Early apoptotic cells for the treatment of sepsis

Abstract

The compositions and methods of use disclosed herein include compositions and methods of use for treating or preventing sepsis in subjects in need thereof, the methods including methods of extending the survival of subjects with sepsis and reducing organ dysfunction or failure caused by sepsis. The methods of treating or preventing sepsis in subjects in need thereof include administering a composition comprising early apoptotic cells or early apoptotic cell supernatants. The compositions and methods of use thereof can reduce the negative pro-inflammatory effects associated with sepsis. Further, anti-inflammatory cytokine release can be reduced. In some cases, the compositions can include additional agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application Serial No. 16 / 672,547, filed on November 4, 2019, and claims the benefit of U.S. Patent Application Serial No. 16 / 594,463, filed on October 7, 2019, and claims the benefit of U.S. Patent Application Serial No. 16 / 194,417, filed on November 19, 2018. All of these applications are hereby incorporated by reference in their entirety. Technical Field

[0003] Disclosed herein are compositions and methods for suppressing cytokine release syndrome (CRS) or cytokine storm or reducing the incidence of the CRS or cytokine storm in subjects undergoing CAR T cell cancer therapy. Further, disclosed herein are compositions and methods for reducing or suppressing cytokine production in subjects experiencing cytokine release syndrome or cytokine storm. Further, the compositions disclosed herein can be used to treat, prevent cancer or tumors in subjects, inhibit the growth of the cancer or tumor, or reduce the incidence of the cancer or tumor. The composition can be used to increase the survival of subjects with cancer or tumors. The composition used can be administered alone or in combination with other chemotherapy. The methods disclosed herein include those comprising administering alone or in combination with CAR T cell therapy a composition comprising apoptotic cells or apoptotic cell supernatants. The methods disclosed herein include those methods for treating sepsis. Background Art

[0004] Although the standard treatment for cancer is surgery, chemotherapy and radiotherapy, improved methods such as targeted immunotherapy are currently being developed and tested. A promising technology uses adoptive cell transfer (ACT) in which immune cells are modified to identify and attack their tumors. An example of ACT is when the patient's own or donor's cytotoxic T cells are engineered to express a chimeric antigen receptor (CAR T cell) targeting a tumor-specific antigen expressed on the surface of a tumor cell. These CAR T cells then have cytotoxicity only to cells expressing tumor-specific antigens. Clinical trials have shown that CAR T cell therapy has great potential in controlling aspects such as late acute lymphoblastic leukemia (ALL) and lymphoma.

[0005] However, some patients receiving CAR T-cell therapy and other immunotherapies experience a dangerous and sometimes life-threatening side effect known as cytokine release syndrome (CRS), in which the infused activated T cells produce a systemic inflammatory response in which cytokines are rapidly and massively released into the bloodstream, leading to dangerously low blood pressure, high fever, and shivering.

[0006] In severe cases of CRS, patients experience a cytokine storm (also known as a cytokine cascade or hypercytokinemia), in which a positive feedback loop exists between cytokines and white blood cells with highly elevated cytokine levels. This can lead to potentially life-threatening complications, including cardiac dysfunction, adult respiratory distress syndrome, neurotoxicity, renal and / or liver failure, pulmonary edema, and disseminated intravascular coagulation.

[0007] For example, in a recent Phase I trial, six patients administered TGN1412, a monoclonal antibody that binds to the CD28 receptor on T cells, developed severe cases of cytokine storm and multi-organ failure. This occurred despite the fact that the dose of TGN1412 was 500 times lower than that found to be safe in animals (St. Clair EW: The calm after the cytokine storm: Lessons from the TGN1412 trial. J Clin Invest 118:1344-1347, 2008).

[0008] So far, corticosteroids, biological therapies (such as anti-IL6 therapy) and anti-inflammatory drugs are being evaluated to control cytokine release syndrome in patients who have been administered CAR T cell therapy. However, steroids may affect the activity and / or proliferation of CAR T cells and put patients at risk of sepsis and opportunistic infections. Anti-inflammatory drugs may not be able to effectively control cytokine release syndrome or cytokine storms because cytokine storms contain very large amounts of cytokines, and the ability to infuse anti-inflammatory drugs into patients is limited. New strategies are needed to control cytokine release syndrome (and specifically, cytokine storms) in order to realize the potential of CAR T cell therapy.

[0009] Cytokine storms remain a problem after other infectious and non-infectious stimuli. In a cytokine storm, many proinflammatory cytokines (such as interleukin-1 (IL-1), IL-6, g-interferon (g-IFN) and tumor necrosis factor-α (TNFα)) are released, resulting in hypotension, bleeding and ultimately multiple organ failure. In the 1918 H1N1 influenza pandemic and the recent avian influenza H5N1 infection, the relatively high mortality rate of young people with healthy immune systems was attributed to cytokine storms. This syndrome is also known to occur in late and terminal cases of severe acute respiratory syndrome (SARS), Epstein-Barr virus-related hemophagocytic lymphohistiocytosis, sepsis, gram-negative sepsis, malaria and many other infectious diseases (including Ebola infection).

[0010] Cytokine storms may also arise from noninfectious causes, such as acute pancreatitis, severe burns or trauma, or acute respiratory distress syndrome. Therefore, novel strategies are needed to control cytokine release syndrome, and cytokine storms in particular.

[0011] Cancer is an abnormal state in which the uncontrolled proliferation of one or more cell populations interferes with normal biological function. Proliferative changes are often accompanied by other changes in cell properties, including reversion to a less differentiated, more primitive state of development. The in vitro correlates of cancer are called cell transformation. Transformed cells often display several or all of the following characteristics: spherical morphology, expression of embryonic antigens, growth factor independence, lack of contact inhibition, anchorage independence, and high-density growth.

[0012] The main cause of lethality of malignant diseases such as lung cancer and skin cancer is due to metastatic spread. In many cases, the onset of metastatic disease cannot be prevented because the cancer is usually already metastatic at the time of diagnosis, and even if the cancer is diagnosed before this stage, it may not be feasible to completely surgically remove or destroy the primary lesion tissue that may eventually lead to metastasis. Due to the small size of metastatic lesions and / or the absence of reliable markers in primary lesions that can reliably predict their presence, metastatic disease may not be diagnosed in the early stages. Because such lesions are difficult to access, diffuse and / or uncertain in location, it may be difficult or impossible to treat them by ablative methods. Chemotherapy / radiotherapy, the current method of choice for treating certain metastatic malignancies, is often ineffective or suboptimal and has significant disadvantages associated with particularly harmful and / or potentially lethal side effects.

[0013] Immunotherapeutic cancer treatment approaches, such as those involving antigen presenting cell (APC) vaccination, may be most effective for treating inaccessible, diffuse, microscopic, recurrent and / or poorly localized cancer lesions. A promising immunotherapy approach involves the use of professional APCs, such as dendritic cells (DCs), to elicit systemic anticancer immunity.

[0014] Dendritic cells are antigen-producing and presenting cells of the mammalian immune system that process antigenic material and present it on the cell surface to the T cells of the immune system, and thereby enable T cells to be sensitive to both new and recalled antigens. DCs are the most potent antigen-producing cells, acting as messengers between the innate and adaptive immune systems. DC cells can be used to induce specific anti-tumor immunity by generating effector cells that attack and lyse tumors.

[0015] Sepsis is the body's overwhelming and life-threatening response to infection that can lead to tissue damage, organ failure, and death. In other words, it is the body's overactive and toxic response to infection.

[0016] The immune system normally works to fight off any microbes (bacteria, viruses, fungi or parasites) to prevent infection. If an infection does occur, the immune system will try to fight it off, although it may need help from drugs such as antibiotics, antivirals, antifungals and antiparasitics. However, for reasons that researchers don't understand, the immune system sometimes stops fighting off the "invader" and starts attacking itself. This is the beginning of sepsis.

[0017] People who are at higher risk for developing sepsis are generally people who are at higher risk for contracting an infection. These people include the very young, the very old, people with chronic illnesses, and people with weakened or compromised immune systems. A patient is diagnosed with sepsis when they develop a set of signs and symptoms associated with sepsis. Sepsis cannot be diagnosed based on the infection itself. A doctor will likely suspect sepsis if someone has more than one symptom of sepsis, especially if there are signs of infection or if the person is in one of the higher risk groups.

[0018] Sepsis, which has been identified as a global health priority by the World Health Organization (WHO), has no proven medical treatment other than appropriate antibiotics, fluids, vasopressors as needed, and possibly corticosteroids (Venkatesh, B., Finfer, S., Cohen, J., Rajbhandari, D, Arabi, Y., Bellomo, R., Billot, L., Correa, M., Glass, P., Harward, M. et al. (2018). Adjunctive Glucocorticoid Therapy in Patients with Septic Shock. N. Engl. J. Med. 378, 797-808). Reported mortality rates for hospitalized patients range from 30% to 45% (Finfer, S., Bellomo, R., Lipman, J., French, C., Dobb, G., and Myburgh, J. (2004). Adult-population incidence of severe sepsis in Australian and New Zealand intensive care units. Intensive Care Med. 30, 589-596; Fleischmann, C., Scherag, A., Adhikari, NKJ, Hartog, CS, Tsaganos, T., Schlattmann, P., Angus, DC, Reinhart, K., and International Forum of Acute Care Trialists (2016). Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Sepsis). Current Estimates and Limitations. Am. J. Respir. Crit. Care Med. 193, 259-272; Liu, V., Escobar, GJ, Greene, JD, Soule, J., Whippy, A., Angus, DCand Iwashyna, TJ (2014). Hospital Deaths in Patients With Sepsis From 2 Independent Cohorts. JAMA 312, 90; Machado, FR, Cavalcanti, AB, Bozza, FA, Ferreira, EM, Angotti Carrara, FS, Sousa, JL, Caixeta, N., Salomao, R., Angus, DC, Pontes Azevedo, LC, et al. (2017). The epidemiology of sepsis in Brazilian intensive care units (the Sepsis PREvalence Assessment Database, SPREAD): an observational study. Lancet Infectious Diseases. Infect. Dis. 17, 1180-1189; Reinhart, K., Daniels, R., Kissoon, N., Machado, FR, Schachter, RD, & Finfer, S. (2017). Recognizing Sepsis as a Global Health Priority—A WHO Resolution. New England Journal of Medicine 377, 414-417; Rhee, C., Dantes, R., Epstein, L., Murphy, DJ, Seymour, CW, Iwashyna, TJ, Kadri, SS, Angus, DC, Danner, RL, Fiore, AE, et al. (2017). Incidence and Trends of Sepsis in US Hospitals Using Clinical vs Claims Data, 2009-2014. Data, 2009-2014). Journal of the American Medical Association 318, 1241).

[0019] Sepsis is often caused by the simultaneous recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by components of the innate immune system, including complement proteins, Toll-like receptors, NOD-like receptors, RIG-like receptors, mannose-binding lectins, and scavenger receptors (HOT Shkiss, RS, Moldawer, LL, Opal, SM, Reinhart, K., Turnbull, IR, and Vincent, J.-L. (2016). Sepsis and septic shock. Nat. Rev. Dis. Prim. 2, 16045). Recognition induces a complex intracellular signaling system with redundant and complementary activities, and the activation of these multiple signaling pathways ultimately leads to the expression of several common classes of genes involved in inflammation, adaptive immunity, and cellular metabolism (Tang, D., Kang, R., Coyne, CB, Zeh, HJ, and Lotze, MT (2012). PAMPs and DAMPs: signal 0s that spur autophagy and immunity. Immunol. Rev. 249, 158-175).

[0020] The dysregulated immune response triggered by sepsis is manifested by elevated cytokines / chemokines (also known as a “cytokine storm”), which is closely associated with disease severity and poor prognosis (Chaudhry, H., Zhou, J., Zhong, Y., Ali, MM, McGuire, F., Nagarkatti, P.S., and Nagarkatti, M (2015). Role of cytokines as a double-edged sword in sepsis. In Vivo 27, 669-684; Matsumoto, H., Ogura, H., Shimizu, K., Ikeda, M., Hirose, T., Matsuura, H., Kang, S., Takahashi, K., Tanaka, T., and Shimazu, T. (2018). The clinical importance of a cytokine network in the acute phase of sepsis. in the acute phase of sepsis.) "Sci. Rep.", 8, 1-4). This excessive immune response adversely affects the physiological homeostasis of vital organs including the kidneys, liver, lungs and heart and often develops into multiple organ failure (also known as multiple organ dysfunction syndrome (MODS)) (Marshall, JC, Cook, DJ, Christou, NV, Bernard, GR, Sprung, CL and Sibbald, WJ (1995). Multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome. Crit. Care Med. 23, 1638-16525; Vincent, J.-L. (2006). Organ Dysfunction in Patients with Severe Sepsis. Surg. Infect. Larchmt. 7, s-69-s-72).

[0021] Sepsis progresses to severe sepsis when the patient experiences signs of organ dysfunction in addition to the physical signs of sepsis, such as difficulty breathing (lungs), low or no urine output (kidneys), abnormal liver tests (liver), and changes in mental status (brain). Almost all patients with severe sepsis require treatment in an intensive care unit (ICU). Septic shock is the most severe level and is diagnosed when a patient's blood pressure drops to dangerous levels.

[0022] Current treatment for sepsis involves the administration of antibiotics and, when indicated, surgical or interventional radiologic procedures to eliminate or at least control the source of infection; the administration of intravenous fluids (crystalloid solutions such as 0.9% sodium chloride solution or colloid solutions such as 5% albumin solution) to restore and maintain adequate intravascular volume; the infusion of titratable vasoconstrictors and / or inotropic drugs (such as vasopressin or norepinephrine) as needed to alter the strength of the heart's contractions; and, when indicated, mechanical ventilation, various forms of renal replacement therapy, and, in rare cases, venovenous or venoarterial extracorporeal membrane oxygenation.

[0023] Due to the high mortality and morbidity caused by sepsis and the associated economic burden, there is a clear need for new pharmacological therapies. Unfortunately, a recent study examining the effects of glucocorticoids in patients with septic shock undergoing mechanical ventilation found that administration of a continuous infusion of hydrocortisone did not reduce 90-day mortality compared with placebo.

[0024] It seems that previous attempts to find a treatment for sepsis have failed due to the parallel progression of biological activity that occurs in sepsis patients. When the medical team applies the best standard of care (mainly antibiotics), cytokine release syndrome also rises simultaneously. Cytokine release syndrome is difficult to treat with traditional small molecules or biotechnology drugs because the symptoms involve dozens of cytokines in multiple biological pathways that induce hyperimmune activity. This hyperimmune activity may cause immune killer cells (e.g., T cells, B cells, natural killer cells) to attack the patient's healthy organs (such as heart, brain, lungs, liver, kidneys, etc.). This result of this attack may cause organ damage, multi-organ failure and death. If cytokine release syndrome can be prevented, the medical team will have enough time to eradicate the core source of sepsis (i.e., antibiotic-resistant bacteria) and is very likely to significantly increase the patient's chance of survival and survival statistics.

[0025] Apoptotic cells present a physiological cell death pathway (most often occurring through apoptosis), which triggers a series of molecular homeostasis mechanisms, including recognition, immune response and removal processes. Moreover, apoptotic cells are immunoregulatory cells that can directly and indirectly induce immune tolerance to dendritic cells and macrophages. It has been shown that apoptotic cells regulate dendritic cells and macrophages and can cause them to develop tolerance and inhibit pro-inflammatory activities, such as the secretion of pro-inflammatory cytokines and the expression of co-stimulatory molecules.

[0026] In the human body, up to 3×10 8 Each cell undergoes apoptosis. One of the primary "eat me" signals expressed by apoptotic cells is phosphatidylserine (PtdSer) membrane exposure. Apoptotic cells themselves are the main contributors to the "non-inflammatory" nature of the phagocytic process, and some apoptotic cells carry out the phagocytic process by secreting thrombospondin-1 (TSP-1) or adenosine monophosphate and other immunomodulatory "calming" signals that may interact with macrophages and DCs. Apoptotic cells also produce "find me" signals and "tolerate me" signals to attract and immunomodulate macrophages and DCs that express specific receptors for some of these signals.

[0027] The homeostatic nature of apoptotic cell interactions with the immune system is demonstrated in known apoptotic cell signaling events in macrophages and DCs associated with Toll-like receptors (TLRs), NF-κB, inflammasomes, lipid-activated nuclear receptors, Tyro3, ​​Axl, and Mertk receptors. In addition, induction of signal transducer, activated transcription 1, and suppression of cytokine signaling lead to immune system silencing and DC tolerance Trahtemberg, U. and Mevorach, D. (2017). Apoptotic cells induced signaling for immune homeostasis in macrophages and dendritic cells. Frontiers in Immunology 8).

[0028] As recently summarized (Trahtemberg and Mevorach, 2017, supra), in both animal and in vitro models, apoptotic cells may have beneficial effects on aberrant immune responses in the context of downregulation of both anti-inflammatory and proinflammatory cytokines derived from PAMPs and DAMPs.

[0029] There is still an unmet need for compositions and methods for treating, preventing, inhibiting the growth of, or reducing the incidence of cancer or tumors in a subject. The apoptotic cell preparations, compositions, and uses described below meet this need by providing an early apoptotic cell population that can be used to treat, prevent, inhibit the growth of, or reduce the incidence of cancer or tumors in a subject. Further, the methods of use described herein address the need to increase the survival of subjects with cancer and tumors (including increasing the remission of cancer or tumors).

[0030] Further, there remains an unmet need for compositions and methods for treating sepsis, including preventing organ failure and death in patients suffering from sepsis.

[0031] The methods of use described herein address the need to increase survival of subjects with sepsis and provide an unexpected solution for treating sepsis, including preventing organ failure. Summary of the invention

[0032] In one aspect, disclosed herein is a method of treating, preventing, inhibiting, reducing the incidence of sepsis in a subject in need thereof, improving or alleviating sepsis in a subject, or any combination thereof, the method comprising the step of administering to the subject a composition comprising an early apoptotic cell population, wherein the administration treats, prevents, inhibits, reduces the incidence of sepsis in the subject, improves or alleviating sepsis in the subject.

[0033] In related aspects, the sepsis comprises mild or severe sepsis. In some embodiments, the source of sepsis comprises pneumonia, intravascular methicillin-resistant Staphylococcus aureus (MRSA) infection, or urinary tract infection (UTI).

[0034] In another related aspect, the method increases the survival of the subject. In another related aspect, the subject treated with the method has a reduced incidence of organ failure or organ dysfunction or organ damage or a combination thereof. In another related aspect, the organ failure comprises acute multiple organ failure.

[0035] In a related aspect, the early apoptotic cell population comprises

[0036] (a) an enriched mononuclear cell population; or (b) an apoptotic population that is stable for more than 24 hours; or (c) a mononuclear apoptotic cell population that includes a reduction in non-quiescent non-apoptotic cells, suppressed cell activation of any live non-apoptotic cells, or a reduced proliferation of any live non-apoptotic cells, or any combination thereof; any combination thereof. In a related aspect, the early apoptotic cell population includes a pooled group of early apoptotic cells.

[0037] In related aspects, the subject in need thereof is a human subject.

[0038] In related aspects, the administration comprises a single infusion of the early apoptotic cell population. In further related aspects, the administration comprises multiple infusions of the apoptotic cell population. In further related aspects, the administration comprises intravenous administration.

[0039] In a related aspect, the method further comprises administering an additional therapy. In another related aspect, the additional therapy is administered before, simultaneously with, or after administering the early apoptotic cells.

[0040] In a related aspect, the method comprises first-line therapy.In another related aspect, the method comprises adjuvant therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0042] The subject matter disclosed herein is particularly pointed out and distinctly claimed in the concluding portion of this specification. However, the compositions and methods disclosed herein (both as to organization and method of operation), together with objects, features and advantages thereof, may be best understood by reference to the following detailed description when read in conjunction with the accompanying drawings.

[0043] Figure 1A-1B : Schematic diagram showing standard CAR T cell therapy ( Figure 1A ) and embodiments of methods of administering safe and effective CAR T cell cancer therapy to a patient using the patient's own cells (autologous) ( Figure 1B ) to produce apoptotic cells or apoptotic cell supernatant.

[0044] Figure 2 : Schematic diagram illustrating an embodiment of a method for safe and effective CAR T cell cancer therapy for a patient using donor cells to produce apoptotic cells or apoptotic supernatant.

[0045] Figure 3 : Flowchart presents the steps during one embodiment of a process for the preparation of an early apoptotic cell population, wherein an anticoagulant is included in the process.

[0046] Figures 4A-4J : Apoptotic cells prevent cytokine storm induced in an in vitro cytokine storm model in the LPS-free model of macrophage activation syndrome in a cancer setting. Figure 4AIt is shown that LPS-induced IL-10 levels in the macrophage activation syndrome model were reduced in the presence of cancer at two time periods (6 hours and 24 hours) after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:8 and 1:16. Figure 4B It is shown that LPS-induced IL-6 levels in the macrophage activation syndrome model were reduced in the presence of cancer and CAR-19 at two time periods (6 hours and 24 hours) after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:8 and 1:16. Figure 4C It is shown that LPS-induced MIP-1α levels in the macrophage activation syndrome model were reduced in the presence of cancer and CAR-19 at two time periods (6 hours and 24 hours) after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:8 and 1:16. Figure 4D It is shown that LPS-induced IL-8 levels in the macrophage activation syndrome model were reduced in the presence of cancer and CAR-19 at two time periods (6 hours and 24 hours) after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:8 and 1:16. Figure 4E It is shown that LPS-induced TNF-α levels in the macrophage activation syndrome model were reduced in the presence of cancer and CAR-19 at two time periods (6 hours and 24 hours) after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:8 and 1:16. Figure 4F It is shown that LPS-induced MIP-1β levels in the macrophage activation syndrome model were reduced at 24 hours after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:4, 1:8, 1:16, 1:32, and 1:64 in the presence of cancer and CAR-19. Figure 4G LPS-induced MCP-1 levels in the macrophage activation syndrome model were shown to be reduced at 24 hours after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:4, 1:8, 1:16, 1:32, and 1:64 in the presence of cancer and CAR-19. Figure 4H It is shown that LPS-induced IL-9 levels in the macrophage activation syndrome model were reduced in the presence of cancer and CAR-19 at two time periods (6 hours and 24 hours) after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:8 and 1:16. Fig. 4ILPS-induced IL-2R levels in the macrophage activation syndrome model are shown to be increased at 24 hours after administration of Apocells at macrophage / monocyte:Apocell ratios of 1:4, 1:8, 1:16, 1:32, and 1:64 in the presence of cancer and CAR-19. Figure 4J Apoptotic cells were shown to not downregulate IL-2 release from cells. Apoptotic cells were incubated with macrophages / monocytes in the presence of cancer and CAR-19 for a period of more than 24 hours at increasing doses of apoptotic cells (n=3). Empty bars (outline only) - 2.5×10 6 apoptotic cells; black - 5×10 per well 6 apoptotic cells; gray - 10×10 per well 6 apoptotic cells.

[0047] Figure 5 : Validation of T cell transduction showed that the transduced T4 + Flow cytometry results of anti-CD124 analysis performed on CAR-T cells.

[0048] Figure 6 : T4 + CAR T cells reduce proliferation of SKOV3-luc ovarian adenocarcinoma cells. The bar graph presents the results of cytotoxicity assays in which monolayers of SKOV3-luc cells were cultured with either untransduced T cells or T4+CAR-T cells.

[0049] Figure 7 : Apoptotic cells do not eliminate T4 + CAR-T cell antitumor activity. Results are based on cytotoxicity assays in which monolayers of SKOV3-luc cells were incubated with untransduced T cells or with T4 in the presence of vehicle (Hartmann solution) or apoptotic cells (Apocell) or apoptotic cell supernatant (ApoSup) or supernatant of a co-culture of apoptotic cells and monocytes / macrophages (ApoMon Sup). + CAR-T cell culture.

[0050] Figure 8 : 11-6, secreted at high levels during cytotoxicity, is downregulated by apoptotic cells. The results shown here confirm the effect of SKOV3-luc co-culture and human monocytes / macrophages exposed to apoptotic cells (ApoCell) or ApoCell supernatant (ApoSup) or apoptotic cells and monocytes / macrophages co-culture (ApoMon Sup).

[0051] Fig. 9:The effect of apoptotic cells or apoptotic cell supernatant or co-culture of apoptotic cells and monocytes after LPS exposure during CAR-T cell therapy. The document records that the secretion of IL-6 is very high when lipopolysaccharide (LPS) is added to the cytotoxicity assay. The results show that exposure to apoptotic cells (Apocell) or apoptotic cell supernatant (ApoSup) or supernatant of apoptotic cells and monocytes / macrophages co-culture (ApoMon Sup) downregulates IL-6, where IL-6 is reduced to an acceptable level.

[0052] Fig.10 :The effect of apoptotic cells or apoptotic cell supernatant or co-culture of apoptotic cells and monocytes after LPS exposure during CAR T cell therapy simulating CART T cell clinical therapy. The document records that the secretion of IL-6 is extremely high when lipopolysaccharide (LPS) is added to the cytotoxicity assay. The results show that exposure to apoptotic cells (Apocell) or apoptotic cell supernatant (ApoSup) or apoptotic cells and monocyte / macrophage co-culture supernatant (ApoMon Sup) downregulates IL-6, wherein IL-6 is reduced to an acceptable level.

[0053] Figures 11A-11B : Mouse weights and tumor sizes at the time of culling. Fig.11A The weight change during the experimental period is shown. Blue - no administration 4.5×10 6 SKOV3-luc cells control. Red - 0.5 × 10 6 SKOV3-luc cells. Green - 1.0×10 6 SKOV3-luc cells. Purple - 4.5 × 10 6 SKOV3-luc cells. Fig. 11B The presented acceptance was 4.5×10 6 Representative SKOV3-luc tumors from mice injected with 10 SKOV3-luc cells 39 days after injection.

[0054] Fig.12 : SKOV3-luc tumor growth. Mice bearing SKOV3-luc tumors imaged by bioluminescent imaging (BLI) are presented, showing control (PBS) versus mice inoculated with 0.5×10 6 , 1×10 6 and 4.5×10 6 The differences between the SKOV3-luc cells.

[0055] Figures 13A-13D : SKOV3-luc tumor burden. Quantification of bioluminescence (BLI) of SKOV3-luc tumors in vivo (see Fig.12). A cutoff of 600 photon counts was implemented following the manufacturer's instructions. Fig.13A , mice were inoculated with 0.5×106 SKOV3-luc. Fig. 13B , mice were inoculated with 1×106 SKOV3-luc. Fig. 13C , mice were inoculated with 4.5×106 SKOV3-luc. Fig.13D , Mean SKOV3-luc tumor growth.

[0056] Fig.14 : Cytotoxicity calibration performed on Raji Burkett lymphoma cells. Raji cells were plated at various cell densities and cell lysis was performed immediately before centrifugation. The results show the number of Raji cells (x-axis) versus the absorbance at 492nm (y-axis). All cell numbers showed significant readings relative to their un-cracked counterparts.

[0057] Fig.15 : Adding early apoptotic cells does not affect the anti-tumor activity of CAR T cells. The E / T ratio shows the ratio of CD19+CART cells to HeLa cells. Survival rate is for CD19+ tumor cells. Solid circle: CD19+HeLa; hollow triangle: CD19+HeLa+naive T cells; solid triangle: CD19+HeLa+CAR T-CD19; hollow circle: CD19+HeLa+CART-CD19+ApoCell.

[0058] Fig.16 :Cytokine analysis (GM-CSF) of Raji Burkett lymphoma cells in the presence and absence of apoptotic cells.Bar graph presents cytokine GM-CSF (pg / ml) found in the culture supernatant of Raji cells incubated in the presence of monocytes and LPS, adding naive T cells (Raji+naive T), adding CD19+CART cells (Raji+CAR T), and adding CD19+CAR T cells and apoptotic cells (ApoCell) (Raji+CAR T+ApoCell 1:8) at a ratio of 1:8CAR T cells:ApoCell, adding CD19+CAR T cells and apoptotic cells (ApoCell) (Raji+CAR T+ApoCell 1:32) at a ratio of 1:32CAR T cells:ApoCell, and adding CD19+CAR T cells and apoptotic cells (ApoCell) (Raji+CAR T+ApoCell 1:32) at a ratio of 1:64CAR T cells:ApoCell, and adding CD19+CAR T cells and apoptotic cells (ApoCell) (Raji+CAR T+ApoCell 1:64) at a ratio of 1:64CAR T cells:ApoCell.

[0059] Fig.17: Cytokine analysis (TNF-α) of Raji Burkett lymphoma cells in the presence and absence of apoptotic cells. The bar graph presents the concentration measurements of the cytokine TNF-α (TNF-α) (pg / ml) found in the culture supernatant of Raji cells incubated in the presence of monocytes and LPS after the addition of naive T cells (Raji+naive T), the addition of CD19+CAR T cells (Raji+CAR T), and the addition of CD19+CAR T cells and apoptotic cells (ApoCell) at a ratio of 1:8CAR T cells:ApoCell (Raji+CAR T+ApoCell 1:8), the addition of CD19+CAR T cells and apoptotic cells (ApoCell) at a ratio of 1:32CAR T cells:ApoCell (Raji+CAR T+ApoCell 1:32), and the addition of CD19+CAR T cells and apoptotic cells (ApoCell) at a ratio of 1:64CAR T cells:ApoCell (Raji+CAR T+ApoCell1:64).

