparison to use of either an apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PDL-1 antibody, delays the progression of a cancer, in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PDL-1 antibody, delays the growth of a tumor, in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti- PDL-1 antibody, extends the survival time of a subject suffering from a cancer or tumor in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PDL-1 antibody, extends the survival time of a subject suffering from a solid tumor in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-PDL-1 antibody, extends the survival time of a subject suffering from a non-solid in comparison to use of either the apoptotic cell population or the antibody alone.

[00232] In some embodiments, methods of use comprising administration of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-CTLA-4 antibody, delays the onset of cancer or the appearance of a tumor, in comparison to use of either an apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-CTLA-4 antibody, delays the progression of a cancer, in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-CTLA-4 antibody, delays the growth of a tumor, in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti- CTLA-4 antibody, extends the survival time of a subject suffering from a cancer or tumor in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-CTLA-4 antibody, extends the survival time of a subject suffering from a solid tumor in comparison to use of either the apoptotic cell population or the antibody alone. In some embodiments, methods of use of an apoptotic cell population in combination with a checkpoint inhibitor comprising an anti-CTLA-4 antibody, extends the survival time of a subject suffering from a non-solid in comparison to use of either the apoptotic cell population or the antibody alone.

[00233] In some embodiments, methods of use comprising administration of a combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising one or more checkpoint inhibitors, reduces the number of cancer cells in a subject, reduces the size of a tumor in a subject, or reduces the amount of cancer in the body of a subject, or any combination thereof compared with a subject not administered combination therapy. In some embodiments, methods of use comprising administration of a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising one or more checkpoint inhibitors, reduces the number of cancer cells in a subject, reduces the size of a tumor in a subject, or reduces the amount of cancer in the body of a subject, or any combination thereof, compared with a subject not administered combination therapy.

[00234] In some embodiments, methods of use comprising administration of an apoptotic cell population in combination with an anti-PDL-1 antibody or fragment thereof reduces the number of cancer cells in a subject, reduces the size of a tumor in a subject, or reduces the amount of cancer in the body of a subject, or any combination thereof, compared with a subject not administered an apoptotic cell population in combination with an anti-PDL-1 antibody. In some embodiments, methods of use comprising administration of an apoptotic cell population in combination with an anti-PD-1 antibody or fragment thereof reduces the number of cancer cells in a subject, reduces the size of a tumor in a subject, or reduces the amount of cancer in the body of a subject, or any combination thereof, compared with a subject not administered an apoptotic cell population in combination with an anti-PD-1 antibody. In some embodiments, methods of use comprising administration of an apoptotic cell population in combination with an anti-CTLA-4 antibody or fragment thereof reduces the number of cancer cells in a subject, reduces the size of a tumor in a subject, or reduces the amount of cancer in the body of a subject, or any combination thereof, compared with a subject not administered an apoptotic cell population in combination with an anti-CTLA-4 antibody.

[00235] In some embodiments of the methods disclosed herein, following administration of the combination therapy, the subject remains disease free for a time period longer than a subject administered either composition alone.

[00236] A skilled artisan would appreciate that the term “disease free” as used herein, may refer to the subject remaining alive, without return of the cancer or tumor, for a defined period of time such as about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, or more from initiation of treatment or from initial diagnosis.

[00237] In some embodiments, the subject remains disease free for at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, or 10 years. In some embodiments, the subject remains disease free for at least 1 year. In some embodiments, the subject remains disease free for at least 5 years. In some embodiments, the subject remains disease free for at least 10 years.

[00238] In some embodiments, the methods disclosed herein reduce the tumor load, or reduce the incidence of the cancer or a tumor in said subject, compared with a subject not administered the combination therapy. In some embodiments, the methods disclosed herein reduce the minimal residual disease, increase remission, increase remission duration, reduce tumor relapse rate, prevent metastasis of said tumor or said cancer, or reduce the rate of metastasis of said tumor or said cancer, or any combination thereof.

[00239] In another embodiment, disclosed herein is a method of reducing the tumor load in a subject, compared with a subject not administered the combination therapy, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of reducing the incidence of the cancer or a tumor in a subject, compared with a subject not administered the combination therapy, said method comprising the step of administering to said subject any of the combination therapies as described herein.

[00240] In another embodiment, disclosed herein is a method of reducing the minimal residual disease in a subject, compared with a subject not administered the combination therapy, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of reducing tumor relapse rate in a subject, compared with a subject not administered the combination therapy, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of reducing the rate of metastasis of said tumor or said cancer in a subject, compared with a subject not administered the combination therapy, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of preventing metastasis of said tumor or said cancer in a subject, compared with a subject not administered the combination therapy, said method comprising the step of administering to said subject any of the combination therapies as described herein.

[00241] In another embodiment, disclosed herein is a method of increasing the remission of said tumor or said cancer in a subject, compared with a subject not administered the combination therapy, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of increasing remission duration of said tumor or said cancer in a subject, compared with a subject not administered the combination therapy, said method comprising the step of administering to said subject any of the combination therapies as described herein. [00242] In some embodiments, administering any of the combination therapies described herein upregulates human leukocyte antigen (HLA) on cancer or tumor cells. In some embodiments, administering to a subject any of the combination therapies described herein upregulates HLA class I on cancer or tumor cells. In some embodiments, administering any of the combination therapies described herein upregulates HLA class II on cancer or tumor cells. In some embodiments, upregulating HLA class I on cancer or tumor cells increases immunogenicity of the cancer or tumor cells. In some embodiments, upregulating HLA class II on cancer or tumor cells increases immunogenicity of the cancer or tumor cells. In some embodiments, administering any of the combination therapies described herein upregulates expression of HLA in cancer or tumor cells. In some embodiments, administering any of the combination therapies described herein upregulates expression of HLA class I in cancer or tumor cells. In some embodiments, administering any of the combination therapies described herein upregulates expression of HLA class II in cancer or tumor cells. In some embodiments, administering any of the combination therapies described herein upregulates expression of MHC class I in cancer or tumor cells. In some embodiments, administering any of the combination therapies described herein upregulates expression of MHC class II in cancer or tumor cells. In some embodiments, upregulating the expression of MHC class I in cancer or tumor cells increases immunogenicity of the cancer or tumor cells. In some embodiments, upregulating the expression of MHC class II in cancer or tumor cells increases immunogenicity of the cancer or tumor cells. [00243] In some embodiments, methods herein upregulate the expression of MHC class I in cancer or tumor cells compared with the level of expression of MHC class I in a subject receiving one or more checkpoint inhibitors and not administered an apoptotic mononuclear- enriched cell population. In some embodiments, methods herein upregulate the expression of MHC class II in cancer or tumor cells compared with the level of expression of MHC class II in a subject receiving one or more checkpoint inhibitors and not administered an apoptotic mononuclear-enriched cell population. In some embodiments, methods herein upregulate the expression of MHC class I in cancer or tumor cells compared with the level of expression of MHC class I in a subject not administered combination therapies disclosed herein. In some embodiments, methods herein upregulate the expression of MHC class II in cancer or tumor cells compared with the level of expression of MHC class II in a subject not administered combination therapies disclosed herein.

[00244] In some embodiments, administering any of the combination therapies described herein, increase cell surface protein levels of HLA in cancer or tumor cells compared with the level of cell surface protein levels of HLA in a subject not administered combination therapies disclosed herein. In some embodiments, administering any of the combination therapies described herein, increase cell surface protein levels of HLA class I in cancer or tumor cells compared with the level of cell surface protein levels of HLA class I in a subject not administered combination therapies disclosed herein. In some embodiments, administering any of the combination therapies described herein, increase cell surface protein levels of HLA class II in cancer or tumor cells compared with the level of cell surface protein levels of HLA class II in a subject not administered combination therapies disclosed herein. In some embodiments, administering any of the combination therapies described herein, increase cell surface protein levels of HLA class I in cancer or tumor cells compared with the level of cell surface protein levels of HLA class I in a subject receiving one or more checkpoint inhibitors and not administered an apoptotic mononuclear-enriched cell population. In some embodiments, administering any of the combination therapies described herein, increase cell surface protein levels of HLA class II in cancer or tumor cells compared with the level of cell surface protein levels of HLA class II in a subject receiving one or more checkpoint inhibitors and not administered an apoptotic mononuclear-enriched cell population.

[00245] In another embodiment, methods of treating, inhibiting, reducing the incidence of, ameliorating, or alleviating a cancer or tumor treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, or any combination thereof, decrease or inhibit cytokine production in a subject, said methods comprising the step of administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising one or more checkpoint inhibitors.

[00246] In another embodiment, methods of treating, inhibiting, reducing the incidence of, ameliorating, or alleviating a cancer or tumor treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, or any combination thereof, increase cytokine production in a subject, said methods comprising the step of administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising one or more checkpoint inhibitors.

[00247] In another embodiment, disclosed herein is a method of treating a cancer or a tumor in a subject, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of preventing a cancer or a tumor in a subject, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of inhibiting a cancer or a tumor in a subject, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of reducing a cancer or a tumor in a subject, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of ameliorating a cancer or a tumor in a subject, said method comprising the step of administering to said subject any of the combination therapies as described herein. In another embodiment, disclosed herein is a method of alleviating a cancer or a tumor in a subject, said method comprising the step of administering to said subject any of the combination therapies as described herein.

[00248] In some embodiment, “treating” comprises therapeutic treatment and “preventing” comprises prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove. Thus, in some embodiments, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in some embodiments, “treating,” “ameliorating,” and “alleviating” refer inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In some embodiments, “preventing” refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In some embodiments, “suppressing” or “inhibiting”, refers inter alia to 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, prolonging patient survival, or a combination thereof.

[00249] In some embodiments, methods described herein reduce the size or reduce the growth rate of a cancer or a tumor, and comprise administering a combination therapy as disclosed herein, to said subject, wherein the method reduces the size or the growth rate of a cancer or tumor. In some embodiments, disclosed herein is a method of reducing the growth rate of a diffuse cancer, comprising the step of administering a combination therapy as disclosed herein, to said subject, wherein the method reduces the growth rate of the cancer. In some embodiments, disclosed herein is a method of reducing the size or reducing the growth rate of a solid cancer or tumor, comprising the step of administering a combination therapy as disclosed herein, to a subject, wherein the method reduces the size or reduces the growth rate of the solid cancer or tumor.

[00250] In some embodiments, methods described herein increase the survival of a subject suffering from a cancer or a tumor, and comprise administering a combination therapy as disclosed herein to said subject, wherein the method increases the survival of the subject.

[00251] A skilled artisan would appreciate that the term "disease" may encompass any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include a cancer, tumor, neoplasia or pathogen infection of cell. [00252] A skilled artisan would appreciate that the term "neoplasia" may encompass a disease characterized by the pathological proliferation of a cell or tissue and its subsequent migration to or invasion of other tissues or organs. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasia can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof. Neoplasia include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumor of the plasma cells).

[00253] A skilled artisan would appreciate that the term "pathogen" may encompass a virus, bacteria, fungi, parasite or protozoa capable of causing disease.

[00254] A skilled artisan would appreciate that the term "tumor antigen" or “tumor associated antigen” may encompass an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on a tumor cell compared to a normal or non-cancerous neoplastic cell. With reference to the compositions and methods disclosed herein, a tumor antigen includes any polypeptide expressed by a tumor that is capable of activating or inducing an immune response via an antigen recognizing receptor (e.g., CD 19, MUCI) or capable of suppressing an immune response via receptor-ligand binding (e.g., CD47, PDL-1/2, B7.1/2). [00255] A skilled artisan would appreciate that the term "virus antigen" may encompass a polypeptide expressed by a virus that is capable of inducing an immune response.

[00256] A skilled artisan would appreciate that the term "treatment" may encompass clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder. [00257] A skilled artisan would appreciate that the term "subject" may encompass a vertebrate, in some embodiments, to a mammal, and in some embodiments, a human. Subject may also refer, in some embodiments, to domesticated animals such as cows, sheep, horses, cats, dogs and laboratory animals such as mice, rats, gerbils, hamsters, etc. In some embodiments, the subject is a human subject. In some embodiments, the subject is a child. In one embodiment, the child is an infant. In some embodiments, the subject is an adult.

[00258] According to any of the methods of the present disclosure and in one embodiment, a subject as described herein is human.

[00259] A skilled artisan would appreciate that therapeutic effectiveness may in some instances be examined in an animal model prior to use in a human. In some embodiments, a mouse model is used. In some embodiments, a rat model is used. In some embodiments, a mouse solid tumor model is used. In some embodiments, a rat model is used. In some embodiments, a rat solid tumor model is used. In some embodiments, the solid tumor model comprises a model for a lung tumor, a prostrate tumor, a breast tumor, a colon tumor, a stomach tumor, liver tumor, a kidney tumor, pancreatic tumor, skin tumor, ovarian tumor, bone tumor, or any solid tumor. In other embodiments, an animal model, for example but not limited to a rat or mouse model, provides a model for a non-solid cancer. In some embodiments, an animal model of mesothelioma may be employed.

[00260] In some embodiments the therapeutic effectiveness may in some instances be examined in a cell line model prior to use in a human. In some embodiments, the cell line comprises a mouse cell line. In some embodiments, the cell line comprises a human cell line. In some embodiments, the cell line comprises a cancer cell line. In some embodiments, the cell line comprises an immunogenic cell line. In some embodiments, the cell line comprises an immunocompetent cell line. In some embodiments, the cell line comprises a mouse cell line. In some embodiments, the cell line comprises a melanoma cell line. In some embodiments, the cell line comprises a colon adenocarcinoma cell line. In some embodiments, the cell line is especially adapted for examining immune checkpoint inhibition. In some embodiments, the cell line is especially adapted for examining anti-tumor responses. In some embodiments, the cell line is especially adapted for examining immunotherapy .

[00261] In some embodiments, disclosed herein is a method of reducing a tumor burden in a subject, said method comprising the step of administering to said subject any of the combination therapies as described herein. In some embodiments, reducing the tumor burden comprises reducing the number of tumor cells in the subject. In another embodiment, reducing the tumor burden comprises reducing tumor size in the subject. In another embodiment, reducing the tumor burden comprises eradicating the tumor in the subject.

[00262] In another embodiment, disclosed herein is a method of inducing tumor cell death in a subject, said method comprising the step of administering to said subject any of the combination therapies as described herein.

[00263] In another embodiment, disclosed herein is a method of increasing, extending, or lengthening the survival of a subject having neoplasia, comprising the step of administering to said subject any of the combination therapies as described herein.

[00264] In another embodiment, disclosed herein is a method of increasing, extending, or lengthening the survival of a subject having neoplasia, comprising the step of administering to said subject any of the combination therapies as described herein.

[00265] In some embodiments, disclosed herein is a method of delaying cancer progression in a subject, comprising a step of administering to the subject any of the combination therapies described herein. In some embodiments, disclosed herein is a method of delaying progression of a leukemia or lymphoma in a subject, comprising a step of administering to the subject any of the combination therapies described herein. In some embodiments, disclosed herein is a method of increasing, extending, or prolonging the survival of a subject suffering from a cancer or a tumor, comprising a step of administering to the subject any of the combination therapies described herein. In some embodiments, disclosed herein is a method of increasing, extending, or prolonging the survival of a subject suffering from a leukemia or lymphoma, comprising administering to the subject any of the combination therapies described herein. In some embodiments, disclosed herein is a method of reducing the tumor cell burden in a subject, comprising administering to the subject any of the combination therapies described herein. In some embodiments, tumor burden is reduced in the liver and bone marrow.

[00266] In some embodiments, the cancer or tumor comprises a non-solid cancer or tumor. In some embodiments, the non-solid cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non- Hodgkin lymphoma, or plasma cell leukemia.

[00267] In another embodiment, disclosed herein is a method of preventing neoplasia in a subject, said method comprising the step of administering to the subject any of the combination therapies described herein. In some embodiments, the neoplasia is selected from the group consisting of blood cancer, B cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, ovarian cancer, or a combination thereof.

[00268] In another embodiment, disclosed herein is a method of treating blood cancer in a subject in need thereof, comprising the step of administering to said subject any of the combination therapies as described herein. In some embodiments, the blood cancer is selected from the group consisting of B cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

[00269] In some embodiments, the cancer or tumor comprises a solid tumor. In some embodiments, the cancer or tumor comprises a metastasis of a cancer or tumor. In some embodiments, the solid tumor comprises a sarcoma or a carcinoma, a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endothelio sarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilm's tumor, a cervical cancer or tumor, a uterine cancer or tumor, a testicular cancer or tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma.

[00270] In some embodiments, the solid tumor comprises a peritoneal cancer. In some embodiments, the solid tumor comprises a peritoneal cancer or a peritoneal metastases. In some embodiments, the solid tumor comprises an ovarian/fallopian tube/primary peritoneal cancer, gastric cancer, colorectal cancer, pancreatic cancer, or other rare peritoneal tumors, or any combination thereof, with no or minimal extraperitoneal disease.

[00271] In some embodiments, a solid tumor comprises a mesothelioma. In some embodiments, a solid tumor comprises a mesothelioma or a colon adenocarcinoma. In some embodiments, a solid tumor comprises a mesothelioma or a colon adenocarcinoma or an ovarian cancer. In some embodiments, a solid tumor comprises a mesothelioma or a colon adenocarcinoma or an ovarian cancer or a peritoneal cancer or a peritoneal metastases, or any combination thereof.

[00272] In some embodiments, said cancer or tumor comprises a solid cancer or tumor, a non-solid cancer , or comprises a metastasis of a cancer or tumor, or any combination thereof.

[00273] In another embodiment, disclosed herein is a method of treating a solid tumor in a subject in need thereof, comprising the step of administering to said subject any of the combination therapies as described herein. In some embodiments, the solid tumor is selected from the group comprising any tumor of cellular or organ origin including a tumor of unknow origin; any peritoneal tumor either primary or metastatic; a tumor of gynecological origin or gastrointestinal origin or pancreatic origin or blood vessel origin, any solid tumor, i.e., adeno carcinoma, hematological solid tumor, melanoma etc. In some embodiments, a solid tumor comprises a sarcoma or a carcinoma, a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endothelio sarcoma, a lymphangiosarcoma, a lymphangioendothelio sarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilm's tumor, a cervical cancer or tumor, a uterine cancer or tumor, a testicular cancer or tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma. In another related aspect, the tumor or cancer comprises a metastasis of a tumor or cancer.

[00274] In some embodiments, methods of use described herein reduce tumor load. A skilled artisan would appreciate that the term “tumor load” may refer to the number of cancer cells, the size of a tumor, or the amount of cancer in the body. The term “tumor load” may be used interchangeably with the term “tumor burden” having all the same meanings and qualities.

[00275] In some embodiments, the combination therapy, as disclosed herein may be used to treat, inhibit the growth of, or reduce the incidence of, any solid tumor known in the art. In some embodiments, the combination therapy, as disclosed herein may be used to treat, inhibit the growth of, or reduce the incidence of, any hematological tumor known in the art. In some embodiments, the combination therapy, as disclosed herein may be used to treat, inhibit the growth of, or reduce the incidence of, any diffuse cancer known in the art.

