CN114729314A

CN114729314A - Combination cancer therapy and cytokine control therapy for cancer treatment

Info

Publication number: CN114729314A
Application number: CN202080077886.3A
Authority: CN
Inventors: S·诺维克; D·梅沃拉赫
Original assignee: Ilwix Therapeutics Rhodes Co ltd
Current assignee: Ilwix Therapeutics Rhodes Co ltd
Priority date: 2015-02-18
Filing date: 2020-09-16
Publication date: 2022-07-08

Abstract

The compositions disclosed herein and methods of use thereof include those that inhibit or reduce the incidence of Cytokine Release Syndrome (CRS) or cytokine storm in a subject undergoing CAR T cell therapy, methods of treating cancer or tumors, methods of reducing tumor burden, methods of reducing the size or growth rate of a cancer or tumor, and methods of extending the survival of a subject having cancer or tumor, wherein the subject is administered a composition comprising apoptotic cells or an apoptotic cell supernatant. The compositions and methods of use thereof can increase the efficacy of CAR T cell cancer therapy. Also disclosed herein are methods of slowing, reducing, inhibiting, or eliminating the metastatic spread of a cancer or tumor in a subject comprising administering a combination therapy comprising an early apoptotic cell population and one or more cancer therapeutic agents.

Description

Combination cancer therapy and cytokine control therapy for cancer treatment

Technical Field

Disclosed herein are compositions and methods thereof for inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing cancer therapy, such as but not limited to chimeric antigen receptor expressing T cell (CAR T cell) cancer therapy. Further, disclosed herein are compositions and methods thereof for reducing or inhibiting cytokine production in a subject experiencing cytokine release syndrome or cytokine storm. Further, the compositions disclosed herein may be used to treat, prevent, inhibit the growth of, or reduce the incidence of a cancer or tumor in a subject. The compositions can be used to increase the survival of a subject having a cancer or tumor. The compositions used may be administered alone or in combination with other cancer therapeutics or chemotherapy. The methods disclosed herein include those comprising administering a combination therapy comprising an early apoptotic cell population or supernatant and one or more cancer therapeutic agents (such as CAR T cells) to slow, reduce, inhibit, or eliminate metastatic spread of cancer or tumor in a subject.

Background

While the standard therapies for cancer are surgery, chemotherapy, and radiation therapy, improved approaches, such as targeted immunotherapy, are currently being developed and tested. One promising technology uses Adoptive Cell Transfer (ACT) in which immune cells are modified to recognize and attack their tumors. One 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 cells) that targets a tumor-specific antigen expressed on the surface of the tumor cells. These CAR T cells are then cytotoxic only to cells expressing the tumor specific antigen. Clinical trials have shown that CAR T cell therapy has great potential in controlling advanced Acute Lymphoblastic Leukemia (ALL) and lymphoma, among others.

However, some patients given CAR T cell therapy and other immunotherapy experience a dangerous and sometimes even life-threatening side effect known as Cytokine Release Syndrome (CRS) in which infused activated T cells produce a systemic inflammatory response in which cytokines are released rapidly and in large quantities into the bloodstream, resulting in dangerous hypotension, high fever and shivering.

In severe cases of CRS, patients experience a cytokine storm (also known as 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 hepatic failure, pulmonary edema, and disseminated intravascular coagulation.

For example, in a recent phase I trial, six patients administered the monoclonal antibody TGN1412 that binds to the CD28 receptor on T cells showed severe cases of cytokine storm and multiple organ failure. This occurs despite The fact that The TGN1412 dose is 500 times lower than that found to be safe for animals (St. Clair EW: The lime after The cytokine storm: Lessons from The TGN1412 triel. J. Clin Invest 118:1344-1347, 2008).

Corticosteroids, biologic therapies (such as anti-IL 6 therapies), and anti-inflammatory drugs are being evaluated to date to control cytokine release syndrome in patients administered CAR T cell therapy. However, steroids may affect the activity and/or proliferation of CAR T cells and put patients at risk for sepsis and opportunistic infections. Anti-inflammatory drugs may not be effective in controlling cytokine release syndrome or cytokine storm because cytokine storm contains 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 in particular, cytokine storm) in order to realize the potential of CAR T cell therapy.

Cytokine storm remains a problem after other infectious and non-infectious stimuli. In cytokine storms, many proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, g-interferon (g-IFN), and tumor necrosis factor- α (TNF α), are released, resulting in hypotension, bleeding, and ultimately multi-organ failure. The relatively high mortality rate in young people with presumably healthy immune systems in the H1N1 influenza pandemic and the recent avian influenza H5N1 infection in 1918 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-associated hemophagocytic lymphohistiocytosis, gram-negative sepsis (gram-negative sepsis), malaria, and many other infectious diseases, including Ebola infection.

Cytokine storms may also originate from non-infectious causes, such as acute pancreatitis, severe burns or trauma, or acute respiratory distress syndrome. Therefore, new strategies are needed to control cytokine release syndrome, and in particular cytokine storm.

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

The main cause of lethality in 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 usually has metastasized at the time of diagnosis, and even in cases where the cancer was diagnosed prior to this stage, complete surgical removal or destruction of the primary diseased tissue that may ultimately lead to metastasis may not be feasible. Metastatic disease may not be diagnosed at an early stage due to the small size of the metastatic lesion and/or the absence of reliable markers in the primary lesion that can reliably predict its presence. Such lesions may be difficult or impossible to treat by ablative methods due to their difficult accessibility, diffuse nature, and/or uncertain location. Chemotherapy/radiation therapy, the current method of choice for treating certain metastatic malignancies, is generally ineffective or sub-optimal and has significant drawbacks associated with particularly harmful and/or potentially lethal side effects.

Immunotherapeutic cancer therapies, such as those involving Antigen Presenting Cell (APC) vaccination, may have optimal efficacy for treating inaccessible, diffuse, microscopic, recurrent and/or poorly localized cancer lesions. One promising approach to immunotherapy involves the use of professional APCs, such as Dendritic Cells (DCs), to elicit systemic anti-cancer immunity.

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 T cells of the immune system and thereby enable sensitivity of T cells to both new and recalled antigens. DCs are the most efficient antigen producing cells, which act as messengers between the innate and adaptive immune systems. DC cells can be used to elicit specific anti-tumor immunity by generating effector cells that attack and lyse the tumor.

Apoptotic cells present a physiological cell death pathway (most commonly occurring via apoptosis) that triggers a range of molecular homeostatic mechanisms including recognition, immune response, and elimination processes. Furthermore, apoptotic cells are immunoregulatory cells capable of directly and indirectly inducing immune tolerance to dendritic cells and macrophages. Apoptotic cells have been shown to regulate dendritic cells and macrophages and to cause them to produce tolerance and inhibit pro-inflammatory activities such as the secretion of pro-inflammatory cytokines and the expression of co-stimulatory molecules.

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

Disclosure of Invention

In one aspect, disclosed herein is a method of slowing, reducing, inhibiting, or eliminating metastatic spread of a cancer or tumor, or any combination thereof, in a subject, the method comprising the step of administering to the subject a combination therapy comprising an early apoptotic cell population and one or more cancer therapeutic agents, wherein the method slows, reduces, inhibits, or eliminates metastatic spread of a cancer or tumor, or any combination thereof, in the subject. In related aspects, the cancer therapeutic comprises a T cell expressing a chimeric antigen receptor (CAR T cell).

In another related aspect, in the methods disclosed herein, the survival of the subject is increased. In a further related aspect, the subject is a human subject.

In yet another related aspect, the population of early apoptotic cells comprises an enriched population of monocytes; stabilizing the apoptotic population for more than 24 hours; an apoptotic population that is free of cell aggregates; an early apoptotic cell population irradiated after the induction of apoptotic cells; a pooled population of early apoptotic cells; or a mononuclear apoptotic cell population (comprising reduced non-quiescent non-apoptotic cells, suppressed cellular activation of any living non-apoptotic cells, or reduced proliferation of any living non-apoptotic cells, or any combination thereof); or any combination thereof.

In a related aspect, the cancer or tumor comprises a solid tumor or a non-solid tumor. In further related aspects, the non-solid cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, multiple myeloma (plasma cell myeloma), acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, or plasma cell leukemia. In yet another further related aspect, the solid tumor comprises a sarcoma or carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma angiosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic carcinoma or tumor, breast carcinoma or tumor, ovarian carcinoma or tumor, prostate carcinoma or tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma or tumor, uterine carcinoma or tumor, testicular carcinoma or tumor, lung carcinoma, choriocarcinoma, and carcinoma of the like, Small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

In a related aspect, the cancer therapeutic and the early apoptotic cells are included in separate compositions. In a related aspect, the CAR T cell and the early apoptotic cell are included in separate compositions. In a still further related aspect, a composition comprising a cancer therapeutic is administered prior to, concurrently with, or after administration of the early apoptotic cells. In a still further related aspect, a composition comprising a CAR T cell is administered prior to, concurrently with, or after administration of the early apoptotic cell.

In one aspect, disclosed herein is a method of slowing, reducing, inhibiting, or eliminating metastatic spread of a cancer or tumor, or any combination thereof, in a subject undergoing cancer therapy, the method comprising the step of administering to the subject an early apoptotic cell population, wherein the method slows, reduces, inhibits, or eliminates metastatic spread of a cancer or tumor, or any combination thereof, in the subject as compared to a subject undergoing cancer therapy and not administered an early apoptotic cell population. In a related aspect, the cancer therapy includes radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormonal therapy, photodynamic therapy, or surgery, or a combination thereof. In a further related aspect, the cancer therapy comprises chimeric antigen receptor T cell (CAR T-cell) therapy.

In one aspect, disclosed herein is a method of improving a cancer therapy in a subject, the method comprising the step of administering to the subject a population of early apoptotic cells, wherein improving a cancer therapy comprises increasing the survival time of the subject, and wherein the method improves the cancer therapy compared to a subject undergoing a cancer therapy and not administered a population of early apoptotic cells. In related aspects, cancer therapy includes radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormonal therapy, photodynamic therapy, or surgery, or a combination thereof. In a further related aspect, the cancer therapy comprises a chimeric antigen receptor expressing T cell (CAR T-cell) therapy.

Drawings

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

This patent application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

Figures 1A-1B schematic diagrams show embodiments of standard CAR T cell therapy (figure 1A) and methods of performing safe and effective CAR T cell cancer therapy in patients using the patient's own cells (autologous) (figure 1B) to produce apoptotic cells or apoptotic cell supernatants.

Figure 2. schematic diagrams show embodiments of methods of performing safe and effective CAR T cell cancer therapy in a patient using donor cells to produce apoptotic cells or apoptotic supernatants.

Figure 3. a flow chart presents steps during one embodiment of a process for preparing a population of early apoptotic cells, wherein an anticoagulant is included in the process.

Figures 4A-4 j. apoptotic cells prevent cytokine storm in an in vitro cytokine storm model induced in LPS sterile model of macrophage activation syndrome in a cancer environment. Figure 4A shows that LPS-induced IL-10 levels in a model of macrophage activation syndrome were reduced at two time periods (6 hours and 24 hours) after administration of Apocell at macrophage/monocyte: Apocell ratios of 1:8 and 1:16 in the presence of cancer. Figure 4B shows that LPS-induced IL-6 levels in the macrophage activation syndrome model were reduced at two time periods (6 hours and 24 hours) after Apocell administration at macrophage/monocyte: Apocell ratios of 1:8 and 1:16 in the presence of cancer and CAR-19. Figure 4C shows that LPS-induced MIP-1 α levels in a model of macrophage activation syndrome were reduced at two time periods (6 hours and 24 hours) after Apocell administration at macrophage/monocyte: Apocell ratios of 1:8 and 1:16 in the presence of cancer and CAR-19. Figure 4D shows that LPS-induced IL-8 levels in a model of macrophage activation syndrome were reduced at two time periods (6 hours and 24 hours) after Apocell administration at macrophage/monocyte: Apocell ratios of 1:8 and 1:16 in the presence of cancer and CAR-19. Figure 4E shows that LPS-induced TNF- α levels in a model of macrophage activation syndrome were reduced at two time periods (6 hours and 24 hours) after Apocell administration at macrophage/monocyte: Apocell ratios of 1:8 and 1:16 in the presence of cancer and CAR-19. Figure 94 shows that LPS-induced MIP-1 β levels in a model of macrophage activation syndrome were reduced at 24 hours following Apocell administration 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. FIG. 94 shows that LPS-induced MCP-1 levels in a model of macrophage activation syndrome are reduced at 24 hours after Apocell administration 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. FIG. 94 shows in a model of macrophage activation syndrome LPS-induced IL-9 levels were reduced at two time periods (6 and 24 hours) after administration of Apocell at macrophage/monocyte: Apocell ratios of 1:8 and 1:16 in the presence of cancer and CAR-19. Figure 4I shows that LPS-induced IL-2R levels in a model of macrophage activation syndrome increased at 24 hours after administration of Apocell 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 shows that apoptotic cells do not down-regulate IL-2 release from cells. In the case of an increased dose of apoptotic cells (n ═ 3), apoptotic cells were incubated with macrophages/monocytes in the presence of cancer and CAR-19 for a period of more than 24 hours. Blank strip (outline only) -2.5 × 10 holes per hole⁶An apoptotic cell; Black-5X 10 pores per well⁶An apoptotic cell; Grey-10X 10 per well⁶And (4) apoptotic cells.

FIG. 5 validation of transduction of T cells shows transduction of T4⁺Flow cytometry results of anti-CD 124 assays performed by CAR-T cells.

FIG. 6.T4⁺CAR T cells reduced proliferation of SKOV3-luc ovarian adenocarcinoma cells. The results of the cytotoxicity assay are presented in bar graph, where a monolayer of SKOV3-luc cells was cultured from non-transduced T cells or T4+ CAR-T cells.

FIG. 7. apoptotic cells did not eliminate T4⁺CAR-T cell anti-tumor activity. The results are based on a cytotoxicity assay in which a monolayer of SKOV3-luc cells was cultured with untransduced T cells or with T4 in the presence of vehicle (Hartmann's solution) or apoptotic cells (Apocell) or apoptotic cell supernatant (ApoSup) or supernatant of co-culture of apoptotic cells and monocytes/macrophages (ApoMon Sup)⁺CAR-T cell culture.

FIG. 8 Il-6 secreted at high levels during cytotoxicity is down-regulated by apoptotic cells. The results shown here demonstrate the effect of SKOV3-luc co-culture and exposure of human monocytes/macrophages to apoptotic cells (ApoCell) or ApoCell supernatant (ApoSup) or apoptotic cell and monocyte/macrophage co-culture (ApoMon Sup).

FIG. 9 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 file records that 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 supernatant of apoptotic cells and monocyte/macrophage co-culture (ApoMon Sup) down-regulates IL-6, with IL-6 reduced to acceptable levels.

Figure 10 effects of apoptotic cells or apoptotic cell supernatants or co-cultures of apoptotic cells and monocytes after LPS exposure during CAR T cell treatment mimicking CAR-T cell clinical therapy. The file records that 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 supernatant of apoptotic cells and monocyte/macrophage co-culture (ApoMon Sup) down-regulates IL-6, with IL-6 reduced to acceptable levels.

11A-11B. weight and tumor size of mice at the time of knockout. Fig. 11A shows the weight change over the experimental period. blue-No application of 4.5X 10⁶Control of SKOV3-luc cells. Red-0.5X 10⁶SKOV3-luc cells. green-1.0X 10⁶SKOV3-luc cells. purple-4.5X 10⁶SKOV3-luc cells. FIG. 11B presents a sample receiving 4.5X 10⁶Representative SKOV3-luc tumors 39 days post injection in mice with SKOV3-luc cells.

FIG. 12.SKOV3-luc tumor growth. SKOV3-luc tumor-bearing mice imaged by bioluminescence imaging (BLI) are presented, showing control (PBS) versus inoculation with 0.5 × 10⁶、1×10⁶And 4.5X 10⁶Differences between individual SKOV3-luc cells.

Skov3-luc tumor burden, fig. 13A-13d. Quantification of Bioluminescence (BLI) of SKOV3-luc tumors in vivo (see FIG. 12). A 600 photon count cutoff was implemented as instructed by the manufacturer. FIG. 13A, mice were inoculated with 0.5X 106 SKOV 3-luc. FIG. 13B, mice were inoculated with 1X 106 SKOV 3-luc. FIG. 13C, mice were inoculated with 4.5X 106 SKOV 3-luc. FIG. 13D, mean SKOV3-luc tumor growth.

FIG. 14 cytotoxicity calibration on Raji Burkett lymphoma cells. Raji cells were plated at various cell densities and lysed immediately prior to centrifugation. The results show the number of Raji cells (x-axis) and the absorbance at 492nm (y-axis). All cell numbers showed significant readings relative to the unlysed counterparts.

Figure 15 addition of early apoptotic cells did not affect CAR T cell anti-tumor activity. The E/T ratio shows the ratio of CD19+ CAR T cells to HeLa cells (HeLa cells). Survival was for CD19+ tumor cells. Solid circle: CD19+ hela; hollow triangle: CD19+ hela + naive T cells; solid triangle: CD19+ Hela + CAR T-CD 19; hollow circle: CD19+ Hela + CAR T-CD19+ ApoCell.

FIG. 16 cytokine analysis (GM-CSF) on Raji Burkett lymphoma cells in the presence and absence of apoptotic cells. The bar graph presents the concentration measurements of the cytokine GM-CSF (pg/ml) found in the culture supernatant of Raji cells incubated in the presence of monocytes and LPS after addition of naive T cells (Raji + naive T), addition of CD19+ CAR T cells (Raji + CAR T) and addition of CD19+ CAR T cells and apoptotic cells (ApoCell) at a ratio of 1:8CAR T cells to ApoCell (Raji + CAR T + ApoCell1: 8), CD19+ CAR T cells and apoptotic cells (ApoCell) (Raji + CAR T + cell1:32) at a ratio of 1:32CAR T cells to ApoCell, and CD19+ CAR T cells and apoptotic cells (ApoCell) at a ratio of 1:64CAR T cells to ApoCell (Raji + CAR T + ApoCell1: 64).

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

Fig. 18A and 18b. Figure 18A presents an experimental protocol for analyzing the effect of apoptotic cells on CAR T cell therapy. SCID mice were injected with Raji cancer cells on day 1, followed by CAR T-CD19 cells (CAR T cell therapy) and apoptotic cells on day 6. Figure 18B shows CAR T cell therapy was not negatively affected by ApoCell co-administration. Survival rate curve: SCID mice were injected with CD19+ Raji cells with or without the addition of early apoptotic cells.

19A, 19B, and 19℃ in a solid tumor in vivo model, the release of proinflammatory cytokines from the tumor was increased. Figure 19A shows a slight increase in IL-6 release from solid tumors present in the peritoneum of BALB/c and SCID mice, with IL-6 release significantly increased in the presence of hela CAR-CD-19CAR T cells. Similarly, figure 19B shows a slight increase in IP-10 release from solid tumors present in the peritoneum of BALB/C and SCID mice, with a significant increase in IP-10 release in the presence of hela CAR-CD-19CAR T cells, and figure 19C shows that even TNF-a release surprisingly increases in the presence of hela CAR-CD-19CAR T cells.

Figures 20A and 20b. test the efficacy of CD19-CAR-T cells in the IP model of hela CD19 (leukemia) in the presence or absence of ApoCell cells. hela-CD 19-blue; hela CD19+ pseudo-green; hala CD19+ CAR-T-purple; and Hela CD19+ CAR-T + ApoCell-orange. FIG. 20A uses 0.5X 10⁶Individual CAR-T positive cells. FIG. 20B uses 2.2X 10⁶Individual CAR-T positive cells.

FIG. 21 survival curves of in vivo diffuse tumor SCID mouse model. The curves show that administration of early apoptotic cells (APO; thick dashed line- - - - - -) prolongs survival compared to mice that were not administered apoptotic cells (no APO; dot-dash line- - - - - -), with control SCID mice showing 100% survival (solid line ______).

22A-22D. apoptotic cell infusion increased the lifespan of leukemic mice and increased the number of mice that achieved complete remission. Queue: no leukemia (control-striped pattern); leukemia + early apoptotic cells (speckle pattern); leukemia only (solid grey). Total n is 51(p < 0.001). FIG. 22A: apoptotic cell infusion increased the percentage of mice that survived to life expectancy after leukemia induction. FIG. 22B: apoptotic cell infusion increased the percentage of mice that survived to 12% of life expectancy after leukemia induction. FIG. 22C: apoptotic cell infusion increased the percentage of mice that survived to 30% of life expectancy after leukemia induction. FIG. 22D: apoptotic cell infusion increased the percentage of mice that survived to 100% of life expectancy and achieved complete remission after leukemia induction.

Fig. 23A-23e. apoptotic cell infusion increased the lifespan of leukemic mice, increased the number of mice that achieved complete remission, and enhanced anti-CD 20 monoclonal antibody (mAb) treatment. And (3) queue: leukemia only (solid grey); leukemia + early apoptotic cells (striped pattern); leukemia + anti-CD 20 mAb (checkerboard); leukemia + anti-CD 20+ early apoptotic cells (spots). Total n is 28(p < 0.002). FIG. 23A: the percentage (%) of survival to the expected life span of mice after induction of leukemia with Raji cells is shown. FIG. 23B: apoptotic cell infusion increased the percentage of mice that survived leukemia induction to 24% longer than life expectancy. FIG. 23C: apoptotic cell infusion increased the percentage of mice that survived leukemia induction to 59% longer than life expectancy and enhanced the effect of anti-CD 20 mAb on the life expectancy of leukemia mice. FIG. 23D: apoptotic cell infusion increased the percentage of mice that survived leukemia induction to 76% longer than life expectancy and enhanced the effect of anti-CD 20 mAb on the life expectancy of leukemia mice. FIG. 23E: apoptotic cell infusion increased the percentage of mice that achieved complete remission.

FIG. 24. Kaplan-Meier survival curves for 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-orange only; raji + ApoCell-blue.

Fig. 25A-25c. kaplan-meier survival curves. Figure 25A presents data from the following study: wherein 7 weeks old female SCID-Bg mice (ENVIGO, Jerusalem, Israel, Inc.) were injected intravenously into each mouse0.1×10⁶Raji cells (each group n 10, three groups). Mice received three intravenous doses (

days

5, 8, 11) of 30X 10⁶And (4) ApoCell. (RPMI-light blue; Raji-orange; and Raji + ApoCell-dark blue) FIG. 25B presents data from the following study: wherein 7-week-old female SCID-Bg mice (ENVIGO, Sjogren.) were injected intravenously at 0.1X 10/mouse⁶Raji cells (n-10 per group, three groups). Mice received three intravenous doses (

days

5, 8, 11) of 30X 10⁶And (4) ApoCell. (RPMI-black; Raji-orange; and Raji + ApoCell-dark blue) FIG. 25C presents data from the following study: wherein 8-9 week old female SCID-Bg mice (ENVIGO, Sjogren.) were injected intravenously with 0.1X 10 injections per mouse ⁶Raji cells (10, 2 groups of n). Mice received three intravenous doses (

days

5, 8, 12) of 30X 10⁶And (4) ApoCell. (Raji-orange; and Raji + ApoCell-deep blue)

FIG. 26. Kaplan-Meier survival curves for SCID-Bg mice with Raji leukemia/lymphoma receiving RtX and ApoCell. (only Raji-orange; Raji + ApoCell-blue; Raji + RtX 2 mg-green; Raji + RtX 2mg + ApoCell-yellow; Raji + RtX 5 mg-violet; Raji + RtX 5mg + ApoCell-grey.)

FIG. 27. Kaplan-Meier survival curves for SCID-Bg mice with Raji leukemia/lymphoma receiving rtX and ApoCell. (only Raji-orange; Raji + ApoCell-blue; Raji + RtX 2 mg-green; Raji + RtX 2mg + ApoCell-yellow.)

Figure 28 effect of pooled ApoCell preparations. Figure 28 presents a graph showing the clear effect (p <0.01) of a single injection of apoptotic cell preparation from multiple individual donors (blue) on survival. The presented figures are kaplan-meier survival curves in GvHD mouse models treated with single doses of irradiated pooled apoptotic cell preparations from multiple individual donors.

Figure 29 effect of pooled ApoCell preparations. Figure 29 presents a graph showing the clear effect (p <0.01) of a single injection of apoptotic cell preparations from multiple individual donors (blue) on the percent weight loss of 2 comparative groups.

Fig. 30 comparison of single donors to pooled ApoCell preparations. Figure 30 presents graphs showing a comparison of single dose administration of single donor apoptotic cell preparation +/-irradiation versus multiple donor apoptotic cell preparation +/-irradiation in terms of% survival using a mouse model of induced GvHD.

Fig. 31A-31b. Fig. 31A-31B present the results of the potency assay, which shows inhibition of maturation of Dendritic Cells (DCs) following interaction with apoptotic cells as measured by expression of HLA-DR. Fig. 31A: average fluorescence of HLA DR of fresh end product a (t 0). Fig. 31B: average fluorescence of HLADR after 24 hours at 2-8 ℃ for final product a.

Fig. 32A-32b. Fig. 32A-32B present the results of the potency assay, which shows inhibition of maturation of Dendritic Cells (DCs) after interaction with apoptotic cells, as measured by expression of CD 86. Fig. 32A: mean fluorescence of CD86 for fresh end product a (t 0). FIG. 32B: mean fluorescence of CD86 after 24 hours at 2-8 ℃ for final product A.

Figure 33, a schematic of the example 17 protocol, showing the general flow of treatment of solid tumors by CAR T-CD19 and ApoCell administration, and cytopathology at the endpoint.

34A-34d. survival following CAR T cell therapy alone or co-administration with apoptotic cells. FIG. 34A shows the survival curves for peritoneal solid tumors HeLa-CD19 treated by CAR T cell therapy alone (SCID-Bg-HeLa-CD 19: no treatment control; SCID-Bg-HeLa-CD 19+ CAR-T: treatment with CAR T cells; SCID-Bg-HeLa-CD 19+ pseudo-T: pseudo-T addition control FIG. 34B represents 5 separate experiments as described in example 17 and shows the survival curves for peritoneal solid tumors HeLa-CD19 treated by CAR T cell therapy alone or with co-administration of apoptotic cells (HeLa-CD 19: no treatment control; HeLa-CD19 + CAR-T: CAR treatment with CAR T cells; HeLa-CD19 + pseudo-T: pseudo-T addition control; HeLa-CD19 + T-T + T-ALC-CAR-4K: treatment with CAR T cell therapy alone CAR T cells and "off-the-shelf apoptotic cells irradiated with 4000 rad). FIG. 34C presents representative results as described in example 17 and shows the survival curves for peritoneal solid tumor Hela-CD19 treated by CAR T cell therapy alone or CAR T cell therapy plus apoptotic cell co-administration (SCID-Bg-Hela-CD 19: no treatment control; SCID-Bg-Hela-CD 19+ CAR-T: treatment including use of CAR T cells; SCID-Bg-Hela-CD 19+ mock-T: mock T cell addition control; SCID-Bg-Hela-CD 19+ CAR-T + Allocetra-OTS: treatment of "off-the-shelf" apoptotic cells using CAR T cells and irradiated with 4000 rad). Fig. 34D (in vivo luciferase imaging system (IVIS) results) shows tumor progression visualized by IVIS, which represents the progression leading to the survival curve results presented in fig. 34B. Tumor spread could already be visualized on day 15 (hela-CD 19-Luc; no treatment control), whereas mice treated with CAR T cells did not show any tumor spread until day 43. When mice were treated with apoptotic cells in combination with CAR T cells, it was unexpected that most mice did not show tumor spread until day 50, and that tumor size (see the relevant ratios) was significantly smaller.

35A-35B. survival following CAR T cell therapy alone or co-administration with apoptotic cells. Figure 35A shows the survival curve for peritoneal solid tumor hela-CD 19 treated by CAR T cell therapy alone. FIG. 35B shows the survival curves for peritoneal solid tumor hala-CD 19 treated by CAR T cell therapy alone or CAR T cell therapy plus apoptotic cell co-administration (SCID-Bg-hala-CD 19: no treatment control; SCID-Bg-hala-CD 19+ CAR-T: treatment including use of CAR T cells; SCID-Bg-hala-CD 19+ CAR-T + Allocetra-OTS: treatment of "off-the-shelf apoptotic cells using CAR T cells and irradiation with 4000 rad).

FIGS. 36A-36C FACS analysis of macrophages. Figure 36A shows FACS analysis of colonizing peritoneal macrophage markers F4/80, CD11b, Tim4, and MerTK in SCID-bg mice (control, no tumor), SCID-bg-hela-CD 19 mice (control with tumor, no treatment) and SCID-bg-hela-CD 19-CAR T (including treatment with CAR T cells). The results seen in SCID-bg mice provide a resident macrophage feature. Data comparison shows that resident peritoneal macrophages disappear during tumor progression. Figure 36B shows FACS analysis of tumor-associated macrophage (TAM) markers CCR2, Ly6c, CD206, CD64, CD169, and CD74 in SCID-bg mice (control, no tumor), SCID-bg-hela-CD 19 mice (control with tumor, no treatment), and SCID-bg-hela-CD 19-CAR T (including treatment with CAR T cells) during tumor progression. The results seen in SCID-bg mice provide a feature of infiltrating macrophages. Data comparison shows TAM infiltration during tumor progression. Fig. 36C presents FAC results showing that the resident macrophages in SCID mice are mostly Large Peritoneal Macrophages (LPM), whereas during tumor progression most resident macrophages disappear and TAMs, monocytes and dendritic cells appear. Macrophage markers F4/80, CD11b, TIM4, MER-TK, rIgG2b, and rIgG2a were measured, as were the percentages of MHCII positive cells.

Figure 37 percent (%) curves after CAR T cell therapy alone or co-administration with apoptotic cells or opsonized apoptotic cells. The figure shows the survival curves of peritoneal solid tumor hela-CD 19 treated by CAR T cell therapy alone or CAR T cell therapy plus apoptotic cells or opsonized apoptotic cells coadministration (SCID-Bg-hela-CD 19: no treatment control; SCID-Bg-hela-CD 19+ CAR-T: treatment involving use of CAR T cells; SCID-Bg-hela-CD 19+ CAR-T + allocenta-OTS: treatment of "off-the-shelf apoptotic cells using CAR T cells and irradiated with 4000 rad; SCID-Bg-hela-CD 19+ CAR-T + D89E _ allocenta-OTS: treatment of" off-the-shelf opsonized apoptotic cells using CAR T cells and irradiated with 4000 rad).

Detailed Description

In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the methods disclosed herein. However, it will be understood by those skilled in the art that the methods may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the methods disclosed herein.

It is well known that genetic modification of immune cells is a strategy for immune cell therapy against cancer. These immune cell therapies are based on the manipulation of autologous or allogeneic immune cells and their administration to a subject in need thereof. 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 (tregs)).

In some embodiments, disclosed herein are compositions comprising genetically modified immune cells. 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+ T cell. In another embodiment, the T cell is a naive T cell. In another embodiment, the T cell is a naive CD8+ T cell. In another embodiment, the genetically modified immune cell is a Natural Killer (NK) cell. In another embodiment, the genetically modified immune cell is a dendritic cell. In yet another embodiment, the genetically modified T cell is a cytotoxic T lymphocyte (CTL cell). In another embodiment, the genetically modified T cell is a regulatory T cell (Treg). In another embodiment, the genetically modified T cell is a Chimeric Antigen Receptor (CAR) T cell. In another embodiment, the genetically modified T cell is a genetically modified T Cell Receptor (TCR) cell.

In some embodiments, disclosed herein are compositions comprising genetically modified immune cells and apoptotic cells. In another embodiment, disclosed herein is a composition comprising a genetically modified immune cell and a supernatant from an apoptotic cell. In another embodiment, the genetically modified immune cell is a T cell. In another embodiment, the genetically modified immune cell is a Natural Killer (NK) cell. In yet another embodiment, the genetically modified immune cell is a cytotoxic T lymphocyte (CTL cell). In another embodiment, the genetically modified immune cell is a regulatory T lymphocyte (Treg cell).

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

In related embodiments, the method does not reduce or inhibit the efficacy of the CAR T cell cancer therapy. In related embodiments, the methods improve the efficacy of the CAR T cell cancer therapy. In another related embodiment, the incidence of Cytokine Release Syndrome (CRS) or cytokine storm is inhibited or reduced in said subject compared to a subject not administered said apoptotic cells or said apoptotic cell supernatant.

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

In some embodiments, disclosed herein is a method of increasing the efficacy of a chimeric antigen receptor T cell (CAR T cell) cancer therapy, the method comprising the step of administering a CAR T cell and an additional agent selected from an apoptotic cell, an apoptotic cell supernatant, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof, wherein the efficacy of the CAR T cell is increased in a subject undergoing a CAR T cell cancer therapy in comparison to the subject without administration of the additional agent. In related embodiments, 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 receiving CAR T cell cancer therapy and not administered a composition comprising the agent. In another related embodiment, the proinflammatory cytokine comprises IL-6.

In a related embodiment, when apoptotic cells or an apoptotic cell supernatant are administered, the method increases the level of IL-2 in the subject compared to a subject undergoing CAR T cell cancer therapy without administration of the apoptotic cells or the apoptotic cell supernatant. In another embodiment, the method maintains the level of IL-2 in the subject when administering apoptotic cells or apoptotic cell supernatant as compared to a subject undergoing CAR T cell cancer therapy without administration of said apoptotic cells or said apoptotic cell supernatant. In another embodiment, the method maintains or increases the level of IL-2 in the subject when administering apoptotic cells or an apoptotic cell supernatant as compared to a subject undergoing CAR T cell cancer therapy without administration of said apoptotic cells or said apoptotic cell supernatant. In another related embodiment, the subject has an inhibited or reduced incidence of Cytokine Release Syndrome (CRS) or a cytokine storm as compared to a subject not administered the additional agent.

In related embodiments, the CAR T cell and the additional agent, or any combination thereof, are included in a single composition. In another related embodiment, the CAR T cell and the additional cancer therapeutic agent, or any combination thereof, are included in at least two compositions.

In some embodiments, disclosed herein is a method of treating, preventing, inhibiting, reducing the incidence, ameliorating, or alleviating a cancer or tumor in a subject, the method comprising the step of administering T cells expressing a chimeric antigen receptor (CAR T cells) and a further agent comprising apoptotic cells, apoptotic cell supernatant, or CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, tellurium-based compound or immunomodulator or any combination thereof, wherein the method treats, prevents, inhibits, reduces the incidence of, ameliorates, or alleviates a cancer or tumor in the subject as compared to a subject administered CAR T cells and not administered the additional agent.

In related embodiments, the method has increased efficacy in treating, preventing, inhibiting, reducing the incidence, ameliorating, or alleviating the cancer or tumor in the subject as compared to a subject administered CAR T cells and not administered the additional agent.

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

In related embodiments, administering the additional agent occurs prior to, concurrently with, or after administering the CAR T cells. In another related embodiment, the apoptotic cells comprise 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.

In a related embodiment, the apoptotic cell supernatant is obtained by a method comprising: (a) providing apoptotic cells, (b) culturing said cells of step (a), and (c) isolating said supernatant from said cells. In another related embodiment, the apoptotic cell supernatant is an apoptotic cell-white blood cell supernatant and the method further comprises the steps of: (d) providing white blood cells, (e) optionally washing said apoptotic cells and said white blood cells, (f) co-culturing said apoptotic cells and said white blood cells, wherein steps (d) - (f) replace step (b). In another related embodiment, the provided white blood cells are selected from the group consisting of phagocytes, macrophages, dendritic cells, monocytes, B cells, T cells, and NK cells. Thus, in some embodiments, apoptotic supernatant comprises supernatant produced by culturing apoptotic cells with macrophages, wherein said macrophages take up said apoptotic cells, and using said supernatant produced by such co-culturing. In some embodiments, the apoptotic supernatant comprises a supernatant produced by culturing apoptotic cells, wherein said supernatant is produced from material secreted by said apoptotic cells.

In some embodiments, disclosed herein are compositions comprising early apoptotic cells. In some embodiments, disclosed herein are compositions comprising early apoptotic cells in combination with an additional agent. 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 (rituximab) or a functional fragment thereof.

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

In some embodiments, disclosed herein are compositions comprising genetically modified T cells and apoptotic cells. In another embodiment, disclosed herein is a composition comprising genetically modified T cells and a supernatant of apoptotic cells. In another embodiment, the genetically modified T cell is a Chimeric Antigen Receptor (CAR) T cell. In another embodiment, the genetically modified T cell is a genetically modified T Cell Receptor (TCR) cell.

In some embodiments, disclosed herein are compositions comprising a CAR T cell and an apoptotic cell. In another embodiment, disclosed herein are compositions comprising a genetically modified T cell receptor cell (TCR) and an apoptotic cell. In another embodiment, disclosed herein are compositions comprising CAR T cells and supernatant from apoptotic cells. In another embodiment, disclosed herein is a composition comprising a genetically modified T cell receptor cell (TCR) and a supernatant of apoptotic cells.

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.

In some embodiments, the present disclosure provides a pooled mononuclear apoptotic cell preparation comprising mononuclear cells in an early apoptotic state, wherein said pooled mononuclear apoptotic cell preparation comprises a pooled mononuclear cell population, and wherein said pooled mononuclear apoptotic 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. In another embodiment, the pooled mononuclear apoptotic cells have been irradiated. In another embodiment, the present disclosure provides a pooled mononuclear apoptotic cell preparation that in some embodiments uses white blood cell fractions (WBCs) obtained from donated blood. Typically, such WBC fractions will be discarded at the blood bank or targeted for study.

In some embodiments, the population of cells disclosed herein is inactivated. In another embodiment, the inactivation comprises irradiation. In another embodiment, the inactivation comprises T cell receptor inactivation. In another embodiment, inactivation comprises T cell receptor editing. In another embodiment, inactivating comprises suppressing or eliminating an immune response in the formulation. In another embodiment, inactivating comprises suppressing or eliminating cross-reactivity between a plurality of individual populations included in the preparation. In other embodiments, inactivating comprises reducing or eliminating T cell receptor activity between a plurality of individual populations included in the preparation. In another embodiment, the inactivated cell preparation comprises a reduced percentage of live non-apoptotic cells, a suppressed cell activation of any live non-apoptotic cells, or a reduced proliferation of any live non-apoptotic cells, or any combination thereof.

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

In some embodiments, disclosed herein is a method of 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 living cells; suppressed cellular activation of any viable non-apoptotic cells; or reduced proliferation of any living non-apoptotic cells; or any combination thereof, the method comprising the steps of:

obtaining an enriched monocyte population of peripheral blood;

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

thawing the enriched monocyte population;

incubating the enriched monocyte population in an apoptosis-inducing incubation medium comprising methylprednisolone (methylprednisolone) at a final concentration of about 10-100 μ g/mL and an anticoagulant;

resuspending the population of apoptotic cells in an administration medium; and

inactivating the enriched mononuclear population, wherein the inactivation occurs after induction,

wherein the method produces a mononuclear apoptotic cell population comprising a reduced percentage of non-quiescent non-apoptotic cells; suppressed cellular activation of any living non-apoptotic cells; or reduced proliferation of any living non-apoptotic cells; or any combination thereof.

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 as compared to an unirradiated cell preparation.

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.

In some embodiments, the pooled blood comprises 3 rd party blood from an HLA-matched or HLA-mismatched source with respect to the recipient.

In some embodiments, disclosed herein are compositions comprising a genetically modified immune cell (such as, but not limited to, a CAR T cell) and an additional agent selected from an apoptotic cell, an apoptotic cell supernatant, a CTLA-4 blocker, an alpha-1 antitrypsin or fragment or analog thereof, a telluril-based compound, or an immunomodulator, or any combination thereof.

In some embodiments, the present disclosure provides a method of producing a pharmaceutical composition comprising a pooled mononuclear apoptotic cell preparation comprising a pooled mononuclear population of mononuclear cells in an early apoptotic state, wherein the composition comprises a reduced percentage of live non-apoptotic cells, a preparation with suppressed cell activation of any live non-apoptotic cells, or a preparation with reduced proliferation of any live 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 single mononuclear cell population in an early apoptotic state, wherein the composition comprises a reduced percentage of non-quiescent non-apoptotic cells.

In some embodiments, disclosed herein is a method of treating, preventing, inhibiting growth of, reducing incidence of, or any combination thereof, a cancer or tumor in a subject, the method comprising the step of administering to the subject an early apoptotic cell population, wherein the method treats, prevents, inhibits growth of, reduces incidence of, or any combination thereof, the cancer or tumor in the subject. In some embodiments, the methods herein comprise treating, preventing, inhibiting the growth of, delaying the progression of a disease in, reducing the load of, or reducing the incidence of a cancer or tumor in a subject, or any combination thereof, in a subject comprising the step of 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 immunomodulatory agent, or any combination thereof. In some embodiments, the additional immunotherapy, chemotherapeutic agent or immunomodulatory agent is administered prior to, concurrently with, or after the administration of the early apoptotic cells.

In some embodiments, disclosed herein is a method of increasing survival of a subject having a cancer or tumor, the method comprising the step of administering to the subject an early apoptotic cell population, wherein the method increases survival of the subject. In some embodiments, the method further comprises administering to the subject an additional cancer therapy. In some embodiments, the additional cancer therapy comprises radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormonal therapy, photodynamic therapy, immunomodulators or surgery, or any combination thereof. In some embodiments, the cancer therapy comprises CAR T cell therapy. In some embodiments, the additional cancer therapy or cancer therapeutic is administered prior to, concurrently with, or subsequent to the administration of the early apoptotic cells.

In some embodiments, disclosed herein is a method of reducing the size of or reducing the growth rate of a cancer or tumor in a subject, or a combination thereof, 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 immunomodulatory agent, or any combination thereof. In some embodiments, the additional immunotherapy, chemotherapeutic agent, or immunomodulatory agent is administered prior to, concurrently with, or after the administration of the early apoptotic cells.

In some embodiments, administration of a composition comprising apoptotic cells does not affect the efficacy of CAR T cell therapy, prevention, inhibition, reduction in incidence, amelioration, reduction in tumor burden, or remission of the cancer or tumor. In another embodiment, administration of the composition comprising apoptotic cells does not reduce the efficacy of CAR T cell therapy, prevention, inhibition, reduction in incidence, amelioration, reduction in load, or remission of the cancer or tumor by more than about 5%. In another embodiment, administration of the composition comprising apoptotic cells does not reduce the efficacy of CAR T cell therapy, prevention, inhibition, reduction in incidence, amelioration, reduction in load, or remission of the cancer or tumor by more than about 10%. In another embodiment, administration of the composition comprising apoptotic cells does not reduce the efficacy of CAR T cell therapy, prevention, inhibition, reduction in incidence, amelioration, reduction in load, or remission of the cancer or tumor by more than about 15%. In another embodiment, administration of the composition comprising apoptotic cells does not reduce the efficacy of CAR T cell therapy, prevention, inhibition, reduction in incidence, amelioration, reduction in load, or remission of the cancer or tumor by more than about 20%.

In some embodiments, administration of apoptotic cells increases the efficacy of CAR T cells. In some embodiments, administration of the apoptotic cells increases the efficacy of the 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%.

In another embodiment, administration of the composition comprising an apoptotic cell supernatant does not reduce the efficacy of CAR T cells to treat, prevent, inhibit, reduce the incidence of, ameliorate or ameliorate the cancer or the tumor by more than about 5%. In another embodiment, administration of the composition comprising an apoptotic cell supernatant does not reduce the efficacy of CAR T cells to treat, prevent, inhibit, reduce the incidence of, ameliorate or ameliorate the cancer or the tumor by more than about 10%. In another embodiment, administration of the composition comprising an apoptotic cell supernatant does not reduce the efficacy of CAR T cells to treat, prevent, inhibit, reduce the incidence of, ameliorate or ameliorate the cancer or the tumor by more than about 15%. In another embodiment, administration of the composition comprising an apoptotic cell supernatant does not reduce the efficacy of CAR T cells to treat, prevent, inhibit, reduce the incidence of, ameliorate or ameliorate the cancer or the tumor by more than about 20%. In another embodiment, administration of a composition comprising the apoptotic cell supernatant does not affect the efficacy of CAR T cell therapy, prevention, inhibition, reduction in incidence, amelioration, or palliation of the cancer or the tumor. In another embodiment, administration of a composition comprising the apoptotic cell supernatant does not reduce the efficacy of CAR T cells to treat, prevent, inhibit, reduce the incidence of, ameliorate, or ameliorate the cancer or the tumor.

In some embodiments, disclosed herein are methods of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing CAR T cell cancer therapy. In another embodiment, the methods disclosed herein reduce or prevent cytokine production in a subject undergoing CAR T cell cancer therapy, thereby inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in the subject. In another embodiment, the methods disclosed herein of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing a CAR T cell cancer therapy comprise the step of administering to the subject undergoing the cancer therapy a composition comprising apoptotic cells. In another embodiment, the methods disclosed herein for reducing or inhibiting cytokine production in a subject undergoing a CAR T cell cancer therapy comprise the step of administering to the subject undergoing the cancer therapy a composition comprising apoptotic cells. In another embodiment, administration of a composition comprising apoptotic cells does not affect the efficacy of CAR T cell therapy. In another embodiment, administration of a composition comprising apoptotic cells or an apoptotic supernatant does not reduce the efficacy of CAR T cell therapy. In another embodiment, administration of a composition comprising apoptotic cells or an 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 an 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 an 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 an apoptotic cell supernatant does not reduce the efficacy of CAR T cell therapy by more than about 20%.

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

In some embodiments, the methods disclosed herein for reducing or inhibiting cytokine production in a subject undergoing a CAR T cell cancer therapy comprise the step of administering to the subject undergoing the cancer therapy a composition comprising an apoptotic cell supernatant. In another embodiment, administration of a composition comprising apoptotic cell supernatant does not affect the efficacy of CAR T cell therapy. In another embodiment, administration of the composition comprising apoptotic cell supernatant does not decrease the efficacy of CAR T cell therapy.

In some embodiments, a method of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing a chimeric antigen receptor expressing T cell (CAR T cell) cancer therapy comprises the step of administering to the subject a composition comprising apoptotic cells or an apoptotic supernatant. In another embodiment, the method of inhibiting or reducing the incidence of a Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing a chimeric antigen receptor expressing T cell (CAR T cell) cancer therapy reduces or inhibits the production of at least one pro-inflammatory cytokine in the subject.

In another embodiment, the present disclosure provides a method of treating, preventing, ameliorating, inhibiting, or reducing the incidence of an immune disease, an autoimmune disease, an inflammatory disease, Cytokine Release Syndrome (CRS), a cytokine storm, or infertility in a subject in need thereof comprising using a pooled mononuclear apoptotic cell preparation of monocytes in an early apoptotic state as described herein. In another embodiment, disclosed herein are pooled mononuclear apoptotic cell preparations, wherein in certain embodiments, use of such cell preparations does not require matching of the donor and recipient, for example, by HLA typing.

Genetically modified immune cells

In one embodiment, disclosed herein are methods comprising genetically modified immune cellsA composition is provided. 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 naive CD4⁺T cells. In another embodiment, the T cell is a naive T cell. In another embodiment, the T cell is naive CD8⁺T cells. In another embodiment, the genetically modified immune cell is a Natural Killer (NK) cell. In another embodiment, the genetically modified immune cell is a dendritic cell. In yet another embodiment, the genetically modified T cell is a cytotoxic T lymphocyte (CTL cell). In another embodiment, the genetically modified T cell is a regulatory T cell (Treg). In another embodiment, the genetically modified T cell is a Chimeric Antigen Receptor (CAR) T cell. In another embodiment, the genetically modified T cell is a genetically modified T Cell Receptor (TCR) cell. .

In one embodiment, disclosed herein is a composition comprising a genetically modified immune cell and an additional agent selected from an apoptotic cell, an apoptotic cell supernatant, a CTLA-4 blocker, an alpha-1 antitrypsin or fragment thereof or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof. In another embodiment, disclosed herein is a composition comprising a genetically modified immune cell, an apoptotic cell, and an additional agent selected from a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof. In another embodiment, disclosed herein is a composition comprising a genetically modified immune cell, an apoptotic cell supernatant, and an additional agent selected from a CTLA-4 blocker, an alpha-1 antitrypsin or fragment or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof.

In one embodiment, the immune cell is cytotoxic. In another embodiment, the genetically modified cytotoxic cells may be obtained from bone marrow of a subject (autologous) or donor (allogeneic). In other cases, the cells are obtained from stem cells. For example, the cytotoxic cells may 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 (IPSCs), such pluripotent stem T cells may be obtained using somatic cells from a subject to which genetically modified cytotoxic cells are to be provided. In one embodiment, the immune cells may be obtained from a subject or donor by: cells are collected by venipuncture, by apheresis methods, by leukocyte mobilization followed by apheresis or venipuncture, or by bone marrow aspiration.

In one embodiment, immune cells (e.g., T cells) are produced and expanded by the presence of specific factors in the body. In another embodiment, T cell production and maintenance is affected by cytokines in vivo. In another embodiment, cytokines that affect the production 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, the Treg cells are generated from naive T cells by in vivo cytokine induction. In yet another embodiment, TGF- β and/or IL-2 play a role in differentiating naive T cells into Treg cells.

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 TGF β maintains or increases the rate of proliferation of T cells in vivo, or both. In another embodiment, the presence of the cytokines IL-2 and/or TGF β maintains or increases 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 TGF β maintains or increases the rate of proliferation of CAR T cells in vivo or both. In another embodiment, the presence of the cytokines IL-2 and/or TGF β maintains or increases or both the proliferation rate of the CAR T cells in vivo. 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 TGF β maintains or increases the rate of proliferation of TCR T cells in vivo or both. In another embodiment, the presence of the cytokines IL-2 and/or TGF β results in the maintenance or increase or both of the proliferation rate of TCR T cells in vivo. 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 TGF β maintains or increases the rate of proliferation of T-reg cells in vivo, or both. In another embodiment, the presence of the cytokines IL-2 and/or TGF β results in the maintenance or improvement or both of the proliferation rate of T-reg cells in vivo.

In one embodiment, T cells having altered expression or form of a STAT 5B-encoded protein or a BACH 2-encoded protein 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 the expression of a BACH2 polypeptide.