[0060] Fig.18A and Fig.18B : Experimental plan. Fig.18A An experimental protocol for analyzing the effect of apoptotic cells on CAR T cell therapy is presented. SCID mice were injected with Raji cancer cells on day 1, followed by administration of CAR T-CD19 cells (CAR T cell therapy) and apoptotic cells on day 6. Fig.18B It shows that CAR T cell therapy is not negatively affected by co-administration of ApoCell. Survival curves: SCID mice were injected with CD19+Raji cells with or without the addition of early apoptotic cells.

[0061] Fig.19A , 19B and 19C: increased release of pro-inflammatory cytokines from tumors in in vivo models of solid tumors. Fig.19A The IL-6 release from solid tumors present in the peritoneum of BALB / c and SCID mice was slightly increased, with IL-6 release significantly increased in the presence of HeLa CAR-CD-19 CAR T cells. Fig.19B showed a slight increase in IP-10 release from solid tumors present in the peritoneum of BALB / c and SCID mice, with IP-10 release significantly increased in the presence of HeLa CAR-CD-19 CAR T cells, and Fig.19C It was shown that even TNF-α release was surprisingly increased in the presence of HeLa CAR-CD-19 CAR T cells.

[0062] Fig. 20A and 20B : Testing the efficacy of CD19-CAR-T cells in the IP model (leukemia) of HeLa CD19 in the presence or absence of ApoCell cells. HeLa-CD19-blue; HeLa CD19+sham-green; HeLa CD19+CAR-T-purple; and HeLa CD19+CAR-T+ApoCell-orange. Fig. 20A Use 0.5×10 6 CAR-T positive cells. Fig. 20B Use 2.2×10 6 CAR-T positive cells.

[0063] Fig.21 Survival curves of the in vivo diffuse tumor SCID mouse model. The curves show that administration of early apoptotic cells (APO; thick dashed line ----) prolonged survival compared to mice not administered apoptotic cells (no APO; dotted line ......), where control SCID mice showed 100% survival (solid line ______).

[0064] Figures 22A-22D : Apoptotic cell infusion increases the lifespan of leukemic mice and increases the number of mice that achieve complete remission. Cohorts: no leukemia (control - striped pattern); leukemia + early apoptotic cells (speckled pattern); leukemia only (solid grey). Total n = 51 (p < 0.001). Fig.22A : Apoptotic cell infusion increases the percentage of mice that survive to their expected lifespan after leukemia induction. Fig. 22B : Apoptotic cell infusion increased the percentage of mice that survived to 12% of the expected lifespan after leukemia induction. Fig. 22C : Apoptotic cell infusion increases the percentage of mice that survive to 30% of their expected lifespan after leukemia induction. Fig.22D : Apoptotic cell infusion increases the percentage of mice that survive to 100% of expected lifespan and achieve complete remission after leukemia induction.

[0065] Figures 23A-23E : Apoptotic cell infusion increases the lifespan of leukemic mice, increases the number of mice that achieve complete remission, and enhances the effect of anti-CD20 monoclonal antibody (mAb) treatment. Cohorts: leukemia only (solid gray); leukemia + early apoptotic cells (striped pattern); leukemia + anti-CD20 mAb (squares); leukemia + anti-CD20 + early apoptotic cells (spots). Total n = 28 (p < 0.002). Fig.23A : Shows the percentage (%) of mice surviving to the expected lifespan after leukemia was induced with Raji cells. Fig. 23B: Apoptotic cell infusion increased the percentage of mice that survived after leukemia induction to 24% longer than the expected lifespan. Fig.23C : Apoptotic cell infusion increased the percentage of mice surviving to 59% longer than the expected lifespan after leukemia induction and enhanced the effect of anti-CD20 mAb on the lifespan of leukemic mice. Fig.23D : Apoptotic cell infusion increased the percentage of mice surviving to 76% longer than the expected lifespan after leukemia induction and enhanced the effect of anti-CD20 mAb on the lifespan of leukemic mice. Fig.23E : Apoptotic cell infusion increased the percentage of mice achieving complete remission.

[0066] Fig.24 : Kaplan-Meier survival curves of SCID-Bg mice with Raji leukemia / lymphoma receiving ApoCell. (RPMI group, n=15; Raji group, n=23; Raji+ApoCell group, n=24) RPMI (control) - black; Raji only - orange; Raji+ApoCell - blue.

[0067] Figures 25A-25C : Kaplan-Meier survival curves. Fig.25A Data are presented from a study in which 7-week-old female SCID-Bg mice (ENVIGO, Jerusalem, Israel) were injected intravenously with 0.1×10 6 Raji cells (n=10 per group, three groups). Mice received three intravenous doses (days 5, 8, and 11) of 30×10 6 ApoCell. (RPMI-light blue; Raji-orange; and Raji+ApoCell-dark blue) Fig.25B Data are presented from a study in which 7-week-old female SCID-Bg mice (ENVIGO, Jerusalem, Israel) were injected intravenously with 0.1×10 6 Raji cells (n=10 per group, three groups). Mice received three intravenous doses (days 5, 8, and 11) of 30×10 6 ApoCell. (RPMI-black; Raji-orange; and Raji+ApoCell-dark blue) Fig.25C Data are presented from a study in which 8-9 week old female SCID-Bg mice (ENVIGO, Jerusalem, Israel) were injected intravenously with 0.1×10 6Raji cells (n=10 per group, 2 groups). Mice received three intravenous doses (days 5, 8, and 12) of 30×10 6 ApoCell. (Raji-orange; and Raji+ApoCell-dark blue)

[0068] Fig.26 : Kaplan-Meier survival curves of SCID-Bg mice with Raji leukemia / lymphoma receiving RtX and ApoCell. (Raji alone - orange; Raji + ApoCell - blue; Raji + RtX 2mg - green; Raji + RtX 2mg + ApoCell - yellow; Raji + RtX 5mg - purple; Raji + RtX 5mg + ApoCell - gray.)

[0069] Fig. 27 : Kaplan-Meier survival curves of SCID-Bg mice with Raji leukemia / lymphoma receiving rtX and ApoCell. (Raji alone - orange; Raji + ApoCell - blue; Raji + RtX 2mg - green; Raji + RtX 2mg + ApoCell - yellow.)

[0070] Fig.28 : Effects of pooled ApoCell preparations. Fig.28 A graph showing a significant effect (p<0.01) of a single injection of a preparation of apoptotic cells from multiple individual donors (blue) on survival is presented. The graph presented is a Kaplan-Meier survival curve in a GvHD mouse model treated with a single dose of irradiated pooled apoptotic cell preparations from multiple individual donors.

[0071] Fig.29 : Effects of pooled ApoCell preparations. Fig.29 Graphs showing the significant effect (p<0.01) of a single injection of apoptotic cell preparations from multiple individual donors (blue) on the percentage of body weight loss in the two comparison groups are presented.

[0072] Fig.30 : Comparison of single donor and pooled ApoCell preparations. Fig.30 Graphs showing comparison of administration of a single dose of a single donor apoptotic cell preparation versus administration of a multiple donor apoptotic cell preparation + / - irradiation in terms of % survival using a mouse model of induced GvHD are presented.

[0073] Figures 31A-31B : Effectiveness test. Figures 31A-31BResults of efficacy tests showing inhibition of maturation of dendritic cells (DCs) following interaction with apoptotic cells as measured by expression of HLA-DR are presented. Fig.31A : Mean fluorescence of HLA DR of fresh final product A(t0). Fig.31B : Mean fluorescence of HLA DR of final product A after 24 hours at 2-8°C.

[0074] Figures 32A-32B : Effectiveness test. Figures 32A-32B Results of efficacy tests showing inhibition of maturation of dendritic cells (DCs) following interaction with apoptotic cells as measured by expression of CD86 are presented. Fig.32A : Mean fluorescence of CD86 of fresh final product A(t0). Fig.32B : Mean fluorescence of CD86 in final product A after 24 hours at 2-8°C.

[0075] Figures 33A-33E : Results of a preclinical analysis of the use of early apoptotic cells to treat sepsis. Fig.33A Increased survival was shown for mice that received antibiotics and Allocetra-OTS (early apoptotic cells as described herein). Fig.33B Clinical scores of different cohorts are shown, wherein clinical scores are correlated with the survival rate of mice in CLP-induced sepsis model subjects. Fig.33C Allocetra was shown to prevent uncontrolled cytokine signaling events following sepsis induction (ie, cytokine storm), which resulted in increased survival of subjects in the CLP-induced sepsis model. Fig.33D The increase in survival of CLP-induced sepsis model subjects treated with Allocetra was shown to be dose-dependent. Fig.33E It was also shown that the increase in survival of CLP-induced sepsis model subjects treated with Allocetra was dose-dependent.

[0076] Figures 34A-34F :CLP mice showed signs of respiratory and cardiovascular dysfunction that correlated with the severity of sepsis. Fig.34A ) 24 hours after CLP, naive mice (n=21) showed no signs of disease, while the majority of CLP mice (n=40) had severe clinical signs (median MSS clinical score 13; 95% CI median 9-14); **P<0.0001 by two-tailed Mann-Whitney test. ( Fig.34B )The lung-to-body weight ratio increased significantly with the severity of sepsis. Fig.34C) Representative 2D echocardiograms of naive mice (upper panel) and CLP mice (lower panel), showing time lapse view (M-mode) and top view (B-mode). To calculate various parameters of LV structure and function (including ( Fig.34D ) heart rate, ( Fig.34E )LV volume and ( Fig.34F ) Cardiac output) and measured LV intra-distance, heart rate and posterior wall thickness. Data are presented as median within interquartile range (IQR); mean values ​​are marked with '+' symbols; error bars represent 5-95 percentile ranges; group size (N) is indicated below its corresponding legend; *P≤0.01, **P≤0.001, ***P≤0.0001, by Kruskal-Wallis nonparametric ANOVA, with multiple comparisons adjusted using Dunn's test. P values ​​above the bars indicate significant differences from the control group, and P values ​​above the brackets indicate significant differences between the other two groups.

[0077] Figures 35A-35C : CLP mice showed signs of renal dysfunction that correlated with the severity of sepsis. Renal dysfunction was assessed by ( Fig.35A )blood urea, ( Fig.35B ) Neutrophil gelatinase-associated lipocalin (NGAL) and ( Fig.35C ) serum potassium concentration. Data are presented as median with interquartile range (IQR); mean values ​​are marked with a '+' symbol; error bars indicate 5-95 percentile range; group sizes (N) are indicated below their respective legends; *P≤0.01, **P≤0.001, ***P≤0.0001 by Kruskal-Wallis nonparametric ANOVA with multiple comparisons adjusted using Dunnett's test. P values ​​above the bars indicate differences from the control group, and P values ​​above the brackets indicate differences between the other two groups.

[0078] Figures 36A-36G :In CLP mice, markers of liver dysfunction strongly correlated with MSS clinical scores. Fig.36A ) 24 h after CLP, total bilirubin serum concentrations in mice with severe sepsis (MSS clinical score > 13) were slightly and non-significantly (p > 0.93), whereas ( Fig.36B ) alanine aminotransferase (ALT) level and ( Fig.36C )Aspartate aminotransferase (AST) levels decreased significantly with the severity of sepsis. Fig.36D ) alkaline phosphatase level and ( Fig.36E ) albumin levels decreased significantly with the severity of sepsis, while ( Fig.36F There were no significant changes in serum globulin concentrations. Figure 36G ) septic mice (notably, mice with mild sepsis (MSS clinical score 1-4)) had lower glucose levels than naive mice. Data are presented as median within interquartile range (IQR); mean values ​​are marked with a '+' symbol; error bars represent 5-95 percentile range; group sizes (N) are indicated below their respective legends; *P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001 by Kruskal-Wallis nonparametric ANOVA with multiple comparisons adjusted using Dunnett's test. P values ​​above the bars indicate differences from the control group, and P values ​​above the brackets indicate differences between the other two groups.

[0079] Figures 37A-37E : Significant thrombocytopenia and lymphocytopenia and abnormal complement activation in septic mice. Fig.37A ) 24 hours after CLP, platelet counts in septic mice were significantly lower than those in naive mice. ( Fig.37B )WBC count and ( Fig.37C ) Decreased lymphocyte count. Fig.37D ) C5a serum concentrations were higher in septic mice regardless of their clinical scores. Fig.37E )C3a serum concentrations were lower in septic mice and correlated with clinical scores. Data are presented as medians within interquartile ranges (IQR); means are marked with a '+' symbol; error bars represent 5-95 percentile ranges; group sizes (N) are indicated below the relevant legends; *P≤0.01, **P≤0.001, ***P≤0.0001 by Kruskal-Wallis parametric ANOVA with multiple comparisons adjusted using Dunnett's test. P values ​​above the bars indicate significant differences from the control group, and P values ​​above the brackets indicate significant differences between the other two groups.

[0080] Figures 38A-38F :CLP mice showed adverse metabolic changes. ( Fig.38A ) 24 hours after CLP, blood pH decreased significantly with the severity of sepsis. Fig.38B ) OCR measurements of PBMCs from naive and CLP mice showed abnormal mitochondrial respiration mainly in severe septic mice (MSS clinical score>10), with abnormal mitochondria mainly manifested by ( Fig.38C )The coupling efficiency is reduced. Fig.38D) Extracellular acidification rate (ECAR) measurements of PBMCs from naive and CLP mice showed only mild changes in overall glycolytic function, which was slightly increased in moderately septic mice (MSS clinical score 7-8.5); ( Fig.38E ) The glycolytic reserve of PBMCs in this assay was significantly reduced in severely septic mice (MSS clinical score >14). Fig.38F ) Blood lactate concentration was slightly lower in CLP mice. Fig.38A , 38C Data in 38A, 38E, and 38F are presented as medians with interquartile ranges (IQR); mean values ​​are marked with a '+' symbol; error bars represent 5-95 percentile ranges; Fig.38B and 38D Data in are presented as mean ± SD; group sizes (N) are indicated below their corresponding legends; *P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001 by Kruskal-Wallis nonparametric ANOVA with multiple comparisons adjusted using Dunnett's test. P values ​​above the bars indicate differences from the control group, and P values ​​above the brackets indicate differences between the other two groups.

[0081] Figures 39A-39D : Beneficial effects of Allocetra-OTS on CLP mice. Unless otherwise indicated, mice were injected intravenously with ertapenem and Hartmann's solution (vehicle) or 20×10 6 Allocetra-OTS. The health of mice was monitored and they were euthanized when the MSS clinical score was >15. Fig.39A ) Kaplan-Meier survival curves of CLP mice treated with Ertapenem+Vehicle or Ertapenem+Allocetra-OTS. ( Fig.39B ) Median survival time was increased in Allocetra-OTS treated mice; error bars represent 95% CI; *P ≤ 0.01 by Kruskal-Wallis nonparametric ANOVA with multiple comparisons adjusted using Dunnett's test. ( Fig.39C ) Mean MSS clinical scores were reduced in Allocetra-OTS treated mice; error bars represent standard error; ***P ≤ 0.0001 by ordinary one-way ANOVA with nonlinear curve fitting. ( Fig.39D ) Kaplan-Meier survival curves of CLP mice treated with Ertapenem + various doses of Allocetra-OTS. The number of mice in each group (N) is indicated next to its corresponding legend.

[0082] Figures 40A-40L: Allocetra-OTS treatment attenuates cytokine / chemokine release after CLP. Blood samples were collected from C57BL / 6 mice before CLP and 24, 48, and 72 hours after CLP following treatment with Ertapenem or Ertapenem+Allocetra-OTS (OTS-ALC). Untreated CLP mice did not survive more than 24 hours and therefore data are not shown. ( Figures 40A-40L ) Cytokine / chemokine levels measured by Luminex, including: IL-6 ( Fig.40A )、TNF-α( Fig.40B ), IL-1β( Fig.40C )、IL-10( Fig.40D )、MIP-1a( Fig.40E )、MIP-1b( Fig.40F )、RANTES( Figure 40G )、ENA-78( Figure 40H )、IL-17a( Fig.40I )、IP-10( Fig.40J )、VEGF-α( Figure 40K ) and IL-12p70( Figure 40L ). Figures 40A-40L It was demonstrated that both pro-inflammatory and anti-inflammatory cytokines were reduced after treatment with Allocetra-OTS. Data are presented as mean ± standard deviation.

[0083] Figures 41A-41C Patient characteristics - All historical matched controls hospitalized in the Medical Intensive Care Unit of Hadassah Ein Kerem Hospital, Jerusalem, Israel, between 2016 and 2018 were reviewed. Historical controls were matched to patients based on age (±3 years), sex matching, Sequential Organ Failure Assessment (SOFA) score at admission (±2), and source of sepsis. The survival probability of the treatment arm of 6 patients based on APACHE II scores obtained in the first 24 hours of admission (scores predict mortality based on general conditions and chronic diseases) was 52.95%. However, no patient died in the treated group. Fig.41A SOFA scores of patients (treated and controls) at admission are shown. (black - matched controls; light grey - treated). Fig.41B The age distribution of patients at admission is shown. (black - matched controls; stripes - treated). Fig.41C Shown are the percentages of patients and the source of their sepsis (treated and control) from pneumonia, intravascular methicillin-resistant Staphylococcus aureus (MRSA), or urinary tract infection (UTI). Fig.41B and41C The Y-axis is the percentage of patients. Fig.41B The x-axis is age. Fig.42 : Comparative interim data: treated and untreated matched control patient populations. Fig.42 Comparative interim data for six (6) treated patients and thirty-seven (37) matched control patients are presented in tabular form.

[0084] Fig.43 : Comparative interim data: SOFA at admission and sepsis-related mortality. Fig.43 It is shown that for the matched control group, mortality was most associated with a low SOFA score on admission.

[0085] Fig.44A and 44B : Comparative interim data: Recovery from sepsis. Fig.44A and 44B Allocetra-OTS was shown to be extremely effective in treating sepsis. Fig.44A It was shown that 100% of all patients treated with Allocetra-OTS recovered from sepsis within 28 days, independent of the source of sepsis, which included pneumonia, intravascular methicillin-resistant Staphylococcus aureus (MRSA), or urinary tract infection (UTI), compared to only 48% of matched controls. Fig.44B Shown that 100% of sepsis patients (sepsis sources: pneumonia and intravascular methicillin-resistant Staphylococcus aureus (MRSA)) treated with Allocetra-OTS recovered from sepsis within 28 days compared to only 45% of matched controls. (Black - matched controls; Light grey - treated with Allocetra-OTS)

[0086] Fig.45 : Comparative Interim Data: Recovery from Sepsis: Following hospital admission, Allocetra-OTS was extremely effective in rapidly treating sepsis. Fig.45 The % of patients fully recovered from sepsis based on days from hospital admission is shown, with 100% of treated patients (dark grey line) recovering by day 8 compared to less than 40% of matched controls (light grey line).

[0087] Fig.46A and 46B : Comparative Top Line Data: Recovery from Sepsis: The mortality data showed that Allocetra-OTS was extremely effective in preventing sepsis-related mortality. Fig.46AIt was shown that there were no deaths in Allocetra-treated patients, independent of the source of sepsis, which included pneumonia, intravascular methicillin-resistant Staphylococcus aureus (MRSA), or urinary tract infection (UTI), compared with mortality rates of 29% (matched controls) and 23% (literature). Fig.46B Shown is the absence of mortality in Allocetra-treated patients when the source of sepsis included pneumonia and intravascular methicillin-resistant Staphylococcus aureus (MRSA), compared to mortality rates of 34% (matched controls) and 28% (literature). (black - matched controls; light grey - literature)

[0088] Fig.47A and 47B : Comparative interim data: Duration of ICU stay showed that Allocetra-OTS improved patients' clinical status and accelerated discharge from the ICU. Fig.47A Shown that after 6 days, only 43% of all matched controls were discharged from the ICU compared to 100% of patients treated with Allocetra-OTS. (treated - black dashed line; matched controls - dotted line) Fig.47B Shown that after 6 days, only 35% of matched controls (excluding UTI controls) were discharged from the ICU compared to 100% of patients treated with Allocetra-OTS. (treated - black dashed line; matched controls - grey solid line; data collection started at 100%)

[0089] Figures 48A-48D : Comparative interim data: Organ dysfunction and failure showed that Allocetra-OTS prevented organ dysfunction and failure. Fig.48A Shown are mean baseline SOFA and maximum SOFA for all subjects (treated and matched controls), independent of the source of sepsis including pneumonia, intravascular methicillin-resistant Staphylococcus aureus (MRSA), or urinary tract infection (UTI). Fig.48B Shown are mean baseline SOFA and maximum SOFA for subjects (treated and matched controls) excluding patients with UTI. Fig.48C Median baseline SOFA and maximum SOFA are shown for all subjects (treated and matched controls), independent of the source of sepsis, which included pneumonia, intravascular methicillin-resistant Staphylococcus aureus (MRSA), or urinary tract infection (UTI). There was no difference between treated patients and historical controls at baseline SOFA, but there was a significant difference between maximum SOFA, indicating that treated patients did not progress in their sepsis course. Fig.48D Median baseline SOFA and maximum SOFA of subjects (treated and matched controls) excluding UTI patients are shown. (Black - matched controls; Light grey - treated with Allocetra-OTS)

[0090] Fig.49A and 49B : Comparative interim data: Organ Dysfunction and Failure showed that treatment with Allocetra-OTS prevented increases in SOFA scores. Fig.49A Shown are the percentages of all patients whose SOFA scores increased from the time of admission, independent of the source of sepsis, which included pneumonia, intravascular methicillin-resistant Staphylococcus aureus (MRSA), or urinary tract infection (UTI). Fig.49B The percentage of patients whose SOFA scores increased from the time of admission is shown, with sepsis sources including pneumonia and intravascular methicillin-resistant Staphylococcus aureus (MRSA). (black - matched controls; light grey - Allocetra-OTS treated)

[0091] Fig.50A and 50B : Interim interim data: Organ Dysfunction and Failure showed that preventing an increase in SOFA of 4 points or more is critical to preventing death and that treatment with Allocetra-OTS prevents an increase in SOFA of 4 points or more. Fig.50A The percentage of death for patients whose SOFA increase was greater than or equal to 4 is shown. Fig.50B The percentage of patients whose SOFA increase was greater than or equal to 4 is shown. (Black - matched controls; Light grey - Allocetra-OTS treated) DETAILED DESCRIPTION

[0092] In the following detailed description, many specific details are set forth in order to provide a thorough understanding of the methods disclosed herein. However, it will be appreciated by those skilled in the art that these methods can be practiced without these specific details. In other cases, well-known methods, procedures, and components are not described in detail to avoid obscuring the methods disclosed herein.

[0093] As is well known, genetic modification of immune cells is a strategy for immune cell therapy for cancer. These immune cell therapies are based on the manipulation of autologous or allogeneic immune cells and their administration to subjects in need. Immune cell-based therapies include natural killer cell therapy, dendritic cell therapy, and T cell immunotherapy (including therapies utilizing naive T cells, effector T cells (also known as T helper cells), cytotoxic T cells, and regulatory T cells (Treg)).

[0094] In some embodiments, disclosed herein are compositions comprising genetically modified immune cells. In another embodiment, the genetically modified immune cells are T cells. In another embodiment, the T cells are naive T cells. In another embodiment, the T cells are naive CD4 + In another embodiment, the T cell is a naive T cell. In another embodiment, the T cell is a naive CD8 + T cells. In another embodiment, the genetically modified immune cells are natural killer (NK) cells. In another embodiment, the genetically modified immune cells are dendritic cells. In yet another embodiment, the genetically modified T cells are cytotoxic T lymphocytes (CTL cells). In another embodiment, the genetically modified T cells are regulatory T cells (Treg). In another embodiment, the genetically modified T cells are chimeric antigen receptor (CAR) T cells. In another embodiment, the genetically modified T cells are genetically modified T cell receptor (TCR) cells.

[0095] In some embodiments, disclosed herein are compositions comprising genetically modified immune cells and apoptotic cells. In another embodiment, disclosed herein are compositions comprising genetically modified immune cells and supernatants from apoptotic cells. In another embodiment, the genetically modified immune cells are T cells. In another embodiment, the genetically modified immune cells are natural killer (NK) cells. In still another embodiment, the genetically modified immune cells are cytotoxic T lymphocytes (CTL cells). In another embodiment, the genetically modified immune cells are regulatory T lymphocytes (Treg cells).

[0096] In some embodiments, disclosed herein is a method of maintaining or increasing the proliferation rate of chimeric antigen receptor-expressing T cells (CAR T cells) during CAR T cell cancer therapy, the method comprising the step of administering to the subject a composition comprising apoptotic cells or apoptotic cell supernatant, and wherein the proliferation rate is maintained or increased in the subject compared to a subject undergoing CAR T cell cancer therapy and not administered the apoptotic cells or the apoptotic cell supernatant.

[0097] In related embodiments, the method does not reduce or inhibit the efficacy of the CAR T cell cancer therapy. In related embodiments, the method improves the efficacy of the CAR T cell cancer therapy. In another related embodiment, the incidence of cytokine release syndrome (CRS) or cytokine storm in the subject is inhibited or reduced compared to a subject to whom the apoptotic cells or the apoptotic cell supernatant are not administered.

[0098] In some embodiments, CRS occurs spontaneously. In another embodiment, CRS occurs in response to LPS. In another embodiment, CRS occurs in response to IFN-γ.

[0099] In some embodiments, disclosed herein is a method for increasing the efficacy of chimeric antigen receptor T cell (CAR T cell) cancer therapy, the method comprising administering CAR T cells and an additional agent selected from a group including apoptotic cells, apoptotic cell supernatants, CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators or any combination thereof, wherein the efficacy of the CAR T cells is increased in the subject compared to a subject undergoing CAR T cell cancer therapy and not administering the additional agent. In a related embodiment, the level of production of at least one proinflammatory cytokine is reduced compared to the level of the proinflammatory cytokine in a subject receiving CAR T cell cancer therapy and not administering a composition including the agent. In another related embodiment, the proinflammatory cytokine includes IL-6.

[0100] In a related embodiment, when apoptotic cells or apoptotic cell supernatants are administered, the method increases the level of IL-2 in the subject compared to a subject who has undergone CAR T cell cancer therapy and has not administered the apoptotic cells or the apoptotic cell supernatants. In another embodiment, when apoptotic cells or apoptotic cell supernatants are administered, the method maintains the level of IL-2 in the subject compared to a subject who has undergone CART cell cancer therapy and has not administered the apoptotic cells or the apoptotic cell supernatants. In another embodiment, when apoptotic cells or apoptotic cell supernatants are administered, the method maintains or increases the level of IL-2 in the subject compared to a subject who has undergone CAR T cell cancer therapy and has not administered the apoptotic cells or the apoptotic cell supernatants. In another related embodiment, the incidence of cytokine release syndrome (CRS) or cytokine storm in the subject is suppressed or reduced compared to a subject to whom the additional agent is not administered.

[0101] In a related embodiment, the CAR T cells and the additional agent or any combination thereof are included in a single composition. In another related embodiment, the CAR T cells and the additional agent or any combination thereof are included in at least two compositions.

[0102] In some embodiments, disclosed herein is a method for treating, preventing, inhibiting, reducing the incidence of, improving or alleviating cancer or tumors in a subject, the method comprising administering T cells expressing a chimeric antigen receptor (CAR T cells) and an additional agent (the additional agent comprises apoptotic cells, apoptotic cell supernatant, or a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium compound or an immunomodulator or any combination thereof), wherein the method treats, prevents, inhibits, reduces the incidence of, improves or alleviating cancer or tumors in the subject, compared to a subject administered CAR T cells and not administered the additional agent.

[0103] In related embodiments, the method provides improved efficacy in treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating the cancer or tumor in the subject, compared to a subject administered CAR T cells and not administered the additional agent.

[0104] In another related embodiment, the level of production of at least one pro-inflammatory cytokine is reduced compared to the level of the pro-inflammatory cytokine in a subject to which the CAR T cells are administered and a composition comprising the agent is not administered. In another related embodiment, the pro-inflammatory cytokine includes IL-6. In another related embodiment, when the additional agent includes apoptotic cells or apoptotic cell supernatant, the method increases the level of IL-2 in the subject compared to a subject to which the CAR T cells are administered and the apoptotic cells or the apoptotic cell supernatant are not administered. In another related embodiment, the CAR T cells and the additional agent or any combination thereof are included in a single composition. In yet another related embodiment, the CAR T cells and the additional agent or any combination thereof are included in at least two compositions.

[0105] In a related embodiment, the administration of the additional agent occurs before, simultaneously with, or after the administration of the CAR T cells. In another related embodiment, the apoptotic cells include apoptotic cells in an early apoptotic state. In another related embodiment, the apoptotic cells are autologous to the subject, or are pooled third-party donor cells.