[00276] In some embodiments, combination therapy, as disclosed herein may be used to extend the survival time of any solid tumor known in the art. In some embodiments, combination therapy, as disclosed herein may be used to extend the survival time of any hematological tumor known in the art. In some embodiments, the combination therapy, as disclosed herein, may be used to extend the survival time of any diffuse cancer known in the art.

[00277] In some embodiments, combination therapy, as disclosed herein may be used to increase the survival of a subject suffering from any solid tumor known in the art. In some embodiments, combination therapy, as disclosed herein may be used to increase the survival of a subject suffering from any hematological tumor known in the art. In some embodiments, combination therapy, as disclosed herein may be used to increase the survival of a subject suffering from any diffuse cancer known in the art.

[00278] In some embodiments, combination therapy, as disclosed herein may be used to reduce the growth rate of any solid tumor known in the art. In some embodiments, combination therapy, as disclosed herein may be used to reduce the growth rate of any hematological tumor known in the art. In some embodiments, the combination therapy, as disclosed herein may be used to reduce the growth rate of any diffuse cancer known in the art.

[00279] In some embodiments, the tumor or cancer being treated comprises a metastasis of a tumor or cancer. In some embodiments, methods of use herein prevent or reduce metastasis of a tumor or cancer. In some embodiments, methods of use herein inhibit the growth or reduce the incidence of metastasis.

[00280] In some embodiments, a method disclosed herein comprises administering combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising one or more checkpoint inhibitors, as described in detail herein. In some embodiments, the apoptotic mononuclear- enriched cell population is stable for greater than 24 hours. Stable populations of apoptotic cells have been described in detail herein. In some embodiments, a method disclosed herein comprises administering the apoptotic mononuclear-enriched cell population comprising a population of cells devoid of cell aggregates, apoptotic cell populations devoid of aggregates and methods of making them are described herein in detail. In some embodiments, the first composition comprises a thawed frozen formulation comprising an apoptotic mononuclear- enriched cell population.

[00281] In some embodiments, a method disclosed herein comprises administering an autologous apoptotic cell population to a subject in need. In some embodiments, a method disclosed herein comprises administering an allogeneic apoptotic cell population to a subject in need.

[00282] In some embodiments, methods disclosed herein comprise a first-line therapy.

[00283] A skilled artisan would appreciate that the term “first-line therapy” may encompass the first treatment given for a disease. It is often part of a standard set of treatments, such as surgery followed by chemotherapy and radiation. When used by itself, first-line therapy is the one accepted as the best treatment. If it doesn't cure the disease or it causes severe side effects, other treatment may be added or used instead. Also called induction therapy, primary therapy, and primary treatment.

[00284] In some embodiments, methods disclosed herein comprise an adjuvant therapy.

[00285] A skilled artisan would appreciate that the term “adjuvant therapy” may encompass a treatment that is given in addition to the primary or initial treatment. In some embodiments, adjuvant therapy may comprise an additional cancer treatment given prior to the primary treatment in preparation of a further treatment. In some embodiments, adjuvant therapy may comprise an additional cancer treatment given after the primary treatment to lower the risk that the cancer will come back. Adjuvant therapy may include chemotherapy, radiation therapy, hormone therapy, targeted therapy, or biological therapy.

[00286] In some embodiments, a method disclosed herein, reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, decreases the size of said tumor, decreases growth rate of said tumor or said cancer, prevents metastasis of said tumor or said cancer, or reduces the rate of metastasis of said tumor or said cancer, or any combination thereof.

[00287] A skilled artisan would appreciate that the term “minimal residual disease” may encompass small numbers of cancer cells that remain in the patient during treatment or after treatment when the patient has no symptoms or signs of disease.

[00288] Additionally, the term “remission” may encompass a decrease or disappearance of signs and symptoms of cancer, though cancer may still be in the body. In some embodiments, remission may comprise partial remission, wherein some, but not all, signs and symptoms of cancer have disappeared. In some embodiments, remission comprises complete remission, wherein all signs and symptoms of cancer have disappeared, although cancer still may be in the body. In some embodiments, methods disclosed herein may be comprise a remission induction therapy, wherein the initial treatment with apoptotic cells or compositions thereof decreases the signs or symptoms of cancer or make them disappear.

[00289] A skilled artisan would appreciate that the term “relapse” may encompass the return of a disease or the signs and symptoms of a disease after a period of improvement. In some embodiments, methods used herein lead to a relapse-free survival, wherein the relapse- free survival encompasses the length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer.

[00290] A skilled artisan would appreciate that the term “metastasis” encompasses the spread of cancer cells from the place where they first formed to another part of the body. In metastasis, cancer cells break away from the original (primary) tumor, travel through the blood or lymph system, and form a new tumor in other organs or tissues of the body. In some embodiments, the new, metastatic tumor is the same type of cancer as the primary tumor. For example, if breast cancer spreads to the lung, the cancer cells in the lung are breast cancer cells, not lung cancer cells.

[00291] Malignancies [00292] In some embodiments, the methods or use of the combination therapy disclosed herein increase the survival of the subject. In some embodiments, disclosed herein is a method of increasing or lengthening the survival of a subject having a diffuse cancer, comprising the step of administering a combination therapy to said subject, wherein the method increases the survival of the subject. In some embodiments, disclosed herein is a method of increasing or lengthening the survival of a subject having a solid tumor, comprising the step of administering a combination therapy to said subject, wherein the method increases the survival of the subject.

[00293] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the solid tumor comprises an abdominal tumor.

[00294] In some embodiments, a cancer may comprise a solid tumor. In some embodiments, a solid tumor comprises an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer), or malignant (cancer). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors. In some embodiments, a solid tumor comprises a sarcoma or a carcinoma.

[00295] In some embodiments, solid tumors are neoplasms (new growth of cells), or lesions (damage of anatomic structures or disturbance of physiological functions) formed by an abnormal growth of body tissue cells other than blood, bone marrow or lymphatic cells. In some embodiments, a solid tumor consists of an abnormal mass of cells which may stem from different tissue types such as liver, colon, breast, or lung, and which initially grows in the organ of its cellular origin. However, such cancers may spread to other organs through metastatic tumor growth in advanced stages of the disease.

[00296] In some embodiments, examples of solid tumors comprise sarcomas, carcinomas, and lymphomas. In some embodiments, a solid tumor comprises a sarcoma or a carcinoma. In some embodiments, the solid tumor is an intra-peritoneal tumor.

[00297] In some embodiments, a solid tumor comprises, but is not limited to, lung cancer, breast cancer, ovarian cancer, stomach cancer, esophageal cancer, cervical cancer, head and neck cancer, bladder cancer, liver cancer, and skin cancer. In some embodiments, a solid tumor comprises a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilm's tumor, a cervical cancer or tumor, a uterine cancer or tumor, a testicular cancer or tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma.

[00298] In some embodiments, the solid tumor comprises an Adrenocortical Tumor (Adenoma and Carcinoma), a Carcinoma, a Colorectal Carcinoma, a Desmoid Tumor, a Desmoplastic Small Round Cell Tumor, an Endocrine Tumor, an Ewing Sarcoma, a Germ Cell Tumor, a Hepatoblastoma a Hepatocellular Carcinoma, a Melanoma, a Neuroblastoma, an Osteosarcoma, a Retinoblastoma, a Rhabdomyosarcoma, a Soft Tissue Sarcoma Other Than Rhabdomyosarcoma, and a Wilms Tumor. In some embodiments, the solid tumor is a breast tumor. In another embodiment, the solid tumor is a prostate cancer. In another embodiment, the solid tumor is a colon cancer. In some embodiments, the tumor is a brain tumor. In another embodiment, the tumor is a pancreatic tumor. In another embodiment, the tumor is a colorectal tumor.

[00299] In some embodiments, a combination therapy as disclosed herein, has a therapeutic and/or prophylactic efficacy against a cancer or a tumor, for example sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelio sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[00300] In some embodiments, the combination therapy as disclosed herein, may be used to treat, inhibit the growth of, or reduce the incidence of, any solid tumor known in the art.

[00301] In some embodiments, the combination therapy as disclosed herein, may be used to increase the survival of a subject suffering from any solid tumor as disclosed herein or known in the art.

[00302] In some embodiments, the combination therapy as disclosed herein, may be used to reduce the size or reduce the growth rate of any solid tumor as disclosed herein or known in the art.

[00303] In some embodiments, a cancer may be a diffuse cancer, wherein the cancer is widely spread; not localized or confined. In some embodiments, a diffuse cancer may comprise a non-solid tumor. Examples of diffuse cancers include leukemias. Leukemias comprise a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream.

[00304] In some embodiments, a diffuse cancer comprises a B-cell malignancy. In some embodiments, the diffuse cancer comprises leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is large B-cell lymphoma.

[00305] In some embodiments, the diffuse cancer or tumor comprises a hematological tumor. In some embodiments, hematological tumors are cancer types affecting blood, bone marrow, and lymph nodes. Hematological tumors may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages, and masT -cells, whereas the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas (e.g., Hodgkin's Lymphoma), lymphocytic leukemias, and myeloma are derived from the lymphoid line, while acute and chronic myelogenous leukemia (AML, CML), myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[00306] In some embodiments, a non-solid (diffuse) cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non-Hodgkin lymphoma, or plasma cell leukemia.

[00307] In another embodiment, the combination therapy as disclosed herein, have therapeutic and/or prophylactic efficacy against diffuse cancers, for example but not limited to leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocyte leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease.

[00308] The compositions and methods as disclosed herein may be used to treat, inhibit, ameliorate, reduce the incidence of, or alleviate any solid tumor known in the art.

Administration

[00309] In some embodiments, administration comprises administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor. In some embodiments, administration comprises administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising one or more checkpoint inhibitors. In some embodiments, administration comprises administering a combination therapy comprising a first composition comprising an inactivated apoptotic mononuclear-enriched cell population and a second composition comprising one or more checkpoint inhibitors, wherein said inactivated apoptotic mononuclear-enriched cell population comprises

(a) a decreased number of non-quiescent non-apoptotic cells;

(b) a suppressed cellular activation of any living non-apoptotic cells; or

(c) a reduced proliferation of any living non-apoptotic cells;

(d) or any combination thereof compared with a population comprising a non-irradiated apoptotic cell population.

[00310] In some embodiments of the methods and combinations disclosed herein, an apoptotic cell population comprises an irradiated, apoptotic mononuclear-enriched cell population, wherein said irradiation is post induction of apoptosis.

[00311] In some embodiments, administration comprises administering a first composition comprising an apoptotic cell population. In some embodiments, administration comprises administering a second composition comprising a checkpoint inhibitor. In some embodiments, administration comprises administering a second composition comprising one or more checkpoint inhibitors. In some embodiments, administration comprises administering a combination therapy of compositions described herein. In some embodiments, administration comprises administering one or more checkpoint inhibitors and an apoptotic cell population in the same or different compositions. In some embodiments, administration comprises administering one or more checkpoint inhibitors in combination with an apoptotic mononuclear-enriched cell population, as described herein. In some embodiments, administration comprises administering an apoptotic mononuclear- enriched cell population and an antibody or fragment thereof in the same or different compositions.

[00312] For treatment, the amount administered is an amount effective in producing the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

[00313] A skilled artisan would recognize that an "effective amount" (or, "therapeutically effective amount") may encompass an amount sufficient to effect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. In some embodiments, an "effective amount" is an amount sufficient to arrest, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion, or migration) of a cancer, tumor or neoplasia. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the antigen-binding fragment administered.

[00314] In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising one or more checkpoint inhibitors, for example but not limited to a PD- 1 , a PDL- 1 , a CTLA-4, or a TIM3 checkpoint inhibitor. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising one or more checkpoint inhibitors comprising an antibody, for example but not limited to a PD-1, a PDL-1, a CTLA- 4, or a TIM3, wherein the checkpoint inhibitor comprises an antibody.

[00315] In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising atezolizumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising avelumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising durvalumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising cosibelimab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising cemiplimab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising nivolumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising pembrolizumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising cemiplimab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising tislelizumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising dostarlimab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising retifanlimab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising spartalizumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising camrelizumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising sintilimab.

[00316] In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear- enriched cell population and a second composition comprising Ipilimumab. In some embodiments, methods disclosed herein comprise administering a combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising Tremelimumab.

[00317] In some embodiments, methods disclosed herein administer compositions comprising apoptotic mononuclear-enriched cell populations and compositions comprising one or more checkpoint inhibitors, as disclosed herein.

[00318] In some embodiments, administration comprises autologous or heterologous administration. For example, apoptotic cell populations can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived apoptotic cell populations disclosed herein or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration.

[00319] In some embodiments, methods of administration of compositions comprising apoptotic cell populations comprise administering a single infusion of said compositions comprising irradiated apoptotic cell populations. In some embodiments, a single infusion may be administered as a prophylactic to a subject predetermined to be at risk for a cancer or tumor. In some embodiments, a single infusion may be administered to a subject having a cancer or tumor on a regular basis as a part of the subject therapeutic treatment. In some embodiments, a single infusion may be administered as a prophylactic to a subject having a cancer or tumor in order to prevent, reduce the risk of, or delay the onset of metastatic cancer. In some embodiments, a single infusion may be administered as part of a combination therapy.

[00320] In some embodiments, methods of administration of compositions comprising apoptotic cell populations comprise administering multiple infusions of said compositions comprising apoptotic cell populations. In some embodiments, multiple infusions may be administered as a prophylactic to a subject predetermined to be at risk for a cancer or tumor. In some embodiments, multiple infusions may be administered to a subject having a cancer or tumor on a regular basis as a part of the subject therapeutic treatment. In some embodiments, multiple infusions may be administered as a prophylactic to a subject having a cancer or tumor in order to prevent, reduce the risk of, or delay the onset of metastatic cancer. In some embodiments, multiple infusions may be administered as part of a combination therapy.

[00321] In some embodiments, multiple infusions comprise at least two infusions. In some embodiments, multiple infusions comprise 2 infusions. In some embodiments, multiple infusions comprise more than 2 infusions. In some embodiments, multiple infusions comprise at least 3 infusions. In some embodiments, multiple infusions comprise 3 infusions. In some embodiments, multiple infusions comprise more than 3 infusions. In some embodiments, multiple infusions comprise at least 4 infusions. In some embodiments, multiple infusions comprise 4 infusions. In some embodiments, multiple infusions comprise more than 4 infusions. In some embodiments, multiple infusions comprise at least 5 infusions. In some embodiments, multiple infusions comprise 5 infusions. In some embodiments, multiple infusions comprise more than 5 infusions. In some embodiments, multiple infusions comprise at least six infusions. In some embodiments, multiple infusions comprise 6 infusions. In some embodiments, multiple infusions comprise more than 6 infusions. In some embodiments, multiple infusions comprise at least 7 infusions. In some embodiments, multiple infusions comprise 7 infusions. In some embodiments, multiple infusions comprise more than 7 infusions. In some embodiments, multiple infusions comprise at least 8 infusions. In some embodiments, multiple infusions comprise 8 infusions. In some embodiments, multiple infusions comprise more than 8 infusions. In some embodiments, multiple infusions comprise at least nine infusions. In some embodiments, multiple infusions comprise 9 infusions. In some embodiments, multiple infusions comprise more than 9 infusions. In some embodiments, multiple infusions comprise at least 10 infusions. In some embodiments, multiple infusions comprise 10 infusions. In some embodiments, multiple infusions comprise more than 10 infusions.

[00322] In some embodiments, multiple infusions comprise smaller amounts of apoptotic cell populations, wherein the total dosage of cells administered is the sum of the infusions. [00323] In some embodiments, multiple infusions are administered over a period of hours. In some embodiments, multiple infusions are administered over a period of days. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least 12 hours between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least 24 hours between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least a day between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least two days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least three days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least four days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least five days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least six days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least seven days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least a week between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least two weeks between infusions.

[00324] In some embodiments, the quantity of cells in multiple infusions is essentially equivalent one to the other. In some embodiments, the number of cells in multiple infusions is different from one to the other. [00325] In some embodiments, methods of administration of compositions comprising one or more checkpoint inhibitors comprise administering a single infusion of said compositions comprising one or more checkpoint inhibitors. In some embodiments, a single infusion may be administered as a prophylactic to a subject predetermined to be at risk for a cancer or tumor. In some embodiments, a single infusion may be administered to a subject having a cancer or tumor on a regular basis as a part of the subject therapeutic treatment. In some embodiments, a single infusion may be administered as a prophylactic to a subject having a cancer or tumor in order to prevent, reduce the risk of, or delay the onset of metastatic cancer. In some embodiments, a single infusion may be administered as part of a combination therapy.

[00326] In some embodiments, methods of administration of compositions comprising one or more checkpoint inhibitors comprise administering multiple infusions of said compositions comprising one or more checkpoint inhibitors. In some embodiments, multiple infusions may be administered as a prophylactic to a subject predetermined to be at risk for a cancer or tumor. In some embodiments, multiple infusions may be administered to a subject having a cancer or tumor on a regular basis as a part of the subject therapeutic treatment. In some embodiments, multiple infusions may be administered as a prophylactic to a subject having a cancer or tumor in order to prevent, reduce the risk of, or delay the onset of metastatic cancer. In some embodiments, multiple infusions may be administered as part of a combination therapy.

[00327] In some embodiments, multiple infusions comprise at least two infusions. In some embodiments, multiple infusions comprise 2 infusions. In some embodiments, multiple infusions comprise more than 2 infusions. In some embodiments, multiple infusions comprise at least 3 infusions. In some embodiments, multiple infusions comprise 3 infusions. In some embodiments, multiple infusions comprise more than 3 infusions. In some embodiments, multiple infusions comprise at least 4 infusions. In some embodiments, multiple infusions comprise 4 infusions. In some embodiments, multiple infusions comprise more than 4 infusions. In some embodiments, multiple infusions comprise at least 5 infusions. In some embodiments, multiple infusions comprise 5 infusions. In some embodiments, multiple infusions comprise more than 5 infusions. In some embodiments, multiple infusions comprise at least six infusions. In some embodiments, multiple infusions comprise 6 infusions. In some embodiments, multiple infusions comprise more than 6 infusions. In some embodiments, multiple infusions comprise at least 7 infusions. In some embodiments, multiple infusions comprise 7 infusions. In some embodiments, multiple infusions comprise more than 7 infusions. In some embodiments, multiple infusions comprise at least 8 infusions. In some embodiments, multiple infusions comprise 8 infusions. In some embodiments, multiple infusions comprise more than 8 infusions. In some embodiments, multiple infusions comprise at least nine infusions. In some embodiments, multiple infusions comprise 9 infusions. In some embodiments, multiple infusions comprise more than 9 infusions. In some embodiments, multiple infusions comprise at least 10 infusions. In some embodiments, multiple infusions comprise 10 infusions. In some embodiments, multiple infusions comprise more than 10 infusions.

[00328] In some embodiments, multiple infusions comprise smaller amounts of one or more checkpoint inhibitors, wherein the total dosage of cells administered is the sum of the infusions.