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

In another embodiment, T cells having altered expression of the protein encoded by STAT5B maintain or increase their rate of proliferation in vivo for greater than 1 year. In another embodiment, T cells having altered expression of the protein encoded by STAT5B maintain or increase their rate of proliferation in vivo for greater than 2 years. In another embodiment, T cells having altered expression of the protein encoded by STAT5B maintain or increase their rate of proliferation in vivo for greater than 3 years. In another embodiment, T cells having altered expression of the protein encoded by STAT5B maintain or increase their rate of proliferation in vivo for greater than 4 years. In another embodiment, T cells having altered expression of the protein encoded by STAT5B maintain or increase their rate of proliferation in vivo for greater than 5 years. In another embodiment, T cells having altered expression of the protein encoded by STAT5B maintain or increase their rate of proliferation in vivo for greater than 10 years. In another embodiment, T cells having altered expression of the protein encoded by STAT5B maintain or increase their rate of proliferation in vivo for greater than 20 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 greater than 1 year. In another embodiment, T cells having altered expression of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 2 years. In another embodiment, T cells having altered expression of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 3 years. In another embodiment, T cells having altered expression of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 4 years. In another embodiment, T cells having altered expression of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 5 years. In another embodiment, T cells having altered expression of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 10 years. In another embodiment, T cells having altered expression of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 20 years.

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

In another embodiment, T cells having an altered form of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 1 year. In another embodiment, T cells having an altered form of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 2 years. In another embodiment, T cells having an altered form of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 3 years. In another embodiment, T cells having an altered form of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 4 years. In another embodiment, T cells having an altered form of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 5 years. In another embodiment, T cells having an altered form of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 10 years. In another embodiment, T cells having an altered form of a protein encoded by BACH2 maintain or increase their rate of proliferation in vivo for greater than 20 years.

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

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

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

In one embodiment, disclosed herein is a method for maintaining or increasing the proliferation rate of a genetically modified immune cell, wherein the method comprises the step of administering apoptotic cells or an apoptotic supernatant. In another embodiment, disclosed herein is a method for increasing the efficacy of a genetically modified immune cell, wherein the method comprises the step of administering an additional agent comprising an apoptotic cell, an apoptotic supernatant, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a telluril-based compound, or an immunomodulator, or any combination thereof. In another embodiment, the method for treating, preventing, inhibiting, reducing the incidence, ameliorating, or alleviating a cancer or tumor disclosed herein, administers genetically modified immune cells and an additional agent, wherein the additional agent comprises apoptotic cells, apoptotic supernatants, CTLA-4 blockers, alpha-1 antitrypsin or fragments or analogs thereof, tellurium-based compounds, or immunomodulators, or any combination thereof.

Chimeric antigen receptor expressing T cells (CAR T cells)

In some embodiments, the Chimeric Antigen Receptor (CAR) is a type of antigen-targeting receptor composed of an intracellular T cell signaling domain (most typically a single-chain variable fragment (scFv) from a monoclonal antibody) fused to an extracellular tumor-binding moiety. CARs recognize cell surface antigens directly, independent of MHC-mediated presentation, allowing the use of a single receptor construct specific for any given antigen in all patients. The original CAR fused the antigen recognition domain to the CD3 zeta activation chain of the T Cell Receptor (TCR) complex. While these first generation CARs induced T cell effector functions in vitro, they were largely limited by poor anti-tumor efficacy in vivo. Subsequent CAR iterations had secondary costimulatory signals in tandem with CD3 ζ, including intracellular domains from CD28 or various TNF receptor family molecules, such as 4-1BB (CD137) and OX40(CD 134). Further, in addition to CD3 ζ, the third generation receptor comprises two costimulatory signals, most commonly from CD28 and 4-1 BB. Second and third generation CARs significantly improved antitumor efficacy, in some cases inducing complete remission in patients with advanced cancer.

In some embodiments, the CAR T cell is an immunoresponsive cell that includes an antigen receptor, which immunoresponsive cell is activated when its receptor binds its antigen.

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

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

In another embodiment, the CAR T cell as disclosed herein is a second generation CAR T cell. In another embodiment, a CAR T cell as disclosed herein comprises a triple chimeric receptor (TPCR). In some embodiments, a CAR T cell as disclosed herein comprises one or more signaling moieties that activate naive T cells in a costimulatory-independent manner. In another embodiment, the CAR T cells further encode 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.

Third generation CAR T cells attempt to exploit the signaling potential of 2 co-stimulatory 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 as disclosed herein further encode a co-stimulatory signaling domain (which in one embodiment is CD 28). In another embodiment, the signaling domain is a CD3 zeta chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, CD28 signaling domain, or a combination thereof.

In some embodiments, telomere length and replicative capacity are correlated with engraftment efficiency and anti-tumor efficacy of adoptively transferred T cell lines. In some embodiments, CD28 stimulation maintains telomere length in T cells.

In some embodiments, CAR-modified T cell potency may be further enhanced by the introduction of additional genes, including those encoding proliferative cytokines (i.e., IL-12) or co-stimulatory ligands (i.e., 4-1BBL), thus generating "armored" fourth generation CAR-modified T cells. In some embodiments, an "armored CAR T cell" is a CAR T cell that is protected from inhibitory tumor microenvironments. In another embodiment, the "armored" CAR technology incorporates local secretion of soluble signaling proteins to amplify the immune response 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 the microenvironment and effective immunosuppressive mechanisms have the potential to make establishment of robust anti-tumor responses more challenging.

In some embodiments, the CAR T cells are genetically modified to encode molecules of chemokine receptors that are involved in preventing apoptosis, remodeling the tumor microenvironment, inducing homeostatic proliferation, and promoting homing of targeted T cells.

In another embodiment, CAR T cell therapy as used in the compositions and methods disclosed herein is enhanced using expression of cytokine transgenes, combination therapy with small molecule inhibitors, or monoclonal antibodies. In another embodiment, other strategies aimed at improving CAR T cell therapy (including the use of dual CARs and chemokine receptors to more specifically target tumor cells) are considered as part of the CAR T cells and CAR T cell therapies as disclosed herein.

In some embodiments, the CAR T cells of the compositions and methods as disclosed herein include a second binding domain that can result in an inhibitory or amplifying signal, so as to increase the specificity of the CAR T cells for cancer cells relative to normal cells. For example, a CAR T cell can be engineered such that it will be triggered in the presence of one target protein, but will be inhibited if a second protein is present. Alternatively, it may also be engineered such that maximum activation would require both target proteins. These methods can increase the specificity of the CAR for tumor versus normal tissue.

In some embodiments, the CAR T cells used in the compositions and methods as disclosed herein encode an antibody-based external receptor structure and a cytoplasmic domain that encodes a signaling module consisting of an immunoreceptor tyrosine-based activation motif.

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

In some embodiments, the CAR T cell further encodes a single chain variable fragment (scFv) that binds a polypeptide having immunostimulatory activity. In another embodiment, the polypeptide having immunostimulatory activity is CD28, OX-40, 4-1BB, or a combination thereof. In another embodiment, the CAR T cells further encode a CD40 ligand (CD40L), which in some embodiments, enhances the immunostimulatory activity of the antigen.

In some embodiments, the immune cell is cytotoxic. In another example, cytotoxic cells for genetic modification may be obtained from bone marrow of a subject or donor. In other cases, the cells are obtained from stem cells. For example, the cytotoxic cells may 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 (IPSCs), such pluripotent T cells may be obtained using somatic cells from a subject to whom the genetically modified cytotoxic cells are provided. In some embodiments, the immune cells may be obtained from a subject or donor by: cells are collected by venipuncture, by apheresis methods, by leukocyte mobilization followed by apheresis or venipuncture, or by bone marrow aspiration.

In some embodiments, a method as disclosed herein comprises obtaining an immune cell from a subject and genetically modifying the immune cell to express a chimeric antigen receptor. In another embodiment, the methods as disclosed herein comprise obtaining immune cells from a subject, genetically modifying the immune cells to express a chimeric antigen receptor, and combining with an apoptotic cell population, thereby resulting in a reduction in cytokine production by the subject, but with substantially unaffected cytotoxicity relative to CAR-expressing immune cells that are not administered with an apoptotic cell population (fig. 1A-1B and 2). In another embodiment, the methods as disclosed herein comprise obtaining an immune cell from a subject, genetically modifying the immune cell to express a chimeric antigen receptor, and combining with an apoptotic cell supernatant or a composition comprising the supernatant, thereby resulting in a reduction in cytokine production by the subject, but with substantially unaffected cytotoxicity relative to CAR-expressing immune cells that are not administered with an apoptotic cell supernatant. In another embodiment, administration of the population of apoptotic cells or the supernatant from apoptotic cells does not result in a decrease in the efficacy of immune cells expressing the chimeric antigen receptor.

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 cell is heterologous to the subject. In some embodiments, the CAR T cell is allogeneic. In some embodiments, the CAR T cell is a universal allogeneic CAR T cell. In another embodiment, the T cells may be autologous, allogeneic, or derived in vitro from engineered progenitor or stem cells.

In another embodiment, both the CAR T cells and apoptotic cells described herein are derived from the same source. In further embodiments, both the CAR T cells and apoptotic cells described herein are derived from a subject (fig. 1). In alternative embodiments, 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 (fig. 2). The skilled person will appreciate that similarly, apoptotic cell supernatant may be prepared from cells derived from the same source as CAR T cells (which in one embodiment may be autologous cells), or apoptotic cell supernatant may be prepared from cells derived from a different source than the source of CAR T cells.

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

Thus, one embodiment as disclosed herein relates to a cytotoxic immune cell (e.g., NK cell or T cell) comprising a Chimeric Antigen Receptor (CAR), whereby the cell retains its cytotoxic function. In another embodiment, the chimeric antigen receptor is exogenous to a T cell. In another embodiment, the CAR is expressed recombinantly. In another embodiment, the CAR is expressed from a vector.

In some embodiments, the T cell used to generate the CAR T cell is a naive CD4+ T cell. In another embodiment, the T cell used to generate the CAR T cell is a naive CD8+ T cell. In another embodiment, the T cell used to generate the CAR T cell is an effector T cell. In another embodiment, the T cell used to generate the CAR T cell is a regulatory T cell (Treg). In another embodiment, the T cell used to generate the CAR T cell is a cytotoxic T cell.

Sources of genetically modified immune cells (e.g., T cells) have been widely described in the literature, see, e.g., the melli et al (2015) New Cell Sources for T Cell Engineering and adaptive immunity. Cell Stem 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-. CAR T cells can be ordered from commercial sources, such as the Creative laboratory (Creative Biolabs) (new york, usa), which provides customized construction and production services for Chimeric Antigen Receptors (CARs) and also provides a stock of pre-prepared CAR constructs that can induce protective immune encoding by recombinant adenovirus vaccines. Customized CAR T cells are also available from Promab Biotechnologies (Promab Biotechnologies), ca, usa, which can provide specially designed CAR T cells.

T Cell Receptor (TCR) cells

In one embodiment, the compositions and methods as disclosed herein utilize designer T Cell Receptor (TCR) cells in addition to or in place of CAR T cells. TCRs are multi-subunit transmembrane complexes that mediate antigen-specific activation of T cells. The TCR is composed of two different polypeptide chains. TCRs confer antigen specificity on T cells by recognizing an epitope on a target cell (e.g., a tumor or cancer cell). After contact with antigens present on the tumor or cancer cells, the T cells proliferate and acquire a phenotype and function that allows them to eliminate the cancer or tumor cells.

In one embodiment, TCR T cell therapy comprises introducing into T cells a T Cell Receptor (TCR) specific for an epitope of a protein of interest. In another embodiment, the protein of interest is a tumor associated antigen. In another embodiment, the genetically engineered TCR recognizes a tumor epitope presented by the Major Histocompatibility Complex (MHC) and the T cell activation domain on a tumor cell. In another embodiment, the T cell receptor recognizes an antigen, whether it is in intracellular or membrane location. In another embodiment, the TCR recognizes a tumor cell expressing a tumor-associated antigen within the cell. In one embodiment, the TCR recognizes an internal antigen. In another embodiment, the TCR recognizes an angiogenic factor. In another embodiment, the angiogenic factor is a molecule involved in the formation of new blood vessels. Various genetically modified T cell receptors and methods for their production are known in the art.

In one embodiment, the TCR T cell therapy is used to treat, prevent, inhibit, ameliorate, reduce the incidence, or ameliorate a cancer or tumor. In one embodiment, TCR T cell therapy is used to treat, prevent, inhibit, ameliorate, reduce the incidence, or ameliorate late metastatic disease, including diseases with blood (lymphomas and leukemias) and solid tumors (refractory melanoma, sarcomas). In one embodiment, TCR T cell therapies as used in the compositions and methods disclosed herein treat malignancies listed in table 1 of the following references: sadelain et al, (Cancer Discov.2013Apr; 3(4): 388-.

In another embodiment, the T cell receptor is genetically modified to bind an 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 the HPV-16E6 epitope and the TCR-engineered T cell is anti-HPV-16E 6. In another embodiment, the T cell receptor is genetically modified to bind the HPV-16E7 epitope and the TCR-engineered T cell is anti-HPV-16E 7. 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/a 6. In another embodiment, the T cell receptor is genetically modified to bind the MAGE A3 epitope and the TCR-engineered T cell is anti-MAGE A3. In another embodiment, the T cell receptor is genetically modified to bind to the SSX2 epitope and the TCR-engineered T cell is anti-SSX 2. 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, the skilled artisan will appreciate that T cell receptors can be genetically modified to bind to a target antigen present on a cancer or tumor cell, wherein TCR-engineered T cells comprise anti-tumor or anti-cancer cells.

In one embodiment, a method as disclosed herein comprises obtaining an immune cell from a subject, and genetically modifying the immune cell to express a recombinant T Cell Receptor (TCR). In another embodiment, a method as disclosed herein comprises obtaining an immune cell from a subject, genetically modifying the immune cell to express a recombinant TCR, and combining with an additional agent, wherein the additional agent comprises an apoptotic cell population, an apoptotic cell supernatant, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a telluril-based compound, or an immunomodulator, or any combination thereof.

In one embodiment, the T cell used to generate the TCR T cell is naive CD4⁺T cells. In another embodiment, the T cell used to generate the TCR T cell is naive CD8⁺T cells. In another embodiment, the T cell used to generate the TCR T cell is an effector T cell. In another embodiment, the T cell used to generate the TCR T cell is a regulatory T cell (Treg). In another embodiment, the T cells used to generate TCR T cells are cytotoxic T cells.

TCR T cells have been widely described in the literature, see, e.g., sharp and Mount (2015) ibid; essand M, Loskog ASI (2013) genetic engineered T cells for the treatment of cancer (Review). J Intern Med 273: 166-; and Kershaw et al (2014) Clinical application of genetic modified T cells in cancer therapy, Clinical & Translational Immunology 3: 1-7.

Targeting antigens

In some embodiments, the CAR binds to an 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).

In one embodiment, the TCR binds to an epitope of an antigen via a genetically modified T cell receptor.

In another embodiment, the CAR T cells of the composition as disclosed herein bind 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 mutant in early tumors (Armet), thermal 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 (BAGE-1), aberrant transcripts 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 protooncogene, Aft, MMP-2, prostate specific membrane antigen (PSM A), Telomerase-related protein-2, Prostatic Acid Phosphatase (PAP), urolytic protein II, or protease 3.

In another embodiment, the CAR binds to CD19 or CD20 to target B cells where it is desired to destroy the B cells (as in leukemia). CD19 is a B cell lineage specific surface receptor whose 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 GD 2. In another embodiment, the CAR binds to NY-ESO-1. In another embodiment, the CAR binds to a MAGE family protein. In another embodiment, the CAR binds to mesothelin. In another embodiment, the CAR is conjugated to c-erbB 2. In another embodiment, the CAR binds to a tumor-specific mutant antigen, such as a BRAFV600E mutation and a BCR-ABL translocation. In another embodiment, the CAR binds to a tumor-specific viral antigen, such as EBV in HD, HPV in cervical cancer, and polyomavirus in Merkel cancer (Merkel cancer). In another embodiment, the CAR T cells bind to Her 2/neu. In another embodiment, the CAR T cell binds to EGFRvIII.

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 CD 20. In another embodiment, the CAR binds to CD 23. In another embodiment, the CAR binds to CD 24. In another embodiment, the CAR binds to CD 30. In another embodiment, the CAR binds to CD 33. In another embodiment, the CAR binds to CD 38. In another embodiment, the CAR binds to CD44v 6. In another embodiment, the CAR binds to CD44v 7/8. In another embodiment, the CAR binds to CD 123. In another embodiment, the CAR binds to CD 171. 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 EphA 2. In another embodiment, the CAR binds to Erb-B2. In another embodiment, the CAR binds to Erb-B2, 3, 4. In another embodiment, the CAR binds to Erb-B3/4. In another embodiment, the CAR binds to FBP. In another embodiment In embodiments, the CAR binds to a fetal acetylcholinesterase receptor. In another embodiment, the CAR and G_D2And (4) combining. In another embodiment, the CAR and G_D3And (4) combining. In another embodiment, the CAR binds to HER 2. In another embodiment, the CAR binds to HMW-MAA. In another embodiment, the CAR binds to IL-11 Ra. In another embodiment, the CAR binds to IL-13R α 1. In another embodiment, the CAR binds to a 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 an 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 MUC 1. In another embodiment, the CAR binds to MUC 16. In another embodiment, the CAR binds to NKG2D ligand. In another embodiment, the CAR binds to NY-ESO-1 (amino acids 157 and 165). In another embodiment, the CAR binds to carcinoembryonic antigen (h5T 4). In another embodiment, the CAR binds to PSCA. In another embodiment, the CAR binds to PSMA. In another embodiment, the CAR binds to ROR 1. In another embodiment, the CAR binds to TAG-72. In another embodiment, the CAR binds to VEGF-R2 or other VEGF receptor. In another embodiment, the CAR binds to B7-H6. In another embodiment, the CAR binds to CA 9. In another embodiment, the CAR and alpha _vβ₆The integrins bind. In another embodiment, the CAR binds to 8H 9. In another embodiment, the CAR binds to NCAM. In another embodiment, the CAR binds to a fetal acetylcholinesterase receptor.

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 cell targetTo the CD22 antigen, and has therapeutic effects 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 a subject having ovarian or epithelial cancer. In another embodiment, the CAR T cell targets CAIX or G250/CAIX and has a therapeutic effect on a subject having renal cell carcinoma. In another embodiment, the CAR T cells target CD20 and have a therapeutic effect on a subject having lymphoma, a B cell malignancy, a 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 a subject with CLL. In another embodiment, the CAR T cells target CD24 and have a therapeutic effect on a subject with pancreatic cancer. In another embodiment, the CAR T cells target CD30 and have a therapeutic effect on a subject having lymphoma or Hodgkin lymphoma. In another embodiment, the CAR T cells target CD33 and have a therapeutic effect on AML in a subject. In another embodiment, the CAR T cells target CD38 and have a therapeutic effect on a subject having Non-Hodgkin lymphoma. In another embodiment, the CAR T cells target CD44v6 and have a therapeutic effect in a subject with a severe malignancy. In another embodiment, the CAR T cells target CD44v7/8 and have a therapeutic effect on a subject having cervical cancer. In another embodiment, the CAR T cells target CD123 and have a therapeutic effect on a subject having a myeloid malignancy. In another embodiment, the CAR T cells target CEA and have a therapeutic effect on a subject having colorectal cancer. In another embodiment, the CAR T cells target EGFRvIII and have a therapeutic effect on a subject having glioblastoma. In another embodiment, the CAR T cells target EGP-2 and have a therapeutic effect on a subject having multiple malignancies. In another embodiment, the CAR T cells target EGP-40 and have treatment for a subject with colorectal cancer And (4) acting. In another embodiment, the CAR T cells target EphA2 and have a therapeutic effect on a subject having glioblastoma. In another embodiment, the CAR T cells target Erb-B2 or ErbB3/4 and have therapeutic effect in subjects with breast and other cancers, prostate cancer, colon cancer, various tumors. In another embodiment, the CAR T cells target Erb-B2, 3, 4 and have a therapeutic effect in a subject with breast and other cancers. In another embodiment, the CAR T cell targets FBP and has a therapeutic effect on a subject having ovarian cancer. In another embodiment, the CAR T cells target fetal acetylcholinesterase receptors and have a therapeutic effect in a subject with rhabdomyosarcoma. In another embodiment, the CAR T cell targets G_D2And has therapeutic effects on subjects having neuroblastoma, melanoma or Ewing's sarcoma. In another embodiment, the CAR T cell targets G_D3And has a therapeutic effect on a subject suffering from melanoma. In another embodiment, the CAR T cell targets HER2 and has a therapeutic effect on a subject having medulloblastoma, pancreatic cancer, glioblastoma, osteosarcoma or ovarian cancer. In another embodiment, the CAR T cell targets HMW-MAA and has a therapeutic effect in a subject having melanoma. In another embodiment, the CAR T cells target IL-11 ra and have a therapeutic effect on a subject with osteosarcoma. In another embodiment, the CAR T cells target IL-13 ra 1 and have a therapeutic effect on a subject having a glioma, glioblastoma, or medulloblastoma. In another embodiment, the CAR T cells target IL-13 receptor alpha 2 and have a therapeutic effect in a subject with a severe malignancy. In another embodiment, the CAR T cells target KDR and have a therapeutic effect on a subject having a tumor by targeting tumor neovasculature. In another embodiment, the CAR T cells target the kappa light chain and have a therapeutic effect on a subject with a B cell malignancy (B-NHL, CLL). In another implementation In the examples, the CAR T cells target Lewis Y and have therapeutic effects on subjects with various cancers or tumors of epithelial origin. In another embodiment, the CAR T cells target the L1 cell adhesion molecule and have a therapeutic effect on a subject having neuroblastoma. In another embodiment, the CAR T cells target MAGE-a1 or HLA-a1 MAGE a1 and have a therapeutic effect on a subject having melanoma. In another embodiment, the CAR T cells target mesothelin and have a therapeutic effect on a subject having mesothelioma. In another embodiment, the CAR T cells target CMV-infected cells and have a therapeutic effect on a subject with CMV. In another embodiment, the CAR T cells target MUC1 and have a therapeutic effect in a subject having breast or ovarian cancer. In another embodiment, the CAR T cells target MUC16 and have a therapeutic effect in a subject having ovarian cancer. In another embodiment, the CAR T cells target NKG2D ligand and have a therapeutic effect on subjects with myeloma, ovarian 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 in a subject 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 a subject having prostate cancer. In another embodiment, the CAR T cells target PSMA and have a therapeutic effect on a subject having prostate cancer/tumor vasculature. In another embodiment, the CAR T cells target ROR1 and have a therapeutic effect on a subject having B-CLL and mantle cell lymphoma. In another embodiment, the CAR T cells target TAG-72 and have a therapeutic effect on a subject having adenocarcinoma or gastrointestinal cancer. In another embodiment, the CAR T cells target VEGF-R2 or other VEGF receptors and have a therapeutic effect on a subject having a tumor by targeting tumor neovasculature. In another embodiment, the CAR T cell targets CA9, and Has therapeutic effect on a subject suffering from renal cell carcinoma. In another embodiment, the CAR T cells target CD171 and have a therapeutic effect in a subject with renal neuroblastoma. In another embodiment, the CAR T cells target NCAM and have a therapeutic effect in a subject having neuroblastoma. In another embodiment, the CAR T cells target fetal acetylcholinesterase receptors and have a therapeutic effect in a subject with rhabdomyosarcoma. In another embodiment, the CAR binds to one of the target antigens listed in Table 1 of Sadelain et al, (Cancer Discov.2013Apr; 3(4): 388-398), incorporated herein by reference in its entirety. In another embodiment, the CAR T cell is associated with a carbohydrate or glycolipid structure.

In one embodiment, the CAR T cells bind to an angiogenic factor, thereby targeting tumor vasculature. In some embodiments, the angiogenic factor is VEGFR 2. 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); a leptin; a midkine; a placental growth factor; platelet-derived endothelial cell growth factor (PD-ECGF); platelet-derived growth factor-BB (PDGF-BB); pleiotropic growth factor (PTN); a granule protein precursor; a proliferation protein; transforming growth factor-alpha (TGF- α); transforming growth factor-beta (TGF-beta); tumor necrosis factor-alpha (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 a Fibroblast Growth Factor (FGF); VEGF; VEGFR and neuropilin 1 (NRP-1); angiopoietin 1(Ang1) and Tie 2; platelet-derived growth factor (PDGF; BB-homodimer) and PDGFR; transforming growth factor beta (TGF-beta), endoglin, and TGF-beta receptor; monocyte chemotactic protein-1 (MCP-1); integrins α V β 3, α V β 5 and α 5 β 1; VE-cadherin and CD 31; ephrin; a plasminogen activator; plasminogen activator inhibitor-1; nitric Oxide Synthase (NOS) and COX-2; an AC 133; or Id1/Id 3. In some embodiments, the angiogenic protein used in the compositions and methods disclosed herein is angiogenin, which in one embodiment is angiogenin 1, angiogenin 3, angiogenin 4, or angiogenin 6. In some embodiments, the endoglin is referred to as CD 105; EDG; HHT 1; ORW; or ORW 1. In some embodiments, the endoglin is a TGF β co-receptor.

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 (Mycobacterium tuberculosis). In some embodiments, the mycobacterium tuberculosis-associated antigen is: antigen 85B, lipoprotein IpqH, ATP-dependent helicase postulate, uncharacterized protein Rv0476/MTO4941 precursor or uncharacterized protein Rv1334/MT1376 precursor.

In another embodiment, the CAR T cell binds to 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, CAR T cells with antibody receptors are administered before, after, or simultaneously with the administration of the antibody, and then bind to the antibody, bringing the T cells into close proximity to the tumor or cancer. In another embodiment, the antibody is directed against a tumor cell antigen. In another embodiment, the antibody is directed to CD 20. In another embodiment, the antibody is rituximab (rituximab).

In another embodiment, the antibody is Trastuzumab (Trastuzumab) (Herceptin (Herceptin); Gene Take (Genentech)): humanized IgG1 against ERBB 2. In another embodiment, the antibody is Bevacizumab (Bevacizumab) (Avastin); gene tach/Roche (Roche)): humanized IgG1 directed against VEGF. In another embodiment, the antibody is Cetuximab (Cetuximab) (Erbitux; Bristol-Myers Squibb): chimeric human-murine IgG1 directed against EGFR. In another embodiment, the antibody is Panitumumab (Panitumumab) (Vectibix); Adam): human IgG2 directed against EGFR. In another embodiment, the antibody is Ipilimumab (Ipilimumab) (yirvoy; bevervay, behcet, precious corporation): IgG1 against CTLA 4.

In another embodiment, the antibody is Alemtuzumab (Alemtuzumab) (capas (Campath); jianzan (Genzyme)): humanized IgG1 against CD 52. In another embodiment, the antibody is Ofatumumab (Ofatumumab) (Azara; Genmab), human IgG1 directed against CD 20. In another embodiment, the antibody is Gemtuzumab ozogamicin (Gemtuzumab ozogamicin) (Mylotarg; Whitman (Wyeth)): humanized IgG4 against CD 33. In another embodiment, the antibody is a Brentuximab vedotin (Brentuximab vedotin) (adtrass (addetris); Seattle Genetics (Seattle Genetics)): chimeric IgG1 against CD 30. In another embodiment, the antibody is 90Y-labeled tematopimozumab (ibritumomab tiuxetan) (Zevalin); IDEC pharmaceutical): murine IgG1 directed against CD 20. In another embodiment, the antibody is 131I-labeled tositumomab (tositumomab) ((Bexxar); GlaxoSmithKline)): murine IgG2 directed against CD 20.

In another embodiment, the antibody is Ramucirumab (Ramucirumab), which is directed against vascular endothelial growth factor receptor 2 (VEGFR-2). In another embodiment, the antibody is ramucirumab (ramucirumab Injection (Cyramza Injection), liensin (Eli Lilly and Company), brinumomab (BLINCYTO, ann), parboluzumab (pembrolizumab) (kezhuda (KEYTRUDA), merchard Sharp & Dohme Corp.), obilizumab (obinutuzumab) (Jiashiwa (GAZYVA), Gentack, formerly known as GA101), pertuzumab (pertuzumab) Injection (parjietat (PERTA), Gentikka), or denomab (denosumab) (Diknoxia, Xgeva, ann). In another embodiment, the antibody is Basiliximab (sumuleximab) (sulley (simulent); nova (Novartis)). In another embodiment, the antibody is Daclizumab (Daclizumab) (zenipax.; Roche).

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

In another embodiment, the CAR T cell-conjugated antibody is directed against a tumor or cancer antigen described herein and/or known in the art, or a fragment thereof.

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

Cytokine storm and cytokine release syndrome

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

In one embodiment, the additional agent for reducing the release of a deleterious cytokine comprises apoptotic cells or a composition comprising said apoptotic cells. In another embodiment, the additional agent for reducing the release of a deleterious cytokine comprises an apoptotic cell supernatant or a composition comprising said supernatant. In another embodiment, the additional agent for reducing the release of an adverse cytokine comprises a CTLA-4 blocking agent. In another embodiment, the additional agent for reducing the release of the deleterious cytokines comprises apoptotic cells or apoptotic cell supernatants or a combination thereof and a CTLA-4 blocking agent. In another embodiment, the additional agent for reducing the release of an adverse cytokine comprises alpha-1 antitrypsin or a fragment or analog thereof. In another embodiment, the additional agent for reducing the release of harmful cytokines comprises apoptotic cells or an apoptotic cell supernatant or a composition thereof and alpha-1 antitrypsin or a fragment or analog thereof. In another embodiment, the additional agent for reducing the release of a deleterious cytokine comprises a tellurium-based compound. In another embodiment, the additional agent for reducing the release of a deleterious cytokine comprises apoptotic cells or apoptotic cell supernatant or a combination thereof and a tellurium-based compound. In another embodiment, the additional agent for reducing the release of an adverse cytokine comprises an immunomodulatory agent. In another embodiment, the additional agent for reducing the release of a deleterious cytokine comprises apoptotic cells or an apoptotic cell supernatant or a composition thereof and an immunomodulatory agent. In another embodiment, the additional agent for reducing the release of harmful cytokines comprises Treg cells. In another embodiment, the additional agent for reducing the release of harmful cytokines comprises apoptotic cells or an apoptotic cell supernatant or a composition thereof and Treg cells.

The skilled person will understand that reducing the release of a toxic cytokine or reducing the level of a toxic cytokine includes reducing the level of a toxic cytokine or inhibiting the production of a toxic cytokine in a subject, or inhibiting the cytokine release syndrome or cytokine storm in a subject or reducing the incidence of said cytokine release syndrome or cytokine storm. In another embodiment, toxic cytokine levels are reduced during CRS or cytokine storm. In another embodiment, reducing the level of toxic cytokines or inhibiting the production of toxic cytokines comprises treating CRS or a cytokine storm. In another embodiment, reducing the level of toxic cytokines or inhibiting the production of toxic cytokines comprises preventing CRS or a cytokine storm. In another embodiment, reducing the level of toxic cytokines or inhibiting the production of toxic cytokines comprises mitigating CRS or a cytokine storm. In another embodiment, reducing the level of a toxic cytokine or inhibiting the production of a toxic cytokine comprises ameliorating CRS or a cytokine storm. In another embodiment, the toxic cytokine comprises a pro-inflammatory cytokine. In another embodiment, the proinflammatory cytokine comprises IL-6. In another embodiment, the proinflammatory cytokine comprises IL-1 β. In another embodiment, the proinflammatory cytokine comprises TNF- α. In another embodiment, the proinflammatory cytokine comprises IL-6, IL-1 β, or TNF- α, or any combination thereof.

In one embodiment, the 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, the inflammatory cytokines include IL-6, IL-1 β, and TNF- α. In another embodiment, the CRS is characterized by an elevated level of IL-6, IL-1 β, or TNF- α, or any combination thereof. In another embodiment, the CRS is characterized by an elevated level of IL-8 or IL-13, or any combination thereof. In another embodiment, the cytokine storm is characterized by an increase in TNF- α, IFN- γ, IL-1 β, IL-2, IL-6, IL-8, IL-10, IL-13, GM-CSF, IL-5, fractalkine (fractalkine), or a combination or subset thereof. In yet another embodiment, IL-6 constitutes a marker for CRS or cytokine storm.

In another example, the increased cytokines in CRS or cytokine storms of humans and mice may include any combination of the cytokines listed in tables 1 and 2 below.

Table 1: increased sets of cytokines in CRS or cytokine storm in humans and/or mice

In some embodiments, the cytokines Flt-3L, a fractal, GM-CSF, IFN- γ, IL-1 β, IL-2R α, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, and IL-13 of Table 1 are considered significant in CRS or cytokine storm. In another example, IFN- α, IFN- β, IL-1, and IL-1 Ra of Table 1 appear to be important in CRS or cytokine storms. In another embodiment, M-CSF is of unknown importance. In another embodiment, any of the cytokines listed in table 1, or a combination thereof, may be used as a marker for CRS or a cytokine storm.

Table 2: increased cytokine sets in CRS or cytokine storm in humans and/or mice

In one embodiment, IL-15, IL-17, IL-18, IL-21, IL-22, IP-10, MCP-1, MIP-1 α, MIP-1 β, and TNF- α of Table 2 are considered significant in CRS or cytokine storm. In another example, IL-27, MCP-3, PGE2, RANTES, TGF- β, TNF- α R1, and MIG of Table 2 appear to be important in CRS or cytokine storm. In another embodiment, IL-23 and IL-25 are of unknown importance. In another embodiment, any of the cytokines listed in table 2, or a combination thereof, may be used as a marker for CRS or a cytokine storm. In another example, the mouse cytokines IL-10, IL-1 β, IL-2, IP-10, IL-4, IL-5, IL-6, IFN α, IL-9, IL-13, IFN- γ, IL-12p70, GM-CSF, TNF- α, MIP-1 β, IL-17A, IL-15/IL-15R, and IL-7 appear to be important in CRS or cytokine storms.

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

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

In another embodiment, the cytokine release syndrome is characterized by any or all of the following symptoms: fever, malaise, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, headache, rash, nausea, vomiting, diarrhea, tachypnea, hypoxemia, cardiovascular tachycardia, broadening of pulse pressure, hypotension, increased cardiac output (early stage), reduced potential cardiac output (late stage), increased D-dimer, hypofibrinogenemia with or without bleeding, azotemia, increased hepatic transaminase, hyperbilirubinemia, headache, change in mental state, confusion, delirium, aphasia with difficulty or marked symptoms, hallucinations, tremor, delusions, dysdiscrimination, seizure, change in gait, 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 the cytokine released by the CAR T cells. In another embodiment, the cytokine storm is characterized by an increase in cytokine release by cells other than CAR T cells.

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

The skilled artisan will appreciate that the characteristic of Cytokine Release Syndrome (CRS) or cytokine storm is estimated to occur days to weeks after triggering CRS or cytokine storm. In one embodiment, the CAR T cell is a CRS or a trigger for a cytokine storm. In another embodiment, the trigger for a CRS or cytokine storm is not a CAR T cell.

In one embodiment, the measure of cytokine level or concentration as an indication of a cytokine storm may be expressed as a fold increase, a percent (%) increase, a net increase, or a rate of change in cytokine level or concentration. In another example, an absolute cytokine level or concentration above a certain level or concentration may indicate that the subject is experiencing or is about to 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 that has not undergone CAR-T cell therapy) may be indicative of a method for inhibiting or reducing the incidence of a cytokine storm in a subject that has undergone CAR T cells.

The skilled person will appreciate that the term "cytokine level" may encompass a measure of concentration, a measure of fold change, a measure of percent (%) 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.

In one embodiment, although it is recognized that cytokine storms are associated with elevations of 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 against cytokine storms. The skilled artisan will appreciate that other cytokines may be used as markers of cytokine storm, such as TNF- α, IB-1 α, IL-8, IL-13, or INF- γ. Further, assay methods for measuring cytokines are well known in the art. The skilled person will appreciate that methods of affecting cytokine storm may similarly affect cytokine release syndrome.

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 predisposed to experiencing cytokine release syndrome or cytokine storm. In another embodiment, the methods disclosed herein reduce or inhibit cytokine production in a subject experiencing cytokine release syndrome or cytokine storm, wherein the production of any cytokine or group of cytokines 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- β 1 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, the production of the cytokine TNF- α is reduced or inhibited. In another embodiment, cytokine IL-6 production, IL-1 β production, or TNF- α production, or any combination thereof, is reduced or inhibited.

In one embodiment, cytokine release syndrome is graded. In another example, level 1 describes a cytokine release syndrome where symptoms are not life threatening and only symptomatic treatment is required, e.g., fever, nausea, fatigue, headache, myalgia, malaise. In another embodiment, grade 2 symptoms require moderate intervention (e.g., oxygen, fluid, or vasopressors for hypotension) and will respond to it. In another embodiment, grade 3 symptoms require active intervention and will respond thereto. In another embodiment, the grade 4 symptom is a life-threatening symptom and requires a ventilator, and the patient exhibits organ toxicity.

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 of the cytokines listed in tables 1 and 2, or a combination thereof. In another embodiment, the cytokine storm is characterized by the release of any cytokine or combination thereof known in the art.

In one embodiment, symptom onset begins minutes to hours after infusion begins. In another embodiment, the symptoms are consistent with peak cytokine levels.

In one embodiment, a method of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing CAR T cell cancer therapy comprises administering a population of apoptotic cells or an apoptotic cell supernatant or a composition thereof. In another embodiment, the apoptotic cell population or apoptotic cell supernatant or composition thereof may contribute to CAR T cell therapy. In another embodiment, the apoptotic cell population or apoptotic cell supernatant or composition thereof may contribute to the inhibition or reduction in the incidence of the CRS or cytokine storm. In another embodiment, the apoptotic cell population or apoptotic cell supernatant or composition thereof may be helpful in treating the CRS or cytokine storm. In another embodiment, the apoptotic cell population or apoptotic cell supernatant or composition thereof may contribute to the prevention of the CRS or cytokine storm. In another embodiment, the apoptotic cell population or apoptotic cell supernatant or composition thereof may contribute to amelioration of the CRS or cytokine storm. In another embodiment, the apoptotic cell population or apoptotic cell supernatant or composition thereof may contribute to the alleviation of the CRS or cytokine storm.

In one embodiment, a method of inhibiting or reducing the incidence of a Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing CAR T cell cancer therapy and administered an apoptotic cell population or an apoptotic cell supernatant or composition thereof comprises administering an additional agent. In another embodiment, the additional agent may contribute to CAR T cell therapy. In another embodiment, the additional agent may contribute to the inhibition of the CRS or cytokine storm or reduce the incidence of the CRS or cytokine storm. In another embodiment, the additional agent may contribute to the treatment of the CRS or cytokine storm. In another embodiment, the additional agent may contribute to the prevention of the CRS or cytokine storm. In another embodiment, the additional agent may contribute to ameliorating the CRS or cytokine storm. In another embodiment, the additional agent may help to alleviate the CRS or cytokine storm.

In one embodiment, a method of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing CAR T cell cancer therapy comprises administering an additional agent. In another embodiment, the additional agent may contribute to CAR T cell therapy. In one embodiment, a method of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing TCR T cell cancer therapy comprises administering an additional agent. In another embodiment, the additional agent may contribute to TCR T cell therapy. In one embodiment, a method of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject experiencing the CRS or the cytokine storm comprises administering an additional agent. In another embodiment, the additional agent may contribute. In one embodiment, a method of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing NK cell therapy comprises administering an additional agent. In another embodiment, the additional agent may contribute to NK cell therapy.

In another embodiment, the additional agent may contribute to inhibiting or reducing the incidence of the CRS or cytokine storm. In another embodiment, the additional agent may contribute to the treatment of the CRS or cytokine storm. In another embodiment, the additional agent may contribute to the prevention of the CRS or cytokine storm. In another embodiment, the additional agent may contribute to ameliorating the CRS or cytokine storm. In another embodiment, the additional agent may help to alleviate the CRS or cytokine storm.

In one embodiment, the additional agent for reducing the release of a deleterious cytokine comprises apoptotic cells or a composition comprising said apoptotic cells. In another embodiment, the additional agent for reducing the release of a deleterious cytokine comprises an apoptotic cell supernatant or a composition comprising said supernatant. In another embodiment, the additional agent for reducing the release of an adverse cytokine comprises a CTLA-4 blocking agent. In another embodiment, the additional agent for reducing the release of the deleterious cytokines comprises apoptotic cells or apoptotic cell supernatants or a combination thereof and a CTLA-4 blocking agent. In another embodiment, the additional agent for reducing the release of an unwanted cytokine comprises alpha-1 antitrypsin or a fragment or analog thereof. In another embodiment, the additional agent for reducing the release of deleterious cytokines comprises apoptotic cells or an apoptotic cell supernatant or a composition thereof and alpha-1 antitrypsin or a fragment or analog thereof. In another embodiment, the additional agent for reducing the release of an adverse cytokine comprises a tellurium-based compound. In another embodiment, the additional agent for reducing the release of a deleterious cytokine comprises an apoptotic cell or an apoptotic cell supernatant or a combination thereof and a tellurium-based compound. In another embodiment, the additional agent for reducing the release of an adverse cytokine comprises an immunomodulatory agent. In another embodiment, the additional agent for reducing the release of a deleterious cytokine comprises apoptotic cells or an apoptotic cell supernatant or a composition thereof and an immunomodulatory agent.

In another embodiment, the compositions and methods disclosed herein utilize a combination therapy of CAR T cells with one or more CTLA-4 blockers (e.g., ipilimumab). In another embodiment, the compositions and methods disclosed herein utilize a combination therapy comprising 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 with one or more CTLA-4 blockers (e.g., ipilimumab). In another embodiment, the compositions and methods disclosed herein utilize a combination therapy comprising 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 with one or more CTLA-4 blocking agents (e.g., ipilimumab). In another embodiment, the compositions and methods disclosed herein utilize a combination therapy comprising apoptotic cells, dendritic cells, and one or more CTLA-4 blockers. In another embodiment, the compositions and methods disclosed herein utilize NK cells in combination therapy with one or more CTLA-4 blockers (e.g., ipilimumab). In another embodiment, the compositions and methods disclosed herein utilize a combination therapy comprising apoptotic cells, NK cells, and one or more CTLA-4 blockers.

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 blocking agent (which in another embodiment is an antibody) results in a net effect of T cell activation.

In another embodiment, other toxicities arising from CAR T cell, TCR T cell, dendritic cell or NK cell administration that may be treated, prevented, inhibited, ameliorated, reduced in incidence or alleviated by the compositions and methods disclosed herein include B cell regeneration disorders or Tumor Lysis Syndrome (TLS).

In one embodiment, the method of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing CAR T cell cancer therapy does not affect the efficacy of the CAR T cell therapy. In another embodiment, the method of inhibiting CRS or a cytokine storm or reducing the incidence of CRS or a cytokine storm in a subject undergoing a CAR T cell cancer therapy reduces the efficacy of the CAR T cell therapy by more than about 5%. In another embodiment, the method of inhibiting CRS or a cytokine storm or reducing the incidence of CRS or a cytokine storm in a subject undergoing a CAR T cell cancer therapy reduces the efficacy of the CAR T cell therapy by more than about 10%. In another embodiment, the method of inhibiting CRS or a cytokine storm or reducing the incidence of CRS or a cytokine storm in a subject undergoing a CAR T cell cancer therapy reduces the efficacy of the CAR T cell therapy by more than about 15%. In another embodiment, the method of inhibiting CRS or a cytokine storm or reducing the incidence of CRS or a cytokine storm in a subject undergoing a CAR T cell cancer therapy reduces the efficacy of the CAR T cell therapy by more than about 20%. In another embodiment, the method of inhibiting CRS or a cytokine storm or reducing the incidence of CRS or a cytokine storm in a subject undergoing a CAR T cell cancer therapy increases the efficacy of the CAR T cell therapy by more than about 5%. In another embodiment, the method of inhibiting CRS or a cytokine storm or reducing the incidence of CRS or a cytokine storm in a subject undergoing a CAR T cell cancer therapy increases the efficacy of the CAR T cell therapy by more than about 10%. In another embodiment, the method of inhibiting CRS or a cytokine storm or reducing the incidence of CRS or a cytokine storm in a subject undergoing a CAR T cell cancer therapy increases the efficacy of the CAR T cell therapy by more than about 15%. In another embodiment, the method of inhibiting CRS or a cytokine storm or reducing the incidence of CRS or a cytokine storm in a subject undergoing a CAR T cell cancer therapy increases the efficacy of the CAR T cell therapy by more than about 20%.

Any suitable method of quantifying cytotoxicity can be used to determine whether activity in immune cells modified to express the CAR remains substantially unchanged. For example, cell culture-based assays (e.g., cytotoxicity assays as described in the examples) can be used to quantify cytotoxicity. The cytotoxicity assay may use a dye that preferentially stains dead cell DNA. In other cases, fluorescence and luminescence assays that measure 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 viable 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.

In another embodiment, the measure of cytotoxicity may be qualitative. In another embodiment, the measure of cytotoxicity can be quantitative. In another example, the measure of cytotoxicity can be related to a change in the expression of a cytotoxic cytokine. In another example, a measure of cytotoxicity can be determined by a survival curve and tumor burden in bone marrow and liver.

In one embodiment, the methods disclosed herein include additional steps that can be used to overcome the rejection of allogeneic donor cells. In one embodiment, the method comprises the step of performing a complete or partial lymphodepletion prior to administration of the CAR T cells (which in one embodiment are allogeneic CAR T cells). In another embodiment, lymphocyte clearance is adjusted such that it delays the host versus graft response for a period of time sufficient for the allogeneic T cells to attack the tumor to which they are directed, but somewhat insufficient to require rescue of the host immune system by bone marrow transplantation. In another embodiment, the agent that delays egress of allogeneic T cells from the 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-l]1,2,4-

Oxadiazole (SEW2871), 3- (2- (-hexylphenylamino) -2-oxyethylamino) propionic acid (W123), 2-ammonio-4- (2-chloro-4- (3-phenoxyphenylthio) phenyl) -2- (hydroxymethyl) butyl biphosphate (KRP-203 phosphate), or other agents known in the art) can be used as part of the compositions and methods disclosed herein to allow for the use of allogeneic CAR-T cells that are potent and do not cause graft-versus-host disease. In one embodiment, MHC expression by the allogeneic T cells is silenced to reduce rejection of the allogeneic cells. In another embodiment, the apoptotic cells prevent rejection of allogeneic cells.

Cytokine release associated with CAR T cell therapy

In one embodiment, cytokine release occurs between days and 2 weeks after administration of immunotherapy (such as CAR T cell therapy). In one embodiment, hypotension and other symptoms follow cytokine release, i.e., from days to weeks. Thus, in one embodiment, apoptotic cells or an apoptotic cell supernatant are administered to a subject as prophylaxis concurrently with immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to subjects 2-3 days after administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to subjects 7 days after administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to subjects 10 days after administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 14 days after administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 2-14 days after administration of immunotherapy.

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

In alternative embodiments, apoptotic cells or apoptotic cell supernatants are administered to a subject as prophylaxis prior to immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 1 day prior to administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 2-3 days prior to administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 7 days prior to administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 10 days prior to administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 14 days prior to administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 2-14 days prior to administration of immunotherapy.

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

In another embodiment, apoptotic cells or apoptotic cell supernatant may be administered therapeutically once cytokine release syndrome has occurred. In one embodiment, apoptotic cells or supernatant may be administered once cytokine release is detected to cause or evidence the onset of cytokine release syndrome. In one embodiment, apoptotic cells or supernatant may cause an increase in cytokine levels or the end of cytokine release syndrome or avoid its sequelae.

In another example, apoptotic cells or apoptotic cell supernatant may be therapeutically administered at multiple time points. In another embodiment, apoptotic cells or apoptotic cell supernatant are administered at least two time points described herein. In another embodiment, apoptotic cells or apoptotic cell supernatant are administered at least three time points described herein. In another embodiment, apoptotic cells or apoptotic cell supernatant are administered prior to CRS or a cytokine storm, and once cytokine release syndrome has occurred and any combination thereof.

In one embodiment, T cell (CAR T cell) therapy expressing a chimeric antigen receptor and apoptotic cell therapy or supernatant are administered together. 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 prior to apoptotic cell therapy or supernatant. According to this aspect, and in one embodiment, apoptotic cell therapy or supernatant is administered about 2-3 weeks after the CAR T cell therapy. In another embodiment, the apoptotic cell therapy or supernatant is administered about 6-7 weeks after the CAR T cell therapy. In another embodiment, the apoptotic cell therapy or supernatant is administered about 9 weeks after the CAR T cell therapy. In another embodiment, apoptotic cell therapy is administered up to several months after the CAR T cell therapy.

Thus, in one embodiment, apoptotic cells or an apoptotic cell supernatant are administered to a subject as prophylaxis concurrently with immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 2-3 days after administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to subjects 7 days after administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 10 days after administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 14 days after administration of immunotherapy. In another embodiment, apoptotic cells or supernatant are administered to the subject 2-14 days after administration of immunotherapy.

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

In other embodiments, the additional agent is administered to the subject concurrently with immunotherapy as prophylaxis. In one embodiment, the additional agent comprises apoptotic cells, apoptotic cell supernatant, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a telluril-based compound, or an immunomodulatory compound, or any combination thereof. In another embodiment, the additional agent is administered to the subject 2-3 days after administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 7 days after administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 10 days after administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 14 days after administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 2-14 days after administration of the immunotherapy.

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

In alternative embodiments, the subject is administered an additional agent as prophylaxis prior to immunotherapy. In another embodiment, the additional agent is administered to the subject 1 day prior to administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 2-3 days prior to administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 7 days prior to administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 10 days prior to administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 14 days prior to administration of the immunotherapy. In another embodiment, the additional agent is administered to the subject 2-14 days prior to administration of the immunotherapy.

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

In another embodiment, once cytokine release syndrome has occurred, the additional agent is administered therapeutically. In one embodiment, the additional agent is administered once cytokine release is detected to cause or evidence the onset of cytokine release syndrome. In one embodiment, the additional agent may increase cytokine levels or end or avoid sequelae of cytokine release syndrome.