[0106] In a related embodiment, the apoptotic cell supernatant is obtained by a method comprising the following steps: (a) providing apoptotic cells, (b) culturing the cells of step (a), and (c) separating the supernatant from the cells. In another related embodiment, the apoptotic cell supernatant is an apoptotic cell-white blood cell supernatant, and the method further comprises the following steps: (d) providing white blood cells, (e) optionally washing the apoptotic cells and the white blood cells, (f) co-culturing the apoptotic cells and the white blood cells, wherein steps (d)-(f) replace step (b). In another related embodiment, the white blood cells provided are selected from the group consisting of phagocytes, macrophages, dendritic cells, monocytes, B cells, T cells and NK cells. Therefore, in some embodiments, the apoptotic supernatant includes a supernatant produced by culturing apoptotic cells with macrophages, wherein the macrophages ingest the apoptotic cells, and use the supernatant produced by this co-culture. In some embodiments, the apoptotic supernatant includes a supernatant produced by culturing apoptotic cells, wherein the supernatant is produced by materials secreted by the apoptotic cells.

[0107] In some embodiments, disclosed herein are compositions comprising early apoptotic cells. In some embodiments, disclosed herein are compositions comprising early apoptotic cells and additional agents. In some embodiments, the additional agent may be a CAR T cell. In some embodiments, the additional agent may be an antibody. In some embodiments, the antibody comprises rituximab or a functional fragment thereof.

[0108] In some embodiments, the composition of early apoptotic cells includes a population of mononuclear apoptotic cells comprising mononuclear cells in an early apoptotic state, wherein the population of mononuclear apoptotic cells includes: a reduced percentage of non-quiescent non-apoptotic live cells; suppressed cellular activation of any live non-apoptotic cells; or reduced proliferation of any live non-apoptotic cells; or any combination thereof.

[0109] In some embodiments, disclosed herein are compositions comprising genetically modified T cells and apoptotic cells. In another embodiment, disclosed herein are compositions comprising supernatants of genetically modified T cells and apoptotic cells. In another embodiment, the genetically modified T cells are chimeric antigen receptor (CAR) T cells. In another embodiment, the genetically modified T cells are genetically modified T cell receptor (TCR) cells.

[0110] In some embodiments, disclosed herein are compositions comprising CAR T cells and apoptotic cells. In another embodiment, disclosed herein are compositions comprising genetically modified T cell receptor cells (TCR) and apoptotic cells. In another embodiment, disclosed herein are compositions comprising CAR T cells and supernatants from apoptotic cells. In another embodiment, disclosed herein are compositions comprising genetically modified T cell receptor cells (TCR) and supernatants from apoptotic cells.

[0111] In certain embodiments, the genetically modified immune cells and apoptotic cells or apoptotic cell supernatants are included in a single composition. In other embodiments, the genetically modified immune cells and apoptotic cells or apoptotic cell supernatants are included in separate compositions.

[0112] In some embodiments, the disclosure provides a mononuclear apoptotic cell preparation comprising a collection of monocytes in an early apoptotic state, wherein the mononuclear apoptotic cell preparation collected includes a single mononuclear cell population collected, and wherein the mononuclear apoptotic cell preparation collected includes a percentage of a reduction in live non-apoptotic cells, cell activation of any live non-apoptotic cells contained, or proliferation of any live non-apoptotic cells or any combination thereof. In another embodiment, the mononuclear apoptotic cells collected have been irradiated. In another embodiment, the disclosure provides a mononuclear apoptotic cell preparation collected, which in some embodiments uses a white blood cell fraction (WBC) obtained from donated blood. Typically, this WBC fraction is discarded at a blood bank, or its goal is to be used for research.

[0113] In some embodiments, the cell populations disclosed herein are 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 curbing or eliminating an immune response in the preparation. In another embodiment, inactivation comprises curbing or eliminating cross-reactivity between multiple individual populations included in the preparation. In other embodiments, inactivation comprises reducing or eliminating T cell receptor activity between multiple individual populations included in the preparation. In another embodiment, the inactivated cell preparation comprises a reduced percentage of live non-apoptotic cells, suppressed cell activation of any live non-apoptotic cells, or reduced proliferation of any live non-apoptotic cells, or any combination thereof.

[0114] In another embodiment, the inactivated cell population comprises a reduced number of non-quiescent non-apoptotic cells compared to a non-irradiated cell preparation. In some embodiments, the inactivated cell population comprises 50 percent (%) of live non-apoptotic cells. In some embodiments, the inactivated cell population comprises 40% of live non-apoptotic cells. In some embodiments, the inactivated cell population comprises 30% of live non-apoptotic cells. In some embodiments, the inactivated cell population comprises 20% of live non-apoptotic cells. In some embodiments, the inactivated cell population comprises 100% of live non-apoptotic cells. In some embodiments, the inactivated cell population comprises 0% of live non-apoptotic cells.

[0115] In some embodiments, disclosed herein is a method for preparing an inactivated early apoptotic cell population. In some embodiments, disclosed herein is a method for producing a mononuclear apoptotic cell population comprising a reduced percentage of non-quiescent non-apoptotic live cells; suppressed cell activation of any live non-apoptotic cells; or reduced proliferation of any live non-apoptotic cells; or any combination thereof, the method comprising the following steps:

[0116] Obtaining an enriched mononuclear cell population from peripheral blood;

[0117] freezing the enriched monocyte population in a freezing medium comprising an anticoagulant;

[0118] thawing the enriched monocyte population;

[0119] incubating the enriched monocyte population in an apoptosis-inducing incubation medium comprising methylprednisolone at a final concentration of about 10-100 μg / mL and an anticoagulant;

[0120] resuspending the apoptotic cell population in administration medium; and

[0121] inactivating the monocyte-enriched population, wherein the inactivation occurs after induction,

[0122] Wherein the method produces a population of mononuclear apoptotic cells comprising a reduced percentage of non-quiescent non-apoptotic cells; suppressed cellular activation of any viable non-apoptotic cells; or reduced proliferation of any viable non-apoptotic cells; or any combination thereof.

[0123] 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 to a non-irradiated preparation of cells.

[0124] In another embodiment, the pooled mononuclear apoptotic cells have undergone T cell receptor inactivation. In another embodiment, the pooled mononuclear apoptotic cells have undergone T cell receptor editing.

[0125] In some embodiments, the pooled blood includes 3rd party blood from a source that is HLA matched or HLA mismatched with respect to the recipient.

[0126] In some embodiments, disclosed herein are compositions comprising genetically modified immune cells (e.g., but not limited to, CAR T cells) and additional agents selected from the group consisting of apoptotic cells, apoptotic cell supernatants, CTLA-4 blockers, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium compound, or an immunomodulator, or any combination thereof.

[0127] In some embodiments, the present disclosure provides a method of producing a pharmaceutical composition comprising a pooled mononuclear apoptotic cell preparation comprising a pooled population of single mononuclear cells in an early apoptotic state, wherein the composition comprises a reduced percentage of viable non-apoptotic cells, a preparation having suppressed cell activation of any viable non-apoptotic cells, or a preparation having reduced proliferation of any viable non-apoptotic cells, or any combination thereof. In another embodiment, the method provides a pharmaceutical composition comprising a pooled mononuclear apoptotic cell preparation comprising a pooled population of single mononuclear cells in an early apoptotic state, wherein the composition comprises a reduced percentage of non-quiescent non-apoptotic cells.

[0128] In some embodiments, disclosed herein is a method for treating, preventing, inhibiting the growth of a subject's cancer or tumor, reducing the incidence of a subject's cancer or tumor, or any combination thereof, the method comprising administering to the subject an early apoptotic cell population, wherein the method treats, prevents, inhibits the growth of a subject's cancer or tumor, reduces the incidence of a subject's cancer or tumor, or any combination thereof. In some embodiments, the methods herein include treating, preventing, inhibiting the growth of a subject's cancer or tumor, delaying disease progression, reducing tumor load, or reducing the incidence of a subject's cancer or tumor, or any combination thereof, the method comprising administering to the subject a composition comprising an early apoptotic cell population. In some embodiments, the method further comprises administering to the subject an additional immunotherapy, chemotherapeutic agent, or immunomodulator, or any combination thereof. In some embodiments, the additional immunotherapy, chemotherapeutic agent, or immunomodulator is administered before, simultaneously, or after administering the early apoptotic cells.

[0129] In some embodiments, disclosed herein is a method of increasing the survival of a subject with cancer or a tumor, the method comprising the step of administering to the subject an early apoptotic cell population, wherein the method increases the survival of the subject. In some embodiments, the method further comprises administering to the subject an additional immunotherapy, chemotherapeutic agent, or immunomodulator, or any combination thereof. In some embodiments, the additional immunotherapy, chemotherapeutic agent, or immunomodulator is administered before, simultaneously with, or after administering the early apoptotic cells.

[0130] In some embodiments, disclosed herein is a method of reducing the size of a cancer or tumor in a subject or reducing the growth rate of the cancer or tumor, or a combination thereof, the method comprising the step of administering to the subject an early apoptotic cell population, wherein the method reduces the size or reduces the growth rate. In some embodiments, the method further comprises administering to the subject an additional immunotherapy, chemotherapeutic agent, or immunomodulator, or any combination thereof. In some embodiments, the additional immunotherapy, chemotherapeutic agent, or immunomodulator is administered before, simultaneously with, or after administering the early apoptotic cells.

[0131] In some embodiments, the administration of a composition comprising apoptotic cells does not affect the efficacy of CAR T cells in treating, preventing, inhibiting cancer or tumors, reducing the incidence of the cancer or tumor, improving the cancer or tumor, reducing tumor load, or alleviating the cancer or tumor. In another embodiment, the administration of a composition comprising apoptotic cells does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting cancer or tumors, reducing the incidence of the cancer or tumor, improving the cancer or tumor, reducing tumor load, or alleviating the cancer or tumor by more than about 5%. In another embodiment, the administration of a composition comprising apoptotic cells does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting cancer or tumors, reducing the incidence of the cancer or tumor, improving the cancer or tumor, reducing tumor load, or alleviating the cancer or tumor by more than about 10%. In another embodiment, the administration of a composition comprising apoptotic cells does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting cancer or tumors, reducing the incidence of the cancer or tumor, improving the cancer or tumor, reducing tumor load, or alleviating the cancer or tumor by more than about 15%. In another embodiment, administration of a composition comprising apoptotic cells does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting, reducing the incidence of, ameliorating, reducing tumor burden, or alleviating cancer or tumors by more than about 20%.

[0132] In some embodiments, administration of apoptotic cells improves the efficacy of CAR T cells. In some embodiments, administration of apoptotic cells improves the efficacy of CAR T cells by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45, or at least 50%.

[0133] In another embodiment, the administration of a composition comprising apoptotic cell supernatant does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting the cancer or the tumor, reducing the incidence of the cancer or the tumor, improving or alleviating the cancer or the tumor by more than about 5%. In another embodiment, the administration of a composition comprising apoptotic cell supernatant does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting the cancer or the tumor, reducing the incidence of the cancer or the tumor, improving or alleviating the cancer or the tumor by more than about 10%. In another embodiment, the administration of a composition comprising apoptotic cell supernatant does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting the cancer or the tumor, reducing the incidence of the cancer or the tumor, improving or alleviating the cancer or the tumor by more than about 15%. In another embodiment, the administration of a composition comprising apoptotic cell supernatant does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting the cancer or the tumor, reducing the incidence of the cancer or the tumor, improving or alleviating the cancer or the tumor by more than about 20%. In another embodiment, the administration of a composition comprising the apoptotic cell supernatant does not affect the efficacy of CAR T cells in treating, preventing, inhibiting, reducing the incidence of, improving or alleviating the cancer or the tumor. In another embodiment, the administration of a composition comprising the apoptotic cell supernatant does not reduce the efficacy of CAR T cells in treating, preventing, inhibiting, reducing the incidence of, improving or alleviating the cancer or the tumor.

[0134] In some embodiments, disclosed herein is a method for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm. In another embodiment, the method disclosed herein reduces or prevents the cytokine production of a subject experiencing CAR T cell cancer therapy, thereby suppressing the cytokine release syndrome (CRS) or cytokine storm of the subject or reducing the incidence of the CRS or cytokine storm. In another embodiment, the method disclosed herein for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm includes the step of applying a composition including apoptotic cells to the subject experiencing the cancer therapy. In another embodiment, the method disclosed herein for reducing or suppressing the cytokine production of a subject experiencing CAR T cell cancer therapy includes applying a composition including apoptotic cells to the subject experiencing the cancer therapy. In another embodiment, the composition including apoptotic cells is administered without affecting the efficacy of CAR T cell therapy. In another embodiment, the composition including apoptotic cells or apoptotic supernatant is administered without reducing the efficacy of CAR T cell therapy. In another embodiment, administration of a composition comprising apoptotic cells or apoptotic cell supernatant does not reduce the efficacy of CAR T cell therapy by more than about 5%. In another embodiment, administration of a composition comprising apoptotic cells or apoptotic cell supernatant does not reduce the efficacy of CAR T cell therapy by more than about 10%. In another embodiment, administration of a composition comprising apoptotic cells or apoptotic cell supernatant does not reduce the efficacy of CAR T cell therapy by more than about 15%. In another embodiment, administration of a composition comprising apoptotic cells or apoptotic cell supernatant does not reduce the efficacy of CAR T cell therapy by more than about 20%.

[0135] In some embodiments, disclosed herein is a method of reducing or inhibiting cytokine production in a subject experiencing cytokine release syndrome or cytokine storm or susceptible to cytokine release syndrome or cytokine storm, the method comprising the step of administering an apoptotic cell supernatant as disclosed herein or a composition comprising the apoptotic cell supernatant. In another embodiment, the apoptotic cell supernatant comprises an apoptotic cell-phagocytic cell supernatant.

[0136] In some embodiments, the method disclosed herein for reducing or inhibiting cytokine production in a subject undergoing CAR T cell cancer therapy comprises the step of administering a composition comprising apoptotic cell supernatant to the subject undergoing the cancer therapy. In another embodiment, administering a composition comprising apoptotic cell supernatant does not affect the efficacy of CAR T cell therapy. In another embodiment, administering a composition comprising apoptotic cell supernatant does not reduce the efficacy of CAR T cell therapy.

[0137] In some embodiments, the method for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing T cells (CAR T cells) cancer therapy expressing a chimeric antigen receptor or reducing the incidence of the CRS or cytokine storm includes administering a composition comprising apoptotic cells or apoptotic supernatant to the subject. In another embodiment, suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing T cells (CAR T cells) cancer therapy expressing a chimeric antigen receptor or reducing the incidence of the CRS or cytokine storm reduces or suppresses the production of at least one proinflammatory cytokine of the subject.

[0138] In another embodiment, the present disclosure provides a method for using a pooled mononuclear apoptotic cell preparation comprising mononuclear cells in an early apoptotic state, as described herein, the pooled mononuclear apoptotic cell preparation is used to treat, prevent, improve, inhibit immune diseases, autoimmune diseases, inflammatory diseases, cytokine release syndrome (CRS), cytokine storm or infertility in a subject in need thereof or reduce the incidence of the above diseases. In another embodiment, a pooled mononuclear apoptotic cell preparation is disclosed herein, wherein in certain embodiments, the use of such a cell preparation does not require matching of donors and recipients, for example, by HLA typing.

[0139] Genetically modified immune cells

[0140] As is well known, genetic modification of immune cells is a strategy for immune cell therapy for cancer. These immune cell therapies are based on the manipulation of autologous or allogeneic immune cells and their administration to subjects in need. Immune cell-based therapies include natural killer cell therapy, dendritic cell therapy, and T cell immunotherapy (including therapies utilizing naive T cells, effector T cells (also known as T helper cells), cytotoxic T cells, and regulatory T cells (Treg)).

[0141] In one embodiment, disclosed herein is a composition comprising a genetically modified immune cell. In another embodiment, the genetically modified immune cell is a T cell. In another embodiment, the T cell is a naive T cell. In another embodiment, the T cell is a naive CD4+ In another embodiment, the T cell is a naive T cell. In another embodiment, the T cell is a naive CD8 + T cells. In another embodiment, the genetically modified immune cells are natural killer (NK) cells. In another embodiment, the genetically modified immune cells are dendritic cells. In yet another embodiment, the genetically modified T cells are cytotoxic T lymphocytes (CTL cells). In another embodiment, the genetically modified T cells are regulatory T cells (Treg). In another embodiment, the genetically modified T cells are chimeric antigen receptor (CAR) T cells. In another embodiment, the genetically modified T cells are genetically modified T cell receptor (TCR) cells.

[0142] In one embodiment, disclosed herein are compositions comprising genetically modified immune cells and additional agents selected from the group comprising apoptotic cells, apoptotic cell supernatants, CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators, or any combination thereof. In another embodiment, disclosed herein are compositions comprising genetically modified immune cells, apoptotic cells, and additional agents selected from the group comprising CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators, or any combination thereof. In another embodiment, disclosed herein are compositions comprising genetically modified immune cells, apoptotic cell supernatants, and additional agents selected from the group comprising CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators, or any combination thereof.

[0143] In one embodiment, the immune cell is cytotoxic. In another embodiment, genetically modified cytotoxic cells can be obtained from the bone marrow (autologous) or donor (allogeneic) of an individual. In other cases, cells are obtained from stem cells. For example, cytotoxic cells can be derived from human pluripotent stem cells, such as human embryonic stem cells or human induced pluripotent T cells. In the case of induced pluripotent stem cells (IPSC), such pluripotent stem T cells can be obtained using somatic cells from the individual to which genetically modified cytotoxic cells will be provided. In one embodiment, immune cells can be obtained from an individual or a donor in the following manner: by venipuncture, by apheresis, by leukocytes moving followed by apheresis or venipuncture or by bone marrow puncture to collect cells.

[0144] In one embodiment, immune cells (such as T cells) are produced and amplified by the presence of specific factors in vivo. In another embodiment, T cells produce and maintain the influence of intracellular cytokines. In another embodiment, the cytokines affecting the generation and maintenance of T helper cells in vivo include IL-1, IL-2, IL-4, IL-6, IL-12, IL-21, IL-23, IL-25, IL-33 and TGF β. In another embodiment, Treg cells are produced from primary T cells by cytokine induction in vivo. In still another embodiment, TGF-β and / or IL-2 work in differentiation primary T cells called Treg cells.

[0145] In another embodiment, the presence of a cytokine selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-12, IL-21, IL-23, IL-25, IL-33 and TGFβ maintains or improves the proliferation rate of T cells in vivo or both. In another embodiment, the presence of cytokines IL-2 and / or TGFβ maintains or improves the proliferation rate of T cells in vivo or both. In another embodiment, the presence of a cytokine selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-12, IL-21, IL-23, IL-25, IL-33 and TGFβ maintains or improves the proliferation rate of CAR T cells in vivo or both. In another embodiment, the presence of cytokines IL-2 and / or TGFβ maintains or improves the proliferation rate of CAR T cells in vivo or both. In another embodiment, the presence of a cytokine selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-12, IL-21, IL-23, IL-25, IL-33 and TGFβ maintains or improves the proliferation rate of TCR T cells in vivo, or both. In another embodiment, the presence of the cytokine IL-2 and / or TGFβ maintains or improves the proliferation rate of TCR T cells in vivo, or both. In another embodiment, the presence of a cytokine selected from the group consisting of IL-1, IL-2, IL-4, IL-6, IL-12, IL-21, IL-23, IL-25, IL-33 and TGFβ maintains or improves the proliferation rate of T-reg cells in vivo, or both. In another embodiment, the presence of the cytokine IL-2 and / or TGFβ maintains or improves the proliferation rate of T-reg cells in vivo, or both.

[0146] In one embodiment, T cells with altered expression or form of a protein encoded by STAT5B or a protein encoded by BACH2 are maintained for an extended period of time or have an increased proliferation rate or both. In another embodiment, the altered expression increases expression of a STAT5B polypeptide. In another embodiment, the altered expression increases expression of a BACH2 polypeptide.

[0147] In another embodiment, T cells with altered expression of a protein encoded by STAT5B are maintained in vivo for an extended period of time or have an increased proliferation rate. In another embodiment, T cells with altered expression of a protein encoded by BACH2 are maintained in vivo for an extended period of time or have an increased proliferation rate. In another embodiment, T cells with an altered form of a protein encoded by STAT5B are maintained in vivo for an extended period of time or have an increased proliferation rate. In another embodiment, T cells with an altered form of a protein encoded by BACH2 are maintained in vivo for an extended period of time or have an increased proliferation rate.

[0148] In another embodiment, the T cells with altered expression of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 1 year. In another embodiment, the T cells with altered expression of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 2 years. In another embodiment, the T cells with altered expression of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 3 years. In another embodiment, the T cells with altered expression of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 4 years. In another embodiment, the T cells with altered expression of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 5 years. In another embodiment, the T cells with altered expression of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 10 years. In another embodiment, the T cells with altered expression of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 20 years.

[0149] In another embodiment, a T cell having altered expression of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 1 year. In another embodiment, a T cell having altered expression of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 2 years. In another embodiment, a T cell having altered expression of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 3 years. In another embodiment, a T cell having altered expression of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 4 years. In another embodiment, a T cell having altered expression of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 5 years. In another embodiment, a T cell having altered expression of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 10 years. In another embodiment, a T cell having altered expression of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 20 years.

[0150] In another embodiment, the T cells having an altered form of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 1 year. In another embodiment, the T cells having an altered form of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 2 years. In another embodiment, the T cells having an altered form of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 3 years. In another embodiment, the T cells having an altered form of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 4 years. In another embodiment, the T cells having an altered form of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 5 years. In another embodiment, the T cells having an altered form of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 10 years. In another embodiment, the T cells having an altered form of the protein encoded by STAT5B maintain or increase their proliferation rate in vivo for more than 20 years.

[0151] In another embodiment, a T cell having an altered form of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 1 year. In another embodiment, a T cell having an altered form of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 2 years. In another embodiment, a T cell having an altered form of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 3 years. In another embodiment, a T cell having an altered form of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 4 years. In another embodiment, a T cell having an altered form of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 5 years. In another embodiment, a T cell having an altered form of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 10 years. In another embodiment, a T cell having an altered form of a protein encoded by BACH2 maintains or increases its proliferation rate in vivo for more than 20 years.

[0152] In another embodiment, CAR T cells with altered expression of a protein encoded by STAT5B are maintained in vivo for an extended period of time or have an increased proliferation rate. In another embodiment, CAR T cells with altered expression of a protein encoded by BACH2 are maintained in vivo for an extended period of time or have an increased proliferation rate. In another embodiment, CAR T cells with an altered form of a protein encoded by STAT5B are maintained in vivo for an extended period of time or have an increased proliferation rate. In another embodiment, CAR T cells with an altered form of a protein encoded by BACH2 are maintained in vivo for an extended period of time or have an increased proliferation rate.

[0153] In another embodiment, TCR T cells with altered expression of a protein encoded by STAT5B maintain an extended period of time in vivo or have an increased proliferation rate. In another embodiment, TCR T cells with altered expression of a protein encoded by BACH2 maintain an extended period of time in vivo or have an increased proliferation rate. In another embodiment, TCR T cells with an altered form of a protein encoded by STAT5B maintain an extended period of time in vivo or have an increased proliferation rate. In another embodiment, TCR T cells with an altered form of a protein encoded by BACH2 maintain an extended period of time in vivo or have an increased proliferation rate.

[0154] In another embodiment, Treg cells with altered expression of a protein encoded by STAT5B maintain or increase their proliferation rate in vivo. In another embodiment, Treg cells with altered expression of a protein encoded by BACH2 maintain or increase their proliferation rate in vivo. In another embodiment, Treg cells with an altered form of a protein encoded by STAT5B maintain or increase their proliferation rate in vivo. In another embodiment, Treg cells with an altered form of a protein encoded by BACH2 maintain or increase their proliferation rate in vivo.

[0155] In one embodiment, disclosed herein is a method for maintaining or increasing the proliferation rate of genetically modified immune cells, wherein the method includes the step of administering apoptotic cells or apoptotic supernatants. In another embodiment, disclosed herein is a method for improving the efficacy of genetically modified immune cells, wherein the method includes the step of administering additional agents, wherein the additional agents include apoptotic cells, apoptotic supernatants, CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators or any combination thereof. In another embodiment, a method for treating, preventing, inhibiting, reducing the incidence of, improving or alleviating a cancer or tumor disclosed herein administers genetically modified immune cells and additional agents, wherein the additional agents include apoptotic cells, apoptotic supernatants, CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators or any combination thereof.

[0156] Chimeric Antigen Receptor-Expressing T Cells (CAR T Cells)

[0157] In some embodiments, a chimeric antigen receptor (CAR) is a type of antigen targeting receptor consisting of an intracellular T cell signaling domain (most commonly a single chain variable fragment (scFv) from a monoclonal antibody) fused to an extracellular tumor binding portion. CAR directly recognizes cell surface antigens independently of MHC-mediated presentation, thereby allowing the use of a single receptor construct specific for any given antigen in all patients. The initial CAR fuses the antigen recognition domain to the CD3ζ activation chain of the T cell receptor (TCR) complex. Although these first-generation CARs induce T cell effector functions in vitro, they are largely limited by poor anti-tumor efficacy in vivo. Subsequent CAR iterations have secondary costimulatory signals in series with CD3ζ, including intracellular domains from CD28 or various TNF receptor family molecules, such as 4-1BB (CD137) and OX40 (CD134). Further, in addition to CD3ζ, the third-generation receptor includes two costimulatory signals, most commonly from CD28 and 4-1BB. Second- and third-generation CARs can significantly improve antitumor efficacy, in some cases inducing complete remissions in patients with advanced cancer.

[0158] In some embodiments, a CAR T cell is an immune response cell that includes an antigen receptor that is activated when its receptor binds to its antigen.

[0159] In some embodiments, the CAR T cells used in the compositions and methods disclosed herein are first generation CAR T cells. In another embodiment, the CAR T cells used in the compositions and methods disclosed herein are second generation CAR T cells. In another embodiment, the CAR T cells used in the compositions and methods disclosed herein are third generation CAR T cells. In another embodiment, the CAR T cells used in the compositions and methods disclosed herein are fourth generation CAR T cells. In some embodiments, each generation of CAR T cells is more effective than the previous generations of CAR T cells.

[0160] In some embodiments, the first generation CAR has one signaling domain, typically the cytoplasmic signaling domain of the CD3 TCR zeta chain.

[0161] In another embodiment, the CAR T cell as disclosed herein is a second generation CAR T cell. In another embodiment, the CAR T cell as disclosed herein includes a triple chimeric receptor (TPCR). In some embodiments, the CAR T cell as disclosed herein includes one or more signal transduction parts that activate the original T cell in a costimulation-independent manner. In another embodiment, the CAR T cell further encodes one or more members of the tumor necrosis factor receptor family, which in some embodiments is CD27, 4-1BB (CD137) or OX40 (CD134) or a combination thereof.

[0162] The third generation CAR T cells attempt to utilize the signaling potential of 2 costimulatory domains: in some embodiments, the CD28 domain is followed by a 4-1BB or OX-40 signaling domain. In another embodiment, the CAR T cells used in the compositions and methods disclosed herein further encode a costimulatory signaling domain (which is CD28 in one embodiment). In another embodiment, the signaling domain is a CD3 ζ chain, CD97, GDI 1a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, CD28 signaling domain, or a combination thereof.

[0163] In some embodiments, telomere length and replication capacity correlate with engraftment efficiency and anti-tumor efficacy of adoptively transferred T cell lines. In some embodiments, CD28 stimulation maintains telomere length in T cells.

[0164] In some embodiments, the potency of CAR-modified T cells can be further enhanced by introducing additional genes (including those encoding proliferative cytokines (ie, IL-12) or co-stimulatory ligands (ie, 4-1BBL)), thus producing "armored" fourth-generation CAR-modified T cells. In some embodiments, "armored CAR T cells" are CAR T cells that are protected from suppressive tumor microenvironments. In another embodiment, "armored" CAR technology combines local secretion of soluble signaling proteins to amplify immune responses within the tumor microenvironment, with the goal of minimizing systemic side effects. In some embodiments, the signaling protein signal is IL-12, which can stimulate T cell activation and recruitment. In some embodiments, "armored" CAR technology is particularly useful in solid tumor indications, where microenvironment and effective immunosuppression mechanisms have the potential to make the establishment of robust anti-tumor responses more challenging.

[0165] In some embodiments, CAR T cells are genetically modified to encode molecules involved in preventing apoptosis, remodeling the tumor microenvironment, inducing homeostatic proliferation, and promoting directed T cell homing.

[0166] In another embodiment, the CAR T cell therapy used in the compositions and methods disclosed herein is enhanced using expression of a cytokine transgene, combination therapy with a small molecule inhibitor, or a monoclonal antibody. In another embodiment, other strategies intended to improve CAR T cell therapy (including the use of dual CARs and chemokine receptors to more specifically target tumor cells) are considered as part of CAR T cells and CAR T cell therapy as disclosed herein.