[00329] In some embodiments, multiple infusions are administered over a period of hours. In some embodiments, multiple infusions are administered over a period of days. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least 12 hours between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least 24 hours between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least a day between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least two days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least three days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least four days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least five days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least six days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least seven days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least a week between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least two weeks between infusions. [00330] In some embodiments, compositions comprising apoptotic cells and one or more checkpoint inhibitors are administered concurrently. In some embodiments, compositions comprising apoptotic cells and one or more checkpoint inhibitors are administered at different time points. In some embodiments, compositions comprising apoptotic cells and one and more checkpoint inhibitors are administered wherein the first composition administered comprises apoptotic cells and the second composition administered comprises one or more checkpoint inhibitors. In some embodiments, compositions comprising apoptotic cells and one and more checkpoint inhibitors are administered wherein the first composition administered comprises one or more checkpoint inhibitors and the second composition administered comprises apoptotic cells. In some embodiments of a combination therapy, compositions comprising apoptotic cells and one and more checkpoint inhibitors are administered wherein the composition comprising one or more checkpoint inhibitors and the composition administered comprises apoptotic cells are each administered on a different schedule.

[00331] In some embodiments, in the methods of the present disclosure, the administration of a second composition comprising one or more checkpoint inhibitors occurs prior to the administration of the first composition comprising an apoptotic mononuclear-enriched cell population. In some embodiments, the administration of a second composition comprising one or more checkpoint inhibitors occurs concurrent with the administration of the first composition comprising an apoptotic mononuclear-enriched cell population. In some embodiments, the administration of a second composition comprising one or more checkpoint inhibitors occurs following the administration of the first composition comprising an apoptotic mononuclear-enriched cell population. In one embodiment, concurrent administration comprises administering a single combination comprising the second composition comprising one or more checkpoint inhibitors and the first composition comprising an apoptotic mononuclear-enriched cell population. In another embodiment, concurrent administration comprises administering separate compositions, an apoptotic mononuclear-enriched cell population.

[00332] In one embodiment, the administration of the second composition comprising one or more checkpoint inhibitors occurs at the same site as the administration of the first composition comprising an apoptotic mononuclear-enriched cell population.

[00333] In one embodiment, the first composition comprising an apoptotic mononuclear- enriched cell population is administered several days before or after the administration of the second composition comprising one or more checkpoint inhibitors. In one embodiment, the first composition comprising an apoptotic mononuclear-enriched cell population is administered 1, 2, 3, 4, or 5 days prior to the administration of the second composition comprising one or more checkpoint inhibitors. In one embodiment, the first composition comprising an apoptotic mononuclear-enriched cell population is administered 1, 2, 3, 4, or 5 days subsequent to the administration of the second composition comprising one or more checkpoint inhibitors.

[00334] In some embodiments, the first and second compositions of the present disclosure are administered at least once during a treatment cycle. In some embodiments, the first and second compositions are administered to the subject on the same days. In some embodiments, the first and second compositions are administered to the subject on the different days. In some embodiments, first and second compositions are administered to the subject on the same days and on different days according to treatment schedules.

[00335] In some embodiments, the first and second compositions are administered to the subject over one or more treatment cycles. A treatment cycle can be at least two, at least three, at least four, at least five, at least six, at least seven, at least 14, at least 21, at least 28, at least 48, or at least 96 days or more. In one embodiment, a treatment cycle is 28 days. In certain embodiments, the first and second compositions are administered over the same treatment cycle or concurrently over different treatment cycles assigned for each composition. In various embodiments, the treatment cycle is determined by a health care professional based on the condition and needs of the subject.

[00336] In some embodiments, the first and second compositions are administered on at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days, at least eleven days, at least twelve days, at least 13 days, at least 14 days, at least 21 days, or all 28 days of a 28-day treatment cycle. In some embodiments, the first and second compositions are administered to a subject once a day. In other some embodiments, the first and second compositions are administered twice a day.

[00337] In some embodiments, in the methods of the present disclosure, the first composition comprising an apoptotic mononuclear-enriched cell population is administered once per week. In some embodiments, the first composition comprising an apoptotic mononuclear-enriched cell population is administered once every two weeks.

[00338] In some embodiments, in the methods disclosed herein, the first composition comprising an apoptotic mononuclear-enriched cell population or the second composition comprising one or more checkpoint inhibitors are intravenously administered to the subject. In some embodiments, the first composition comprising an apoptotic mononuclear-enriched cell population or the second composition comprising one or more checkpoint inhibitors are orally administered to the subject. In some embodiments, the first composition and said second composition are administered intravenously or orally, or a combination thereof. In some embodiments, the first composition is administered orally. In some embodiments, the second composition is administered orally. In some embodiments, the first composition is administered intravenously. In some embodiments, the second composition is administered intravenously. In some embodiments, the first composition is administered intraperitoneally (i.p.). In some embodiments, the second composition is administered intraperitoneally.

[00339] In some embodiments of methods disclosed herein, the first composition comprising intraperitoneally or the second composition comprising one or more checkpoint inhibitors are non-systemically administered to the subject. In some embodiments of methods disclosed herein, the first composition comprising intraperitoneally or the second composition comprising one or more checkpoint inhibitors are administered by local delivery to the subject.

[00340] In some embodiments, administration comprises co-administration of the first composition and the second composition in the same or separate compositions. In some embodiments, administration comprises administration of the first composition and the second composition at different time points. In some embodiments, the first composition and the second composition are administered at separate sites or at the same sites. In some embodiments, the first composition and the second composition are administered at separate sites. In some embodiments, the first composition and the second composition are administered at the same sites.

[00341] In some embodiments, the first composition comprising intraperitoneally and the second composition comprising one or more checkpoint inhibitors are administered together. In some embodiments, the first composition comprising intraperitoneally and the second composition comprising one or more checkpoint inhibitors are administered at separate sites or at separate times. In some embodiments, the first composition comprising intraperitoneally and the second composition comprising one or more checkpoint inhibitors are administered at separate sites. In some embodiments, the first composition comprising intraperitoneally and the second composition comprising one or more checkpoint inhibitors are administered at separate times.

[00342] In one embodiment, one or more of the first and second compositions described herein are administered in one to four doses per day. In one embodiment, one or more of the first and second compositions as described herein are administered once per day. In another embodiment, one or more of the first and second compositions as described herein are administered twice per day. In another embodiment, one or more of the first and second compositions as described herein are administered three times per day. In another embodiment, one or more of the first and second compositions as described herein are administered four times per day. In another embodiment, one or more of the first and second compositions as described herein are administered once every two days, once every three days, twice a week, once a week, once every 2 weeks, once every 3 weeks.

[00343] In one embodiment, one or more of the first and second compositions as described herein are administered for 7 days to 28 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 7 days to 8 weeks. In another embodiment, one or more of the first and second compositions as described herein are administered for 7 days to 50 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 7 days to six months. In another embodiment, one or more of the first and second compositions as described herein are administered for 7 days to one and half years. In another embodiment, one or more of the first and second compositions as described herein are administered for 14 days to 12 months. In another embodiment, one or more of the first and second compositions as described herein are administered for 14 days to 3 years. In another embodiment, one or more of the first and second compositions as described herein are administered for several years. In another embodiment, one or more of the first and second compositions as described herein are administered for one month to six months.

[00344] In one embodiment, one or more of the first and second compositions as described herein are administered for 7 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 14 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 21 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 28 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 50 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 56 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 84 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 90 days. In another embodiment, one or more of the first and second compositions as described herein are administered for 120 days.

[00345] The number of times a first or second composition is administered to a subject in need thereof depends on the discretion of a medical professional, the disorder, the severity of the disorder, and the subject's response to the compositions. In some embodiments, the first and second compositions disclosed herein are administered once to a subject in need thereof with a mild acute condition. In some embodiments, the first and second compositions disclosed herein are administered more than once to a subject in need thereof with a moderate or severe acute condition. In the case wherein the subject's condition does not improve, upon the doctor's discretion the first or second composition may be administered chronically, that is, for an extended period of time, including throughout the duration of the subject's life in order to ameliorate or otherwise control or limit the symptoms of the subject's disease or condition.

[00346] In the case wherein the subject's status does improve, upon the doctor's discretion the first or second composition may be administered continuously; or the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dose reduction during a drug holiday may be from 10%- 100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[00347] Unless specifically stated otherwise herein, references made in the singular may also include the plural. For example, “a” and “an” may refer to either one, or one or more. [00348] As used herein, the terms "administering," "administer," or "administration" encompass delivering one or more compounds or compositions to a subject.

[00349] In some embodiments, a composition of the present disclosure comprises a pharmaceutically acceptable composition or compositions. In some embodiment, the phrase “pharmaceutically acceptable” encompasses those compounds, materials, compositions, combinations, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00350] In some embodiments, a combination of the present disclosure is administered in a therapeutically effective amount. In one embodiment, a “therapeutically effective amount” is intended to include an amount of a compound of the present disclosure alone or an amount of the combination of compounds claimed or an amount of a compound of the present disclosure in combination with other active ingredients effective to treat or prevent proliferative diseases such as cancer. In one embodiment, a "therapeutically effective amount" of a composition of the disclosure is that amount of composition which is sufficient to provide a beneficial effect to the subject to which the composition is administered.

[00351] In some embodiments, the term “decreasing the size of the tumor” as used herein is assessed using the “Response Evaluation Criteria in Solid Tumors” (RECIST). In one embodiment, RECIST measures reduction in tumor size by measuring the longest dimension of a target lesion. In one embodiment, the target lesion is selected on the basis of its size (lesion with the longest diameter) and its suitability for accurate repeated measurements (either by imaging techniques or clinically). In one embodiment, all other lesions (or sites of disease) are identified as non-target lesions and are also recorded at baseline. Measurements of these lesions are not required, but the presence or absence of each is noted throughout follow-up.

[00352] In some embodiments, the term “decreasing the volume of the tumor” as used herein is assessed using the radiological tumor response evaluation criteria. In one embodiment, the maximum diameter (width) of the tumor is measured in two dimensions in the translation plane and its largest perpendicular diameter on the same image (thickness), according to the World Health Organization (WHO).

[00353] According to any of the methods of the present disclosure and in certain embodiments, a subject as described herein is human. In another embodiment, the subject is a mammal. In another embodiment, the subject is a primate, which in one embodiment, is a non-human primate. In another embodiment, the subject is murine, which in one embodiment is a mouse, and, in another embodiment is a rat.

[00354] It is to be understood that the compositions, combinations, and methods of the present disclosure comprising the elements or steps as described herein may, in another embodiment, consist of those elements or steps, or in another embodiment, consist essentially of those elements or steps. In some embodiments, the term “comprise” refers to the inclusion of the indicated active agents, as well as inclusion of other active agents, and pharmaceutically or physiologically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some embodiments, the term “consisting essentially of' refers to a composition, whose only active ingredients are the indicated active ingredients. However, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredients. In some embodiments, the term “consisting essentially of' may refer to components which facilitate the release of the active ingredient. In some embodiments, the term “consisting of' refers to a composition which contains the active ingredients and a pharmaceutically acceptable carrier or excipient.

[00355] A skilled artisan would appreciate that the term “about”, may encompass a deviance of between 0.0001-5% from the indicated number or range of numbers. Further, it may encompass a deviance of between 1 -10% from the indicated number or range of numbers. In addition, it may encompass a deviance of up to 25% from the indicated number or range of numbers.

[00356] 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 disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. [00357] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicated number and a second indicated number and “ranging/ranges from” a first indicated number “to” a second indicated number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[00358] Abbreviations found throughout this application include CTLA-4 (Cytotoxic T Lymphocyte Associated protein 4); AB12-luc (Luciferase - expressing AB 12 tumor cells); CAR-T (Chimeric antigen receptor - T); N/A (Not Applicable); i.p. (Intra Peritoneal); IV (Intravenous); SC (subcutaneous); RT (Room Temperature); TB (Trypan Blue); h or hr (Hour); and AUC (Area under the ROC curve).

EXAMPLES

EXAMPLE 1: Apoptotic Cell Production

[00359] Objective: To produce an apoptotic cell population from an enriched mononuclear cell population, wherein the population of cells produced has unique properties including decreased number of non-quiescent non-apoptotic cells, suppressed cellular activation of any living non-apoptotic cells, and reduced proliferation of any living non- apoptotic cells within the population.

[00360] Methods: Methods of making populations of apoptotic cells have been well documented in International Publication No. WO 2014/087408 and United States Application Publication No. US2015/0275175-A1, see for example, the Methods section preceding the Examples at “Apoptotic cell population Preparation” and “Generation of apoptotic cells” (paragraphs [0223] through [0288]), and Examples 11, 12, 13, and 14, which are incorporated herein in their entirety).

[00361] The flow chart presented in Figure 1A provides an overview of some embodiments of the steps used during the process of producing a population of apoptotic cells, wherein anticoagulants were included in the thawing and induction of apoptosis steps. As is described in detailed in Example 14 of International Publication No. WO 2014/087408 and United States Application Publication No. US US-2015-0275175-Al, apoptotic cell populations were prepared wherein anti-coagulants were added at the time of freezing, or at the time of incubation, or at the time of freezing and at the time of incubation. The anticoagulant used was acid-citrate dextrose, NIH Formula A (ACD formula A) was supplemented with 10 U/ml heparin to a final concentration of 5% ACD of the total volume and 0.5 U/ml heparin.

[00362] Briefly, the cells were collected and then frozen with addition of 5% anticoagulant citrate dextrose formula A and lOU/ml heparin (ACDhep) to the freezing media. Thawing, incubation in an apoptosis induction media containing 5% ACDhep, and final product preparation were performed in a closed system.

[00363] Apoptosis and viability analysis, potency assay, and cell population characterization were performed in each experiment. In order to establish consistency in production of the apoptotic cell product, the final product (FP) of initial batches of apoptotic cells were stored at 2-8°C and examined at tO, t24h, t48h and t72h. At each point apoptosis analysis, short potency assay (Applicants CD14+ frozen cells), trypan blue measurement and cell population characterization were performed. The FP was tested for cell count to assess average cell loss during storage and apoptosis and viability analysis.

[00364] The methods sections cited above and Example 11 of International Publication No. WO 2014/087408 and United States Application Publication No. US US-2015- 0275175-Al provide details of preparing other embodiment of apoptotic cell populations in the absence of anti-coagulants, and are incorporated herein in full.

[00365] Methods of preparing irradiated apoptotic cells: Similar methods were used to prepare an irradiated (inactivated) apoptotic cell population, wherein a mononuclear apoptotic cell population comprises a decreased percent of non-quiescent_non-apoptotic cells, or a population of cells having a suppressed cellular activation of any living non- apoptotic cells, or a population of cells having a reduced proliferation of any living non- apoptotic cells, or any combination thereof.

[00366] Briefly, an enriched mononuclear cell fraction was collected via leukapheresis procedure from healthy, eligible donors. Following apheresis completion, cells were washed and resuspended with freezing media comprising 5% Anticoagulant Citrate Dextrose Solution-Formula A (ACD-A) and 0.5U\ml heparin. Cells were then gradually frozen and transferred to liquid nitrogen for long term storage.

[00367] For preparation of irradiated apoptotic cells, cryopreserved cells were thawed, washed and resuspended with apoptosis induction media comprising 5% ACD-A, 0.5U\ml heparin sodium and 50μg/ml methylprednisolone. Cells were then incubated for 6 hours at 37°C in 5% CO₂. At the end of incubation, cells were collected, washed and resuspended in Hartmann's solution using a cell processing system (Fresenius Kabi, Germany). Following manufacturing completion, apoptotic cells were irradiated at 4000 cGy using g-camera at the radiotherapy unit, Hadassah Ein Kerem. Apoptosis and viability of apoptotic cells determined using AnnexinV and PI (MBL, MA, USA) staining (≥ 40% and < 15%, respectively) via Flow cytometer. Results analyzed using FCS express software. Thus, the apoptotic cells were irradiated after they were prepared (after induction of apoptosis).

[00368] This irradiated apoptotic cell population is considered to include apoptotic cells, wherein any viable cells present have suppressed cellular activity and reduced or no proliferation capabilities. In certain cases, the apoptotic cells population has no viable non- apoptotic cells.

[00369] Results: The stability of the FP produced with inclusion of anticoagulant at freezing and incubation (apoptotic induction) and then stored at 2-8 °C are shown below in Table 1.

[00370] Table 1: Cell count*- performed using a MICROS 60 hematology analyzer.

* Results Representative of 6 (six) experiments.

[00371] When manufacturing the cells without including an anticoagulant in the induction medium, cells were stable for 24 hours and less stable thereafter. Use of anticoagulants unexpectedly extended the stability of the apoptotic cell population for at least 72 hours, as shown in Table 1.

[00372] Table 2: Trypan blue measurement

[00373] The results of Table 2 show viability of the FP remained high for at least 72 hours.

[00374] Table 3: Apoptosis analysis- (AnPI staining) performed using Flow Cytometry

[00375] The data in Table 3 confirms that the majority of cells in the population produced are in apoptosis, wherein the percent of cells in the population in apoptosis (An+PI-) was greater than 50% and in some instances greater than 60%. The cell population produced comprises a minimal percent of cells in late apoptosis or dead cells (less than or equal to 6%). See also Table 3.

[00376] The results of Table 3 show that the percent apoptotic cells versus necrotic cells was maintained over at extended time period of at least 72 hours post preparation of the cells, as was the percentage of apoptotic cells.

[00377] Inclusion of anticoagulants both at the time of freezing and during induction of apoptosis resulted in the most consistently high yield of stable early-apoptotic cells (average yield of apoptotic cells 61.3+2.6% % versus 48.4+5.0%, wherein 100% yield is based on the number of cells at freezing). This high yield was maintained even after 24 hours storage at 2-8°C.

[00378] Next a comparison was made between the inclusion of the anticoagulant at freezing or thawing or both, wherein percent (%) recovery was measured as well as stability. Anticoagulant was included in the apoptotic incubation mix for all populations. Table 4 presents the results of these studies.

[00379] Table 4: Yield and stability comparison of final products (FP) manufactured from cells collected, with ("+") or without ("-") addition of anticoagulant during freezing ("F") and thawing (“Tha”)

[00380] Additional population analysis comparisons of apoptotic cell populations (batches of cells) prepared with and without anti-coagulant added, show the consistency of these results.

[00381] Table 5: Cell population analysis comparison between batches prepared with and without anticoagulant.

[00382] Percentage of final product cells (yield) in the presence or absence of anticoagulants. Similar to the results presented above at Table 1 , the data presented in Table 4 demonstrates that apoptotic cells manufactured from cells frozen in the presence of anticoagulant had a beneficial effect on average yield of fresh final product (FP tO) as compared to cells frozen without anticoagulant. The beneficial effect was seen when anticoagulant was used while freezing only (61.3+2.6% versus 48.4+5.0%), or both freezing and thawing (56.5+5.2% versus 48.4+5.0%). The beneficial effect was less significant when anticoagulant was used upon thawing only (44.0+8.5% versus 48.4+5.0%). These were non- high triglyceride samples.

[00383] Effect of anticoagulants on aggregation. No cell aggregations were seen in these 3 non-high triglyceride samples, or in 21 additional samples (data not shown). However, in 41 other non-high triglyceride samples manufactured without anticoagulants (data not shown), mild aggregates were seen in 10 (24.4%) and severe aggregates in 5 (12.2%); thus, anticoagulants avoid completely cell aggregates.