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

In one embodiment, a T cell expressing a chimeric antigen receptor (CAR T cell) therapy is administered with an additional agent. In another embodiment, the CAR T cell therapy is administered to an additional agent. In another embodiment, the CAR T cell therapy is administered prior to the additional agent. According to this aspect, and in one embodiment, the additional agent is administered about 2-3 weeks after the CAR T cell therapy. In another embodiment, the additional agent is administered about 6-7 weeks after the CAR T cell therapy. In another embodiment, the additional agent is administered about 9 weeks after the CAR T cell therapy. In another embodiment, the additional agent is administered up to several months after the CAR T cell therapy.

In one embodiment, the CAR T cell is 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 cell is a universal allogeneic CAR T cell. In another embodiment, the CAR T cell is a syngeneic CAR T cell. In another embodiment, the CAR T cell is from a mismatched third party donor. 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 comprise one or more MHC non-restricted tumor-targeted chimeric receptors. In one example, 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.

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

In one embodiment, the apoptotic cells are heterologous 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, 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 a mismatched third party donor. In one embodiment, the apoptotic cells are universal allogeneic apoptotic cells. In another embodiment, the apoptotic cells are from syngeneic donors. 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 example, the patient's own cells are used.

According to some embodiments, the enriched therapeutic monocyte preparation or apoptotic cell supernatant disclosed herein is administered systemically to the subject. In another embodiment, administration is by intravenous route. Alternatively, the enriched therapeutic monocytes or supernatant may be administered to the subject according to various other routes, including but not limited to parenteral, intraperitoneal, intraarticular, intramuscular, and subcutaneous routes.

According to some embodiments, the enriched therapeutic monocyte preparation or additional agent disclosed herein is administered systemically to the subject. In another embodiment, administration is by intravenous route. Alternatively, the enriched therapeutic monocytes or additional agents may be administered to the subject according to various other routes, including but not limited to parenteral, intraperitoneal, intra-articular, intramuscular, and subcutaneous routes.

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 region comprises a tumor or cancer.

In another embodiment, the enriched therapeutic monocytes or supernatant are administered to the 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 supplements that help maintain cell viability. In another embodiment, the additional agent is administered to the subject suspended in a suitable physiological buffer (such as, but not limited to, saline solution, PBS, HBSS, etc.).

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 a plurality of 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.

In one embodiment, any suitable method of providing a modified CAR-expressing immune cell to a subject can be used in the methods described herein. In one embodiment, the method for providing cells to a subject comprises Hematopoietic Cell Transplantation (HCT), infusion of donor source NK cells into a cancer patient, or a combination thereof.

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

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

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

In certain embodiments, the CAR T cell therapy comprises administering a composition disclosed herein comprising a CAR T cell and an apoptotic cell or an apoptotic cell supernatant or another additional agent disclosed herein or a combination thereof. In alternative embodiments, the CAR T cell therapy comprises administering a composition comprising a CAR T cell disclosed herein and a composition comprising an apoptotic cell or an apoptotic cell supernatant or an additional agent disclosed herein or a combination thereof.

Cytokine release associated with non-CAR T cell applications

In one 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 apoptotic cells or an 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 experiencing cytokine release syndrome or cytokine storm or susceptible to cytokine release syndrome or cytokine storm and not administered apoptotic cells or an apoptotic supernatant. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits pro-inflammatory cytokine production. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits the production of at least one pro-inflammatory cytokine. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits at least the production of cytokine IL-6. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits the production of at least the cytokine IL-1 β. In another embodiment, the method for reducing or inhibiting cytokine production reduces or inhibits the production of at least the cytokine TNF- α. In another embodiment, the methods disclosed herein for reducing or inhibiting cytokine production result in reduced or inhibited production of the cytokine IL-6, IL-1 β, or TNF- α, or any combination, in the subject as compared to a subject experiencing cytokine release syndrome or cytokine storm or being vulnerable to cytokine release syndrome or cytokine storm and not being administered apoptotic cells or an apoptotic supernatant.

Cancer or tumors can also affect the absolute levels of cytokines including pro-inflammatory cytokines. The level of tumor burden in a subject may affect cytokine levels, particularly proinflammatory cytokines. The skilled artisan will appreciate that the phrase "reduce or inhibit" or grammatical variants thereof can encompass a fold reduction or inhibition of cytokine production, or a net reduction or inhibition or percent (%) reduction or inhibition of cytokine production, or can encompass a rate of change of reduction or inhibition of cytokine production.

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, comprising the step of administering to the subject apoptotic cells or a composition comprising the apoptotic cells.

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, 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.

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

In one embodiment, the infection causes a cytokine release syndrome or a cytokine storm in the subject. In one embodiment, the infection is influenza infection. In one embodiment, the influenza infection is H1N 1. 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 has hemophagocytic lymphohistiocytosis associated with epstein barr virus (HLH). 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.

In one embodiment, the cause of the cytokine release syndrome or cytokine storm in the subject is Hemophagocytic Lymphohistiocytosis (HLH). In another embodiment, the HLH is episodic HLH. In another embodiment, the HLH is Macrophage Activation Syndrome (MAS). In another embodiment, the cause of cytokine release syndrome or cytokine storm in the subject is MAS.

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

In one embodiment, the cytokine release syndrome or cytokine storm in the subject is due to cold imidacloprid (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 comprises chronic infantile neurocutaneous joint (CINCA) syndrome. In yet another embodiment, CAPS comprises FCAS, FCU, MWS, or CINCA syndrome, or any combination thereof. In another embodiment, the subject's cytokine release syndrome or cytokine storm is due to FCAS. In another embodiment, the subject's cytokine release syndrome or cytokine storm is due to FCU. In another embodiment, the cause of the cytokine release syndrome or cytokine storm in the subject is MWS. In another embodiment, the cytokine release syndrome or cytokine storm in the subject is due to CINCA syndrome. In yet another embodiment, the cytokine release syndrome or cytokine storm in the subject is due to FCAS, FCU, MWS, or CINCA syndrome, or any combination thereof.

In another embodiment, the cause of the cytokine release syndrome or cytokine storm in the subject is cryopyridoxine disease (cryopyridopathy), which includes a genetic or functionally-recovering mutation in the NLRP3 gene (also known as the CIASI gene).

In one embodiment, the cause of the cytokine release syndrome or cytokine storm in the subject is an inherited autoinflammatory disorder.

In one embodiment, the trigger for inflammatory cytokine release is modulation of Lipopolysaccharide (LPS), gram-positive toxin, mycotoxin, Glycosylphosphatidylinositol (GPI), or RIG-1 gene expression.

In another embodiment, the subject experiencing cytokine release syndrome or cytokine storm does not have an infectious disease. In one embodiment, the subject has acute pancreatitis. In another embodiment, the subject has tissue damage, which in one embodiment is a severe burn or wound. In another embodiment, the subject has acute respiratory distress syndrome. In another embodiment, the subject has a cytokine release syndrome or cytokine storm secondary to drug use. In another embodiment, the subject has a cytokine release syndrome or cytokine storm secondary to toxin inhalation.

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 superagonic CD 28-specific monoclonal antibody (CD28 SA). In one embodiment, the CD28SA is TGN 1412. In another embodiment, the immunotherapy is CAR T cell therapy.

In another embodiment, apoptotic cells or supernatant or CTLA-4 blocking agent, alpha-1 antitrypsin or fragment thereof or analog thereof, telluril-based compound or immunomodulator or any combination thereof may be used to control cytokine release syndrome or cytokine storm due to administration of the pharmaceutical composition. In one embodiment, the pharmaceutical composition is oxaliplatin (oxaliplatin), cytarabine (cytarabine), lenalidomide (lenalidomide), or a combination thereof.

In another embodiment, apoptotic cells or supernatant or CTLA-4 blocking agent, alpha-1 antitrypsin or fragments or analogs thereof, tellurium-based compounds or immunomodulators or any combination thereof may be used to control cytokine release syndrome or cytokine storm due to 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 (molobumab-CD 3). In another embodiment, the antibody is alemtuzumab, toltuzumab, CP-870,893, LO-CD2a/BTI-322, or TGN 1412.

In another example, examples of diseases in which control of inflammatory cytokine production may be beneficial include cancer, allergy, any type of infection, toxic shock syndrome, sepsis, any type of autoimmune disease, arthritis, Crohn's disease, lupus, psoriasis, or any disease characterized by release of toxic cytokines that produce a deleterious effect in a subject.

Alpha-1-antitrypsin (AAT)

Alpha-1-antitrypsin (AAT) is a circulating 52-kDa glycoprotein produced primarily by the liver. AAT is mainly called serpin and is encoded by the gene SERPINA 1. AAT inhibits neutrophil elastase, and genetic defects in circulating AAT can lead to deterioration of lung tissue and liver disease. During inflammation, serum AAT concentrations in healthy individuals increase by a factor of two.

There is a negative 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.

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.

Indeed, the addition of AAT to human Peripheral Blood Mononuclear Cells (PBMC) inhibits LPS-induced TNF- α and IL-1 β release, but increases IL-1 receptor antagonist (IL-1Ra) and IL-10 production.

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

Monotherapy with clinical grade human aat (haat) reduces circulating proinflammatory cytokines, reduces the severity of graft versus host disease (GvHD) and prolongs animal survival following experimental allogeneic bone marrow transplantation (Tawara et al, Proc Natl Acad Sci U S a.2012jan 10; 109(2):564-9), incorporated herein by reference. AAT treatment reduces the expansion of alloreactive T effector cells but enhances the recovery of T regulatory T cells (tregs), thereby altering the ratio of donor T effector cells to T regulatory cells, which is beneficial in reducing the pathological process. In vitro, AAT suppresses LPS-induced in vitro secretion of proinflammatory cytokines (e.g., TNF- α and IL-1 β), enhances production of the anti-inflammatory cytokine IL-10 and impairs NF- κ B translocation in host dendritic cells. Marcondes, blood.2014(Oct 30; 124(18):2881-91) showed that treatment with AAT not only improved GvHD, but also retained and possibly even enhanced graft-versus-leukemia (GVL) effects, which are incorporated herein by reference.

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

In another embodiment, disclosed herein is a method of treating, preventing, inhibiting, or reducing the incidence, ameliorating, or alleviating a cancer or tumor in a subject, the method comprising the step of 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 an apoptotic cell supernatant.

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

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, comprising the step of administering to the subject a composition comprising alpha-1-antitrypsin (AAT).

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.

Immune modulator

It will be understood by those skilled in the art that immune modulators may encompass extracellular mediators, receptors, mediators of intracellular signaling pathways, modulators of translation and transcription, and immune cells. In one embodiment, the additional agents disclosed herein are immunomodulatory agents known in the art. In another embodiment, the use of an immunomodulatory agent in the methods disclosed herein reduces the level of at least one cytokine. In another embodiment, CRS or a cytokine storm is reduced or suppressed using an immunomodulatory agent in the methods disclosed herein. In some embodiments, an immunomodulatory agent is used in a method disclosed herein to treat, prevent, inhibit growth, delay progression, reduce tumor burden, or reduce the incidence of a tumor or cancer, or any combination thereof. In some embodiments, an immunomodulator is used in combination with another composition disclosed herein (e.g., without limitation, a composition comprising early apoptotic cells or comprising CAR T cells).

In one embodiment, the immunomodulator comprises a compound that blocks, inhibits or reduces cytokine or chemokine release. In another embodiment, the immunomodulator comprises a compound that blocks, inhibits or reduces the release of IL-21 or IL-23 or a combination thereof. In another embodiment, the immunomodulator comprises an antiretroviral drug from the chemokine receptor-5 (CCR5) receptor antagonist class, such as maraviroc (maraviroc). In another embodiment, the immunomodulator comprises an anti-DNAM-1 antibody. In another embodiment, the immunomodulator comprises an injury/pathogen associated molecule (DAMP/PAMP) selected from heparin sulfate, ATP, and uric acid, or any combination thereof. In another embodiment, the immunomodulator comprises a sialic acid-binding Ig-like lectin (Siglecs). In another embodiment, the immunomodulatory agent comprises a tolerogenic cellular mediator, such as regulatory CD4+ CD25+ T cells (tregs) or constant natural killer T cells (inkt cells). In another embodiment, the immunomodulator comprises a dendritic cell. In another embodiment, the immunomodulator comprises a monocyte. In another embodiment, the immunomodulator comprises a macrophage. In another embodiment, the immunomodulatory agent comprises a JAK2 or JAK3 inhibitor selected from ruxolitinib and tofacitinib. In another embodiment, the immunomodulatory agent comprises an inhibitor of spleen tyrosine kinase (Syk), such as fostamatinib. In another embodiment, the immunomodulator comprises histone deacetylase inhibitor vorinostat (vorinostat) acetylated STAT 3. In another embodiment, the immune modulator comprises an ubiquitination (neddylation) inhibitor, such as MLN 4924. In another embodiment, the immunomodulator comprises a miR-142 antagonist. In another embodiment, the immunomodulator comprises a chemical analog of cytidine, such as Azacitidine (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).

Tellurium-based compound

Tellurium is a trace element found in the human body. Various tellurium compounds have immunomodulatory properties and have been shown to have beneficial effects in various preclinical and clinical studies. In, for example, U.S. patent nos. 4,752,614; 4,761,490, No. 4,761,490; a particularly effective family of tellurium-containing compounds is disclosed in 4,764,461 and 4,929,739. The immunomodulatory properties of this family of tellurium-containing compounds are described, for example, in U.S. patent nos. 4,962,207, 5,093,135, 5,102,908 and 5,213,899, all incorporated by reference as if fully set forth herein.

One promising compound is ammonium trichloro (dioxyethylene-O, O') tellurate, which is also referred to herein and in the art AS 101. As representative examples of the tellurium-containing compound family discussed above, AS101 exhibits antiviral activity (Nature. 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.

In one embodiment, compositions comprising tellurium-containing immunomodulator compounds, wherein the tellurium-based compounds stimulate the innate and acquired part of the immune response (arm) can be used in the methods disclosed herein. For example, AS101 has been shown to be effective activators of Interferon (IFN) in mice (J.Natl.cancer Inst.88(18): 1276. sup. 84. 1996) and in humans (nat.Immun.cell Growth Regul.9(3): 182. sup. 90. 1990; Immunology 70(4): 473. sup. 7. 1990; J.Natl.cancer Inst.88(18): 1276. sup. 84. sup. 1996).

In another embodiment, the tellurium-based compound induces the secretion of a range of cytokines (e.g., IL-1 α, IL-6, and TNF- α).

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

In one embodiment, the tellurium-based compound inhibits secretion of at least one cytokine. In another embodiment, the tellurium-based compound reduces secretion of at least one cytokine. In another embodiment, the tellurium-based compound inhibits or reduces Cytokine Release Syndrome (CRS) of a cytokine storm.

In one embodiment, disclosed herein are compositions comprising a T cell expressing a chimeric antigen receptor (CAR T cell) and a tellurium-based compound. In another embodiment, the CAR T cell and the tellurium-based compound are in separate compositions. In another embodiment, the AAT comprises a full-length AAT or a functional fragment thereof. In another embodiment, AA includes analogs of full-length AAT or functional fragments thereof.

In another embodiment, disclosed herein is a method of treating, preventing, inhibiting, or reducing the incidence of, ameliorating, or alleviating a cancer or tumor in a subject, comprising the step of administering to the subject a chimeric antigen receptor-expressing T cell (CAR T cell) and a composition comprising a tellurium-based compound.

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

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, comprising the step of administering to the subject a composition comprising a tellurium-based compound.

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

Dendritic cell

In one embodiment, Dendritic Cells (DCs) are antigen producing and presenting cells of the mammalian immune system that process and present antigenic material to T cells of the immune system on the cell surface and thereby enable sensitivity of T cells to both neo-and recall antigens. In another example, DCs are the most efficient antigen producing cells that act as messengers between the innate and adaptive immune systems. In one embodiment, DC cells can be used to elicit specific anti-tumor immunity by generating effector cells that attack and lyse the tumor.

Dendritic cells are present in those tissues in contact with the external environment, such as the skin (where there is a specialized dendritic cell type known as Langerhans cells) and the lining of the nose, lungs, stomach, and intestines. They can also be found in an immature state in the blood. Once activated, they migrate to lymph nodes where they interact with T cells and B cells to initiate and shape adaptive immune responses. At some developmental stage they will grow branched processes, i.e. dendrites giving the cell its name. Dendritic cells can be engineered to express specific tumor antigens.

The three signals required for T cell activation are: (i) presentation of homologous antigens in self MHC molecules; (ii) co-stimulation of membrane-bound receptor-ligand pairs; and (iii) direct depolarization of soluble factors with consequent immune responses. Dendritic Cells (DCs) are able to provide all three signals required for T cell activation, making them an excellent cancer vaccine platform.

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

In another embodiment, disclosed herein is a method of treating, preventing, inhibiting, reducing the incidence, ameliorating or alleviating a cancer or tumor in a subject, the method comprising the step of administering to the subject dendritic cells and a composition comprising an additional agent, wherein the agent comprises apoptotic cells, an apoptotic supernatant, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a telluro-based compound or an immunomodulator, or any combination thereof.

Genetic modification

In some embodiments, genetic modification of T cells, dendritic cells, and/or apoptotic cells may be accomplished using RNA, DNA, recombinant viruses, or a combination thereof. In some embodiments, vectors derived from gammaretrovirus or lentivirus are used in the compositions and methods disclosed herein. In another example, these vectors may be integrated into a host genome with potentially permanent expression of the transgene and have low innate immunogenicity. In another embodiment, another vector that is integrated into the host genome and/or has low innate immunogenicity may be used in the compositions and methods disclosed herein. In another embodiment, a non-viral vector mediated sleeping beauty (sleeping beauty) transposon system is used to insert the CAR and other genes into the T cell. In another embodiment, a "suicide gene" is integrated into a T cell, wherein expression of the pro-apoptotic gene is under the control of an inducible promoter responsive to systemically delivered drugs.

In some embodiments, the genetic modification may be transient. In another example, the genetic modification may utilize messenger rna (mrna). In another example, a large number of cells can be infused multiple times onto transiently engineered T cells (e.g., mRNA transfected T cells). In another embodiment, RNA-based electroporation of lymphocytes with in vitro transcribed mRNA mediates transient expression of proteins for about one week and avoids the risk of integrating viral vectors. In another embodiment, mRNA transduced dendritic cells or mRNA electroporated T and NK lymphocytes.

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.

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

Apoptotic cells

The generation of apoptotic cells ("ApoCell") for use 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, apoptotic cells for use in the compositions and methods disclosed herein are autologous to the subject undergoing therapy. In another embodiment, apoptotic cells for use in the compositions and methods disclosed herein are allogeneic to the subject undergoing therapy. In another embodiment, a composition comprising apoptotic cells comprises apoptotic cells as disclosed herein or known in the art.

The skilled artisan will appreciate that the term "autologous" may encompass tissues, cells, nucleic acid molecules, or polypeptides in which the donor and recipient are the same person.

The skilled artisan will appreciate that the term "allogeneic" may encompass tissues, cells, nucleic acid molecules, or polypeptides derived from a single individual of the same species. In some embodiments, the allogeneic donor cell is genetically different from the recipient.

In some embodiments, obtaining an enriched monocyte composition according to the production methods disclosed herein is accomplished by leukapheresis. The skilled person will appreciate that the term "leukapheresis" may encompass apheresis procedures in which leukocytes are separated from the donor's blood. In some embodiments, the donor's blood is subjected to leukopheresis and thus an enriched monocyte composition is obtained according to the production methods disclosed herein. It should be noted that at least one anticoagulant is required during leukopheresis, as is known in the art, in order to prevent coagulation of the collected cells.

In some embodiments, the leukapheresis procedure is configured to allow collection of the enriched monocyte composition according to the production methods disclosed herein. In some embodiments, the collection of cells obtained by leukopheresis comprises at least 65%. In other embodiments, at least 70% or at least 80% monocytes as disclosed herein. In some embodiments, in the production methods disclosed herein, plasma from the cell donor is collected in parallel to obtain the enriched monocyte composition. In some embodiments, about 300-600ml of plasma from a cell donor is collected in parallel to obtain an enriched monocyte composition according to the production methods disclosed herein. In some embodiments, plasma collected in parallel to obtain an enriched monocyte composition according to the production methods disclosed herein is used as part of the freezing and/or incubation media. Additional details of methods for obtaining enriched apoptotic cell populations for use in the compositions and methods disclosed herein may be found in WO 2014/087408, which is incorporated herein by reference in its entirety.

In some embodiments, early apoptotic cells for use in the methods disclosed herein comprise at least 85% monocytes. In further embodiments, the early apoptotic cells for use in the methods disclosed herein comprise at least 85% monocytes, 90% monocytes or alternatively more than 90% monocytes. In some embodiments, early apoptotic cells for use in the methods disclosed herein comprise at least 90% monocytes. In some embodiments, early apoptotic cells for use in the methods disclosed herein comprise at least 95% monocytes.

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

In certain embodiments, the enriched monocyte preparation used to produce the composition of early apoptotic cells for use in the methods disclosed herein comprises at least 50% monocytes at the time of cell collection. In certain embodiments, disclosed herein are methods for generating a drug population, wherein the methods comprise obtaining an enriched monocyte preparation from peripheral blood of a donor, the enriched monocyte preparation comprising at least 50% monocytes. In certain embodiments, disclosed herein are methods for producing a drug population, wherein the methods comprise freezing an enriched monocyte preparation comprising at least 50% monocytes.

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^{High (a)}An expression cell.

The skilled person will appreciate that the term "early apoptotic state" may encompass cells that show early signs of apoptosis without late signs of apoptosis. Examples of early signs of apoptosis include Phosphatidylserine (PS) exposure and mitochondrial membrane potential loss. Examples of late events include Propidium Iodide (PI) entry into the cell and eventual DNA cleavage. To demonstrate that cells are in An "early apoptotic" state, in some embodiments, PS exposure detection by annexin-V and PI staining is used, and cells stained by annexin V but not PI or only minimally PI are considered "early apoptotic cells" (An)⁺PI^-). In some embodiments, minimal IP staining comprises less than or equal to (≦) 15% PI + cells within the cell population. In some embodiments, minimal IP staining comprises less than or equal to (≦) 10% PI + cells within the cell population. In some embodiments, minimal IP staining comprises less than or equal to (≦) 5% PI + cells within the cell population. In another example, cells stained with both annexin-V FITC and high PI are considered "late apoptotic cells". In some embodiments, high IP staining comprises greater than: (a) within a population of cells >) 15% of PI + cells. In some embodiments, the high IP staining comprises greater than or equal to (≧) 16% PI + cells within the cell population. In another embodiment, cells that are not stained with annexin-V or PI are considered non-apoptotic, viable cells.

In some embodiments, at least 40% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 45% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 50% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 55% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 60% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 65% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 70% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 75% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 80% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 85% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 90% of the cells in the formulation are in an early apoptotic state. In some embodiments, at least 95% of the cells in the formulation are in an early apoptotic state.

In some embodiments, the early apoptotic cell preparation comprises less than or equal to (≦) 15% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 10% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 9% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 8% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 7% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 6% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 5% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 4% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 3% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 2% PI⁺A cell. In some embodiments, the preparation of early apoptotic cells comprises ≦ 1% PI⁺A cell.

In some embodiments, at least 40% of the cells in the preparation are in An early apoptotic state (An)⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI ⁺. In some embodiments, at least 45% of the cells in the formulation are in an early apoptotic state(An⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 50% of the cells in the preparation are in An early apoptotic state (An)⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 55% of the cells in the preparation are in An early apoptotic state (An)⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 60% of the cells in the preparation are in An early apoptotic state (An)⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 65% of the cells in the preparation are in An early apoptotic state (An)⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 70% of the cells in the preparation are in An early apoptotic state (An) ⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 75% of the cells in the preparation are in An early apoptotic state (An)⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 80% of the cells in the formulation are in An early apoptotic state (An)⁺) Wherein less than or equal to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 85% of the cells in the formulation are in An early apoptotic state (An)⁺) Wherein<15% or less than 14%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 90% of the cells in the preparation are in An early apoptotic state (An)⁺) Wherein<10% or less than or equal to 9%, 8%, 7%, 6%, 5%, 4%, 3%% 2%, 1% or 0% of the cells are PI⁺. In some embodiments, at least 95% of the cells in the preparation are in An early apoptotic state (An)⁺) Wherein<5%, < 4%, 3%, 2%, 1% or 0% of the cells are PI ⁺。

The skilled artisan will appreciate that, in some embodiments, the terms "apoptotic cell," "early apoptotic cell," "Allocetra," "Autocetra," "ALC," and "ApoCell," and grammatical variants thereof, may be used interchangeably to represent a population of "early apoptotic cells," wherein the population of cells is enriched for monocytes and has unique properties (see, e.g., example 1). The skilled artisan will appreciate that, in some embodiments, the compositions and methods described herein include early apoptotic cells.

In some embodiments, allocenta comprises a population of early apoptotic cells obtained from a single allogeneic donor. In some embodiments, allocenta comprises a population of early apoptotic cells obtained from multiple allogeneic donors. In some embodiments, allocenta comprises a pooled population of early apoptotic cells obtained from multiple allogeneic donors or cells obtained from a blood bank. In some embodiments, allocenta comprises a pooled population of early apoptotic cells obtained from the same allogeneic donor. In some embodiments, Allocetra comprises an irradiated population of early apoptotic cells. In some embodiments, the term "allocenta" may be used interchangeably with the term "allocenta-OTS". In some embodiments, the terms "Allocetra" and "Allocetra-OTS" encompass mononuclear early apoptotic cells, prepared as described in example 1, regardless of the source of the cells.

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

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

In some embodiments, the early apoptotic cells are stable. The skilled artisan will appreciate that in some embodiments, stability encompasses maintaining early apoptotic cell characteristics over time, e.g., maintaining early apoptotic cell characteristics upon storage at about 2-8 ℃. In some embodiments, stability comprises maintaining early apoptotic cell characteristics upon storage at freezing temperatures (e.g., temperatures at or below 0 ℃).

In some embodiments, the enriched monocyte population obtained according to the methods for generation of early apoptotic cells for use in 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 frozen. In some embodiments, the cell preparation is subjected to at least one washing step in a washing medium after cell collection and before freezing.

As used herein, the terms "obtaining cells" and "cell collection" are 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 up to 6 hours of cell collection. 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 for up to 8 hours, 12 hours, 24 hours, 48 hours, 72 hours of collection. In other embodiments, after collection, the cells are maintained at 2-8 ℃ until frozen.

In some embodiments, freezing based on the generation of the early apoptotic cell population comprises: the cell preparation is frozen at about-18 ℃ to-25 ℃, then the cell preparation is frozen at about-80 ℃, and finally the cell preparation is frozen in liquid nitrogen until thawing. In some embodiments, freezing based on the generation of the early apoptotic cell population comprises: the cell preparation is frozen at about-18 ℃ to-25 ℃ for at least 2 hours, then the cell preparation is frozen at about-80 ℃ for at least 2 hours, and finally the cell preparation is frozen in liquid nitrogen until thawing. In some embodiments, the cells are maintained in liquid nitrogen for at least 8 hours, 10 hours, or 12 hours prior to thawing. In some embodiments, the cells of the cell preparation are maintained in liquid nitrogen until thawed and incubated with an apoptosis-inducing incubation medium. In some embodiments, the cells of the cell preparation are maintained in liquid nitrogen until the day of hematopoietic stem cell transplantation. In a non-limiting example, the time from cell collection and freezing to preparation of the final population 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.

In some embodiments, freezing based on the generation of the early apoptotic cell population comprises freezing the cell preparation at about-18 ℃ to-25 ℃ for at least 0.5 hour, 1 hour, 2 hours, 4 hours. In some embodiments, freezing based on the generation of the early apoptotic cell population comprises freezing the cell preparation at about-18 ℃ to-25 ℃ for about 2 hours. In some embodiments, freezing in the generation of the early apoptotic cell population comprises freezing the cell preparation at about-80 ℃ for at least 0.5 hour, 1 hour, 2 hours, 4 hours, 12 hours.

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.

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 (at about-18 ℃ to-25 ℃, at about-80 ℃ and in liquid nitrogen) of the enriched monocyte composition is at least 8 hours, 10 hours, 12 hours, 18 hours, 24 hours.

In some embodiments, freezing induces an early apoptotic state at least in part in the cells of the enriched monocyte composition. In some embodiments, the freezing medium comprises 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 that donates enriched monocytes from the population. In some embodiments, the freezing medium comprises RPMI 1640 medium comprising 2mM L-glutamine, 10mM Hepes, 5% Hes, 10% dimethyl sulfoxide, and 20% v/v plasma.

In some embodiments, the freezing medium comprises an anticoagulant. In certain embodiments, at least some of the media (including freezing media, incubation media, and wash media) used during the generation of the early apoptotic cell population comprises an anticoagulant. In certain embodiments, all media (which includes an anticoagulant) used during the generation of the early apoptotic cell population includes the same concentration of anticoagulant. In some embodiments, the anticoagulant is not added to the final suspension medium of the cell population.

In some embodiments, adding an anticoagulant to at least the freezing medium increases the yield of the cell preparation. In other embodiments, addition of anticoagulant to the freezing medium increases the yield of cell preparation in the presence of high triglyceride levels. As used herein, an increase in the yield of a cell preparation involves an increase in at least one of: a percentage of viable cells in the frozen cells, a percentage of early state apoptotic cells in the viable cells, and combinations thereof.

In some embodiments, the 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.

The skilled person will understand that the term "stable" covers apoptotic cells that remain PS positive (phosphatidylserine positive) while the percentage of PI positive (propidium iodide positive) is very small. PI positive cells provide an indication of membrane stability, where PI positive cells are allowed into the cells, which shows poor membrane stability. In some embodiments, the stable early apoptotic cells remain in early apoptosis for at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. In another embodiment, the stable early apoptotic cells remain in early apoptosis for 24 hours, 36 hours, 48 hours, or 72 hours. In another embodiment, the stable early apoptotic cells remain in early apoptosis for more than 24 hours, more than 36 hours, more than 48 hours, or more than 72 hours. In another embodiment, the stable early apoptotic cells maintain their status for an extended period of time.

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 number of cell aggregates of the population of apoptotic cells is reduced as compared to a population of apoptotic cells prepared without the addition of an anticoagulant in a step other than the collection of cells from a donor (leukopheresis). In some embodiments, the population of apoptotic cells or a composition thereof comprises an anticoagulant.

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

The skilled person will appreciate that the term "aggregate" or "cell aggregate" may encompass reversible agglutination of blood cells under low shear forces or under stasis. Cell aggregates can be visually observed during the incubation step resulting from apoptotic cells. Cell aggregation can be measured by any method known in the art, for example by visually imaging the sample under a light microscope or using flow cytometry.

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.

In some embodiments of the methods of preparing the population of early apoptotic cells and compositions thereof, an anticoagulant is added to at least one culture medium used during preparation of the population. In some embodiments, the at least one culture medium used during the preparation of the population is selected from: freezing medium, washing medium, apoptosis-inducing incubation medium, and any combination thereof.

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, fondaparinux (fondaparinux), Bivalirudin (Bivalirudin), and Argatroban (Argatroban).

In some embodiments, at least one of the media used during population preparation contains a 5% ACD formula a solution comprising 10U/ml heparin. In some embodiments, the 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.

In some embodiments, at least one culture medium used during population preparation comprises heparin at a concentration between 0.1-2.5U/ml. In some embodiments, at least one culture medium used during population preparation comprises ACD formula a at a concentration between 1% -15% v/v. In some embodiments, the freezing medium comprises an anticoagulant. In some embodiments, the incubation medium comprises an anticoagulant. In some embodiments, both the freezing medium and the incubation medium comprise an anticoagulant. In some embodiments the anticoagulant is selected from: heparin, ACD formula a, and combinations thereof.

In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5U/ml. In some embodiments, the concentration of ACD formula a in the freezing medium is between 1% and 15% v/v. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5U/ml. In some embodiments, the ACD formula a concentration in the incubation medium is between 1-15% v/v. In some embodiments, the anticoagulant is a solution of acid-citrate-dextrose (ACD) formulation a. In some embodiments, the anticoagulant added to at least one of the media used during the population preparation is ACD formula a containing heparin at a concentration of 10U/ml.

In some embodiments, the apoptosis-inducing incubation medium used to generate the early apoptotic cell population comprises an anticoagulant. In some embodiments, both the freezing medium and the apoptosis-inducing incubation medium used to generate the early apoptotic cell population comprise an anticoagulant. Without wishing to be bound by any theory or mechanism, to maintain high and stable cell yields in different cell compositions, regardless of the cell collection protocol, in some embodiments, adding an anticoagulant comprises adding anticoagulant to both the freezing medium and the apoptosis-inducing incubation medium during generation of the apoptotic cell population. In some embodiments, a high and stable cell yield within the composition comprises a cell yield of at least 30%, preferably at least 40%, typically at least 50% of the cells in the initial population of cells used to induce apoptosis.

In some embodiments, both the freezing medium and the incubation medium comprise an anticoagulant. In some embodiments, the addition of anticoagulant to both the incubation medium and the freezing medium results in high and stable cell yields between different preparations of the population regardless of cell collection conditions (such as, but not limited to, the timing and/or type of anticoagulant added during cell collection). In some embodiments, the addition of anticoagulant to both the incubation medium and the freezing medium results in high and stable yields of cell preparation regardless of the timing and/or type of anticoagulant added during leukopheresis. In some embodiments, production of a cell preparation results in low and/or unstable cell yields between different preparations in the presence of high triglyceride levels. In some embodiments, production of the cell preparation from the blood of a donor having a high triglyceride level results in low and/or unstable cell yields of the cell preparation. In some embodiments, the term "high triglyceride levels" refers to triglyceride levels that are higher than normal levels in healthy subjects of the same gender and age. In some embodiments, the term "high triglyceride levels" refers to triglyceride levels above about 1.7 mmoles/liter. As used herein, high and stable yield refers to a yield of cells in a population that is sufficiently high to enable preparation of a dose that will exhibit therapeutic efficiency when administered to a subject. In some embodiments, therapeutic efficacy refers to the ability to treat, prevent, or ameliorate an immune disease, an autoimmune disease, or an inflammatory disease in a subject. In some embodiments, the high and stable cell yield is that of at least 30%, possibly at least 40%, typically at least 50% of the cells in the population that were initially frozen.

In some embodiments, if the cell preparation is obtained from a donor with a high triglyceride level, the donor will take at least one action selected from the group consisting of: administration of triglyceride-lowering drugs (such as, but not limited to, statins and/or bezafibrates) prior to donation; fasting for a period of at least 8 hours, 10 hours, 12 hours prior to donation; feeding an appropriate diet for at least 24 hours, 48 hours, 72 hours prior to donation to reduce blood triglyceride levels; and any combination thereof.

In some embodiments, the cell yield in the population is related to the number of cells in the composition in the initial number of cells undergoing apoptosis induction. As used herein, the terms "induce an early apoptotic state" and "induce apoptosis" may be used interchangeably.

In some embodiments, after freezing and thawing, the enriched monocyte composition is incubated in an incubation medium. In some embodiments, there is at least one wash step between thawing and incubation. As used herein, the terms "incubation medium" and "apoptosis-inducing incubation medium" are used interchangeably. In some embodiments, the incubation medium comprises RPMI 1640 medium supplemented with L-glutamine, Hepes, methylprednisolone, and plasma. In some embodiments, the wash medium comprises 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, at 37 ℃ and 5% CO ₂The incubation was performed.

In some embodiments, the incubation medium comprises methylprednisolone. In some embodiments, methylprednisolone within said incubation medium further induces cells in the enriched monocyte composition into an early apoptotic state. In some embodiments, the cells in the enriched monocyte composition are induced to enter an early apoptotic state by freezing and incubating in the presence of methylprednisolone. In some embodiments, the generation of an early apoptotic cell population advantageously allows for the induction of an early apoptotic state without substantially inducing necrosis, wherein the cells remain stable in said early apoptotic state about 24 hours after production.

In some embodiments, the incubation medium comprises methylprednisolone at a concentration of about 10-100 μ g/ml. In some embodiments, the incubation medium comprises methylprednisolone at a concentration of about 40-60 μ g/ml, optionally about 45-55 μ g/ml. In some embodiments, the incubation medium comprises methylprednisolone at a concentration of 50 μ g/ml.

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

In some embodiments, the incubation medium comprises an anticoagulant. In some embodiments, adding an anticoagulant to the incubation medium increases the yield of the cell preparation. In some embodiments, the concentration of anticoagulant in the incubation medium is the same as the concentration of anticoagulant in the freezing medium. In some embodiments, the incubation 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 incubation medium is ACD formula a containing heparin at a concentration of 10U/ml.

In some embodiments, the incubation medium comprises heparin. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5U/ml. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5U/ml, possibly between 0.3-0.7U/ml, typically about 0.5U/ml. In certain embodiments, the concentration of heparin in the incubation medium is about 0.5U/ml.

In some embodiments, the incubation medium comprises ACD formula a. In some embodiments, the ACD formula a concentration in the incubation medium is between 1-15% v/v. In some embodiments, the ACD formula a concentration in the incubation medium is between 1-15% v/v, possibly between 4-7% v/v, typically about 5% v/v. In some embodiments, the ACD formula a concentration in the incubation medium is about 5% v/v.

In some embodiments, the increase in yield of the cell preparation comprises an increase in the number of early apoptotic living cells of the preparation in the number of frozen cells from which the preparation is produced.

In some embodiments, the addition of 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, the addition of anticoagulant at least to the freezing medium and the incubation medium results in a high and stable yield between different preparations of the pharmaceutical composition regardless of the cell collection protocol used.

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 10U/ml. In some embodiments, the freezing medium comprises a 5% v/v ACD formulation A solution comprising heparin at a concentration of 10U/ml.

In some embodiments, the freezing medium comprises heparin. In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5U/ml. In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5U/ml, possibly between 0.3-0.7U/ml, typically about 0.5U/ml. In certain embodiments, the concentration of heparin in the freezing medium is about 0.5U/ml.

In some embodiments, the freezing medium comprises ACD formula a. In some embodiments, the concentration of ACD formula a in the freezing medium is between 1% to 15% v/v. In some embodiments, the concentration of ACD formula a in the freezing medium is between 1-15% v/v, possibly between 4-7% v/v, typically about 5% v/v. In some embodiments, the concentration of ACD formula a in the freezing medium is about 5% v/v.

In some embodiments, the addition of anticoagulant to the incubation medium and/or the freezing medium results in high and stable cell yield within the population regardless of triglyceride levels in the blood of the donor. In some embodiments, addition of anticoagulant to the incubation medium and/or freezing medium results in high and stable cell yield within the composition when obtained from the blood of a donor with normal or high triglyceride levels. In some embodiments, the addition of anticoagulant at least to the incubation medium results in high and stable cell yield within the composition regardless of triglyceride levels in the blood of the donor. In some embodiments, the addition of anticoagulant to the freezing medium and the incubation medium results in high and stable cell yield within the composition regardless of triglyceride levels in the blood of the donor.

In some embodiments, the freezing medium and/or the incubation medium and/or the washing medium comprises heparin at a concentration of at least 0.1U/ml, possibly at least 0.3U/ml, typically at least 0.5U/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, typically at least 5% v/v.

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 the wash medium.

In some embodiments, the enriched monocyte composition undergoes at least one wash step between each stage of generating an early apoptotic cell population. In some embodiments, the anticoagulant is added to the wash medium during the wash step throughout the process of generating the early apoptotic cell population. In some embodiments, the enriched monocyte composition undergoes at least one washing step after incubation. In some embodiments, the enriched monocyte composition undergoes at least one wash step following incubation using PBS. In some embodiments, no anticoagulant is added to the final wash step prior to resuspending the cell preparation in the administration medium. In some embodiments, no anticoagulant is added to the PBS used in the final wash step prior to resuspending the cell preparation in the administration medium. In certain embodiments, no anticoagulant is added to the administration medium.

In some embodiments, the cell concentration during incubation is about 5x10⁶Individual cells/ml.

In some embodiments, the enriched monocyte composition is suspended in an administration medium after freezing, thawing, and incubation, thereby producing a drug population. In some embodiments, the administration medium comprises a suitable physiological buffer. Non-limiting examples of suitable physiological buffers are: saline Solution, Phosphate Buffered Saline (PBS), Hank's Balanced Salt Solution (HBSS), and the like. In some embodiments, the administration medium comprises PBS. In some embodiments, the administration medium comprises a supplement that helps maintain cell viability. In some embodiments, the enriched monocyte composition is filtered prior to administration. In some embodiments, the enriched monocyte composition is filtered prior to administration using a filter of at least 200 μm.

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

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

In some embodiments, the wash medium comprises an anticoagulant. In some embodiments, the wash medium comprises an anticoagulant selected from the group consisting of: heparin, ACD formula a, and combinations thereof. In some embodiments, the concentration of anticoagulant in the wash medium is the same as the concentration of anticoagulant in the freeze medium. In some embodiments, the concentration of anticoagulant in the wash medium is the same as the concentration of anticoagulant in the incubation medium. In some embodiments, the anticoagulant used in the wash medium is ACD formula a containing heparin at a concentration of 10U/ml.

In some embodiments, the wash medium comprises heparin. In some embodiments, the concentration of heparin in the wash medium is between 0.1-2.5U/ml. In some embodiments, the concentration of heparin in the wash medium is between 0.1-2.5U/ml, possibly between 0.3-0.7U/ml, typically about 0.5U/ml. In certain embodiments, the concentration of heparin in the wash medium is about 0.5U/ml.

In some embodiments, the wash medium comprises ACD formula a. In some embodiments, the concentration of ACD formula a in the wash medium is between 1% and 15% v/v. In some embodiments, the concentration of ACD formula a in the wash medium is between 1-15% v/v, possibly 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.

In some embodiments, the enriched monocyte composition is thawed hours before it is expected to be administered to the subject the population. In some embodiments, the enriched monocyte composition is thawed at about 33 ℃ -39 ℃. In some embodiments, the enriched monocyte composition is thawed for about 30-240 seconds, preferably 40-180 seconds, most preferably 50-120 seconds.

In some embodiments, the enriched monocyte composition is thawed at least 10 hours prior to administration of the intended population, optionally at least 20 hours, 30 hours, 40 hours, or 50 hours prior to administration of the intended population. In some embodiments, the enriched monocyte composition is thawed at least 15-24 hours prior to administration of the intended population. In some embodiments, the enriched monocyte composition is thawed at least about 24 hours prior to administration of the intended population. In some embodiments, the enriched monocyte composition is thawed at least 20 hours prior to administration of the intended population. In some embodiments, the enriched monocyte composition is thawed 30 hours prior to administration of the intended population. In some embodiments, the enriched monocyte composition is thawed at least 24 hours prior to administration of the intended population. In some embodiments, the enriched monocyte composition undergoes at least one wash step in a wash medium before and/or after thawing.

In some embodiments, the composition further comprises methylprednisolone. In some embodiments, the concentration of methylprednisolone is no more than 30 μ g/ml.

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

In some embodiments, apoptotic cells disclosed herein do not comprise necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 1% necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 2% necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 3% necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 4% necrotic cells. In some embodiments, apoptotic cells disclosed herein comprise less than 5% necrotic cells.

In some embodiments, about 140 x 10 is administered⁶-210×10⁶Dose of individual apoptotic cells. In some embodiments, about 10-100X 10 is administered⁶Dose of individual apoptotic cells. In some embodiments, about 20 x 10 is administered⁶Dose of individual apoptotic cells. In some embodiments, about 30 x 10 is administered⁶Dose of individual apoptotic cells. In some embodiments, about 40 x 10 is administered⁶The dose of individual apoptotic cells. In some embodiments, about 50 x 10 is administered⁶The dose of individual apoptotic cells. In some embodiments, 60 x 10 is administered⁶And (4) apoptotic cells. In some embodiments, about 60 x 10 is administered⁶The dose of individual apoptotic cells. In some embodiments, about 70 x 10 is administered⁶The dose of individual apoptotic cells. In some embodiments, about 80 x 10 is administered⁶The dose of individual apoptotic cells. In some embodiments, about 90 x 10 is administered⁶The dose of individual apoptotic cells. In some embodiments, about 1-15X 10 is administered⁷The dose of individual apoptotic cells. In some embodiments, about 10 x 10 is administered⁷The dose of individual apoptotic cells. In some embodiments, about 15 x 10 is administered⁷The dose of individual apoptotic cells.

In some embodiments, 10 x 10 is administered⁶The dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered ⁷Dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered⁸Dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered⁹Dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered¹⁰The dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered¹¹The dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered¹²The dose of individual apoptotic cells. In another embodiment, administration 10 is prepared10⁵The dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered⁴The dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered³The dose of individual apoptotic cells. In another embodiment, 10 × 10 is administered²The dose of individual apoptotic cells.

In some embodiments, a high dose of apoptotic cells is administered. In some embodiments, 35 x 10 is administered⁶The dose of individual apoptotic cells. In another embodiment, 210 x 10 is administered⁶The dose of individual apoptotic cells. In another embodiment, 70 x 10 is administered⁶The dose of individual apoptotic cells. In another embodiment, about 140X 10 is administered⁶The dose of individual apoptotic cells. In another embodiment, 35-210X 10 is administered⁶The dose of individual apoptotic cells.

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.

In some embodiments, the apoptotic cells may 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.

In some embodiments, the apoptotic cells are prepared from cells obtained from a subject other than the subject that will receive the apoptotic cells. In some embodiments, the methods as disclosed herein include methods useful for overcoming allogeneic donor cell rejection Additional steps, including one or more of the steps described in U.S. patent application 20130156794, which is incorporated herein by reference in its entirety. In some embodiments, the method comprises the step of performing a complete or partial lymphocyte clearance prior to administration of the apoptotic cells (which in some embodiments are allogeneic apoptotic cells). In some embodiments, the lymphocyte clearance is adjusted such that it delays the host versus graft response for a period of time sufficient to allow the allogeneic apoptotic cells to control cytokine release. In some embodiments, the method comprises administering an agent that delays egress of allogeneic apoptotic T cells from the 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-l]1,2,4-

Oxadiazole (SEW2871), 3- (2- (-hexylphenylamino) -2-oxyethylamino) 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) that can be used as part of the compositions and methods disclosed herein to allow the use of allogeneic apoptotic cells that have potency and do not cause graft-versus-host disease. In another embodiment, MHC expression by the allogeneic apoptotic T cells is silenced to reduce rejection of allogeneic cells.

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

In some embodiments, the method comprises the steps of: prior to administering the apoptotic cell population to the subject, the apoptotic cell population derived from the same subject is irradiated (autologous ApoCell). In some embodiments, the method comprises the steps of: prior to administering the population of apoptotic cells to the recipient, the apoptotic cells derived from the subject are irradiated (allogeneic ApoCell).

In some embodiments, the cells are irradiated in a manner that will reduce proliferation and/or activation of residual viable cells within the apoptotic cell population. In some embodiments, the cells are irradiated in a manner that reduces the percentage of viable, non-apoptotic cells in the population. In some embodiments, the percentage of viable, 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 viable, 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 viable, 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 viable, 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 viable, 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 viable, non-apoptotic cells in the inactivated early apoptotic cell population is reduced to 0% of the population.

In another embodiment, the irradiated apoptotic cells retain all of their early apoptotic, immunomodulatory, and stability properties. In another embodiment, the irradiating step uses UV radiation. In another embodiment, the irradiating step uses gamma radiation. In another embodiment, the apoptotic cells comprise a reduced percentage of viable non-apoptotic cells, comprise a preparation with suppressed cellular activation of any viable non-apoptotic cells present within the apoptotic cell preparation, or comprise a preparation with reduced proliferation of any viable non-apoptotic cells present within the apoptotic cell preparation, or any combination thereof.

In some embodiments, irradiation of apoptotic cells does not increase the population of dead cells (PI +) 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 1% 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 2% 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 3% 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 4% 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 5% 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 6% 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 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.

In some embodiments, a cell population comprising viable non-apoptotic cells with a reduced or absent fraction may in one embodiment provide a mononuclear, early apoptotic cell population without any viable (living/viable) cells. In some embodiments, a population of cells comprising live non-apoptotic cells with a reduced or absent fraction may, in one embodiment, provide a population of mononuclear apoptotic cells that do not elicit GVHD in a recipient.

In some embodiments, using irradiated ApoCell to remove possible graft-versus-leukemia effects, the use of an apoptotic population (which comprises a minimal fraction of living cells) may result in demonstrating that the effects shown in the examples (see example 8) are caused by apoptotic cells and not by a viable cell proliferative population with cellular activity present within the apoptotic cell population.

In another embodiment, the method comprises the 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 properties. In another embodiment, the irradiating step uses UV radiation. In another embodiment, the irradiating step uses gamma radiation. In another embodiment, the apoptotic cells comprise a reduced percentage of viable non-apoptotic cells, comprise a preparation of suppressed cell activation of any viable non-apoptotic cells present within a preparation of apoptotic cells, or comprise a preparation of reduced proliferation of any viable non-apoptotic cells present within a preparation of apoptotic cells, or any combination thereof.

In some embodiments, the apoptotic cells comprise a pooled mononuclear apoptotic cell preparation. In some embodiments, the pooled mononuclear apoptotic cell preparation comprises mononuclear cells in an early apoptotic state, wherein said pooled mononuclear apoptotic cells comprise a reduced percentage of viable non-apoptotic cells, a preparation with suppressed cell activation of any viable non-apoptotic cells, or a preparation with reduced proliferation of any viable non-apoptotic cells, or any combination thereof. In another embodiment, the pooled mononuclear apoptotic cells have been irradiated. In another embodiment, disclosed herein is a pooled mononuclear apoptotic cell preparation, which in some embodiments is derived from white blood cell fractions (WBCs) obtained from donated blood.

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 as 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 a population of non-irradiated apoptotic cells.

In some embodiments, the pooled blood comprises a 3 rd party blood that is mismatched between the donor and the recipient.

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

In some embodiments, the pooled apoptotic cell preparation is derived from between about 2 and 25 cells present in a unit of blood. In another embodiment, the pooled apoptotic cell preparation comprises between 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 cells present in a unit of blood. In another embodiment, the pooled apoptotic cell preparation consists of 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 recovery of WBCs from the blood. For example, low efficiency WBC recovery would result in the need for additional units, while high efficiency WBC recovery would result in fewer units being needed. In some embodiments, each unit is a bag of blood. In another embodiment, the pooled apoptotic cell preparation consists of cells present in at least 25 units of blood, at least 50 units of blood, or at least 100 units of blood.