[0167] In some embodiments, the CAR T cells of the compositions and methods disclosed herein include a second binding domain that can cause an inhibitory or amplified signal to increase the specificity of the CAR T cells for cancer cells relative to normal cells. For example, the CAR T cell can be engineered so that it will be triggered in the presence of one target protein, but if there is a second protein, it will be inhibited. Alternatively, it can also be engineered so that maximum activation will require two target proteins. These methods can increase the specificity of CAR for tumors relative to normal tissues.

[0168] In some embodiments, the CAR T cells used in the compositions and methods disclosed herein encode an antibody-based external receptor structure and a cytoplasmic domain encoding a signaling module composed of an immunoreceptor tyrosine-based activation motif.

[0169] In some embodiments, the CAR T cell further encodes a single chain variable fragment (scFv) that binds a polypeptide with immunosuppressive activity. In another embodiment, the polypeptide with immunosuppressive activity is CD47, PD-1, CTLA-4, or a combination thereof.

[0170] In some embodiments, CAR T cells further encode a single-chain variable fragment (scFv) binding to a polypeptide with immunostimulatory activity. In another embodiment, the polypeptide with immunostimulatory activity is CD28, OX-40, 4-1BB or a combination thereof. In another embodiment, CAR T cells further encode CD40 ligand (CD40L), which, in some embodiments, enhances the immunostimulatory activity of the antigen.

[0171] In some embodiments, the immune cell is cytotoxic. In another embodiment, the cytotoxic cells for genetic modification can be obtained from the bone marrow of a subject or a donor. In other cases, the cell is obtained from a stem cell. For example, the cytotoxic cells can be derived from human pluripotent stem cells, such as human embryonic stem cells or human induced pluripotent T cells. In the case of induced pluripotent stem cells (IPSC), such pluripotent T cells can be obtained using somatic cells from a subject to which genetically modified cytotoxic cells are provided. In some embodiments, immune cells can be obtained from a subject or a donor by venipuncture, by apheresis method, by mobilizing leukocytes after apheresis or venipuncture, or by bone marrow aspiration.

[0172] In some embodiments, the methods disclosed herein include obtaining immune cells from a subject and genetically modifying the immune cells to express a chimeric antigen receptor. In another embodiment, the methods disclosed herein include obtaining immune cells from a subject, genetically modifying the immune cells to express a chimeric antigen receptor, and combining with a population of apoptotic cells, thereby resulting in a decrease in cytokine production in the subject, but relative to CAR-expressing immune cells not administered with apoptotic cell populations, cytotoxicity is substantially unaffected ( Figure 1A-1B And 2). In another embodiment, the method disclosed herein includes obtaining immune cells from a subject, genetically modifying the immune cells to express a chimeric antigen receptor, and combining with apoptotic cell supernatant or a composition comprising the supernatant, thereby resulting in a decrease in cytokine production in the subject, but relative to CAR-expressing immune cells not administered with apoptotic cell supernatant, cytotoxicity is substantially unaffected. In another embodiment, administration of apoptotic cell populations or supernatants from apoptotic cells does not reduce the efficacy of immune cells expressing chimeric antigen receptors.

[0173] In one embodiment, disclosed herein are immune cells (in some embodiments, CAR T cells), wherein the T cells are autologous to the subject. In another embodiment, the CAR T cells are heterologous to the subject. In some embodiments, the CAR T cells are allogeneic. In some embodiments, the CAR T cells are universal allogeneic CAR T cells. In another embodiment, the T cells can be autologous, allogeneic, or derived in vitro from engineered progenitor cells or stem cells.

[0174] In another embodiment, the CAR T cells and apoptotic cells described herein are both derived from the same source. In another embodiment, the CAR T cells and apoptotic cells described herein are both derived from a subject (Fig. 1). In an alternative embodiment, the CAR T cells and apoptotic cells described herein are derived from different sources. In yet another embodiment, the CAR T cells are autologous, and the apoptotic cells described herein are allogeneic ( Figure 2 ). The skilled person will understand that, similarly, the apoptotic cell supernatant can be prepared from cells derived from the same source as the CAR T cells (which in one embodiment can be autologous cells), or the apoptotic cell supernatant can be prepared from cells derived from a source different from that of the CAR T cells.

[0175] The skilled artisan will appreciate that the term "heterologous" can encompass tissues, cells, nucleic acid molecules or polypeptides derived from different organisms. In some embodiments, a heterologous protein is a protein that was originally cloned or derived from a different T cell type or species than the recipient and is not normally present in the cell or in a sample obtained from the cell.

[0176] Therefore, as disclosed herein, one embodiment relates to a cytotoxic immune cell (e.g., NK cell or T cell), which includes a chimeric antigen receptor (CAR), whereby the cell maintains its cytotoxic function. In another embodiment, the chimeric antigen receptor is exogenous to the T cell. In another embodiment, the CAR is expressed in a recombinant manner. In another embodiment, the CAR is expressed from a vector.

[0177] In some embodiments, the T cells used to generate CAR T cells are naive CD4 + In another embodiment, the T cells used to generate CAR T cells are naive CD8 + T cells. In another embodiment, the T cells used to produce CAR T cells are effector T cells. In another embodiment, the T cells used to produce CAR T cells are regulatory T cells (Treg). In another embodiment, the T cells used to produce CAR T cells are cytotoxic T cells.

[0178] Sources of genetically modified immune cells (e.g., T cells) have been extensively described in the literature, see, e.g., Themelli et al. (2015) New Cell Sources for T Cell Engineering and Adoptive Immunotherapy. Cell Stem Cell 16:357-366; Han et al. (2013) Journal of Hematology & Oncology 6:47-53; Wilkie et al. (2010) J Bio Chem 285(33):25538-25544; and van der Stegen et al. (2013) J. Immunol 191:4589-4598. CAR T cells can be ordered from commercial sources such as Creative Laboratories. Biolabs) (New York, USA), which provides customized construction and production services for chimeric antigen receptors (CARs) and also provides a pre-prepared CAR construct inventory that can be induced by recombinant adenovirus vaccines to encode protective immunity. Customized CAR T cells can also be obtained from Promab Biotechnologies (California, USA), which can provide specially designed CAR T cells.

[0179] T cell receptor (TCR) cells

[0180] In one embodiment, in addition to or in place of CAR T cells, compositions and methods as disclosed herein also utilize designer T cell receptor (TCR) cells. TCR is a multi-subunit transmembrane complex that mediates antigen-specific activation of T cells. TCR is composed of two different polypeptide chains. TCR confers antigen specificity to T cells by recognizing antigen epitopes on target cells (e.g., tumors or cancer cells). After contact with antigens present on tumors or cancer cells, T cells proliferate and acquire phenotypes and functions that allow them to eliminate cancer or tumor cells.

[0181] In one embodiment, TCR T cell therapy comprises introducing a T cell receptor (TCR) specific for an epitope of a protein of interest into a T cell. In another embodiment, the protein of interest is a tumor-associated antigen. In another embodiment, a genetically engineered TCR recognizes tumor antigen epitopes presented by the major histocompatibility complex (MHC) on tumor cells and T cell activation domains. In another embodiment, the T cell receptor recognizes antigens, regardless of their intracellular or membrane localization. In another embodiment, TCR recognizes tumor cells expressing tumor-associated antigens in cells. In one embodiment, TCR recognizes internal antigens. In another embodiment, TCR recognizes angiogenic factors. In another embodiment, angiogenic factors are molecules involved in the formation of new blood vessels. Various genetically modified T cell receptors and methods for producing them are known in the art.

[0182] In one embodiment, TCR T cell therapy is used to treat, prevent, inhibit, improve, reduce the incidence of cancer or tumors, or alleviate cancer or tumors. In one embodiment, TCR T cell therapy is used to treat, prevent, inhibit, improve, reduce the incidence of advanced metastatic disease, or alleviate advanced metastatic disease, including diseases with blood (lymphoma and leukemia) and solid tumors (refractory melanoma, sarcoma). In one embodiment, the TCR T cell therapy used in the compositions and methods disclosed herein treats the malignancies listed in Table 1 of the following documents: Sadelain et al., Cancer Discov. April 2013; 3(4): 388-398).

[0183] In another embodiment, the T cell receptor is genetically modified to bind to the NY-ESO-1 epitope, and the TCR engineered T cell is anti-NY-ESO-1. In another embodiment, the T cell receptor is genetically modified to bind to the HPV-16E6 epitope, and the TCR engineered T cell is anti-HPV-16E6. In another embodiment, the T cell receptor is genetically modified to bind to the HPV-16E7 epitope, and the TCR engineered T cell is anti-HPV-16E7. In another embodiment, the T cell receptor is genetically modified to bind to the MAGE A3 / A6 epitope, and the TCR engineered T cell is anti-MAGE A3 / A6. In another embodiment, the T cell receptor is genetically modified to bind to the MAGE A3 epitope, and the TCR engineered T cell is anti-MAGEA3. In another embodiment, the T cell receptor is genetically modified to bind to the SSX2 epitope, and the TCR engineered T cell is anti-SSX2. In another embodiment, the T cell receptor is genetically modified to bind to a target antigen disclosed herein. Using tools well known in the art, one of skill will appreciate that a T cell receptor can be genetically modified to bind a target antigen present on a cancer or tumor cell, wherein the TCR engineered T cell comprises an anti-tumor or anti-cancer cell.

[0184] In one embodiment, the method as disclosed herein comprises obtaining immune cells from an individual, and genetically modifying the immune cells to express a recombinant T cell receptor (TCR). In another embodiment, the method as disclosed herein comprises obtaining immune cells from a subject, genetically modifying the immune cells to express a recombinant TCR, and combining with another agent, wherein the other agent comprises an apoptotic cell population, an apoptotic cell supernatant, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or an analog thereof, a tellurium compound or an immunomodulator or any combination thereof.

[0185] In one embodiment, the T cells used to generate TCR T cells are naive CD4 + In another embodiment, the T cells used to generate TCR T cells are naive CD8 + T cells. In another embodiment, the T cells used to produce TCR T cells are effector T cells. In another embodiment, the T cells used to produce TCR T cells are regulatory T cells (Treg). In another embodiment, the T cells used to produce TCR T cells are cytotoxic T cells.

[0186] TCR T cells have been extensively described in the literature, see, e.g., Sharpe and Mount (2015) supra; Essand M, Loskog ASI (2013) Genetically engineered T cells for the treatment of cancer (Review). J Intern Med 273: 166-181; and Kershaw et al. (2014) Clinical application of genetically modified T cells in cancer therapy. Clinical & Translational Immunology 3: 1-7.

[0187] Targeting Antigen

[0188] In some embodiments, the CAR binds to the epitope of the antigen through an antibody or antibody fragment directed against the antigen. In another embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. In another embodiment, the antibody fragment is a single chain variable fragment (scFv).

[0189] In one embodiment, the TCR binds to an epitope of an antigen through a genetically modified T cell receptor.

[0190] In another embodiment, the CAR T cells of the composition disclosed herein are bound to a tumor-associated antigen (TAA). In another embodiment, the tumor-associated antigen is: mucin 1, cell surface-associated (MUC1) or polymorphic epithelial mucin (PEM), arginine-rich mutated in early tumors (Armet), heat shock protein 60 (HSP60), calnexin (CANX), methenyltetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2), fibroblast activation protein (FAP), matrix metallopeptidase (MMP6), B melanoma antigen-1 (B AGE-1), aberrant transcript of N-acetylglucosaminyltransferase V (GnTV), Q5H943, carcinoembryonic antigen (CEA), Pmel, kallikrein-4, mammaglobin-1, MART-1, GPR143-OA1, prostate-specific antigen (PSA), TRP1, tyrosinase, FGP-5, NEU proto-oncogene, Aft, MMP-2, prostate-specific membrane antigen (PSMA), telomerase-associated protein-2, prostatic acid phosphatase (PAP), uroprotein II, or proteinase 3.

[0191] In another embodiment, the CAR binds to CD19 or CD20 to target B cells when it wants to destroy B cells (such as in leukemia). CD19 is a B cell lineage-specific surface receptor, and its widespread expression from progenitor B cells to early plasma cells makes it an attractive target for immunotherapy of B cell malignancies. In another embodiment, the CAR binds to ROR1, CD22 or GD2. In another embodiment, CAR binds to NY-ESO-1. In another embodiment, the CAR binds to MAGE family proteins. In another embodiment, the CAR binds to mesothelin. In another embodiment, the CAR binds to c-erbB2. In another embodiment, CAR binds to tumor-specific mutant antigens, such as BRAFV600E mutations and BCR-ABL translocations. In another embodiment, CAR binds to tumor-specific viral antigens, such as EBV in HD, HPV in cervical cancer, and polyomavirus in Merkel cancer. In another embodiment, CAR T cells bind to Her2 / neu. In another embodiment, the CAR T cells bind to EGFRvIII.

[0192] In some embodiments, the chimeric antigen receptor (CAR) T cell binds to the CD19 antigen. In another embodiment, the CAR binds to the CD22 antigen. In another embodiment, the CAR binds to the alpha folate receptor. In another embodiment, the CAR binds to CAIX. In another embodiment, the CAR binds to CD20. In another embodiment, the CAR binds to CD23. In another embodiment, the CAR binds to CD24. In another embodiment, the CAR binds to CD30. In another embodiment, the CAR binds to CD33. In another embodiment, the CAR binds to CD38. In another embodiment, the CAR binds to CD44v6. In another embodiment, the CAR binds to CD44v7 / 8. In another embodiment, the CAR binds to CD123. In another embodiment, the CAR binds to CD171. In another embodiment, the CAR binds to carcinoembryonic antigen (CEA). In another embodiment, the CAR binds to EGFRvIII. In another embodiment, the CAR binds to EGP-2. In another embodiment, the CAR binds to EGP-40. In another embodiment, the CAR binds to EphA2. In another embodiment, the CAR binds to Erb-B2. In another embodiment, the CAR binds to Erb-B 2, 3, 4. In another embodiment, the CAR binds to Erb-B3 / 4. In another embodiment, the CAR binds to FBP. In another embodiment, the CAR binds to fetal acetylcholinesterase receptor. In another embodiment, the CAR binds to G D2 In another embodiment, the CAR is combined with G D3In another embodiment, the CAR binds to HER2. In another embodiment, the CAR binds to HMW-MAA. In another embodiment, the CAR binds to IL-11Rα. In another embodiment, the CAR binds to IL-13Rα1. In another embodiment, the CAR binds to KDR. In another embodiment, the CAR binds to a kappa light chain. In another embodiment, the CAR binds to Lewis Y. In another embodiment, the CAR binds to L1 cell adhesion molecule. In another embodiment, the CAR binds to MAGE-A1. In another embodiment, the CAR binds to mesothelin. In another embodiment, the CAR binds to CMV infected cells. In another embodiment, the CAR binds to MUC1. In another embodiment, the CAR binds to MUC16. In another embodiment, the CAR binds to NKG2D ligands. In another embodiment, the CAR binds to NY-ESO-1 (amino acids 157-165). In another embodiment, the CAR binds to carcinoembryonic antigen (h5T4). In another embodiment, the CAR binds to PSCA. In another embodiment, the CAR binds to PSMA. In another embodiment, the CAR binds to ROR1. In another embodiment, the CAR binds to TAG-72. In another embodiment, the CAR binds to VEGF-R2 or other VEGF receptors. In another embodiment, the CAR binds to B7-H6. In another embodiment, the CAR binds to CA9. In another embodiment, the CAR binds to α v β 6 Integrin binding. In another embodiment, the CAR binds to 8H9. In another embodiment, the CAR binds to NCAM. In another embodiment, the CAR binds to fetal acetylcholinesterase receptor.

[0193] In another embodiment, the chimeric antigen receptor (CAR) T cells target the CD19 antigen and have a therapeutic effect on subjects with B cell malignancies, ALL, follicular lymphoma, CLL and lymphoma. In another embodiment, the CAR T cells target the CD22 antigen and have a therapeutic effect on subjects with B cell malignancies. In another embodiment, the CAR T cells target the alpha folate receptor or folate receptor alpha and have a therapeutic effect on subjects with ovarian cancer or epithelial cancer. In another embodiment, the CAR T cells target CAIX or G250 / CAIX and have a therapeutic effect on subjects with renal cell carcinoma. In another embodiment, the CAR T cells target CD20 and have a therapeutic effect on subjects with lymphoma, B cell malignancies, B cell lymphoma, mantle cell lymphoma, indolent B cell lymphoma. In another embodiment, the CAR T cells target CD23 and have a therapeutic effect on subjects with CLL. In another embodiment, the CAR T cells target CD24 and have a therapeutic effect on subjects with pancreatic cancer. In another embodiment, the CAR T cells target CD30 and have a therapeutic effect on subjects with lymphoma or Hodgkin lymphoma. In another embodiment, the CAR T cells target CD33 and have a therapeutic effect on subjects with AML. In another embodiment, the CAR T cells target CD38 and have a therapeutic effect on subjects with non-Hodgkin lymphoma. In another embodiment, the CAR T cells target CD44v6 and have a therapeutic effect on subjects with severe malignancies. In another embodiment, the CAR T cells target CD44v7 / 8 and have a therapeutic effect on subjects with cervical cancer. In another embodiment, the CAR T cells target CD123 and have a therapeutic effect on subjects with myeloid malignancies. In another embodiment, the CAR T cells target CEA and have a therapeutic effect on subjects with colorectal cancer. In another embodiment, the CAR T cells target EGFRvIII and have a therapeutic effect on subjects with glioblastoma. In another embodiment, the CAR T cells target EGP-2 and have a therapeutic effect on a subject with multiple malignancies. In another embodiment, the CAR T cells target EGP-40 and have a therapeutic effect on a subject with colorectal cancer. In another embodiment, the CAR T cells target EphA2 and have a therapeutic effect on a subject with glioblastoma.In another embodiment, the CAR T cells target Erb-B2 or ErbB3 / 4 and have a therapeutic effect on subjects with breast cancer and other cancers, prostate cancer, colon cancer, and various tumors. In another embodiment, the CAR T cells target Erb-B 2, 3, 4, and have a therapeutic effect on subjects with breast cancer and other cancers. In another embodiment, the CAR T cells target FBP and have a therapeutic effect on subjects with ovarian cancer. In another embodiment, the CAR T cells target fetal acetylcholinesterase receptor and have a therapeutic effect on subjects with rhabdomyosarcoma. In another embodiment, the CAR T cells target G. D2 , and has a therapeutic effect on subjects with neuroblastoma, melanoma or Ewing's sarcoma. In another embodiment, the CAR T cells target G D3, and has a therapeutic effect on subjects with melanoma. In another embodiment, the CAR T cells target HER2 and have a therapeutic effect on subjects with medulloblastoma, pancreatic cancer, glioblastoma, osteosarcoma or ovarian cancer. In another embodiment, the CART cells target HMW-MAA and have a therapeutic effect on subjects with melanoma. In another embodiment, the CAR T cells target IL-11Rα and have a therapeutic effect on subjects with osteosarcoma. In another embodiment, the CAR T cells target IL-13Rα1 and have a therapeutic effect on subjects with glioma, glioblastoma or medulloblastoma. In another embodiment, the CAR T cells target IL-13 receptor α2 and have a therapeutic effect on subjects with severe malignancies. In another embodiment, the CAR T cells target KDR and have a therapeutic effect on subjects with tumors by targeting tumor neovasculature. In another embodiment, the CAR T cells target kappa light chains and have a therapeutic effect on subjects with B cell malignancies (B-NHL, CLL). In another embodiment, the CAR T cells target Lewis Y and have a therapeutic effect on subjects with various cancers or epithelial tumors. In another embodiment, the CAR T cells target L1 cell adhesion molecules and have a therapeutic effect on subjects with neuroblastoma. In another embodiment, the CAR T cells target MAGE-A1 or HLA-A1 MAGEA1 and have a therapeutic effect on subjects with melanoma. In another embodiment, the CAR T cells target mesothelin and have a therapeutic effect on subjects with mesothelioma. In another embodiment, the CAR T cells target CMV infected cells and have a therapeutic effect on subjects with CMV. In another embodiment, the CAR T cells target MUC1 and have a therapeutic effect on subjects with breast cancer and ovarian cancer. In another embodiment, the CAR T cells target MUC16 and have a therapeutic effect on subjects with ovarian cancer. In another embodiment, the CAR T cells target NKG2D ligands and have a therapeutic effect on subjects with myeloma, ovarian tumors, and other tumors. In another embodiment, the CAR T cells target NY-ESO-1 (157-165) or HLA-A2 NY-ESO-1 and have a therapeutic effect on subjects with multiple myeloma. In another embodiment, the CAR T cells target carcinoembryonic antigen (h5T4) and have a therapeutic effect on subjects with various tumors. In another embodiment, the CAR T cells target PSCA and have a therapeutic effect on subjects with prostate cancer.In another embodiment, the CAR T cells target PSMA and have a therapeutic effect on subjects with prostate cancer / tumor vasculature. In another embodiment, the CAR T cells target ROR1 and have a therapeutic effect on subjects with B-CLL and mantle cell lymphoma. In another embodiment, the CAR T cells target TAG-72 and have a therapeutic effect on subjects with adenocarcinoma or gastrointestinal cancer. In another embodiment, the CAR T cells target VEGF-R2 or other VEGF receptors and have a therapeutic effect on subjects with tumors by targeting tumor neovasculature. In another embodiment, the CAR T cells target CA9 and have a therapeutic effect on subjects with renal cell carcinoma. In another embodiment, the CAR T cells target CD171 and have a therapeutic effect on subjects with renal neuroblastoma. In another embodiment, the CAR T cells target NCAM and have a therapeutic effect on subjects with neuroblastoma. In another embodiment, the CAR T cells target fetal acetylcholinesterase receptor and have a therapeutic effect on subjects with rhabdomyosarcoma. In another embodiment, the CAR binds to one of the target antigens listed in Table 1 of the following document, Sadelain et al. Cancer Discov. April 2013; 3(4):388-398), which is incorporated herein by reference in its entirety. In another embodiment, the CAR T cell binds to a carbohydrate or glycolipid structure.

[0194] In one embodiment, the CAR T cells bind to angiogenic factors, thereby targeting tumor vasculature. In some embodiments, the angiogenic factor is VEGFR2. In another embodiment, the angiogenic factor is endoglin. In another embodiment, the angiogenic factor disclosed herein is angiogenin; angiopoietin 1; Del-1; fibroblast growth factor; acidic (aFGF) and basic (bFGF); follistatin; granulocyte colony stimulating factor (G-CSF); hepatocyte growth factor (HGF) / scatter factor (SF); interleukin-8 (IL-8); leptin; midkine; placental growth factor; platelet-derived endothelial cell growth factor (PD-ECGF); platelet-derived growth factor-BB (PDGF-BB); pleiotropic growth factor (PTN); progranulin; proliferative protein; transforming growth factor-α (TGF-α); transforming growth factor-β (TGF-β); tumor necrosis factor-α (TNF-α); vascular endothelial growth factor (VEGF) / vascular permeability factor (VPF). In another embodiment, the angiogenic factor is an angiogenic protein. In some embodiments, the growth factor is an angiogenic protein. In some embodiments, the angiogenic protein used in the compositions and methods disclosed herein is fibroblast growth factor (FGF); VEGF; VEGFR and neuropilin 1 (NRP-1); angiopoietin 1 (Ang1) and Tie2; platelet-derived growth factor (PDGF; BB-homodimer) and PDGFR; transforming growth factor β (TGF-β), endoglin and TGF-β receptor; monocyte chemoattractant protein-1 (MCP-1); integrins αVβ3, αVβ5 and α5β1; VE-cadherin and CD31; ephrins; plasminogen activator; plasminogen activator inhibitor-1; nitric oxide synthase (NOS) and COX-2; AC133; or Id1 / Id3. In some embodiments, the angiogenic protein used in the compositions and methods disclosed herein is an angiogenin, which in one embodiment is angiogenin 1, angiogenin 3, angiogenin 4, or angiogenin 6. In some embodiments, endoglin is referred to as CD105; EDG; HHT1; ORW; or ORW1. In some embodiments, endoglin is a TGFβ co-receptor.

[0195] In another embodiment, the CAR T cell binds to an antigen associated with an infectious agent. In some embodiments, the infectious agent is Mycobacterium tuberculosis. In some embodiments, the Mycobacterium tuberculosis-associated antigen is: antigen 85B, lipoprotein IpqH, ATP-dependent helicase putative, uncharacterized protein Rv0476 / MTO4941 precursor, or uncharacterized protein Rv1334 / MT1376 precursor.

[0196] In another embodiment, the CAR T cell is combined with an antibody. In some embodiments, the CAR T cell is an "antibody-coupled T cell receptor" (ACTR). According to this embodiment, the CAR T cell is a universal CAR T cell. In another embodiment, a CAR T cell with an antibody receptor is administered before, after, or simultaneously with the administration of an antibody, and then binds to the antibody, bringing the T cell into close proximity with a tumor or cancer. In another embodiment, the antibody is directed against a tumor cell antigen. In another embodiment, the antibody is directed against CD20. In another embodiment, the antibody is rituximab.

[0197] In another embodiment, the antibody is Trastuzumab (Herceptin; Genentech): a humanized IgG1 against ERBB2. In another embodiment, the antibody is Bevacizumab (Avastin; Genentech / Roche): a humanized IgG1 against VEGF. In another embodiment, the antibody is Cetuximab (Erbitux; Bristol-Myers Squibb): a chimeric human-mouse IgG1 against EGFR. In another embodiment, the antibody is Panitumumab (Vectibix; Amgen): a human IgG2 against EGFR. In another embodiment, the antibody is Ipilimumab (Yervoy; Bristol-Myers Squibb): an IgG1 against CTLA4.

[0198] In another embodiment, the antibody is Alemtuzumab (Campath; Genzyme): a humanized IgG1 against CD52. In another embodiment, the antibody is Ofatumumab (Arzerra; Genmab), a human IgG1 against CD20: In another embodiment, the antibody is Gemtuzumab ozogamicin (Mylotarg; Wyeth): a humanized IgG4 against CD33. In another embodiment, the antibody is Brentuximab vedotin (Adcetris; Seattle Genetics): a chimeric IgG1 against CD30. In another embodiment, the antibody is 90Y-labeled ibritumomab tiuxetan (Zevalin; IDEC Pharmaceuticals): murine IgG1 against CD20. In another embodiment, the antibody is 131II-labeled tositumomab (Bexxar; GlaxoSmithKline): murine IgG2 against CD20.

[0199] In another embodiment, the antibody is Ramucirumab, which is directed against vascular endothelial growth factor receptor 2 (VEGFR-2). In another embodiment, the antibody is Ramucirumab (Cyramza Injection, Eli Lilly and Company), blinatumomab (BLINCYTO, Amgen), pembrolizumab (KEYTRUDA, Merck Sharp & Dohme Corp.), obinutuzumab (GAZYVA, Genentech; formerly known as GA101), pertuzumab injection (PERJETA, Genentech) or denosumab (Xgeva, Amgen). In another embodiment, the antibody is Basiliximab (Simulect; Novartis). In another embodiment, the antibody is Daclizumab (Zenapax; Roche).

[0200] In another embodiment, the antibody coupled to the CAR T cell is directed against a tumor or cancer antigen or a fragment thereof described herein and / or known in the art. In another embodiment, the antibody coupled to the CAR T cell is directed against a tumor-associated antigen. In another embodiment, the antibody coupled to the CAR T cell is directed against a tumor-associated antigen or a fragment thereof that is an angiogenic factor.

[0201] In another embodiment, the antibody coupled to the CAR T cell is directed against a tumor or cancer antigen or a fragment thereof described herein and / or known in the art.

[0202] In some embodiments, the antibodies described herein can be used in combination with the compositions described herein, such as, but not limited to, compositions comprising CAR-T cells or early apoptotic cells or any combination thereof.

[0203] Cytokine Storm and Cytokine Release Syndrome

[0204] In one embodiment, the methods disclosed herein comprise providing immune cells (such as NK cells, dendritic cells, TCR T cells, or T cells comprising an engineered chimeric antigen receptor (CAR T cells) and at least one additional agent to reduce toxic cytokine release or "cytokine release syndrome" (CRS) or "severe cytokine release syndrome" (sCRS) or "cytokine storm" that may occur in a subject. In another embodiment, the CRS, sCRS or cytokine storm occurs as a result of administering immune cells. In another embodiment, the CRS, sCRS or cytokine storm is the result of a stimulus, condition or syndrome separate from the immune cells (see below). In another embodiment, a cytokine storm, cytokine cascade or hypercytokinemia is a more severe form of cytokine release syndrome.