[00384] Effect of anticoagulants on stability. Fresh FPs manufactured with- or without anticoagulants were stored at 2-8°C for 24 hours to determine whether addition of ACDhep to the manufacturing procedure impairs the stability of the FP. Cells were sampled following 24 hours of storage and yield was calculated in cell count. Similar to the results shown in Table 1 for extended time periods (up to 72 hours), Table 4 shows that the beneficial effect was kept and observed when anticoagulant was used while freezing only (59.8+2.1 % versus 47.5±4.7%), or both freezing and thawing (56.4±5.3% versus 47.5±4.7%). The beneficial effect was less significant when anticoagulant was added only upon thawing (42.4+6.1% versus 47.5+4.7%). These were all non-high triglyceride samples. These results show minimal cell loss following 24 hours of FP storage in all treatments with significant advantage to cells treated with anticoagulant during both freezing and thawing. Average loss of cells treated with anticoagulant during freezing only was 2.3+3.2% compared to 1.9+3.3% without anticoagulants, upon thawing only was 3.0+4.7 compared to 1.9+3.3% without anticoagulants, and 0.2+0.4% compared to 1.9+3.3% without anticoagulants when cells were both frozen and thawed with ACDhep. In summary, the beneficial effect of anticoagulants on yield was kept for at least 24 hours.

[00385] The characteristics of a representative cell population of the FP are shown below in Table 6.

[00386] Table 6: Characterization of the cell population of fresh (tO) FP manufactured from cells collected with ("+") or without

addition of anticoagulant during freezing ("F") and thawing (“Tha”

*Induction of apoptosis was performed using a medium containing anticoagulant for all batches.

[00387] The results of Table 6 show the cell characteristics of the final products (FP) manufactured with or without anticoagulant at freezing and thawing. Batches were sampled, stained for mononuclear markers, and analyzed via flow cytometry to determine the cell distribution in each sample and to examine whether the addition of anticoagulant affected the cell population. As presented in Table 5, there were no significant differences detected in cell populations manufactured with or without anticoagulants at freezing or thawing. The average T cell population (CD3+ cells) in fresh FP was 62.3+1.2% between treatments compared to 62.9+1.1% before freezing; the average B cell population (CD 19+ cells) was 8.3+2.5% between treatments compared to 3.1+0.8% before freezing; the average natural killer cell population (CD56+ cells) was 9.5+0.7% between treatments compared to 12.9+0.5% before freezing; the average monocyte cell population (CD14+ cells) was 13.8+0.5% between treatments compared to 17.5+0.3% before freezing; and the average granulocyte population (CD15+ cells) was 0.0% in the fresh FP compared to 0.35+0.2% at freezing.

[00388] The potency of the apoptotic population was also examined.

[00389] Table 7: Potency analysis of fresh (tO) FP manufactured from cells with ("+") or without ("-") addition of anticoagulant during freezing ("F") and thawing ("Tha") procedures.

[00390] The results presented in Table 7 are from a potency assay performed to determine the ability of each final product to enhance a tolerogenic state in immature dendritic cells (iDCs) following stimulation with (LPS). The tolerogenic effect was determined by assessing downregulation of co-stimulatory molecule HLA-DR and CD86 expression on iDCs following interaction with the apoptotic cell populations and different treatments leading to LPS upregulation. The analysis was performed on DCsign+ cells. Results represent the percent delay in maturation following interaction with apoptotic cell population and following addition of LPS versus LPS-induced maturation. The experiment tested the potency of fresh FP (tO) manufactured with- or without anticoagulant. Results presented in Table 7 show that apoptotic cells manufactured with or without anticoagulant enhance the tolerance effect of both co-stimulatory markers in a dose-dependent manner.

[00391] The apoptotic cells produced herein were from non-high triglyceride samples. This consistent high yield of stable apoptotic cells was produced even in the cases when the donor plasma is high in triglycerides (See for example, Examples 12 and 13 of International Publication No. WO 2014/087408 and United States Application Publication No. US US- 2015-0275175-Al). Note that anti-coagulants were not added to the PBS media used for formulation of the final apoptotic cell dose for infusion.

[00392] Improved delineation of Apoptotic cell populations

[00393] The distinction between apoptotic sub-populations (early, mature, and late), as indicated in Figure IB, was achieved following optimization of the analytical method and improvement in the flow cytometry sensitivity. The optimized method, with a more accurate gate strategy allows a more accurate measurement of the apoptotic population. This led to an update in release specifications. Figure IB shows the distribution, prevalence, and state of the apoptosis of cells within a thawed frozen formulation. Similar results are observed for a freshly prepared formulation of apoptotic cells (Figure 1C). Alignment of late-stage apoptotic cells and a control necrotic population is shown in Example 2 below.

[00394] Summary:

[00395] The objective of this study was to produce a stable, high yield apoptotic cell population. The rational for use of anticoagulants was that aggregates were seen first in patients with high triglycerides, but later in a significant portion of other patients. A concern here was the disclosure in United States Patent No. US 6,489,311 that the use of anticoagulants prevented cell apoptosis.

[00396] In short, with minimal impact on the composition, viability, stability, and the apoptotic nature of the cells, there was a significant improvement of at least 10-20% in the number of collected cells in the final product (Yield) when anticoagulant was added. In this study an up to 13% increase in yield was shown, which represents 26.8% augmentation in yield in controlled conditions but in real GMP conditions it went up to 33% and more augmentations in cell number then can be produced in a single collection. This effect is crucial, since it may avoid the need for a second apheresis from a donor.

[00397] This effect was surprising because the anticipated impact was expected to be dissolution of mild aggregates. It had been hypothesized that thawing cells with anticoagulant would reduce the number of aggregates. When formed, these aggregates eventually lead to massive cell loss. Cells collected and frozen without anticoagulant demonstrated aggregate formation at thawing, immediately after washing. Furthermore, a high level of aggregates was also detected in cells that were frozen without anticoagulant and resuspended with media containing anticoagulant. No aggregates were seen in cells that were both frozen and resuspended with media containing anticoagulant. Taken together, it was concluded that the addition of anticoagulants during freezing and apoptosis induction is of high importance, and did not appear to negatively impact the induction of apoptosis on the cell population.

[00398] Recovery of apoptotic cells was further tested, for example, following 24 hours of storage at 2-8°C, for stability purposes, during which an average cell loss of 3^4.7% was measured, regardless of manufacturing conditions, with favorable results for cells that were both frozen and thawed with media containing anticoagulant (0.2±0.4% cell loss following 24 hours of FP storage), suggesting that addition of anticoagulant is critical during freezing and thawing, but once finally formulated, the apoptotic cell population is stable. Extended time point studies showed this stability to at least 72 hours.

[00399] Apoptosis and viability, as well as cell composition of the FP product were not significantly affected by the addition of anticoagulant at the freezing and/or thawing stage. Values measured from a wide variety of characteristics were similar, indicating the ACDhep did not change the apoptotic cell characteristics and the final product met the acceptance criteria of ≥40% apoptotic cells.

[00400] The assay used to test apoptotic cells potency was based on immature dendritic cells (iDCs), DCs that are characterized by functions such as phagocytosis, antigen presentation, and cytokine production.

[00401] The HLA-DR (MHC class II) membrane molecule and co-stimulatory molecule CD86 were selected as markers to detect the tolerogenic effects of antigen-presenting cells (APCs). Using flow cytometry, changes in expression of HLA-DR and CD86 on iDCs were measured following stimulation with LPS, as well as in the presence of the apoptotic cell population manufactured with- or without anticoagulant and stimulated with LPS. Apoptotic cell populations were offered to DCs in ascending ratios of 1:2, 1:4, and 1:8 iDCs: apoptotic cell population. As presented in Table 4, it was shown that apoptotic cell population enhanced the tolerogenic effect over stimulated DCs in a dose-dependent manner, with slightly better results for apoptotic cell population manufactured with anticoagulant both at freezing and apoptosis induction.

[00402] Taken together, it was concluded that addition of anticoagulant to both freezing and apoptosis media is of high importance to increase cell recovery and avoid massive cell loss due to aggregates, and to avoid in many cases a second round of apheresis from a donor. It was shown that all cells met acceptance criteria for the validated FP, indicating that the addition of anticoagulant does not impair the FP.

Example 2: Analysis of Late- Stage apoptotic cells.

[00403] Objective-. To compare the appearance on the AnV/PI flow cytometry histogram of the late-stage apoptotic cells population with control necrotic cells and determine overlapping gating in flow cytometry.

[00404] Methods'.

[00405] Naive PBMCs were received from the leukapheresis process and were induced to necrosis process as follow. Cells were incubated overnight under 5% CO₂ at 37°C and then transferred to the liquid nitrogen tank for prolonged storage before the AnV/PI staining. Cells were thawed at 37°C for 2 minutes. The thawed cells were stained with Annexin V (AnV) and Propidium Iodide (PI) to assess the prevalence and state of the necrotic cells. Necrosis induction promoted all of the cells to a necrotic state.

[00406] Results'.

[00407] The Figure ID shows that following the necrosis induction, more than 85% of the total PBMCs were induced to necrosis phase and appeared in the AnV+, PI+ necrosis control gate.

[00408] The necrotic cell control was used as reference cells to characterize the flow cytometry pattern of late apoptotic cells, for example late apoptotic cells as identified in Figure IB of Example 1. Analysis showed that the late apoptotic cells overlapped by at least 80% with the generated control necrotic cells (Figure ID and data not shown).

[00409] Figure ID shows that about -90% of the total late-stage apoptotic/ necrosis cells colocalized with the defined necrosis control gate. Thus, the late-stage apoptotic cells identified as part of the total Allocetra/product population produced (See, Example 1) overlap with flow cytometry sorted necrotic cells. Further, necrotic cells have significantly reduced activity compared with apoptotic cells (Figure IE).

[00410] Summary:

[00411] Based on the results of the flow cytometry comparison presented here, Allocetra late apoptotic cells are those cells that localized at the same gate as greater than (>) 85% necrotic cells are localized under the same setting of AnV/PI staining and same setting for flow cytometry acquisition and voltages.

EXAMPLE 3: Effect of Irradiation on Final Apoptotic Cell Product

[00412] Apoptotic cells are increasingly used in novel therapeutic strategies because of their intrinsic immunomodulatory and anti-inflammatory properties. Apoptotic cell preparations may contain as much as 20-40% viable cells (as measured by lack of PS exposure and no PI admission; Annexin V negative and Propidium iodide negative) of which some may be rendered apoptotic after use in a transfusion, but some will remain viable. In the case of bone marrow transplantation from a matched donor, the viable cells do not represent a clinical issue as the recipient is already receiving many more viable cells in the actual transplant. However, in the case of a third-party transfusion, (or fourth party or more as may be represented in a pooled mononuclear apoptotic cell preparation) use of an apoptotic cell population that includes viable cells may introduce a second GvHD inducer. Furthermore, the implication of irradiation on the immunomodulatory potential of apoptotic cells has so far been not assessed. A skilled artisan may consider that additional irradiation of an apoptotic cell population may lead cells to progress into later stages of apoptosis or necrosis. As this appears to be a particularly relevant question with regard to clinical applications, the experiments presented below were designed to address this issue, with at least one goal being to improve the biosafety of functional apoptotic cells.

[00413] Thus, the aim was to facilitate the clinical utilization of apoptotic cells for many indications wherein the potency of apoptotic cells may rely on a bystander effect rather than engraftment of the transplanted cells.

[00414] Objective'. Examine the effect of irradiation on apoptotic cells, wherein irradiation occurs following induction of apoptosis.

[00415] Methods: (in brief): Three separate apoptotic cell batches were prepared on different dates (collections 404-1, 0044-1 and 0043-1). Each final product was divided into three groups: (1) Untreated; (2) 2500rad; and (3) 4000rad.

[00416] Following irradiation, apoptotic cells were tested immediately (to) for cell count, Annexin V positive-PI negative staining, cell surface markers (% population of different cell types) and potency (dendritic cells (DCs)). Following examination at to, apoptotic cells were stored at 2-8°C for 24 hours, and examined the next day using the same test panel (t24h) (cell count, Annexin V positive-PI negative staining, and cell surface markers and potency).

[00417] Previously, a post-release potency assay was developed, which assesses the ability of donor mononuclear apoptotic cells (Apoptotic Cells) to induce tolerance (Mevorach et al, BBMT 2014 ibid). The assay is based on using flow cytometric evaluation of MHC -class II molecules (HLA-DR) and costimulatory molecule (CD86) expression on iDC membranes after exposure to LPS. As previously and repeatedly shown, tolerogenic DCs can be generated upon interaction with apoptotic cells (Verbovetsky et al., J Exp Med 2002, Krispin et al., Blood 2006), and inhibition of maturation of LPS-treated DCs (inhibition of DR and CD86 expression), occurs in a dose dependent manner.

[00418] During phase l/2a of the apoptotic cell clinical study, the post-release potency assay was conducted for each apoptotic cell batch (overall results n=13) in order to evaluate the ability of each batch to induce tolerance (Results are shown in Figure 1, Mevorach et al. (2014) Biology of Blood and Marrow Transplantation 20(1): 58-65).

[00419] DCs were generated for each apoptotic cell batch from fresh buffy coat, collected from an unknown and unrelated healthy donor, and were combined with apoptotic cells at different ratios (1:2, 1:4 and 1:8 DC:Apoptotic Cells, respectively). After incubation with apoptotic cells and exposure to LPS, potency was determined based on downregulation of DC membrane expression of either HLA DR or CD86 at one or more ratios of DC: apoptotic cells. In all 13 assays, apoptotic cells demonstrated a tolerogenic effect, which was seen with preparations at most DC: apoptotic cells ratios, and for both markers, in a dose dependent manner.

[00420] Monocyte obtained immature DCs (iDCs) were generated from peripheral blood PBMCs of healthy donors and cultured in the presence of 1% autologous plasma, G-CSF and IL-4. iDCs were then pre-incubated for 2 hours at 1 ;2, 1;4 and 1;8 ratios with apoptotic cells either freshly prepared final product or final product stored at 2-8°C for 24 hours. The two final products were examined simultaneously in order to determine whether storage affects potency ability of apoptotic cells. Following incubation, LPS was added to designated wells and were left for additional 24 hours. At the end of incubation, iDCS were collected, washed and stained with both DC-sign and HLA-DR or CD86 in order to determine changes in expression. Cells were analyzed using flow cytometer and analysis performed using FCS-express software from DC-sign positive cells gate to assure analysis on DCs only.

[00421] Figures 2A and 2B and Figures 3A and 3B show potency test of irradiated pooled apoptotic cells compared to non-irradiated single donor cell.

[00422] Results'.

[00423] Single Donor preparations

[00424] Table 8 presents the comparative results of non-radiated and irradiated apoptotic cells; Average cell loss (%) at 24 hours; Annexin positive (⁺) Propidium Iodide (PI) negative (') % at 0 hours and 24 hrs (% of apoptotic cells; Annexin positive (⁺) Propidium Iodide (PI) positive (⁺) % at 0 hours and 24 hrs (% of late apoptotic cells); presence of cell surface antigens CD3 (T cells), CD19 (B cells), CD56 (NK cells), CD14 (monocytes), and CD15^hlgh (granulocyte), at 0 hours and 24 hours.

[00425] Table 8:

[00426] The results in Table 8 show that both non-irradiated apoptotic cells and irradiated apoptotic cells had comparable percentages of (rows 2 and 3) and late (rows 4 and 5) apoptotic cells. Thus, 25 or 40 Gy irradiation did not accelerate the apoptotic or necrotic process induced prior to this high level of gamma-irradiation. Further, there was consistency between irradiated cell populations vs. control non-irradiated population with regard to cell type.

[00427] The results of potency assays, presented in Figures 2A-2B (HLA-DR expression) and Figures 3A-3B (CD86 expression) show that there was no change in the immune modulatory capacity of fresh (Figure 2A, Figure 3A) and 24 hour-stored (Figure 2B and Figure 3B) irradiate apoptotic cells when compared with non-irradiated apoptotic cells.

[00428] In both Figures 2A-2B and Figures 3A-3B there is a clear upregulation in both HLA-DR and CD86 expression, following exposure to maturation agent LPS. Significant (p<0.01), dose-dependent down regulation of both co- stimulatory markers was observed in the presence of freshly prepared apoptotic cells both from a single donor or irradiated pooled donors. In addition, dose dependent down regulation was maintained in both markers in the presence of apoptotic cells stored at 2-8°C for 24 hours, indicating final product stability and potency following 24 hours of storage.

[00429] Effect on dendritic cells. In order to test the immunomodulatory capacity of apoptotic cells a post release potency assay was used (Mevorach et al., (2014) BBMT, ibid). No change in immune modulatory assay in dendritic cells was observed. (Data not shown) [00430] Effect on Mixed Lymphocyte Reaction (MLR). In order to further test the immunomodulatory effect a standardized MLR assay was established. Here, co-cultivation of stimulator and responder cells, i.e., a MLR, yielded strong and reliable proliferation. Upon addition of non-irradiated apoptotic cells to the MLR, the lymphocyte proliferation was significantly reduced by >5- fold, demonstrating cell inhibition of proliferation. Inhibition of lymphocyte proliferation in MLRs mediated by irradiated apoptotic cells was completely comparable. (Data not shown)

[00431] The next step was to evaluate in vivo, irradiated and non-irradiated apoptotic cells in a completely mismatched mouse model. As shown, irradiated and non-irradiated apoptotic cell preparations had comparable in vivo beneficial effects.

[00432] Single Donor Preparations Conclusion'.

[00433] In conclusion, irradiation of 25 Gy or 40 Gy did not significantly accelerate apoptosis or induced necrosis in populations of apoptotic cells. Significantly, these populations maintained the immunomodulatory effect of apoptotic cells both in vitro and in vivo.

[00434] Multiple Donor preparations

[00435] Next, experiments were performed to verify that the phenomenon observed with single donor, third party preparation was also true for multiple third-party donors. Unexpectedly, when using pooled individual donor apoptotic cell preparations, the beneficial effect of a single unmatched donor was lost. This was not due to GvHD, as the beneficial effect of each donor separately was maintained (test results no shown). One possibility is that the beneficial effect of the apoptotic cell preparation was lost due to the interaction of the individual donor cells among themselves. It was further examined whether this possible interaction of different donors could be avoided by gamma irradiation.

[00436] As shown, the beneficial effect of a single donor was completely restored following gamma irradiation, wherein the irradiated multiple donor preparation and the single donor preparation (irradiated or non-irradiated) had similar survival patterns.

[00437] Conclusion:

[00438] It is shown here for the first time that surprisingly irradiation (and possibly any method leading to T-cell Receptor inhibition) not only avoided unwanted proliferation and activation of T-cells but also allowed for the beneficial effects of immune modulation when using a preparation of multiple donor third party apoptotic cells.

EXAMPLE 4: Effect of Allocetra-OTS on peritoneal mesothelioma, alone and in combination with immune checkpoint inhibitor.

[00439] Malignant mesothelioma is an aggressive tumor arising from the cells lining the pleural, peritoneal, and pericardial cavity. This tumor is highly resistance to current conventional therapies.

[00440] CTLA-4 is a member of the immunoglobulin superfamily that functions as an immune checkpoint that regulates T cell activation. Treatment of human mesothelioma with anti-CTLA4 to boost generation and activation of T cells, have thus far shown minor improvements, emphasizing the need for combined treatments. Since apoptotic cells harbor an immune-modulatory potential, a combined therapy with anti-CTLA4 has a possible therapeutic potential.