In some embodiments, the unit blood comprises a White Blood Cell (WBC) fraction from a blood donation. In another embodiment, the donation may be from a blood center or blood bank. In another example, the donation may be from a donor who has accumulated in a hospital in preparation for a pooled apoptotic cell preparation. In another embodiment, a unit of blood comprising WBCs from multiple donors is preserved and maintained in a separate blood bank created for the purposes of the compositions and methods disclosed herein. In another embodiment, a blood bank developed for the purposes of the compositions and methods disclosed herein is capable of supplying a unit of blood comprising WBCs from multiple donors and comprises a leukapheresis unit.

In some embodiments, the unit of pooled WBCs is not restricted by HLA matching. Thus, the resulting pooled apoptotic cell preparation comprises a population of cells that are not restricted by HLA matching. Thus, in certain embodiments, the pooled mononuclear apoptotic cell preparation comprises allogeneic cells.

The advantage of pooled mononuclear apoptotic cell preparations derived from pooled WBCs that are not restricted by HLA matching is that WBC sources are readily available and the cost of obtaining WBCs is reduced.

In some embodiments, the pooled blood comprises blood from multiple donors that is HLA-matched independent. In another embodiment, the pooled blood comprises blood from multiple donors, wherein HLA matching with the recipient has been taken into account. For example, where 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 recipient. In another embodiment, multiple donors are partially matched, e.g., 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 recipients. Each possibility includes embodiments as disclosed herein.

In certain embodiments, some viable non-apoptotic cells (anti-apoptotic) may remain after the apoptosis-inducing step described below (example 1). In some embodiments, the presence of these viable, non-apoptotic cells is observed prior to the irradiation step. These living non-apoptotic cells may be capable of proliferating or being activated. In some embodiments, pooled mononuclear apoptotic cell preparations derived from multiple donors may be activated against the host, against each other, or both.

In some embodiments, the cell activation of the irradiated cell preparation as disclosed herein is arrested and proliferation is reduced compared to a non-irradiated cell preparation. In another embodiment, the irradiation comprises gamma irradiation or UV irradiation. In another embodiment, the irradiated cell preparation has a reduced number of non-apoptotic cells as compared to a non-irradiated cell preparation. In another embodiment, the irradiation comprises about 15 gray units (Gy). In another embodiment, the irradiation comprises about 20 gray units (Gy). In another embodiment, the irradiation comprises about 25 gray units (Gy). In another embodiment, the irradiation comprises about 30 gray units (Gy). In another embodiment, the irradiation comprises about 35 gray units (Gy). In another embodiment, the irradiation comprises about 40 gray units (Gy). In another embodiment, the irradiation comprises about 45 gray units (Gy). In another embodiment, the irradiation comprises about 50 gray units (Gy). In another embodiment, the irradiation comprises 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 apoptosis profile, stability and efficacy as the unirradiated pooled apoptotic cell preparation.

In some embodiments, a pooled mononuclear apoptotic cell preparation as disclosed herein is stable for up to 24 hours. In another embodiment, the pooled mononuclear apoptotic cell preparation is stable for at least 24 hours. In another embodiment, the pooled mononuclear apoptotic cell preparation is stable for more than 24 hours. In yet another embodiment, a pooled mononuclear apoptotic cell preparation as disclosed herein is stable for up to 36 hours. In yet another embodiment, the pooled mononuclear apoptotic cell preparation is stable for at least 36 hours. In further embodiments, the pooled mononuclear apoptotic cell preparation is stable for more than 36 hours. In another embodiment, a pooled mononuclear apoptotic cell preparation as disclosed herein is stable for up to 48 hours. In another embodiment, the pooled mononuclear apoptotic cell preparation is stable for at least 48 hours. In another embodiment, the pooled mononuclear apoptotic cell preparation is stable for more than 48 hours.

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 single matched donors, wherein the cell preparation may not comprise an irradiation step. In another embodiment, a pooled mononuclear apoptotic cell preparation as disclosed herein does not elicit a Graft Versus Host Disease (GVHD) response.

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

In another embodiment, the percentage of apoptotic cells in a pooled mononuclear apoptotic cell preparation as disclosed herein is close to 100%, thereby reducing the fraction of viable, non-apoptotic cells in the cell preparation. 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 yet another embodiment, the percentage of apoptotic cells is at least 70%. In further embodiments, the percentage of apoptotic cells is at least 80%. In another embodiment, the percentage of apoptotic cells is at least 90%. In yet another embodiment, the percentage of apoptotic cells is at least 99%. Thus, in one embodiment, a cell preparation comprising live non-apoptotic cells with reduced or absent fractions may provide a pooled mononuclear apoptotic cell preparation that does not elicit GVHD in a recipient. Each possibility represents an embodiment as disclosed herein.

Optionally, in another embodiment, the percentage of viable, non-apoptotic WBCs is reduced by specifically removing the viable cell population (e.g., by targeted precipitation). In another example, magnetic beads bound to phosphatidylserine can be used to reduce the percentage of viable, non-apoptotic cells. In another example, magnetic beads that bind markers on the cell surface of non-apoptotic cells rather than apoptotic cells may be used to reduce the percentage of viable non-apoptotic cells. In another example, apoptotic cells may be selected for further preparation using magnetic beads that bind to markers on the cell surface of apoptotic cells rather than non-apoptotic cells. In yet another embodiment, the percentage of viable non-apoptotic WBCs is reduced by using ultrasound.

In one embodiment, the apoptotic cells are from a pooled third party donor.

In some embodiments, the pooled cell preparation comprises at least one cell type selected from the group consisting of: lymphocytes, monocytes, and natural killer cells. In another embodiment, the pooled cell preparation comprises an enriched monocyte population. In some embodiments, the pooled monocytes are an enriched monocyte preparation comprising a cell type selected from the group consisting of: lymphocytes, monocytes, and natural killer cells. In another embodiment, the enriched monocyte preparation comprises no more than 15%, optionally no more than 10%, typically no more than 5% polymorphonuclear leukocytes, also known as granulocytes (i.e., neutrophils, basophils, and eosinophils). In another embodiment, the pooled monocyte preparation is devoid of granulocytes.

In another embodiment, the pooled enriched monocyte preparation comprises no more than 15%, optionally no more than 10%, typically no more than 5% CD15^{High (a)}An expression cell. In some embodiments, the pooled apoptotic cell preparation comprises less than 15% CD15 high expressing cells.

In some embodiments, the pooled enriched monocyte preparation disclosed herein comprises at least 80% monocytes, at least 85% monocytes, optionally at least 90% monocytes, or at least 95% monocytes, wherein each possibility is a separate embodiment disclosed herein. According to some embodiments, the pooled enriched monocyte preparation disclosed herein comprises at least 85% monocytes.

In another embodiment, any pooled cell preparation having a final pooled percentage of monocytes of at least 80% is considered a pooled enriched monocyte preparation as disclosed herein. Thus, pooling a cell preparation with increased polymorphonuclear cells (PMNs) with a cell preparation with high monocytes (the resulting "pool" has at least 80% monocytes) includes a preparation as disclosed herein. According to some embodiments, the monocytes comprise lymphocytes and monocytes.

The skilled person will appreciate that the term "monocyte" may encompass leukocytes having a single lobe nucleus. In another embodiment, a pooled apoptotic cell preparation as disclosed herein comprises less than 5% polymorphonuclear leukocytes.

In some embodiments, the apoptotic cell is a T cell. In another embodiment, the apoptotic cells are derived from the same pooled third party donor T cells as CAR T cells. In another embodiment, the apoptotic cells are derived from a population of CAR T cells.

Surprisingly, the apoptotic cells reduced the production of cytokines associated with cytokine storm, including but not limited to IL-6 and interferon-gamma (IFN- γ), alone or in combination, while maintaining the effectiveness of CAR T cell therapy (example 2). In one embodiment, the apoptotic cells affect cytokine expression levels in macrophages. In another embodiment, the apoptotic cells decrease cytokine expression levels in macrophages. In one embodiment, the apoptotic cells suppress cytokine expression levels in macrophages. In one embodiment, the apoptotic cells inhibit cytokine expression levels in macrophages. In one embodiment, the apoptotic cells maintain IFN- γ levels that match or nearly match the levels present prior to CAR-T cell administration. In another embodiment, the apoptotic cells affect cytokine expression levels in macrophages but do not affect cytokine expression levels in CAR T cells. In another embodiment, the apoptotic cells affect cytokine expression levels in DCs but not CAR T cells. Thus, it was unexpected that apoptotic cells would be useful in maintaining the effectiveness of CAR T cell therapy.

In another embodiment, the effect of apoptotic cells on the level of cytokine expression in macrophages, DCs, or a combination thereof results in a reduction in CRS. In another embodiment, the effect of apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof results in a reduction in severe CRS. In another embodiment, the effect of apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof results in suppression of CRS. In another embodiment, the effect of apoptotic cells on cytokine expression levels in macrophages, DCs, or a combination thereof results in suppression of severe CRS. In another embodiment, the effect of apoptotic cells on the level of cytokine expression in macrophages, DCs, or a combination thereof results in the inhibition of CRS. In another embodiment, the effect of apoptotic cells on the level of cytokine expression in macrophages, DCs, or a combination thereof results in the inhibition of severe CRS. In another embodiment, the effect of apoptotic cells on the level of cytokine expression in macrophages, DCs, or a combination thereof results in prevention of CRS. In another embodiment, the effect of apoptotic cells on the level of cytokine expression in macrophages, DCs, or a combination thereof results in prevention of severe CRS.

In another embodiment, apoptotic cells trigger the death of T cells, but not by a change in the level of cytokine expression.

In another embodiment, apoptotic cells antagonize the priming of macrophages and dendritic cells to secrete cytokines that would otherwise amplify the cytokine storm. In another embodiment, apoptotic cells increase tregs that suppress the inflammatory response and/or prevent excessive cytokine release.

In some embodiments, the apoptotic cells stabilize the presence of resident macrophages in the tumor region. In some embodiments, the method of treating cancer by combining cancer therapy with administration of early apoptotic cells results in stabilization of resident macrophages in the tumor region. In some embodiments, the method of treating cancer by combining a cancer therapeutic with administration of early apoptotic cells results in stabilization of resident macrophages in the tumor region. In some embodiments, the method of treating cancer by combining CAR-T cell therapy with administration of early apoptotic cells results in stabilization of resident macrophages in the tumor region.

In some embodiments, administration of apoptotic cells inhibits one or more proinflammatory cytokines. In some embodiments, the proinflammatory cytokine comprises IL-1 β, IL-6, TNF- α, or IFN- γ, or any combination thereof. In another embodiment, administration of apoptotic cells promotes secretion of one or more anti-inflammatory cytokines. In some embodiments, the anti-inflammatory cytokine comprises TGF- β, IL10, or PGE2, or any combination thereof.

In another embodiment, administration of apoptotic cells inhibits dendritic cell maturation following exposure to a TLR ligand. In another embodiment, administration of apoptotic cells results in potentially tolerogenic dendritic cells, which in some embodiments are capable of migrating, and in some embodiments, migration is due to CCR 7. In another embodiment, administration of apoptotic cells triggers various signaling events, which in one embodiment are TAM receptor signaling (Tyro3, Axl, and Mer), which in some embodiments inhibit inflammation in antigen presenting cells.

In some embodiments, Tyro-3, Axl, and Mer constitute the TAM family of Receptor Tyrosine Kinases (RTKs) characterized by conserved sequences within the kinase domain and the adhesion molecule-like extracellular domain. In another embodiment, administration of apoptotic cells activates signaling through MerTK. In another embodiment, administration of apoptotic cells activates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway which in some embodiments negatively regulates NF-. kappa.B. In another embodiment, the administration of apoptotic cells down-regulates in one embodiment an inflammasome that inhibits pro-inflammatory cytokine secretion, DC maturation, or a combination thereof. In another embodiment, administration of apoptotic cells upregulates expression of anti-inflammatory genes (e.g., Nr4a, Thbs1, or a combination thereof). In another embodiment, administration of apoptotic cells induces high levels of AMP that accumulate in some embodiments in a pannexin 1-dependent manner. In another embodiment, administration of apoptotic cells suppresses inflammation.

Apoptotic cell supernatant (ApoSup and ApoSup Mon)

In some embodiments, the compositions for use in methods and treatments as disclosed herein comprise apoptotic cell supernatants as disclosed herein.

In some embodiments, the apoptotic cell supernatant is obtained by a method comprising: a) providing apoptotic cells, b) culturing said apoptotic cells of step a), and c) isolating said supernatant from said cells.

In some embodiments, the apoptotic cells used to prepare apoptotic cell supernatants as disclosed herein are autologous to the subject undergoing therapy. In another embodiment, the apoptotic cells used to prepare the apoptotic cell supernatants disclosed herein are allogeneic to the subject undergoing therapy.

The "apoptotic cells" from which apoptotic cell supernatants are obtained may be cells of any cell type selected from the subject, or any commercially available cell line subjected to a method of inducing apoptosis known to those of skill in the art. The method of inducing apoptosis may be hypoxia, ozone, heat, radiation, chemicals, osmotic pressure, pH change, X-ray irradiation, gamma ray irradiation, UV irradiation, serum deprivation, corticoids, or combinations thereof or any other method described herein or known in the art. In another embodiment, the method of inducing apoptosis produces apoptotic cells in an early apoptotic state.

In some embodiments, the apoptotic cell is a leukocyte.

In embodiments, the apoptotic leukocytes are derived from Peripheral Blood Mononuclear Cells (PBMCs). In another embodiment, the leukocytes are from a pooled third party donor. In another embodiment, the white blood cells are allogeneic.

According to some embodiments, the apoptotic cells are provided by selecting non-adherent leukocytes and subjecting them to apoptosis induction, followed by a cell culture step in culture medium. The "leukocytes" used to prepare the apoptotic cell-phagocyte supernatant can be derived from any lineage or sub-lineage of nucleated cells of the immune system and/or hematopoietic system, including but not limited to dendritic cells, macrophages, masT cells, basophils, hematopoietic stem cells, bone marrow cells, natural killer cells, and the like. The leukocytes can be derived or obtained in any of a variety of suitable manners from any of a variety of suitable anatomical chambers, depending on the application and purpose, desired leukocyte lineage, and the like, according to any of a variety of commonly practiced methods. In some embodiments, the source leukocytes are primary leukocytes. In another embodiment, the source leukocytes are primary peripheral blood leukocytes.

Primary lymphocytes and monocytes may conveniently be derived from peripheral blood. Peripheral blood leukocytes contain 70-95% lymphocytes and 5-25% monocytes.

Methods for obtaining specific types of source leukocytes from blood are routinely practiced. Obtaining source lymphocytes and/or monocytes can be accomplished, for example, by collecting blood in the presence of an anticoagulant such as heparin or citrate. The collected blood was then centrifuged over a Ficoll pad to separate lymphocytes and monocytes at the gradient interface and neutrophils and erythrocytes in the pellet.

Leukocytes can be separated from each other by standard immunomagnetic selection or immunofluorescence flow cytometry techniques based on their specific surface markers or by centrifugal elutriation. For example, monocytes may be selected as the CD14+ fraction, T lymphocytes may be selected as the CD3+ fraction, B lymphocytes may be selected as the CD19+ fraction, and macrophages as the CD206+ fraction.

Lymphocytes and monocytes can be isolated from each other by subjecting these cells to matrix adherent conditions (e.g., by static culture in a tissue culture-treated culture recipient), which results in the selective adherence of monocytes to the cell adherent matrix rather than lymphocytes.

Leukocytes can also be obtained from Peripheral Blood Mononuclear Cells (PBMCs) that can be isolated as described herein.

One of ordinary skill in the art will have the necessary expertise to properly culture primary leukocytes to produce the desired amount of cultured source leukocytes as disclosed herein, and sufficient guidance for practicing such culture methods is available in the literature in the art.

One of ordinary skill in the art will further have the necessary expertise to establish, purchase, or otherwise obtain a suitable established leukocyte cell line from which to derive apoptotic leukocytes. Suitable leukocyte cell lines can be obtained from commercial suppliers, such as the American Tissue Type Collection (ATCC). It will be apparent to those skilled in the art that the source leukocytes should not be obtained by techniques that would significantly interfere with their ability to produce apoptotic leukocytes.

In another embodiment, the apoptotic cell may be an apoptotic lymphocyte. Apoptosis of lymphocytes, such as primary lymphocytes, can be induced by treating the primary lymphocytes with serum deprivation, corticosteroids, or irradiation. In another embodiment, inducing apoptosis of primary lymphocytes by treatment with a corticosteroid is achieved by treating primary lymphocytes with dexamethasone (dexamethasone). In another embodiment, the concentration of dexamethasone is about 1 micromolar. In another embodiment, inducing apoptosis of the primary lymphocytes by irradiation is achieved by treating the primary lymphocytes with gamma irradiation. In another embodiment, the dose is about 66 rad. Such treatment results in the production of apoptotic lymphocytes suitable for the co-culture step with phagocytes.

In another embodiment, the apoptotic cell may be an apoptotic monocyte, such as a primary monocyte. To generate apoptotic monocytes, the monocytes are subjected to in vitro matrix/surface adherent conditions under serum deprivation conditions. Such treatment results in the production of non-pro-inflammatory apoptotic monocytes suitable for co-culture steps with phagocytes.

In other embodiments, the apoptotic cell may be any apoptotic cell described herein, including allogeneic apoptotic cells, third party apoptotic cells, and apoptotic cell pools.

In other embodiments, the apoptotic cell supernatant may be obtained by co-culturing apoptotic cells with other cells.

Thus, in some embodiments, the apoptotic cell supernatant is an apoptotic cell supernatant obtained by a method comprising: a) providing apoptotic cells, b) providing further cells, c) optionally washing said cells from steps a) and b), d) co-culturing said cells of steps a) and b) and optionally e) isolating said supernatant from said cells.

In some embodiments, the other cells co-cultured with the apoptotic cells are white blood cells.

Thus, in some embodiments, the apoptotic cell supernatant is an apoptotic white blood cell supernatant obtained by a method comprising: a) providing apoptotic cells, b) providing white blood cells, c) optionally washing the cells from steps a) and b), d) co-culturing the cells of steps a) and b) and optionally e) separating the supernatant from the cells.

In some embodiments, the white blood cells may be phagocytic cells, such as macrophages, monocytes, or dendritic cells.

In some embodiments, the white blood cells may be B cells, T cells, or natural killer cells (NK cells).

Thus, in some embodiments, compositions for use in methods and treatments as disclosed herein comprise apoptotic-phagocyte supernatants as described in WO2014/106666, which is incorporated herein by reference in its entirety. In another embodiment, apoptotic cell-phagocyte supernatant for use in compositions and methods as disclosed herein is produced in any manner known in the art.

In some embodiments, the apoptotic-phagocyte supernatant is obtained from co-culturing phagocytes with apoptotic cells.

In some embodiments, the apoptotic cell-phagocyte supernatant is obtained by a method comprising: a) providing a phagocytic cell, b) providing an apoptotic cell, c) optionally washing the cell from steps a) and b), d) co-culturing the cells of steps a) and b), and optionally e) isolating the supernatant from the cell.

The term "phagocytic cell" refers to a cell that protects the body by uptake (phagocytosis) of harmful foreign particles, bacteria, and dead or dying cells. Phagocytic cells include, for example, cells known as neutrophils, monocytes, macrophages, dendritic cells and mast T cells, preferably dendritic cells and monocytes/macrophages. Phagocytes can be dendritic cells (CD4+ HLA-DR + lineage-BDCA 1/BDCA3+), macrophages (CD14+ CD206+ HLA-DR +), or derived from monocytes (CD14 +). Techniques for distinguishing these different phagocytes are known to those skilled in the art.

In embodiments, the monocytes are obtained by a plastic adherence step. The monocytes can be distinguished from B and T cells with the marker CD14+, whereas unwanted B cells express CD19+ and T cell CD3 +. In some embodiments, macrophages obtained after maturation induction by macrophage colony stimulating factor (M-CSF) are positive for markers CD14+, CD206+, HLA-DR +.

In one embodiment, the phagocytic cells are derived from Peripheral Blood Mononuclear Cells (PBMCs).

Phagocytes can be provided by any method known in the art for obtaining phagocytes. In some embodiments, phagocytic cells (such as macrophages or dendritic cells) can be isolated directly from a subject or derived from precursor cells by a maturation step.

In some embodiments, macrophages may be isolated directly from the peritoneal cavity of a subject and cultured in complete RRPMI medium. Macrophages can also be isolated from the spleen.

Phagocytes can also be obtained from peripheral blood mononuclear cells. In the example, upon addition of, but not limited to, macrophage colony-stimulating factor (M-CSF) to the cell culture medium, monocytes differentiate in culture into monocyte-derived macrophages.

For example, phagocytes can be derived from Peripheral Blood Mononuclear Cells (PBMCs). For example, PBMCs can be isolated from cell purification bags from individuals by Ficoll gradient centrifugation, plated in complete RPMI medium (10% FBS, 1% penicillin/streptomycin) for 90 minutes in a cell attachment step. Nonadherent T cells are removed by a plastic adherence step and adherent T cells are cultured in a complete RPMI environment supplemented with recombinant human M-CSF. After the culture period, monocyte-derived macrophages were obtained.

Phagocytes can be selected by a cell attachment procedure. The "cell attachment step" refers to the selection of phagocytes or cells that can mature into phagocytes by culture conditions that allow the cultured cells to attach to a surface (a cell attachment surface, e.g., a tissue culture dish, a substrate, a capsule or bag with an appropriate type of nylon or plastic). The skilled person will appreciate that the term "cell adhesion surface" may encompass hydrophilic and negatively charged and may be obtained in any of various ways known in the art. In another embodiment, the polystyrene surface is modified by using, for example, corona discharge or gas plasma. These processes generate energetic oxygen ions that are grafted onto surface polystyrene chains, causing the surface to become hydrophilic and negatively charged. Culture recipients designed to promote adherence to their cells are available from various commercial suppliers (e.g., Corning, Perkin-Elmer, Fisher Scientific, Evergreen Scientific, Nunc, etc.).

B cells, T cells and NK cells may be provided by any method known in the art for obtaining such cells. In some embodiments, B cells, T cells, or NK cells may be isolated directly from a subject or derived from precursor cells by a maturation step. In another embodiment, the B cell, T cell, or NK cell can be from a B cell line, a T cell line, or an NK cell line. One of ordinary skill in the art will have the necessary expertise to establish, purchase, or otherwise obtain suitable established B cell lines, T cell lines, and NK cell lines. Suitable cell lines are available from commercial suppliers such as the American tissue type Collection (ATCC).

In embodiments, the apoptotic cells and the white blood cells (such as phagocytes, B cells, T cells or NK cells) are cultured separately prior to the co-culturing step d).

Cell maturation of phagocytes occurs during cell culture, for example, due to the addition of maturation factors to the culture medium. In one embodiment, the maturation factor is M-CSF, which can be used, for example, to obtain monocyte-derived macrophages.

The culturing step for maturation or selection of phagocytes may take hours to days. In another embodiment, the premature phagocytes are cultured in an appropriate medium for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours.

Phagocytic cell culture media are known to those skilled in the art and can be, for example, but are not limited to, RPMI, DMEM, X-vivo, and Ultraculture mileus.

In embodiments, co-culturing of apoptotic cells and phagocytes occurs in physiological solution.

Prior to this "co-cultivation", the cells may be subjected to a washing step. In some embodiments, the white blood cells (e.g., phagocytic cells) and apoptotic cells are washed prior to the co-culturing step. In another example, the cells are washed with PBS.

During the co-culture, white blood cells (e.g., phagocytes (e.g., macrophages, monocytes, or phagocytes) or B cells, T cells, or NK cells) and apoptotic cells may be mixed in a ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1 or in a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (white blood cells: apoptotic cells). In one example, the ratio of white blood cells to apoptotic cells is 1: 5.

Co-culturing of cells may last from hours to days. In some embodiments, the apoptotic cells are cultured for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours. One skilled in the art can assess the optimal time for co-culturing by measuring the presence of anti-inflammatory compounds, the amount of viable white blood cells, and the amount of apoptotic cells that have not been eliminated so far. The elimination of apoptotic cells by phagocytes due to their disappearance can be observed with a light microscope.

In some embodiments, culturing of apoptotic cells, such as co-culturing with culturing of white blood cells (e.g., phagocytes (such as macrophages, monocytes, or phagocytes) or B cells, T cells, or NK cells), occurs in culture medium and/or in a physiological solution compatible with administration (e.g., injection) to a subject.

The skilled person will appreciate that "physiological solution" may cover solutions that do not cause the white blood cells to die within the incubation time. In some embodiments, the physiological solution does not cause death within 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours. In other embodiments, 48 hours or 30 hours.

In some embodiments, the white blood cells (e.g., phagocytes (such as macrophages, monocytes, or phagocytes) or B cells, T cells, or NK cells) and the apoptotic cells are incubated in the physiological solution for at least 30 minutes. This incubation time allows for the induction of phagocytosis and secretion of cytokines and other beneficial substances.

In embodiments, such physiological solutions do not inhibit apoptotic leukocyte depletion by leukocyte-derived macrophages.

At the end of the culturing or co-culturing step, the supernatant is optionally isolated from the cultured apoptotic or co-cultured cells. Techniques for separating supernatants from cells are known in the art. For example, the supernatant may be collected and/or filtered and/or centrifuged to eliminate cells and debris. For example, the supernatant can be centrifuged at 3000pm for 15 minutes at room temperature to separate it from the cells.

The supernatant may be "inactivated" prior to use, for example by irradiation. Thus, the method for preparing the apoptotic cell supernatant may comprise an optional further irradiation step f). The "irradiation" step may be considered as a sterilization process using X-ray irradiation (25-45Gy) to kill microorganisms at a sufficient rate, as is conventionally done to inactivate blood products.

Irradiation of the supernatant is considered safe in the art. Currently, irradiation procedures are routinely performed on donated blood to prevent reactions to WBCs.

In embodiments, the apoptotic cell supernatant is formulated into a pharmaceutical composition suitable for administration to a subject as described in detail herein.

In some embodiments, the final product is stored at +4 ℃. In another embodiment, the final product is used over the next 48 hours.

In some embodiments, apoptotic cell supernatants (e.g., apoptotic cell-phagocyte supernatants or pharmaceutical compositions comprising supernatants) may be lyophilized, e.g., stored at-80 ℃.

In one particular example, apoptotic cell-phagocyte supernatant may be prepared using thymocytes as apoptotic cells as described in example 1 of WO 2014/106666. After isolation, thymocytes (e.g., irradiated with 35X gray) are irradiated and cultured in complete DMEM medium for, e.g., 6 hours to allow apoptosis to occur. In parallel, macrophages were isolated from the peritoneal cavity, washed and cultured in complete RPMI (10% FBS, Peni-Strepto, EAA, Hepes, NaP and 2-mercaptoethanol). The macrophages and apoptotic cells were then washed and co-cultured in phenol-free X-vivo medium at a macrophage/apoptotic cell ratio of 1/5 for an additional 48 hour period. The supernatant is then collected, centrifuged to remove debris, and may be frozen or lyophilized for storage. Macrophage enrichment can be confirmed by FACS using F4/80 positive staining. Apoptosis can be confirmed by FACS using annexin-V positive staining and 7AAD exclusion.

In one embodiment, the apoptotic cell supernatant is enriched in the active and potential forms of TGF- β at the level of TGF- β as compared to supernatants obtained from macrophages or apoptotic cells cultured alone. In one embodiment, IL-10 levels are also increased compared to macrophages cultured alone, and levels are significantly increased compared to apoptotic cells cultured alone. In another embodiment, no inflammatory cytokines (e.g., IL-6) are detected, and IL-1 β and TNF are not detected or are present at very low levels.

In the examples, the apoptotic cell supernatants have increased levels of IL-1ra, TIMP-1, CXCL1/KC and CCL2/JE/MCP1 in addition to TGF- β and IL-10 when compared to supernatants from macrophages or apoptotic cells cultured alone, which may be associated with the tolerogenic effects of the supernatants in controlling inflammation.

In another embodiment, human apoptotic cell-phagocyte supernatant may be prepared from a co-culture of macrophages derived from Peripheral Blood Mononuclear Cells (PBMCs) cultured with apoptotic PBMCs, as described in example 3 of WO 2014/106666. Thus, PBMCs are isolated from cell purification bags from healthy volunteers by e.g. Ficoll gradient centrifugation. PBMCs were then plated in complete RPMI medium (10% FBS, 1% penicillin/streptomycin) for 90 minutes. Non-adherent T cells are then removed and allowed to apoptosis, for example using X-ray irradiation at a dose of 35Gy, and cultured in an intact RPMI environment for 4 days (including cell washing after the first 48 hours of culture) to allow apoptosis to occur. In parallel, adherent T cells were cultured for 4 days in intact RPMI environment supplemented with 50 μ g/mL of recombinant human M-CSF, including cell washes after the first 48 hours. At the end of the 4-day culture period, monocyte-derived macrophages and apoptotic cells were washed and cultured together in X-vivo medium for a further 48 hours at a ratio of one macrophage to 5 apoptotic cells. The supernatant from the latter culture is then collected, centrifuged to remove cells and debris, and may be frozen or lyophilized for storage and subsequent use.

In an example, human apoptotic cell-phagocyte supernatant can be obtained from Peripheral Blood Mononuclear Cells (PBMCs) within 6 days as described in WO 2014/106666. PBMC-derived macrophages were obtained four days in culture using M-CSF supplementation and were co-cultured with apoptotic cells for an additional 2 days, which corresponds to non-adherent PBMC isolation at day 0.

In an example, as described in WO 2014/106666, standardized human apoptotic cell-phagocyte supernatants can be obtained independently of the donor or source of PBMCs (cell purification or buffy coat). The plastic adherence step was sufficient to obtain a significant starting population of enriched monocytes (20% to 93% of CD14+ cells after adherence to a plastic dish). In addition, such adherent T cells demonstrated very low presence of B cells and T cells (1.0% CD19+ B cells and 12.8% CD3+ T cells). After 4 days of culture of adherent T cells in the presence of M-CSF, the proportion of monocyte-derived macrophages increased significantly from 0.1% of CD14+ CD206+ HLA-DR + macrophages to 77.7%. At that time, monocyte-derived macrophages can be co-cultured with apoptotic nonadherent PBMCs (47.6% apoptosis as shown by annexin V staining and 7AAD exclusion) to produce apoptotic cell-phagocyte supernatants during 48 hours.

In the examples, the apoptotic-phagocyte supernatant collected contained significantly more latent TGF than monocyte-derived macrophages alone or culture supernatants of monocyte-derived macrophages treated under inflammatory conditions (+ LPS) and contained only trace or low levels of inflammatory cytokines (such as IL-1 β or TNF).

In some embodiments, the composition comprising apoptotic cell supernatant further comprises an anticoagulant. In some embodiments, the anticoagulant is selected from the group consisting of: heparin, Acid Citrate Dextrose (ACD) formula a, and combinations thereof.

In another embodiment, the anticoagulant is added during the process of preparing apoptotic cells. In another embodiment, the added anticoagulant is selected from ACD and heparin or any combination thereof. In another embodiment, the concentration of ACD is 1%. In another embodiment, the concentration of ACD is 2%. In another embodiment, the concentration of ACD is 3%. In another embodiment, the concentration of ACD is 4%. In another embodiment, the concentration of ACD is 5%. In another embodiment, the concentration of ACD is 6%. In another embodiment, the concentration of ACD is 7%. In another embodiment, the concentration of ACD is 8%. In another embodiment, the concentration of ACD is 9%. In another embodiment, the concentration of ACD is 10%. In another embodiment, the concentration of ACD is between about 1-10%. In another embodiment, the concentration of ACD is between about 2-8%. In another embodiment, the concentration of ACD is between about 3-7% in another embodiment, the concentration of ACD is between about 1-5%. In another embodiment, the concentration of ACD is between about 5-10%. In another embodiment, the final concentration of heparin is 0.5U/ml. In another embodiment, the final concentration of heparin is between about 0.1U/ml and 1.0U/ml. In another embodiment, the final concentration of heparin is about 0.2U/ml to 0.9U/ml. In another embodiment, the final concentration of heparin is between about 0.3U/ml and 0.7U/ml. In another embodiment, the final concentration of heparin is between about 0.1U/ml and 0.5U/ml. In another embodiment, the final concentration of heparin is between about 0.5U/ml and 1.0U/ml. In another embodiment, the final concentration of heparin is between about 0.01U/ml and 1.0U/ml. In another embodiment, the final concentration of heparin is 0.1U/ml. In another embodiment, the final concentration of heparin is 0.2U/ml. In another embodiment, the final concentration of heparin is 0.3U/ml. In another embodiment, the final concentration of heparin is 0.4U/ml. In another embodiment, the final concentration of heparin is 0.5U/ml. In another embodiment, the final concentration of heparin is 0.6U/ml. In another embodiment, the final concentration of heparin is 0.7U/ml. In another embodiment, the final concentration of heparin is 0.8U/ml. In another embodiment, the final concentration of heparin is 0.9U/ml. In another embodiment, the final concentration of heparin is 1.0U/ml. In another embodiment, the concentration of ACD is 5% and the final concentration of heparin is 0.5U/ml.

In some embodiments, the composition comprising apoptotic cell supernatant further comprises methylprednisolone. In some embodiments, the methylprednisolone concentration is no more than 30 μ g/ml.

In some embodiments, the composition may be used in a total dose or aliquot of apoptotic cell supernatant derived from about 14 x 10 obtained by cell decontamination⁹A co-culture of CD45+ cells, equaling about 2 million cells per kilogram body weight (subject 70 kg). In an embodiment, such total dose is administered in a unit dose of supernatant derived from about 1 million cells per kilogram body weight, and/or in a unit dose at weekly intervals, in another embodiment, both. A suitable total dose according to this embodiment comprises a total dose derived from supernatant of about 1000 to about 40 million cells per kilogram body weight. In another implementationIn an example, the supernatant is derived from about 4000 to about 10 million cells per kilogram body weight. In another embodiment, the supernatant is derived from about 8000 thousands to about 5 billion cells per kilogram body weight. In another embodiment, the supernatant is derived from about 1.6 to about 2.5 million cells per kilogram body weight. A suitable unit dose according to this embodiment comprises a unit dose derived from supernatant of about 400 to about 4 million cells per kilogram body weight. In another embodiment, the supernatant is derived from about 800 to about 2 million cells per kilogram body weight. In another embodiment, the supernatant is derived from about 1600 to about 1 million cells per kilogram body weight. In yet another embodiment, the supernatant is derived from about 3200 to about 5000 million cells per kilogram body weight.

In another embodiment, administration is from about 10 × 10⁶Dose of apoptotic cell supernatant of co-culture of individual apoptotic cells. In another embodiment, administration is from 10 x 10⁷The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10⁸The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10⁹The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10¹⁰The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10¹¹The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10¹²The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10⁵The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10⁴The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10³The dose of individual apoptotic cells. In another embodiment, administration is from 10 x 10²The dose of individual apoptotic cells.

In some embodiments, administration is from 35 x 10⁶Dose of apoptotic cell supernatant of individual apoptotic cells. In another embodiment, administration is from 210 x 10⁶Dose of individual apoptotic cells. In another embodiment, administration is from 70 x 10 ⁶Dose of individual apoptotic cells. In another embodiment, administration is derived from 140-10⁶Dose of individual apoptotic cells. In another embodiment, administration is from 35-210X 10⁶Dose of individual apoptotic cells.

In some embodiments, the apoptotic cell supernatant or a composition comprising the apoptotic cell supernatant may be administered by any method known in the art as discussed in detail herein (including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intraperitoneally, and directly to the thymus).

Surprisingly, the apoptotic cell supernatant (e.g., apoptotic-phagocyte supernatant) reduces the production of cytokines (e.g., IL-6) associated with cytokine storm. Another cytokine, IL-2, although secreted by DCs and macrophages in small amounts, is not involved in cytokine release syndrome. However, it is required for survival and proliferation of CAR-T cells and is mostly produced by these T cells. Unexpectedly, the apoptotic cell supernatant (e.g., apoptotic-phagocyte supernatant) does not sufficiently reduce IL-2 levels to adversely affect survival of CAR T cells.

In some embodiments, the apoptotic cell supernatant (e.g., apoptotic cell-phagocyte supernatant) affects cytokine expression levels in macrophages and DCs, but does not affect cytokine expression levels in T cells themselves. Thus, unexpectedly, the apoptotic cell supernatant can be used to enhance CAR T cell therapy or dendritic cell therapy.

In another embodiment, apoptotic cell supernatant triggers death of T cells, but not by a change in cytokine expression levels.

In another embodiment, apoptotic cell supernatants (e.g., apoptotic cell-phagocyte supernatants) antagonize the priming of macrophages and dendritic cells to secrete cytokines that would otherwise amplify the cytokine storm. In another embodiment, apoptotic cell supernatant increases tregs that suppress inflammatory responses and/or prevent excessive cytokine release.

In some embodiments, administration of apoptotic cell supernatant (e.g., apoptotic cell-phagocyte supernatant) inhibits one or more proinflammatory cytokines. In some embodiments, the proinflammatory cytokine comprises IL-1 β, IL-6, TNF- α, or IFN- γ, or any combination thereof. In another embodiment, administration of the apoptotic cell supernatant promotes secretion of one or more anti-inflammatory cytokines. In some embodiments, the anti-inflammatory cytokine comprises TGF- β, IL10, or PGE2, or any combination thereof.

In another embodiment, administration of apoptotic cell supernatant (e.g., apoptotic cell-phagocyte supernatant) inhibits dendritic cell maturation following exposure to a TLR ligand. In another embodiment, administration of the apoptotic cell supernatant results in potentially tolerogenic dendritic cells that are capable of migrating in some embodiments, and in some embodiments, migration is due to CCR 7. In another embodiment, administration of apoptotic cell supernatant triggers various signaling events, which in one embodiment are TAM receptor signaling (Tyro3, Axl, and Mer), which in some embodiments inhibit inflammation in antigen presenting cells. In some embodiments, Tyro-3, Axl, and Mer constitute the TAM family of Receptor Tyrosine Kinases (RTKs) characterized by conserved sequences within the kinase domain and the adhesion molecule-like extracellular domain. In another embodiment, administration of the apoptotic cell supernatant activates signaling through MerTK. In another embodiment, administration of apoptotic cell supernatant activates phosphatidylinositol 3-kinase (PI3K)/AKT pathway that in some embodiments negatively regulates NF- κ B. In another embodiment, the administration of the apoptotic cell supernatant down-regulates, in one embodiment, the inflammatory corpuscles that result in inhibition of pro-inflammatory cytokine secretion, DC maturation, or a combination thereof. In another embodiment, administration of the apoptotic cell supernatant upregulates expression of an anti-inflammatory gene (e.g., Nr4a, Thbs1, or a combination thereof). In another embodiment, administration of apoptotic cell supernatant induces high levels of AMP that accumulate in a pannexin 1-dependent manner in some embodiments. In another embodiment, administration of apoptotic cell supernatant suppresses inflammation.

Composition comprising a fatty acid ester and a fatty acid ester

As used herein, the terms "composition" and "pharmaceutical composition" may be used interchangeably in some embodiments, having all the same properties and meanings. In some embodiments, disclosed herein are pharmaceutical compositions for treating a condition or disease as described herein.

In another embodiment, the pharmaceutical compositions disclosed herein are used to maintain or increase the proliferation rate of genetically modified immune cells. In another embodiment, the method for maintaining or increasing the proliferation rate of a genetically modified immune cell further comprises reducing or inhibiting the incidence of Cytokine Release Syndrome (CRS) or cytokine storm. In another embodiment, disclosed herein is a pharmaceutical composition for increasing the efficacy of a genetically modified immune cell therapy. In another embodiment, the composition for use in a method for increasing the efficacy of an immune cell therapy further comprises reducing or inhibiting the incidence of CRS or cytokine storm. In another embodiment, disclosed herein are compositions for use in methods of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating a cancer of a tumor in a subject. In another embodiment, the composition for use in a method for treating, preventing, reducing the incidence, ameliorating or alleviating a cancer or tumor in a subject further comprises reducing or inhibiting the incidence of CRS or cytokine storm.

In another embodiment, the pharmaceutical composition comprises a genetically modified immune cell or a genetically modified receptor thereof. In another embodiment, the genetically modified immune cell comprises a T cell. In another embodiment, the genetically modified immune cell comprises a chimeric antigen receptor CAR T cell. In another embodiment, the genetically modified immune cell comprises a chimeric antigen receptor TCR T cell. In another embodiment, the genetically modified immune cells comprise cytotoxic T lymphocytes. In another embodiment, the genetically modified immune cell comprises a dendritic cell. In another embodiment, the genetically modified immune cell comprises a natural killer cell. In another embodiment, the genetically modified receptor comprises a genetically modified T cell receptor.

In another embodiment, the pharmaceutical composition comprises an early apoptotic cell population. In another embodiment, the pharmaceutical composition comprises an apoptotic supernatant.

In yet another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of a genetically modified immune cell or a genetically modified receptor thereof as described herein in a pharmaceutically acceptable excipient. In another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of a CAR T cell as described herein and a pharmaceutically acceptable excipient. In another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of TCR T cells as described herein and a pharmaceutically acceptable excipient. In another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of cytotoxic T cells as described herein and a pharmaceutically acceptable excipient. In another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of a genetically modified dendritic cell as described herein and a pharmaceutically acceptable excipient. In another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of genetically modified natural killer cells as described herein and a pharmaceutically acceptable excipient. In another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of a genetically modified T cell receptor as described herein and a pharmaceutically acceptable excipient. In yet another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of an early apoptotic cell population as described herein in a pharmaceutically acceptable excipient. In another embodiment, a pharmaceutical composition for treating a condition or disease as described herein comprises an effective amount of an apoptotic supernatant as described herein in a pharmaceutically acceptable excipient.

In another embodiment, the condition or disease as described herein is a tumor or cancer. In another embodiment, disclosed herein is a composition comprising a genetically modified immune cell or receptor thereof (e.g., a CAR T cell) that binds to a protein or peptide of interest as described herein. In another embodiment, disclosed herein is a composition comprising a genetically modified immune cell or receptor thereof (e.g., a TCR T cell) that recognizes and binds a protein or peptide of interest as described herein. In another embodiment, the protein or peptide of interest comprises a tumor antigen or fragment thereof.

In another embodiment, the composition disclosed herein and used in the methods disclosed herein comprises apoptotic cells or an apoptotic cell supernatant and a pharmaceutically acceptable excipient. In some embodiments, a composition comprising apoptotic cells or an apoptotic cell supernatant is used in a method disclosed herein, e.g., for treating, preventing, inhibiting growth, delaying progression of a disease, reducing tumor burden, or reducing the incidence of a cancer or tumor in a subject, or any combination thereof.

In yet another embodiment, the composition comprising an effective amount of a genetically modified immune cell or a genetically modified receptor thereof can be the same as the composition comprising an apoptotic cell population or an apoptotic cell supernatant. In another embodiment, the composition comprising an effective amount of CAR T cells, TCR T cells, or cytotoxic T cells or genetically modified dendritic cells or genetically modified natural killer cells thereof can be the same as the composition comprising the population of apoptotic cells or the supernatant of apoptotic cells. In yet another embodiment, the composition comprising an effective amount of a genetically modified T cell receptor may be the same as the composition comprising a population of apoptotic cells or a supernatant of apoptotic cells. In yet another embodiment, the composition comprising an effective amount of a genetically modified immune cell selected from a CAR T cell, a TCR T cell, a cytotoxic T cell, a natural killer cell, or a dendritic cell may not be the same as the composition comprising an apoptotic cell population or an apoptotic cell supernatant. In another embodiment, a composition comprises a chimeric antigen receptor-expressing T cell (CAR T cell) and apoptotic cells or an apoptotic cell supernatant and a pharmaceutically acceptable excipient. In another embodiment, a composition comprises T cells expressing a genetically modified T cell receptor (TCR T cells) and apoptotic cells or an apoptotic cell supernatant and a pharmaceutically acceptable excipient. In another embodiment, the composition comprising an effective amount of a genetically modified T cell receptor is not the same as the composition comprising an apoptotic cell population or an apoptotic cell supernatant.

In another embodiment, the apoptotic cells included in the composition comprise apoptotic cells in an early apoptotic state. In another embodiment, the apoptotic cells included in the composition are pooled third party donor cells. In another embodiment, apoptotic cell supernatants included in compositions disclosed herein are collected from early apoptotic cells. In another embodiment, apoptotic cell supernatant included in a composition disclosed herein is a pooled third party donor cell collected.

In one embodiment, the composition comprising the genetically modified immune cell (e.g., CAR T cell) further comprises an additional pharmaceutical composition for preventing, suppressing, or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm. In another embodiment, the composition comprising the genetically modified immune cell (e.g., CAR T cell) and the apoptotic cell further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm. In another embodiment, the composition comprising the genetically modified immune cell (e.g., CAR T cell) and apoptotic cell supernatant further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm.

In one embodiment, the composition comprising the genetically modified immune cell (e.g., TCR T cell) further comprises an additional pharmaceutical composition for preventing, suppressing, or modulating cytokine release in a patient having a cytokine release syndrome or experiencing a cytokine storm. In another embodiment, the composition comprising the genetically modified immune cell (e.g., TCR T cell) and the apoptotic cell further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm. In another embodiment, the composition comprising the genetically modified immune cell (e.g., TCR T cell) and apoptotic cell supernatant further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm.

In one embodiment, the composition comprising the genetically modified immune cell (e.g., dendritic cell) further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm. In another embodiment, the composition comprising the genetically modified immune cell (e.g., dendritic cell) and the apoptotic cell further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm. In another embodiment, the composition comprising the genetically modified immune cell (e.g., dendritic cell) and apoptotic cell supernatant further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm.

In one embodiment, the composition comprising the genetically modified immune cell (e.g., NK cell) further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm. In another embodiment, the composition comprising genetically modified immune cells (e.g., NK cells) and apoptotic cells further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm. In another embodiment, the composition comprising genetically modified immune cells (e.g., NK cells) and apoptotic cell supernatant further comprises an additional pharmaceutical composition for preventing, suppressing or modulating cytokine release in a patient suffering from a cytokine release syndrome or experiencing a cytokine storm.

In one embodiment, the additional pharmaceutical composition comprises a CTLA-4 blocking agent, which in one embodiment is ipilimumab. In another embodiment, the additional pharmaceutical composition comprises alpha-1 antitrypsin or a fragment or analog thereof as disclosed herein. In another embodiment, the additional pharmaceutical composition comprises a tellurium-based compound as disclosed herein. In another embodiment, the additional pharmaceutical composition comprises an immunomodulatory agent as disclosed herein. In another embodiment, the additional pharmaceutical composition comprises a CTLA-4 blocking agent, alpha-1 antitrypsin or fragments or analogs thereof, a telluril-based compound, or an immunomodulatory compound, or any combination thereof.

In one embodiment, a composition comprising a genetically modified immune cell (e.g., a CAR T cell) and a pharmaceutical composition comprising any one of a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell, or an apoptotic cell supernatant, a tellurium-based compound, or an immunomodulator comprises a single composition. In another embodiment, a composition comprising a genetically modified immune cell (e.g., a CAR T cell) and a pharmaceutical composition comprising any one of a CTLA-4 blocker, an alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell, or an apoptotic cell supernatant, a telluribased compound or an immunomodulator or any combination thereof comprises a plurality of compositions, wherein each of the genetically modified immune cell (which in one embodiment is a CAR T cell), the CTLA-4 blocker, the alpha-1 antitrypsin or a fragment or analog thereof, the apoptotic cell, or the apoptotic cell supernatant, the telluribased compound or the immunomodulator or any combination thereof is comprised in a separate composition. In yet another embodiment, a composition comprising a genetically modified immune cell (which in one embodiment is a CAR T cell) and a pharmaceutical composition comprising any one of a CTLA-4 blocking agent, alpha-1 antitrypsin or fragments or analogs thereof, apoptotic cells, apoptotic cell supernatants, tellurium-based compounds, or immunomodulators, or any combination thereof, comprises a plurality of compositions, wherein the genetically modified immune cell (which in one embodiment is a CAR T cell), the CTLA-4 blocker, or the alpha-1 antitrypsin or fragment or analog thereof, the tellurium-based compound, or the immunomodulator, or any combination thereof, is present in a genetically modified immune cell (e.g., CAR T cell) composition, and the apoptotic cells or the apoptotic cell supernatant are included in a separate composition.

In one embodiment, a composition comprising a genetically modified immune cell (e.g., a TCR T cell) and a pharmaceutical composition comprising any of a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell, or an apoptotic cell supernatant, a tellurium-based compound, or an immunomodulator comprises a single composition. In another embodiment, a composition comprising a genetically modified immune cell (e.g., a TCR T cell) and a pharmaceutical composition comprising any one of a CTLA-4 blocker, an alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell, or an apoptotic cell supernatant, a tellurium-based compound, or an immunomodulator, or any combination thereof, comprises a plurality of compositions, wherein each of the genetically modified immune cell (which in one embodiment is a TCR T cell), the CTLA-4 blocker, the alpha-1 antitrypsin or a fragment or analog thereof, the apoptotic cell, or the apoptotic cell supernatant, the tellurium-based compound, or the immunomodulator, or any combination thereof, is comprised in a separate composition. In yet another embodiment, a composition comprising a genetically modified immune cell (which in one embodiment is a TCR T cell) and a pharmaceutical composition comprising any one of a CTLA-4 blocking agent, alpha-1 antitrypsin or fragments or analogs thereof, apoptotic cells, apoptotic cell supernatants, tellurium-based compounds or immunomodulators or any combination thereof comprises a plurality of compositions, wherein the genetically modified immune cell (which in one embodiment is a TCR T cell), the CTLA-4 blocker, or the alpha-1 antitrypsin or fragment or analog thereof, the tellurium-based compound, or the immunomodulator, or any combination thereof, is present in a composition of genetically modified immune cells (e.g., TCR T cells), and the apoptotic cells or the apoptotic cell supernatant are included in a separate composition.