[0205] In one embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cells or compositions comprising said apoptotic cells. In another embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cell supernatants or compositions comprising said supernatants. In another embodiment, additional agents for reducing the release of harmful cytokines include CTLA-4 blockers. In another embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cells or apoptotic cell supernatants or compositions thereof and CTLA-4 blockers. In another embodiment, additional agents for reducing the release of harmful cytokines include alpha-1 antitrypsin or fragments thereof or analogs thereof. In another embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cells or apoptotic cell supernatants or compositions thereof and alpha-1 antitrypsin or fragments thereof or analogs thereof. In another embodiment, additional agents for reducing the release of harmful cytokines include tellurium compounds. In another embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cells or apoptotic cell supernatants or compositions thereof and tellurium compounds. In another embodiment, the additional agent for reducing the release of harmful cytokines includes an immunomodulator. In another embodiment, the additional agent for reducing the release of harmful cytokines includes apoptotic cells or apoptotic cell supernatant or a composition thereof and an immunomodulator. In another embodiment, the additional agent for reducing the release of harmful cytokines includes Treg cells. In another embodiment, the additional agent for reducing the release of harmful cytokines includes apoptotic cells or apoptotic cell supernatant or a composition thereof and Treg cells.

[0206] It should be understood by the technician that reducing the release of toxic cytokines or reducing the level of toxic cytokines includes reducing the level of toxic cytokines in the subject or inhibiting the production of toxic cytokines, or inhibiting the cytokine release syndrome or cytokine storm of the subject or reducing the incidence of the cytokine release syndrome or cytokine storm. In another embodiment, the level of toxic cytokines is reduced during CRS or cytokine storm. In another embodiment, reducing the level of toxic cytokines or inhibiting the production of toxic cytokines includes treating CRS or cytokine storm. In another embodiment, reducing the level of toxic cytokines or inhibiting the production of toxic cytokines includes preventing CRS or cytokine storm. In another embodiment, reducing the level of toxic cytokines or inhibiting the production of toxic cytokines includes alleviating CRS or cytokine storm. In another embodiment, reducing the level of toxic cytokines or inhibiting the production of toxic cytokines includes improving CRS or cytokine storm. In another embodiment, the toxic cytokines include proinflammatory cytokines. In another embodiment, proinflammatory cytokines include IL-6. In another embodiment, proinflammatory cytokines include IL-1β. In another embodiment, proinflammatory cytokines include TNF-α. In another embodiment, the proinflammatory cytokine comprises IL-6, IL-1β, or TNF-α, or any combination thereof.

[0207] In one embodiment, cytokine release syndrome is characterized by elevated levels of several inflammatory cytokines and adverse physical reactions in the subject, such as hypotension, high fever and shivering. In another embodiment, inflammatory cytokines include IL-6, IL-1β and TNF-α. In another embodiment, CRS is characterized by elevated levels of IL-6, IL-1β or TNF-α or any combination thereof. In another embodiment, CRS is characterized by elevated levels of IL-8 or IL-13 or any combination thereof. In another embodiment, cytokine storms are characterized by an increase in TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-13, GM-CSF, IL-5, fracktalkine or a combination thereof or a subgroup thereof. In yet another embodiment, IL-6 includes markers of CRS or cytokine storms.

[0208] In another embodiment, the cytokines increased in CRS or cytokine storm in humans and mice may include any combination of the cytokines listed in Tables 1 and 2 below.

[0209] Table 1: Cytokine panels increased in CRS or cytokine storm in humans and / or mice

[0210]

[0211] In some embodiments, the cytokines Flt-3L, fractin, GM-CSF, IFN-γ, IL-1β, IL-2, IL-2Rα, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL-13 of Table 1 are considered to be significant in CRS or cytokine storm. In another embodiment, IFN-α, IFN-β, IL-1 and IL-1Rα of Table 1 appear to be important in CRS or cytokine storm. In another embodiment, M-CSF has unknown importance. In another embodiment, any cytokine listed in Table 1 or a combination thereof can be used as a marker for CRS or cytokine storm.

[0212] Table 2: Cytokine panels increased in CRS or cytokine storm in humans and / or mice

[0213]

[0214] In one embodiment, IL-15, IL-17, IL-18, IL-21, IL-22, IP-10, MCP-1, MIP-1α, MIP-1β and TNF-α of Table 2 are considered to be significant in CRS or cytokine storm. In another embodiment, IL-27, MCP-3, PGE2, RANTES, TGF-β, TNF-αR1 and MIG of Table 2 appear to be important in CRS or cytokine storm. In another embodiment, IL-23 and IL-25 have unknown importance. In another embodiment, any cytokine listed in Table 2 or a combination thereof can be used as a marker for CRS or cytokine storm. In another embodiment, mouse cytokines IL-10, IL-1β, IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15 / IL-15R and IL-7 appear to be important in CRS or cytokine storm.

[0215] The skilled artisan will appreciate that the term "cytokine" may encompass cytokines (e.g., interferon gamma (IFN-γ), granulocyte macrophage colony stimulating factor, tumor necrosis factor α), chemokines (e.g., MIP 1α, MIP 1β, RANTES), and other soluble mediators of inflammation (such as reactive oxygen species and nitric oxide).

[0216] In one embodiment, the increase in the release of a specific cytokine (whether significant, important, or of unknown importance) does not a priori mean that the specific cytokine is part of a cytokine storm. In one embodiment, the increase in at least one cytokine is not the result of a cytokine storm or CRS. In another embodiment, CAR T cells can be the source of elevated levels of a specific cytokine or a group of cytokines.

[0217] In another embodiment, the cytokine release syndrome is characterized by any or all of the following symptoms: fever with or without chills, malaise, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, headache, rash, nausea, vomiting, diarrhea, tachypnea, hypoxemia, cardiovascular tachycardia, widened pulse pressure, hypotension, increased cardiac output (early), potential decreased cardiac output (late), elevated D-dimers, hypofibrinogenemia with or without bleeding, azotemia, elevated liver transaminases, hyperbilirubinemia, headache, mental state changes, confusion, delirium, dysphasia or symptomatic aphasia, hallucinations, tremors, illusions, dysmetria, seizures, gait changes, epilepsy. In another embodiment, the cytokine storm is characterized by IL-2 release and lymphoproliferation. In another embodiment, the cytokine storm is characterized by an increase in cytokines released by CAR T cells. In another embodiment, the cytokine storm is characterized by an increase in cytokines released by cells other than CAR T cells.

[0218] In another embodiment, cytokine storm can lead to potentially life-threatening complications including cardiac dysfunction, adult respiratory distress syndrome, neurotoxicity, renal and / or hepatic failure, and disseminated intravascular coagulation.

[0219] It should be understood by the technician that the characteristics of cytokine release syndrome (CRS) or cytokine storm are estimated to occur a few days to a few weeks after triggering CRS or cytokine storm. In one embodiment, CAR T cells are triggers of CRS or cytokine storm. In another embodiment, the trigger of CRS or cytokine storm is not CAR T cells.

[0220] In one embodiment, the measurement of the cytokine level or concentration as an indication of a cytokine storm can be expressed as a doubling of the cytokine level or concentration, a percentage (%) increase, a net increase, or a rate of change. In another embodiment, an absolute cytokine level or concentration above a certain level or concentration can indicate that the subject is experiencing or will experience a cytokine storm. In another embodiment, an absolute cytokine level or concentration at a certain level or concentration (e.g., a level or concentration typically found in a control subject who has not experienced CAR-T cell therapy) can indicate a method for suppressing a cytokine storm in a subject who has experienced CAR T cells or reducing the incidence of the cytokine storm.

[0221] The skilled person will appreciate that the term "cytokine level" may encompass a measure of concentration, a measure of fold change, a measure of percentage (%) change, or a measure of rate change. Further, methods for measuring cytokines in blood, saliva, serum, urine, and plasma are well known in the art.

[0222] In one embodiment, although it is recognized that cytokine storms are associated with increases in several inflammatory cytokines, IL-6 levels can be used as a common measure of cytokine storms and / or as a common measure of the effectiveness of treatment for cytokine storms. It will be appreciated by the skilled person that other cytokines can be used as markers of cytokine storms, such as TNF-α, IB-1α, IL-8, IL-13, or INF-γ. Further, assays for measuring cytokines are well known in the art. It will be appreciated by the skilled person that methods affecting cytokine storms can similarly affect cytokine release syndrome.

[0223] In one embodiment, disclosed herein is a method of reducing or inhibiting cytokine production in a subject experiencing cytokine release syndrome or cytokine storm. In another embodiment, disclosed herein is a method of reducing or inhibiting cytokine production in a subject susceptible to cytokine release syndrome or cytokine storm. In another embodiment, the method disclosed herein reduces or inhibits cytokine production in a subject experiencing cytokine release syndrome or cytokine storm, wherein the production of any cytokine or cytokine group listed in Table 1 and / or Table 2 is reduced or inhibited. In another embodiment, cytokine IL-6 production is reduced or inhibited. In another embodiment, cytokine IL-β1 production is reduced or inhibited. In another embodiment, cytokine IL-8 production is reduced or inhibited. In another embodiment, cytokine IL-13 production is reduced or inhibited. In another embodiment, cytokine TNF-α production is reduced or inhibited. In another embodiment, cytokine IL-6 production, IL-1β production or TNF-α production or any combination thereof is reduced or inhibited.

[0224] In one embodiment, cytokine release syndrome is graded. In another embodiment, 1 grade describes cytokine release syndrome in which symptoms are not life-threatening and only require symptomatic treatment, such as fever, nausea, fatigue, headache, myalgia, discomfort. In another embodiment, 2 grade symptoms require moderate intervention (such as oxygen, fluid or vasopressor for hypotension) and will respond to it. In another embodiment, 3 grade symptoms require active intervention and will respond to it. In another embodiment, 4 grade symptoms are life-threatening symptoms and require a ventilator, and the patient shows organ toxicity.

[0225] In another embodiment, the cytokine storm is characterized by IL-6 and interferon gamma release. In another embodiment, the cytokine storm is characterized by the release of any cytokine listed in Tables 1 and 2, or a combination thereof. In another embodiment, the cytokine storm is characterized by the release of any cytokine known in the art, or a combination thereof.

[0226] In one embodiment, the onset of symptoms begins minutes to hours after the start of the infusion. In another embodiment, symptoms coincide with peak cytokine levels.

[0227] In one embodiment, the method for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm includes administering apoptotic cell groups or apoptotic cell supernatants or compositions thereof. In another embodiment, the apoptotic cell groups or apoptotic cell supernatants or compositions thereof may contribute to CAR T cell therapy. In another embodiment, the apoptotic cell groups or apoptotic cell supernatants or compositions thereof may contribute to suppressing the CRS or cytokine storm or reducing the incidence of the CRS or cytokine storm. In another embodiment, the apoptotic cell groups or apoptotic cell supernatants or compositions thereof may contribute to treating the CRS or cytokine storm. In another embodiment, the apoptotic cell groups or apoptotic cell supernatants or compositions thereof may contribute to preventing the CRS or cytokine storm. In another embodiment, the apoptotic cell groups or apoptotic cell supernatants or compositions thereof may contribute to improving the CRS or cytokine storm. In another embodiment, the apoptotic cell groups or apoptotic cell supernatants or compositions thereof may contribute to alleviating the CRS or cytokine storm.

[0228] In one embodiment, suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject who experiences CAR T cell cancer therapy and administers apoptotic cell group or apoptotic cell supernatant or its composition or reducing the incidence of the CRS or cytokine storm includes administering another agent. In another embodiment, the other agent can contribute to CAR T cell therapy. In another embodiment, the other agent can contribute to suppressing the CRS or cytokine storm or reducing the incidence of the CRS or cytokine storm. In another embodiment, the other agent can contribute to treating the CRS or cytokine storm. In another embodiment, the other agent can contribute to preventing the CRS or cytokine storm. In another embodiment, the other agent can contribute to improving the CRS or cytokine storm. In another embodiment, the other agent can contribute to alleviating the CRS or cytokine storm.

[0229] In one embodiment, the method for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm includes administering another agent. In another embodiment, the other agent may contribute to CAR T cell therapy. In one embodiment, the method for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing TCR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm includes administering another agent. In another embodiment, the other agent may contribute to TCR T cell therapy. In one embodiment, the method for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing TCR T cell therapy or reducing the incidence of the CRS or cytokine storm includes administering another agent. In one embodiment, the method for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing NK cell therapy or reducing the incidence of the CRS or cytokine storm includes administering another agent. In another embodiment, the other agent may contribute to NK cell therapy.

[0230] In another embodiment, the additional agent can help suppress the CRS or cytokine storm or reduce the incidence of the CRS or cytokine storm. In another embodiment, the additional agent can help treat the CRS or cytokine storm. In another embodiment, the additional agent can help prevent the CRS or cytokine storm. In another embodiment, the additional agent can help improve the CRS or cytokine storm. In another embodiment, the additional agent can help alleviate the CRS or cytokine storm.

[0231] In one embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cells or compositions comprising said apoptotic cells. In another embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cell supernatants or compositions comprising said supernatants. In another embodiment, additional agents for reducing the release of harmful cytokines include CTLA-4 blockers. In another embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cells or apoptotic cell supernatants or compositions thereof and CTLA-4 blockers. In another embodiment, additional agents for reducing the release of harmful cytokines include alpha-1 antitrypsin or fragments thereof or analogs thereof. In another embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cells or apoptotic cell supernatants or compositions thereof and alpha-1 antitrypsin or fragments thereof or analogs thereof. In another embodiment, additional agents for reducing the release of harmful cytokines include tellurium compounds. In another embodiment, additional agents for reducing the release of harmful cytokines include apoptotic cells or apoptotic cell supernatants or compositions thereof and tellurium compounds. In another embodiment, the additional agent for reducing the release of harmful cytokines comprises an immunomodulator. In another embodiment, the additional agent for reducing the release of harmful cytokines comprises apoptotic cells or apoptotic cell supernatant or a combination thereof and an immunomodulator.

[0232] In another embodiment, the compositions and methods disclosed herein utilize a combination therapy of CAR T cells and one or more CTLA-4 blockers (such as ipilimumab). In another embodiment, the compositions and methods disclosed herein utilize a combination therapy including apoptotic cells, CAR T cells, and one or more CTLA-4 blockers. In another embodiment, the compositions and methods disclosed herein utilize a combination therapy of TCR T cells and one or more CTLA-4 blockers (such as ipilimumab). In another embodiment, the compositions and methods disclosed herein utilize a combination therapy including apoptotic cells, TCR T cells, and one or more CTLA-4 blockers. In another embodiment, the compositions and methods disclosed herein utilize a combination therapy of dendritic cells and one or more CTLA-4 blockers (such as ipilimumab). In another embodiment, the compositions and methods disclosed herein utilize a combination therapy including apoptotic cells, dendritic cells, and one or more CTLA-4 blockers. In another embodiment, the compositions and methods disclosed herein utilize a combination therapy including apoptotic cells, dendritic cells, and one or more CTLA-4 blockers. In another embodiment, the compositions and methods disclosed herein utilize a combination therapy of NK cells and one or more CTLA-4 blockers (such as ipilimumab). In another embodiment, the compositions and methods disclosed herein utilize combination therapy including apoptotic cells, NK cells, and one or more CTLA-4 blocking agents.

[0233] In another embodiment, CTLA-4 is a potent inhibitor of T cell activation that helps maintain self-tolerance. In another embodiment, administration of an anti-CTLA-4 blocker (which is an antibody in another embodiment) produces a net effect of T cell activation.

[0234] In another embodiment, other toxicities resulting from the administration of CAR T cells, TCR T cells, dendritic cells or NK cells that can be treated, prevented, inhibited, improved, reduced in incidence, or alleviated by the compositions and methods disclosed herein include B cell aplasia or tumor lysis syndrome (TLS).

[0235] In one embodiment, the method for suppressing the cytokine release syndrome (CRS) or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm will not affect the efficacy of CAR T cell therapy. In another embodiment, the method for suppressing the CRS or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm will not reduce the efficacy of CAR T cell therapy by more than about 5%. In another embodiment, the method for suppressing the CRS or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm will not reduce the efficacy of CAR T cell therapy by more than about 10%. In another embodiment, the method for suppressing the CRS or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm will not reduce the efficacy of CAR T cell therapy by more than about 15%. In another embodiment, the method for suppressing the CRS or cytokine storm of a subject experiencing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm will not reduce the efficacy of CAR T cell therapy by more than about 20%. In another embodiment, the method of suppressing CRS or cytokine storm in a subject undergoing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm increases the efficacy of CAR T cell therapy by more than about 5%. In another embodiment, the method of suppressing CRS or cytokine storm in a subject undergoing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm increases the efficacy of CAR T cell therapy by more than about 10%. In another embodiment, the method of suppressing CRS or cytokine storm in a subject undergoing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm increases the efficacy of CAR T cell therapy by more than about 15%. In another embodiment, the method of suppressing CRS or cytokine storm in a subject undergoing CAR T cell cancer therapy or reducing the incidence of the CRS or cytokine storm increases the efficacy of CAR T cell therapy by more than about 20%.

[0236] Any suitable method for quantifying cytotoxicity can be used to determine whether the activity in the immune cells modified to express CAR remains substantially unchanged. For example, cell culture-based assays (cytotoxicity assays as described in the examples) can be used to quantify cytotoxicity. Cytotoxicity assays can use dyes that preferentially stain dead cell DNA. In other cases, fluorescence and luminescence analysis of the relative number of live and dead cells in a cell population can be used. For such assays, protease activity serves as a marker of cell viability and cytotoxicity, and labeled cell permeable peptides produce a fluorescent signal proportional to the number of live cells in the sample. For example, cytotoxicity assays can use 7-AAD in flow cytometry analysis. Kits for various cytotoxicity assays are commercially available from manufacturers (such as Promega, Abcam, and Life Technologies).

[0237] In another embodiment, the measure of cytotoxicity can be qualitative. In another embodiment, the measure of cytotoxicity can be quantitative. In another embodiment, the measure of cytotoxicity can be related to changes in the expression of cytotoxic cytokines. In another embodiment, the measure of cytotoxicity can be determined by survival curves and tumor burden in the bone marrow and liver.

[0238] In one embodiment, the method disclosed herein includes additional steps that can be used to overcome allogeneic donor cell rejection. In one embodiment, the method includes a step of complete or partial lymphocyte clearance before administering CAR T cells (which are allogeneic CAR T cells in one embodiment). In another embodiment, lymphocyte clearance is adjusted so that it delays the host-versus-graft response enough to allow the allogeneic T cells to attack the tumor they are directed against, but to a certain extent, it is not enough to require a time period for saving the host immune system by bone marrow transplantation. In another embodiment, agents that delay the egress of allogeneic T cells from lymph nodes (such as 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720), 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl-1]1,2,4-oxadiazole (SEW2871), 3-(2-(-hexylphenylamino)-2-oxoethylamino)propionic acid (W123), 2-ammonio-4-(2-chloro-4-(3-phenoxyphenylthio)phenyl)-2-(hydroxymethyl)butyl hydrogen phosphate (KRP-203 phosphate), or other agents known in the art) can be used as part of the compositions and methods disclosed herein to allow the use of allogeneic CAR-T cells that are effective and do not induce graft-versus-host disease. In one embodiment, MHC expression by the allogeneic T cells is silenced to reduce rejection of allogeneic cells. In another embodiment, the apoptotic cells prevent rejection of allogeneic cells.

[0239] Cytokine Release Associated with CAR T Cell Therapy

[0240] In one embodiment, cytokine release occurs between a few days to 2 weeks after administering immunotherapy (such as CAR T cell therapy). In one embodiment, hypotension and other symptoms occur after cytokine release, that is, from a few days to a few weeks. Therefore, in one embodiment, apoptotic cells or apoptotic cell supernatant are administered to the subject as prevention while immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 2-3 days after immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to the subject 7 days after immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to the subject 10 days after immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to the subject 14 days after immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to the subject 2-14 days after immunotherapy is administered.

[0241] In another embodiment, apoptotic cells or apoptotic cell supernatant are administered to a subject 2-3 hours after the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 7 hours after the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 10 hours after the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 14 hours after the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-14 hours after the administration of immunotherapy.

[0242] In an alternative embodiment, apoptotic cells or apoptotic cell supernatant are administered to a subject as prevention before immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 1 day before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-3 days before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 7 days before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 10 days before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 14 days before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-14 days before immunotherapy is administered.

[0243] In another embodiment, apoptotic cells or apoptotic cell supernatant are administered to a subject 2-3 hours prior to the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 7 hours prior to the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 10 hours prior to the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 14 hours prior to the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-14 hours prior to the administration of immunotherapy.

[0244] In another embodiment, apoptotic cells or apoptotic cell supernatant can be administered therapeutically once cytokine release syndrome has occurred. In one embodiment, apoptotic cells or supernatant can be administered once cytokine release is detected leading to or demonstrating the onset of cytokine release syndrome. In one embodiment, apoptotic cells or supernatant can increase cytokine levels or end cytokine release syndrome or avoid its sequelae.

[0245] In another embodiment, apoptotic cells or apoptotic cell supernatants can be administered therapeutically at multiple time points. In another embodiment, apoptotic cells or apoptotic cell supernatants are administered at least at two time points described herein. In another embodiment, apoptotic cells or apoptotic cell supernatants are administered at least at three time points described herein. In another embodiment, before CRS or cytokine storm, and once cytokine release syndrome has occurred, apoptotic cells or apoptotic cell supernatants and any combination thereof are administered.

[0246] In one embodiment, T cells (CAR T cells) expressing chimeric antigen receptors are administered together with apoptotic cell therapy or supernatant. In another embodiment, the CAR T cell therapy is administered after apoptotic cell therapy or supernatant. In another embodiment, the CAR T cell therapy is administered before apoptotic cell therapy or supernatant. According to this aspect, and in one embodiment, apoptotic cell therapy or supernatant is administered approximately 2-3 weeks after the CAR T cell therapy. In another embodiment, apoptotic cell therapy or supernatant is administered approximately 6-7 weeks after the CAR T cell therapy. In another embodiment, apoptotic cell therapy or supernatant is administered approximately 9 weeks after the CAR T cell therapy. In another embodiment, apoptotic cell therapy or supernatant is administered up to several months after the CAR T cell therapy.

[0247] Thus, in one embodiment, apoptotic cells or apoptotic cell supernatant are administered to a subject as a preventive at the same time as immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-3 days after immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 7 days after immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 10 days after immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 14 days after immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-14 days after immunotherapy is administered.

[0248] In another embodiment, apoptotic cells or apoptotic cell supernatant are administered to a subject 2-3 hours after the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 7 hours after the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 10 hours after the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 14 hours after the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-14 hours after the administration of immunotherapy.

[0249] In an alternative embodiment, apoptotic cells or apoptotic cell supernatant are administered to a subject as prevention before immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 1 day before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-3 days before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 7 days before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 10 days before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 14 days before immunotherapy is administered. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-14 days before immunotherapy is administered.

[0250] In another embodiment, apoptotic cells or apoptotic cell supernatant are administered to a subject 2-3 hours prior to the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 7 hours prior to the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 10 hours prior to the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 14 hours prior to the administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to a subject 2-14 hours prior to the administration of immunotherapy.

[0251] In another embodiment, apoptotic cells or apoptotic cell supernatant can be administered therapeutically once cytokine release syndrome has occurred. In one embodiment, apoptotic cells or supernatant can be administered once cytokine release is detected leading to or demonstrating the onset of cytokine release syndrome. In one embodiment, apoptotic cells or supernatant can increase cytokine levels or end cytokine release syndrome or avoid its sequelae.

[0252] In another embodiment, apoptotic cells or apoptotic cell supernatants can be administered therapeutically at multiple time points. In another embodiment, apoptotic cells or apoptotic cell supernatants are administered at least at two time points described herein. In another embodiment, apoptotic cells or apoptotic cell supernatants are administered at least at three time points described herein. In another embodiment, before CRS or cytokine storm, and once cytokine release syndrome has occurred, apoptotic cells or apoptotic cell supernatants and any combination thereof are administered.

[0253] In one embodiment, T cells (CAR T cells) expressing chimeric antigen receptors are administered together with apoptotic cell therapy or supernatant. In another embodiment, the CAR T cell therapy is administered after apoptotic cell therapy or supernatant. In another embodiment, the CAR T cell therapy is administered before apoptotic cell therapy or supernatant. According to this aspect, and in one embodiment, apoptotic cell therapy or supernatant is administered approximately 2-3 weeks after the CAR T cell therapy. In another embodiment, apoptotic cell therapy or supernatant is administered approximately 6-7 weeks after the CAR T cell therapy. In another embodiment, apoptotic cell therapy or supernatant is administered approximately 9 weeks after the CAR T cell therapy. In another embodiment, apoptotic cell therapy or supernatant is administered up to several months after the CAR T cell therapy.

[0254] In other embodiments, additional agents are administered to the subject as prevention while immunotherapy is being administered. In one embodiment, the additional agents include apoptotic cells, apoptotic cell supernatants, CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulatory compounds or any combination thereof. In another embodiment, additional agents are administered to the subject 2-3 days after immunotherapy is administered. In another embodiment, additional agents are administered to the subject 7 days after immunotherapy is administered. In another embodiment, additional agents are administered to the subject 10 days after immunotherapy is administered. In another embodiment, additional agents are administered to the subject 14 days after immunotherapy is administered. In another embodiment, additional agents are administered to the subject 2-14 days after immunotherapy is administered.

[0255] In another embodiment, the additional agent is administered to the subject 2-3 hours after the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 7 hours after the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 10 hours after the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 14 hours after the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 2-14 hours after the administration of the immunotherapy.

[0256] In another embodiment, the additional agent is administered to the subject as a prophylaxis prior to the immunotherapy. In another embodiment, the additional agent is administered to the subject 1 day prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 2-3 days prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 7 days prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 10 days prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 14 days prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 2-14 days prior to the administration of the immunotherapy.

[0257] In another embodiment, the additional agent is administered to the subject 2-3 hours prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 7 hours prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 10 hours prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 14 hours prior to the administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 2-14 hours prior to the administration of the immunotherapy.

[0258] In another embodiment, once cytokine release syndrome has occurred, additional agents are administered therapeutically. In one embodiment, additional agents are administered once cytokine release is detected leading to or demonstrating the onset of cytokine release syndrome. In one embodiment, additional agents can increase cytokine levels or end cytokine release syndrome or avoid its sequelae.

[0259] In another embodiment, additional agents are administered therapeutically at multiple time points. In another embodiment, additional agents are administered at least at two time points described herein. In another embodiment, additional agents are administered at least at three time points described herein. In another embodiment, additional agents and any combination thereof are administered prior to CRS or cytokine storm, and once cytokine release syndrome has occurred.

[0260] In one embodiment, T cells expressing chimeric antigen receptors (CAR T cells) therapy are administered together with another agent. In another embodiment, the CAR T cell therapy is administered to another agent. In another embodiment, the CAR T cell therapy is administered before another agent. According to this aspect, and in one embodiment, another agent is administered about 2-3 weeks after the CAR T cell therapy. In another embodiment, another agent is administered about 6-7 weeks after the CAR T cell therapy. In another embodiment, another agent is administered about 9 weeks after the CAR T cell therapy. In another embodiment, another agent is administered up to several months after the CAR T cell therapy.

[0261] In one embodiment, the CAR T cells are heterologous to the subject. In one embodiment, the CAR T cells are derived from one or more donors. In one embodiment, the CAR T cells are derived from one or more bone marrow donors. In another embodiment, the CAR T cells are derived from one or more blood bank donations. In one embodiment, the donor is a matched donor. In one embodiment, the CAR T cells are universal allogeneic CAR T cells. In another embodiment, the CAR T cells are isogenic CAR T cells. In another embodiment, the CAR T cells are from unmatched third-party donors. In another embodiment, the CAR T cells are from pooled third-party donor T cells. In one embodiment, the donor is a bone marrow donor. In another embodiment, the donor is a blood bank donor. In one embodiment, the CAR T cells of the compositions and methods disclosed herein include one or more MHC non-restricted tumor-directed chimeric receptors. In one embodiment, non-autologous T cells can be engineered or administered according to protocols known in the art to prevent or minimize autoimmune reactions, as described in U.S. Patent Application No. 20130156794, which is incorporated herein by reference in its entirety.

[0262] In another embodiment, the CAR T cells are autologous to the subject. In one embodiment, the patient's own cells are used. In this embodiment, if the patient's own cells are used, the CAR T cell therapy is administered after the apoptotic cell therapy.

[0263] In one embodiment, the apoptotic cells are allogeneic to the subject. In one embodiment, the apoptotic cells are derived from one or more donors. In one embodiment, the apoptotic cells are derived from one or more bone marrow donors. In another embodiment, the apoptotic cells are derived from one or more blood bank donations. In one embodiment, the donor is a matched donor. In another embodiment, the apoptotic cells are from an unmatched third party donor. In one embodiment, the apoptotic cells are universal allogeneic apoptotic cells. In another embodiment, the apoptotic cells are from a syngeneic donor. In another embodiment, the apoptotic cells are from pooled third party donor cells. In one embodiment, the donor is a bone marrow donor. In another embodiment, the donor is a blood bank donor. In another embodiment, the apoptotic cells are autologous to the subject. In this embodiment, the patient's own cells are used.