[00441] Previous data showed synergistic effect when using Allocetra-OTS with CAR-T therapy in peritoneal tumor model (See for example, the Examples in both WO 2021/053667 and WO 2018/225072; which are incorporated herein in their entirety).

[00442] Objective: In the following experiments, the effect of combined treatment using Allocetra-OTS and an anti-CTLA4 antibody was tested in a BALB/c AB12-luc peritoneal mesothelioma syngeneic immune-competent model. Analysis was of tumor progression and model subject survival rates. Immune cells were characterized as macrophages and T cell population at different time points.

[00443] Specific objectives included: Testing the effect of mono and combined therapy of Allocetra-OTS (Liquid and frozen) and anti-CTLA4 in AB12-luc BALB/c mesothelioma model; Testing the effect of Allocetra-OTS (liquid) dose on combined therapy with anti- CTLA4 in AB 12 mesothelioma model; and testing the effect of frozen formulation of Allocetra-OTS on combined therapy with anti-CTLA4 in AB 12 mesothelioma model.

[00444] Methods: Figure 4 provides an overview schematic showing the time-frame and actions for reprogramming peritoneal pro-tumor macrophages using apoptotic cells, in an mesothelioma syngeneic mouse model (AB 12), wherein synergistic effects of apoptotic cells and immune checkpoint inhibitors was observed (See results below)

[00445] Tables 9A-9C present the study details including dose administered, route of administration, regime used, and trial groups. [00446] Table 9A:

*i.p. intraperitoneal.

[00447] Table 9B - In vivo 136:

[00448] Table 9C -In vivo 137:

* Group C was modified after the start. One mouse developed s.c. tumor (instead of i.p. ) and was excluded from experiment.

[00449] In one series of experiments, the effect of combined administration of anti- CTLA4 and 20x10⁶ Allocetra-OTS was used. Single administration of anti-CTLA4 or 20x10⁶ Allocetra-OTS were tested as controls.

[00450] In follow-up experiments, the effect of combined administration of anti-CTLA4 and 5x10⁶ or 20x10⁶ Allocetra-OTS was used. Single administration of anti-CTLA4 or 20x10⁶ Allocetra-OTS were included as monotherapy controls.

[00451] Animals and animal facility

[00452] In Vivo 136

[00453] BALB/c female mice, 7 weeks old, were purchased from Envigo (formerly known as Harlan). Mice were kept in an SPF free animal facility in compliance with institutional International Animal Care and Use Committee (IACUC) Guidelines. Mice were monitored daily and assigned a clinical score according to the abdominal tumors scoring table (according to EV Paster et al, 2009; Penn Animal Welfare; Table 10). Mice were weighed twice a week at the beginning of the experiment and daily when they mice reached score 2 in abdominal swelling. Tumor burden was evaluated periodically by IVIS imaging. In addition, abdominal circumference was measured and calculated. Mice were measured at day 35 and twice a week from day 54 to the end of experiment (day 83). Measurements were made for width and height using a caliper (MRC, MT-141253) or measuring tape. Abdominal circumference with the caliper was calculated using the ellipse circumference formula

(according to Sapi j. et al, 2015; PLOS ONE). Mice were sacrificed when reaching score 15.

[00454] In Vivo 137

[00455] BALB/c female mice, 7 weeks old, were purchased from Envigo (formerly known as Harlan). Mice were kept in an SPF free animal facility in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines. Mice were monitored daily and assigned a clinical score according to the abdominal tumor scoring table (EV Paster et al, 2009; Penn Animal Welfare; Table 10). Mice were weighed twice a week at the beginning of the experiment and daily starting once they had reached a score of 2 in abdominal swelling. Tumor burden was evaluated periodically by IVIS imaging. In addition, abdominal circumference was measured and calculated. Mice were measured at day 27 and twice a week from day 33. Measurements were made for width and height by measuring tape or using a caliper (MRC, MT-141253). When the caliper was used, abdominal circumference calculated using the ellipse circumference formula (7t* ((0.5*width)²+(0.5*height)²) (Sapi j. et al, 201510(11) PLOS ONE). Mice were sacrificed upon reaching score 15.

[00456] Table 10: Sacrifice criteria in a peritoneal tumor model.

[00457] Clinical Scoring table (Table 10) was based on EV Paster et al, (2009) (ibid), with modifications for better evaluation of animal welfare.

[00458] AB 12 Mesothelioma model

[00459] A schematic figure outlining the treatment regime used in this Example is presented in Figure 4. Mice received 0.1x10^{^6} luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated i.p. with 200μg anti- CTLA-4 (BioXcell, BE0164) and/ or 20x10^{^6} Allocetra-OTS (Liquid or frozen formulation) or 5x10^{^6} Allocetra-OTS cells (Liquid formulation) in 1 hour interval, wherein the anti- CTLA-4 was added 1 hour prior to the addition of Allocetra. Control mice were treated with equivalent amounts of anti-CTLA-4 or Allocetra-OTS in control monotherapies. Mice were monitored by IVIS imaging for tumor progression.

[00460] Abdominal Circumference

[00461] In-vivo 136

[00462] BALB/c mice (female, 7 weeks) received 0.1x10^6 luciferase- expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated i.p with 200μg anti-CTLA-4 (BioXcell, BE0164) and 20x10^6 Allocetra-OTS cells in 1 hour interval, wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. Control mice were treated with equivalent amounts of anti-CTLA-4 or Allocetra-OTS in control monotherapies. Abdominal circumference was measured and calculated, starting day 35 and twice a week from day 54 by two methods - circumference of abdomen by measuring tape, or measurements of width and height using caliper and calculating circumference using the ellipse circumference formula

(Figure 9) [00463] In-vivo 137.

[00464] BALB/c mice (female, 7 weeks) received 0.1x10^6 luciferase- expressing AB 12 tumor cells (AB12-luc) on day 0. On days 12, 15, 19, and 22, mice were treated i.p with 200μg anti-CTLA-4 (BioXcell, BE0164) 20x10^6 or 5x10^6 Allocetra-OTS cells in 1 hour interval, wherein the anti-CTLA-4 was added 1 hour prior to the addition of Allocetra. Control mice were treated with equivalent amounts of anti-CTLA-4 or Allocetra-OTS in control monotherapies. Abdominal circumference was measured and calculated, starting day 27 and twice a week from day 33 by two methods - circumference of abdomen by measuring tape, or measurements of width and height using caliper and calculating circumference using the ellipse circumference formula

(Figure 10A)

[00465] In-vivo imaging - IVIS

[00466] Mice were monitored periodically for tumor burden by IVIS (Perkin-Elmer, Lumina III). 10 minutes before imaging, mice were injected interperitoneally (IP) with a luciferin solution (150mg/kg, Perkin-Elmer, 122799). 7 minutes after luciferin injection, mice were anesthetized by isoflurane in an induction chamber. The mice were placed in a supine position (anterior facing upwards) inside the IVIS chamber. 10 minutes after luciferin injection, the mice were imaged (exposure: 10 sec, F-stop: 2, Binning: medium). Analysis was performed by Living Image® software (Perkin Elmer, version 4.5.5). Total flux (photon/ sec) was measured at each time point. Average radiance was calculated by the sum of the radiance from each pixel inside the ROI/numbcr of pixels or super pixels (photons/sec/cm²/sr). Results are presented as group median ± IQR.

[00467] Allocetra-OTS - In vivo 136

[00468] An enriched mononuclear cell fraction was collected via leukapheresis procedure from healthy, eligible donors. Following apheresis completion, cells were washed and resuspended with freezing media composed of PlasmaLyte A pH 7.4, 5% Human Serum Albumin, 10% dimethyl sulfoxide (DMSO), 5% Anticoagulant Citrate Dextrose Solution- Formula A (ACD-A) and 0.5U\ml heparin. Cells were then gradually frozen and transferred to liquid nitrogen for long term storage.

[00469] For preparation of Allocetra-OTS - liquid formulation, cryopreserved cells were thawed, washed and resuspended with apoptosis induction media, composed of RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM Hepes, 10% autologous plasma, 5% ACD- A, 0.5U\ml heparin sodium and 50μg/ml methylprednisolone. Cells were then incubated for 6 hours at 37°C in 5% CO₂. At the end of incubation, cells were collected, washed and resuspended in appropriate solution using the LOVO cell processing system (Fresenius Kabi, Germany). Following manufacturing completion, Allocetra product was irradiated at 4000 cGy (by X-ray with a maximum potential of 160kV). Apoptosis and viability of Allocetra-OTS were determined using AnnexinV and PI staining (MBL, MA, USA) by flow-cytometry. Results were analyzed using FCS express software.

[00470] For Frozen formulation, the preparation of each frozen drug product (DP) was performed separately in a 50mL corning centrifugation tube. The commercial CryoStor-10 cryopreservation medium and the DP were mixed homogeneously in 3:1 ratio to create 20mL of frozen DP composed formulated to a final concentration of 2.5% DMSO. The tube was mixed until homogeneous and its contents were transferred to its respective cryobag using a port and a syringe serving as funnel. All cryobags were immediately gradually frozen and kept in liquid nitrogen until use. Frozen formulation bags were thawed for injection quickly in 37°C water bath.

[00471] Prior to injection, Allocetra-OTS cells were filtered through 200pm filter (pluriSelect, 43-50200-01) and centrifuged at 290g, for 10 min (liquid formulation at 2-8°C; frozen formulation at RT). Cells were resuspended with Hartmann solution and counted by trypan blue exclusion using light microscopy. Cells were diluted to injected concentration and filtered again through 200pm filter. Allocetra-OTS cells were injected within 1-1.5 h from preparation. Allocetra-OTS AnPI and Trypan blue viability parameters are presented in Tables 11A, 11B, and 11C.

[00472] Table 11A: Allocetra-OTS AnV-PI analysis and Trypan blue viability - In- vivo 136

* product time is time from preparation to use. For liquid formulations it was up to 96 hours and for frozen formulations it was up to 3 hours.

# TB- Trypan Blue.

[00473] Table 11B: Allocetra-OTS AnV-PI analysis and Trypan blue viability - In- vivo 137 (Liquid Formulation)

[00474] Table 11C: Allocetra-OTS AnV-PI analysis and Trypan blue viability - In- vivo 137 (Frozen Formulation)

- AnPI analysis was performed at t=0 for Liquid formulation and at t=0 + t=2h for Frozen formulation; Cell Viability was by TB exclusion (t=0 for Frozen Formulation; and Cells were injected 0.5-1 hr from thawing for Frozen formulation and 1-1.5 hr from preparation for Liquid Formulation.

[00475] 20x10^{^6} or 5x10^{^6} Allocetra-OTS cells (Liquid or frozen formulation) were injected i.p on days 12, 15, 19, and 22, 1 hour after anti-CTLA4 injections.

[00476] Allocetra-OTS - Frozen Formulation

[00477] Enriched mononuclear cell fraction was collected via leukapheresis procedure from healthy, eligible donors. Following apheresis completion, cells were washed and resuspended with freezing media composed of PlasmaLyte A pH 7.4, 5% Human Serum Albumin, 10% dimethyl sulfoxide (DMSO), 5% Anticoagulant Citrate Dextrose Solution- Formula A (ACD-A) and 0.5U\ml heparin. Cells were then gradually frozen and transferred to liquid nitrogen for long term storage.

[00478] For preparation of Allocetra-OTS - Frozen Formulation, cryopreserved cells were thawed, washed and resuspended with apoptosis induction media, composed of RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM Hepes, 10% autologous plasma, 5% ACD-A, 0.5U\ml heparin sodium and 50μg/ml methylprednisolone. Cells were then incubated for 6 hours at 37° C in 5% CO₂. At the end of incubation, cells were collected, washed, and resuspended in appropriate solution using the LOVO cell processing system (Fresenius Kabi, Germany). Following manufacturing completion, Allocetra product was irradiated at 4000 cGy (by X-ray with a maximum potential of 160kV).

[00479] For the frozen formulation, the preparation of each frozen drug product (DP) was performed separately in a 50mL Coming® centrifugation tube. The commercial CryoStor- 10 cryopreservation medium and a fresh batch of Allocetra cells were mixed homogeneously in 3: 1 ratio to create 20mL of frozen Allocetra Frozen Formulation Drug Product formulated to a final concentration of 2.5% DMSO. The tube was mixed until homogeneous and the contents were transferred to its respective cryobag using a port and a syringe serving as funnel. All cryobags were immediately gradually frozen and kept in liquid nitrogen until use. Frozen formulation bags were thawed for injection quickly in 37°C water bath.

[00480] Prior to injection, the thawed Allocetra-OTS cells were filtered through 200pm filter (pluriSelect, 43-50200-01) and centrifuged at 290g, for 10 min at RT. Cells were resuspended with Hartmann solution and counted by trypan blue exclusion using a light microscopy. Cells were diluted to injected concentration and filtered again through 200pm filter. Frozen Allocetra-OTS cells were injected within 1-1.5h from preparation.

[00481] Figure IE provides a comparison flowchart for the preparation of apoptotic cell formulations as either Fresh (liquid) Formulations or as Frozen Formulations.

[00482] For In vivo 136: 20x10^{^6} Allocetra-OTS cells (liquid or frozen formulation) were injected IP on days 12, 15, 19, and 22, 1 hour after anti-CTLA4 injections. IP with 200μg anti-CTLA-4 (BioXcell, BE0164).

[00483] For In vivo 137: 20x10^{^6} or 5x10^{^6} Allocetra-OTS cells (liquid or frozen formulation) were injected IP on days 12, 15, 19, and 22, 1 hour after anti-CTLA4 injections. [00484] Anti CTLA4

[00485] On days 12, 15, 19, and 22, mice were treated i.p with 200μg anti-CTLA-4 (BioXcell, BE0164).

[00486] Characterization of peritoneal and spleen immune cells

[00487] Scheduled sacrifices were performed on day 12, before treatment, for naive and tumor bearing mice (n=2), and on day 19, 3 days after 2^nd treatment, for naive and all treated mice (n=l). Peritoneal cells and splenocytes were isolated and tested for macrophage panel - CD19, CD11c, CD1 lb, F4/80 and MHC-II, and for T cells panel - CD4, CD8, CD25 and Foxp3.

[00488] Clinical score

[00489] Murine clinical score was evaluated daily starting at day 19 according to sacrifice criteria in peritoneal tumor model (Table 10 above). Briefly, each animal was individually evaluated on 6 parameters: Appearance (i.e., coat piloerection and mouse emaciation), level of activity, response to stimulus, respiration quality, posture, and abdominal swelling (ascites). Each parameter was given a score between 0-4 and the total score was calculated. Animals were sacrificed upon reaching a total score of 15, a score of 3 in the “respiration quality” category, or a maximal score in any other category.

[00490] Survival

[00491] Mice were monitored daily for wellbeing and clinical scores. Mice were sacrificed when reaching score 15.

[00492] Pathology [00493] Upon sacrifice on schedule, as well as due to clinical score (reaching sacrificing parameters), spleen length, and spleen and liver weight were measured. Spleen, liver, and tumor samples were collected for pathology evaluation (hematoxylin and eosin [H&E] staining).

[00494] Statistics

[00495] Survival analysis was performed according to the Kaplan-Meier methods. Logrank statistical test was evaluated. The correlation between ant two parameters was evaluated by a Pearson's correlation coefficient. All statistical analysis were performed using GraphPad Prism software (San Diego, CA, USA) [00496] Results:

[00497] Results shown are as of day 67 for the “In vivo 136” experiment, and as of day 46 for the “In vivo 137” experiment.

[00498] Survival analysis

[00499] In-vivo 136

[00500] Kaplan- Meier analysis of mouse survival is represented in Figures 5 and 6. n=8 mice per group. The experiment ended on day 83.

[00501] Mice in Group A, who received only PBS as expected for a negative control, did not develop cancer signs and symptoms, and did not show any luminescence signal on IVIS. [00502] Mice in Group B, the AB12-luc cancer positive control group, developed cancer signs and symptoms well as IVIS staining as expected, and died between days 26 and 35.

[00503] Mice in Group C, which included AB12-luc mice treated with anti-CTLA4 monotherapy, died between days 29 and 42.

[00504] Mice in Group D, which included AB12-luc mice treated with Allocetra-OTS monotherapy (liquid formulation), died between days 26 and 79.

[00505] Mice in Group E, which included AB 12-luc mice treated with both anti-CTLA4 and Allocetra-OTS, (combination therapy, liquid formulation) had 62.5% survival; 3 mice died on days 32, 35, and 83. 5 mice were healthy at the end of experiment (score 0).

[00506] Mice in Group F, which included AB 12-luc mice treated with both anti-CTLA4 and Allocetra-OTS, (combination therapy, frozen formulation) had 50% survival; 4 mice had died on days 29, 56, 64, and 74 and 4 mice were healthy et the end of the experiment (score 0). [00507] Taken together, anti-CTLA4 monotreatment (Group C) significantly increased mice survival compared to cancer control with no treatment (Group B, p=0.006, log rank). No mouse survived at the end of the experiment.

[00508] Allocetra-OTS monotreatment (Group D) significantly increased survival compared to cancer control with no treatment (Group B, p=0.0206, log rank). No mouse survived at the end of the experiment.

[00509] Allocetra-OTS and anti CTLA-4 mono-treatments did not differ significantly in their effect on survival (0.1210, log rank); however, deterioration differed, as evidenced from the clinical scores (See Figures 7A and 7B for further details).

[00510] Notably, combined treatment with anti-CTLA4 and Allocetra-OTS using the liquid formulation (Group E), not only significantly increased survival in comparison to the control cancer population (Group B, p=0.0005, log rank), but also significantly increased survival in comparison to anti-CTLA4 monotreatment (p=0.0077, log rank) and Allocetra- OTS monotreatment (p= 0.0058, log rank). Furthermore, 62.5% of mice treated with combined treatment had tumor-free survival at the end of experiment (cure) on day 83, compared to 0% in Groups B, C, and D.

[00511] Similarly, combined treatment with anti-CTLA4 and Allocetra-OTS using the frozen formulation (Group F), not only significantly increased survival in comparison to the control cancer population (Group B, p=0.0007, log rank), but also significantly increased survival in comparison to anti-CTLA4 monotreatment (p= 0.0011, log rank) and Allocetra- OTS monotreatment (p= 0.0422, log rank). Fifty percent (50%) of mice treated by combined treatment (frozen formulation) had tumor-free survival at the end of experiment (cure) on day 83. Compared to 0% in group B, C and D.

[00512] No significant differences were seen between combined treatment using the liquid vs the frozen formulations (p=0.6269, log rank)

[00513] Preliminary results presented above, showed significant therapeutic effect of anti- CTEA4 and Allocetra-OTS monotherapy and combined therapy in BAEB/c mice AB 12 mesothelioma model (In-vivo 136), wherein the combined therapy provided a synergistic beneficial effect on life expectancy (survival).

[00514] In-vivo 137.

[00515] The Kaplan- Meier survival analysis is presented in Figures 5 and 6. The study ended on day 95. [00516] All Group A, AB 12-luc cancer positive control group mice died between days 26 and 64; 7/8 mice were dead by day 41, and 1 mouse extended survival to day 64.