In one embodiment, a composition comprising a genetically modified immune cell (e.g., a dendritic cell) and a pharmaceutical composition comprising any of a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell, or an apoptotic cell supernatant, a tellurium-based compound, or an immunomodulator comprises a single composition. In another embodiment, a composition comprising a genetically modified immune cell (e.g., a dendritic cell) and a pharmaceutical composition comprising any one of a CTLA-4 blocker, an alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell, or an apoptotic cell supernatant, a tellurium-based compound, or an immunomodulator, or any combination thereof, comprises a plurality of compositions, wherein each of the genetically modified immune cell (which in one embodiment is a dendritic cell), the CTLA-4 blocker, the alpha-1 antitrypsin or a fragment or analog thereof, the apoptotic cell, or the apoptotic cell supernatant, the tellurium-based compound, or the immunomodulator, or any combination thereof, is comprised in a separate composition. In yet another embodiment, a composition comprising a genetically modified immune cell (which in one embodiment is a dendritic cell) and a pharmaceutical composition comprising any one of a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell supernatant, a tellurium-based compound, or an immunomodulator, or any combination thereof, comprises a plurality of compositions, wherein the genetically modified immune cell (which in one embodiment is a dendritic cell), the CTLA-4 blocker, or the alpha-1 antitrypsin or fragment or analog thereof, the tellurium-based compound, or the immunomodulator, or any combination thereof, is present in a genetically modified immune cell (e.g., dendritic cell) composition, and the apoptotic cells or the apoptotic cell supernatant are included in a separate composition.

In one embodiment, a composition comprising a genetically modified immune cell (e.g., an NK cell) and a pharmaceutical composition comprising any of a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell, or an apoptotic cell supernatant, a tellurium-based compound, or an immunomodulator comprises a single composition. In another embodiment, a composition comprising a genetically modified immune cell (e.g., an NK cell) and a pharmaceutical composition comprising any one of a CTLA-4 blocker, an alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell, or an apoptotic cell supernatant, a tellurium-based compound or an immunomodulator or any combination thereof, wherein each of said genetically modified immune cell (which in one embodiment is an NK cell), said CTLA-4 blocker, said alpha-1 antitrypsin or a fragment or analog thereof, said apoptotic cell, or said apoptotic cell supernatant, said tellurium-based compound or said immunomodulator or any combination thereof is included in a separate composition, comprises a plurality of compositions. In yet another embodiment, a composition comprising a genetically modified immune cell (which in one embodiment is an NK cell) and a pharmaceutical composition comprising any of a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, an apoptotic cell supernatant, a tellurium-based compound, or an immunomodulator, or any combination thereof, comprises a plurality of compositions, wherein the genetically modified immune cell (which in one embodiment is an NK cell), the CTLA-4 blocker, or the alpha-1 antitrypsin or fragment or analog thereof, the tellurium-based compound, or the immunomodulator, or any combination thereof, is present in a genetically modified immune cell (e.g., NK cell) composition, and the apoptotic cells or the apoptotic cell supernatant are included in a separate composition.

In some embodiments, the composition comprises apoptotic cells and an additional agent. In some embodiments, the composition comprises apoptotic cells and an antibody or functional fragment thereof. In some embodiments, the composition comprises an apoptotic cell and RtX antibody or functional fragment thereof. In some embodiments, the apoptotic cells and the antibody or functional fragment thereof may be included in separate compositions. In some embodiments, the apoptotic cells and the antibody or functional fragment thereof may be included in the same composition.

The skilled person will appreciate that "pharmaceutical compositions" may encompass the formulation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compound to an organism.

In some embodiments, disclosed herein are pharmaceutical compositions for treating, preventing, inhibiting the growth of, or reducing the incidence of a cancer or tumor. In some embodiments, disclosed herein are pharmaceutical compositions for increasing survival of a subject having a cancer or tumor. In some embodiments, disclosed herein are pharmaceutical compositions for reducing the size of a tumor or cancer or reducing the growth rate of the tumor or cancer. In some embodiments, disclosed herein are medicaments comprising a pharmaceutical composition for reducing tumor burden in a subject having a cancer or tumor. In some embodiments, disclosed herein are medicaments comprising a pharmaceutical composition for delaying disease progression in a subject having a cancer or tumor. In some embodiments, disclosed herein are medicaments comprising a pharmaceutical composition for reducing the incidence of a cancer or tumor in a subject having the cancer or tumor. In some embodiments, disclosed herein are medicaments comprising a composition for reducing the size of and/or reducing the growth rate of a cancer or tumor in a subject having the cancer or tumor.

In some embodiments, the pharmaceutical composition comprises an early apoptotic cell population as described herein. In some embodiments, the pharmaceutical composition comprises an early apoptotic cell population as described herein and a pharmaceutically acceptable excipient.

The skilled artisan will appreciate that the phrases "physiologically acceptable carrier," "pharmaceutically acceptable carrier," "physiologically acceptable excipient," and "pharmaceutically acceptable excipient" can be used interchangeably and can encompass carriers, excipients, or diluents that do not cause significant irritation to an organism and do not abrogate the biological activity and properties of the administered active ingredient.

The skilled person will appreciate that "excipients" may encompass inert substances added to the pharmaceutical composition to further facilitate administration of the active ingredient. In some embodiments, the excipient comprises calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Techniques for formulating and administering drugs are found in "Remington's Pharmaceutical Sciences," Mack Publishing co., Easton, PA, latest edition, which are incorporated herein by reference.

In some embodiments, the compositions are administered simultaneously. In alternative embodiments, the compositions are administered at different times. In another embodiment, the composition comprising apoptotic cells is administered prior to infusion or genetic modification of the immune cells or their receptors. In another embodiment, the composition comprising apoptotic cells is administered prior to CAR-T cell infusion. In another embodiment, the composition comprising apoptotic cells is administered prior to cytotoxic T cell infusion. In another embodiment, the composition comprising apoptotic cells is administered prior to infusion of natural killer cells. In another embodiment, the composition comprising apoptotic cells is administered prior to dendritic cell infusion. In another embodiment, the composition comprising apoptotic cells is administered prior to infusion of the genetically modified T cell receptor.

In another embodiment, the composition comprising apoptotic cell supernatant is administered prior to infusion or genetically modified immune cells or their recipients. In another embodiment, the composition comprising apoptotic cell supernatant is administered prior to CAR-T cell infusion. In another embodiment, the composition comprising apoptotic cell supernatant is administered prior to cytotoxic T cell infusion. In another embodiment, the composition comprising apoptotic cell supernatant is administered prior to natural killer cell infusion. In another embodiment, the composition comprising apoptotic cell supernatant is administered prior to dendritic cell infusion. In another embodiment, the composition comprising apoptotic cell supernatant is administered prior to infusion of the genetically modified T cell receptor.

In another embodiment, the composition comprising apoptotic cell supernatant is administered prior to infusion of the genetically modified immune cell or its receptor. In another embodiment, the composition comprising apoptotic cells is administered about 24 hours prior to infusion of the genetically modified immune cell or a receptor thereof. In another embodiment, the composition comprising apoptotic cells is administered about 24 hours prior to infusion of CAR T cells or cytotoxic T cells or natural killer cells or dendritic cells or genetically modified T cell receptors. In another embodiment, the composition comprising an apoptotic cell supernatant is administered about 24 hours prior to infusion of the CAR T cells or cytotoxic T cells or natural killer cells or dendritic cells or genetically modified T cell receptors.

In some embodiments, the compositions are administered simultaneously. In alternative embodiments, the compositions are administered at different times. In another embodiment, the composition comprising apoptotic cells is administered prior to administration of the antibody or fragment thereof or the composition comprising said antibody or fragment thereof.

In another embodiment, the composition comprising apoptotic cells is administered about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours before the antibody or fragment thereof or the composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cell supernatant is administered about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours before the antibody or fragment thereof or composition comprising the antibody or fragment thereof.

In another embodiment, the composition comprising apoptotic cells is administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days before the antibody or fragment thereof or the composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cell supernatant is administered about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks before the antibody or functional fragment thereof or the composition comprising said antibody or functional fragment thereof.

In another embodiment, the composition comprising apoptotic cells is administered after infusion of the antibody or fragment thereof or the composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cells is administered after the antibody or fragment thereof or the composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cell supernatant is administered after the administration of the antibody or fragment thereof or the composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cell supernatant is administered after the administration of the antibody or fragment thereof or the composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cells is administered about 24 hours after the antibody or fragment thereof or the composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cells is administered after administration of the antibody or fragment thereof or the composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cells is administered about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours after the administration of the antibody or fragment thereof or composition comprising said antibody or fragment thereof. In another embodiment, the composition comprising apoptotic cell supernatant is administered about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours after the antibody or fragment thereof or composition comprising the antibody or fragment thereof is administered.

In another embodiment, the composition comprising apoptotic cells is administered at about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or 15 days after the antibody or fragment thereof or the composition comprising said antibody or functional fragment thereof. In another embodiment, the composition comprising apoptotic cell supernatant is administered about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks after the antibody or functional fragment thereof or the composition comprising said antibody or functional fragment thereof.

In some embodiments, the composition comprising apoptotic cells is administered independently of CAR T cells. In some embodiments, the composition comprising apoptotic cells is administered in combination with an additional agent. In some embodiments, the additional agent is an antibody.

In some embodiments, the compositions as disclosed herein comprise therapeutic compositions. In some embodiments, a composition as disclosed herein comprises therapeutic efficacy.

In some embodiments, a composition as disclosed herein is administered once. In another embodiment, the composition is applied twice. In another embodiment, the composition is administered three times. In another embodiment, the composition is administered four times. In another embodiment, the composition is administered at least four times. In another embodiment, the composition is administered more than four times.

In some embodiments, the CAR T cell as disclosed herein is administered once. In another embodiment, the CAR T cells are administered twice. In another embodiment, the CAR T cells are administered three times. In another embodiment, the CAR T cells are administered four times. In another embodiment, the CAR T cells are administered at least four times. In another embodiment, the composition is administered more than four times.

In some embodiments, the composition as disclosed herein is a therapeutic composition. In another embodiment, the composition as disclosed herein has therapeutic efficacy.

In some embodiments, disclosed herein are compositions that provide reduced release of inflammatory cytokines or chemokines compared to compositions comprising only CAR T cells, but are considerably cytotoxic compared to compositions comprising only CAR T cells.

Combination therapy/therapy

In some embodiments, disclosed herein are combination therapies comprising the early apoptotic cell populations described herein and one or more cancer therapeutics. In some embodiments, the cancer therapeutic agent comprises a T cell expressing a chimeric antigen receptor (CAR T cell) described herein.

In some embodiments, disclosed herein are combination therapies comprising an early apoptotic cell population and one or more chemotherapeutic agents as detailed herein. In some embodiments, the term "chemotherapeutic agent" may encompass any chemical agent that has therapeutic utility in the treatment of a proliferative disease (such as cancer).

In some embodiments, a "cancer therapeutic agent" or "chemotherapeutic agent" includes a drug selected from one or more of the following: alkylating agents (including mustard, nitrogen mustard, methanesulfonate, busulfan, alkylsulfonate, nitrosourea, ethyleneimine derivatives and triazenes, or combinations thereof); anti-angiogenic agents (including matrix metalloproteinase inhibitors); antimetabolites (including adenosine deaminase inhibitors, folic acid antagonists, purine analogs, and pyrimidine analogs); antibiotics or antibodies (including monoclonal antibodies, CTLA-4 antibodies, anthracyclines); an aromatase inhibitor; a cell cycle response modifier; an enzyme; farnesyl protein transferase inhibitors; hormones and anti-hormonal agents and steroids (including synthetic analogs, glucocorticoids, estrogens/anti-estrogens [ e.g., SERMs ], androgens/anti-androgens, progestins, progesterone receptor agonists, and luteinizing hormone releasing [ LHRH ] agonists and antagonists); insulin-like growth factor (IGF)/insulin-like growth factor receptor (IGFR) system modulators (including IGFR1 inhibitors); an integrin signaling inhibitor; kinase inhibitors (including multi-kinase inhibitors and/or inhibitors of Src kinase or Src/ab1, cyclin-dependent kinase [ CDK ] inhibitors, panHer, Her-1 and Her-2 antibodies, VEGF inhibitors (including anti-VEGF antibodies), EGFR inhibitors, PARP (poly ADP-ribose polymerase) inhibitors, mitogen-activated protein [ MAP ] inhibitors, MET inhibitors, aurora kinase inhibitors, PDGF inhibitors, and other tyrosine kinase or serine/threonine kinase inhibitors, microtubule disruptors, such as ecteinascidins or analogs and derivatives thereof, microtubule stabilizing agents, such as taxanes, platinum antineoplastic agents (platins), e.g., cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthroline, picoplatin and satraplatin, as well as naturally occurring epothilones and synthetic and semisynthetic analogs thereof, microtubule binding, destabilizing agents (including vinca alkaloids); a topoisomerase inhibitor; an isoprene protein transferase inhibitor; a platinum coordination compound; a signal transduction inhibitor; histone Deacetylase Inhibitors (HDI), inhibitors of bromodomains and terminal exomotifs (BET), inhibitors of protein arginine methyltransferase 5(PRMT5), retinoic acid, and other agents useful as anti-cancer and cytotoxic agents, such as biological response modifiers, growth factors, and immunomodulators.

In some embodiments, a "cancer therapeutic agent" or "chemotherapeutic agent" includes an antibody or antigen-binding fragment thereof.

In some embodiments, the methods of using early apoptotic cells as described herein comprise using early apoptotic cells or compositions thereof in combination with an antibody. In some embodiments, the antibody is directed against a tumor cell antigen. In another embodiment, the antibody is directed to CD 20. In another embodiment, the antibody is rituximab (Rtx).

In some embodiments, the early apoptotic cells and the antibody are included in the same composition. In some embodiments, the early apoptotic cells and the antibody are included in different compositions. In some embodiments, the administration of the combination of early apoptotic cells and antibodies, or one or more compositions thereof, is simultaneous. In some embodiments, administration of the combination of early apoptotic cells and antibodies or one or more compositions thereof comprises administering apoptotic cells or compositions thereof prior to the antibodies. In some embodiments, administration of the combination of early apoptotic cells and antibodies or one or more compositions thereof comprises administering apoptotic cells or compositions thereof after administration of the antibodies.

In another embodiment, the antibody is trastuzumab (herceptin; gene tag): humanized IgG1 against ERBB 2. In another embodiment, the antibody is Bevacizumab (Bevacizumab) (Avastin); gene tach/Roche (Roche)): humanized IgG1 directed against VEGF. In another embodiment, the antibody is Cetuximab (Cetuximab) (Erbitux; Bristol-Myers Squibb): chimeric human-murine IgG1 directed against EGFR. In another embodiment, the antibody is Panitumumab (Panitumumab) (Vectibix); Adam): human IgG2 directed against EGFR. In another embodiment, the antibody is Ipilimumab (Ipilimumab) (yirvoy; bevervay, behcet, precious corporation): IgG1 against CTLA 4.

In another embodiment, the antibody is Alemtuzumab (Alemtuzumab) (kanpas (Campath); jianzan (Genzyme)): humanized IgG1 against CD 52. In another embodiment, the antibody is Ofatumumab (Ofatumumab) (Azara; Genmab), human IgG1 directed against CD 20. In another embodiment, the antibody is Gemtuzumab ozogamicin (Gemtuzumab ozogamicin) (Mylotarg; Whitman (Wyeth)): humanized IgG4 against CD 33. In another embodiment, the antibody is a Brentuximab vedotin (Brentuximab vedotin) (adtrass (addetris); Seattle Genetics (Seattle Genetics)): chimeric IgG1 against CD 30. In another embodiment, the antibody is 90Y-labeled tematopimozumab (ibritumomab tiuxetan) (Zevalin); IDEC pharmaceutical): murine IgG1 directed against CD 20. In another embodiment, the antibody is 131I-labeled tositumomab (tositumomab) ((Bexxar); GlaxoSmithKline)): murine IgG2 directed against CD 20.

In another embodiment, the antibody is Ramucirumab (Ramucirumab), which is directed against vascular endothelial growth factor receptor 2 (VEGFR-2). In another embodiment, the antibody is ramucirumab (ramucirumab Injection (Cyramza Injection), liensin (Eli Lilly and Company), brinumomab (BLINCYTO, ann), parboluzumab (pembrolizumab) (kezhuda (KEYTRUDA), merchard Sharp & Dohme Corp.), obilizumab (obinutuzumab) (Jiashiwa (GAZYVA), Gentack, formerly known as GA101), pertuzumab (pertuzumab) Injection (parjietat (PERTA), Gentikka), or denomab (denosumab) (Diknoxia, Xgeva, ann). In another embodiment, the antibody is Basiliximab (sumuleximab) (sulley (simulent); nova (Novartis)). In another embodiment, the antibody is daclizumab (cenipine; roche).

In another embodiment, the antibody administered in combination with apoptotic cells is directed to a tumor or cancer antigen or fragment thereof described herein and/or known in the art. In another embodiment, the antibody is directed against a tumor associated antigen. In another embodiment, the antibody is directed against a tumor associated antigen or a fragment thereof that is an angiogenic factor.

In some embodiments, the antibodies described herein can be used in combination with compositions described herein, such as, but not limited to, compositions comprising early apoptotic cells.

In some embodiments, the combination therapy comprises one or more cancer therapeutic agents. In some embodiments, the combination therapy comprises at least one cancer therapeutic agent. In some embodiments, the combination therapy comprises a cancer therapeutic agent. In some embodiments, the combination therapy comprises two cancer therapeutic agents. In some embodiments, the combination therapy comprises 3 cancer therapeutic agents. In some embodiments, the combination therapy comprises 4 cancer therapeutic agents. In some embodiments, the combination therapy comprises 5 cancer therapeutic agents. In some embodiments, the combination therapy comprises 6 cancer therapeutic agents.

In some embodiments, disclosed herein are combination therapies comprising the early apoptotic cell populations described herein and one or more cancer therapies. In some embodiments, the cancer therapy comprises radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormonal therapy, photodynamic therapy, or surgery, or a combination thereof.

In some embodiments, the cancer therapy comprises a chimeric antigen receptor expressing T cell (CAR T-cell) therapy described herein. In some embodiments, the cancer therapy comprises immunotherapy comprising administering an antibody, or antigen-binding portion thereof, as described herein. In some embodiments, the cancer therapy comprises radiation therapy. In some embodiments, the cancer therapy comprises chemotherapy. In some embodiments, the cancer therapy comprises transplantation. In some embodiments, the cancer therapy comprises immunotherapy. In some embodiments, the cancer therapy comprises a targeted therapy. In some embodiments, the cancer therapy comprises hormone therapy. In some embodiments, the cancer therapy comprises photodynamic therapy. In some embodiments, the cancer therapy comprises surgery.

Formulation (recipe)

The pharmaceutical compositions disclosed herein, including the population of early apoptotic cells, may conveniently be provided in the form of sterile liquid formulations (e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions) which may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, administration of liquid compositions (especially by injection) is somewhat more convenient. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact periods with a particular tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the early apoptotic cell populations described herein and used in the practice of the methods disclosed herein in the appropriate solvent in the required amount, along with various amounts of other ingredients, as desired. Such compositions may be mixed with suitable carriers, diluents or excipients (e.g., sterile water, physiological saline, glucose, dextrose, and the like). The composition may also be lyophilized. Depending on the route of administration and the desired formulation, the compositions may contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents, and the like. Standard text (e.g., Remington' S PHARMACEUTICAL SCIENCEs, 17 th edition, 1985, incorporated herein by reference) can be queried to prepare suitable formulations without undue experimentation.

Various additives may be added that enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. However, any vehicle, diluent or additive used will have to be compatible with the genetically modified immunoresponsive cell or its progenitor cells in accordance with the disclosure herein.

The compositions or formulations disclosed herein may be isotonic, i.e., they may have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions as disclosed herein can be achieved using sodium chloride or other pharmaceutically acceptable agents (such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes). Sodium chloride is particularly preferred for buffers containing sodium ions.

If desired, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. Methylcellulose may be preferred because it is readily available and economically viable and easy to work with.

Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomers (carbomers), and the like. The preferred concentration of the thickening agent will depend on the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of a suitable carrier and other additives will depend on the particular route of administration and the nature of the particular dosage form (e.g., liquid dosage form) (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a time-release form or liquid-filled form).

One skilled in the art will recognize that the components of the composition or formulation should be selected to be chemically inert and will not affect the viability or efficacy of the early apoptotic cell population as disclosed herein for use in the methods disclosed herein. This will be no problem to the skilled person of chemical and pharmaceutical principles, or it can be easily avoided by reference to standard texts or by simple experiments (without involving undue experimentation) from the present disclosure and the references cited herein.

One consideration regarding the therapeutic use of the genetically modified immunoresponsive cells disclosed herein is the number of cells required to achieve optimal results. The number of cells to be administered will vary for the subject being treated. In some embodiments, between 10 is administered to a human subject ⁴To 10¹⁰Between 10⁵To 10⁹Or between 10⁶An and 10⁸In between the genetically modified immunoresponsive cells disclosed herein. More potent cells can be administered in even smaller numbers. In some embodiments, at least about 1 x 10 is administered to a human subject ⁸2, 2 x 10⁸2, 3 x 10⁸4 x 10 pieces⁸Sum of 5 xl 0⁸A genetically modified immunoresponsive cell disclosed herein. The precise determination of the dose to be considered an effective dose can be based on individual factors per subject, including its size, age, sex, weight and condition of the particular subject. Dosages can be readily determined by those skilled in the art from the present disclosure and knowledge in the art.

The amount of cells and optional additives, vehicles, and/or carriers in the composition can be readily determined by one skilled in the art and administered in the methods disclosed herein. Generally, any additives (other than one or more active cells and/or one or more pharmaceutical agents) are present in phosphate buffered saline in an amount of 0.001% to 50% by weight solution, and the active ingredient is present in the order of micrograms to milligrams (e.g., about 0.0001 to about 5% by weight). In another embodiment, from about 0.0001 to about 1 wt%. In yet another embodiment, from about 0.0001 to about 0.05 wt%, or from about 0.001 to about 20 wt%. In further embodiments, from about 0.01 to about 10 wt%. In another embodiment, from about 0.05 to about 5 wt%. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine: toxicity, such as by determining the Lethal Dose (LD) and LD50 in a suitable animal model (e.g., a rodent, such as a mouse); and determining the dosage of the one or more compositions, the concentration of the components therein, and the timing of administration of the one or more compositions to elicit a suitable response. Such assays do not require undue experimentation based on the knowledge of those skilled in the art, the present disclosure, and the references cited herein. Also, the timing of sequential administration can be determined without undue experimentation.

Nucleic acid sequences, vectors, cells

In some embodiments, disclosed herein are isolated nucleic acid sequences encoding a Chimeric Antigen Receptor (CAR) as described herein for use in the compositions and methods as disclosed herein.

In another embodiment, disclosed herein is a vector comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) as described herein.

In some embodiments, disclosed herein are isolated nucleic acid sequences encoding a genetically modified T Cell Receptor (TCR) as described herein for use in the compositions and methods as disclosed herein. In another embodiment, disclosed herein is a vector comprising a nucleic acid sequence encoding a genetically modified T Cell Receptor (TCR) as described herein.

Genetic modification of immune responsive cells (e.g., T cells, CTL cells, NK cells) can be accomplished by transducing a substantially homogeneous composition of cells with a recombinant DNA construct. In some embodiments, a retroviral vector (gamma-retrovirus or lentivirus) is used to introduce the DNA construct into the cell. For example, a polynucleotide encoding a receptor for a binding antigen (e.g., a tumor antigen or variant or fragment thereof) can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific for a target T cell type of interest. Non-viral vectors may also be used.

Non-viral methods may also be used for expression of proteins in cells. Nucleic acid molecules can be introduced into cells, for example, by microinjection in the presence of lipofection (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413,1987; Ono et al, Neuroscience Letters 17:259,1990; Brigham et al, J.Med. Sci. North America, 298:278,1989; Staubinger et al, Methods in Enzymology 101:512,1983), asialoglycoprotein-polylysine conjugate (Wu et al, Journal of Biochemical Chemistry 263:14621,1988; Wu et al, biochemistry 264:16985,1989) or under surgical conditions (Wolff et al, Science 247:1465,1990). Other non-viral means of gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Liposomes may also have potential benefits for delivery of DNA into cells. Transplantation of normal genes into the affected tissue of a subject can also be accomplished by ex vivo transfer of normal nucleic acids into culturable cell types (e.g., autologous or heterologous primary cells or progeny thereof), followed by injection of the cells (or progeny thereof) into the targeted tissue or systemic injection. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression can be obtained by RNA electroporation. cDNA expression for polynucleotide therapy methods can be directed from any suitable promoter (e.g., human Cytomegalovirus (CMV), simian virus 40(SV40), or metallothionein promoters) and regulated by any suitable mammalian regulatory elements or introns (e.g., elongation factor Ia enhancer/promoter/intron constructs). For example, enhancers known to preferentially direct gene expression in a particular cell type can be used to direct expression of a nucleic acid, if desired. The enhancer used may include, but is not limited to, those characterized as tissue or cell specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation may be mediated by homologous regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source comprising any of the promoters or regulatory elements described above.

In another embodiment, disclosed herein is a cell comprising a vector comprising a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) as disclosed herein.

Method of use

In some embodiments, disclosed herein are methods of slowing, reducing, inhibiting, or eliminating metastatic spread of a cancer or tumor, or any combination thereof, in a subject comprising the step of administering to the subject a combination therapy described herein comprising an early apoptotic cell population and one or more cancer therapeutic agents. In some embodiments, the cancer therapeutic comprises a CAR T cell. In some embodiments, the cancer therapeutic comprises an antibody or antigen-binding portion thereof. In some embodiments, the method slows, reduces, inhibits, or eliminates metastatic spread of the cancer or tumor, or any combination thereof, in the subject compared to a subject not administered the early apoptotic cell population.

The skilled person will understand that the term "metastasis" encompasses the spread of cancer cells from where they first form to another part of the body. During metastasis, cancer cells detach from the original (primary) tumor, pass through the blood or lymphatic system, and form new tumors in other organs or tissues of the body. In some embodiments, the new metastatic tumor is of the same type of cancer as the primary tumor. For example, if breast cancer spreads to the lung, the cancer cells in the lung are breast cancer cells, not lung cancer cells.

The skilled artisan will be familiar with methods for determining the effectiveness of slowing, reducing, inhibiting or eliminating metastatic spread. For example, imaging methods can be used to determine whether a tumor of a subject has spread to a new or second part or organ of the body.

In some embodiments, disclosed herein is a method of slowing, reducing, inhibiting, or eliminating metastatic spread of a cancer or tumor, or any combination thereof, in a subject undergoing cancer therapy, the method comprising the step of administering to the subject an early apoptotic cell population, wherein the method slows, reduces, inhibits, or eliminates metastatic spread of a cancer or tumor, or any combination thereof, in the subject as compared to a subject that did not undergo cancer therapy and was not administered an early apoptotic cell population. In some embodiments, the cancer therapy comprises radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormonal therapy, photodynamic therapy, or surgery, or a combination thereof. In some embodiments, the cancer therapy comprises CAR T cell therapy. In some embodiments, the cancer therapy comprises radiation therapy. In some embodiments, the cancer therapy comprises chemotherapy. In some embodiments, the cancer therapy comprises transplantation. In some embodiments, the cancer therapy comprises immunotherapy. In some embodiments, the cancer therapy comprises a targeted therapy. In some embodiments, the cancer therapy comprises hormone therapy. In some embodiments, the cancer therapy comprises photodynamic therapy. In some embodiments, the cancer therapy comprises surgery.

In some embodiments, a subject undergoing cancer therapy comprises a subject being treated with chimeric antigen receptor T cell (CAR T-cell) therapy, radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormone therapy, photodynamic therapy, or has been previously treated with chimeric antigen receptor T cell (CAR T-cell) therapy, radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormone therapy, photodynamic therapy, has undergone surgical treatment, or any combination thereof.

In some embodiments, the cancer therapy comprises CAR T cell therapy. In some embodiments, the cancer therapy comprises radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormonal therapy, photodynamic therapy, having undergone surgical treatment, or any combination thereof.

In some embodiments, slowing, reducing, inhibiting, or eliminating metastatic spread of the cancer or tumor comprises stabilizing resident macrophages in the region of the cancer or tumor. In some embodiments, slowing, reducing, inhibiting, or eliminating metastatic spread of the cancer or tumor comprises increasing the number of resident macrophages in the region of the cancer or tumor. In some embodiments, slowing, reducing, inhibiting, or eliminating metastatic spread of the cancer or tumor comprises increasing the population of anti-tumor macrophages in the cancer or tumor region.

In some embodiments, slowing, reducing, inhibiting, or eliminating metastatic spread of the cancer or tumor comprises reducing the incidence of metastasis to lymph nodes. In some embodiments, slowing, reducing, inhibiting, or eliminating metastatic spread of the cancer or tumor comprises reducing the incidence of angiogenesis. In some embodiments, slowing, reducing, inhibiting, or eliminating the cancer or metastatic spread of the cancer comprises reducing the metastatic rate of the tumor or the cancer. In some embodiments, slowing, reducing, inhibiting, or eliminating the metastatic spread of the cancer or tumor comprises reducing the formation of cancer cells with metastatic potential. In some embodiments, slowing, reducing, inhibiting, or eliminating metastatic spread of the cancer or tumor comprises reducing migration of cancer cells.

In some embodiments, slowing, reducing, inhibiting, or eliminating the metastatic spread of the cancer or tumor comprises a 10% -100% reduction in the incidence of metastasis. In some embodiments, slowing, reducing, inhibiting, or eliminating metastatic spread of the cancer or tumor comprises reducing the incidence of metastasis by up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

In some embodiments, the cancer therapeutic comprises a T cell expressing a chimeric antigen receptor (CAR T cell). In some embodiments, the CAR T cell and the early apoptotic cell are included in separate compositions. In some embodiments, the CAR T cells and early apoptotic cells are administered together. In some embodiments, the CAR T cells and the early apoptotic cells are administered simultaneously. In some embodiments, the CAR T cells are administered prior to early apoptotic cells. In some embodiments, the CAR T cells are administered after administration of the early apoptotic cells.

In some embodiments, administration of the combination therapy comprising the early apoptotic cell population and the one or more cancer therapeutics occurs at the same site of administration. In some embodiments, the early apoptotic cell population and the one or more cancer therapeutics are administered at different sites of administration. In some embodiments, the population of early apoptotic cells and the one or more cancer therapeutics are administered intravenously to the subject. In some embodiments, the population of early apoptotic cells and the one or more cancer therapeutics are administered orally to the subject.

In some embodiments, the one or more cancer therapeutic agents are administered days before administration of the early apoptotic cell population. In some embodiments, the one or more cancer therapeutic agents are administered 1, 2, 3, 4, or 5 days prior to administration of the early apoptotic cell population.

In some embodiments, the one or more cancer therapeutic agents are administered days after administration of the early apoptotic cell population. In some embodiments, the one or more cancer therapeutic agents are administered 1, 2, 3, 4, or 5 days after administration of the early apoptotic cell population.

In some embodiments, the early apoptotic cell population and the one or more cancer therapeutics are administered at least once during a treatment cycle. In some embodiments, the early apoptotic cell population and the one or more cancer therapeutics are administered to the subject on the same day. In some embodiments, the early apoptotic cell population and the one or more cancer therapeutics are administered to the subject on different days. In some embodiments, the early apoptotic cell population and the one or more cancer therapeutic agents are administered to the subject on the same day or within different days according to a treatment regimen (or treatment cycle).

In some embodiments, the treatment cycle comprises at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, at least 21 days, at least 28 days, at least 48 days, or at least 96 days or more. In one embodiment, the treatment cycle is 7 days. In one embodiment, the treatment cycle is 14 days. In one embodiment, the treatment cycle is 28 days. In some embodiments, the population of early apoptotic cells and the one or more cancer therapeutic agents are administered within the same treatment cycle or concurrently administered within different treatment cycles assigned to each. In some embodiments, the treatment period is determined by a healthcare professional according to the condition and need of the subject.

In some embodiments, the early apoptotic cell population and the one or more cancer therapeutic agents are administered on at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, or all 28 days of a 28 day treatment cycle. In some embodiments, the early apoptotic cell population and the one or more cancer therapeutics are administered to the subject once a day. In some embodiments, the early apoptotic cell population and the one or more cancer therapeutics are administered twice a day.

In some embodiments, the early apoptotic cell population and the one or more cancer therapeutics are administered to the subject within one or more treatment cycles. In some embodiments, the subject is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. In some embodiments, the early apoptotic cell population and one or more cancer therapeutics described herein are administered for several years. In some embodiments, the population of early apoptotic cells and one or more cancer therapeutics described herein are administered between one or up to six months.

The number of times the population of early apoptotic cells and the one or more cancer therapeutic are administered to a subject in need thereof depends on the judgment of the medical professional, the disorder, the severity of the disorder, and the subject's response to the combination therapy. In the case where the disorder in the subject is not ameliorated, the early apoptotic cell population and the one or more cancer therapeutic agents are administered chronically, i.e., for an extended period of time, including throughout the life cycle of the subject, at the discretion of the physician, to slow, reduce, inhibit or eliminate metastatic spread of the cancer or tumor, or otherwise control or limit the symptoms of the disease or disorder in the subject.

Where the subject's condition does improve, the early apoptotic cell population and the one or more cancer therapeutic are administered sequentially, based on the judgment of a physician; or the dose administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday varies between 2 days and 1 year, including, for example, only 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dose reduction during a drug holiday can be 10% -100%, including for example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

In one embodiment, disclosed herein is a method for treating, preventing, inhibiting, reducing the incidence, ameliorating, or alleviating a cancer or tumor, comprising the step of administering a composition as disclosed herein.

In some embodiments, disclosed herein are methods of treating, preventing, inhibiting, reducing the incidence, ameliorating, or alleviating a cancer or tumor in a subject, comprising the step of administering a composition comprising apoptotic cells. In some embodiments, disclosed herein are methods of treating, preventing, inhibiting the growth of, delaying the progression of, reducing the burden of, or reducing the incidence of a cancer or tumor in a subject, or any combination thereof. In some embodiments, the methods disclosed herein reduce the size of a tumor or cancer and/or reduce the growth rate of the tumor or cancer. In some embodiments, the methods disclosed herein increase survival of a subject having a tumor or cancer. In some embodiments, the use of apoptotic cells or compositions thereof increases the efficacy of genetically modified immune cell therapies (such as, but not limited to, CAR T cell therapies).

In another embodiment, disclosed herein is a method for treating, preventing, inhibiting, reducing the incidence, ameliorating, or alleviating a cancer or tumor, the method comprising the step of administering a genetically modified immune cell and a composition comprising an additional agent, wherein the additional agent comprises apoptotic cells, a supernatant from apoptotic cells, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof, wherein the method treats, prevents, inhibits, reduces the incidence, ameliorates, or alleviates a cancer or tumor in a subject compared to a subject administered the genetically modified immune cell and not administered the additional agent. In another embodiment, the genetically modified immune cell comprises a genetically modified T cell, cytotoxic T cell, Treg cell, effector T cell, helper T cell, NK cell, or dendritic cell.

In another embodiment, disclosed herein is a method for treating, preventing, inhibiting, reducing the incidence, ameliorating, or alleviating a cancer or tumor, the method comprises the step of administering T cells expressing a chimeric antigen receptor (CAR T cells) and a composition comprising an additional agent, wherein the additional agent comprises apoptotic cells, supernatant from apoptotic cells, a CTLA-4 blocking agent, alpha-1 antitrypsin or fragments or analogues thereof, a tellurium-based compound or an immunomodulator or any combination thereof, wherein in comparison to a subject administered the genetically modified immune cell and not administered the additional agent, the methods treat, prevent, inhibit, reduce the incidence, ameliorate, or alleviate a cancer or tumor in the subject.

In another embodiment, disclosed herein is a method for treating, preventing, inhibiting, reducing the incidence, ameliorating, or alleviating a cancer or tumor, the method comprising the step of administering a genetically modified T cell receptor cell (TCR T cell) and a composition comprising an additional agent, wherein the additional agent comprises apoptotic cells, supernatant from apoptotic cells, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound or an immunomodulator or any combination thereof, wherein in comparison to a subject administered the genetically modified immune cell and not administered the additional agent, the methods treat, prevent, inhibit, reduce the incidence, ameliorate, or ameliorate the cancer or tumor in the subject.

In another embodiment, administration of apoptotic cells or apoptotic supernatant or composition thereof does not result in said administration of T cells expressing chimeric antigen receptors for treating, preventing, inhibiting, reducing the incidence, ameliorating or alleviating a cancer or tumor with reduced efficacy. In another embodiment, administration of an additional agent or composition thereof comprising apoptotic cells, supernatant from apoptotic cells, CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, tellurium-based compound or immunomodulator, or any combination thereof, does not result in said administration of T cells expressing chimeric antigen receptors for treating, preventing, inhibiting, reducing the incidence, ameliorating or ameliorating the efficacy of a cancer or tumor.

In another embodiment, administration of apoptotic cells or apoptotic supernatant or composition thereof results in increased efficacy of said administration of chimeric antigen receptor-expressing T cells for treating, preventing, inhibiting, reducing the incidence, ameliorating or alleviating a cancer or tumor. In another embodiment, administration of an additional agent or composition thereof comprising apoptotic cells, supernatant from apoptotic cells, CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, tellurium-based compound or immunomodulator, or any combination thereof increases the efficacy of said administration of chimeric antigen receptor-expressing T cells for treating, preventing, inhibiting, reducing the incidence, ameliorating or alleviating a cancer or tumor.

In one embodiment, a method of increasing the efficacy of a genetically modified immune cell cancer therapy comprises administering the genetically modified immune cell and an additional agent comprising an apoptotic cell, a supernatant from an apoptotic cell, a CTLA-4 blocker, an alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof, or a composition thereof, wherein the efficacy is increased compared to a subject not administered the additional agent. 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 naive CD4 ⁺T cells. In another embodiment, the T cell is a naive T cell. In another embodiment, the T cell is naive CD8⁺T cells. In another embodiment, the genetically modified immune cell is a Natural Killer (NK) cell. In another embodiment, the genetically modified immune cell is a dendritic cell. In yet another embodiment, the genetically modified T cell is a cytotoxic T lymphocyte (CTL cell). In another embodiment, the genetically modified T cell is a regulatory T cell (Treg). In another embodiment, the genetically modified T cell is a Chimeric Antigen Receptor (CAR) T cell. In another embodiment, the genetically modified T cell is a genetically modified T cell receptor cell (TCR T cell). In another embodiment, a method of increasing the efficacy of a CAR T cell cancer therapy comprises administering the genetically modified immune cell and an additional agent or composition thereof comprising an apoptotic cell, a supernatant from an apoptotic cell, a CTLA-4 blocker, an alpha-1 antitrypsin or fragment or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof, wherein the efficacy is increased compared to a subject not administered the additional agent.

In another embodiment, the methods herein reduce the level of production of at least one pro-inflammatory cytokine compared to the level of said pro-inflammatory cytokine in a subject receiving an immune cancer therapy without administration of an additional agent. In another embodiment, the methods herein inhibit or reduce the incidence of a cytokine release syndrome or cytokine storm in a subject undergoing a genetically modified immune cell cancer therapy and not administered an additional agent.

In another embodiment, the methods disclosed herein reduce IL-6.

In another embodiment, the methods herein increase the production of at least one cytokine compared to the level of the cytokine in a subject receiving an immune cancer therapy and not administered an additional agent. In some embodiments, the additional agent is an apoptotic cell, and in other embodiments, the additional agent is an apoptotic cell supernatant. In another embodiment, the methods disclosed herein increase IL-2.

The skilled person will understand that the term "production" as used herein in relation to a cytokine may encompass the expression of the cytokine as well as the secretion of the cytokine from a cell. In one embodiment, increased production of the cytokine results in increased secretion of the cytokine from the cell. In alternative embodiments, decreased cytokine production results in decreased secretion of cytokines from the cell. In another embodiment, the methods disclosed herein reduce the secretion of at least one cytokine. In another embodiment, the methods disclosed herein reduce the secretion of IL-6. In another embodiment, the methods disclosed herein increase the secretion of at least one cytokine. In another embodiment, the methods disclosed herein increase the secretion of IL-2.

In another embodiment, the cell that secretes the at least one cytokine is a tumor cell. In another embodiment, the cell that secretes the at least one cytokine is a T cell. In another embodiment, the cell that secretes the at least one cytokine is an immune cell. In another embodiment, the cell that secretes the at least one cytokine is a macrophage. In another embodiment, the cell that secretes the at least one cytokine is a B cell lymphocyte. In another embodiment, the cell that secretes at least one cytokine is a mast cell. In another embodiment, the cell that secretes the at least one cytokine is an endothelial cell. In another embodiment, the cell that secretes the at least one cytokine is a fibroblast. In another embodiment, the cell that secretes the at least one cytokine is a stromal cell. The skilled artisan will recognize that the level of a cytokine in a cytokine-secreting cell may be increased or decreased depending on the environment surrounding the cell.

In yet another embodiment, the additional agent used in the methods disclosed herein increases the secretion of at least one cytokine. In yet another embodiment, the additional agent used in the methods disclosed herein maintains the secretion of at least one cytokine. In yet another embodiment, the additional agent used in the methods disclosed herein does not decrease the secretion of at least one cytokine. In another embodiment, the additional agent used in the methods disclosed herein increases the secretion of IL-2. In another embodiment, the additional agent used in the methods disclosed herein increases the secretion of IL-2R. In another embodiment, the additional agent used in the methods disclosed herein maintains the level of secretion of IL-2. In another embodiment, the additional agent used in the methods disclosed herein maintains the level of secretion of IL-2R. In another embodiment, the additional agent used in the methods disclosed herein does not decrease the level of secretion of IL-2R. In another embodiment, the additional agent used in the methods disclosed herein maintains or increases the level of secretion of IL-2. In another embodiment, the additional agent used in the methods disclosed herein maintains or increases the level of secretion of IL-2R. In another embodiment, the additional agent used in the methods disclosed herein does not decrease the level of secretion of IL-2R.

In still further embodiments, the additional agent used in the methods disclosed herein reduces the secretion of IL-6. In another embodiment, the additional agent used in the methods disclosed herein maintains, increases, or does not decrease the level of secretion of IL-2 while decreasing the secretion of IL-6. In another embodiment, the additional agent used in the methods disclosed herein maintains, increases, or does not decrease the level of secretion of IL-2R while decreasing the secretion of IL-6.

In one embodiment, a method of increasing the efficacy of a CAR T cell cancer therapy disclosed herein comprises decreasing IL-6 levels in the subject, the method comprising administering a CAR T cell and an additional agent or composition thereof comprising an apoptotic cell, a supernatant from an apoptotic cell, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound or an immunomodulator, or any combination thereof, wherein the efficacy is increased compared to a subject not administered the additional agent. In another embodiment, a method of increasing the efficacy of a CAR T cell cancer therapy disclosed herein comprises increasing IL-2 levels in the subject, the method comprising administering a CAR T cell and an additional agent or composition thereof comprising an apoptotic cell, a supernatant from an apoptotic cell, a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound or an immunomodulator, or any combination thereof, wherein the efficacy is increased compared to a subject not administered the additional agent. In another embodiment, a method of increasing the efficacy of a CAR T cell cancer therapy disclosed herein comprises increasing proliferation of said CAR T cell, said method comprising administering a CAR T cell and an additional agent or composition thereof comprising an apoptotic cell, a supernatant from an apoptotic cell, a CTLA-4 blocker, an alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound or an immunomodulator, or any combination thereof, wherein said efficacy and proliferation of said CAR T cell is increased compared to a subject not administered said additional agent.

In one embodiment, a method of increasing the efficacy of a CAR T cell cancer therapy, comprising the step of administering a composition comprising a CAR T cell and a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound or an immunomodulator, or any combination thereof or composition thereof, reduces or inhibits cytokine production in a subject. In another embodiment, a method for treating, preventing, inhibiting, reducing the incidence, ameliorating, or ameliorating a cancer or tumor, comprising the step of administering a composition comprising a CAR T cell and a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof or combination thereof, or a composition thereof, reduces or inhibits cytokine production in a subject.

In another embodiment, disclosed herein are methods of treating a cytokine release syndrome or cytokine storm in a subject undergoing CAR T cell cancer therapy.

In another embodiment, a method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or ameliorating a cancer or tumor, comprising the step of administering a composition comprising a CAR T cell and a CTLA-4 blocker, alpha-1 antitrypsin or a fragment or analog thereof, a tellurium-based compound, or an immunomodulator, or any combination thereof or combination thereof, or a composition thereof, reduces or inhibits cytokine production in a subject.

In another embodiment, disclosed herein is a method of treating cancer or tumor in a subject comprising the step of administering to the subject any of the compositions as described herein. In another embodiment, disclosed herein is a method of preventing cancer or tumor in a subject, the method comprising the step of administering to the subject any of the compositions as described herein. In another embodiment, disclosed herein is a method of inhibiting a cancer or tumor in a subject, the method comprising the step of administering to the subject any of the compositions as described herein. In another embodiment, disclosed herein is a method of reducing cancer or tumor in a subject, comprising the step of administering to the subject any of the compositions as described herein. In another embodiment, disclosed herein is a method of ameliorating a cancer or tumor in a subject, the method comprising the step of administering to the subject any of the compositions as described herein. In another embodiment, disclosed herein is a method of ameliorating a cancer or tumor in a subject, the method comprising the step of administering to the subject any of the compositions as described herein.

In one embodiment, disclosed herein is a method of maintaining or increasing the rate of proliferation of a genetically modified immune cell during immunotherapy, the method comprising the step of administering a composition comprising apoptotic cells or an apoptotic supernatant during immunotherapy. In another embodiment, the genetically modified immune cell comprises a T cell, a naive CD4+ T cell, a naive CD8+ T cell, a Natural Killer (NK) cell, a dendritic cell, a cytotoxic T lymphocyte (CTL cell), a regulatory T cell (Treg), a Chimeric Antigen Receptor (CAR) T cell, or a genetically modified T Cell Receptor (TCR) cell. In another embodiment, disclosed herein is a method of maintaining or increasing the rate of proliferation of CAR T cells during immunotherapy, the method comprising the step of administering a composition comprising apoptotic cells or an apoptotic supernatant during immunotherapy.

In another embodiment, the method of maintaining or increasing the proliferation rate of a genetically modified immune cell does not reduce or inhibit the efficacy of immunotherapy. For example, in another embodiment, the method of maintaining or increasing the proliferation rate of CAR T cells does not decrease or inhibit the efficacy of the CAR T cell cancer therapy. In another embodiment, the method of maintaining or increasing the rate of proliferation of a genetically modified immune cell (e.g., a CAR T cell) reduces or inhibits cytokine production in a subject.

In another embodiment, the compositions and methods as disclosed herein utilize combination therapy with apoptotic cells or apoptotic supernatants as disclosed herein and one or more CTLA-4 blockers (such as ipilimumab). In one embodiment, CTLA-4 is a potent inhibitor of T cell activation that helps maintain self-tolerance. In one embodiment, administration of an anti-CTLA-4 blocking agent (which in another embodiment is an antibody) results in a net effect of T cell activation. In another embodiment, the compositions and methods disclosed herein utilize a combination therapy comprising apoptotic cells, CAR T cells, and one or more CTLA-4 blockers.

In some cases, the polypeptides disclosed herein and the polypeptides used in the methods disclosed herein include at least one conservative amino acid substitution relative to the unmodified amino acid sequence. In other cases, the polypeptide includes non-conservative amino acid substitutions. In such cases, polypeptides having such modifications exhibit increased stability or longer half-life relative to polypeptides lacking such amino acid substitutions.

In some embodiments, "treating" includes therapeutic treatment, and "preventing" includes prophylactic (preventative) measures, wherein the object is to prevent or alleviate the targeted pathological condition or disorder as described above. Thus, in some embodiments, treating may comprise directly affecting or curing, suppressing, inhibiting, preventing, lessening the severity of a disease, disorder, or condition, delaying the onset of the disease, disorder, or condition, reducing symptoms associated with the disease, disorder, or condition, or a combination thereof. Thus, in some embodiments, "treating," "improving," and "ameliorating" refer, inter alia, to delaying progression, accelerating remission, inducing remission, increasing remission, accelerating recovery, increasing efficacy, or decreasing resistance to alternative therapy, or a combination thereof. In some embodiments, "preventing" refers to, inter alia, delaying the onset of symptoms, preventing disease recurrence, reducing the number or frequency of recurring events, increasing the time delay between symptomatic events, or a combination thereof. In some embodiments, "suppressing" or "inhibiting" refers to, inter alia, reducing the severity of a symptom, reducing the severity of an acute event, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the time delay of a symptom, ameliorating a symptom, reducing a secondary infection, extending the survival of a patient, or a combination thereof.

The skilled artisan will appreciate that the term "antigen recognizing receptor" can encompass receptors capable of activating immune cells (e.g., T cells) in response to antigen binding. Exemplary antigen recognition receptors can be native or endogenous T cell receptors or chimeric antigen receptors in which a tumor antigen binding domain is fused to an intracellular signaling domain capable of activating an immune cell (e.g., a T cell).

In some embodiments, the methods described herein increase survival of a subject having a cancer or tumor, and comprise administering to the subject a population of early apoptotic cells, wherein the method increases survival of the subject.

The skilled artisan will appreciate that the term "disease" can encompass any condition or disorder that impairs or interferes with the normal function of a cell, tissue or organ. Examples of diseases include neoplasia or pathogen infection of a cell.

The skilled person will appreciate that the term "effective amount" may encompass an amount sufficient to have a therapeutic effect. In some embodiments, an "effective amount" is an amount sufficient to prevent, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasia.

The skilled artisan will appreciate that the term "neoplasia" refers to a disease characterized by pathological proliferation of cells or tissues and their subsequent migration to or invasion of other tissues or organs. Neoplasia growth is generally uncontrolled and progressive, and occurs under conditions that will not initiate or will cause cessation of normal cell proliferation. Neoplasias may affect a variety of cell types, tissues or organs, including but not limited to organs selected from the group consisting of: bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, gall bladder, heart, intestine, kidney, liver, lung, lymph node, neural tissue, ovary, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof. Neoplasias include cancers such as sarcomas, carcinomas or plasmacytomas (malignant tumors of plasma cells).

The skilled person will appreciate that the term "pathogen" may encompass viruses, bacteria, fungi, parasites or protozoa capable of causing disease.