[0264] According to some embodiments, the cell preparations or apoptotic cell supernatants enriched with therapeutic molecules disclosed herein are administered systemically to a subject. In another embodiment, the administration is by intravenous route. Alternatively, the cells or supernatants enriched with therapeutic mononuclear cells can be administered to a subject according to various other routes (including but not limited to parenteral, intraperitoneal, intraarticular, intramuscular and subcutaneous routes).

[0265] According to some embodiments, the therapeutic mononuclear enriched cell preparation disclosed herein or another agent is administered systemically to a subject. In another embodiment, it is administered by an intravenous route. Alternatively, the therapeutic mononuclear enriched cells or another agent can be administered to a subject according to various other routes (including but not limited to parenteral, intraperitoneal, intraarticular, intramuscular and subcutaneous routes).

[0266] In one embodiment, the formulation is administered in a local rather than systemic manner, for example, by injecting the formulation directly into a specific area of ​​the patient's body. In another embodiment, the specific area includes a tumor or cancer.

[0267] In another embodiment, the cells or supernatant enriched with therapeutic mononuclear cells are administered to a subject suspended in a suitable physiological buffer (such as, but not limited to, saline solution, PBS, HBSS, etc.). In addition, the suspension medium may further include a supplement that helps maintain cell viability. In another embodiment, an additional agent is administered to a subject suspended in a suitable physiological buffer (such as, but not limited to, saline solution, PBS, HBSS, etc.).

[0268] According to some embodiments, the pharmaceutical composition is administered intravenously. According to another embodiment, the pharmaceutical composition is administered in a single dose. According to an alternative embodiment, the pharmaceutical composition is administered in multiple doses. According to another embodiment, the pharmaceutical composition is administered in two doses. According to another embodiment, the pharmaceutical composition is administered in three doses. According to another embodiment, the pharmaceutical composition is administered in four doses. According to another embodiment, the pharmaceutical composition is administered in five or more doses. According to some embodiments, the pharmaceutical composition is formulated for intravenous injection.

[0269] In one embodiment, any suitable method of providing a modified CAR-expressing immune cell to a subject can be used for the methods described herein. In one embodiment, the method for providing cells to a subject includes hematopoietic cell transplantation (HCT), infusing donor-derived NK cells into a cancer patient, or a combination thereof.

[0270] In another embodiment, disclosed herein is a method of inhibiting or reducing the incidence of cytokine release syndrome or cytokine storm in a subject undergoing chimeric antigen receptor-expressing T cell (CAR T cell) therapy, the method comprising the step of administering to the subject a composition comprising apoptotic cells.

[0271] In another embodiment, disclosed herein is a method for inhibiting cytokine release syndrome or cytokine storm or reducing the incidence of cytokine release syndrome or cytokine storm in a subject undergoing chimeric antigen receptor-expressing T cell (CAR T cell) therapy, the method comprising the step of administering an apoptotic cell supernatant (such as an apoptotic cell-phagocyte supernatant) to the subject.

[0272] In another embodiment, disclosed herein is a method of inhibiting or reducing the incidence of cytokine release syndrome or cytokine storm in a subject undergoing chimeric antigen receptor-expressing T cell (CAR T cell) therapy, the method comprising the step of administering to the subject at least one additional agent.

[0273] In certain embodiments, CAR T cell therapy comprises administering a composition comprising CAR T cells and apoptotic cells or apoptotic cell supernatant or another additional agent disclosed herein or a combination of the additional agents disclosed herein. In alternative embodiments, CAR T cell therapy comprises administering a composition comprising CAR T cells disclosed herein and a composition comprising apoptotic cells or apoptotic cell supernatant or another agent disclosed herein or a combination thereof.

[0274] Cytokine release associated with non-CAR T cell applications

[0275] In one embodiment, disclosed herein is a method for reducing or inhibiting cytokine production in a subject who experiences cytokine release syndrome or cytokine storm or is susceptible to cytokine release syndrome or cytokine storm, the method comprising administering to the subject a composition comprising apoptotic cells or apoptotic supernatant, wherein the administration reduces or inhibits cytokine production in the subject. In another embodiment, cytokine production is reduced or inhibited compared to a subject who experiences cytokine release syndrome or cytokine storm or is susceptible to cytokine release syndrome or cytokine storm and is not administered apoptotic cells or apoptotic supernatant. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits proinflammatory cytokine production. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits the production of at least one proinflammatory cytokine. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits the production of at least cytokine IL-6. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits the production of at least cytokine IL-1β. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits the production of at least cytokine TNF-α. In another embodiment, the method for reducing or inhibiting cytokine production disclosed herein results in a reduction or inhibition of the production of any combination of cytokines IL-6, IL-1β, or TNF-α in the subject as compared to a subject experiencing cytokine release syndrome or cytokine storm or susceptible to cytokine release syndrome or cytokine storm and not administered apoptotic cells or apoptotic supernatant.

[0276] Cancer or tumors can also affect the absolute level of cytokines including proinflammatory cytokines. The level of tumor burden in a subject may affect cytokine levels, particularly proinflammatory cytokines. It will be appreciated by the skilled artisan that the phrase "reduce or inhibit" or its grammatical variants can encompass a fold reduction or inhibition of cytokine production, or a net reduction or inhibition or percentage (%) reduction or inhibition of cytokine production, or a rate of change of reduction or inhibition of cytokine production can be encompassed.

[0277] In another embodiment, disclosed herein is a method of reducing or inhibiting cytokine production in a subject experiencing or susceptible to cytokine release syndrome or cytokine storm, the method comprising the step of administering to the subject apoptotic cells or a composition comprising the apoptotic cells.

[0278] In another embodiment, disclosed herein is a method for reducing or inhibiting cytokine production in a subject experiencing or susceptible to cytokine release syndrome or cytokine storm, the method comprising the step of administering to the subject an apoptotic cell supernatant (such as an apoptotic cell-phagocyte supernatant) or a composition comprising the supernatant.

[0279] In another embodiment, disclosed herein is a method of reducing or inhibiting cytokine production in a subject experiencing cytokine release syndrome or cytokine storm or susceptible to cytokine release syndrome or cytokine storm, the method comprising the step of administering to the subject an apoptotic cell supernatant (such as an additional agent selected from the group consisting of apoptotic cells, apoptotic cell supernatant, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium compound or an immunomodulator or any combination thereof) or a composition comprising the supernatant.

[0280] In one embodiment, the infection causes a cytokine release syndrome or cytokine storm in the subject. In one embodiment, the infection is an influenza infection. In one embodiment, the influenza infection is H1N1. In another embodiment, the influenza infection is H5N1 avian influenza. In another embodiment, the infection is severe acute respiratory syndrome (SARS). In another embodiment, the subject suffers from hemophagocytic lymphohistiocytosis (HLH) associated with Epstein-Barr virus. In another embodiment, the infection is sepsis. In one embodiment, the sepsis is gram-negative. In another embodiment, the infection is malaria. In another embodiment, the infection is an Ebola virus infection. In another embodiment, the infection is smallpox virus. In another embodiment, the infection is a systemic gram-negative bacterial infection. In another embodiment, the infection is Jarisch-Herxheimer syndrome.

[0281] In one embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is hemophagocytic lymphohistiocytosis (HLH). In another embodiment, HLH is sporadic HLH. In another embodiment, HLH is macrophage activation syndrome (MAS). In another embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is MAS.

[0282] In one embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is chronic arthritis. In another embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is systemic juvenile idiopathic arthritis (sJIA), also known as Still's Disease.

[0283] In one embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is Cryopyrin-associated periodic syndrome (CAPS). In another embodiment, CAPS includes familial cold autoinflammatory syndrome (FCAS), also known as familial cold urticaria (FCU). In another embodiment, CAPS includes Muckle-Well Syndrome (MWS). In another embodiment, CAPS includes chronic infantile neurological skin and joint (CINCA) syndrome. In another embodiment, CAPS includes FCAS, FCU, MWS or CINCA syndrome or any combination thereof. In another embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is FCAS. In another embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is FCU. In another embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is MWS. In another embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is CINCA syndrome. In yet another embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is FCAS, FCU, MWS or CINCA syndrome, or any combination thereof.

[0284] In another embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is a cryopyrinopathy comprising an inherited mutation or a gain-of-function mutation in the NLRP3 gene (also known as the CIASI gene).

[0285] In one embodiment, the cause of the subject's cytokine release syndrome or cytokine storm is a genetic autoinflammatory disorder.

[0286] In one embodiment, the trigger for the release of inflammatory cytokines is lipopolysaccharide (LPS), Gram-positive toxins, fungal toxins, glycosylphosphatidylinositol (GPI), or modulation of RIG-1 gene expression.

[0287] In another embodiment, the subject experiencing cytokine release syndrome or cytokine storm does not have an infectious disease. In one embodiment, the subject suffers from acute pancreatitis. In another embodiment, the subject suffers from tissue damage, which in one embodiment is severe burns or trauma. In another embodiment, the subject suffers from acute respiratory distress syndrome. In another embodiment, the subject suffers from cytokine release syndrome or cytokine storm secondary to drug use. In another embodiment, the subject suffers from cytokine release syndrome or cytokine storm secondary to toxin inhalation.

[0288] In another embodiment, the subject has a cytokine release syndrome or cytokine storm secondary to receiving immunotherapy, which in one embodiment is immunotherapy with a super-agonist CD28-specific monoclonal antibody (CD28SA). In one embodiment, the CD28SA is TGN1412. In another embodiment, the immunotherapy is CAR T cell therapy.

[0289] In another embodiment, apoptotic cells or supernatant or CTLA-4 blocker, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators or any combination thereof can be used to control cytokine release syndrome or cytokine storm caused by administration of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is oxaliplatin, cytarabine, lenalidomide or a combination thereof.

[0290] In another embodiment, apoptotic cells or supernatants or CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators or any combination thereof can be used to control cytokine release syndrome or cytokine storm caused by administration of antibodies. In one embodiment, the antibody is monoclonal. In another embodiment, the antibody is polyclonal. In one embodiment, the antibody is rituximab. In another embodiment, the antibody is Orthoclone OKT3 (Muromonab-CD3). In another embodiment, the antibody is alemtuzumab, totuzumab, CP-870,893, LO-CD2a / BTI-322 or TGN1412.

[0291] In another embodiment, examples of diseases for which controlling the production of inflammatory cytokines may be beneficial include cancer, allergies, any type of infection, toxic shock syndrome, sepsis, any type of autoimmune disease, arthritis, Crohn's disease, lupus, psoriasis, or any disease characterized by the release of toxic cytokines that produce deleterious effects on the subject.

[0292] septicemia

[0293] In some embodiments, disclosed herein is a method for treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject, the method comprising the step of administering to the subject a composition comprising an early apoptotic cell population, wherein the administration treats, prevents, inhibits sepsis in the subject, reduces the incidence of sepsis in the subject, improves or alleviates sepsis in the subject. In some embodiments, disclosed herein is a method for treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject, the method comprising the step of administering to the subject a composition comprising an apoptotic supernatant, wherein the administration treats, prevents, inhibits sepsis in the subject, reduces the incidence of sepsis in the subject, improves or alleviates sepsis in the subject.

[0294] In some embodiments, disclosed herein is a method for treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject, the method comprising the step of administering to the subject a composition comprising an early apoptotic cell population in combination with an antibiotic, wherein the administration treats, prevents, inhibits sepsis in the subject, reduces the incidence of sepsis in the subject, improves or alleviates sepsis in the subject. In some embodiments, disclosed herein is a method for treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject, the method comprising the step of administering to the subject a composition comprising apoptotic cell supernatant in combination with an antibiotic, wherein the administration treats, prevents, inhibits sepsis in the subject, reduces the incidence of sepsis in the subject, improves or alleviates sepsis in the subject.

[0295] In some embodiments, the use of early apoptotic cells to treat, prevent, inhibit, reduce the incidence of, ameliorate or alleviate sepsis in a subject in need thereof is part of a combination therapy, such as, but not limited to, also administering antibiotics to the subject.

[0296] In some embodiments, sepsis includes severe sepsis. In some embodiments, sepsis includes mild sepsis. In some embodiments, sepsis includes acute sepsis. In some embodiments, sepsis includes highly aggressive sepsis.

[0297] In some embodiments, the source of sepsis includes pneumonia. In some embodiments, the source of sepsis includes intravascular methicillin-resistant Staphylococcus aureus (MRSA). In some embodiments, the source of sepsis includes urinary tract infection (UTI). In some embodiments, the source of sepsis includes biliary tract infection.

[0298] In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing organ failure, reducing the incidence of said organ failure. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing organ dysfunction, reducing the incidence of said organ dysfunction. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing organ failure, reducing the incidence of said organ failure. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing organ damage, reducing the incidence of said organ damage. In some embodiments, treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating sepsis in a subject in need thereof includes preventing, inhibiting, or reducing the incidence of acute multiple organ failure.

[0299] In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of organ failure, or reduction in the incidence of said organ failure. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of organ dysfunction, or reduction in the incidence of said organ dysfunction. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of organ failure, or reduction in the incidence of said organ failure. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of organ damage, or reduction in the incidence of said organ damage. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of acute multiple organ failure, or reduction in the incidence of said acute multiple organ failure.

[0300] In some embodiments, organ failure during sepsis includes failure of vital organs such as, but not limited to, lungs, heart, kidneys, liver, and blood organs. In some embodiments, multi-organ failure as a component of sepsis includes failure of a combination of lungs, heart, kidneys, liver, and blood. In some embodiments, hematological aberrations during sepsis include thrombocytopenia, lymphopenia, neutropenia, or neutrophilia, or any combination thereof. In some embodiments, organ failure can be measured using standards known in the art, including, but not limited to, sequential organ failure assessment (SOFA) scores. In some embodiments, sepsis is measured using standards known in the art, including, but not limited to, Glasgow coma scale (GCS).

[0301] In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing organ dysfunction, reducing the incidence of said organ dysfunction. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing multiple organ dysfunction, reducing the incidence of said multiple organ dysfunction. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing important organ dysfunction, reducing the incidence of said important organ dysfunction.

[0302] In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of organ dysfunction, or reduction in the incidence of said organ dysfunction. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of multiple organ dysfunction, or reduction in the incidence of said multiple organ dysfunction. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of vital organ dysfunction, or reduction in the incidence of said vital organ dysfunction. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention of an increase in vital organ dysfunction compared to a subject not administered early apoptotic cells.

[0303] In some embodiments, administering early apoptotic cells to a subject with sepsis is extremely effective in treating sepsis. In some embodiments, a measure of effective treatment of sepsis includes the percentage of patients who have recovered from sepsis within a given time frame. In some embodiments, a measure of effective treatment of sepsis includes the percentage of patients who have been discharged from intensive care compared to patients who have not been administered early apoptotic cells. In some embodiments, subjects with sepsis administered early apoptotic cells recover faster than subjects with sepsis who have not been administered early apoptotic cells. In some embodiments, subjects with sepsis administered early apoptotic cells recover more completely than subjects with sepsis who have not been administered early apoptotic cells. In some embodiments, patients with sepsis treated with early apoptotic cells have a reduced mortality rate compared to patients who have not been administered early apoptotic cells.

[0304] In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing cardiovascular dysfunction, reducing the incidence of said cardiovascular dysfunction. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing acute kidney injury, reducing the incidence of said acute kidney injury. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing pulmonary dysfunction, reducing the incidence of said pulmonary dysfunction. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing liver dysfunction, reducing the incidence of said liver dysfunction. In some embodiments, treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject comprises preventing, inhibiting hematological aberrations, reducing the incidence of said hematological aberrations. In some embodiments, treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject comprises preventing, inhibiting a combination of any one of cardiovascular dysfunction, acute kidney injury, pulmonary dysfunction and hematological aberrations, reducing the incidence of a combination of any one of said cardiovascular dysfunction, acute kidney injury, pulmonary dysfunction and hematological aberrations.

[0305] In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of cardiovascular dysfunction, or reduction in the incidence of the cardiovascular dysfunction. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of acute kidney injury, or reduction in the incidence of the acute kidney injury. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of pulmonary dysfunction, or reduction in the incidence of the pulmonary dysfunction. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of liver dysfunction, or reduction in the incidence of the liver dysfunction. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of hematological aberrations, or reduction in the incidence of hematological aberrations. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of any combination of cardiovascular dysfunction, acute kidney injury, pulmonary dysfunction, and hematological abnormalities, or reduction in the incidence of any combination of cardiovascular dysfunction, acute kidney injury, pulmonary dysfunction, and hematological abnormalities.

[0306] In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing cytokine storm, reducing the incidence of the cytokine storm. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing chemokine storm, reducing the incidence of the chemokine storm. In some embodiments, treating, preventing, suppressing sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject includes preventing, suppressing cytokine and chemokine storm, reducing the incidence of the cytokine and chemokine storm.

[0307] In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of cytokine storm, or reduction in the incidence of said cytokine storm. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of chemokine storm, or reduction in the incidence of said chemokine storm. In some embodiments, administering early apoptotic cells to a subject with sepsis results in prevention, inhibition of cytokine and chemokine storm, or reduction in the incidence of said cytokine and chemokine storm.

[0308] In some embodiments, treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject comprises rebalancing the subject's immune response. In some embodiments, treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject comprises reducing the secretion of pro-inflammatory cytokines. In some embodiments, treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject comprises reducing the secretion of pro-inflammatory cytokines / chemokines and anti-inflammatory cytokines / chemokines.

[0309] In some embodiments, administering early apoptotic cells to a subject with sepsis results in rebalancing the subject's immune response. In some embodiments, administering early apoptotic cells to a subject with sepsis results in reduced secretion of pro-inflammatory cytokines. In some embodiments, administering early apoptotic cells to a subject with sepsis results in reduced secretion of pro-inflammatory cytokines / chemokines and anti-inflammatory cytokines / chemokines.

[0310] In some embodiments, treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject comprises reducing the mortality rate of a subject with sepsis. In some embodiments, treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject comprises increasing the survival time of a subject in need thereof.

[0311] In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by more than 60% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by more than 70% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by more than 80% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by more than 90% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting, reducing the incidence of, ameliorating or alleviating sepsis in a subject in need thereof increases the survival time of the subject by greater than 100%, compared to a subject not administered apoptotic cells.

[0312] In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 50%-100% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 80%-100% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 80%, 90% or 100% compared to a subject not administered with apoptotic cells.

[0313] In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 100%-2000% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 200%-300% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by greater than 100% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by greater than 200% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by more than 300% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by more than 400% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by more than 500%, 600%, 700%, 800%, 900% or 1000% compared to a subject not administered with apoptotic cells.

[0314] In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 100% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need thereof, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% compared to a subject not administered with apoptotic cells.

[0315] In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 100%-1000% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 100%-500% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 500%-1000% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 70%-80% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 50% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 60% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 70% compared to a subject not administered with apoptotic cells. In some embodiments, the method of treating, preventing, inhibiting sepsis in a subject in need, reducing the incidence of sepsis in a subject, improving or alleviating sepsis in a subject increases the survival time of the subject by about 80% compared to a subject not administered with apoptotic cells.

[0316] In another embodiment, treating, preventing, inhibiting, reducing the incidence of sepsis in a subject in need thereof, ameliorating or alleviating sepsis in a subject is compared to a subject who experienced sepsis and was not administered apoptotic cells. In another embodiment, treating, preventing, inhibiting, reducing the incidence of sepsis in a subject in need thereof, ameliorating or alleviating sepsis in a subject is compared to a subject who experienced sepsis and was not administered apoptotic supernatant.

[0317] In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering intravenously. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering intravenously after initial standard of care treatment with antibiotics, fluids, and vasopressors.

[0318] In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering between 12-24 hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering between 12-36 hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering between 24-36 hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering between 12-18 hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering between 18-24 hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering between 18-30 hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering between 24-30 hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering between 24-36 hours after diagnosis of sepsis.

[0319] In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering at about 12 hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering at about 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours or hours after diagnosis of sepsis. In some embodiments, administering apoptotic cells to a subject experiencing sepsis comprises administering within 24 hours ± 6 hours after diagnosis of sepsis.

[0320] In some embodiments, the response of a subject having sepsis and administered a composition comprising apoptotic cells comprises a dose response. In some embodiments, the response of a subject having sepsis and administered a composition comprising apoptotic cell supernatant comprises a dose response.

[0321] Alpha-1-antitrypsin (AAT)

[0322] Alpha-1-antitrypsin (AAT) is a circulating 52-kDa glycoprotein mainly produced by the liver. AAT is mainly known as a serine protease inhibitor and is encoded by the gene SERPINA1. AAT inhibits neutrophil elastase, and hereditary defects in circulating AAT can lead to lung tissue deterioration and liver disease. During inflammation, the serum AAT concentration of healthy individuals increases by two times.

[0323] There is an inverse correlation between AAT levels and the severity of several inflammatory diseases. For example, reduced levels or activity of AAT have been described in patients with HIV infection, diabetes, chronic liver disease caused by hepatitis C infection, and several types of vasculitis.

[0324] There is increasing evidence that human serum-derived alpha-1-antitrypsin (AAT) reduces the production of pro-inflammatory cytokines, induces anti-inflammatory cytokines, and interferes with the maturation of dendritic cells.

[0325] Indeed, addition of AAT to human peripheral blood mononuclear cells (PBMCs) inhibited LPS-induced TNF-α and IL-1β release but increased the production of IL-1 receptor antagonist (IL-1Ra) and IL-10.

[0326] AAT reduces IL-1β-mediated islet toxicity in vitro, and AAT monotherapy prolongs islet allograft survival, promotes antigen-specific immune tolerance in mice, and delays the development of diabetes in non-obese diabetic (NOD) mice. In experimental models, AAT was shown to inhibit LPS-induced acute lung injury. Recently, in a mouse model of acute myocardial ischemia-reperfusion injury, AAT was shown to reduce infarct size and the severity of heart failure.

[0327] Monotherapy with clinical-grade human AAT (hAAT) reduced circulating proinflammatory cytokines, reduced the severity of graft-versus-host disease (GvHD) and prolonged animal survival after experimental allogeneic bone marrow transplantation (Tawara et al., Proc Natl Acad Sci USA, January 10, 2012; 109(2):564-9), which is incorporated herein by reference. AAT treatment reduced the expansion of allogeneic reactive T effector cells, but enhanced the recovery of T regulatory T cells (Treg), thereby changing the ratio of donor T effector cells to T regulatory cells, which is conducive to reducing pathological processes. In vitro, AAT suppressed the in vitro secretion of LPS-induced proinflammatory cytokines (such as TNF-α and IL-1β), enhanced the production of anti-inflammatory cytokines IL-10 and impaired NF-κB translocation in host dendritic cells. Marcondes, Blood. 2014 (Oct 30;124(18):2881-91), incorporated herein by reference, showed that treatment with AAT not only improved GvHD but also preserved and possibly even enhanced the graft-versus-leukemia (GVL) effect.

[0328] In one embodiment, disclosed herein are compositions comprising T cells (CAR T cells) expressing chimeric antigen receptors and alpha-1-antitrypsin (AAT). In another embodiment, CAR T cells and alpha-1-antitrypsin (AAT) are in separate compositions. In another embodiment, AAT includes full-length AAT or a functional fragment thereof. In another embodiment, AA includes an analog of full-length AAT or a functional fragment thereof. In another embodiment, the composition comprising AAT further includes apoptotic cells or apoptotic cell supernatant.

[0329] In another embodiment, disclosed herein is a method for treating, preventing, inhibiting a subject's cancer or tumor, or reducing the incidence of the cancer or tumor, improving or alleviating the cancer or tumor, the method comprising administering to the subject a T cell expressing a chimeric antigen receptor (CAR T cell) and a composition comprising alpha-1-antitrypsin (AAT). In another embodiment, the method further comprises apoptotic cells or apoptotic cell supernatant.

[0330] In another embodiment, disclosed herein is a method for inhibiting cytokine release syndrome or cytokine storm in a subject experiencing T cell (CAR T cell) therapy expressing a chimeric antigen receptor or reducing the incidence of the cytokine release syndrome or cytokine storm, the method comprising administering to the subject a composition comprising alpha-1-antitrypsin (AAT). In another embodiment, the method for treating cytokine release syndrome or cytokine storm in a subject experiencing T cell (CAR T cell) therapy expressing a chimeric antigen receptor comprises administering to the subject a composition comprising alpha-1-antitrypsin (AAT). In another embodiment, the method for preventing cytokine release syndrome or cytokine storm in a subject experiencing T cell (CAR T cell) therapy expressing a chimeric antigen receptor comprises administering to the subject a composition comprising alpha-1-antitrypsin (AAT). In another embodiment, the method for improving cytokine release syndrome or cytokine storm in a subject experiencing T cell (CAR T cell) therapy expressing a chimeric antigen receptor comprises administering to the subject a composition comprising alpha-1-antitrypsin (AAT). In another embodiment, a method of ameliorating cytokine release syndrome or cytokine storm in a subject undergoing chimeric antigen receptor-expressing T cell (CAR T cell) therapy comprises the step of administering to the subject a composition comprising alpha-1-antitrypsin (AAT).

[0331] In another embodiment, disclosed herein is a method of reducing or inhibiting cytokine production in a subject experiencing or susceptible to cytokine release syndrome or cytokine storm, the method comprising the step of administering to the subject a composition comprising alpha-1-antitrypsin (AAT).

[0332] In one embodiment, AAT is administered alone to control cytokine release. In another embodiment, both AAT and apoptotic cells or a composition thereof or apoptotic cell supernatant or a composition thereof are administered to control cytokine release.

[0333] Immunomodulators

[0334] It will be appreciated by those skilled in the art that immunomodulators may encompass extracellular mediators, receptors, mediators of intracellular signaling pathways, regulators of translation and transcription, and immune cells. In one embodiment, another agent disclosed herein is an immunomodulator known in the art. In another embodiment, an immunomodulator is used in the method disclosed herein to reduce the level of at least one cytokine. In another embodiment, an immunomodulator is used in the method disclosed herein to reduce or inhibit CRS or cytokine storm. In some embodiments, an immunomodulator is used in the method disclosed herein to treat, prevent tumors or cancers, inhibit the growth of the tumors or cancers, delay disease progression, reduce tumor load, or reduce the incidence of the tumors or cancers, or any combination thereof. In some embodiments, an immunomodulator is used in combination with another composition disclosed herein (for example, but not limited to, a composition comprising early apoptotic cells or comprising CAR T cells).

[0335] In one embodiment, the immunomodulator includes a compound that blocks, inhibits or reduces the release of cytokines or chemokines. In another embodiment, the immunomodulator includes a compound that blocks, inhibits or reduces the release of IL-21 or IL-23 or a combination thereof. In another embodiment, the immunomodulator includes an antiretroviral drug in the chemokine receptor-5 (CCR5) receptor antagonist class, such as maraviroc. In another embodiment, the immunomodulator includes an anti-DNAM-1 antibody. In another embodiment, the immunomodulator includes a damage / pathogen-associated molecule (DAMP / PAMP) selected from a group including heparin sulfate, ATP and uric acid or any combination thereof. In another embodiment, the immunomodulator includes sialic acid-binding Ig-like lectins (Siglecs). In another embodiment, the immunomodulator includes a cell mediator with tolerance, such as a regulatory CD4 + CD25 +In another embodiment, the immunomodulator includes a dendritic cell. In another embodiment, the immunomodulator includes a monocyte. In another embodiment, the immunomodulator includes a macrophage. In another embodiment, the immunomodulator includes a JAK2 or JAK3 inhibitor selected from the group including ruxolitinib and tofacitinib. In another embodiment, the immunomodulator includes an inhibitor of spleen tyrosine kinase (Syk), such as fostamatinib. In another embodiment, the immunomodulator includes STAT3 acetylated by the histone deacetylase inhibitor vorinostat. In another embodiment, the immunomodulator includes a neddylation inhibitor, such as MLN4924. In another embodiment, the immunomodulator includes a miR-142 antagonist. In another embodiment, the immunomodulator includes a chemical analog of cytidine, such as azacitidine. In another embodiment, the immunomodulator comprises an inhibitor of histone deacetylase, such as vorinostat. In another embodiment, the immunomodulator comprises an inhibitor of histone methylation. In another embodiment, the immunomodulator comprises an antibody. In another embodiment, the antibody is rituximab (RtX).

[0336] Tellurium compounds

[0337] Tellurium is a trace element found in the human body. Various tellurium compounds have immunomodulatory properties and have shown beneficial effects in various preclinical and clinical studies. A particularly effective family of tellurium-containing compounds is disclosed in, for example, U.S. Pat. Nos. 4,752,614; 4,761,490; 4,764,461 and 4,929,739. The immunomodulatory properties of this family of tellurium-containing compounds are described in, for example, U.S. Pat. Nos. 4,962,207, 5,093,135, 5,102,908 and 5,213,899, all of which are incorporated by reference as if fully set forth herein.