[00517] In Group B, there was 12.5% survival at day 95, with 7 AB 12-luc mice treated with anti-CTLA4 monotherapy dead between days 32 and 95 (6/7 by day 50 and 1 mouse on day 95). 1 mouse was still alive and healthy on day 95.

[00518] In Group C, there was 28.5% survival on day 95 for AB 12-luc mice treated with 20x106 Allocetra-OTS (liquid formulation) monotherapy. 5 mice had died at days 29, 31, 33, 35 and 49; 2 mice were still alive and healthy (score 0) at day 95.

[00519] In Group D, there was 89% survival on day 95 among AB 12-luc mice treated with combined therapy of anti-CTLA4 and 20x106 Allocetra-OTS, liquid formulation. 1 mouse out of 9 was found dead on day 88 with no abdominal swelling and no signs of tumor in peritoneum. 8/9 mice were still alive and healthy at day 95 (score 0).

[00520] In Group E, there was 66.6% survival among AB 12-luc mice treated with combined therapy of anti-CTLA4 and 5x106 Allocetra-OTS, liquid formulation on day 95. 3 mice died on days 31, 54, and 64; 6/9 mice were still alive and healthy on day 95 (score 0).

[00521] In Group F, there was 75% survival among AB 12-luc mice who had combined therapy of anti-CTLA4 and 20x106 Allocetra-OTS, frozen formulation on day 95. 2 mice died on days 44 and 74; 6/8 mice were still alive and healthy at day 95 (score 0).

[00522] Anti-CTLA4 mono treatment (Group B) increased survival compared to cancer control, but the difference did not reach statistical significance (Group A, p=0.1291, log rank). However, this treatment prolonged survival in one mouse to 95 days.

[00523] Allocetra-OTS monotreatment (Group C) increased survival compared to cancer control (Group A, p=0.3028, log rank) or anti-CTLA4 monotherapy (Group B, p=0.9823, log rank), but these differences did not reach statistical significance. However, 28.5% of mice treated with Allocetra-OTS mono treatment survived and were tumor free (cure) at the end of experiment (day 95), compared to 0% in Groups A, and B.

[00524] Notably, combined treatment with anti-CTLA4 and 20x10^{^6} Allocetra-OTS, liquid formulation (Group D), not only significantly increased survival in comparison to the cancer control mice (Group A, p<0.0001, log rank), but also significantly increased survival in comparison to anti-CTLA4 monotreatment (Group B, p=0.0008, log rank) and Allocetra- OTS mono treatment (Group C, p=0.0064, log rank). 89% of mice treated with combined therapy of 20x10^{^6} Allocetra-OTS (liquid formulation) and anti-CTLA4 (Group D) survived and were tumor free at the end of experiment (day 95), while one mouse died at day 88 with no signs of tumor.

[00525] Similarly, combined treatment of anti-CTLA4 and 5x10^{^6} Allocetra-OTS (25% of the first dose), liquid formulation (Group E), not only significantly increased survival in comparison to cancer control (Group A, p=0.001, log rank) but also significantly increased survival in comparison to anti-CTLA4 monotreatment (Group B, p=0.0213, log rank) and Allocetra-OTS monotreatment (Group C, p=0.07, log rank). 66.6% of mice treated with combined therapy with 5x10^{^6} Allocetra-OTS (liquid) (Group E) survived and were tumor- free at the end of experiment (day 95).

[00526] Similarly, combined treatment of anti-CTLA4 and 20x10^{^6} Allocetra-OTS, frozen formulation (Group F), not only significantly increased survival in comparison to cancer control (Group A, p=0.0009, log rank) but also significantly increased survival in comparison to anti-CTLA4 monotreatment (Group B, p=0.0091, log rank) and Allocetra- OTS monotreatment (Group C, p=0.036, log rank). 75% of mice treated with combined therapy with 20x10^{^6} Allocetra-OTS (frozen) (Group F) survived and were tumor-free at the end of experiment (day 95).

[00527] Combined treatment with 20x10^{^6} Allocetra-OTS (Group D) cells had better survival rate than those treated with 5x10^{^6} cells (Group E), but the difference did not reach statistical significance (p=0.23, log rank).

[00528] No substantial differences were seen between combined treatment with liquid and combined treatment with frozen formulation (20x10^{^6}) (Group D and F, respectively) (p=0.4197, log rank), although the liquid formulation had a slight advantage.

[00529] These results show that the combined therapy provided a synergistic beneficial effect on life expectancy (survival).

[00530] Similarly, combined treatment of anti-CTLA4 and 20x10^{^6} Allocetra-OTS, frozen formulation (Group F), not only significantly increased survival in comparison to cancer control (Group A, p=0.0009, log rank) but also significantly increased survival in comparison to anti-CTLA4 mono treatment (Group B, p=0.0091, log rank) and Allocetra- OTS mono treatment (Group C, p=0.036, log rank). 75% of mice treated with combined therapy with 20x10^{^6} Allocetra-OTS (Frozen formulation) (Group F) survived and were tumor-free at the end of experiment (day 95). These results showed that the combined therapy utilizing a frozen Allocetra formulation also provided a synergistic beneficial effect on life expectancy (survival).

[00531 ] Clinical score

[00532] Mice were assigned clinical scores daily, based on mice appearance, activity, response to stimulus, respiration quality, posture, and abdominal swelling (Table 10).

[00533] In-vivo 136

[00534] Mean (Figure 7A) and median (Figure 7B) clinical score are presented. Mice that were sacrificed or died, received a maximal clinical score of 15. Once no living mice were included in the group, the plotted mean or median was halted.

[00535] The clinical scores for all AB 12-luc cancer control (Group B) and all anti-CTLA- 4 monotherapy-treated mice (Group C) clinical score gradually increased until death. The pattern was similar with an interval of several days. However, all mice treated with Allocetra-OTS monotherapy (Group D) had low clinical scores up to day 69, when their clinical scores rapidly increased. This may indicate a favorable effect of Allocetra-OTS monotherapy when compared to anti-CTLA4 monotherapy.

[00536] Combined treatment with anti-CTLA4 and Allocetra-OTS (Groups E and F) resulted in a much lower clinical score compared to cancer control and monotherapies.

[00537] Of note, in the combined group there was a large SE or STD because some of the mice were cured and received clinical scores of 0, while the clinical scores of other mice in the group were elevated. These results correlated with the success of treatment.

[00538] In-vivo 137

[00539] The clinical scores were correlated with survival results.

[00540] Mean (Figure 8A) and median (Figure 8B) clinical scores are presented. Mice that were sacrificed or died received a maximal clinical score of 15. Once no living mice were included in the group, the plots were stopped.

[00541] The mean and median group clinical scores varied significantly between the AB 12-luc cancer control group (Group A), the anti-CTLA4 (Group B) and Allocetra-OTS (Group C) monotherapy groups, and the combined-therapy groups (D-F). There was a steady increase in clinical scores of the AB 12 control mice (Group A) compared with the Allocetra-OTS monotherapy (Group B) and anti-CTLA4 monotherapy (Group C) groups. Combined anti-CTLA4 and Allocetra-OTS therapy (Groups D-F), however, resulted in very low clinical score from the beginning of the experiment. [00542] Of note, in the AB12-luc cancer control (Group A) and monotherapy groups (B-C) there were large SE or STD due to variability in the course of disease progression, while in combined the therapy groups (D-F) the SE and STD were lower since most mice were cured (score 0).

[00543] Abdominal circumference

[00544] In-vivo 136.

[00545] Since abdominal swelling due to edema is a prime parameter of wellbeing in this model, abdominal circumference was measured and calculated once a week starting on day 35 and twice a week from day 54. Abdominal circumference was measured using two methods: 1. Measuring tape (Figure 9 lower panel), and 2. Measurements of width and height using a caliper and calculating circumference using the ellipse circumference formula (according Sapi j. et al. PLOS ONE 2015; 10(11)) (Figure

9, upper panel). Abdominal size was substantially greater for mice in the AB12-luc cancer control (Group B), and anti-CTLA4 monotherapy (Group C) groups compared to those in the Allocetra-OTS monotherapy (Group D) and combined anti-CTLA4 and Allocetra-OTS therapy (Groups E and F) groups. Results obtained from the two methods were comparable in trend. Furthermore, the Pearson correlation coefficient found there was a high correlation between the methods as well as between abdomen size and clinical score (>0.7; p<0.0001). [00546] In-vivo 137.

[00547] Since abdominal swelling due to edema is a prime parameter of mice wellbeing in this model, abdominal circumference was measured and calculated once a week starting at day 27 and then twice a week from day 33. Abdominal circumference was measured by two methods: 1. measuring tape (Figure 10A), 2. measurements of width and height using a caliper and circumference calculation using the ellipse circumference formula (Sapi j. et al. PLOS ONE 2015; 10(11)) (Figure 10B). At

the beginning of the experiment, results from the two methods had different absolute numbers, but the trend was similar. From day 39 the measurements were similar with both methods since we improved and adjusted our technique for caliper measurements. On day 71 the measurements were made using only the caliper method due to similar findings with the methods. [00548] As in clinical score, abdominal circumference was substantially greater in the AB 12-luc control mice (Group A) compared to mice in the anti-CTLA4 monotherapy group (B), the Allocetra-OTS monotherapy group (C), and the combined therapy groups (D-F).

[00549] Luminescence imaging - IVIS

[00550] In vivo 136

[00551] Mice were monitored by IVIS at days 6, 11, 18, 25, 34, 48, 62, and 74. A solid luminescence signal can be seen as early as day 6 (Data not shown). On day 11, the mice were regrouped according to their IVIS signal for homogeneous spreading before treatment. Figure 11A shows the IVIS signal at day 11, 1 day before treatment, and at day 25, 3 days after the end of treatment. The luminescence signal was increased at day 25 in all mice from the AB 12-luc cancer control group (B), whereas in other treated groups (C-F), a substantial decrease in the luminescence signal at day 25 was observed in many of the mice. Of note, in some mice from treatment groups (C-F), the signal was relatively high at day 11, and did not show a decrease.

[00552] On day 74 after tumor inoculation, combined treatment of anti-CTLA4 and Allocetra-OTS with the liquid formulation (Group E) resulted in 75% survival, and at day 83 (the end of experiment) there was 66% survival. On IVIS of the mice in Group E, there was no luminescence signal at day 74 in 5 mice, one mouse had luminescence signal, and 2 mice had died (Figure 11B).

[00553] On day 74 after tumor inoculation, combined treatment of anti-CTLA4 and Allocetra-OTS with the frozen formulation (Group F) resulted in 50% survival. On IVIS there was no luminescence signal in any of the 4/8 surviving mice (Figure 11A).

[00554] All mice who received anti-CTLA-4 monotherapy treatment (Group C) died prior to IVIS examination (0% survival). However, 25% of the mice treated with Allocetra-OTS monotherapy (Group D) survived (2 mice). Both surviving mice showed abdominal swelling, but only one of them showed a luminescence signal on IVIS examination on day 74 (Figure 11A).

[00555] Figure 11B shows a graphic presentation of total flux (photons/sec) in IVIS signals for the entire experiment. Results are presented as group median ± IQR. The findings are consistent with previous results, an increase in the luminescence signal in mice in the AB 12-luc cancer control group (Group B); a signal that remained the same in the anti- CTLA4 monotherapy-treated group (Group C), and decreased signal in response to combined treatment of Allocetra-OTS and anti-CTLA4 (liquid and frozen formulation, groups E and F, respectively).

[00556] IVIS signals were generally a good indicator of tumor progression, but were not continuously accurate across all time points. For example, tumor signal had decreased between days 6 and 11 before any treatment in some groups. In addition, some mice presented low or basal levels of luminescence even when their abdomens were enlarged (see clinical scores), which indicates obvious tumor accumulation. For that reason, only the combination of the clinical score, IVIS findings, and survival were compatible with tumor progression. Possible reasons for variability in bioluminescence imaging of mice at advanced stages of tumor growth include:

[00557] Silencing of the Luc gene; and

[00558] Masking of the luminescent signal by increasing tissue formed between the signal source and the camera.

[00559] In vivo 137

[00560] Mice were monitored by IVIS (Perkin-Elmer, Lumina III) on days 6, 11, 18, 25, 39, 53, 67, and 81 (Figures 12A-12H). Solid luminescence signal can be seen as early as day 6 (Figure 12A). Figure 12B shows an IVIS signal at day 11, a day before treatment, and at day 25, 3 days after the end of treatment.

[00561] The luminescence signal was increased in all mice from AB 12-luc cancer control group (A), whereas we observed a substantial decrease in luminescence signals in many of mice in the treated groups (B-F). Of note, in some mice from treatment groups (B-F), in which the signal was relatively high at day 11, there was no signal decrease and was even an increase.

[00562] As of day 81, after tumor inoculation, all mice from the AB 12-luc cancer control (Group A) had died prior to IVIS examination (0% survival). There was 25% survival in the anti-CTLA4 (Group B) and Allocetra-OTS (Group C) monotherapy groups, although only 12.5% survival in the anti-CTLA4 monotherapy group at the end of the study period. IVIS results for both groups did not show luminescence signals in the surviving mice (Figure 12H).

[00563] Survival in groups receiving combined treatment with anti-CTLA4 and Allocetra- OTS () was 100%, 66.6%, and 75% survival, respectively, for groups D, E, and F (20x10^{^6}, liquid formulation, Group D; 5x10^{^6}, liquid formulation, Group E; and 20x10^{^6}, frozen formulation, Group F, respectively) at day 81, with no luminescence signal in any of the surviving mice (Figure 12H).

[00564] Figure 13B shows a graphic presentation of total flux (photon/sec) of IVIS signals for the entire experiment. Results are presented as group median ± IQR. Corelated with previous results the luminescence signal was substantially lower in combined treatment groups (group D-F) and in the anti-CTLA4 monotherapy group (Group B), compared to the AB12-luc cancer control group (A) and Allocetra-OTS monotherapy group (Group C), regardless of Allocetra-OTS concentrations that were examined (5x10^{^6} or 20x10^{^6} cells/mouse) or Allocetra formulation (liquid or frozen).

[00565] Due to signal spillover, the following mice were excluded from analysis: A6 and A8 at day 11, and A6, A8, C2, C4, E1, and E3 at day 18. Signal spillovers occur when adjacent mice have high signal. This problem was solved from day 25 by adding dividers between mice.

[00566] IVIS signals were generally a good indicator of tumor progression but were not continuously accurate across time. For example, tumor signal had decreased between days 6 and 11 before any treatment in some groups. In addition, some mice presented low or basal levels of luminescence in spite of an enlarged abdominal circumference (see clinical score), which indicates obvious tumor accumulation. For that reason, only the combination of clinical score, IVIS, and survival were compatible with tumor progression. Possible variability in the findings from bio-luminescence imaging of mice in advanced stages of tumor growth include:

[00567] Silencing of the Luc gene; and

[00568] Masking of the luminescent signal by the increasing tissue formed between the signal source and the camera.

[00569] In vivo 136 and In vivo 137

[00570] Figures 13A and 13B show graphic presentation of total flux (photon/ sec) of IVIS signals for entire experiments (Figure 13A for 136 and Figure 13B for 137), results are presented as group median ± IQR. Signal in AB 12-luc control group had slightly elevated over time for both experiments. Combined treatment for anti-CTLA4 and Allocetra-OTS substantially decreased luminescence signaling, regardless of Allocetra-OTS concentrations that were examined.

[00571] IVIS signals were generally good indicators of tumor progression but were not continuously accurate across time point. For example, tumor signal had decreased between day 6 and 11 before any treatment in some groups. In addition, some mice presented low or basal levels of luminescence while presenting enlarged abdomen (see clinical score) which indicates obvious tumor accumulation. For that reason, only the combination of clinical score, IVIS and survival were compatible with tumor progression. Possible variability in bio-Luminescence imaging of mice in advanced stages of tumor growth include a “Silencing of the Luc gene” and or “Masking of the luminescent signal by the increasing tissue formed between the signal source and the camera”.

[00572] Clinical score as a predictor of mortality

[00573] In vivo 136

[00574] The non-treated, AB 12 tumor-bearing mice, were daily assigned with a clinical score, and retrospectively analyzed for mortality outcome (death within 5 days or 10 days) at each timepoint. One hundred and fourteen (114) data-points were analyzed, from 8 mice. The clinical score was evaluated as a predictor of 5 or 10-day mortality using a simple (univariate) logistic regression (Analysis with GraphPad Prism 9.3). The clinical score strongly predicted mortality with AUCs of ROC of 0.9437 (95% CI: 0.9119-0.9827) for 5- days mortality, and 0.9230 (95% CI: 0.8747-0.9714) for 10-days mortality (p < 0.0001, Figure 14A). The Best-fit values (Clinical score at which probability = 50%) for 5-days mortality and 10-days mortality were 8.8 ± 0.5 and 4.6 ± 0.4, respectively (Figure 14B). TB strengthen this analysis, more data were added (non-treated, AB 12 tumor-bearing mice from In-vivo 137); analysis of 218 data-points from 16 mice reinforced the above results, with AUCs of ROC of 0.9543 (95% CI: 0.9303-0.9784) for 5-days mortality, and 0.9512 (95% CI: 0.9246-0.9777) for 10-days mortality (p < 0.0001, Figure 14C). The Best-fit values of this analysis for 5-days mortality and 10-days mortality were 8.9 ± 0.4 and 4.2 ± 0.3, respectively (Figure 14D).

[00575] Summary and Conclusion:

[00576] Preliminary results showed significant survival therapeutic effect of anti-CTLA4 and Allocetra-OTS combined treatment in BALB/c mice AB 12 mesothelioma model.

[00577] AB 12 mesothelioma cancer cells spread in mice abdomen and lead to death mainly between day 30 to day 40. Mice developed ascites and massive tumors in the peritoneal cavity. Most of the mice had evidence of peritoneal tumor, by IVIS analysis, as seen at day 6. [00578] Anti-CTA4 treatment alone improved survival but only by prolongation and did not prevent mortality. Allocetra-OTS by itself was better than anti-CTLA-4 alone and prevented death in about 40% of mice. Combined treatment of anti-CTLA4 and Allocetra- OTS had a synergistic effect and significantly improved clinical wellbeing and survival of the mice.

EXAMPLE 5: Testing the efficacy of combination therapy with anti-PD-1 and Allocetra in the MC38 syngeneic model.

[00579] Objective: To evaluate the efficacy of Allocetra and Anti-PDl treatment on MC38 tumor cells when the Allocetra is administered by different routes of administration, Intraperitoneal (IP), Intravenous (IV) and subcutaneous (SC).

[00580] Methods'.

[00581] Mice

[00582] Female C57/B1 mice (Evigo CRS (Israel) LTD), aged approximately 8 weeks were used for this study. Water and food were supplied ad libitum during the study period.

[00583] Model Induction

[00584] MC38 cells were injected subcutaneous (SC) to a flank of each mouse according to Table 13:

[00585] Table 13: Model Induction

[00586] Before treatment initiation, animals were assigned into treatment groups based on their tumor size, creating similar tumor size distribution between groups.

[00587] Test Items Administered

[00588] Group Administration is as shown in Table 14.

[00589] Table 14: Test items administration

[00590] Intravenous (IV), Intraperitoneal (IP) and subcutaneous (SC) administration equipment: Standard 27-gauge syringe, 1ml. Treatments began 9 days after subcutaneous engraftment of the tumor cells when palpable tumors reach a volume of 50-80 mm³.