The skilled artisan will appreciate that the term "tumor antigen" or "tumor-associated antigen" may encompass an antigen (e.g., a polypeptide) that IS uniquely or differentially expressed on tumor cells as compared to normal or non-IS tumor cells. With respect to the compositions and methods disclosed herein, a tumor antigen comprises any polypeptide expressed by a tumor that is capable of activating or inducing an immune response via an antigen recognition receptor (e.g., CD19, MUCI) or capable of suppressing an immune response via receptor-ligand binding (e.g., CD47, PD-L1/L2, B7.1/2).

The skilled artisan will appreciate that the term "viral antigen" may encompass polypeptides expressed by the virus that are capable of inducing an immune response.

The terms "comprising", "including" and "comprising" are intended to have the broad meaning attributed to them in U.S. patent law and may mean "including", "comprising" and the like. Similarly, the terms "consisting of … …" and "consisting essentially of … …" have the meaning assigned thereto by the U.S. patent laws. It is contemplated that the compositions and methods disclosed herein comprise, consist of, or consist essentially of the active ingredient or the specified steps.

The skilled artisan will appreciate that the term "treatment" may encompass clinical interventions that attempt to alter the course of the disease in the individual or cell being treated, and may be directed to prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating a disease state, and alleviating or improving prognosis. By preventing the progression of a disease or disorder, treatment can prevent the exacerbation due to the disorder in a subject affected or diagnosed with or suspected of having the disorder, and treatment can prevent the onset of the disorder or symptoms of the disorder in a subject at risk of or suspected of having the disorder.

The skilled artisan will appreciate that the term "subject" can encompass vertebrates, in some embodiments mammals, and in some embodiments humans. In some embodiments, the subject may also refer to domestic, such as cattle, sheep, horses, cats, dogs, and laboratory animals, such as mice, rats, gerbils, hamsters, and the like.

In some embodiments, disclosed herein are CAR T cells, wherein the CAR is directed against a peptide of interest. In some embodiments, the CAR binds to a peptide of interest. In another embodiment, the CAR targets a peptide of interest. In another embodiment, the CAR activates a peptide of interest. In another embodiment, the CAR is a ligand for the peptide of interest. In another embodiment, the peptide of interest is a ligand of the CAR. Each of these embodiments is considered a part of the disclosure herein.

In some embodiments, an immune cell as disclosed herein is not a T cell. In another embodiment, the immune cell as disclosed herein is not an NK cell. In another embodiment, the immune cell as disclosed herein is not a CTL. In another embodiment, the immune cell as disclosed herein is not a regulatory T cell. In another embodiment, the immune cell is not a human embryonic stem cell. In another embodiment, the immune cell is not a pluripotent stem cell from which lymphoid cells can be differentiated.

One approach to immunotherapy involves engineering a patient's own immune cells to produce genetically modified immune cells that will recognize and attack their tumor. As described herein, immune cells are harvested and genetically modified, for example, to produce Chimeric Antigen Receptors (CARs) on their cell surface, which will allow the immune cells (e.g., T cells) to recognize specific protein antigens on tumor or cancer cells. The expanded population of genetically modified immune cells (e.g., CAR T cells) is then administered to the patient. In some embodiments, the administered cells multiply within the body of the patient and recognize and kill cancer and tumor cells having antigens on their surface. In another embodiment, the administered cells multiply within the patient's body and recognize and kill tumor-associated antigens, which results in cancer and tumor cell death.

In some embodiments, disclosed herein are methods of inhibiting or reducing the incidence of cytokine release syndrome or cytokine storm in a subject undergoing CAR T cell cancer therapy, and methods of reducing or inhibiting cytokine production in a subject undergoing cytokine release syndrome or cytokine storm, comprising the step of administering a composition comprising apoptotic cells or an apoptotic cell supernatant. In another embodiment, disclosed herein are methods of treating a cytokine release syndrome or cytokine storm in a subject undergoing CAR T cell cancer therapy. In another embodiment, disclosed herein are methods of preventing a cytokine release syndrome or cytokine storm in a subject undergoing CAR T cell cancer therapy. In another embodiment, disclosed herein are methods of alleviating a cytokine release syndrome or cytokine storm in a subject undergoing CAR T cell cancer therapy. In another embodiment, disclosed herein are methods of improving cytokine release syndrome or cytokine storm in a subject undergoing CAR T cell cancer therapy. In another embodiment, administration of apoptotic cells or apoptotic supernatant or a composition thereof does not decrease the efficacy of CAR T cell therapy.

In some embodiments, disclosed herein are methods of inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm in a subject undergoing a chimeric antigen receptor expressing T cell (CAR T cell) cancer therapy, wherein the method comprises the step of administering to the subject a composition comprising apoptotic cells or an apoptotic cell supernatant or a composition thereof. In another embodiment, inhibiting or reducing the incidence of Cytokine Release Syndrome (CRS) or a cytokine storm is determined by measuring cytokine levels in a subject undergoing a chimeric antigen receptor-expressing T cell cancer therapy and administered apoptotic cells or an apoptotic supernatant. In another embodiment, the measured cytokine level is compared to the cytokine level in a subject that has not undergone CAR T cell cancer therapy. In another embodiment, the measured cytokine level is compared to the cytokine level in a subject not administered apoptotic cells or apoptotic supernatant. In another embodiment, the measured cytokine levels are compared to control subjects.

In another embodiment, the level of a proinflammatory cytokine in the subject is reduced compared to a subject undergoing CAR T cell cancer therapy and not administered the apoptotic cells or the apoptotic cell supernatant or the composition thereof. In another embodiment, the methods disclosed herein result in a reduction or inhibition in the level of production of at least one pro-inflammatory cytokine as compared to a subject undergoing CAR T cell cancer therapy and not administered the apoptotic cells or the apoptotic cell supernatant or composition thereof.

In another embodiment, the methods disclosed herein may further comprise administering an additional agent. Alternatively, the methods disclosed herein may comprise administering an additional agent and not administering apoptotic cells or apoptotic cell supernatant. In still further embodiments, the additional agent can be those compounds or compositions that enhance or improve CAR T cell cancer therapy, or any combination thereof. In yet further embodiments, the additional agent that enhances or improves CAR T cell cancer therapy comprises a CTLA-4 blocker, alpha-1 antitrypsin or a functional fragment or analog thereof, a telluro compound, or an immunomodulator, or any combination thereof. In another embodiment, the additional agent comprises apoptotic cells or an apoptotic supernatant. In another embodiment, the administration of the additional agent described herein is prior to, concurrent with, or subsequent to the CAR T cell cancer therapy.

In some embodiments, an IL-6 receptor antagonist (which in one embodiment is truzumab) is used with the compositions and methods as disclosed herein.

In some embodiments, adoptively transferred T cells are transplanted and more efficiently expanded in a lymphopenic host. Thus, in some embodiments, the subject is lymphodepleted prior to transferring the CAR T cells or other modified immune cells. In another embodiment, the subject receiving the CAR T cells is administered a T cell supporting cytokine.

In some embodiments, the T cell is an effector T cell. In another embodiment, the T cell is a naive T cell. In another embodiment, the T cell is central memory (T)_CM) T cells. In another embodiment, the T cell is a Th17 cell. In another embodiment, the T cell is a T stem memory cell. In another embodiment, the T cell has a high replication capacity. In another embodiment, T cell expansion occurs in the patient. In another example, a small number of cells may be transferred to a patient. In another embodiment, T cell expansion occurs in vitro. In another example, a large number of cells can be transferred to a patient, cells and/or supernatant can be transferred to a patient multiple times, or a combination thereof.

In some embodiments, CAR T cells are advantageous in that because they are specific for cell surface molecules, they overcome the limitations of MHC-restricted TCR recognition and avoid tumor escape through impairment of antigen presentation or human leukocyte antigen expression.

In some embodiments, disclosed herein is a method of reducing tumor burden in a subject, the method comprising the step of administering to the subject any of the compositions as described herein.

In some embodiments, reducing the tumor burden comprises reducing the number of tumor cells in the subject. In another embodiment, reducing the tumor burden comprises reducing the size of the tumor in the subject. In another embodiment, reducing the tumor burden comprises eradicating the tumor in the subject.

In another embodiment, disclosed herein is a method of inducing tumor cell death in a subject, comprising the step of administering to the subject any of the compositions as described herein. In another embodiment, a method for inducing tumor cell death in a subject as disclosed herein comprises administering an immune cell (such as an NK cell or a T cell comprising an engineered chimeric antigen receptor) and at least one additional agent to reduce toxic cytokine release or "cytokine release syndrome" (CRS) or "severe cytokine release syndrome" (CRS) or "cytokine storm" in the subject.

In another embodiment, disclosed herein is a method of increasing or prolonging survival of a subject having a neoplasia, the method comprising the step of administering to the subject any of the compositions as described herein. In another embodiment, a method for increasing or prolonging survival in a subject as disclosed herein comprises administering an immune cell (such as a NK cell or T cell comprising an engineered chimeric antigen receptor) and at least one additional agent to reduce toxic cytokine release or "cytokine release syndrome" (CRS) or "severe cytokine release syndrome" (CRS) or "cytokine storm" in the subject.

In another embodiment, disclosed herein is a method of increasing or prolonging survival of a subject having a neoplasia, the method comprising the step of administering to the subject any of the compositions as described herein.

In some embodiments, disclosed herein is a method of delaying cancer progression in a subject, the method comprising the step of administering to the subject any of the compositions or combination of compositions described herein. In some embodiments, disclosed herein is a method of delaying the progression of a leukemia or lymphoma in a subject, the method comprising the step of administering to the subject any of the compositions or combination of compositions described herein. In some embodiments, disclosed herein is a method of increasing, extending, or prolonging survival of a subject having a cancer or tumor, the method comprising the step of administering to the subject any of the compositions or combination of compositions described herein. In some embodiments, disclosed herein is a method of increasing, extending, or prolonging (progening) survival of a subject having leukemia or lymphoma, the method comprising administering to the subject any of the compositions or combination of compositions described herein. In some embodiments, disclosed herein is a method of reducing tumor cell burden in a subject, the method comprising administering to the subject any of the compositions or combination of compositions described herein. In some embodiments, the tumor burden of the liver and bone marrow is reduced.

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

In another embodiment, disclosed herein is a method of treating a hematologic cancer in a subject in need thereof, comprising the step of administering to the subject any of the compositions as described herein. In some embodiments, the hematologic cancer is selected from: b cell leukemia, multiple myeloma, Acute Lymphoblastic Leukemia (ALL), chronic lymphocytic leukemia, and non-hodgkin's lymphoma.

In some embodiments, administering comprises administering a composition comprising a CAR T cell. In some embodiments, administering comprises administering a composition comprising early apoptotic cells. In some embodiments, administering comprises administering a composition comprising a supernatant obtained from early apoptotic cells. In some embodiments, administering comprises administering a combination of the compositions described herein. In some embodiments, administering comprises administering the CAR T cell and the apoptotic cell in the same or different compositions. In some embodiments, administering comprises administering the CAR T cells in combination with an additional agent as described herein. In some embodiments, administering comprises administering apoptotic cells and antibodies or fragments thereof in the same or different compositions.

In some embodiments, the combination therapy provides a synergistic effect. In some embodiments, the method of using early apoptotic cells in combination with CAR T cells results in increased CAR T cell efficacy compared to CAR T cells alone. In some embodiments, the method of using early apoptotic cells in combination with CAR T cells results in an increased survival time for a subject having a cancer or tumor as compared to CAR T cells alone. In some embodiments, the method of using early apoptotic cells in combination with CAR T cells results in an increased survival time for a subject with lymphoma or leukemia as compared to CAR T cells alone.

In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof delays the onset of cancer or the appearance of a tumor as compared to using apoptotic cells or antibodies alone. In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof delays progression of cancer as compared to using apoptotic cells or antibodies alone. In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof delays the growth of a tumor compared to using apoptotic cells or antibodies alone. In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof results in an increased survival time of a subject having a cancer or tumor as compared to the use of apoptotic cells or antibodies alone. In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof extends the survival time of a subject with lymphoma or leukemia as compared to the use of apoptotic cells or antibodies alone.

In some embodiments, the method of using (including administering) early apoptotic cells in combination with an antibody or fragment thereof (including RtX) delays the onset of cancer or the appearance of a tumor compared to the use of apoptotic cells or antibodies alone. In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof (including RtX) delays progression of cancer as compared to the use of apoptotic cells or antibodies alone. In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof (including RtX) delays the growth of a tumor compared to using apoptotic cells or antibodies alone. In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof (including RtX) results in an increased lifetime of a subject with cancer or tumor compared to the use of apoptotic cells or antibodies alone. In some embodiments, the method of using early apoptotic cells in combination with an antibody or fragment thereof (including RtX) results in an increased survival time for a subject with lymphoma or leukemia as compared to the use of apoptotic cells or antibodies alone.

In some embodiments, the methods of use described herein result in a reduction in tumor burden. The skilled artisan will appreciate that the term "tumor burden" may refer to the number of cancer cells, the size of the tumor, or the amount of cancer in the body. The term "tumor burden" may be used interchangeably with the term "tumor burden" and has all the same meaning and properties. In some embodiments, the method of using (including administering) early apoptotic cells reduces the number of cancer cells in a subject, reduces the size of a tumor in a subject, or reduces the amount of cancer in a subject, or any combination thereof, as compared to a subject not administered apoptotic cells. In some embodiments, the method of using (including administering) early apoptotic cells in combination with an antibody or fragment thereof reduces the number of cancer cells in the subject, reduces the size of a tumor in the subject, or reduces the amount of cancer in the subject, or any combination thereof, as compared to a subject not administered apoptotic cells or not administered antibodies or combinations thereof. In some embodiments, the method of using (including administering) early apoptotic cells in combination with RtX antibody or fragment thereof reduces the number of cancer cells in the subject, reduces the size of a tumor in the subject, or reduces the amount of cancer in the subject, or any combination thereof, as compared to a subject not administered apoptotic cells, RtX antibody, or a combination thereof.

In some embodiments, a method of reducing or inhibiting cytokine production in a subject experiencing or susceptible to cytokine release syndrome or cytokine storm as disclosed herein reduces or inhibits cytokine production. In another embodiment, the method reduces or inhibits pro-inflammatory cytokine production. In another embodiment, the method reduces or inhibits at least one pro-inflammatory cytokine. In another embodiment, wherein the subject is undergoing CAR T cell cancer therapy, the method does not reduce the efficacy of the CAR T cell therapy.

The methods provided herein comprise administering the T cells, NK cells, or CTL cells disclosed herein in an amount effective to achieve the desired effect (whether to alleviate the existing condition or prevent relapse). For treatment, the amount administered is an amount effective to produce the desired effect. The effective amount may be provided in a single administration or in a series of administrations. The effective amount may be provided in a bolus or by continuous infusion.

The skilled artisan will recognize that an "effective amount" (or "therapeutically effective amount") can encompass an amount sufficient to produce a beneficial or desired clinical result following treatment. An effective amount may be administered to a subject in one or more doses. For treatment, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse or slow the progression of a disease or otherwise reduce the pathological consequences of a disease. An effective amount is generally determined by a physician on a case-by-case basis and is within the abilities of one of skill in the art. Several factors are generally considered in determining the appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the antigen-binding fragment administered.

In one embodiment, the methods disclosed herein comprise administering a composition comprising a genetically modified cell and an additional agent, or a combination thereof, included in a single composition. In another embodiment, the method comprises administering a composition comprising a CAR T cell and an additional agent, or a combination thereof, included in a single composition. In another embodiment, the method comprises administering a composition comprising TCR T cells and an additional agent, or a combination thereof, comprised in a single composition.

In one embodiment, the methods disclosed herein comprise administering a composition comprising a genetically modified cell and an additional agent, or a combination thereof, included in at least two compositions. In another embodiment, the method comprises administering a composition comprising a CAR T cell and an additional agent, or a combination thereof, included in at least two compositions. In another embodiment, the method comprises administering a composition comprising TCR T cells and an additional agent, or a combination thereof, included in at least two compositions.

For adoptive immunotherapy using antigen-specific T cells (e.g., CAR T cells), typically 10 is infused⁶-10¹⁰In (e.g., 10)⁹) The cell dose of (a). Upon administration of the genetically modified cells to a host and subsequent differentiation, T cells specific for a particular antigen are induced. "induction" of T cells may comprise inactivation of antigen-specific T cells, e.g. by deletion or anergy. Inactivation is particularly useful for establishing or re-establishing tolerance, such as in autoimmune disorders. The modified cells can be obtained by any method known in the art (including, but not limited to, intravenous, subcutaneous, intra-nodal, intratumoral, intrathecal, intravenous, intratumoral, and intravenous,Intrapleural, intraperitoneal, and direct administration to the thymus). In some embodiments, the T cells are not administered intraperitoneally. In some embodiments, the T cell is administered intratumorally.

Compositions comprising genetically modified immunoresponsive cells (e.g., T cells, N cells, CTL cells, or progenitors thereof) as disclosed herein can be provided systemically or directly to a subject to treat a neoplasia, pathogen infection, or infectious disease. In some embodiments, the cells disclosed herein are injected directly into an organ of interest (e.g., an organ affected by neoplasia). Alternatively, a composition comprising genetically modified immunoresponsive cells is provided to an organ of interest indirectly, for example, by administration into the circulatory system (e.g., tumor vasculature). The expansion and differentiation agents may be provided before, during or after administration of the cells to increase the in vitro or in vivo production of T cells, NK cells or CTL cells.

As described above, in the methods disclosed herein, a composition comprising an additional agent can be provided prior to, concurrently with, or subsequent to administration of the genetically modified immune cells. In one embodiment, in the methods disclosed herein, the genetically modified immune cell (e.g., CAR T cell) is administered prior to the additional agent as disclosed herein. In another embodiment, in the methods disclosed herein, the genetically modified immune cells (e.g., CAR T cells) are administered at the same time as the additional agent as disclosed herein. In another embodiment, in the methods disclosed herein, the genetically modified immune cells (e.g., CAR T cells) are administered after the administration of the additional agent.

In one embodiment, the methods disclosed herein administer a composition comprising apoptotic cells as disclosed herein. In another embodiment, the methods disclosed herein administer a composition comprising an apoptotic cell supernatant as disclosed herein.

The modified cells may be administered in any physiologically acceptable vehicle (typically intravascular), although they may also be introduced into the bone or the cells may find suitable sites for regeneration and differentiation In other convenient parts of the tract (e.g., the thymus). Typically, at least 1X 10 will be applied⁵One cell, finally reaching 1X 10¹⁰Or more. Genetically modified immunoresponsive cells disclosed herein can include purified cell populations. The percentage of genetically modified immunoresponsive cells in a population can be readily determined by one skilled in the art using a variety of well-known methods, such as Fluorescence Activated Cell Sorting (FACS). In some embodiments, the purity in the population comprising genetically modified immunoresponsive cells ranges from about 50% to about 55%, from about 55% to about 60%, and from about 65% to about 70%. In other embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%. In further embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, from about 95% to about 100%. The dosage can be readily adjusted by one skilled in the art (e.g., a decrease in purity may require an increase in dosage). The cells may be introduced by injection, catheter, or the like. If desired, factors may also be included, including but not limited to interleukins (e.g., IL-2, IL-3, IL-6, IL-11, IL7, IL12, ILIS, IL21, and other interleukins), colony stimulating factors (e.g., G-, M-, and GM-CSF), interferons (e.g., gamma-interferon), and erythropoietin.

The compositions comprise a pharmaceutical composition comprising a genetically modified immunoresponsive cell or progenitor thereof and a pharmaceutically acceptable carrier. Administration may be autologous or heterologous. For example, the immunoresponsive cells or progenitor cells may be obtained from one subject and administered to the same subject or a different compatible subject. The peripheral blood-derived immunoresponsive cells disclosed herein or progeny thereof (e.g., derived in vivo, ex vivo, or in vitro) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition as disclosed herein (e.g., a pharmaceutical composition containing genetically modified immunoresponsive cells) is administered, it is typically formulated in a unit dose injectable form (solution, suspension, emulsion).

In another embodiment, disclosed herein is a method of producing a composition comprising a CAR T cell or other immune cell as disclosed herein and an apoptotic cell or apoptotic cell supernatant, the method comprising introducing into the T cell or immune cell a nucleic acid sequence encoding a CAR that binds to an antigen of interest. In alternative embodiments, a composition comprising a CAR T cell or other immune cell as disclosed herein is isolated from a composition comprising apoptotic cells or an apoptotic supernatant.

In one embodiment, disclosed herein is a method of treating, preventing, inhibiting, reducing the incidence of, ameliorating, or alleviating a malignancy, comprising the step of administering a composition comprising chimeric antigen receptor-expressing T cells (CAR T cells) and apoptotic cells or an apoptotic cell supernatant.

The skilled artisan will appreciate that the anti-tumor immune response elicited by the genetically modified immune cells (e.g., CAR-modified T cells) can be an active immune response or a passive immune response. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy approach, wherein the CAR-modified T cells induce an immune response specific to the antigen-binding portion of the CAR.

The skilled artisan will appreciate that immunotherapy may encompass the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may act as an effector of the therapy, or it may recruit other cells to actually effect cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin a chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

In some embodiments, the early apoptotic cells and compositions thereof as disclosed herein may be used to treat, prevent, inhibit the growth of, or reduce the incidence of any hematologic tumor known in the art. In some embodiments, the early apoptotic cells and compositions thereof as disclosed herein may be used to treat, prevent, inhibit the growth of, or reduce the incidence of any diffuse cancer known in the art (such as, but not limited to, diffuse breast cancer, wherein a solid tumor is not formed in the breast). In some embodiments, early apoptotic cells and compositions thereof as disclosed herein may be used to extend the survival time of any hematological tumor known in the art. In some embodiments, early apoptotic cells and compositions thereof as disclosed herein may be used to prolong the survival time of any diffuse cancer known in the art (such as, but not limited to, diffuse breast cancer, wherein a solid tumor is not formed in the breast).

In some embodiments, early apoptotic cells and compositions thereof as disclosed herein may be used to increase survival of a subject with any hematological tumor known in the art. In some embodiments, early apoptotic cells and compositions thereof as disclosed herein may be used to increase survival in subjects suffering from any diffuse cancer known in the art (such as, but not limited to, diffuse breast cancer, wherein a solid tumor is not formed in the breast).

In some embodiments, early apoptotic cells and compositions thereof as disclosed herein may be used to reduce the growth rate of any hematological tumor known in the art. In some embodiments, early apoptotic cells and compositions thereof as disclosed herein may be used to reduce the growth rate of any diffuse cancer known in the art (such as, but not limited to, diffuse breast cancer, wherein a solid tumor is not formed in the breast).

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

In some embodiments, the subject is a human subject. In some embodiments, the subject is a child. In some embodiments, the subject is an adult. In some embodiments, the subject is a mammal.

In some embodiments, the methods disclosed herein comprise administering an early apoptotic cell population comprising an enriched monocyte population as described in detail above. In some embodiments, the methods disclosed herein comprise administering an early apoptotic cell population comprising a stable population of cells, wherein the cell population is stable for more than 24 hours. The stable population of early apoptotic cells has been described in detail above. In some embodiments, the methods disclosed herein comprise administering an early apoptotic cell population comprising a cell population that is free of cell aggregates. The aggregate-free population of early apoptotic cells and methods for their preparation have been described in detail above.

In some embodiments, the methods disclosed herein comprise administering to a subject in need thereof a population of autologous early apoptotic cells. In some embodiments, the methods disclosed herein comprise administering to a subject in need thereof a population of allogeneic early apoptotic cells.

In some embodiments, the method of administering an early apoptotic cell population or composition thereof comprises administering a single infusion of said apoptotic cell population or composition thereof. In some embodiments, a single infusion may be administered as a prophylactic to a subject scheduled to be at risk of developing a cancer or tumor. In some embodiments, a single infusion may be periodically administered to a subject having a cancer or tumor as part of the subject's therapeutic treatment. In some embodiments, a single infusion may be administered as a prophylactic to a subject having a cancer or tumor in order to prevent, reduce the risk of, or delay the appearance of metastatic cancer.

In some embodiments, the method of administering an early apoptotic cell population or composition thereof comprises administering multiple infusions of said apoptotic cell population or composition thereof. In some embodiments, multiple infusions may be administered as a prophylactic to a subject scheduled to be at risk of developing a cancer or tumor. In some embodiments, multiple infusions can be administered periodically to a subject having a cancer or tumor as part of the subject's therapeutic treatment. In some embodiments, multiple infusions can be administered as a prophylactic agent to a subject having a cancer or tumor in order to prevent, reduce the risk of, or delay the appearance of metastatic cancer.

In some embodiments, the multiple infusions comprise at least two infusions. In some embodiments, the plurality of infusions comprises 2 infusions. In some embodiments, the plurality of infusions comprises more than 2 infusions. In some embodiments, the plurality of infusions comprises at least 3 infusions. In some embodiments, the plurality of infusions comprises 3 infusions. In some embodiments, the multiple infusions comprise more than 3 infusions. In some embodiments, the plurality of infusions comprises at least 4 infusions. In some embodiments, the plurality of infusions comprises 4 infusions. In some embodiments, the multiple infusions comprise more than 4 infusions. In some embodiments, the plurality of infusions comprises at least 5 infusions. In some embodiments, the plurality of infusions comprises 5 infusions. In some embodiments, the plurality of infusions comprises more than 5 infusions. In some embodiments, the plurality of infusions comprises at least six infusions. In some embodiments, the plurality of infusions comprises 6 infusions. In some embodiments, the multiple infusions comprise more than 6 infusions. In some embodiments, the plurality of infusions comprises at least 7 infusions. In some embodiments, the plurality of infusions comprises 7 infusions. In some embodiments, the plurality of infusions comprises more than 7 infusions. In some embodiments, the plurality of infusions comprises at least 8 infusions. In some embodiments, the plurality of infusions comprises 8 infusions. In some embodiments, the multiple infusions comprise more than 8 infusions. In some embodiments, the plurality of infusions comprises at least nine infusions. In some embodiments, the plurality of infusions comprises 9 infusions. In some embodiments, the multiple infusions comprise more than 9 infusions. In some embodiments, the plurality of infusions comprises at least 10 infusions. In some embodiments, the plurality of infusions comprises 10 infusions. In some embodiments, the plurality of infusions comprises more than 10 infusions.

In some embodiments, the multiple infusions comprise a lesser amount of early apoptotic cells, wherein the total dose of cells administered is the sum of the infusions.

In some embodiments, multiple infusions are administered over a period of hours. In some embodiments, multiple infusions are administered over a period of days. In some embodiments, multiple infusions are administered over a period of hours, with at least 12 hours between infusions. In some embodiments, multiple infusions are administered over a period of hours, with at least 24 hours between infusions. In some embodiments, multiple infusions are administered over a period of hours, with at least one day between infusions. In some embodiments, multiple infusions are administered over a period of hours, with at least two days between infusions. In some embodiments, multiple infusions are administered over a period of hours, with at least three days between infusions. In some embodiments, multiple infusions are administered over a period of hours, with at least four days between infusions. In some embodiments, multiple infusions are administered over a period of hours, with at least five days between infusions. In some embodiments, multiple infusions are administered over a period of hours, with at least six days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there are at least seven days between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there is at least one week between infusions. In some embodiments, multiple infusions are administered over a period of hours, wherein there are at least two weeks between infusions.

In some embodiments, the amount of cells in the plurality of infusions are substantially equal to each other. In some embodiments, the amount of cells in the multiple infusions is different from one infusion to another.

In some embodiments, the methods described herein further comprise administering to the subject an additional chemotherapeutic agent or an immunomodulatory agent.

In some embodiments, the additional chemotherapeutic agent or immunomodulatory agent is administered simultaneously or substantially simultaneously with the early apoptotic cells. In some embodiments, the additional chemotherapeutic agent or immunomodulatory agent is included in the same composition as the early apoptotic cells. In some embodiments, the additional chemotherapeutic agent or immunomodulatory agent is included in a different composition than the early apoptotic cells.

In some embodiments, the additional chemotherapeutic agent or immunomodulatory agent is administered prior to administration of the early apoptotic cells. In some embodiments, the additional chemotherapeutic agent or immunomodulatory agent is administered after administration of the early apoptotic cells.

In some embodiments, the chemotherapeutic agent comprises an alkylating agent, nitrogen mustard (nitrogen mustard), nitrosourea, tetrazine, aziridine, cisplatin (cissplatin) and derivatives, non-classical alkylating agents, dichloromethyl diethylamine, cyclophosphamide (cyclophosphamide), melphalam (melphalan), chlorambucil, ifosfamide (ifosfamide), busulfan (busufan), N-nitroso-N-Methylurea (MNU), carmustine (BCNU), lomustine (ccun), semustine (hexamethyl, mecnu), fotemustine (fotemustine), streptozotocin (streptazocin), dacarbazine (dacarbazine), mitozolomide (mitozolomide), temozolomide (temozolomide), mitomycin (mitomycin), propidin (oxazidine), antimetabolite (oxanthine, propidium, propinqrine (oxanthine), antimetabolite (oxanthine), propidium (oxanthine), propidium, propinqim (oxazid), and a, Methotrexate (methotrexate), pemetrexed (pemetrexed), fluoropyrimidine, fluorouracil, capecitabine (capecitabine), deoxynucleoside analogs, cytarabine, gemcitabine (gemcitabine), decitabine (decitabine), azacitidine (azacitidine), fludarabine (fludarabine), nelarabine (nellabine), cladribine (cladribine), clofarabine (clofarabine), and pentostatin (pentostatin), thiopurines (thioprines), thioguanine (thioguanine), mercaptopurine (mercapecitabine), antimicrotubule agents, vinca alkaloids (vinca alkloids), taxanes (taxanes), vincristine (vinblastine), semisynthetic vinblastine (vinblastine), vincristine (vinorelbine), vinorelbine (vinorelbine), docetaxel (paclitaxel), docetaxel (docetaxel), docetaxel (paclitaxel), docetaxel (paclitaxel), paclitaxel (paclitaxel), paclitaxel (vincristine (vinblastine (vincristine), vinblastine (vincristine), vincristine (vincristine), vincristine (semi-L (vincristine), vincristine (semi-L), vincristine (semi-L (semi-paclitaxel), vincristine (semi-L), vincristine (vincristine), vincristine (vincristine), vincristine (vincristine), vincristine (vincristine), vincristine (vincristine), vinblastine (vincristine), vinblastine (docetaxel (vincristine (docetaxel (vincristine), vincristine (docetaxel (vincristine), vinblastine (docetaxel (vincristine, Topotecan (topotecan), camptothecin (camptothecin), etoposide, doxorubicin (doxorubicin), mitoxantrone (mitoxantrone), teniposide (teniposide), catalytic inhibitors, neomycin (novobiocin), mebendazole (merbarone), aclarubicin (aclarubicin), cytotoxic antibiotic, anthracycline (anthracyclines), bleomycin (bleomycin), mitomycin c (mitomycin c), mitoxantrone (mitoxantrone), actinomycin (actinomycin), doxorubicin, daunorubicin (daunorubicin), epirubicin (epirubicin), idarubicin (idarubicin), anthracycline, pirarubicin (pirarubicin), aclarubicin (aclarubicin), mitoxantrone, mitomycin, targeted therapy, a monoclonal antibody, a bispecific antibody, a monoclonal antibody, a conjugated monoclonal antibody, any combination thereof or any combination thereof.

In some embodiments, the immunomodulatory agent comprises an antibody or functional fragment thereof. In some embodiments, the antibody or functional fragment thereof comprises a monoclonal antibody, a single chain antibody, an Fab fragment, an F (ab')2 fragment, or an Fv fragment.

In some embodiments, disclosed herein are active fragments of any of the polypeptides or peptide domains disclosed herein. The skilled person will appreciate that the term "fragment" may encompass at least 5, 10, 13 or 15 amino acids. In other embodiments, a fragment is at least 20 contiguous amino acids. The fragments disclosed herein can be produced by methods known to those skilled in the art or can be produced by normal protein processing (e.g., removal of amino acids from nascent polypeptides that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and specifically refer to polyclonal antibodies, monoclonal antibodies, or any fragment thereof that retains the binding activity of the antibody. In certain embodiments, the methods disclosed herein comprise the use of chimeric, humanized, or human antibodies.

In some embodiments, the term "antibody" refers to intact molecules as well as functional fragments thereof, such as Fab, F (ab')2, and Fv, that are capable of specifically interacting with a desired target (e.g., binding to phagocytes) as described herein. In some embodiments, the antibody fragment comprises:

(1) fab, fragments containing monovalent antigen-binding fragments of antibody molecules that can be produced by digestion of whole antibodies with the enzyme papain to produce a complete light chain and a portion of one heavy chain;

(2) fab', fragments of an antibody molecule that can be obtained by treating the whole antibody with pepsin, followed by reduction to produce a portion of the complete light and heavy chains; obtaining two Fab' fragments per antibody molecule;

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

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

(5) single chain antibodies ("SCAs"), comprising a light chain variable region and a heavy chain variable region, genetically engineered molecules linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods for preparing such fragments are known in the art. (see, e.g., Harlow and Lane, "Antibodies: A Laboratory Manual", Cold spring harbor Laboratory, N.Y., 1988, incorporated herein by reference).

In some embodiments, antibody fragments can be prepared by proteolysis of an antibody or by expression of DNA encoding the fragment in e.coli or mammalian cells (e.g., chinese hamster ovary cell culture or other protein expression systems).

In some embodiments, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of an antibody with pepsin to provide a 5S fragment denoted F (ab') 2. Such fragments can be further cleaved using thiol reducing agents and optionally thiol protecting groups resulting from cleavage of disulfide bonds to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and an Fc fragment. These methods are described, for example, by golden berg, U.S. patent nos. 4,036,945 and 4,331,647, and references contained therein, which are incorporated by reference herein in their entirety. See also Porter, R.R., Biochem.J.), 73:119-126, 1959. Other methods of cleaving antibodies (e.g., separation of heavy chains to form monovalent light chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques) can also be used, so long as the fragments bind to the antigen recognized by the intact antibody.

Fv fragments comprise an association of a VH chain and a VL chain. Such associations may be non-covalent, as described in Inbar et al, Proc. Natl. Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variable chains may be linked by intermolecular disulfide bonds or crosslinked by chemicals such as glutaraldehyde. Preferably, the Fv fragment comprises a VH chain and a VL chain connected by a peptide linker. These single-chain antigen binding proteins (sFv) can be prepared by constructing a structural gene comprising DNA sequences encoding the VH domain and the VL domain linked by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E.coli. Recombinant host cells synthesize a single polypeptide chain by a linker peptide bridging two V domains. Methods for producing sFv are described, for example, by Whitlow and Filpula, "Methods (Methods), 2:97-105,1991; bird et al, science 242:423-426, 1988; pack et al, Biotechnology (Bio/Technology) 11:1271-77, 1993; and Ladner et al, U.S. patent No. 4,946,778, which is incorporated herein by reference in its entirety.

Another form of antibody fragment is a peptide encoding a single Complementarity Determining Region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDRs of an antibody of interest. Such genes are prepared, for example, by synthesizing the variable regions from RNA of antibody-producing cells using the polymerase chain reaction. See, for example, Larrick and Fry, methods, 2: 106-.

In some embodiments, an antibody or fragment as described herein may comprise a "humanized version" of an antibody. In some embodiments, the term "humanized form of an antibody" refers to a non-human (e.g., murine) antibody, which is a chimeric molecule of an immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab ', F (ab'). sub.2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. Humanized antibodies comprise human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in either the recipient antibody or the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of the variable domains of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Humanized antibodies will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin [ Jones et al, Nature, 321:522-525 (1986); riechmann et al, Nature 332:323-329 (1988); and Presta, Current Structure biology review (curr. Op. struct. biol.), 2: 593-.

Methods for humanizing non-human antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced into them from a source that is not human. These non-human amino acid residues are commonly referred to as "import" (import) residues, which are typically taken from an "import" variable domain. Humanization can be carried out essentially as described by Winter and co-workers [ Jones et al, Nature, 321:522-525 (1986); riechmann et al, Nature 332:323-327 (1988); verhoeyen et al, science 239:1534-1536(1988) ] was performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an entire human variable domain has been substituted by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be generated using various techniques known in the art, including phage display libraries [ Hoogenboom and Winter, journal of molecular biology (J.mol.biol.), (227: 381 (1991)); marks et al, molecular biology (mol. biol.), 222:581 (1991). The techniques of Cole et al and Boerner et al can also be used to prepare human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and Cancer therapeutics, Allen R Rich, Inc. (Alan R.Liss), p.77 (1985) and Boerner et al, J.Immunol., 147(1):86-95 (1991)), similarly human immunoglobulin gene loci can be prepared by introducing human immunoglobulin gene loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). after human challenge, human antibody production is observed, which is closely similar in all respects to that seen in humans, including gene rearrangements, assembly and antibody libraries. the methods described in, for example, U.S. Pat. Nos. 5,545,807, 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; and in the following publications: Markss et al, Marnks et al, Biotechnology 779 (Lonber et al, 1992 et al, Lonber et al, 1992, 3, nature 368856 and 859 (1994); morrison, Nature 368812-13 (1994); fishwild et al, Nature Biotechnology 14,845-51 (1996); neuberger, Nature Biotechnology 14,826 (1996); and Lonberg and huskzar, international immunological reviews (inter. rev. immunol.) 1365-93 (1995).

In some embodiments, the immunomodulatory agent comprises an anti-CD 20 monoclonal antibody. In some embodiments, the anti-CD 20 monoclonal antibody is rituximab. Rituximab is commercially available and marketed under the name Bohai healthcare (Biogen) and Genetech Inc. (Genentech USA, Inc.) in combination

And (4) selling.

In some embodiments, the methods disclosed herein comprise first line therapy.

The skilled person will appreciate that the term "first line therapy" may encompass a first treatment for a disease. It is usually part of a standard set of treatments such as surgery followed by chemotherapy and radiation. First line therapy alone is the therapy that is accepted as the best treatment. Other treatments may be added or used instead if they do not cure the disease or they cause severe side effects. Also known as induction therapy, primary therapy and primary treatment.

In some embodiments, the methods disclosed herein comprise adjuvant therapy.

The skilled person will appreciate that the term "adjuvant therapy" may encompass treatments administered in addition to the primary or initial treatment. In some embodiments, adjuvant therapy may include additional cancer treatments administered prior to primary treatment in preparation for further treatment. In some embodiments, adjuvant therapy may include administering additional cancer treatments after the primary treatment to reduce the risk of cancer recurrence. The adjuvant therapy may comprise chemotherapy, radiation therapy, hormonal therapy, targeted therapy or biological therapy.

In some embodiments, the methods disclosed herein reduce minimal residual disease, increase remission, increase duration of remission, decrease tumor recurrence rate, decrease the size of the tumor, decrease the growth rate of the tumor or the cancer, prevent metastasis of the tumor or the cancer, or decrease the rate of metastasis of the tumor or the cancer, or any combination thereof.

The skilled artisan will appreciate that the term "minimal residual disease" can encompass a small number of cancer cells that remain in a patient after treatment, either during treatment or when the patient is free of symptoms or signs of disease.

Additionally, the term "alleviating" can encompass a reduction or disappearance of signs and symptoms of cancer, although the cancer may still be in the body. In some embodiments, remission may include partial remission in which some, but not all, signs and symptoms of cancer have disappeared. In some embodiments, remission includes complete remission, in which all signs and symptoms of cancer have disappeared, although the cancer may still be in the body. In some embodiments, the methods disclosed herein may include remission induction therapy, wherein initial treatment with early apoptotic cells or compositions thereof reduces or eliminates signs or symptoms of cancer.

The skilled artisan will appreciate that the term "recurrence" can encompass the recurrence of disease or signs and symptoms of disease after a period of improvement. In some embodiments, the methods used herein result in relapse-free survival, wherein said relapse-free survival encompasses the length of time that a patient survives without any signs or symptoms of said cancer after the end of primary treatment for the cancer.

Malignant tumor

In some embodiments, the CAR T cells are used in a method of treating, preventing, inhibiting, reducing the incidence, ameliorating, or ameliorating a cancer or tumor, wherein the method comprises the step of administering T cells expressing a chimeric antigen receptor (CAR T cells). As disclosed herein, the methods may further comprise administering an additional agent for the purpose of inhibiting CRS or a cytokine storm or reducing the incidence of said CRS or cytokine storm.

In some embodiments, the methods disclosed herein increase survival in a subject. In some embodiments, disclosed herein is a method of increasing or prolonging survival of a subject having a diffuse cancer, the method comprising the step of administering to the subject an early apoptotic cell population, wherein the method increases survival (survival) of the subject.

In some embodiments, the cancer is a B cell malignancy. In some embodiments, the B cell malignancy is leukemia. In another embodiment, the B cell malignancy is Acute Lymphoblastic Leukemia (ALL). In another embodiment, the B cell malignancy is chronic lymphocytic leukemia.

In some embodiments, the cancer is leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is a large B cell lymphoma.

In some embodiments, the methods described herein reduce the size of or the growth rate of a cancer or tumor, and comprise administering to the subject a population of early apoptotic cells, wherein the methods reduce the size of or the growth rate of the cancer or tumor. In some embodiments, disclosed herein is a method of reducing the growth rate of a diffuse cancer comprising the step of administering to said subject an early apoptotic cell population, wherein said method reduces the growth rate of said cancer. In some embodiments, disclosed herein are methods of reducing the size or reducing the growth rate of a solid cancer or tumor, comprising the step of administering to a subject an early apoptotic cell population, wherein said method reduces the size or reduces the growth rate of said solid cancer or tumor.

In some embodiments, the cancer may comprise a solid tumor. In some embodiments, a solid tumor comprises an abnormal tissue mass that is generally free of cysts or fluid regions. Solid tumors can be benign (not cancer) or malignant (cancer). Different types of solid tumors are named for the type of cell that forms them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Leukemias (hematologic cancers) do not typically form solid tumors. In some embodiments, the solid tumor comprises a sarcoma or carcinoma.

In some embodiments, a solid tumor is a tumor (new growth of cells) or lesion (damage to anatomical structures or disruption of physiological function) formed by abnormal growth of cells of body tissues other than blood, bone marrow, or lymphocytes. In some embodiments, a solid tumor consists of an abnormal mass of cells, which may originate from a different tissue type (e.g., liver, colon, breast, or lung) and initially grow in the organ from which the cells are derived. However, such cancers may spread to other organs by metastatic tumor growth at advanced stages of the disease.

In some embodiments, examples of solid tumors include sarcomas, carcinomas, and lymphomas. In some embodiments, the solid tumor comprises a sarcoma or carcinoma. In some embodiments, the solid tumor is an intraperitoneal tumor.

In some embodiments, solid tumors include, but are not limited to, lung cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, cervical cancer, head and neck cancer, bladder cancer, liver cancer, and skin cancer. In some embodiments, the solid tumor comprises fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer or tumor, breast cancer or tumor, ovarian cancer or tumor, prostate cancer or tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilm' stmoma, cervical cancer or tumor, uterine cancer or tumor, testicular cancer or tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, angiosarcoma, endothelial cancer, breast cancer, cervical cancer, breast cancer, bladder cancer, cervical cancer, breast cancer, bladder cancer, and combinations of the like cancer, in addition of the like, Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

In some embodiments, the solid tumor comprises an adrenocortical tumor (adenoma and carcinoma), a carcinoma, a colorectal cancer, a desmoid tumor, a proliferative small round cell tumor, an endocrine tumor, ewing's sarcoma, a germ cell tumor, a hepatoblastoma, a hepatocellular carcinoma, a melanoma, a neuroblastoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a soft tissue sarcoma other than rhabdomyosarcoma, and a wilms tumor. In some embodiments, the solid tumor is a breast tumor. In another embodiment, the solid tumor is prostate cancer. In another embodiment, the solid tumor is colon cancer. In some embodiments, the tumor is a brain tumor. In another embodiment, the tumor is a pancreatic tumor. In another embodiment, the tumor is a colorectal tumor.

In some embodiments, the early apoptotic cells or compositions thereof as disclosed herein are directed to a cancer or tumor (e.g., sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma), colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonic carcinoma, wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, melanoma, choriocarcinoma, and combinations thereof, Astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma) have therapeutic and/or prophylactic efficacy.

In some embodiments, the early apoptotic cells and compositions thereof as disclosed herein may be used to treat, prevent, inhibit the growth of, or reduce the incidence of any solid tumor known in the art.

In some embodiments, early apoptotic cells as disclosed herein and compositions thereof may be used to increase survival of a subject having any solid tumor as disclosed herein or known in the art.

In some embodiments, early apoptotic cells and compositions thereof as disclosed herein may be used to reduce the size or decrease the growth rate of any solid tumor disclosed herein or known in the art.

In some embodiments, the cancer may be a diffuse cancer, wherein the cancer is widespread; non-topical or occlusive. In some embodiments, the diffuse cancer may comprise a non-solid tumor. Examples of diffuse cancers include leukemia. Leukemias include cancers that begin in blood-forming tissues such as the bone marrow and cause the production of large numbers of abnormal blood cells and enter the bloodstream.

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

In some embodiments, the diffuse cancer or tumor comprises a hematological tumor. In some embodiments, the hematologic tumor is a type of cancer that affects blood, bone marrow, and lymph nodes. Hematological tumors may derive from two major blood cell lineages: myeloid cell lines and lymphoid cell lines. Myeloid cell lines typically produce granulocytes, erythrocytes, platelets, macrophages and masT cells, while lymphoid cell lines produce B cells, T cells, NK cells and plasma cells. Lymphomas (e.g., hodgkin's lymphoma), lymphocytic leukemias, and myelomas are derived from lymphoid lineages, while acute and chronic myelogenous leukemias (AML, CML), myelodysplastic syndromes, and myeloproliferative diseases all originate in the bone marrow.

In some embodiments, the non-solid (diffuse) cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, multiple myeloma (plasma cell myeloma), acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, or plasma cell leukemia.

In another embodiment, the early apoptotic cells and compositions thereof as disclosed herein have therapeutic and/or prophylactic efficacy against diffuse cancer (such as, but not limited to, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, acute myelocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (hodgkin's disease, non-hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease).

The compositions and methods as disclosed herein can be used to treat, prevent, inhibit, ameliorate, reduce the incidence, or ameliorate any hematological tumor known in the art.

The skilled artisan will appreciate that, in certain embodiments, the use of the term "comprising" may be replaced by the term "consisting essentially of … …" or "consisting of … …". The skilled person will understand that the term "comprising" is intended to mean that the system comprises the elements listed, but does not exclude other elements which may be optional. For example, a composition that includes early apoptotic cells but is not limited to this cell population. Further, the term "consisting essentially of … …" can encompass a method that comprises the recited elements (e.g., a composition consisting essentially of early apoptotic cells) but excludes other elements that may have a significant effect on the performance of the method. Thus, such compositions may still comprise pharmaceutically acceptable excipients that do not include essential activity in the treatment of cancer. Further, "consisting of … …" is intended to exclude other elements beyond trace amounts. Thus, such a composition consisting of early apoptotic cells will not contain more than trace amounts of other elements as disclosed herein.

In some embodiments, a method as disclosed herein may be expressed as using a composition as described herein for various therapeutic and prophylactic purposes as described herein, or alternatively, using a composition as described herein in the preparation of a medicament or therapeutic composition or composition for various therapeutic and prophylactic purposes as described herein.

In some embodiments, the compositions and methods as disclosed herein comprise various components or steps. However, in another embodiment, the compositions and methods as disclosed herein consist essentially of various components or steps, which may include other components or steps. In another embodiment, the compositions and methods as disclosed herein consist of various components or steps.

In some embodiments, the term "comprising" may encompass inclusion of other components of the composition that may be known in the art to affect the efficacy of the composition. In some embodiments, the term "consisting essentially of … …" includes compositions having T cells expressing a chimeric antigen receptor (CAR T cells) and apoptotic cells or the supernatant of any apoptotic cells. However, other components not directly related to the use of the composition may be included. In some embodiments, the term "consisting of" encompasses a composition having T cells expressing a chimeric antigen receptor (CAR T cells) and apoptotic cells or an apoptotic cell supernatant as disclosed herein, in any form or embodiment as described herein.

The skilled artisan will appreciate that the term "about" can encompass deviations from the indicated number or range of numbers of between 0.0001% and 5%. Further, the terms may encompass a deviation of between 1% and 10% from the indicated number or range of numbers. In addition, the terms may encompass deviations of up to 25% from the indicated number or range of numbers.

The skilled artisan will appreciate that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "agent" or "at least one agent" may encompass a plurality of agents, including mixtures thereof.

Throughout this application, various embodiments disclosed herein may be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as a non-flexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered to have explicitly disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have explicitly disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc.; and individual numbers within the stated range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is intended to include any referenced number (fractional or integer) within the indicated range. The phrases "a range/range between a first indicated digit and a second indicated digit" and "a range/range" from a first indicated digit "to a second indicated digit are used interchangeably herein and are meant to encompass the first indicated digit and the second indicated digit and all fractions and integers in between.

The following examples are presented in order to more fully demonstrate the embodiments disclosed herein. However, the examples should in no way be construed as limiting the broad scope of the disclosure.

Examples of the invention

Example 1: apoptotic cell production

The purpose is as follows: early apoptotic cells are produced.

The method comprises the following steps: methods for preparing populations of early apoptotic cells have been well documented in international publication No. WO2014/087408 and U.S. application publication No. US2015/0275175-a1, see, e.g., the methods at "preparation of populations of early apoptotic cells" and "generation of apoptotic cells" section [0223] to [0288] and examples 11, 12, 13 and 14 prior to the examples, which disclosures are incorporated herein in their entirety.

The flow chart presented in fig. 1 provides an overview of one embodiment of the steps used during the process of generating an early apoptotic cell population, wherein an anticoagulant is included in the thawing and apoptosis inducing steps. As described in detail in example 14 of International publication No. WO2014/087408 and U.S. application publication No. US-2015-0275175-A1, an early apoptotic cell population was prepared in which an anticoagulant was added at the time of freezing, or at the time of incubation, or at the time of freezing and at the time of incubation. The anticoagulant used was acid citrate dextrose, and NIH formula A (ACD formula A) was supplemented with 10U/ml heparin to a final concentration of 5% ACD and 0.5U/ml heparin of total volume.

Briefly: cells were collected and then frozen with the addition of 5% anticoagulant citrate dextrose formulation a and 10U/ml heparin (ACDhep) to the freezing medium. Thawing, incubation in apoptosis-inducing medium containing 5% ACDhep and final product preparation were performed in a closed system.