[0338] One promising compound is trichloro(dioxyethylene-O,O')ammonium tellurate, also referred to herein and in the art as AS101. As a representative example of the family of tellurium-containing compounds discussed above, AS101 exhibits antiviral (Nat. Immun. Cell Growth Regul. 7(3):163-8, 1988; AIDS Res Hum Retroviruses. 8(5):613-23, 1992) and antitumor activity (Nature 330(6144):173-6, 1987; J. Clin. Oncol. 13(9):2342-53, 1995; J. Immunol. 161(7):3536-42, 1998). Further, AS101 is characterized by low toxicity.

[0339] In one embodiment, a composition comprising a tellurium-containing immunomodulator compound can be used in the methods disclosed herein, wherein the tellurium-based compound stimulates both the innate and acquired arms of the immune response. For example, AS101 has been shown to be a potent activator of interferon (IFN) in mice (J. Natl. Cancer Inst. 88(18):1276-84, 1996) and humans (Natural Immunity and Cell Growth Regulation 9(3):182-90, 1990; Immunology 70(4):473-7, 1990; J. Natl. Cancer Inst. 88(18):1276-84, 1996).

[0340] In another embodiment, the tellurium-based compound induces the secretion of a series of cytokines such as IL-1α, IL-6 and TNF-α.

[0341] In another embodiment, the tellurium compound includes a tellurium compound known in the art to have immunomodulatory properties. In another embodiment, the tellurium compound includes trichloro(dioxyethylene-O,O')ammonium tellurate.

[0342] In one embodiment, the tellurium compound inhibits the secretion of at least one cytokine. In another embodiment, the tellurium compound reduces the secretion of at least one cytokine. In another embodiment, the tellurium compound inhibits or reduces the cytokine release syndrome (CRS) of a cytokine storm.

[0343] In one embodiment, disclosed herein are compositions comprising T cells (CAR T cells) expressing chimeric antigen receptors and tellurium compounds. In another embodiment, CAR T cells and tellurium compounds are in separate compositions. In another embodiment, AAT includes full-length AAT or a functional fragment thereof. In another embodiment, AA includes an analog of full-length AAT or a functional fragment thereof.

[0344] In another embodiment, disclosed herein is a method for treating, preventing, inhibiting, reducing the incidence, improving or alleviating cancer or tumor in a subject, the method comprising administering to the subject a T cell expressing a chimeric antigen receptor (CAR T cell) and a composition comprising a tellurium compound.

[0345] In another embodiment, disclosed herein is a method for inhibiting cytokine release syndrome or cytokine storm in a subject experiencing T cell (CAR T cell) therapy expressing a chimeric antigen receptor or reducing the incidence of the cytokine release syndrome or cytokine storm, the method comprising administering to the subject a composition comprising a tellurium compound. In another embodiment, the method for treating cytokine release syndrome or cytokine storm in a subject experiencing T cell (CAR T cell) therapy expressing a chimeric antigen receptor comprises administering to the subject a composition comprising a tellurium compound. In another embodiment, the method for preventing cytokine release syndrome or cytokine storm in a subject experiencing T cell (CAR T cell) therapy expressing a chimeric antigen receptor comprises administering to the subject a composition comprising a tellurium compound. In another embodiment, the method for improving cytokine release syndrome or cytokine storm in a subject experiencing T cell (CAR T cell) therapy expressing a chimeric antigen receptor comprises administering to the subject a composition comprising a tellurium compound. In another embodiment, a method of alleviating cytokine release syndrome or cytokine storm in a subject undergoing chimeric antigen receptor-expressing T cell (CAR T cell) therapy comprises the step of administering to the subject a composition comprising a tellurium compound.

[0346] In another embodiment, disclosed herein is a method of reducing or inhibiting cytokine production in a subject experiencing or susceptible to cytokine release syndrome or cytokine storm, the method comprising the step of administering to the subject a composition comprising a tellurium compound.

[0347] In one embodiment, the tellurium-based compound is administered alone to control cytokine release. In another embodiment, the tellurium-based compound and apoptotic cells or a composition thereof or apoptotic cell supernatant or a composition thereof are both administered to control cytokine release.

[0348] Dendritic cells

[0349] In one embodiment, dendritic cells (DC) are antigen-producing and presenting cells of the mammalian immune system, which process antigenic substances and present them on the cell surface to the T cells of the immune system, and thereby enable T cells to be sensitive to both new antigens and recall antigens. In another embodiment, DC is the most effective antigen-producing cell, acting as a messenger between the innate immune system and the adaptive immune system. In one embodiment, DC cells can be used to induce specific anti-tumor immunity by producing effector cells that attack and lyse tumors.

[0350] Dendritic cells are found in tissues that come into contact with the external environment, such as the skin (where a specialized dendritic cell type called Langerhans cells is found) and the lining of the nose, lungs, stomach, and intestines. They can also be found in the blood in an immature state. Once activated, they migrate to lymph nodes where they interact with T cells and B cells to initiate and shape adaptive immune responses. At certain stages of development, they grow branch-like projections, the dendrites that give the cells their name. Dendritic cells can be engineered to express specific tumor antigens.

[0351] The three signals required for T cell activation are: (i) presentation of cognate antigens in the context of self-MHC molecules; (ii) co-stimulation of membrane-bound receptor-ligand pairs; and (iii) soluble factors that directly depolarize the ensuing immune response. Dendritic cells (DCs) are able to provide all three signals required for T cell activation, making them an excellent cancer vaccine platform.

[0352] Therefore, in one embodiment, disclosed herein is a composition comprising dendritic cells and an additional agent, wherein the additional agent comprises apoptotic cells, apoptotic supernatant, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium compound or an immunomodulator or any combination thereof.

[0353] In another embodiment, disclosed herein is a method for treating, preventing, inhibiting, reducing the incidence of, improving or alleviating cancer or tumors in a subject, the method comprising the step of administering to the subject a composition comprising dendritic cells and additional agents, wherein the agents comprise apoptotic cells, apoptotic supernatants, CTLA-4 blockers, alpha-1 antitrypsin or fragments thereof or analogs thereof, tellurium compounds or immunomodulators or any combination thereof.

[0354] Genetic modification

[0355] In some embodiments, RNA, DNA, recombinant viruses, or a combination thereof can be used to complete genetic modification of T cells, dendritic cells, and / or apoptotic cells. In some embodiments, vectors derived from gamma retroviruses or lentiviruses are used in the compositions and methods disclosed herein. In another embodiment, these vectors can be integrated into the host genome with potential permanent expression of transgenes and have low inherent immunogenicity. In another embodiment, another vector integrated into the host genome and / or with low inherent immunogenicity can be used in the compositions and methods disclosed herein. In another embodiment, a non-viral vector-mediated sleeping beauty transposon system is used to insert CAR and other genes into T cells. In another embodiment, a "suicide gene" is integrated into T cells, wherein the expression of pro-apoptotic genes is under the control of an inducible promoter that responds to systemically delivered drugs.

[0356] In some embodiments, genetic modification can be transient. In another embodiment, genetic modification can utilize messenger RNA (mRNA). In another embodiment, a large number of cells can be repeatedly infused on transiently engineered T cells (such as mRNA transfected T cells). In another embodiment, the transient expression of electroporation-mediated protein based on RNA performed on lymphocytes using in vitro transcribed mRNA lasts for about a week and avoids the risk of integrating viral vectors. In another embodiment, mRNA transduced dendritic cells or mRNA electroporated T and NK lymphocytes.

[0357] It has been demonstrated that genetically modified T cells can persist for more than ten years after adoptive transfer without side effects, indicating that genetic modification of human T cells is fundamentally safe.

[0358] In another embodiment, the genetic modification in the compositions and methods disclosed herein can be any method known in the art.

[0359] Apoptotic cells

[0360] The generation of apoptotic cells ("ApoCell") used in the compositions and methods disclosed herein has been described in WO2014 / 087408 (which is incorporated herein by reference in its entirety) and is briefly described in Example 1 below. In another embodiment, apoptotic cells for use in the compositions and methods disclosed herein are generated in any manner known in the art. In another embodiment, the apoptotic cells for use in the compositions and methods disclosed herein are autologous to the subject undergoing therapy. In another embodiment, the apoptotic cells for use in the compositions and methods disclosed herein are allogeneic to the subject undergoing therapy. In another embodiment, the composition comprising apoptotic cells comprises apoptotic cells as disclosed herein or known in the art.

[0361] The skilled artisan will appreciate that the term "autologous" may encompass tissues, cells, nucleic acid molecules or polypeptides where the donor and recipient are the same individual.

[0362] The skilled artisan will appreciate that the term "allogeneic" can encompass tissues, cells, nucleic acid molecules or polypeptides derived from separate individuals of the same species. In some embodiments, the allogeneic donor cells are genetically different from the recipient.

[0363] In some embodiments, the enriched mononuclear cell composition obtained according to the production methods disclosed herein is achieved by leukapheresis. The skilled person will understand that the term "leukapheresis" can encompass apheresis procedures in which white blood cells are separated from the blood of the donor. In some embodiments, the blood of the donor undergoes leukapheresis and thus an enriched mononuclear cell composition is obtained according to the production methods disclosed herein. It should be noted that, as is known in the art, at least one anticoagulant needs to be used during leukapheresis in order to prevent the collected cells from clotting.

[0364] In some embodiments, the leukapheresis procedure is configured to allow the collection of enriched mononuclear cell compositions according to the production methods disclosed herein. In some embodiments, the cell collection obtained by leukapheresis includes at least 65%. In other embodiments, at least 70% or at least 80% of monocytes as disclosed herein. In some embodiments, in the production methods disclosed herein, plasma from cell donors is collected in parallel to obtain enriched mononuclear cell compositions. In some embodiments, according to the production methods disclosed herein, plasma of about 300-600ml from cell donors is collected in parallel to obtain enriched mononuclear cell compositions. In some embodiments, plasma is collected in parallel to obtain enriched mononuclear cell compositions according to the production methods disclosed herein as a part of freezing and / or incubation medium. Other detailed methods for obtaining apoptotic cell enriched populations for compositions and methods disclosed herein can be found in WO 2014 / 087408, which is incorporated herein by reference in its entirety.

[0365] In some embodiments, the early apoptotic cells used in the methods disclosed herein include at least 85% monocytes. In further embodiments, the early apoptotic cells used in the methods disclosed herein include at least 85% monocytes, 90% monocytes, or alternatively more than 90% monocytes. In some embodiments, the early apoptotic cells used in the methods disclosed herein include at least 90% monocytes. In some embodiments, the early apoptotic cells used in the methods disclosed herein include at least 95% monocytes.

[0366] It should be noted that in some embodiments, while the enriched monocyte preparation at the time of cell collection includes at least 65%, preferably at least 70%, and most preferably at least 80% monocytes, the final drug population after the method of generating early apoptotic cells for use in the methods disclosed herein includes at least 85%, preferably at least 90%, and most preferably at least 95% monocytes.

[0367] In certain embodiments, the enriched monocyte preparation for producing a composition of early apoptotic cells for use in the methods disclosed herein includes at least 50% monocytes at the time of cell collection. In certain embodiments, disclosed herein is a method for producing a drug population, wherein the method includes obtaining an enriched monocyte preparation from the peripheral blood of a donor, the enriched monocyte preparation including at least 50% monocytes. In certain embodiments, disclosed herein is a method for producing a drug population, wherein the method includes freezing an enriched monocyte preparation including at least 50% monocytes.

[0368] In some embodiments, the cell preparation comprises at least 85% monocytes, wherein at least 40% of the cells in the preparation are in an early apoptotic state, wherein at least 85% of the cells in the preparation are viable cells. In some embodiments, the apoptotic cell preparation comprises no more than 15% CD15 高 Expressing cells.

[0369] Technicians will appreciate that the term "early apoptotic state" can encompass cells showing early signs of apoptosis without late signs of apoptosis. Examples of early signs of apoptosis include exposure of phosphatidylserine (PS) and loss of mitochondrial membrane potential. Examples of late events include the entry of propidium iodide (PI) into cells and final DNA cleavage. In order to prove that cells are in an "early apoptotic" state, in some embodiments, PS exposure detection performed by annexin-V and PI staining is used, and cells stained with annexin V but not PI are considered to be "early apoptotic cells". In another embodiment, cells stained with both annexin-V FITC and PI are considered to be "late apoptotic cells". In another embodiment, cells not stained with annexin-V or PI are considered to be non-apoptotic living cells.

[0370] The skilled person will understand that in some embodiments, the terms "early apoptotic cells", "apoptotic cells", "Allocetra", "ALC" and "ApoCell" and grammatical variants thereof are used interchangeably, with all the same properties and meanings. The skilled person will understand that in some embodiments, the compositions and methods described herein include early apoptotic cells. In some embodiments, as described herein, early apoptotic cells are HLA matched to a recipient (a subject in need) of a composition comprising early apoptotic cells. In some embodiments, as described herein, early apoptotic cells are mismatched to a recipient (a subject in need) of a composition comprising early apoptotic cells. In some embodiments, as described herein in detail, early apoptotic cells that are mismatched to a recipient (a subject in need) of a composition comprising early apoptotic cells are irradiated. In some embodiments, irradiated mismatched cells are referred to as "Allocetra-OTS" or "ALC-OTS".

[0371] In some embodiments, apoptotic cells include cells in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 90% of the cells are in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 80% of the cells are in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 70% of the cells are in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 60% of the cells are in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 50% of the cells are in an early apoptotic state.

[0372] In some embodiments, the composition comprising apoptotic cells further comprises an anticoagulant.

[0373] In some embodiments, early apoptotic cells are stable. It will be appreciated by the skilled artisan that in some embodiments, stability encompasses maintaining early apoptotic cell characteristics over time, such as maintaining early apoptotic cell characteristics when stored at about 2-8° C. In some embodiments, stability includes maintaining early apoptotic cell characteristics when stored at freezing temperatures (e.g., at or below 0° C.).

[0374] In some embodiments, the enriched mononuclear cell population obtained according to the method for producing early apoptotic cells of the methods disclosed herein is subjected to freezing in a freezing medium. In some embodiments, freezing is progressive. In some embodiments, after collection, the cells are maintained at room temperature until freezing. In some embodiments, the cell preparation undergoes at least one washing step in a washing medium after cell collection and before freezing.

[0375] As used herein, the terms "obtaining cells" and "cell collection" can be used interchangeably. In some embodiments, the cells of the cell preparation are frozen within 3-6 hours of collection. In some embodiments, the cell preparation is frozen within 6 hours of cell collection at most. In some embodiments, the cells of the cell preparation are frozen within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours of collection. In other embodiments, the cells of the cell preparation are frozen at most 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours of collection. In other embodiments, after collection, the cells are maintained at 2-8°C until frozen.

[0376] In some embodiments, freezing according to the generation of early apoptotic cell groups includes: freezing the cell preparation at about -18 ° C to -25 ° C, then freezing the cell preparation at about -80 ° C, and finally freezing the cell preparation in liquid nitrogen until thawing. In some embodiments, freezing according to the generation of early apoptotic cell groups includes: freezing the cell preparation at about -18 ° C to -25 ° C for at least 2 hours, then freezing the cell preparation at about -80 ° C for at least 2 hours, and finally freezing the cell preparation in liquid nitrogen until thawing. In some embodiments, before thawing, the cells are kept in liquid nitrogen for at least 8 hours, 10 hours or 12 hours. In some embodiments, the cells of the cell preparation are kept in liquid nitrogen until thawing and incubated with apoptosis-inducing incubation medium. In some embodiments, the cells of the cell preparation are kept in liquid nitrogen until the day of hematopoietic stem cell transplantation. In a non-limiting example, the time from cell collection and freezing to the preparation of the final group can be between 1-50 days, alternatively between 6-30 days. In alternative embodiments, the cell preparation may be maintained in liquid nitrogen for an extended period of time, such as at least several months.

[0377] In some embodiments, freezing in accordance with the generation of an early apoptotic cell population comprises freezing the cell preparation at about -18°C to -25°C for at least 0.5 hours, 1 hour, 2 hours, 4 hours. In some embodiments, freezing in accordance with the generation of an early apoptotic cell population comprises freezing the cell preparation at about -18°C to -25°C for about 2 hours. In some embodiments, freezing in the generation of an early apoptotic cell population comprises freezing the cell preparation at about -80°C for at least 0.5 hours, 1 hour, 2 hours, 4 hours, 12 hours.

[0378] In some embodiments, the enriched monocyte composition can remain frozen for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 20 months. In some embodiments, the enriched monocyte composition can remain frozen for at least 0.5 years, 1 year, 2 years, 3 years, 4 years, 5 years. In certain embodiments, the enriched monocyte composition can remain frozen for at least 20 months.

[0379] In some embodiments, the enriched monocyte composition is frozen for at least 8 hours, 10 hours, 12 hours, 18 hours, 24 hours. In certain embodiments, the enriched monocyte composition is frozen for a period of at least 8 hours. In some embodiments, the enriched monocyte composition is frozen for at least about 10 hours. In some embodiments, the enriched monocyte composition is frozen for at least about 12 hours. In some embodiments, the enriched monocyte composition is frozen for about 12 hours. In some embodiments, the total freezing time of the enriched monocyte composition (at about -18°C to -25°C, at about -80°C and in liquid nitrogen) is at least 8 hours, 10 hours, 12 hours, 18 hours, 24 hours.

[0380] In some embodiments, freezing induces an early apoptotic state at least in part in the cells of the enriched mononuclear cell composition. In some embodiments, the freezing medium comprises an RPMI 1640 medium comprising L-glutamine, Hepes, Hes, dimethyl sulfoxide (DMSO) and plasma. In some embodiments, the plasma in the freezing medium is autologous plasma of a donor of the enriched mononuclear cells of a donation population. In some embodiments, the freezing medium comprises an RPMI 1640 medium comprising 2mM L-glutamine, 10mM Hepes, 5% Hes, 10% dimethyl sulfoxide and 20% v / v plasma.

[0381] In certain embodiments, the freezing medium includes an anticoagulant. In certain embodiments, at least some of the culture media (comprising freezing medium, incubation medium, and wash medium) used during the generation of the early apoptotic cell population include an anticoagulant. In certain embodiments, all culture media (comprising anticoagulants) used during the generation of the early apoptotic cell population include the same concentration of anticoagulants. In certain embodiments, anticoagulants are not added to the final suspension medium of the cell population.

[0382] In certain embodiments, adding an anticoagulant to at least the freezing medium improves the productivity of the cell preparation. In other embodiments, adding an anticoagulant to the freezing medium improves the productivity of the cell preparation in the presence of high triglyceride levels. As used herein, the increase in the productivity of the cell preparation relates to an increase in at least one of the following: the percentage of viable cells in the frozen cells, the percentage of early stage apoptotic cells in the viable cells, and a combination thereof.

[0383] In some embodiments, early apoptotic cells are stable for at least 24 hours. In another embodiment, early apoptotic cells are stable for 24 hours. In another embodiment, early apoptotic cells are stable for more than 24 hours. In another embodiment, early apoptotic cells are stable for at least 36 hours. In another embodiment, early apoptotic cells are stable for 48 hours. In another embodiment, early apoptotic cells are stable for at least 36 hours. In another embodiment, early apoptotic cells are stable for more than 36 hours. In another embodiment, early apoptotic cells are stable for at least 48 hours. In another embodiment, early apoptotic cells are stable for 48 hours. In another embodiment, early apoptotic cells are stable for at least 48 hours. In another embodiment, early apoptotic cells are stable for more than 48 hours. In another embodiment, early apoptotic cells are stable for at least 72 hours. In another embodiment, early apoptotic cells are stable for 72 hours. In another embodiment, early apoptotic cells are stable for more than 72 hours.

[0384] Technicians should understand that the term "stable" encompasses apoptotic cells that maintain PS positive (phosphatidylserine positive) and PI positive (propidium iodide positive) percentage is very small. PI positive cells provide an indication of membrane stability, wherein PI positive cells are allowed to enter the cell, thereby showing that the stability of the membrane is poor. In certain embodiments, stable early apoptotic cells maintain at least 24 hours, at least 36 hours, at least 48 hours or at least 72 hours in early apoptosis. In another embodiment, stable early apoptotic cells maintain 24 hours, 36 hours, 48 ​​hours or 72 hours in early apoptosis. In another embodiment, stable early apoptotic cells maintain more than 24 hours, more than 36 hours, more than 48 hours or more than 72 hours in early apoptosis. In another embodiment, stable early apoptotic cells maintain their state in the extended time period.

[0385] In some embodiments, the apoptotic cell population is free of cell aggregates. In some embodiments, the apoptotic cell population is free of large cell aggregates. In some embodiments, the apoptotic cell population is free of large cell aggregates compared to an apoptotic cell population prepared without adding an anticoagulant in a step other than collecting cells from a donor (leukapheresis). In some embodiments, the apoptotic cell population or a composition thereof comprises an anticoagulant.

[0386] In some embodiments, the apoptotic cells are free of cell aggregates, wherein the apoptotic cells are obtained from a subject with high blood triglycerides. In some embodiments, the subject's blood triglyceride level is above 150 mg / dL. In some embodiments, the apoptotic cell population is free of cell aggregates, wherein the apoptotic cell population is prepared from cells obtained from a subject with normal blood triglycerides. In some embodiments, the subject's blood triglyceride level is equal to or less than 150 mg / dL. In some embodiments, cell aggregates produce cell loss during the apoptotic cell generation method.

[0387] The skilled artisan will appreciate that the term "aggregate" or "cell aggregate" may encompass the reversible agglutination of blood cells under low shear or stagnant conditions. Cell aggregates may be visually observed during the incubation step in which apoptotic cells are generated. Cell aggregation may be measured by any method known in the art, such as by visual imaging of the sample under an optical microscope or using flow cytometry.

[0388] In some embodiments, the anticoagulant is selected from the group consisting of: heparin, acid citrate dextrose (ACD) formula A, and combinations thereof. In some embodiments, the anticoagulant is selected from the group consisting of: heparin, acid citrate dextrose (ACD) formula A, and combinations thereof.

[0389] In some embodiments of the method for preparing early apoptotic cell populations and compositions thereof, an anticoagulant is added to at least one culture medium used during the preparation of the population. In some embodiments, at least one culture medium used during the preparation of the population is selected from the group consisting of: freezing medium, washing medium, apoptosis induction incubation medium, and any combination thereof.

[0390] In some embodiments, the anticoagulant is selected from the group consisting of: heparin, ACD formula A, and combinations thereof. It should be noted that other anticoagulants known in the art may be used, such as but not limited to Fondaparinaux, Bivalirudin, and Argatroban.

[0391] In some embodiments, at least one culture medium used during the preparation of the population contains a 5% ACD Formula A solution including 10 U / ml heparin. In some embodiments, an anticoagulant is not added to the final suspension medium of the cell population. As used herein, the terms "final suspension medium" and "administration medium" are used interchangeably and have all the same properties and meanings.

[0392] In some embodiments, at least one culture medium used during the preparation of the population includes heparin at a concentration of 0.1-2.5 U / ml. In some embodiments, at least one culture medium used during the preparation of the population includes ACD formula A at a concentration between 1%-15% v / v. In some embodiments, the freezing medium includes an anticoagulant. In some embodiments, the incubation medium includes an anticoagulant. In some embodiments, both the freezing medium and the incubation medium include an anticoagulant. In some embodiments, the anticoagulant is selected from the group consisting of heparin, ACD formula A, and combinations thereof.

[0393] In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5 U / ml. In some embodiments, the concentration of ACD Formula A in the freezing medium is between 1%-15% v / v. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5 U / ml. In some embodiments, the concentration of ACD Formula A in the incubation medium is between 1%-15% v / v. In some embodiments, the anticoagulant is a solution of Acid-Citrate-Dextrose (ACD) Formula A. In some embodiments, the anticoagulant added to at least one culture medium used during the preparation of the population is ACD Formula A containing heparin at a concentration of 10 U / ml.

[0394] In some embodiments, the apoptosis-inducing incubation medium for producing an early apoptotic cell population includes an anticoagulant. In some embodiments, the freezing medium and the apoptosis-inducing incubation medium for producing an early apoptotic cell population both include an anticoagulant. Without wishing to be bound by any theory or mechanism, in order to maintain a high and stable cell yield in different cell compositions, regardless of the cell collection scheme, in some embodiments, the addition of an anticoagulant includes adding an anticoagulant to both the freezing medium and the apoptosis-inducing incubation medium during the generation of the apoptotic cell population. In some embodiments, the high and stable cell yield in the composition includes a cell yield of at least 30%, preferably at least 40%, and usually at least 50% of the cells in the initial cell population for inducing apoptosis.

[0395] In certain embodiments, the freezing medium and the incubation medium both include anticoagulants. In certain embodiments, anticoagulants are added to the incubation medium and the freezing medium both resulting in high and stable cell yields between different preparations of the group regardless of cell collection conditions (such as but not limited to the type of anticoagulants added at regular intervals and / or during cell collection). In certain embodiments, anticoagulants are added to the incubation medium and the freezing medium both resulting in high and stable cell yields between different preparations regardless of the type of anticoagulants added at regular intervals and / or during leukapheresis. In certain embodiments, in the presence of high triglyceride levels, the generation of cell preparations results in low and / or unstable cell yields between different preparations. In certain embodiments, the blood of donors with high triglyceride levels produces cell preparations resulting in low and / or unstable cell yields of cell preparations. In certain embodiments, the term "high triglyceride levels" refers to triglyceride levels higher than the normal levels of healthy subjects of the same sex and age. In certain embodiments, the term "high triglyceride levels" refers to triglyceride levels higher than about 1.7 mmoles / liter. As used herein, high and stable yield refers to a cell yield in a population that is high enough to enable the preparation of a dose that will exhibit therapeutic efficiency when administered to a subject. In some embodiments, therapeutic efficiency refers to the ability to treat, prevent, or improve an immune disease, autoimmune disease, or inflammatory disease in a subject. In some embodiments, a high and stable cell rate is a cell yield of at least 30%, possibly at least 40%, and typically at least 50% of the cells initially frozen in the population.

[0396] In some embodiments, if the cell preparation is obtained from a donor with high triglyceride levels, the donor will take at least one measure selected from the group consisting of: taking a triglyceride-lowering medication (such as, but not limited to, a statin and / or bezafibrate) before donation; fasting for at least 8 hours, 10 hours, 12 hours before donation; eating an appropriate diet for at least 24 hours, 48 ​​hours, 72 hours before donation to lower blood triglyceride levels; and any combination thereof.

[0397] In some embodiments, the yield of cells in a population is related to the number of cells in the composition in the initial number of cells subjected to apoptosis induction.As used herein, the terms "induce an early apoptotic state" and "induce apoptosis" are used interchangeably.

[0398] In some embodiments, after freezing and thawing, the enriched mononuclear cell composition is incubated in an incubation medium. In some embodiments, there is at least one washing step between thawing and incubation. As used herein, the terms "incubation medium" and "apoptosis induction incubation medium" are used interchangeably. In some embodiments, the incubation medium includes RPMI 1640 culture medium supplemented with L-glutamine, Hepes, methylprednisolone and plasma. In some embodiments, the wash medium includes 2mM L-glutamine, 10mM Hepes and 10% v / v plasma. In some embodiments, the plasma in the incubation medium is derived from the same donor from which the cells of the cell preparation are derived. In some embodiments, plasma is added to the incubation medium on the day of incubation. In some embodiments, incubation is carried out at 37°C and 5% CO2.

[0399] In some embodiments, the incubation medium includes methylprednisolone. In some embodiments, the methylprednisolone in the incubation medium further induces the cells in the enriched mononuclear cell composition to enter an early apoptotic state. In some embodiments, the cells in the enriched mononuclear cell composition are induced to enter an early apoptotic state by freezing and incubating in the presence of methylprednisolone. In some embodiments, the generation of the early apoptotic cell population advantageously allows the induction of an early apoptotic state without substantially inducing necrosis, wherein the cells remain stable in the early apoptotic state for about 24 hours after preparation.

[0400] In some embodiments, the incubation medium comprises methylprednisolone at a concentration of about 10-100 μg / ml. In some embodiments, the incubation medium comprises methylprednisolone at a concentration of about 40-60 μg / ml, alternatively about 45-55 μg / ml. In some embodiments, the incubation medium comprises methylprednisolone at a concentration of 50 μg / ml.

[0401] In some embodiments, the incubation lasts about 2-12 hours, possibly 4-8 hours, and typically about 5-7 hours. In some embodiments, the incubation lasts about 6 hours. In some embodiments, the incubation lasts at least 6 hours. In a preferred embodiment, the incubation lasts 6 hours.

[0402] In some embodiments, the incubation medium includes an anticoagulant. In some embodiments, adding an anticoagulant to the incubation medium improves the yield of the cell preparation. In some embodiments, the concentration of the anticoagulant in the incubation medium is the same as the concentration of the anticoagulant in the freezing medium. In some embodiments, the incubation medium includes an anticoagulant selected from the group consisting of: heparin, ACD formula A, and a combination thereof. In some embodiments, the anticoagulant used in the incubation medium is an ACD formula A containing heparin at a concentration of 10U / ml.

[0403] In some embodiments, the incubation medium includes heparin. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5 U / ml. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5 U / ml, may be between 0.3-0.7 U / ml, typically about 0.5 U / ml. In certain embodiments, the concentration of heparin in the incubation medium is about 0.5 U / ml.