[00591] Allocetra (Fresh Formulation 3 x 10^A7/dose) and Anti-PDl (40 mg/kg/dose; antimouse CD279 Cat. 114122 Ultra-Leaf™ purified) were given together on the same day, for a total of 5 injections every 4 days. The term “Fresh Formulation” may be used interchangeably with the term “Liquid Formulation” and have the same qualities and meanings.

[00592] A cohort of 50 female mice of C57BL/6J01aHsd strain were designated to 5 different treatment groups according to Table 5Y.

[00593] All treatments started when tumors reached approximately 50 mm3 (day 9) and were given once every 4 days (for a total of 4 treatments).

[00594] Tumor volume

[00595] Tumor volume measurements were taken for each animal three times a week, by measuring tumor length (L) and width (W). Measuring was performed 3 times a week, using a calibrated caliper (0.00 mm).

[00596] The volume was calculated according to the following formula: V = (L x W x W)/2.

[00597] Body weight [00598] Animals were weighed at the following time points:

[00599] Three (3) times a week during the study period and on termination.

[00600] Statistical Analysis

[00601] Statistical analysis was performed using SPSS V 25. Data were evaluated for normality and equality of variance between tested groups. Numerical results were presented as means and standard errors (SEM). Descriptive statistics and group comparisons were performed. The appropriate parametric or non-parametric statistical tests were applied (General Linear Model, repeated measures ANOVA following BONFERRONI, POSTHOC test).

[00602] *A p-value equal to or less than 0.05 is regarded as statistically significant.

[00603] Results'.

[00604] Body weight

[00605] Repeated measures ANOVA revealed no significant changes in body weight. The test was designed to measure the difference between the 5 treatment groups following 11 measurement time points (Figure 15).

[00606] Tumor volume

[00607] Repeated measures ANOVA reveled a significant change in tumor volume was observed between the vehicle group and 3 of the treatment groups; the only exception was group 2 (Anti-PD-1 only), p=0.175 // group 3 (Allocetra IP + Anti-PD-1), p=0.019, group 4 (Allocetra IV + Anti-PD-1), p=0.007, group 5 (Allocetra SC + Anti-PD-1), p=0.008). The test was designed to measure the difference between the 5 treatment groups following the measurement of 10 time points (Figure 15).

[00608] Tumor weight

[00609] One way ANOVA revealed no significant changes in tumor weight. The test was designed to measure the difference between the 5 treatment groups during the termination time point (Figure 16).

[00610] Conclusions :

[00611] Overall, the combination of Allocetra cells and Anti-PDl treatments restricted tumor's growth (volume) as compared to Anti-PDl treatment and the Vehicle group (Figure 15). However, no significant changes were found comparing routes of administration, as observed with groups 3, 4, and 5 (IP, IV and SC routes of administration).

[00612] No significant changes were found in the tumor' s weight. This might be due to a gap between the final tumor's measurements and the termination time point (Figure 17; a total of 7 days). In addition, one must take in to consider the physiology and morphology of tumor cells, tumors might keep their length and width the same but lose inside mass due to cell necrotic death.

[00613] Regarding body weight, repeated measures ANOVA revealed no significant change; there was no significant decrease or increase in body weight during the experiment. This is a good clinical sign and demonstrates the homogeneity between all 5 treatment groups in their body weight (Figure 15).

EXAMPLE 6: Testing the efficacy of combination therapy with anti-PD-1 and Allocetra in the ID-8 Ovarian Cancer syngeneic mouse model.

[00614] Objective-. To evaluate the efficacy of a combination therapy comprising Allocetra (apoptotic cells) and Anti-PDl antibody (Ab) treatment in an ID-8 syngeneic model, when the Allocetra is administered by different routes of administration, Intraperitoneal (IP), Intravenous (IV) and subcutaneous (SC).

[00615] Methods'.

[00616] Mice

[00617] Female albino C5/B1/6 mice were used for this study. Water and food were supplied ad libitum during the study period.

[00618] Model Induction

[00619] ID*-Luc clone #9 cells were injected subcutaneous (SC) to a flank of each mouse according to Table 15:

[00620] Table 15: Model Induction

[00621] Ten million ID-8 Luc cells in a 100 ul volume were implanted into the intraperitoneal cavity of ninety 8-week-old, female C57BL/6 Albino wildtype mice. One week after implantation, the mice were imaged for bioluminescence after intraperitoneal injection of 600 ugs of Luciferin in a 200 ul volume. Seventy of these mice were selected and allocated into the 7 experiment groups after visual confirmation of BLI signal. Mice with low or absent signals were not included in the study. Treatment of the mice with Allocetra and anti PD-1 Ab began on this day (Day 0). Thereafter, the mice were imaged every seven days and their body weight was recorded on the same day. Occasionally, imaging was carried out over a span of 2-3 days (due to logistical and instrument/staff availability reasons).

[00622] Test Items Administered

[00623] Mice were distributed between the 7 test groups, wherein group Administration is as shown in Table 16. Administration of anti-PD-1 Ab (aPD-1) and Allocetra was on days 7, 10, 14, and 18 counting from day 0 of model induction (day 0 = the day ID*-Luc clone #9 cells were injected)

[00624] Table 16: Test items administration

* Intravenous (IV), Intraperitoneal (IP), and subcutaneous (SC) administration equipment. [00625] Anti-PD-1 was administered at a dose of 1.25 mg/kg. Allocetra was administered at doses of 2xlO^A7 or lxlO^A7.

[00626] Tumor volume:

[00627] Tumor burden quantified by bioluminescent imaging once a week.

[00628] Body weight:

[00629] Recorded once a week.

[00630] Results'.

[00631] In these experiments, red is the vehicle and green is the anti PD-1 Ab alone, which act as the baseline for this experiment. Twenty million (20M) cells of Allocetra™ (orange) were injected intraperitoneally (IP) alone showing a strong anti-tumoral effect as monotherapy. Twenty million (20M) cells of Allocetra™ were injected (IP) in combination with anti PD-1 Ab (dark purple). Ten million (10M; Low dose) cells of Allocetra™ were injected (IP) in combination with anti PD-1 Ab (pink). Twenty million (20M) cells of Allocetra™ frozen formulation were injected (IP) in combination with anti PD-1 Ab (turquoise). Twenty million (20M) cells of Allocetra™ injected IV in combination with anti PD-1 Ab (blue). At week 12, one animal with a high tumor burden showed ascites resulting in a very low signal. (Figure 23)

[00632] Figures 18, 19, 20A and 20B, and 21A and 21B provide the preliminary results from this study. Figure 19 graphically summarizes the Bio-Luminescence Imaging results shown in Figure 18. The graphs of Figure 19 clearly show that while (1) the monotherapies (Allocetra alone or anti-PD-1 Ab alone) were more effective than no treatment at all, (2) administration of Allocetra by IV in combination with anti-PD-1 Ab administration (IP) had an effective synergistic effect, and (3) there was a dramatic synergistic effect when Allocetra was administered intraperitoneally (IP) in combination with anti-PD Ab administration (IP). The data of Figures 20A-20B and Figures 21A-21B support this finding, as do the survival curves presented in Figures 22A and 22B.

[00633] The Kaplan-Meier Medium Survival Charts shown in Figures 22A and 22B, with treatment of Allocetra™ shows a median survival of vehicle in red at 6 weeks from start of the experiment as baseline; treatment with anti PD-1 median survival is at 8.5 weeks, treatment with Allocetra™ alone in orange line showed a median survival of 9.5 weeks; in purple line treatment of Allocetra™ in combination with anti PD-1 showed a statistically significant increase of median survival to 11 weeks with a P value < 0.01 vs. a 6-week median survival with the vehicle alone; with half the dose of Allocetra™ in pink line the median survival is 10.5 weeks and also statistically significant; the frozen formulation of Allocetra™ showed an identical median survival of 11 weeks as the liquid formulation and also statistically significant. Finally, the IV administration showed an 8.5-week medial survival, but it was not statistically significant.

[00634] Figure 23 shows that the mice in the bottom left with very high tumor burden at week 11, showed a remarkable body weight gain at week 12, from 23.4 gm to 25.1 gm, confirming a case of ascites, abnormal build-up of fluid in the abdomen. It is known that when ascites happens, the signal goes down.

[00635] Conclusion at 12-week time point (Data not shown):

[00636] The ID8-L model is the flagship model to show strong synergistic effects of Allocetra™ when combining with Check Point Inhibitors. The imaging results at Week- 12, combining 10 million cells or 20 million cells of Allocetra™ with 1.5mg/kg anti-PDl Ab at 11-weeks, across 7 arms (See Table 15 Above) clearly show strong synergistic effect at week-12 with a potential 50% cure using either the Frozen or Liquid Allocetra formulations. [00637] The Kaplan-Meier Medium Survival Charts with treatment of Allocetra™ in combination with Checkpoint inhibition showed a potent synergistic effect with statistically significant increase of median survival of 11 weeks with a P value < 0,01 vs. a 6-week median survival without treatment.

[00638] Summary of Results from Examples 3-5

[00639] These three experiments showed significant effects of monotherapy with Allocetra in a peritoneal solid tumor model. Surprisingly, the effect was synergistically enhanced significant when treatment was in combination with an immune checkpoint inhibitor, for example with an anti-CTLA4 Ab in Example 4 (treating mesothelioma (AB 12) in syngeneic Balb/c); and with an anti-PD-1 Ab in Example 6 (treating ovarian cancer). Additionally, use of a Frozen formulation of Allocetra to treat solid cancers showed a significant effect.

EXAMPLE 7: Effect of anti-PD-1 and Allocetra-OTS on tumor progression in AB-12 mouse mesothelioma model [00640] Malignant mesothelioma is an aggressive tumor arising from the cells lining the pleural, peritoneal, and pericardial cavity. This tumor is highly resistant to current conventional therapies.

[00641] PD-1 (programed cell death protein 1) is a member of the immunoglobulin superfamily that function as an immune checkpoint that regulates T cell activation. Treatment of human mesothelioma with anti-PD-1 to boost generation and activation of T cells, have thus far shown minor improvements, emphasizing the need of combined treatments. Since apoptotic cells harbor an immune- modulatory potential, a combined therapy with anti-PD-1 has a promising therapeutic potential.

[00642] In previous experiments shown in Example 4 above, significant therapeutic effect of Allocetra-OTS alone and in combination with anti-CTLA4 in BALB/c mice AB 12 mesothelioma model was demonstrated.

[00643] In the current experiment the effect of standalone (monotherapy) and combined treatment of Allocetra-OTS and anti-PD-1 was tested in a BALB/c AB12-luc mesothelioma model on tumor progression and mice survival.

[00644] Objective-. To test the effect of different doses of standalone therapy (monotherapy) of anti-PDl or Allocetra-OTS (Frozen Drug Product (FDP)), and combined therapy using Allocetra-OTS and anti- PD-1, in a AB12-luc BALB/c mesothelioma model. [00645] Methods'.

[00646] The study design used is presented below in Tables 17A and 17B, and a schematic of the mesothelioma syngeneic AB 12 model in BALB/c mice is shown in Figure 24. The study was run for 83 days.

[00647] Table 17A: Study design

[00648] Table 17B: Dosages and Products

[00649] Animals and animal facility

[00650] BALB/c female mice, 7 weeks old, were purchased from Envigo. Mice were kept in an SPF free animal facility in compliance with institutional International Animal Care and Use Committee (IACUC) Guidelines. Mice were monitored daily and assigned a clinical score according to the abdominal tumors scoring table (Table 10), according to Paster EV, et al. Endpoints for mouse abdominal tumor models: refinement of current criteria. Comp Med. 2009;59(3):234-241; Penn Animal Welfare).

[00651] AB12 mesothelioma model

[00652] The scheme of the model is shown in Figure 24. Mice received 0.1x10⁶ luciferase-expressing AB 12 tumor cells (AB12-luc) on day 0 (IP, 0.2ml/mouse in PBS). On days 12, 15, 19, and 22, mice were treated IP with 125μg or 250μg anti-PD-1 (BioXcell, BE0146, Lot- 810421S1; InVivoMAb anti-mouse PD-1 (CD279) clone RMP1-14) (0.2ml/ mouse in PBS) and/or 5x10⁶ or 20x10⁶ Allocetra-OTS cells (FDP) (0.2ml/ mouse, in Hartmann solution) at a 1-hour interval. Mice were monitored by IVIS imaging for tumor progression. On day 12, prior to treatment injections, mice were regrouped to enable homogeneous spreading of tumor cells, according to day 11 IVIS results.

[00653] Allocetra-OTS [00654] Allocetra-OTS batch number FDP-21-017 was used in this experiment (FDP). FDP was manufactured about 8 months prior to the start of these experiments, frozen and then thawed on the days of injection in a 37°C water bath.

[00655] Prior to injection, Allocetra-OTS cells were filtered through a 200pm filter (pluriSelect, 43-50200-01) and centrifuged at 290g, for 10 min at RT. Cells were then resuspended with Hartmann solution and counted by Trypan blue (TB) exclusion using light microscopy (injected cell numbers were calculated based on viable cells only, therefore total number of injected cells were 22x10⁶. (See Table 19: Assessment of schedule Summary). Cells were diluted to injected concentration and filtered again through a 200pm filter. Allocetra-OTS cells were injected (0.2ml/ mouse) within 1-1.5h from thawing. Allocetra- OTS AnPI and Trypan blue viability parameters are presented in Table 18.

[00656] Table 18: Injected Allocetra-OTS viability parameters.

[00657] Table 19: Assessment schedule summary

[00658] Weight

[00659] Mice were weighed once a week starting at day 7.

[00660] Clinical score

[00661] The murine clinical score was evaluated daily starting at day 8 according to sacrifice criteria in a peritoneal tumor model (Table 10 above). Briefly, each animal was individually evaluated on 6 parameters: Appearance (i.e., coat piloerection and mouse emaciation), level of activity, response to stimulus, respiration quality, posture, and abdominal swelling (ascites). Each parameter was given a score between 0-4 and the total score was calculated. Animals were sacrificed upon reaching a total score of 15, a score of 3 in the “respiration quality” category, or a maximal score in any other category.

[00662] Survival

[00663] Mice were monitored daily for wellbeing and clinical scores. Mice were sacrificed when reaching score 15. Survival rate was determined by reaching score 15 or occasionally spontaneous death.

[00664] In vivo imaging (IVIS)

[00665] Mice were monitored every other week for tumor imaging by IVIS (Perkin- Elmer, Lumina III). Ten (10) minutes before imaging, mice were injected IP with luciferin solution (150mg/kg, Perkin-Elmer, 122799). Seven (7) minutes after injection, they were anesthetized by isoflurane in an induction chamber. The mice were placed in a supine position (anterior facing upwards) inside the IVIS chamber. Ten (10) minutes after luciferin injection the mice were imaged (exposure: 10 sec, F-stop: 2, Binning: medium). Analysis was performed by Living Image® software (Perkin Elmer, version 4.5.5) and total flux (photon/sec) per time point was measured.

[00666] Characterization of peritoneal and spleen immune cells

[00667] Six (6) mice that were sacrificed on day 83 (end of experiment) were collected for characterization of immune cells. Peritoneal cells and splenocytes were isolated and tested using the macrophage panel - CD19, CD11c, CD1 lb, F4/80, and MHC-II, and the T cell panel - CD4, CD8, CD25, and Foxp3. Sacrificed mice were from 3 experimental groups - Allocetra-OTS standalone therapy, 20x10⁶ (n=l), combined therapy of anti-PD-1 and Allocetra-OTS (20x10⁶, n=4; 5x10⁶, n=l). These data were sent for analysis.

[00668] Pathology

[00669] Upon sacrifice due to clinical score (reaching sacrificing parameters) and at end of experiment, Spleen, liver, and tumor samples were collected for pathology evaluation (hematoxylin and eosin [H&E] staining). This data was sent for pathological evaluation and is not included in this report. Will be provided as an addendum to the final report, once available.

[00670] Statistics [00671] Survival analysis was performed according to the Kaplan-Meier estimator. The log-rank statistical test was calculated. All statistical analyses were performed using GraphPad Prism software (San Diego, CA, USA).

[00672] Results

[00673] Weight

[00674] Mice weight is presented in Figure 25.

[00675] Mice weight were slightly elevated during the experiment, as expected. No substantial increase in average weight was seen.

[00676] Clinical score

[00677] Mice were assigned a clinical score each day based on their appearance, activity, response to stimulus, respiration quality, posture, and abdominal swelling (Data not shown). [00678] Mean (upper panel) and median (lower panel) clinical scores are presented in Figures 26A and 26B. Mice that were sacrificed or died, received a maximal clinical score of 15. Once no living mice were included in the group, the plotted mean or median was halted.

[00679] AB 12-luc cancer control (Group A) started presenting elevated clinical score at day 19, that rapidly increased until death/sacrifice, reaching maximal clinical score at day 44.

[00680] Anti-PD-1 standalone therapy -treated mice (Groups B & C, 250μg and 125μg, respectively) presented moderate elevation in clinical score, starting at days 22-25, that gradually increased until death/sacrifice, with the exception of 1 mouse from group B (250μg anti-PD-1) that survived the experiment. The elevation in clinical score correlated with anti-PD-1 dose, showing lower average clinical score in mice in Group B, receiving 250μg anti-PD-1.

[00681] Mice treated with Allocetra-OTS standalone therapy (Groups D, 20x10⁶ and group E, 5x10⁶) presented moderate elevation in clinical score, starting at day 22, and exhibited delayed progression of clinical score compared to AB 12 cancer control mice.

[00682] Combined treatment of anti-PD-1 (250μg) and Allocetra-OTS (Groups F, 20x10⁶ and Group G, 5x20⁶) resulted in a much lower clinical score compared to cancer control and standalone therapies (monotherapies) throughout the study. [00683] Of note, initial clinical score progression in this experiment was a bit more rapid than anticipated, with several mice presenting a clinical score of 10 as early as day 22 after AB 12 administration.

[00684] Of note there was a large SE in clinical score because some of the mice were cured and received clinical scores of 0, while the clinical scores of other mice in the group were elevated.

[00685] In vivo imaging: IVIS

[00686] Mice were monitored by IVIS (Perkin-Elmer, Lumina III) at days 6, 11, 25, 39, 53, 64, and 78. A solid luminescence signal can be seen as early as day 6 (data not shown). At day 12, the mice were regrouped according to their day 11 IVIS signal for homogeneous spreading before treatment. Figure 27 shows IVIS signal at day 11, 1 day before treatment (after regrouping), and at day 25, 3 days after the end of treatment. The luminescence signal was increased at day 25 in 5/8 mice from the AB12-luc cancer control group (Group A), with 1 animal dying before day 25 imaging, increased luminescence signal was also observed in the Allocetra-OTS standalone therapy groups (D & E), whereas in the anti-PD- 1 standalone therapy (B & C) and in the combined of Allocetra-OTS and anti-PD-1 treated groups (F & G) a substantial decrease in the luminescence signal was observed at day 25 in many of the mice. Of note, in some mice from treatment groups (B-G), the signal was relatively high at day 11, and did not show a decrease.

[00687] At day 78 after tumor inoculation, combined treatment of anti-PD- 1 and high dose Allocetra-OTS (20x10⁶ cells/mouse, Group F) resulted in 50% survival, with no luminescence signal in any of the 4/8 surviving mice (Figure 28).