Apoptosis and viability analysis, potency assay and cell population characterization were performed in each experiment. To establish consistency in the production of early apoptotic cell products, the initial batch of apoptotic cells Final Product (FP) was stored at 2-8 ℃ and examined at t0, t24 hours, t48 hours, and t72 hours. Apoptosis analysis, short potency assay (applicant CD14+ frozen cells), trypan blue measurement and cell population characterization were performed at each point. Cell counts of FP were tested to assess average cell loss during storage and apoptosis as well as viability assays.

The above-cited method section, as well as example 11 of international publication No. WO 2014/087408 and U.S. application publication No. US-2015-0275175-a1, provide details of other examples of preparing apoptotic cell populations in the absence of anticoagulants and are incorporated herein in their entirety.

A method of preparing irradiated apoptotic cells: a similar method is used to prepare an inactivated apoptotic cell population, wherein the mononuclear early apoptotic cell population comprises a reduced percentage of non-quiescent non-apoptotic cells, or a cell population with suppressed cellular activation of any living non-apoptotic cells, or a cell population with reduced proliferation of any living non-apoptotic cells, or any combination thereof.

Briefly, an enriched monocyte fraction is collected from a healthy, qualified donor by a leukapheresis procedure. After apheresis is complete, the cells are washed and resuspended in a freezing medium comprising 5% anticoagulant citrate dextrose solution-formula a (ACD-a) and 0.5U/ml heparin. The cells were then gradually frozen and transferred to liquid nitrogen for long term storage.

To prepare irradiated ApoCell, cryopreserved cells were thawed, washed and treated with a mixture comprising 5% ACD-A, 0.5U/ml heparin sodium and 5Apoptosis induction medium resuspension of 0. mu.g/ml methylprednisolone. Then, the cells were incubated at 37 ℃ in 5% CO₂Incubated for 6 hours. At the end of the incubation, the cells were collected, washed and resuspended in hartmann's solution using a cell processing system (Fresenius Kabi, germany). After preparation, ApoCell was irradiated at 4000cGy using a g-camera at a radiotherapy apparatus (hadamard einkamer). ApoCell apoptosis and viability were determined by flow cytometry using annexin V and PI (MBL, Mass.) staining ≥ 40% and ≤ 15%, respectively. The results were analyzed using FCS Express software.

This irradiated APOcell population is believed to comprise early apoptotic cells, where the cellular activity of any living cells present is suppressed and proliferative capacity is reduced or absent. In some cases, the Apocell population has no viable non-apoptotic cells.

As a result:

the stability of the FP produced with the inclusion of the anticoagulant upon freezing and incubation (apoptosis induction) and then stored at 2-8 ℃ is shown below in table 3.

Table 3: cell counting-performed using a MICROROS 60 hematology Analyzer

FP time Point	Cell concentration (. times.10)⁶Cells/ml)	% cell loss
			t0	20.8	NA
t24h	20.0	-3.85
			t48h	20.0	-3.85
t72h	19.7	-5.3

Results represent 6 (six) experiments.

When cells were prepared without including anticoagulant in the induction medium, the cells were stable for 24 hours and less stable thereafter. As shown in table 3, the use of anticoagulants unexpectedly prolonged the stability of apoptotic cell populations by at least 72 hours.

Table 4: trypan blue measurement

FP time Point	Trypan blue positive cells (%)
		t0	3.0
t24h	5.9
		t48h	5.2
t72h	6.5

The results in table 4 show that the viability of the FP remained high for at least 72 hours.

Table 5: apoptosis analysis- (AnPI staining) was performed using flow cytometry

The results in table 5 show that the percentage of apoptotic cells to necrotic cells was maintained over an extended period of time of at least 72 hours after cell preparation, as was the percentage of early apoptotic cells. .

Inclusion of anticoagulant both at freezing and during induction of apoptosis resulted in the most consistently high yields of stable early apoptotic cells (average yield of early apoptotic cells was 61.3 ± 2.6%% versus 48.4 ± 5.0%, with 100% yield based on the number of cells at freezing). This high yield is maintained even after storage at 2-8 ℃ for 24 hours.

Next, a comparison was made between including anticoagulant either when frozen or when thawed or both, where percent (%) recovery and stability were measured. All populations included an anticoagulant in the apoptosis incubation mixture. Table 6 presents the results of these studies.

Table 6: yield and stability comparisons were made for the Final Product (FP) prepared from harvested cells with or without addition ("+") anticoagulant during freezing ("F") and thawing ("Tha").

Additional population analysis comparisons of early apoptotic cell populations (cell batches) prepared with and without anticoagulant addition showed the consistency of these results.

Table 7: comparison of cell population analysis between batches prepared with and without anticoagulant addition

Percentage of final product cells (yield) in the presence or absence of anticoagulant. Similar to the results presented above at table 3, the data presented in table 6 demonstrate that early apoptotic cells prepared from cells frozen in the presence of anticoagulant produced a beneficial effect on the average yield of fresh end product (FP t0) compared to cells frozen in the absence of anticoagulant. Beneficial effects are seen when anticoagulants are used either frozen alone (61.3 + -2.6% versus 48.4 + -5.0%) or both frozen and thawed (56.5 + -5.2% versus 48.4 + -5.0%). The beneficial effect was less pronounced when the anticoagulant was used when thawing only (44.0 ± 8.5% versus 48.4 ± 5.0%). These are non-high triglyceride samples.

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

Effect of anticoagulant on stability. Fresh FP prepared with or without anticoagulant was stored at 2-8 ℃ for 24 hours to determine if addition of ACDhep to the preparation program would compromise FP stability. Cells were sampled after 24 hours of storage and the yield of cell counts was calculated. Similar to the results for the extended period of time (up to 72 hours) shown in table 3, table 6 shows that the beneficial effect remains and is observed when the anticoagulant is used when freezing alone (59.8 ± 2.1% versus 47.5 ± 4.7%) or both freezing and thawing (56.4 ± 5.3% versus 47.5 ± 4.7%). The beneficial effect was less pronounced when anticoagulant was added only at thawing (42.4 ± 6.1% versus 47.5 ± 4.7%). These are all non-high triglyceride samples. These results show that in all treatments, cell loss was minimal after 24 hours storage of the FP, with significant advantages for cells treated with anticoagulant during both freezing and thawing. The average loss of cells treated with anticoagulant during freezing alone was 2.3 ± 3.2% compared to 1.9 ± 3.3% without anticoagulant; 3.0 ± 4.7% when thawed alone, compared to 1.9 ± 3.3% without anticoagulant; and 0.2 ± 0.4% when cells were both frozen and thawed with ACDhep, compared to 1.9 ± 3.3% without anticoagulant. In summary, the beneficial effect of anticoagulant on yield is maintained for at least 24 hours.

The characteristics of representative cell populations of FPs are shown below in Table 8.

Table 8: characterization of cell populations of fresh (t0) FP prepared from collected cells with or without addition ("+") anticoagulant during the freezing ("F") and thawing ("Tha") procedures. *

Induction of apoptosis was performed in all batches using medium containing anticoagulant.

The results in table 8 show the cellular characteristics of the Final Product (FP) prepared with or without anticoagulant upon freezing and thawing. Batches were sampled, stained for mononuclear markers, and analyzed by flow cytometry to determine the cell distribution in each sample and to check whether addition of anticoagulant affects the cell population. As presented in table 7, no significant differences were detected in cell populations prepared with or without anticoagulant upon freezing or thawing. The mean T cell population (CD3+ cells) in fresh FP was 62.3 ± 1.2% between treatments compared to 62.9 ± 1.1% before freezing; the mean B cell population (CD19+ cells) was 8.3 ± 2.5% between treatments compared to 3.1 ± 0.8% before freezing; the mean natural killer cell population (CD56+ cells) was 9.5 ± 0.7% between treatments compared to 12.9 ± 0.5% before freezing; the mean monocyte cell population (CD14+ cells) was 13.8 ± 0.5% between treatments compared to 17.5 ± 0.3% before freezing; and the mean granulocyte population (CD15+ cells) in fresh FP was 0.0% compared to 0.35 ± 0.2% when frozen.

The efficacy of the early apoptotic population was also examined.

Table 9: potency analysis of fresh (t0) FP prepared from cells with or without addition ("+") anticoagulant during the freezing ("F") and thawing ("Tha") procedures.

The results presented in table 9 are from potency assays performed to determine the ability of each end product to enhance the tolerogenic state in Immature Dendritic Cells (iDC) upon stimulation with (LPS). Tolerogenic effects were determined by assessing the down-regulation of the expression of the co-stimulatory molecules HLA-DR and CD86 on idcs after interaction with early apoptotic cell populations and different treatments leading to LPS up-regulation. Analysis was performed on DCsign + cells. The results represent the percentage of the delay in maturation on LPS-induced maturation after interaction with early apoptotic cell populations and after addition of LPS. Experiments the efficacy of fresh FP (t0) prepared with or without anticoagulant was tested. The results presented in table 9 show that apoptotic cells prepared with or without anticoagulants potentiate the tolerogenic effects of both co-stimulatory markers in a dose-dependent manner.

The early apoptotic cells generated herein are from non-hypertriglyceridemic samples. This consistently high yield of stable early apoptotic cells occurs even in the presence of high triglyceride levels in the donor plasma (see, e.g., examples 12 and 13 of International publication No. WO 2014/087408 and U.S. application publication No. US-2015-0275175-A1). Note that no anticoagulant was added to the PBS medium used to formulate the final early apoptotic cell dose for infusion.

Summary

The aim of this study was to generate a stable, high-yielding population of early apoptotic cells. The rationale for using anticoagulants is that aggregates are seen first in patients with high triglyceride content, but only later in a large proportion of other patients. Of interest here is the disclosure in US patent No. US 6,489,311, that is, the use of anticoagulants to prevent apoptosis.

In short, the number of collected cells in the final product (yield) is significantly increased by at least 10-20% when added with anticoagulant with minimal impact on the composition, viability, stability and apoptotic properties of the cells. In this study it was shown that the yield was increased by up to 13%, which means that under controlled conditions the yield was increased by 26.8%, but under actual GMP conditions the yield was increased by up to 33%, and then a larger increase in the number of cells could be produced in a single collection. This effect is important because it can avoid the need for a second apheresis from the donor.

This effect is surprising, since the expected effect is expected to be dissolution of the mild aggregates. It has been hypothesized that thawing cells in the presence of an anticoagulant reduces the amount of aggregates. These aggregates eventually lead to a large loss of cells when formed. Cells collected and frozen in the absence of anticoagulant demonstrated aggregate formation upon thawing immediately after washing. In addition, high levels of aggregates were also detected in cells frozen in the absence of anticoagulant and resuspended in medium containing anticoagulant. No aggregates were seen in cells frozen and resuspended in medium containing anticoagulant. In summary, it is concluded that: the addition of anticoagulants during freezing and apoptosis induction is very important and does not appear to have a negative impact on the induction of early apoptosis in cell populations.

Recovery of early apoptotic cells was further tested for stability purposes, e.g. after 24 hours of storage at 2-8 ℃, during which an average cell loss of 3-4.7% (regardless of the preparation conditions) was measured, and satisfactory results were obtained for cells frozen and thawed with anticoagulant-containing media (cell loss of 0.2 ± 0.4% after 24 hours of FP storage), suggesting that anticoagulant addition during freezing and thawing is critical, but once finally formulated, the early apoptotic cell population is stable. Prolonged time point studies showed this stability to reach at least 72 hours.

Addition of anticoagulants during the freezing and/or thawing stages did not significantly affect apoptosis and viability of the FP products and cellular composition. The values measured from the various features are similar, indicating that ACDHep does not change the characteristics of early apoptotic cells and that the final product meets the acceptance criteria of ≧ 40% apoptotic cells.

Assays for testing the efficacy of apoptotic cells are based on Immature Dendritic Cells (iDC), which are DCs characterized by functions such as phagocytosis, antigen presentation, and cytokine production.

HLA-DR (MHC class II) membrane molecules and the co-stimulatory molecule CD86 were selected as markers to detect the tolerogenic effects of Antigen Presenting Cells (APC). Changes in HLA-DR and CD86 expression on idcs were measured using flow cytometry after stimulation with LPS and in the presence of early apoptotic cell populations prepared and stimulated with LPS with or without anticoagulants. The population of early apoptotic cells was provided to DCs at increasing ratios of 1:2, 1:4 and 1:8iDC to the population of early apoptotic cells. As presented in table 6, it is shown that the early apoptotic cell population enhances the tolerogenic effect in a dose-dependent manner compared to stimulated DC, with slightly better results for the early apoptotic cell population prepared with anticoagulant at both freezing and apoptosis induction.

In summary, it is concluded that: the addition of anticoagulant to both the freezing medium and the apoptosis medium is important to improve cell recovery and to avoid significant cell loss due to aggregates and in many cases to avoid a second round of apheresis from the donor. All cells are shown to meet the validated acceptance criteria for FP, indicating that addition of anticoagulant does not compromise FP.

Example 2: effect of apoptotic cells on cytokine storm in vitro model of cytokine storm

The purpose is as follows: apoptotic cells were tested for their effect on the levels of cytokine storm markers (cytokines IL-6, IL-10, MIP-1 alpha, IL-8, TNF-alpha, MIP-1 beta, MCP-1 and IL-9) in cytokine storms induced in an LPS sterile model of macrophage activation syndrome.

Method

Cell lines and culture reagents

Human lymphoma cell lines Raji (eCACC, UK, accession number 85011429), human cervical adenocarcinoma cell line Hara (ATCC, USA, accession number: CCL-2) and Hira-CD 19 (Promega Biotechnology Co., Ltd., USA, catalog number PM-Hira-CD 19) were cultured in RPMI 1640(Gibco, Feishal technology Co., Ltd., USA, catalog number 12657-. The hala-CD 19 medium was further supplemented with 1. mu.g/ml puromycin (Sigma Aldrich, USA, Cat. No. P9620) as a selective antibiotic during standard culture.

All cells were maintained under sub-confluent conditions. Raji cells were maintained at 0.3X 10⁶-2×10⁶Concentration range of individual cells/ml. When the recipient was filled to 90% confluence, hela and hela-CD 19 cells were passaged.

Primary monocytes were isolated from the blood-contributed buffy coat (Sheba Medical Center). First, Peripheral Blood Mononuclear Cells (PBMC) were isolated on a Ficoll density gradient (Ficoll-Paque PLUS, GE Healthcare, U.K., catalog No. 17-1440-03). After centrifugation (800x g, 2-8 ℃, 20 min, interruption 0), the PBMC-containing mesophase was transferred to fresh tubes and washed with 2mM L-glutamine (Lonza group (Lonza), switzerland, catalog No. BE17-605E) and 10mM Hepes (Lonza group,switzerland, catalog No. BE17-737B) was washed with RPMI-1640 (Lorsoxa group, Switzerland, catalog No. BE12-918F) and centrifuged (650x g, 2-8 ℃ C., 10 minutes). Resuspend the pelleted cells in "Wash Medium" to 15X 10⁶Concentration of individual cells/ml. Cells were seeded in 0.9ml drops at the center of a 35-mm plate (Corning, U.S. catalog No. 430165). The plates were incubated in a humidified incubator for 1.5 hours (37 ℃, 5% CO) ₂) Monocytes were allowed to adhere and then washed three times with pre-warmed PBS (longsha group, switzerland, catalog No. BE17-516F) to remove other cell types. After washing, the cells were cultured in 2ml RPMI 1640(Gibco, Saimeisheishi technologies, USA, Cat. No. 31870-.

All cell lines were incubated at 37 ℃ and 5% CO₂The humidified incubator of (1) is cultured.

Briefly, and following the manufacturer's guidelines, target cells (hela or hela-CD 19) were cultured alone or in combination with monocytes. After target cells were attached to the plate (6 hours-overnight), cultures were exposed to y × 10⁶ApoCell cells were washed out for 1 hour, after which the cells were washed out by 4-5 RPMI washes. Removal of ApoCell cells was visually confirmed under an optical microscope. 10ng/ml LPS (Sigma Aldrich, USA, catalog number L4391) was introduced into the co-culture and incubated for 1 hour. After incubation, LPS was removed by 3-5 wash cycles with RPMI. Live CD19-CAR T cells or naive T cells were added at one or more of the indicated E/T ratios and incubated for 4 hours. To collect the medium, the plates were centrifuged at 250x g for 4 minutes at 2-25 ℃ (centrifuge 5810R, Eppendorf, germany) to pellet the cells. Transfer 50 μ l of supernatant medium from each well to a fresh flat bottom 96 well microplate well (corning, usa, catalog No. 3596) and add 50 μ l of CytoTox 96 reagent to each well. Will board Incubate in the dark at room temperature for 30 minutes, after which the reaction is stopped by adding 50. mu.l stop solution per well. The absorbance was read at 492nm using Infinite F50 (Tecan, Switzerland) and captured using Magellan F50 software. Data analysis and graph generation were performed using Microsoft Excel 2010.

Following incubation with apoptotic cells or with supernatants from apoptotic cells, analysis of cytokine release was performed using the limiex technique.

As a result: FIGS. 4A to 4H show a significant reduction in the levels of the LPS-induced cytokine storm markers IL-10, IL-6, MIP-1 α, IL-8, TNF- α, MIP-1 β, MCP-1 and IL-9 in an in vitro model of macrophage activation syndrome. While administration of ApoCell to achieve a macrophage Apocell ratio of 1:8 resulted in a significant reduction in the levels of IL-10, IL-6, MIP-1 α, IL-8, TNF- α, MIP-1 β, MCP-1, and IL-9 released into the culture medium (FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H), administration of ApoCell to achieve a macrophage Apocell ratio of 1:16 actually inhibited or nearly inhibited the release of cytokines IL-10, IL-6, MIP-1 α, IL-8, TNF- α, MIP-1 β, MCP-1, and IL-9 in this model.

The addition of apoptotic cells resulted in the inhibition of at least 20 proinflammatory cytokines and chemokines induced in macrophage activation, samples of the results are shown in fig. 4A-4H. A common mechanism of proinflammatory cytokines and chemokines is NF-. kappa.B inhibition.

Inhibition of release of pro-inflammatory cytokines and chemokines appears to be specific, as examination of cytokine IL-2R (IL-2 receptor) levels under similar conditions shows that the levels of released IL-2R are not affected in the same way as pro-inflammatory cytokines. (FIG. 4I). Addition of apoptotic cells at a ratio of 1:4 and 1:8 increased IL-2R release. Further, fig. 4J shows that apoptotic cells had no effect on the release of IL-2 over a 24 hour period. Activation of the IL-2 receptor is thought to play an important role in key functions of the immune system, including tolerance.

And (4) conclusion: the addition of early apoptotic cells to the cytokine storm model of macrophage activation syndrome in the presence of cancer and CAR-19 resulted in a significant reduction and surprisingly even prevention of proinflammatory cytokines (e.g., IL-10, IL-6, MIP-1 alpha, IL-8, TNF-alpha, MIP-1 beta, MCP-1, and IL-9) while cytokine IL-2R levels were elevated or unaffected. Thus, the results herein show that while pro-inflammatory cytokines are reduced by incubation with apoptotic cells, incubation with early apoptotic cells does not affect IL-2 and IL-2R in the same way. Thus, T cell-associated cytokines are not affected by CAR T cell therapy + apoptotic cells, whereas innate immune cytokines (such as those released from monocytes, macrophages, and dendritic cells) are affected.

Example 3: effect of apoptotic cells on cytokine storm without negative effects on CAR-T cell efficacy

The purpose is as follows: apoptotic cells or supernatants derived from apoptotic cells were tested for the effect of cytokine storm marker cytokines and the efficacy of CAR T cells against tumor cells.

The method comprises the following steps:

t4+ CAR T cells

A solid tumor model reported to induce cytokine storm in mice was used (van der Stegen et al, 2013 supra). In this model, T cells were engineered with Chimeric Antigen Receptors (CARs) targeting certain ErbB dimers (T4)⁺CAR-T cells) that are often highly upregulated in specific solid tumors, such as head and neck tumors and ovarian cancer. T cells were isolated from PBMCs isolated from peripheral blood using CD3 microbeads. Vectors containing the chimeric T4+ receptor were constructed and transduced into isolated T cells, thereby generating T4+ CAR T cells. For the experiments performed herein, T4+ CAR T cells were purchased from creative laboratory (new york, usa) or pomi biotechnology limited (ca, usa). Figure 5 presents flow cytometry curves demonstrating surface expression of the 4 α β chimeric receptor on T4+ CAR T cells using an anti-CD 124 monoclonal antibody (Wilkie et al, supra). In addition, a PCR procedure was performed and it verified the presence of the vector in the transduced T cells.

SKOV3-luc cells

SKOV3-luc ovarian adenocarcinoma tissue culture cells were purchased from Cell laboratories (Cell BioLabs) (Cat. No. AKR-232). SKOV3-luc highly expresses the ErbB receptor and is T4⁺CAR-T cell targets (van der Stegen et al, 2013, supra). These cells have been further propagated to constitutively express the firefly luciferase gene, allowing for tracking of cell proliferation in vitro and tumor growth and regression in vivo.

Apoptotic cells

Apoptotic cells were prepared as in example 1.

Apoptotic cell supernatant

Eight (8) million apoptotic cells were seeded per well in 12-well plates. After 24 hours, the cells were centrifuged (290g, 4 ℃ C., 10 minutes). The supernatant was collected and frozen in aliquots at-80 degrees until use. Different numbers of cells were used to prepare supernatants. Some aliquots contained concentrated supernatant.

Monocyte isolation

PBMCs were isolated from peripheral blood/buffy coat obtained from healthy, qualified donors using Ficoll (GE healthcare, uk). Cells were made to 15X 10 in RPMI1640(Gibco, Seimer Feishell science, Mass.) of⁶Concentration of individual cells/ml and was seeded in 0.9ml drops in the middle of a 35mm plate (corning, n.y., usa). The plates were then incubated at 37 ℃ in 5% CO ₂Incubated for 1 hour. At the end of the incubation, the cells were washed three times with PBS (Biological industries ltd), israhbett, and the adherence was determined using an optical microscope. The cells were then incubated with complete medium (RPMI1640+ 10% heat-inactivated FBS + 1% Glutamax + 1% PenStrep, both from Gibco).

An alternative monocyte isolation method has also been used, in which human monocytes are isolated from heparinized peripheral blood by density gradient centrifugation. The isolated monocytes were then separated into monocyte populations, B cell populations, and T cell populations by magnetic bead isolation (Miltenyi Biotec, usa, ontario, ca) by positive selection of monocytes as CD14+ fraction, positive selection of B-cells as CD22+ fraction, and negative selection of T cells as CD14-CD 22-fraction. The purity of the mononuclear cells is more than 95%.

For macrophage differentiation, at the end of adherence, cells were washed three times with PBS and then incubated with RPMI1640+ 1% Glutamax + 1% PenStrep and 10% heat-inactivated human AB serum (sigma, missouri, usa). Cells were incubated at 37 ℃ and 5% for 7-9 days with media changes at day 3 and day 6. Differentiation was determined morphologically by light microscopy.

Supernatants from apo + monocytes

CD14+ monocytes were cultured with apoptotic cells prepared as above at a ratio of 1:16 for 24 hours. The monocyte count was: in 12-well plates 50 ten thousand cells per well and the number of apoptotic cells was: 800 cells per well in 12-well plates. After 24 hours of incubation, the cells were centrifuged (290g, 4 degrees Celsius, 10 minutes). The supernatant was collected and frozen in aliquots at-80 degrees until use. Similar procedures can be performed with different ratios of monocytes to apoptotic cells and/or using other cell sources such as macrophages and dendritic cells.

In vitro culture conditions

Initial experiments were performed by incubating SKOV3-luc cancer cells with apoptotic cells or apoptotic supernatant for 1 hour, followed by co-culture with T4+ CAR T cells (+/-monocytes-macrophages) for 48 hours.

To simulate in vivo conditions, 1X 10⁵Individual THP-1 cells/ml (HTCC, usa) or monocytes or macrophages or dendritic cells will differentiate with 200nM (123.4ng/ml) Phorbol Myristate Acetate (PMA) for 72 hours and then will be cultured in complete medium without PMA for another 24 hours. Next, the cancer or tumor cells (e.g., SKOV3-luc cells) will be at 5X 10 ⁵Individual SKOV3-luc cells/well were plated on differentiated THP-1 cells in 24-well plates. 4X 10 after initial culture of cancer or tumor cells⁵-8×10⁵Individual apoptotic cells (ApoCell) will be added to the culture for 1-3 hours to induce an immune tolerant environment. Will be paired withThe ratio of cancer cells to ApoCell for each cell type was optimized. After washing, the co-culture will be treated with 10ng/ml LPS, after which 1X 10 will be added⁶A T4⁺CAR T cells (or number to be determined by effector/target ratio mapping). The ratio of tumor cells and T4+ CAR T cells will vary in order to generate an effector/target (E/T) ratio map for each tumor or cancer cell type.

To determine the cytotoxicity of SKOV3 cancer cells, lysates were prepared and luciferase activity was determined after a 48 hour incubation period. Additional experiments will be performed to determine the cytotoxicity of cancer or tumor cells of other cancer cell types and will be performed at intervals over a 48 hour incubation period. Alternatively, a CytoTox 96 nonradioactive cytotoxicity assay from plomega corporation (catalog number G1780) will be used.

Lysate preparation

SKOV3-luc monolayers were washed with PBS to remove any residual serum and 70. mu.l CCLR lysis buffer X1/well (for 24-well plates) was added to prepare SKOV3-luc cell lysates. Detachment is further enhanced by physically scraping the bottom of the hole. After vortexing for 15 seconds, the lysate was centrifuged at 12,000g for 2 minutes at 4 ℃. The supernatant was collected and stored at-80 ℃.

In vitro luciferase Activity

To detect luciferase activity in SKOV3-luc cells in culture, a luciferase assay system (promega, catalog No. E1501) was used. Calibration of this kit with a luminometer reader (core facility of Hadamard Carlem medical college at the University of Hebrew University of Jerusalem) was performed by using QuantiLum recombinant luciferase (Promega, Cat. No. E170A). 612 ag-61.2. mu.g (10) was used^-20-10^-9Moles) to determine the detection range and follow the manufacturer's guidelines. Briefly, a 20 μ l volume of each luciferase was placed in a well of a black 96-well plate (Nunc). Each amount was performed in triplicate. Add 100. mu.l LAR (luciferin substrate from luciferase assay System kit) to each well and add to each wellRead immediately after 10 seconds exposure.

For luciferase activity reads, lysates were thawed on ice and 20 μ Ι samples were placed in black 96-well plates (Nunc). Each sample was read in duplicate. 100 μ l LAR was added and the luminescence for the 10 second exposure period was read every 2.5 minutes for 25 minutes and every 40 seconds for the next 10 minutes.

Cytokine analysis

IL-2, IL-2 receptor (IL-2R), IL-6, IL-1 α, IL-4, IL-2, TNF- α were initially assayed. To measure the reduction in cytokine release by IL-2, IL-2 receptor (IL-2R), IL-6, IL-1 α, IL-4, IL-2, TNF- α, and other cytokines, supernatants were collected and examined for selected cytokines using a Luminex MagPix reader and ELISA assay.

As a result:

SKOV3-luc growth

SKOV3-luc growth was followed using luciferase activity as an indicator to determine the number of target SKOV3-luc cells in future experiments. Mixing 3.8X 10⁴-3.8×10⁵Individual SKOV3-luc cells/well were plated in 24-well plates (corning) and luciferase activity was monitored daily for 3 days. 1.9X 10 of plating⁵Cells/well or higher numbers reached confluence and exhibited saturation of growth as indicated by luciferase activity 2 days after plating (figure 6). Note that 3.8 × 10⁴-1.1×10⁵Individual SKOV3-luc cells/well were still in a linear or exponential growth phase three days after plating (fig. 6, orange, blue-green and purple plots). Negative control (3.8X 10 without LAR substrate⁵Individual SKOV3-luc cells) showed only background level readings and confirmed that bioluminescent readings from SKOV3-luc cells were generated due to luciferase activity.

T4 against SKOV3-luc tumor cells⁺Validation of CAR-T cell Activity

To confirm T4⁺CAR-T cell Activity multilayers of SKOV3-luc were exposed to 1,000,000 (one million) T4⁺CAR-T cells or cells exposed to 1,000,000 (one million) untransduced T cells. T4 after 24 hours incubation compared to untransduced T cell controls⁺CAR-T cells reduced SKOV3-luc proliferation by 30% (FIG. 7), which shows T4⁺Anti-tumor activity of CAR-T cells.

Comparing the activity of independent T4+ CAR-T cells against SKOV3-luc tumor cells to the activity after exposure to apoptotic cells

Apoptotic cells (ApoCell) and apoptotic cell supernatants (ApoSup and ApoMon Sup) were tested to determine whether they interfered with T4+ CAR-T cell anti-tumor activity. SKOV3-luc tumor cells were incubated with apoptotic cells for one hour, followed by addition of either T4+ CAR-T cells (500,000, fifty thousand) or T4+ untransduced T cells (500,000, fifty thousand) (T4)⁺CAR-T cell to apoptotic cell ratio of 1: 2). Tumor cells/apoptotic cells/T4⁺CAR T cells were co-cultured for 48 hours. Control SKOV3-luc tumor cells were co-cultured with T4+ CAR-T cells and hartmann's solution (vehicle of apoptotic cells), but without apoptotic cells, for 48 hours.

The results show that after 48 hours of incubation, T4+ CAR-T cells had superior anti-tumor activity compared to incubation with untransduced T cells. Similar incubations were performed with apoptotic cells or apoptotic cell supernatants. Surprisingly, T4+ CAR T cell anti-tumor activity was comparable with or without exposure to apoptotic cells or apoptotic cell supernatants. (FIG. 8).

Effect of apoptotic cells on ameliorating, reducing or inhibiting cytokine storm due to CAR-T therapy

The effect of apoptotic cells on reducing cytokine storm was next examined. IL-6 is the prototypical proinflammatory cytokine released in cytokine storms (Lee DW et al (2014) blood 124(2):188-195) and is commonly used as a marker of cytokine storm responses.

Cultures were established to mimic the CAR T cell therapy environment in vivo. SKOV3-luc tumor cells were cultured in the presence of human monocyte-macrophages and T4+ CAR T cells. The measured Il-6 concentration in the medium was approximately 500-600 pg/ml. This concentration of IL-6 is indicative of a cytokine storm.

Unexpectedly, the level of IL-6 measured in the medium of SKOV3-luc tumor cells, human monocyte-macrophages, T4+ CAR-T cells where the tumor cells had been previously incubated with apoptotic cells for one hour (ratio of T4+ CAR-T cells to apoptotic cells 1:2) was significantly reduced. Similarly, IL-6 levels measured in media of SKOV3-luc tumor cells, human monocyte-macrophages, T4+ CAR-T cells where the tumor cells had been previously incubated with apoptotic cell supernatant for one hour were also significantly reduced. This decrease in IL-6 concentration represents a decrease in cytokine storm (FIG. 9).

The conclusion is drawn: unexpectedly, apoptotic cells and apoptotic supernatants did not abrogate the effect of CAR-T cells on tumor cell proliferation, while at the same time they down-regulated pro-inflammatory cytokines such as IL-6, which is believed to be the major cytokine responsible for pathogenesis.

Assays using a wider range of cytokines

To further evaluate the effect on a potentially greater range and level of cytokines not produced during the experimental procedure but present in the clinical setting during a human cytokine storm, LPS (10ng/ml) was added to SKOV3-luc culture conditions outlined above. The addition of LPS is expected to exponentially increase cytokine storm levels. As expected, the addition of LPS increased the cytokine storm effect and, therefore, the IL-6 level increased to approximately 30,000 pg/ml. Other cytokines known to be expressed at high levels during cytokine storms show elevated levels, for example: TNF-alpha (250-300pg/ml), IL-10(200-300pg/ml), IL 1-alpha (40-50pg/ml) and IL-18(4-5 pg/ml). As shown in figure 10, exposure to apoptotic cells significantly reduced the level of IL-6 even during the exponential state of the cytokine storm to an almost normal level that can be seen in the clinical setting and that is not typically seen in experimental procedures performed with CAR T cells. This effect is similar in the other proinflammatory cytokines TNF- α, IL-10, IL1- α, IL-1 β and IL-18, which show a reduction between 20% and 90%. Similar results were found when apoptotic cell supernatants were used instead of apoptotic cells.

Effect of apoptotic cells on IL-2 and IL-2R

After incubation of SKOV3-luc cells with T4+ CAR T cells, the IL-2 concentration measured in the culture supernatant was 1084 pg/ml. Surprisingly, the concentration of IL-2 increased to 1190pg/ml when SKOC3-luc cells were first incubated with apoptotic cells and then with T4+ CAR T cells. Similarly, after incubation of SKOV3-luc cells with T4+ CAR T cells, the IL-2R concentration measured in the culture supernatant was 3817 pg/ml. Surprisingly, the concentration of IL-2R increased to 4580pg/ml when SKOC3-luc cells were first incubated with apoptotic cells and then with T4+ CAR T cells. In SKOV3-luc only, the concentration of Il-2 was 3.2pg/ml, and in the case of apoptotic cells, the concentration was 10.6 pg/ml. In SKOV3-luc alone, the concentration of Il-2R was 26.3pg/ml, and in the case of apoptotic cells, the concentration was 24.7 pg/ml.

Conclusion

CAR-T cell therapy has been documented as causing cytokine storms in a large number of patients. These results demonstrate that apoptotic cells and apoptotic cell supernatants surprisingly reduce cytokine storm cytokine markers without affecting the efficacy of CAR-T cells against tumor cells. Furthermore, it appears that apoptotic cells increase the cytokine IL-2, which can increase the duration of CAR T cell therapy by maintaining or increasing CAR T cell proliferation.

Example 4: apoptotic cell therapy to prevent cytokine storm in mice administered CAR T cell therapy

The purpose is as follows: apoptotic cells or apoptotic cell supernatants were tested in a solid tumor model (SKOV3 ovarian adenocarcinoma) for in vivo effects in order to determine T4+ CAR T cell efficacy and cytokine storm marker cytokine levels.

Materials and methods

In vitro studies:

methods comprising preparing, culturing and analyzing the results described above have been described above in example 1 and relate to in vitro methods and various assays using T4+ CAR T cells that recognize ErbB target antigens (referred to herein as "T4 + CAR T cells"), SKOV3-luc cells, apoptotic supernatants, monocytes, macrophages. The same method is used herein.

In vivo studies

Mouse

SCID-light brown mice and NSGS mice, 7-8 weeks old, were purchased from Harland (Harlan) (Israel) and stored in the SPF animal facility at the Charite Institute (Sharett Institute).

SKOV3-luc tumor cells (1X 10)⁶2 or 2X 10⁶Individual) were inoculated into SCID light brown mice or NSGS mice by intraperitoneal injection in PBS or subcutaneous injection in 200ml Matrigel (BD Biosciences). At about 14-18 days post injection, tumor engraftment was confirmed by bioluminescence imaging (BLI), and mice were sorted into groups with similar signal intensity prior to administration of T cells.

Mice will be administered 24 hours prior to T4+ CAR T cells or with T4+ CAR T cells (10-30X 10)⁶One T4+ CAR T cell) received 30 × 10 at the same time⁶And (4) apoptotic cells. Bioluminescence imaging (BLI) will be performed after tumor growth and circulating cytokine levels will be determined by Luminex.

In vivo luciferase assay

Tumor growth was monitored weekly by firefly luciferase activity. Briefly, 3mg D-fluorescein (e1605. promegage, usa)/mouse (100 μ Ι of 30mg/ml D-fluorescein) was injected intraperitoneally into isoflurane anesthetized mice and ventral images were acquired 10 minutes after injection using IVIS imaging system and Live Image capture software (both from Perkin Elmer, usa).

Paired 0.5X 10-pass recipients 5 minutes after the D-fluorescein injection "auto" option⁶Mice of SKOV3-luc cells/mouse were imaged to select image acquisition parameters for each image session (session). The capture parameters were set for exposure of bin 4, F/aperture 1.2 and 2-4 minutes using a 24 lens. Data analysis and quantification were performed using Live Image software and graphs were generated using Microsoft's Excel program.

In vivo cytotoxicity

To assess in vivo exclusive use of T cells, organs were collected from mice, formalin-fixed, and histopathological analysis was performed on the organs.

Cytokine analysis

Supernatants and sera were analyzed using a Luminex MagPix reader and/or ELISA kit, flow cytometry bead arrays as described by the manufacturer (Th1/Th2/Th 17; BD biosciences). For example, assays may be performed for proinflammatory cytokines, in one instance the proinflammatory cytokine is IL-6, although in some embodiments any cytokine listed in tables 1 and 2 or known in the art may be assayed herein.

As a result, the

Calibration of SKOV3-luc tumors in vivo

Mixing 0.5X 10⁶1, 1 × 10⁶Or 4.5X 10⁶Individual SKOV3-luc cells were injected intraperitoneally into SCID light brown mice and bioluminescence imaging (BLI) was performed weekly to track tumor growth as described in the methods (data not shown).

Clinical scores of mice

The mice did not show any clinical symptoms within the first 4 weeks. However, 28 days after injection of SKOV3-luc, high doses (4.5X 10)⁶A plurality of; purple line) began to steadily lose weight (fig. 11A), and the overall appearance of the mice deteriorated, manifesting as lethargy, abnormal pacing, and overall loss of activity. This group was removed on day 39 and abdominal necropsy was performed to reveal the appearance and size of the tumor (fig. 11B). SKOV3-luc tumors were large, solid, vascularized and showed a whitish and shiny skin color. The caudal or medullary part of a large tumor in the abdominal cavity dominates on the injection side (left side). This tumor occupies approximately 25-75% of the lumen and significantly compresses and interferes with the intestinal tract. Smaller lesions were also observed at various locations within the abdominal cavity. Tumors were contained within the abdominal cavity, and no other tumors were observed in any other part of the body of any of the mice. Receive a low dose (0.5X 10) ⁶Single) or medium dose (1X 10)⁶Individual) mice of SKOV3-luc stopped weight gain and began to steadily lose weight 40 days after SKOV3-luc injection. The experiment was terminated 50 days after injection of SKOV 3-luc.

SKOV3-luc tumor kinetics

PBS was injected to control SKOV3-luc cells, and these mice did not exhibit any luciferase activity throughout the experiment (figure 12, left panel). Tumor detection and growth is dose dependent. Lower dose (0.5X 10)⁶SKOV3-luc cells) showed tumors beginning 25 days after injection (4/5 animals), medium dose (1 × 10)⁶One) injection showed tumors 18 days after injection (4/5 animals), while at higher doses (4.5 × 10)⁶Individual), tumors were detected in 3/5 animals as early as 11 days post injection, and by day 18 all animals showed established tumors (fig. 12 and fig. 13A-13D).

CAR T cell therapy induces cytokine release syndrome

Increased doses of T4+ CAR T cells (3 × 10) were administered (intraperitoneally or directly into the tumor) to three groups of tumor-free mice as well as to tumor-bearing

mice

⁶2, 10 is multiplied by 10⁶Or 30 x 10⁶One). At the highest dose, both tumor-free and tumor-bearing mice showed suppressed (reduced) behavior, hair uprightness and decreased mobility within 24 hours, with rapid weight loss, and then died within 48 hours. At least human interferon-gamma and mouse IL-6 were detectable in blood samples from mice given the highest dose of CAR T cells. Animals receiving high doses of CAR T cells against different tumor antigens showed no weight loss or behavioral changes.

Administration of apoptotic cells inhibits or reduces the incidence of cytokine release syndrome induced by CAR T cell therapy

Concomitant administration of 2.10 × 10 to a group of mice given the highest dose of CAR T cells⁸One/kg of apoptotic cells, which has previously been demonstrated to be a safe and effective dose. Mice receiving human CAR T + apoptotic cells had significantly reduced levels of IL-6, weight loss, and decreased mortality.

Example 5: effect of combination immunotherapy on in vitro diffuse tumor models

The purpose is as follows: apoptotic cells or supernatants derived from apoptotic cells were tested for effects in a diffuse tumor model, in which the cancer is widespread and non-local or occluded, in order to determine the efficacy of CAR T cells on cancer cells and cytokine storm marker cytokine levels.

The method comprises the following steps:

CD19+ T4+ CAR T cells ("CD 19+ CAR T cells")

CD 19-specific CAR-T cells were purchased from Promega Inc. (batch No. 012916). T cells were 30% positive for CAR (according to manufacturer's FACS data-Fab staining). Briefly, cells were thawed into AimV + 5% heat-inactivated FBS, centrifuged (300g, 5 min, room temperature), and then resuspended in AimV. On day 6 of the experiment, each mouse was injected intravenously at 20X 10 ⁶Individual cells (70% annexin PI negative, 30% CAR positive).

Recombinant hela cells expressing CD19 will be used as a control cell type that also expresses CD19 on their cell surface.

CD123+ CAR T cells

T4+ CAR T cells will also be engineered with CARs that target the CD123 epitope (referred to herein as "CD 123+ CAR T cells").

Raji cells, hela cells expressing CD19 and leukemia cells expressing CD123

Raji cells, purchased from ECACC (catalog No. 85011429), were routinely cultured in complete medium (RPMI-1640 supplemented with 10% h.i. fbs, 1% Glutamax and 1% penicillin/streptomycin) and maintained at 3 × 10⁵-3×10⁶Individual cells/ml. On day 1 of the experiment, each mouse was injected intravenously at 0.1X 10⁶And (4) cells.

Similarly, hela cells expressing CD19 will be generated in the laboratory and used as targets for CD19+ CAR T cells. CD123 expressing leukemia cells will serve as targets for CD123+ CAR T cells. In addition, primary cancer cells will serve as targets for CAR T cells.

Hela cells expressing CD19 were prepared using methods known in the art. The cells will be cultured as is well known in the art.

CD123 is a membrane biomarker and a therapeutic target in hematologic malignancies. As known in the art, CD123 expressing leukemia cells, such as leukemia blast cells and leukemia stem cells, will be cultured.

Apoptotic cells, apoptotic cell supernatants and monocytes prepared as described in example 1 were isolated. The early apoptotic cells produced were at least 50% annexin V-positive cells and less than 5% PI-positive cells.

Macrophages. Produced by adherence from CD14 positive cells.

A dendritic cell. CD14 derived growth in the presence of IL4 and GMCSF.

Flow cytometry. The following antibodies were used: hCD19-PE (e biosciences, Cat. No. 12-0198-42); mIgG1-PE (e biosciences, catalog number 12-0198-42); hCD3-FITC (e biosciences, Cat. No. 11-0037-42); mIgG2a-FITC (e biosciences, Cat. No. 11-4724-82). Acquisition was performed using FACS Calibur for BD.

Naive T cells. Naive T cells were separated from buffy coat using magnetic Beads (BD) and cryopreserved in 90% human AB serum and 10% DMSO. Thawing and injection were the same as CAR-T cells.

In vitro culture conditions

Cell lines and culture reagents

Human lymphoma cell lines Raji (eCACC, UK, accession number 85011429), human cervical adenocarcinoma cell line Hara (ATCC, USA, accession number: CCL-2) and Hira-CD 19 (Promega Biotechnology Co., Ltd., USA, catalog number PM-Hira-CD 19) were cultured in RPMI 1640(Gibco, Sammer Feishi technology Corp., U.S. Pat. No. 35050-038) supplemented with 10% FBS (Gibco, Sammer Feishi technology Corp., south America, catalog number 12657-029), 2mM GlutaMAX (Gibco, Sammer Feishi technology Corp., U.S. Pat. No. 35050-038) and 100U/ml penicillin +100U/ml streptomycin (Gibco, Sammer Feishi technology Corp., U.S. catalog number 15140-122), catalog number 31870-025), hereinafter referred to as "complete medium". The hala-CD 19 medium was further supplemented with 1 μ g/ml puromycin (sigma aldrich, usa, catalog No. P9620) as a selective antibiotic during standard culture.

Primary monocytes were isolated from the blood-contributed buffy coat (israel house medical center). First, Peripheral Blood Mononuclear Cells (PBMC) were isolated on a Ficoll density gradient (Ficoll-Paque PLUS, GE healthcare, UK, Cat. No. 17-1440-03). After centrifugation (800x g, 2-8 ℃, 20 min, interruption 0), the intermediate phase containing PBMC was transferred to fresh tubes and washed with RPMI-1640 (Dragon sand group, Switzerland, catalog No. BE12-918F) supplemented with 2mM L-glutamine (Dragon sand group, Switzerland, catalog No. BE17-605E) and 10mM Hepes (Dragon sand group, Switzerland, catalog No. BE17-737B), hereinafter referred to as "wash medium", and centrifuged (650x g, 2-8 ℃, 10 min). Resuspend the pelleted cells in "Wash Medium" to 15X 10⁶Concentration of individual cells/ml. Cells were seeded in 0.9ml drops at the center of a 35-mm plate (Corning, U.S. catalog No. 430165). The plates were incubated in a humidified incubator for 1.5 hours (37 ℃, 5% CO) ₂) Allowing monocytes to adhere and then washed three times with pre-warmed PBS (longsha group, switzerland, catalog No. BE17-516F) to remove other cell types. After washing, the cells were cultured in 2ml RPMI 1640(Gibco, Saimeisheishi technologies, USA, Cat. No. 31870-.

CD19-CAR T cells (promei ltd, usa, catalog No. FMC63) were delivered in AIM-V medium or frozen form. Cryopreserved CAR T cells for in vitro experiments were thawed in a 35-38 ℃ bath on the day of the experiment and immediately immersed in pre-warmed AIM V medium (Gibco, Sammel Feishell science, USA, catalog No. 12055-. DMSO was removed by centrifugation of the cells (300x g, room temperature, 5 min) and resuspended in pre-warmed AIM V medium. The concentration and viability of the CD19-CAR + cell population were determined by reading anti-FLAG (biog legend, usa, catalog No. 637310) staining and annexin V and PI staining (MEBCYTO apoptosis kit, MBL, usa, catalog No. 4700) with a FACSCalibur flow cytometer (BD, usa).

For naive T cell isolation, PBMCs were extracted from leukapheresis fractions collected from qualified donors signed with informed consent at hadamard medical center (university of einkamer (Ein Kerem campas), jeldahl, israel) or from buffy coats (samson medical center, israel) loaded on Ficoll density gradients using Cobe spectra (tm apheresis BCT, usa) according to SOP of leukapheresis department and centrifuged (800x g, 2-8 ℃, 20 min). T cells were isolated from the positive fraction using the MagniSort human CD3 positive selection kit (e biosciences, U.S. catalog No. 8802-6830-74) following the manufacturer's guidelines. T cells were cryopreserved in "complete medium" (defined above) containing an additional 20% FBS (Gibco, Saimer Feishell technologies, south America, catalog No. 12657-029) and 5% DMSO (Cryosure-DMSO, WAK-Chemie Medical GmbH, Germany, catalog No. WAK-DMSO-70) and thawed in parallel with CD19-CAR T cells on the day of the experiment.

LDH cytotoxicity assays

Lactate Dehydrogenase (LDH) is a stable cytosolic enzyme that is released in a correlated manner by cells undergoing lysis. Therefore, LDH levels in the media can be used to quantify cytotoxic activity. CytoTox 96 nonradioactive cytotoxicity assays (promega, usa, catalog number G1780) are colorimetric assays for quantifying LDH levels in culture media. The tetrazolium salt substrate (iodonitro-tetrazolium violet, INT) was introduced into the culture medium in excess, and LDH converted the substrate to a red formazan product. The amount of red color formed is directly proportional to the number of lysed cells.

Briefly, and following the manufacturer's guidelines, target cells (hela or hela-CD 19) were cultured alone or in combination with monocytes. After target cells were attached to the plate (6 hours-overnight), cultures were exposed to y × 10⁶ApoCell cells were washed out for 1 hour, after which the cells were washed out by 4-5 RPMI washes. Removal of ApoCell cells was visually confirmed under an optical microscope. 10ng/ml LPS (Sigma Aldrich, USA, Cat. No. L4391) was introduced into the co-culture and incubated for 1 hour. After incubation, LPS was removed by 3-5 wash cycles with RPMI. Live CD19-CAR T cells or naive T cells were added at one or more of the indicated E/T ratios and incubated for 4 hours. To collect the medium, the plates were centrifuged at 250x g for 4 minutes at 2-25 ℃ (centrifuge 5810R, Eppendorf, germany) to pellet the cells. 50 μ l of supernatant medium from each well was transferred to a fresh flat-bottom 96-well microplate well (corning, U.S. catalog No. 3596) and 50 μ l of CytoTox 96 reagent was added to each well. The plates were incubated in the dark at room temperature for 30 minutes, after which the reaction was stopped by adding 50. mu.l of stop solution per well. The absorbance was read at 492nm using Infinite F50 (diken, switzerland) and captured using Magellan F50 software. Data analysis and graph generation were performed using Microsoft Excel 2010.

Flow cytometry cytotoxicity assay

hela-CD 19 (target) cells and hela (control) cells were pre-stained with 5 μ M carboxyfluorescein succinimidyl ester (CFSE, life technologies, usa, catalog No. C1157), mixed together, and plated on fresh plates or on plates filled with isolated primary monocytes. After target cells were attached to the plate (6 hours-overnight), cultures were exposed to y × 10⁶ApoCell cells for 1 hour. The plates were washed 3-5 times with RPMI and it was visually verified that suspended ApoCell cells were washedAnd washing the solution. 10ng/ml LPS was introduced into the co-culture and incubated for 1 hour, after which the LPS was removed by 3-5 wash cycles with RPMI. Viable CD19-CAR T cells were then added to the co-culture as indicated by one or more specific E/T ratios and incubated for 4 hours. After the incubation, the cells were collected by adding trypsin-EDTA (Biotech Co., Ltd., Israel, catalog No. 03-052-1B) and incubating at 37 ℃ for 4 minutes. To stop the enzymatic activity, two to four times the volume of "complete medium" was added. Cells were collected, centrifuged at 200x g for 5 minutes at room temperature, and resuspended in 100. mu.l RPMI (Gibco, Saimer Feishell science, USA, Cat. No. 15140-. Subsequently, staining was first performed for anti-CD 19(e biosciences, USA, Cat. No. 12-0198-42) incubated at room temperature for 30 minutes in the dark. After centrifugation (290x g, 1 min, 2-8 ℃) and resuspension in 300. mu.l RPMI, the cells were stained for anti-7 AAD (e biosciences, USA, Cat. No. 00-6993-50). The assay was gated (gate) on 7ADD negative cells (live cells), with live target cells (hela-CD 19) and live control cells (hela) being counted. Percent survival was calculated by dividing the percent of live target cells by the percent of live control cells. To correct for variations in starting cell number and spontaneous target cell death, percent survival was divided by the ratio of percent target cells to percent control cells cultured in the absence of effector cells (CD19-CAR T cells). Finally, the percent cytotoxicity was determined by subtracting the corrected percent survival from 100% ²。

Initial experiments were performed by incubating Raji cancer cells with CD19+ CAR T cells (+/-monocytes-macrophages) for 48 hours in order to determine the optimal ratio of CD19+ CAR T cells to target Raji cancer cells in a 96-well plate at 5 x 10⁴One Raji cell/well was started. An effector/target (E/T) ratio plate was constructed based on the results.