[0404] In some embodiments, the incubation medium includes ACD Formula A. In some embodiments, the concentration of ACD Formula A in the incubation medium is between 1%-15% v / v. In some embodiments, the concentration of ACD Formula A in the incubation medium is between 1%-15% v / v, may be between 4%-7% v / v, typically about 5% v / v. In some embodiments, the concentration of ACD Formula A in the incubation medium is about 5% v / v.

[0405] In some embodiments, the improvement in the yield of a cell preparation comprises an increase in the number of early apoptotic viable cells of the preparation among the number of frozen cells from which the preparation is produced.

[0406] In some embodiments, adding an anticoagulant to the freezing medium helps to make the yield between different preparations of the drug population high and stable. In a preferred embodiment, adding an anticoagulant to at least the freezing medium and the incubation medium causes the yield between different preparations of the pharmaceutical composition to be high and stable regardless of the cell collection scheme used.

[0407] In some embodiments, the freezing medium comprises an anticoagulant selected from the group consisting of: heparin, ACD formula A, and combinations thereof. In some embodiments, the anticoagulant used in the freezing medium is ACD formula A containing heparin at a concentration of 10 U / ml. In some embodiments, the freezing medium comprises a 5% v / v ACD formula A solution comprising heparin at a concentration of 10 U / ml.

[0408] In some embodiments, the freezing medium includes heparin. In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5 U / ml. In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5 U / ml, may be between 0.3-0.7 U / ml, and is typically about 0.5 U / ml. In certain embodiments, the concentration of heparin in the freezing medium is about 0.5 U / ml.

[0409] In some embodiments, the freezing medium includes ACD Formula A. In some embodiments, the concentration of ACD Formula A in the freezing medium is between 1%-15% v / v. In some embodiments, the concentration of ACD Formula A in the freezing medium is between 1%-15% v / v, may be between 4%-7% v / v, and is typically about 5% v / v. In some embodiments, the concentration of ACD Formula A in the freezing medium is about 5% v / v.

[0410] In some embodiments, adding an anticoagulant to the incubation medium and / or the freezing medium results in a high and stable cell yield in the group regardless of the triglyceride level in the donor's blood. In some embodiments, when obtained from the blood of a donor with normal or high triglyceride levels, adding an anticoagulant to the incubation medium and / or the freezing medium results in a high and stable cell yield in the composition of the present invention. In some embodiments, adding an anticoagulant to at least the incubation medium results in a high and stable cell yield in the composition regardless of the triglyceride level in the donor's blood. In some embodiments, adding an anticoagulant to the freezing medium and the incubation medium results in a high and stable cell yield in the composition regardless of the triglyceride level in the donor's blood.

[0411] In some embodiments, the freezing medium and / or the incubation medium and / or the washing medium comprises heparin at a concentration of at least 0.1 U / ml, possibly at least 0.3 U / ml, and usually at least 0.5 U / ml. In some embodiments, the freezing medium and / or the incubation medium and / or the washing medium comprises ACD formula A at a concentration of at least 1% v / v, possibly at least 3% v / v, and usually at least 5% v / v.

[0412] In some embodiments, the enriched monocyte composition undergoes at least one washing step after cell collection and before being resuspended in the freezing medium and frozen. In some embodiments, the enriched monocyte composition undergoes at least one washing step after freezing and thawing. In some embodiments, the washing step comprises centrifuging the enriched monocyte composition, then extracting the supernatant and resuspending it in a washing medium.

[0413] In some embodiments, the monocyte composition of the enrichment undergoes at least one washing step between each stage of producing an early apoptotic cell group. In some embodiments, in the whole process of producing an early apoptotic cell group, an anticoagulant is added to a washing medium during the washing step. In some embodiments, the monocyte composition of the enrichment undergoes at least one washing step after incubation. In some embodiments, the monocyte composition of the enrichment undergoes at least one washing step after incubation using PBS. In some embodiments, before the cell preparation is resuspended in the application medium, an anticoagulant is not added to the final washing step. In some embodiments, before the cell preparation is resuspended in the application medium, an anticoagulant is not added to the PBS used in the final washing step. In certain embodiments, an anticoagulant is not added to the application medium.

[0414] In some embodiments, the cell concentration during incubation is about 5×10 6 cells / ml.

[0415] In some embodiments, after freezing, thawing and incubation, the enriched monocyte composition is suspended in an administration medium, thereby producing a drug population. In some embodiments, the administration medium includes a suitable physiological buffer. Non-limiting examples of suitable physiological buffers are: saline solution, phosphate buffered saline (PBS), Hank's Balanced Salt Solution (HBSS), etc. In some embodiments, the administration medium includes PBS. In some embodiments, the administration medium includes a supplement that helps maintain cell viability. In some embodiments, the enriched monocyte composition is filtered before administration. In some embodiments, the enriched monocyte composition is filtered before administration using a filtrate of at least 200 μm.

[0416] In some embodiments, the enriched monocyte population is resuspended in administration medium such that the final volume of the resulting cell preparation is between 100-1000 ml, possibly between 200-800 ml, and typically between 300-600 ml.

[0417] In some embodiments, cell collection refers to obtaining an enriched mononuclear cell composition. In some embodiments, the washing step performed during the generation of an early apoptotic cell population is performed in a washing medium. In certain embodiments, the washing step performed until the incubation step of the early apoptotic cell population is performed in a washing medium. In some embodiments, the washing medium comprises RPMI 1640 culture medium supplemented with L-glutamine and Hepes. In some embodiments, the washing medium comprises RPMI 1640 culture medium supplemented with 2mM L-glutamine and 10mM Hepes.

[0418] In some embodiments, the wash medium includes an anticoagulant. In some embodiments, the wash medium includes an anticoagulant selected from the group consisting of heparin, ACD formula A, and combinations thereof. In some embodiments, the concentration of the anticoagulant in the wash medium is the same as the concentration of the anticoagulant in the freezing medium. In some embodiments, the concentration of the anticoagulant in the wash medium is the same as the concentration of the anticoagulant in the incubation medium. In some embodiments, the anticoagulant used in the wash medium is an ACD formula A containing heparin at a concentration of 10U / ml.

[0419] In some embodiments, the wash medium includes heparin. In some embodiments, the concentration of heparin in the wash medium is between 0.1-2.5 U / ml. In some embodiments, the concentration of heparin in the wash medium is between 0.1-2.5 U / ml, may be between 0.3-0.7 U / ml, and is typically about 0.5 U / ml. In certain embodiments, the concentration of heparin in the wash medium is about 0.5 U / ml.

[0420] In some embodiments, the wash medium includes ACD Formula A. In some embodiments, the concentration of ACD Formula A in the wash medium is between 1%-15% v / v. In some embodiments, the concentration of ACD Formula A in the wash medium is between 1%-15% v / v, may be between 4%-7% v / v, typically about 5% v / v. In some embodiments, the concentration of ACD Formula A in the wash medium is about 5% v / v.

[0421] In some embodiments, the enriched monocyte composition is thawed several hours prior to the intended administration of the population to a subject. In some embodiments, the enriched monocyte composition is thawed at about 33° C.-39° C. In some embodiments, the enriched monocyte composition is thawed for about 30-240 seconds, preferably 40-180 seconds, most preferably 50-120 seconds.

[0422] In some embodiments, the monocyte composition of the enrichment is thawed at least 10 hours before the expected group is used, alternatively at least 20 hours, 30 hours, 40 hours or 50 hours before the expected group is used. In some embodiments, the monocyte composition of the enrichment is thawed at least 15-24 hours before the expected group is used. In some embodiments, the monocyte composition of the enrichment is thawed at least about 24 hours before the expected group is used. In some embodiments, the monocyte composition of the enrichment is thawed at least 20 hours before the expected group is used. In some embodiments, the monocyte composition of the enrichment is thawed at least 30 hours before the expected group is used. In some embodiments, the monocyte composition of the enrichment is thawed at least 24 hours before the expected group is used. In some embodiments, the monocyte composition of the enrichment is thawed at least 24 hours before the expected group is used. In some embodiments, the monocyte composition of the enrichment is thawed before and / or after thawing in the washing medium.

[0423] In some embodiments, the composition further comprises methylprednisolone. In some embodiments, the concentration of methylprednisolone does not exceed 30 μg / ml.

[0424] In some embodiments, the apoptotic cells are used in high doses. In some embodiments, the apoptotic cells are used in high concentrations. In some embodiments, human apoptotic polymorphonuclear neutrophils (PMN) are used. In some embodiments, a group of cells in which 50% are apoptotic cells are used. In some embodiments, apoptotic cells are verified by cytoprep stained with May-Giemsa. In some embodiments, cell viability is assessed by trypan blue exclusion. In some embodiments, the apoptotic state and necrosis state of the cells are confirmed by annexin V / propidium iodide staining and by FACS detection.

[0425] In some embodiments, the apoptotic cells disclosed herein do not include necrotic cells. In some embodiments, the apoptotic cells disclosed herein include less than 1% necrotic cells. In some embodiments, the apoptotic cells disclosed herein include less than 2% necrotic cells. In some embodiments, the apoptotic cells disclosed herein include less than 3% necrotic cells. In some embodiments, the apoptotic cells disclosed herein include less than 4% necrotic cells. In some embodiments, the apoptotic cells disclosed herein include less than 5% necrotic cells.

[0426] In some embodiments, about 140×10 6 -210×10 6 In some embodiments, about 10-100×10 6 In some embodiments, about 20×10 6In some embodiments, about 30×10 6 In some embodiments, about 40×10 6 In some embodiments, about 50×10 6 In some embodiments, about 60×10 6 In some embodiments, about 60×10 6 In some embodiments, about 70×10 6 In some embodiments, about 80×10 6 In some embodiments, about 90×10 6 In some embodiments, about 1-15×10 7 In some embodiments, about 10×10 7 In some embodiments, about 15×10 7 The dose of apoptotic cells.

[0427] In some embodiments, about 10×10 6 In another embodiment, about 10×10 7 In another embodiment, about 10×10 8 In another embodiment, about 10×10 9 In another embodiment, about 10×10 10 In another embodiment, about 10×10 11 In another embodiment, about 10×10 12 In another embodiment, about 10×10 5 In another embodiment, about 10×10 4 In another embodiment, about 10×10 3 In another embodiment, about 10×10 2 The dose of apoptotic cells.

[0428] In some embodiments, a high dose of apoptotic cells is administered. In some embodiments, 35×10 6 In another embodiment, 210×10 6In another embodiment, 70×10 6 In another embodiment, about 140×10 6 In another embodiment, 35-210×10 6 The dose of apoptotic cells.

[0429] In some embodiments, a single dose of apoptotic cells is administered. In some embodiments, multiple doses of apoptotic cells are administered. In some embodiments, 2 doses of apoptotic cells are administered. In some embodiments, 3 doses of apoptotic cells are administered. In some embodiments, 4 doses of apoptotic cells are administered. In some embodiments, 5 doses of apoptotic cells are administered. In some embodiments, 6 doses of apoptotic cells are administered. In some embodiments, 7 doses of apoptotic cells are administered. In some embodiments, 8 doses of apoptotic cells are administered. In some embodiments, 9 doses of apoptotic cells are administered. In some embodiments, more than 9 doses of apoptotic cells are administered. In some embodiments, multiple doses of apoptotic cells are administered.

[0430] In some embodiments, the apoptotic cells can be administered by any method known in the art, including but not limited to intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and direct administration to the thymus.

[0431] In some embodiments, the apoptotic cells are prepared from cells obtained from a subject different from the subject to which the apoptotic cells will be received. In some embodiments, the methods as disclosed herein include additional steps that can be used to overcome allogeneic donor cell rejection, including one or more steps described in U.S. Patent Application 20130156794, which is incorporated herein by reference in its entirety. In some embodiments, the method includes a step of performing complete or partial lymphocyte clearance before administering apoptotic cells (which in some embodiments are allogeneic apoptotic cells). In some embodiments, the lymphocyte clearance is adjusted so that it delays the host versus graft response for a period of time sufficient to allow allogeneic apoptotic cells to control cytokine release. In some embodiments, the method comprises administering an agent that delays the egress of allogeneic apoptotic T cells from lymph nodes (e.g., 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720), 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl-1]1,2,4-oxadiazole (SEW2871), 3-(2-(-hexylphenylamino)-2-oxoethylamino)propionic acid (W123), 2-ammonio-4-(2-chloro-4-(3-phenoxyphenylthio)phenyl)-2-(hydroxymethyl)butyl hydrogen phosphate (KRP-203 phosphate), or other agents known in the art) can be used as part of the compositions and methods disclosed herein to allow the use of allogeneic apoptotic cells that are effective and do not induce graft-versus-host disease. In another embodiment, MHC expression by said allogeneic apoptotic T cells is silenced to reduce rejection of allogeneic cells.

[0432] In some embodiments, a method comprises producing a population of mononuclear apoptotic cells comprising a reduced percentage of non-quiescent non-apoptotic live cells; suppressed cellular activation of any live non-apoptotic cells; or reduced proliferation of any live non-apoptotic cells; or any combination thereof, the method comprising the steps of obtaining an enriched population of mononuclear cells from peripheral blood; freezing the enriched population of mononuclear cells in a freezing medium comprising an anticoagulant; thawing the enriched population of mononuclear cells; incubating the enriched population of mononuclear cells in an apoptosis-inducing incubation medium comprising methylprednisolone and an anticoagulant at a final concentration of about 10-100 μg / mL; resuspending the population of apoptotic cells in an administration medium; and inactivating the enriched population of mononuclear cells, wherein the inactivation occurs after apoptosis induction, wherein the method produces a population of mononuclear apoptotic cells comprising a reduced percentage of non-quiescent non-apoptotic cells; suppressed cellular activation of any live non-apoptotic cells; or reduced proliferation of any live non-apoptotic cells; or any combination thereof.

[0433] In some embodiments, the method comprises the steps of irradiating apoptotic cells from a subject prior to administering the apoptotic cell population to the subject (autologous ApoCell). In some embodiments, the method comprises the steps of irradiating apoptotic cells from a subject prior to administering the apoptotic cell population to the recipient (allogeneic ApoCell).

[0434] In some embodiments, the cells are irradiated in a manner that will reduce the proliferation and / or activation of the residual live cells in the apoptotic cell population. In some embodiments, the cells are irradiated in a manner that reduces the percentage of live non-apoptotic cells in the population. In some embodiments, the percentage of live non-apoptotic cells in the inactivated early apoptotic cell population is reduced to less than 50% of the population. In some embodiments, the percentage of live non-apoptotic cells in the inactivated early apoptotic cell population is reduced to less than 40% of the population. In some embodiments, the percentage of live non-apoptotic cells in the inactivated early apoptotic cell population is reduced to less than 30% of the population. In some embodiments, the percentage of live non-apoptotic cells in the inactivated early apoptotic cell population is reduced to less than 20% of the population. In some embodiments, the percentage of live non-apoptotic cells in the inactivated early apoptotic cell population is reduced to less than 10% of the population. In some embodiments, the percentage of live non-apoptotic cells in the inactivated early apoptotic cell population is reduced to 0% of the population.

[0435] In another embodiment, the irradiated apoptotic cells retain all of their early apoptotic, immunomodulatory, stability characteristics. In another embodiment, the irradiating step uses UV radiation. In another embodiment, the irradiating step uses gamma radiation. In another embodiment, the apoptotic cells include a reduced percentage of live non-apoptotic cells, a preparation comprising suppressed cell activation of any live non-apoptotic cells presented within the apoptotic cell preparation, or a preparation comprising reduced proliferation of any live non-apoptotic cells presented within the apoptotic cell preparation, or any combination thereof.

[0436] In some embodiments, the irradiation of apoptotic cells does not increase the population (PI+) of dead cells compared to non-irradiated apoptotic cells. In some embodiments, the irradiation of apoptotic cells does not increase the population (PI+) of dead cells by more than about 1% compared to non-irradiated apoptotic cells. In some embodiments, the irradiation of apoptotic cells does not increase the population (PI+) of dead cells by more than about 2% compared to non-irradiated apoptotic cells. In some embodiments, the irradiation of apoptotic cells does not increase the population (PI+) of dead cells by more than about 3% compared to non-irradiated apoptotic cells. In some embodiments, the irradiation of apoptotic cells does not increase the population (PI+) of dead cells by more than about 4% compared to non-irradiated apoptotic cells. In some embodiments, the irradiation of apoptotic cells does not increase the population (PI+) of dead cells by more than about 5% compared to non-irradiated apoptotic cells. In some embodiments, the irradiation of apoptotic cells does not increase the population (PI+) of dead cells by more than about 6% compared to non-irradiated apoptotic cells. In some embodiments, the irradiation of apoptotic cells does not increase the population (PI+) of dead cells by more than about 7% compared to non-irradiated apoptotic cells. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 8% compared to non-irradiated apoptotic cells. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 9% compared to non-irradiated apoptotic cells. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 10% compared to non-irradiated apoptotic cells. In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI+) by more than about 15% compared to non-irradiated apoptotic cells. 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 to non-irradiated apoptotic cells.

[0437] In some embodiments, a cell population comprising a reduced fraction or the absence of live non-apoptotic cells can provide, in one embodiment, a mononuclear early apoptotic cell population without any living (living / viable) cells. In some embodiments, a cell population comprising a reduced fraction or the absence of live non-apoptotic cells can provide, in one embodiment, a mononuclear apoptotic cell population that does not induce GVHD in a recipient.

[0438] In some embodiments, the use of irradiated ApoCells to remove a possible graft-versus-leukemia effect, the use of an apoptotic population (which contains a minimal portion of live cells) can lead to confirmation that the effects shown in the examples (see Example 8) are caused by apoptotic cells and not by a live cell proliferation population with cellular activity presented within the apoptotic cell population.

[0439] In another embodiment, the method includes a step of irradiating apoptotic cells derived from WBCs from a donor prior to administration to a recipient. In some embodiments, the cells are irradiated in a manner that avoids proliferation and / or activation of residual viable cells within the apoptotic cell population. In another embodiment, the irradiated apoptotic cells retain all of their early apoptotic, immunomodulatory, stability characteristics. In another embodiment, the irradiation step uses UV radiation. In another embodiment, the irradiation step uses gamma radiation. In another embodiment, the apoptotic cells include a reduced percentage of live non-apoptotic cells, a preparation comprising suppressed cell activation of any live non-apoptotic cells presented within the apoptotic cell preparation, or a preparation comprising reduced proliferation of any live non-apoptotic cells presented within the apoptotic cell preparation, or any combination thereof.

[0440] In some embodiments, apoptotic cells include mononuclear apoptotic cell preparations collected. In some embodiments, the mononuclear apoptotic cell preparations collected include mononuclear cells in an early apoptotic state, wherein the mononuclear apoptotic cells collected include a percentage of live non-apoptotic cells reduced, a preparation of cell activation with any live non-apoptotic cell containment, or a preparation of proliferation with any live non-apoptotic cell reduction or any combination thereof. In another embodiment, the mononuclear apoptotic cells collected have been irradiated. In another embodiment, the mononuclear apoptotic cell preparations collected are disclosed herein, which in some embodiments are derived from a white blood cell fraction (WBC) obtained from donated blood.

[0441] In some embodiments, the apoptotic cell preparation is irradiated. In another embodiment, the irradiation comprises gamma irradiation or UV irradiation. In yet another embodiment, the irradiated preparation has a reduced number of non-apoptotic cells compared to an unirradiated apoptotic cell preparation. In another embodiment, the irradiated preparation has a reduced number of proliferating cells compared to an unirradiated apoptotic cell preparation. In another embodiment, the irradiated preparation has a reduced number of potentially immunocompetent cells compared to an unirradiated apoptotic cell population.

[0442] In some embodiments, the pooled blood includes 3rd party blood that is not matched between the donor and the recipient.

[0443] The skilled artisan will appreciate that the term "pooled" may encompass blood collected from multiple donors, prepared and possibly stored for later use. This pooled blood pool may then be processed to produce a pooled mononuclear apoptotic cell preparation. In another embodiment, the pooled mononuclear apoptotic cell preparation ensures that an easily accessible supply of mononuclear apoptotic cells is available. In another embodiment, the cells are pooled just before the incubation step in which apoptosis is induced. In another embodiment, the cells are pooled at the resuspension step, after the incubation step. In another embodiment, the cells are pooled just before the irradiation step. In another embodiment, the cells are pooled just after the irradiation step. In another embodiment, the cells are pooled at any step in the preparation method.

[0444] In some embodiments, the apoptotic cell preparations collected are derived from cells present in about 2 to 25 units of blood. In another embodiment, the apoptotic cell preparations collected comprise cells present in about 2-5, 2-10, 2-15, 2-20, 5-10, 5-15, 5-20, 5-25, 10-15, 10-20, 10-25, 6-13 or 6-25 units of blood. In another embodiment, the apoptotic cell preparations collected comprise cells present in about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 units of blood. The number of units of blood required also depends on the efficiency of recovering WBCs from blood. For example, low efficiency WBC recovery will result in the need for additional units, while high efficiency WBC recovery will result in fewer units required. In some embodiments, each unit is a bag of blood. In another embodiment, the pooled apoptotic cell preparation comprises cells present in at least 25 units of blood, at least 50 units of blood, or at least 100 units of blood.

[0445] In some embodiments, the unit blood includes a white blood cell (WBC) fraction from a blood donation. In another embodiment, the donation can be from a blood center or a blood bank. In another embodiment, the donation can be from a donor gathered in a hospital when preparing a pooled apoptotic cell preparation. In another embodiment, a unit blood including WBC from multiple donors is stored and maintained in an independent blood bank created for the purpose of the compositions disclosed herein and methods thereof. In another embodiment, a blood bank developed for the purpose of the compositions disclosed herein and methods thereof can supply a unit blood including WBC from multiple donors and includes a leukapheresis unit.

[0446] In some embodiments, the units of pooled WBCs are not restricted by HLA matching. Thus, the pooled apoptotic cell preparations produced include cell populations that are not restricted by HLA matching. Thus, in certain embodiments, the pooled mononuclear apoptotic cell preparations include allogeneic cells.

[0447] An advantage of a pooled mononuclear apoptotic cell preparation derived from pooled WBCs that is not restricted by HLA matching is that the source of WBCs is readily available and the cost of obtaining WBCs is reduced.

[0448] In some embodiments, the collected blood includes blood from multiple donors independent of HLA matching. In another embodiment, the collected blood includes blood from multiple donors, wherein the HLA matching with the recipient has been taken into account. For example, wherein 1 HLA allele, 2 HLA alleles, 3 HLA alleles, 4 HLA alleles, 5 HLA alleles, 6 HLA alleles or 7 HLA alleles have been matched between the donor and the recipient. In another embodiment, multiple donors are partially matched, such as some donors have been HLA matched, wherein 1 HLA allele, 2 HLA alleles, 3 HLA alleles, 4 HLA alleles, 5 HLA alleles, 6 HLA alleles or 7 HLA alleles have been matched between some donors and the recipient. Each possibility includes embodiments as disclosed herein.

[0449] In certain embodiments, after the apoptosis induction step described below (Example 1), some live non-apoptotic cells (anti-apoptosis) may remain. In some embodiments, the presence of these live non-apoptotic cells is observed before the irradiation step. These live non-apoptotic cells may be able to proliferate or be activated. In some embodiments, the mononuclear apoptotic cell preparations derived from the pooling of multiple donors may be activated against the host, activated against each other, or both.

[0450] In some embodiments, compared to non-irradiated cell preparations, the cell activation of the irradiated cell preparations disclosed herein is suppressed and the proliferation is reduced. In another embodiment, the irradiation includes gamma irradiation or UV irradiation. In another embodiment, compared to non-irradiated cell preparations, the irradiated cell preparation has a reduced number of non-apoptotic cells. In another embodiment, the irradiation includes about 15 Gray units (Gy). In another embodiment, the irradiation includes about 20 Gray units (Gy). In another embodiment, the irradiation includes about 25 Gray units (Gy). In another embodiment, the irradiation includes about 30 Gray units (Gy). In another embodiment, the irradiation includes about 35 Gray units (Gy). In another embodiment, the irradiation includes about 40 Gray units (Gy). In another embodiment, the irradiation includes about 45 Gray units (Gy). In another embodiment, the irradiation includes about 50 Gray units (Gy). In another embodiment, the irradiation includes about 55 Gray units (Gy). In another embodiment, the irradiation comprises about 60 Gray units (Gy). In another embodiment, the irradiation comprises about 65 Gray units (Gy). In another embodiment, the irradiation comprises up to 2500 Gy. In another embodiment, the irradiated pooled apoptotic cell preparation maintains the same or similar apoptotic profile, stability, and efficacy as the non-irradiated pooled apoptotic cell preparation.

[0451] In some embodiments, the pooled mononuclear apoptotic cell preparations as disclosed herein are stable for up to 24 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for at least 24 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for more than 24 hours. In yet another embodiment, the pooled mononuclear apoptotic cell preparations as disclosed herein are stable for up to 36 hours. In still another embodiment, the pooled mononuclear apoptotic cell preparations are stable for at least 36 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for more than 36 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations as disclosed herein are stable for up to 48 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for at least 48 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for more than 48 hours.

[0452] In some embodiments, the method of producing a pooled cell preparation comprising an irradiation step retains the early apoptosis, immunomodulation and stability properties observed in apoptotic preparations derived from a single matched donor, wherein the cell preparation may not comprise an irradiation step. In another embodiment, the pooled mononuclear apoptotic cell preparation as disclosed herein does not elicit a graft versus host disease (GVHD) response.

[0453] Irradiation of cell preparations is considered safe in the art. Currently, irradiation procedures are routinely performed on donated blood to prevent reactions to WBCs.

[0454] In another embodiment, the percentage of apoptotic cells in the mononuclear apoptotic cell preparations collected as disclosed herein is close to 100%, thereby reducing the fraction of live non-apoptotic cells in the cell preparations. In some embodiments, the percentage of apoptotic cells is at least 40%. In another embodiment, the percentage of apoptotic cells is at least 50%. In yet another embodiment, the percentage of apoptotic cells is at least 60%. In still another embodiment, the percentage of apoptotic cells is at least 70%. In another embodiment, the percentage of apoptotic cells is at least 80%. In another embodiment, the percentage of apoptotic c...

Claims

1. Use of a composition comprising an irradiated early apoptotic cell population for preparing a medicament for treating organ failure, organ dysfunction, or organ damage, or a combination thereof, in a subject suffering from sepsis, wherein the irradiated early apoptotic cell population comprises an enriched mononuclear cell population irradiated after induction of apoptosis, the enriched mononuclear cell population being obtained from peripheral blood.

2. The use according to claim 1, wherein sepsis comprises mild, severe, acute or highly invasive sepsis.

3. The use according to claim 1, wherein the survival rate of the subject is increased, or wherein the incidence of organ failure or organ dysfunction or organ damage or a combination thereof is reduced, or a combination thereof.

4. The use according to claim 3, wherein the organ failure comprises acute multiple organ failure.

5. The use according to any one of claims 1 to 4, wherein the irradiated early apoptotic cell population comprises A mononuclear apoptotic cell population comprising a reduction in non-quiescent non-apoptotic cells, suppressed cellular activation of any viable non-apoptotic cells, or reduced proliferation of any viable non-apoptotic cells, or any combination thereof.

6. The use according to any one of claims 1 to 4, wherein the irradiated early apoptotic cell population comprises a pooled population of early apoptotic cells.

7. The use according to any one of claims 1 to 4, wherein the subject is a human subject.

8. The use according to any one of claims 1 to 4, wherein the medicament is a formulation for administration in the form of a single infusion of the early apoptotic cell population or is formulated for administration in the form of multiple infusions of the early apoptotic cell population.

9. The use according to any one of claims 1 to 4, wherein the medicament is formulated for administration in the form of multiple infusions of the apoptotic cell population.

10. The use according to any one of claims 1 to 4, wherein the medicament is formulated for intravenous administration.

11. The use according to any one of claims 1 to 4, further comprising the use of an additional therapy.

12. The use according to claim 11, wherein the use of the additional therapy is before, simultaneously with or after the administration of the early apoptotic cells.

13. The use according to any one of claims 1 to 4, wherein the medicament comprises a first-line therapy.

14. The use according to any one of claims 1 to 4, wherein the medicament comprises an adjunctive therapy.

15. The use according to any one of claims 1 to 4, wherein the treatment of organ failure, organ dysfunction, or organ damage, or a combination thereof, comprises rebalancing the subject's immune response.

16. Use according to claim 15, wherein the rebalancing comprises reducing the secretion of one or more pro-inflammatory cytokines / chemokines.

17. The use according to claim 15, wherein the rebalancing comprises reducing the secretion of one or more anti-inflammatory cytokines / chemokines.

18. The use according to claim 15, wherein the rebalancing comprises reducing the secretion of one or more pro-inflammatory cytokines / chemokines and one or more anti-inflammatory cytokines / chemokines.

19. The use according to any one of claims 1 to 4, wherein the agent prevents, inhibits, reduces the incidence of, or reduces the severity of a cytokine and chemokine storm in the subject.

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