[00688] At day 78 after tumor inoculation, combined treatment of anti-PD-1 and low dose Allocetra-OTS (5x10⁶ cells/mouse, Group G) resulted in 50% survival (4/8). One animal presented a substantial luminescence signal at that time but did not die before the experiment end at day 83 (score 11, Figure 28).

[00689] Mice that received standalone therapy of 250μg anti-PD-1 (Group B) had 12.5% survival (1/8), without presenting luminescence signal.

[00690] Mice that received standalone therapy 125μg anti-PD-1 (Group C) died prior to last IVIS session (0% survival).

[00691] Mice that received standalone therapy of 20x10⁶ Allocetra-OTS cells/mouse (Group D) had 12.5% survival (1/8) and presented a substantial luminescence signal but had a clinical score of 0. Clinical signs upon sacrifice et the end of experiment showed no signs of tumor in the abdomen, but the mouse had enlarged ovaries.

[00692] Mice that received standalone therapy of 5x10⁶ Allocetra-OTS cells/mouse (Group E) died prior to last IVIS session (0% survival).

[00693] Figure 29 shows a graphic presentation of total flux (photons/sec) in IVIS signals for the entire experiment. Results are presented as group median ± IQR. The findings of the AB 12-luc cancer control group (Group A) are consistent with previous results of an increase in the luminescence. In all treatment groups (B-G) luminescence was maintained at a low level or slightly decreased after treatment.

[00694] IVIS signals were generally a good indicator of tumor progression but were not continuously accurate across all time points. In addition, some mice presented low or basal levels of luminescence even when their abdomens were enlarged (see clinical score), which indicates obvious tumor accumulation. For that reason, only the combination of the clinical score, IVIS findings, and survival were compatible with tumor progression. Possible reasons for variability in bioluminescence imaging of mice were discussed previously.

[00695] Survival analysis

[00696] Kaplan-Meier survival analysis is represented in Figures 30A and 30B and descriptive statistics of average survival in Figure 31. The experiment ended on day 83.

[00697] Mice in Group A, the AB 12-luc cancer positive control group, developed elevated clinical score as well as IVIS signal as expected, and died between days 24 and 44 (0% survival, n=8), with mean survival of 29.9 ± 2.1 days.

[00698] Mice in Group B, the AB 12-luc mice treated with 250μg anti-PD-1 standalone therapy, died between days 29 and 63, with 1/8 animal (12.5%) surviving at end of experiment (day 83). Mean survival of the group was 48.0 ± 7.1 days. p=0.0021, log rank, compared to cancer control group.

[00699] Mice in Group C, which the AB 12-luc mice treated with 125μg anti-PD-1 standalone therapy, died between days 31 and 67 (0% survival, n=8), with mean survival of 42.75± 5.0 days. p=0.0072, log rank, compared to cancer control group.

[00700] Mice in Group D, the AB 12-luc mice treated with 20x10⁶ Allocetra-OTS standalone therapy, died between days 27 and 54, with 1/8 (12.5%) animal surviving at end of experiment (day 83). Mean survival of the group was 42.1 ± 7.0 days. p=0.0409, log rank, compared to cancer control group. [00701] Mice in Group E, the AB12-luc mice treated with 5x10⁶ Allocetra-OTS standalone therapy (monotherapy), died between days 27 and 64 (0% survival, n=7), with mean survival of 38.3+ 4.9 days. p=0.1083, log rank, compared to cancer control group.

[00702] Mice in Group F, the AB12-luc mice treated with combined therapy of anti-PD- 1 and 20x10⁶ Allocetra-OTS, (combination therapy, 20x10⁶) had 50% survival; 4/8 mice died between days 27 and 67. 4 mice were healthy at the end of experiment (score 0). Mean survival was 64.25 + 8.5 days. p=0.0026, log rank, compared to cancer control group.

[00703] Mice in Group G, the AB12-luc mice treated with combined therapy anti-PD-1 and 5x10⁶ Allocetra-OTS, (combination therapy, 5x10⁶) had 50% survival; 4/8 mice died between days 26 and 63. Four (4) mice were still alive at end of experiment, 3 mice were healthy (score 0) and 1 mouse exhibited advanced disease as expressed by clinical score 11. Mean survival was 61.9 + 8.8 days. p=0.0040, log rank, compared to cancer control group.

[00704] Taken together, anti-PD-1 standalone therapy (Groups B & C; monotherapies) significantly increased mice survival compared to cancer control group (Group B, p=0.0021. Group C, p=0.0072, log rank). No statistical difference was observed between the two doses of anti-PD-1 standalone therapy groups (p=0.7592, log rank).

[00705] Standalone therapy with high dose of Allocetra-OTS (20x10⁶, Group D; monotherapy) significantly increased survival compared to cancer control group (Group A, p=0.0409, log rank). Allocetra-OTS standalone therapy (20x10⁶, Group D) was comparable to anti-PD-1 standalone therapies of 250μg (Group B, p=0.4654, log rank) or 125μg (Group C, p=0.6892, log rank).

[00706] Standalone therapy with low dose of Allocetra-OTS (5x10⁶, Group E; monotherapy) increased survival, but with no statistical significance, compared to cancer control group (Group A, p=0.1083, log rank), showing dose-dependency of the effect, although no statistical difference was observed between the Allocetra-OTS standalone therapy groups (p=0.6651, log rank). Allocetra-OTS standalone therapy (5x10⁶, Group E) was comparable to anti-PD-1 standalone therapies of 250μg (Group B, p=02201, log rank) or 125μg (Group C, p=0.3452, log rank).

[00707] Combined therapy of anti-PD-1 (250μg) and Allocetra-OTS 20x10⁶ or 5x10⁶ resulted each with 50% survival (p=0.9644, log rank).

[00708] Combined therapy of anti-PD-1 (250μg) and 20x10⁶ Allocetra-OTS significantly increased mice survival compared to AB12-luc cancer control (Group A, p=0.0026, log rank). The combined therapy group increased survival, with borderline statistically significant result, compared to anti-PD-1 standalone therapy (250μg, Group B, p=0.1530, log rank) and compared to Allocetra-OTS standalone therapy (20x10⁶, Group D, p=0.0648, log rank).

[00709] Combined therapy of anti-PD-1 (250μg) and 5x10⁶ Allocetra-OTS significantly increased mice survival compared to AB12-luc cancer control (Group A, p=0.0040, log rank). The combined treatment significantly increased mice survival compared to Allocetra- OTS standalone therapy (5x10⁶, Group E, p-0.0384, log rank) and not significantly compared to anti-PD-1 standalone therapy (250μg, Group B, p=0.1643, log rank).

[00710] Characterization of peritoneal and spleen immune cells

[00711] Six (6) mice that were sacrificed on day 83 (end of experiment) were collected for characterization of immune cells. Peritoneal cells and splenocytes were isolated and tested using the macrophage panel - CD19, CD11c, CD1 lb, F4/80, and MHC-II, and the T cell panel - CD4, CD8, CD25, and Foxp3. Sacrificed mice were from 3 experimental groups - AllocetraOOTS standalone therapy, 20x10⁶ (n=l), combined therapy of anti-PD-1 and Allocetra-OTS (20x10⁶, n=4; 5x106, n=l). This data was sent for analysis.

[00712] Pathology

[00713] Upon sacrifice due to clinical score (reaching sacrificing parameters) and at end of experiment, spleen, liver, and tumor samples were collected for pathology evaluation (hematoxylin and eosin [H&E] staining). This data was sent for pathological evaluation.

[00714] Conclusion

[00715] AB 12-luc mesothelioma cancer cells spread in the abdomen of mice in the study and resulted in death between days 24 and 44 in untreated mice. The mice developed ascites and a massive tumor burden in the peritoneal cavity. All but three mice had peritoneal tumors already by day 6 as judged by IVIS.

[00716] Standalone (monotherapies) therapy with 125 or 250μg anti-PD-1 increased mice survival. The effect of anti-PD-1 showed slight dose tendency and administration of 250μg (IP on days 12, 15, 19, and 22) seems to be the preferred dosage to use in further experiments. [00717] Standalone (monotherapy) therapy with 5x10⁶ or 20x10⁶ Allocetra-OTS (FDP, IP on days 12, 15, 19, and 22) also increased mice survival with dose dependency. Allocetra- OTS standalone therapy was comparable to anti-PD-1 standalone therapy in survival percentage, but anti-PD-1 was slightly more effective in delaying mesothelioma symptoms and mortality deterioration as evaluated by clinical score and survival graph.

[00718] Combination therapy with both anti-PD-1 and Allocetra-OTS, either 20x10⁶ or 5x10⁶ cells/mouse, was significantly more effective (synergistic) than either standalone therapy and resulted with 50% survival at day 83, in both groups.

EXAMPLE 8: A Phase U2a Study Evaluating Allocetra-OTS as Monotherapy or in Combination with Anti-PD-1 Therapy for the Treatment of Advanced Solid Tumor Malignancy

[00719] Objective-. Despite the advent of novel targeted immune-therapeutics for the treatment of solid tumors, many patients remain without cure. Allocetra-OTS is an immunomodulatory cell-based therapy consisting of allogeneic peripheral blood mononuclear cells that have been modified (induced apoptotic cells) to be engulfed by macrophages and reprogram the macrophages into their homeostatic state. The goal of this study is to evaluate the safety and potential efficacy of escalating doses of Allocetra-OTS in the treatment of advanced solid tumor malignancy as monotherapy (Stage 1) or in combination with an anti-PD-1 therapy (Stage 2) [00720] Methods'.

[00721] Allocetra-OTS

[00722] Allocetra-OTS is a cell-based therapy consisting of non-HLA matched allogeneic peripheral blood mononuclear cells, derived from a healthy human donor following a leukapheresis procedure, induced to an apoptotic stable state. Allocetra-OTS was produced essentially as described in Example 1 above. Allocetra-OTS was administered systemically or locally (intravenous [IV] or intraperitoneal [IP]) according to the tumor location.

[00723] Study Subjects

[00724] A total of 48 subject will be enrolled in this study. Currently, 7 patients have been entered and treated in this study. Ages Eligible for Study: 18 Years and older (Adult, Older Adult). Sexes Eligible for Study: All.

[00725] Patient Criteria

[00726] Inclusion Criteria:

[00727] Patients must have histologically or cytologically confirmed locally advanced, unresectable or metastatic solid tumors, that have relapsed or have been refractory to available approved therapies, or patients who are not eligible for or declined additional standard of care systemic therapy.

[00728] Patients with peritoneal carcinomatosis can be eligible if an appropriate IP catheter or port can be placed. Peritoneal tumors or peritoneal spread (peritoneal carcinomatosis) for IP administration of Allocetra-OTS will include ovarian/fallopian tube/primary peritoneal cancer, gastric cancer, colorectal cancer, pancreatic cancer, and other rare peritoneal tumors, with no or minimal extraperitoneal disease.

[00729] Patients must have measurable disease.

[00730] Age ≥ 18 years old.

[00731] ECOG performance status <1.

[00732] Adequate renal function, hepatic function, and bone marrow function.

[00733] Exclusion Criteria:

[00734] Primary central nervous system (CNS) malignancy or CNS involvement, unless stable clinically.

[00735] Clinically significant uncontrolled infection, autoimmune or inflammatory diseases requiring systemic immunosuppression, clinically significant cardiovascular disease, severe pulmonary diseases or additional malignancies.

[00736] [For patients in Stage 2] Patients who previously experienced an ICI-related adverse reaction that resulted in discontinuation of the ICI.

[00737] Dosages and Dose Regimen

[00738] Experimental: Stage 1 (Allocetra-OTS monotherapy)

[00739] Dose escalation of Allocetra-OTS up to 10 x 10^A9 cells by IV or IP administration. Dose escalation of Allocetra-OTS includes a lower dose of 2.5 x 10^9 Allocetra-OTS cells (Cohort 1; IV administration, Cohort 3; IP administration), and a higher dose of 10 x 10^A9 Allocetra-OTS cells (Cohort 2; IV administration, Cohort 4; IP administration).

[00740] Experimental: Stage 2 (Allocetra-OTS in combination with anti-PD-1 therapy) [00741] Dose escalation of Allocetra-OTS up to 10 x 10^A9 cells by IV or IP administration, with IV nivolumab 240 mg. Dose escalation of Allocetra-OTS includes a lower dose of 2.5 x 10^9 Allocetra-OTS cells, and may include a higher dose of 10 x 10^9 Allocetra-OTS cells, wherein treatment includes IV administration of nivolumab (240mg). [00742] Outcome Measures [00743] Primary Outcome Measures :

[00744] Safety of Allocetra-OTS [Time Frame: 3-5 weeks ]

[00745] Characterize the safety of Allocetra-OTS based on the dose-limiting toxicities (DLTs) of Allocetra-OTS as monotherapy or in combination with anti-PDl therapy.

[00746] Secondary Outcome Measures :

[00747] Overall Response Rate (ORR)/Best Overall Response Rate (BORR) [Time Frame: 12 months]

[00748] Overall Response Rate (ORR)/Best Overall Response Rate (BORR) (percentage of patients who achieve best response of complete response [CR] or partial response [PR]). [00749] Clinical benefit rate (CBR) [Time Frame: 12 months]

[00750] Clinical benefit rate (CBR) (percentage of patients who achieve best response of CR, PR or stable disease [SD]).

[00751] Duration of response (DoR) [Time Frame: 12 months]

[00752] Duration of response (DoR), defined as the time from first documented evidence of CR or PR until disease progression or death.

[00753] Time to response (TTR) [Time Frame: 12 months]

[00754] Time to response (TTR), defined as the time to the first documented CR or PR.

[00755] Progression-free survival (PFS) [Time Frame: 12 months]

[00756] Progression-free survival (PFS), defined as the time to disease progression or death due to any cause.

[00757] Overall survival (OS) [Time Frame: 12 months]

[00758] Overall survival (OS) defined as the time to death due to any cause.

[00759] Results'.

[00760] To date, 7 patients have been treated in this study. Three patients were treated with 3 intravenous infusions of Allocetra-OTS 2.5 x 10^9 cells (cohort 1), and 3 additional patients were treated with 3 intravenous infusions of Allocetra-OTS 10 x 10^9 cells (cohort 2; one patient died prior to the first tumor assessment). One additional patient was treated with 2 intravenous infusions of anti-PDl therapy (nivolumab 240 mg) combined with Allocetra-OTS 2.5 x 10^9 cells. Among the first 3 patients treated with Allocetra-OTS 2.5 x 10^9 cells (cohort 1), 2 patients demonstrated stable disease at a follow-up of 14 weeks and 6 weeks following the first Allocetra-OTS infusion, and one patient had progressive disease. A summary of the 7 patients, dosage, and outcomes is provided below in Table 20. [00761] Table 20: Initial Study Design and Results

[00762] While certain features of the combination therapy have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of this combination therapy.

Claims

CLAIMS What is claimed is:

1. A combination therapy comprising a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor.

2. The combination therapy of claim 1, wherein said apoptotic mononuclear- enriched cell population comprises an inactivated apoptotic mononuclear-enriched cell population, wherein said inactivated apoptotic mononuclear-enriched cell population comprises an irradiated population, and wherein said irradiation is post induction of apoptosis.

3. The combination therapy of claim 2, wherein said inactivated apoptotic mononuclear-enriched cell population comprises pooled, apoptotic mononuclear- enriched cell populations, and wherein said pooled, apoptotic mononuclear-enriched cell populations comprise allogeneic cells from HLA matched or HLA unmatched sources, with respect to a recipient subject.

4. The combination therapy of claim 1, wherein said apoptotic mononuclear- enriched cell population comprises a white blood cell (WBC) fraction from a blood donation.

5. The combination therapy of claim 1, wherein said apoptotic 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.

6. The combination therapy of claim 1, wherein said checkpoint inhibitor comprises a CTLA-4, programmed death ligand 1 (PDL-1), PDL-2, programmed cell death protein 1 (PD-1), BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 or CGEN- inhibitor.

7. The combination therapy of claim 6, wherein said checkpoint inhibitor comprises an antibody.

8. The combination therapy of claim 7, wherein when said checkpoint comprises PD-1, said antibody is selected from nivolumab, pembrolizumab, cemiplimab, tislelizumab, dostarlimab, retifanlimab, spartalizumab, camrelizumab, sintilimab; or when said checkpoint comprises PDL-1, said antibody is selected from atezolizumab, avelumab, durvalumab, and cosibelimab.

9. A method of treating, inhibiting the growth of, or delaying disease progression, of a cancer or a tumor in a human subject, comprising a step of administering to a subject in need a first composition comprising an apoptotic mononuclear-enriched cell population and a second composition comprising a checkpoint inhibitor, wherein said method treats, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said human subject.

10. The method of claim 9, wherein said method reduces the tumor load or reduces the incidence of the cancer or a tumor; reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, prevents metastasis of said tumor or said cancer; or reduces the rate of metastasis of said tumor or said cancer; or any combination thereof, in said subject, compared with a subject not administered the combination therapy.

11. The method of claim 9, wherein said apoptotic mononuclear-enriched cell population comprises an inactivated apoptotic mononuclear-enriched cell population, and wherein said inactivated apoptotic mononuclear-enriched cell population comprises an irradiated population, wherein said irradiation is post induction of apoptosis.

12. The method of claim 11, wherein said inactivated apoptotic mononuclear- enriched cell population comprises pooled, apoptotic mononuclear-enriched cell populations, wherein said pooled, apoptotic mononuclear-enriched cell populations comprise allogeneic cells from HLA matched or HLA unmatched sources, with respect to a recipient subject.

13. The method of claim 9, wherein said apoptotic mononuclear-enriched cell population comprises a white blood cell (WBC) fraction from a blood donation.

14. The method of claim 9, wherein said apoptotic 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.

15. The method of claim 9, wherein said checkpoint inhibitor comprises a CTLA-4, programmed death ligand 1 (PDL-1), PDL-2, programmed cell death protein 1 (PD-1), BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 or CGEN-inhibitor.

16. The method of claim 15, wherein said checkpoint inhibitor comprises an antibody.

17. The method of claim 16, wherein when said checkpoint comprises PD-1, said antibody comprises nivolumab, pembrolizumab, cemiplimab, tislelizumab, dostarlimab, retifanlimab, spartalizumab, camrelizumab, or sintilimab. or when said checkpoint comprises PDL-1, said antibody is selected from atezolizumab, avelumab, durvalumab, and cosibelimab.

18. The method of claim 9, wherein said cancer or tumor comprises a solid cancer or tumor, a non-solid cancer, or comprises a metastasis of a cancer or tumor, or any combination thereof.

19. The method of claim 18, wherein said solid tumor comprises a sarcoma or a carcinoma, a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilm's tumor, a cervical cancer or tumor, a uterine cancer or tumor, a testicular cancer or tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma, or wherein said nonsolid cancer comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non-Hodgkin lymphoma, or plasma cell leukemia.

20. The method of claim 9, wherein administration comprises Intravenous (IV), Intraperitoneal (IP), subcutaneous (SC), or oral administration, and wherein said first composition and said second composition may be administered by the same or different routes.

21. The method of claim 9, wherein following administration of said combination therapy, the subject remains disease free for a time period longer than a subject administered either composition alone.