A combination immunotherapy experiment was performed by incubating Raji cancer cells with apoptotic cells or apoptotic supernatant for 1 hour, followed by co-culture with CD19+ CAR T cells (+/-monocytes-macrophages) for 48 hours.

To imitate the bodyInternal Condition, 1X 10⁵Individual THP-1 cells/ml will be differentiated with 200nM (123.4ng/ml) Phorbol Myristate Acetate (PMA) for 72 hours and then will be cultured in complete medium in the absence of PMA for a further 24 hours. Next, Raji cancer cells will be at 5X 10⁵Individual Raji cells/well were plated on differentiated THP-1 cells in 24-well plates.

After initial culture of Raji cancer cells, 4X 10 cells were cultured⁵-8×10⁵Apoptotic cells (ApoCell) were added to the culture for 1-3 hours to induce an immune tolerant environment. The ratio of cancer cells to ApoCell for each cell type will be optimized. After washing, co-cultures were plated with a predetermined amount of CD19 based on the E/T ratio plot ⁺CAR-T cells were processed. In some experiments, 10ng/ml LPS was added to the culture medium before addition of CD19+ CAR T cells. In other experiments, interferon gamma (IFN- γ) was added to the culture medium prior to addition of CD19+ CAR T cells. The addition of LPS or IFN-gamma is expected to exponentially increase the cytokine storm level.

To determine the cytotoxicity of Raji cancer cells, lysates were prepared and viability was determined after a 48 hour incubation period. Additional experiments to determine the cytotoxicity of Raji cells will be performed and will be performed at intervals over a 48 hour incubation period. Alternatively, a CytoTox 96 nonradioactive cytotoxicity assay from plomega corporation (catalog number G1780) will be used.

Similar experiments were run with hela cells expressing CD19 and CD19+ CAR T cells.

Similar experiments were run with CD123 expressing leukemia cells and CD123+ CAR T cells.

Cytokine analysis

Initial cytokine assay the culture supernatants were examined for levels of MIP1a, IL-4, IL-2R, IL-6, IL8, IL-9, IL-10, IL-13, IL-15, INF-gamma, GMCSF, TNF-alpha.

Additional cytokine assays the levels of cytokines IL-10, IL-1 β, IL-2, IP-10, IL-4, IL-5, IL-6, IFN α, IL-9, IL-13, IFN- γ, IL-12p70, GM-CSF, TNF- α, MIP-1 β, IL-17A, IL-15/IL-15R or IL-7 or any combination thereof are examined.

Cultures were established to mimic the CAR T cell therapy environment in vivo. Raji Burkett lymphoma cells were cultured in the presence of human monocyte-macrophages, LPS and CD19+ CAR T cells with and without the addition of apoptotic cells.

Raji cells were incubated in the presence of monocytes and LPS, followed by addition of naive T cells (Raji + naive T), addition of CD19+ CAR T cells (Raji + CAR T) and CD19+ CAR T cells and apoptotic cells (ApoCell) (Raji + CAR T + ApoCell 1:8) at a ratio of 1:8CAR T cells to ApoCell, addition of CD19+ CAR T cells and apoptotic cells (ApoCell) (Raji + CAR T + ApoCell 1:32) at a ratio of 1:32CAR T cells to ApoCell, and addition of CD19+ CAR T cells and apoptotic cells (ApoCell) (Raji + T + CAR + ApoCell 1:64) at a ratio of 1:64CAR T cells to ApoCell. Concentration measurements were performed after GM-CSF and TNF- α (TNF- α).

To measure the reduction in cytokine release of IL-6, IL-8 and IL-13, as well as other cytokines, supernatants were collected and examined for selected cytokines using a Luminex MagPix reader and ELISA assay. Cytokines (mouse or human) can be evaluated by Luminex technology using a MAPIX system analyzer (mercmikey Millipore) and mliplex analysis software (mercmikey Millipore). Mice IL-6R α, MIG (CXCL9) and TGF-. beta.1 were evaluated by Quantikine ELISA (R & D systems).

Tissue analysis

Bone marrow and liver were evaluated using flow cytometry and immunohistochemistry. After sacrifice, liver and bone marrow were collected for histopathological analysis. Tissues were fixed in 4% formalin for 48 hours at room temperature and then submitted to animal facilities at hebrew university for processing. The bone is decalcified before processing. Paraffin sections were stained with hematoxylin and eosin and CD 19.

IFN-gamma Effect

The IFN- γ effect was evaluated by both STAT1 phosphorylation and the biological product.

As a result:

cell number to calibrate cytotoxicity assays

To determine the number of Raji cells to be used in the in vitro model, the sensitivity limits of the cytotoxicity assays were evaluated. Will be 5X 10⁴-20×10⁴Individual Raji cells/well were plated in quadruplicate in 96-well plates. One set of quadruplicates was lysed for comparison with still fully viable cells. Lysis was transient, with lysis solution added immediately prior to centrifugation to mimic cytotoxicity of a fraction of the cells. Virtually all cell numbers showed much higher readings than live cells, of which 5X 10⁴The number of individual cells produced the largest relative reading (FIG. 14; extrapolation of the data). Thus, subsequent experiments will use this cell number as a default value unless the experimental design requires otherwise.

CD19 directed against Raji Burkett lymphoma cells⁺Validation of CAR-T cell Activity

To verify CD19⁺CAR-T cell Activity, exposure of Raji cancer cell monolayers to 1,000,000 (one million) CD19⁺CAR-T cells or exposure to 1,000,000 (one million) untransduced T cells. After 24 hours incubation, CD19⁺CAR-T cells reduced Raji cancer cell proliferation, showing CD19⁺Antitumor activity of CAR-T cells.

Comparing the activity of independent CD19+ CAR-T cells against Raji Burkett lymphoma cells to the activity following exposure to apoptotic cells

Apoptotic cells (ApoCell) and apoptotic cell supernatants (ApoSup and ApoMon Sup) were tested to determine whether they interfered with CD19+ CAR-T cell anti-tumor activity. Raji Burkett lymphoma cells were incubated with apoptotic cells for one hour, and then either CD19+ CAR-T cells (500,000, fifty thousand) or CD19+ untransduced T cells (500,000, fifty thousand) (CD 19) were added⁺CAR-T cell to apoptotic cell ratio of 1: 2). Tumor cells/apoptotic cells/CD 19⁺CAR T cells were co-cultured for 48 hours. Control Raji Burkett lymphoma cells were co-cultured with CD19+ CAR-T cells and Hartmann's solution (a vehicle for apoptotic cells), but without apoptotic cells, for 48 hours.

The results show that after 48 hours of incubation, CD19+ CAR-T cells had superior anti-tumor activity compared to incubation with untransduced T cells. Similar incubations were performed with apoptotic cell supernatants. Surprisingly, CD19+ CAR T cell anti-tumor activity was comparable with or without exposure to apoptotic cells or apoptotic cell supernatants.

No in vitro negative effects of apoptotic cells on CAR-modified T cells against CD19 were seen with comparable results for the E/T ratio of CAR T in the presence or absence of apoptotic cells.

CD19 against Hela leukemia cells⁺Validation of CAR-T cell Activity

Hela cells are specific CD19 expressing cells, which makes them specific for CAR CD19⁺T cell activity is sensitive. In addition, in contrast to Raji cells, which belong to non-adherent cell lines, hela cells are adherent.

To verify CD19⁺CAR-T cell Activity, Hira cancer cell monolayer was exposed to 1,000,000 (one million) CD19⁺CAR-T cells or exposure to 1,000,000 (one million) untransduced T cells. After 24 hours incubation, CD19⁺CAR-T cells reduced Hela cancer cell proliferation, suggesting CD19⁺Anti-tumor activity of CAR-T cells (figure 15CD 19)⁺+ RPMI and CD19 ⁺+ CAR T-19 cells).

Will be directed to CD19⁺Independent CD19 of HeLa cells⁺CAR-T cell Activity compared to Activity after Exposure to apoptotic cells

Apoptotic cells (ApoCell) were tested to determine if they interfered with CD19+ CAR-T cell anti-tumor activity. Heila cells were incubated with apoptotic cells for one hour, and then either CD19+ CAR-T cells (500,000, fifty thousand) or CD19+ untransduced T cells (naive T cells; 500,000, fifty thousand) (CD 19) were added⁺CAR-T cell to apoptotic cell ratio of 1: 2). Tumor cells/apoptotic cells/CD 19⁺CAR T cells were co-cultured for 48 hours. Control hela cells were co-cultured with CD19+ CAR-T cells and RPMI (a vehicle for apoptotic cells), but without apoptotic cells, for 48 hours. CD19⁺CAR-T cells to hela cells ratio (E/T ratio) ranged from 5-20 (figure 15).

Figure 15 shows that after 48 hours of incubation, CD19+ CAR-T cells had superior anti-tumor activity compared to incubation with untransduced T cells (naive cells) or buffer only. Similar incubations were performed with apoptotic cells. Surprisingly, CD19 with or without exposure to apoptotic cells⁺CAR T cell antitumor activity was comparable. Similar experiments were performed using apoptotic cell supernatants. Figure 15 shows that CAR T-CD19 therapy has the same in vitro cytotoxic effect with or without ApoCell added.

No negative effect of apoptotic cells on CAR modified T cells against CD19+ hela cells was observed with comparable E/T ratios in the presence or absence of apoptotic cells.

Thus, CD19 was observed⁺CAR T cells have the same in vitro cytotoxic effect with or without the addition of early apoptotic cells.

Effect of apoptotic cells on ameliorating, reducing, or inhibiting cytokine storm due to CAR-T therapy

Cytokines IL-8 and IL-13 were measured in culture media before or after addition of CD19+ CAR T cells and are showing concentrations consistent with cytokine storm. Addition of apoptotic cells or apoptotic cell supernatants showed a decrease in IL-8 and IL-13 concentrations in the media.

Assays using a wider range of cytokines

To further evaluate the effect on a potentially greater range and level of cytokines not produced during the experimental procedure but present in the clinical setting during a human cytokine storm, LPS (10ng/ml) was added to Raji cell culture conditions outlined above in the presence of cancer and CAR-19. The addition of LPS is expected to exponentially increase cytokine storm levels. Exposure to apoptotic cells significantly reduced the levels of cytokines. The results presented in fig. 16 and 17 show that although addition of CD19+ CAR T cells greatly increased the cytokine concentrations (pg/ml) of GM-CSF and TNF-a in the culture medium, both GM-CSF and TNF-a were significantly reduced in the presence of apoptotic cells. The decrease in cytokine concentration is dose-dependent relative to the apoptotic cell ratio of CAR T cells to apoptotic cells.

And (4) conclusion:

apoptotic cells are able to down-regulate cytokine markers of cytokine storm associated with CAR T cell clinical programs. Notably, apoptotic cells did not show an effect on the tumor activity of CAR T cells. Apoptotic cells reduce pro-inflammatory cytokines derived from innate immunity and suppress IFN- γ effects without compromising IFN- γ levels and CAR-T cytotoxicity.

Example 6: apoptotic cell therapy to prevent cytokine storm in the in vivo model of diffuse cancer to which Car T cell therapy is administered

The purpose is as follows: apoptotic cells or apoptotic cell-derived supernatants were tested for in vivo effects in a diffuse tumor model to determine the efficacy of CAR T cells on cancer cells and cytokine storm marker cytokine levels.

Materials and methods

In vitro study

See the method described in example 5 for in vitro studies.

Cells and cell culture

Raji Burkitt lymphoma cells (sigma aldrich, catalog No. 85011429) were cultured according to the manufacturer's instructions. CD19+ CAR T cells, cell culture, apoptotic cells, apoptotic cell supernatants, monocyte isolation and in vitro measurements are shown in the examples above. The early apoptotic cells produced were at least 50% annexin V-positive cells and less than 5% PI-positive cells.

In vivo studies

Mouse

SCID light brown mice 7-8 weeks old were purchased from Envigo (formerly harland). Mice were maintained in an SPF-free animal facility according to institutional IACUC guidelines. During the course of the experiment, mice were monitored daily and weighed 3 times per week. Mice showing hind limb paralysis were sacrificed. After sacrifice, bone marrow and liver were collected for FACS analysis and histological processing, and serum was frozen at-80 ℃ for cytokine profiling.

In vivo experiments

SCID light brown mice (C.B-17/IcrHsd-Prkdc-SCID-Lyst-bg, Harland, Israel) were housed under SPF conditions and according to the Association for laboratory animal Care assessment and certification (AAALAC) at the animal facilities administration (AAF) of Jubrew university (Eineka university, Israel). This study was approved by the university of hebrew animal research ethics committee and minimized the pain experienced by the animals as much as possible.

(FIG. 18A) for disseminated tumor model, 7-8 week old female SCID light brown mice were injected intravenously into 1X 10 mice each suspended in 200. mu.l RPMI (Gibco, Saimer Feishell science, USA, catalog number 15140-⁵Individual Raji cells (day 1). On day 6, mice of the relevant group were inoculated intravenously with a vaccine containing 30X 10 cells per mouse ⁶A200. mu.l lactated ringer's injection of Hartmann's solution of individual cells ApoCell (Teva Medical, Israel, Cat. No. AWN 2324). On day 6, mice of the relevant group were inoculated intravenously with a 10X 10-containing vaccine per mouse⁶200 μ l AIM V of live CD19-CAR T cells or naive T cells. Control mice received equal volume of RPMI in each treatment.

Mice were examined for clinical indications and weighed twice weekly and sacrificed when hind limb paralysis developed. The animal facilities department of hebrew university of yersinia prepared pathological samples of bone and liver and stained for human CD20 (Cell Marque, usa, clone L26, catalog number 120M-84) to detect Raji cells and human CD3 (Cell Signaling Technology, usa, catalog number 85061) to detect human T cells.

In certain experiments, LPS will be administered to animal subjects prior to the addition of CD19+ CAR T cells. In other experiments, interferon-gamma (IFN- γ) will be administered prior to the addition of CD19+ CAR T cells. The addition of LPS or IFN-gamma is expected to exponentially increase cytokine storm levels.

Cytokine assays cytokine levels including but not limited to IL-10, IL-1 beta, IL-2, IP-10, IL-4, IL-5, IL-6, IFN alpha, IL-9, IL-13, IFN-gamma, IL-12p70, GM-CSF, TNF-alpha, MIP-1 beta, IL-17A, IL-15/IL-15R or IL-7 or any combination thereof are examined. Cytokines (mouse or human) were evaluated by Luminex technology using a MAPIX system analyzer (merck millipore) and mliplex analysis software (merck millipore). Mice IL-6R α, MIG (CXCL9) and TGF-. beta.1 were evaluated by Quantikine ELISA (R & D systems).

Tissue analysis

IFN-gamma Effect

Results

CAR T cell therapy induces cytokine release syndrome

Increased doses of CD19+ CAR T cells (3 x 10) were administered (intraperitoneally or directly into the tumor) to three groups of tumor-free mice as well as to tumor-bearing

mice

⁶2, 10 is multiplied by 10⁶Or 30 x 10⁶One). At the highest dose, both tumor-free and tumor-bearing mice showed suppressed behavior, hair uprightness and decreased mobility within 24 hours, with rapid weight loss, and then died within 48 hours. Human interferon-gamma and mouse IL-6, IL-8 and IL-13 were detectable in blood samples from mice given the highest dose of CD19+ CAR T cells. Animals receiving high doses of CD19+ CAR T cells against different tumor antigens showed no weight loss or behavioral changes.

Concomitant administration of 2.10 × 10 to a group of mice given the highest dose of CD19+ CAR T cells⁸Per kg of apoptotic cells, which has previously been shown to be a safe and effective dose. Mice receiving human CD19+ CAR T + apoptotic cells had significantly reduced levels of at least one mouse proinflammatory cytokine, reduced body weight, and reduced mortality.

Administration of apoptotic cells in combination with CAR T cell administration did not affect CAR T cell anti-tumor activity

FIG. 18B shows CD19 not being administered⁺Injection of CD19 in the case of CAR T cells⁺SCID mice with Raji cells are expected to die for 18-21 days. Received CD19⁺Forty percent (40%) of mice in CAR T cells survived to at least day 30 (fig. 18 dash-dot-and-dot-dash line). The percentage of survivors was independent of the addition of apoptotic cells (figure 18). Surviving mice were sacrificed on day 30.

And (4) conclusion: survival was comparable and there were no in vivo negative effects of apoptotic cells on CAR-modified T cells against CD 19.

The document records a significant down-regulation (p <0.01) of proinflammatory cytokines including IL-6, IP-10, TNF- α, MIP-1 β. IFN- γ was not down-regulated, but its effect on macrophages and dendritic cells was inhibited both in the level of phosphorylated STAT1 and in IFN- γ -induced CXCL10 and CXCL9 expression.

And (4) conclusion:

apoptotic cells reduce pro-inflammatory cytokines derived from innate immunity and inhibit IFN- γ effects without compromising IFN- γ levels and CAR-T cytotoxicity.

Example 7: apoptotic cell therapy to prevent cytokine storm in vivo model of solid tumor cancer administered CAR T cell therapy

The purpose is as follows: apoptotic cells or supernatants derived from apoptotic cells were tested for in vivo effects in solid tumor models in order to determine the efficacy of CAR T cells on cancer cells and cytokine storm marker cytokine levels.

Materials and methods

In vitro study

Cells and cell culture

CD19+ CAR T cells, second generation CAR-T-CD19 cells containing TMCD28, were used, cell culture, apoptotic cells, apoptotic cell supernatants, monocyte isolation and in vitro measurements as shown in examples 2 and 4 and 6 above. As detailed in example 1, the early apoptotic cells produced were at least 50% annexin V-positive cells and less than 5% PI-positive cells.

In vivo studies

Mouse

SCID light brown mice or NSGS mice were inoculated with CD19 expressing hela cells that can adhere to the peritoneum to form solid intraperitoneal tumors. Prior to T cell administration, mice were divided into several groups.

Six days after intravenous inoculation, mice were administered 10 × 10 on day 5 with and without preconditioning of apoptotic cells (ApoCell)⁶Individual CD19+ CAR T cells. Administration of 5X 10 to mice receiving preconditioning⁶Or 30 x 10⁶And (4) ApoCell. Tumors were investigated once a week and circulating cytokine levels were monitored weekly and determined by the Luminex system. 25 mouse cytokines and 32 human cytokines were evaluated using the Luminex technique. After termination of the experiment, mice were culled and organs (bone marrow, liver and spleen) were examined for the presence/size of tumors (by FACS and immunohistochemistry).

Cytokine assays cytokine levels including but not limited to GM-CSF, IFN gamma, IL-1 beta, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, MIP-1 alpha, TNF alpha, MIP-1 beta, IFN alpha and IP-10 were examined. Cytokines (mouse or human) were evaluated by Luminex technology using a MAPIX system analyzer (merck millipore) and mliplex analysis software (merck millipore). Mice IL-6R α, MIG (CXCL9) and TGF-. beta.1 were evaluated by Quantikine ELISA (R & D systems).

Tissue analysis

Bone marrow and liver were evaluated using flow cytometry and immunohistochemistry.

IFN-gamma Effect

Results

CAR T cell therapy induces cytokine release syndrome

mice

⁶2, 10 is multiplied by 10⁶Or 30 x 10⁶One). At the highest dose, both tumor-free and tumor-bearing mice showed suppressed behavior, hair uprightness and decreased mobility within 24 hours, with rapid weight loss, and then died within 48 hours.

Figures 19A-19C graphically show that the levels of IL-6, IP-10, and surprisingly even TNF-alpha cytokines released from tumors were increased even before the presence of CAR T cells. Figures 19A-19C show that IL-6, IP-10 and TNF-a are surprisingly increased by the presence of cancer cells even in the absence of CAR T cell therapy. In the presence of CAR T cell therapy (hela-CAR T cell CD-19), cytokine release was significantly increased. These results show that tumors release pro-inflammatory cytokines themselves.

To evaluate the benefit of adding early apoptotic cells, the cytokines GM-CSF, IFN γ, IL-1 β, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, MIP-1 α, TNF α, MIP-1 β, IFN α, and IP-10 were measured in three experiments, where the results showed that macrophage-associated cytokines were down-regulated in the presence of ApoCell administration, while T-cell-associated cytokine levels were not significantly changed (Table 10).

Table 10: cytokine levels from an intraperitoneal in vivo model containing Hela cell solid tumor expressing CD19, +/-CAR T cell CD19 therapy and +/-ApoCell

Table 10 shows cytokine measurements twenty-four (24) hours after administration of the CAR T cells +/-ApoCell. Cytokines (including GM-CSF, IL-10, IL-12p70, IL-6, MIP-1 α, TNF α, MIP-1 β, and IP-10) were elevated due to cytotoxicity generated by CAR T cell therapy, and their levels were significantly down-regulated in the presence of ApoCell (p < 0.05-0.0001). These cytokines are primarily associated with macrophages. In contrast, there was no significant change in the levels of cytokines associated with T cells (e.g., IL-2, IL-4, IL-13, and IL 15).

The results presented in fig. 19A-C and table 10 demonstrate that CRS has several components in the context of cancer and CAR: tumors that can secrete cytokines; innate immunity in response to tumors and CARs and other factors; CAR-T cells secreting cytokines that affect innate immunity lead to death. ApoCell is interacting with innate immunity (primarily macrophages, monocytes and dendritic cells) to down-regulate the response of these macrophages, monocytes and dendritic cells without interacting with T cells or CAR T cells.

Animals receiving high doses of CD19+ CAR T cells against different tumor antigens showed no weight loss or behavioral changes.

Concomitant administration of 2.10 × 10 to a group of mice given the highest dose of CD19+ CAR T cells⁸One/kg of apoptotic cells, which has previously been demonstrated to be a safe and effective dose. Apoptotic cells had no negative effects in vitro or in vivo on CAR-modified T cells specific for CD 19. In the presence/absence of apoptotic cells, the in vitro E/T ratios of CAR T cells were comparable and the in vivo survival curves were comparable (data not shown).

Mice receiving human CD19+ CAR T + apoptotic cells had significantly reduced levels of at least one mouse proinflammatory cytokine, reduced body weight, and reduced mortality.

No in vivo negative effects of apoptotic cells on CAR-modified T cells against CD19 were seen with comparable results for the E/T ratio of CAR T and comparable in vivo survival curves in the presence or absence of apoptotic cells.

The file recorded significant down-regulation (p <0.01) of proinflammatory cytokines (including IL-6, IP-10, TNF- α, MIP-1 β) (data not shown). IFN- γ was not down-regulated, but its effect on macrophages and dendritic cells was inhibited both in the level of phosphorylated STAT1 and IFN- γ -induced expression of CXCL10 and CXCL9 (data not shown).

And (4) conclusion:

CRS evolves from a variety of factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and response to CAR T cell effects and expansion. Apoptotic cells reduce pro-inflammatory cytokines derived from innate immunity and inhibit the effects of IFN-g on monocytes/macrophages/dendritic cells without compromising IFN- γ levels or CAR-T cytotoxicity. Thus, apoptotic cells reduce pro-inflammatory cytokines derived from innate immunity and inhibit IFN- γ effects without compromising IFN- γ levels and CAR-T cytotoxicity. These results support the safe use of ApoCell to prevent CRS in clinical studies using CAR-T cell therapy.

Example 8: apoptotic cells down-regulate Cytokine Release Syndrome (CRS) and increase CAR-T cell efficacy

The purpose is as follows: to test the effect of early apoptotic cells on cytotoxicity of cytokines and CAR T cells over an extended period of time. To demonstrate the in vivo efficacy of CD19-CAR T cells. To demonstrate the synergistic effect of early apoptotic cells and CD19-CAR T cells.

The method comprises the following steps: in vitro and intraperitoneal experiments were performed in mice using hela cells expressing CD19 (pomi ltd) alone or after co-incubation with human macrophages. Raji was used in vivo for leukemia induction. LPS and IFN-g were used to trigger additional cytokine release. Anti-tumor effects against CD 19-bearing cells were performed using second generation, CD 28-bearing, CD 19-specific CAR-modified cells (produced by pomi ltd or using fibronectin (retronectin) or polyene preparation protocols). Cytotoxicity assays were examined in vivo (7-AAD flow cytometry) and in vitro (survival curves; tumor burden in bone marrow and liver; flow cytometry and immunohistochemistry). CRS occurs spontaneously or in response to LPS and IFN-g. Mouse IL-10, IL-1 β, IL-2, IP-10, IL-4, IL-5, IL-6, IFN- α, IL-9, IL-13, IFN-g, IL-12p70, GM-CSF, TNF-a, MIP-1 β, IL-17A, IL-15/IL-15R, IL-7, and 32 human cytokines were evaluated (Luminex technology, MAPIX system; MILLIPLEX Analyzer, Mercindex). Evaluation of IL-6R α, MIG (CXCL9) and TGF-. beta.1 in mice (Quantikine ELISA, R & D system). The IFN- γ effect (STAT1 phosphorylation, bioproduct) was evaluated. Human macrophages and dendritic cells are produced by monocytes. Generating early apoptotic cells generally as presented in example 1 above; more than or equal to 40 percent of cells are positive for annexin V; less than or equal to 15 percent of the total is positive by PI.

And (3) mice: SCID-Bg mice (female, 7-8 weeks old) were injected intraperitoneally (i.p) with 2 consecutive doses of 0.25X 10 on

days

1 and 2 of the experiment⁶Individual hela-CD 19 cells. On day 9, mice received a dose of 10X 10 intraperitoneally⁶4000-rad (cGy) irradiated ApoCell (using a cell processing system) and received a dose comprising 0.5X 10 intraperitoneally one day thereafter⁶A CAR-T positive cell or 2.2X 10⁶ 10X 10 of individual CAR-T positive cells⁶A population of CAR-T cells. As a control, mice received 10X 10⁶Activated monocytes or pseudo-T cells. Mice were maintained in an SPF animal facility according to institutional IACUC guidelines. Mice were weighed twice weekly and monitored daily for clinical signs and peritonitis. Endpoints are defined as severe peritonitis, manifested by abdominal swelling and tension, lethargy, decreased mobility, or increased respiratory effort. Survival analysis was performed according to the kaplan-meier method.

As a result: the file recorded significant down-regulation (p <0.01) of proinflammatory cytokines (including IL-6, IP-10, TNF- α, MIP-1a, MIP-1 β) (data not shown). IFN-g was not down-regulated, but its effect on macrophages and dendritic cells was inhibited in terms of the level of phosphorylated STAT1 (data not shown). IFN- γ -induced reduction in CXCL10 and CXCL9 expression in macrophages (data not shown).

In use of 0.5 × 10⁶In each CAR-T positive cell experiment, 2 mice were sacrificed per group on

days

17 and 21. HeLa-CD19 treated mice showed peritonitis, manifested by blood accumulation in the peritoneum, splenomegaly and tumor foci (data not shown). Mice treated with control MNCs had little blood in the peritoneum and few tumor lesions. Mice treated with CAR-T or CAR-T and ApoCell had no signs of peritonitis. This observation correlates with the survival curve presented in fig. 20A.

Therein use 2.2X 10⁶In an experiment with CAR-T positive cells, the same pattern of effects was observed with four-fold less CAR T cells (fig. 20B). CAR-T treatment extended survival of mice with peritoneal HeLa-CD19 (p ═ 0.0002). The effect was likely due to a larger number of infused CAR T cells (2.2 vs 0.5 x 10)⁶CAR-T positive cells) and more significant in this experiment. Even with the more pronounced and prolonged effects, early apoptotic cells have synergistic effects and prolonged survival (p ═ 0.0032). The in vitro E/T ratio of CAR T was comparable in the presence/absence of apoptotic cells. Surprisingly, CAR T cell therapy administered in the presence of apoptotic cells improves survival of mice with a significant and reproducible increase (p) of at least 12 days compared to CAR therapy alone <0.0032, fig. 20A and 20B).

And (4) conclusion: CAR-T cell therapy extended survival of mice with peritoneal HeLa-CD19 cells. Administration of irradiated early apoptotic cells one day prior to CAR-T had a synergistic effect and extended the survival of mice by more than 10 days compared to CAR-T cell therapy alone (p <0.044, log rank test).

In treating hela cells carrying CD19 in SCID mice, irradiated apoptotic cell infusion had a significant synergistic effect on CD19 specific CAR T cells. In this example, the results observed are similar to those presented in examples 5 and 6. By using irradiated apoptotic cells in this example, the potential for a "graft versus leukemia effect" has been removed compared to examples 5 and 6. Thus, this surprising synergistic effect appears to be mediated by the provided irradiated apoptotic cells.

CRS evolves from a variety of factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and response to CAR T cell effects and expansion. Apoptotic cells reduce pro-inflammatory cytokines derived from innate immunity and inhibit the effects of IFN- γ on monocytes/macrophages/dendritic cells without compromising IFN- γ levels or CAR-T cytotoxicity and with significantly improved CAR-T cell efficacy. Unexpectedly, CAR-T cell therapy was supplemented with treatment with irradiated apoptotic cells, effectively augmenting the anti-cancer effects of CAR-T cell therapy.

Example 9: effect of apoptotic cell therapy on non-solid tumor models

The purpose is as follows: apoptotic cells were tested for their effect on a model of non-solid tumors, where the cancer is widespread and not localized or blocked, in order to determine the efficacy of apoptotic cells on cancer survival.

Method

Raji cell

Raji cells were purchased from ECACC (catalog No. 85011429) and routinely cultured in complete medium (RPMI-1640 supplemented with 10% h.i.fbs, 1% Glutamax and 1% penicillin/streptomycin) and maintained at 3 × 10⁵-3×10⁶Individual cells/ml.

Apoptotic cells were prepared as described in example 1. The early apoptotic cells produced were at least 50% annexin V-positive cells and less than 5% PI-positive cells.

Non-solid (diffuse) tumor model

SCID mice received 10 on day 1 of the experiment⁵A single intravenous injection of individual Raji cells. SCID mice in the control group received saltA single intravenous injection of the aqueous solution. (3 cohorts were tested; leukemia was induced in 2 cohorts using Raji cells, and 1 cohort was maintained as a control).

Solid tumor model

SCID mice will receive 10 on day 1 of the experiment⁵A single IP injection of individual Raji cells, wherein the control group will receive a single IP injection of saline solution.

Apoptotic cell therapy

In the preliminary study, mice received an infusion of early apoptotic cells 6 days after the infusion of Raji cells. In subsequent studies, mice from one of the above leukemia cohorts began to receive early apoptotic cells (30 × 10) 6 days after infusion of Raji cells⁶One cell) were infused 3 times.

Results

SCID mice have no T cells and therefore cannot recover from leukemia without therapy.

Surprisingly, in preliminary studies and as shown in fig. 21, apoptotic cell infusion (APO) performed 6 days after infusion of Raji led to a significant prolongation of tumor-free death of SCIDs injected with CD19+ Raji compared to mice that did not receive apoptotic cell infusion (APO-free).

In the leukemic (APO-free) cohort, 70% of mice receiving Raji cells survived to their lifespan compared to 94% of mice receiving both Raji and apoptotic cells (total n ═ 51 animals, p < 0.001). As expected, 100% of the control mice survived to their expected lifespan (fig. 22A). In the leukemic cohort, 9% of mice receiving Raji cells and no apoptotic cells survived to more than 12% of the expected lifespan compared to 47% of mice receiving both Raji cells and apoptotic cells (fig. 22B). Mice that did not receive Raji cells and were devoid of apoptotic cells survived to over 30% of life expectancy compared to 41% in mice that received both Raji cells and apoptotic cells (fig. 22C). Mice that did not receive Raji cells and did not have apoptotic cells achieved complete remission, compared to 10% in mice that received both Raji cells and apoptotic cells (fig. 22D).

And (4) conclusion:

administration of apoptotic cell infusions maintained and increased the lifespan of leukemic mice, with complete remission in mice administered with early apoptotic cells in some cases (fig. 2 and fig. 3A-3D).

Example 10: effect of combined apoptotic cell and anti-CD 20 mAb treatment on diffuse tumor models

The purpose is as follows: to test the effect of administering a combination of early apoptotic cells and anti-CD 20 mAb on a diffuse (non-solid) tumor model, where the cancer is widespread and not localized or blocked, in order to determine the efficacy of this combination therapy on survival.

Raji cells, apoptotic cells, non-solid (diffuse) tumor models, solid tumor models, and apoptotic cell therapy were as described in examples 1 and 9 above.

anti-CD 20 mAb

Commercially available anti-CD 20 mAb was obtained from roche.

anti-CD 20 mAb treatment

Mice received an intravenous infusion of 5mg of anti-CD 20 mAb.

Combined apoptotic cell and anti-CD 20 mAb treatment

Starting on day 6 after Raji cell administration, mice received three intravenous infusions, 30 × 10 per infusion⁶And (4) apoptotic cells. In addition, mice received an intravenous infusion of 5mg of anti-CD 20 mAb.

Results

100% of mice receiving Raji cells, Raji cells + anti-CD 20 mAb and Raji cells + anti-CD 20+ apoptotic cells survived to the life expectancy of leukemic mice compared to 86% of mice receiving both Raji cells and apoptotic cells (total n ═ 28 animals, p <0.0002) (fig. 23A). Mice that did not receive Raji cells survived over 24% of life expectancy compared to 29% and 100% of mice that received both Raji cells + apoptotic cells, either Raji cells + anti-CD 20 mAb or Raji cells + anti-CD 20+ apoptotic cells (fig. 23B). Mice that did not receive Raji cells survived 59% of the life expectancy compared to 29% in mice that received both Raji cells + apoptotic cells, 57% in mice that received Raji cells + anti-CD 20 mAb, and 100% in mice that received Raji cells + anti-CD 20+ apoptotic cells (fig. 23C). Mice that did not receive Raji cells survived 76% of the life expectancy compared to 29% in mice that received both Raji cells + apoptotic cells, 14% in mice that received Raji cells + anti-CD 20 mAb, and 85% in mice that received Raji cells + anti-CD 20+ apoptotic cells (fig. 23D). Mice that did not receive Raji cells or Raji cells + anti-CD 20 mAb survived over 100% of the life expectancy of the mice compared to 29% in mice that received Raji cells + apoptotic cells or Raji cells + anti-CD 20+ apoptotic cells (fig. 23E).

And (4) conclusion:

apoptotic cell infusion increased the lifespan of leukemic mice, increased the number of mice that achieved complete remission, and enhanced the anti-CD 20 mAb treatment (fig. 23A-23E).

Example 11: effect of ApoCell (early apoptotic cells) on leukemia/lymphoma

The purpose is as follows: the work presented here has three main goals: (1) evaluating the effect of ApoCell in a leukemia-lymphoma mouse model in terms of disease onset, progression and subsequent death; (2) assessing the distribution of tumor cells in a leukemia-lymphoma mouse model following treatment with ApoCell; and (3) evaluation of the possible synergistic effects of ApoCell and rituximab (RtX) in the treatment of leukemia-lymphoma in SCID-Bg mice. As part of the work to achieve these objectives, measurement of survival of leukemic mice following ApoCell administration was measured. Also, the distribution of tumor cells was measured after treatment with ApoCell.

The method comprises the following steps:

a mouse. 7-week-old female SCID-Bg mice (ENVIGO, Yersinia, Israel) were injected intravenously with Raji cells, 0.1X 10 cells per mouse⁶And (4) cells. On

days

5, 8 and 11 of the experiment, mice received 3 doses of 30X 10 intravenously ^{6 are provided with}ApoCell. For combination therapy, mice received a dose (day 8) of RtX (2 or 5 mg/kg; rituximab (Mabthera), Roche, Basel, Switzerland) 1.5 hours after ApoCell administration.

Mice were followed daily and weighed twice weekly. The endpoint was defined as death or sacrifice due to the development of any of the following symptoms: paraplegia (lower limb paralysis), initial weight loss of 20% compared to mice, lethargy, decreased mobility or increased respiratory effort.

Survival analysis was performed according to the kaplan-meier method. Mice were maintained in a Specific Pathogen Free (SPF) animal facility according to Institutional Animal Care and Use Committee (IACUC) guidelines.

Raji cell line. This human Burkitt's lymphoma cell line was purchased from the european collection of certified cell cultures (ECACC, catalog No. 85011429) and routinely cultured in complete medium (RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% glutamax, 1% penicillin/streptomycin).

ApoCell. Essentially as described in example 1. Briefly, an enriched monocyte fraction is collected from a healthy, qualified donor by leukapheresis. After apheresis was completed, the cells were washed and resuspended in a freezing medium consisting of PlasmaLyte a, 5% human serum albumin, 10% dimethyl sulfoxide (DMSO), 5% anticoagulant citrate dextrose solution formulation a (ACD-a) and 0.5U/ml heparin, ph 7.4. The cells were then gradually frozen and transferred to liquid nitrogen for long term storage.

To prepare ApoCell, cryopreserved cells were thawed, washed, and resuspended in apoptosis-inducing medium consisting of RPMI 1640 supplemented with 2mM L-glutamine and 10mM heparin, 10% autologous plasma, 5% ACD-A, 0.5U/ml heparin sodium, and 50. mu.g/ml methylprednisolone. The cells were then incubated at 37 ℃ in 5% CO₂Incubated for 6 hours. At the end of the incubation, the cells were collected, washed and resuspended in hartmann's solution using the cell processing system. ApoCell was centrifuged at 290g at 2-8 ℃ for 10 min and resuspended in Hartmann's solution for injection. Using FCS express software annexin V and propidium iodide (PI, Medical and biological laboratories)&Biological Laboratories), republic of japan) to determine apoptosis and viability of ApoCell.

Flow cytometry. Mice spleen, liver and bone marrow (after exacerbation of clinical signs as defined above) were collected from sacrificed mice and analyzed for the presence of Raji tumors (anti-CD 20) by flow cytometry (FACSCalibur, BD, franklin lake, nj, usa).

As a result:

part A: ApoCell delays the onset and subsequent death of leukemic mice

Fig. 24 presents kaplan-meier survival curves (RPMI group, n 15; Raji group, n 23; Raji + ApoCell group, n 24) for 3 separate experiments. In each experiment, female SCID-Bg mice (7-8 weeks old) were injected intravenously with 0.1X 10 ⁶Individual Raji cells, and RPMI was injected to the control group. Then, on

days

5, 8 and 11, mice were administered three doses of 30 × 10 by intravenous administration (IV)⁶And (4) ApoCell. Mice were followed daily and weighed twice weekly. The endpoint was defined as death or sacrifice due to the development of any of the following symptoms: paraplegia (lower limb paralysis), initial weight loss of 20% compared to mice, lethargy, decreased mobility or increased respiratory effort. The experimental details are given in table 11. A significant beneficial effect of ApoCell was seen (p ═ 0.002, log rank (Mantel-Cox) test).

Table 11: experimental details of graph a

Disease-free survival of DFS

Data from the individual studies are presented in FIGS. 25A-25C.

As depicted above, after administration of Raji cells, mice treated with 3 doses of ApoCell had slower disease progression and significantly later death compared to untreated mice (p ═ 0.0020).

The leukemic mice treated with ApoCell had a significant delay in onset of symptoms, exhibited slower disease progression and died later than untreated control mice. Interestingly, about 10% of the mice administered with Raji cells and ApoCell did not develop any of the expected symptom signatures in this leukemia/lymphoma model and remained healthy until termination of the experiment (day 53 or day 60).

ApoCell reduces tumor burden in leukemic mice

After sacrifice, following worsening of clinical signs as described above, organs of interest were collected for analysis (i.e., liver, spleen, and bone marrow). Cells of these target organs were analyzed by flow cytometry for the presence of human tumor cells (Raji cells positive for CD 20).

The following data (table 12) describe the average percentage of cell population in the target organs of sacrificed mice from the 3 experiments described above; values for individual mice in each experiment can be found in tables 13-18 below.

Table 12: mean percentage of tumor population in spleen, bone marrow and liver (flow cytometry)

In vivo experiment 011

FACS analysis of CD20+ cells in bone marrow, spleen and liver:

at day 60 of death, one mouse receiving three doses of ApoCell was healthy.

Liver, spleen and bone marrow cells were collected and targeted to human CD20-FITC (bio-legend, cat. No. 302206) by flow cytometry (FACSCalibur, BD); mIgG1-FITC (Biogesso, Cat. No. 400110) was analyzed for its presence.

In vivo experiment 019

FACS analysis of tumor cells in bone marrow, spleen and liver:

mouse spleen, liver and bone marrow cells were collected from mice sacrificed after clinical exacerbation as defined in the method and analyzed for the presence of human CD20(FITC) by flow cytometry (FACSCalibur, BD).

The results of the spleen, bone marrow and liver analyses are presented in tables 13-15 below.

Table 13: spleen

Table 14: bone marrow

Table 15: liver disease

In vivo experiment 023

Expression (%) of CD20 tumor cells in spleen, bone marrow and liver as determined by flow cytometry. Mouse spleen, liver and bone marrow cells were collected from mice that were sacrificed (after clinical exacerbations as defined in the method) and analyzed for the presence of human CD20(FITC) by flow cytometry (FACSCalibur, BD). The results for spleen, bone marrow, and liver of individual mice are presented in tables 16-18 below.

Table 16: spleen

Table 17: bone marrow

Table 18: liver disease

And (4) preliminary conclusion: taken together, tumor distribution in mouse organs correlated with the beneficial effects seen in survival curves and was significantly reduced in bone marrow and liver of treated mice.

And part B: synergistic effect of ApoCell and rituximab (RtX) in treatment of leukemia/lymphoma

Next, whether ApoCell treatment was synergistic with other conventional treatments for leukemia/lymphoma was examined by evaluating the combined effect of RtX and ApoCell on leukemic mice in two experiments.

The purpose is as follows: survival of the leukemic mice after RtX and ApoCell administration was measured and tumor cells in bone marrow, liver and spleen were detected in the leukemic mice.

The method comprises the following steps: the following work is representative of the results obtained in the combination therapy experiments (ApoCell and rtx). Briefly, female SCID light brown mice were injected intravenously with 0.1X 10⁶Raji cells (all groups were 7 for n). Mice received three doses of 30 x 10 intravenously on

days

5, 8 and 12⁶And (4) ApoCell. On day 8, mice received a single intravenous dose of 2 or 5mg/kg RtX 1.5 hours after ApoCell injection. Mice were followed daily and weighed twice weekly. EndpointIs defined as death or sacrifice due to the development of one or more of the following symptoms: paraplegia (lower limb paralysis), 20% loss of weight compared to the starting, lethargy, decreased mobility or increased respiratory effort.

As a result:

as shown in fig. 26, ApoCell has beneficial effects, confirming the results presented in fig. 24. Rituxan (rtx) alone had superior effects compared to ApoCell at both 2mg and 5mg, but the combination of ApoCell and Rituxan had synergistic effects at both 2mg (p ═ 0.104) and 5mg, although the synergistic effect seen at 5mg did not reach statistical significance. Tumor distribution in mouse organs correlated with beneficial effects (table 19).

Table 19: statistical analysis of survival distribution (Log rank (Mantel-Cox) test)

End of experiment-day 57

After the experiment, one mouse (Raji + RtX 2mg/kg + ApoCell) was declared disease-free

In additional experiments, the synergistic effect of a 2mg RtX dose was measured. As shown explicitly in this experiment (fig. 27), the synergistic effect of ApoCell and RtX was again verified to be significant (p ═ 0.01).

As shown in table 20, the spleen was not filled with tumor cells (0.1-0.3 for background staining) and was used as a control. In contrast, bone marrow and liver are tumor targets. Following treatment by ApoCell and RtX alone, the tumor population (e.g., Rajji cells measured using CD20 marker) in bone marrow and liver decreased, and when both were administered in combination, the benefit increased; for Raji + rtx (2mg/Kg) + ApoCell, p is 0.0034(×), and for Raji + rtx (5mg/Kg) + ApoCell, p is 0.0031(×) (T-test). As expected, RtX significantly reduced tumor burden in the target organs of leukemic mice. Interestingly, treatment with ApoCell alone reduced tumor cells in those organs to a level comparable to treatment with conventional RtX therapy.

Table 20: mean tumor cell population in spleen, bone marrow and liver (flow cytometry)

And (4) conclusion: in summary, the survival rate curves (fig. 24, 26 and 27) clearly demonstrate the beneficial effects of ApoCell and Rtx alone and the synergistic effect of ApoCell and RtX in combination. Notably, when conventional RtX treatment was combined with ApoCell, survival time was significantly increased regardless of RtX dose, indicating a synergistic effect of the two therapies. A supportive clinical observation is the reduction of tumor cell populations in bone marrow and liver (table 12).

Surprisingly, the ApoCell formulation has a significant beneficial effect on disease progression and survival in a leukemic mouse model, independent of any other treatment. 10-20% of mice had prolonged survival in the kaplan-meier assay (fig. 24, 26, and 27) and tumor cell burden in liver and bone marrow was reduced. Furthermore, ApoCell and RtX, when administered in combination, had a clear synergistic effect, further delaying disease onset and progression and improving survival.

Example 12: use of pooled apoptotic cell preparations in GVHD leukemia/lymphoma model

In the following preliminary work, the effect of the same infusion in the GvHD leukemia/lymphoma model was examined. The safety and efficacy of irradiated multi-donor single apoptotic cell infusions (pooled irradiated mononuclear apoptotic cell preparations) for preventing acute GvHD in mice undergoing Bone Marrow Transplantation (BMT) was examined. In this model, BMT rescued irradiated mice (80-100%).

The problem with the possible loss of graft-versus-leukemia (GvL) effects occurs with every successful treatment that potentially avoids high grade aGVHD, as this effect was found to correlate with the severity of GVHD.

Method

Apoptotic cells were prepared as in example 1 above, except that: in the current experiment, preparations were performed from 4 donors simultaneously. After preparation from 4 donors, the cell preparations were combined at the last step (before irradiation), immediately after irradiation and injected immediately after irradiation. The irradiation was 25 Gy.

Results

The two graphs presented in fig. 28 and 29 show the clear effect of a single injection of apoptotic cells from multiple individual donors (dot-dash line) on both survival and weight loss (p < 0.01). Figure 28 is a Kaplan-Meier survival curve in a GvHD mouse model treated with a single dose of irradiated apoptotic cells from multiple individual donors with a significant improvement in survival. Figure 29 is the percentage of weight loss for the 2 comparative groups that followed and correlated with the findings of figure 28.

In summary, a single infusion of multiple donors of irradiated apoptotic cells successfully and significantly improved life expectancy in the GvHD mouse model.

Example 13: stability criteria for apoptotic cells from multiple individual donors

The objective of this study was to establish stability criteria for apoptotic cells from multiple individual donors and to conduct a comparable study with unirradiated HLA-matched apoptotic cells (Mevorach et al (2014) Biology of Blood and Marrow Transplantation 20(1): 58-65; Mevorach et al (2015) Biology of Blood and Marrow Transplantation 21(2): S339-S340).

After the apoptotic cell end product formulations were stored at 2-8 ℃ for 8 hours, 24 hours, 48 hours, and 60 hours and sampled at each time point, their cell number, viability, apoptotic phenotype, and potency were evaluated. Apoptotic cell end product batches will be prepared following Standard Operating Procedures (SOPs) (example 1; example 5) and batch records (BR; i.e. specific preparation procedures). For efficacy evaluation, samples of the early apoptotic cell preparation end product batches were tested for inhibition of upregulation of MHC-II expression on Lipopolysaccharide (LPS) -induced immature dendritic cells (time points 0-24 hours) or monocytes (time points 0-6) and were performed according to SOP and recorded on BR. These series of tests will be performed on pooled and non-pooled products in preparations derived from multiple individual donors and from a single donor, respectively.

In addition, CD3(T cell), CD19(B cell), CD14 (monocyte), CD15 (monocyte) will be treated^{High (a)}Flow cytometric analysis of (granulocytes) and CD56(NK cells) were documented. The purpose of these studies was to demonstrate the consistency of the results within a narrow range. Preliminary results are consistent with these goals, no deviation from SOP was noted, and no technical issues were reported. However, further studies are needed to summarize the range and stability of effective treatments. Preliminary results show that all these parameters are equivalent. Further, single donor stability studies showed stability over a period of at least 48 hours (see example 1).

Example 14: safety and efficacy of multi-donor irradiated apoptotic cells as prophylaxis against acute graft versus host disease

The purpose is as follows: phase 1/2a, multicenter, open study evaluated the following: single dose administration of irradiated apoptotic cells from multiple mismatched donors prevents the safety, tolerance and primary efficacy of graft-versus-host disease in hematopoietic malignancies of human leukocyte antigen-matched related (related) and non-related (unrelated) patients undergoing allogeneic hla-matched hematopoietic stem cell transplantation.

The main purpose is as follows: to determine the safety and tolerability of multiple donor irradiated apoptotic cell therapy.

For the secondary purposes: to determine the efficacy of irradiated apoptotic cells from multiple individual donors as a preventative measure for acute GVHD (aGVHD) in patients with hematopoietic malignancies who are scheduled to undergo Hematopoietic Stem Cell Transplantation (HSCT). For the purposes of this study, HSCT can be Bone Marrow Transplantation (BMT) or Peripheral Blood Stem Cell Transplantation (PBSCT).

Treatment indication: post-transplant anti-host disease (GVHD) in hematopoietic malignancies in Human Leukocyte Antigen (HLA) -matched, parental and non-parental patients.

Research and design: this is an open, multicenter, phase 1/2a study of patients diagnosed with hematopoietic malignancies who are scheduled to undergo HSCT (bone marrow transplantation or peripheral blood stem cell transplantation) from HLA-matched parental or non-parental donors following a complete or reduced intensity myeloablative conditioning regimen.

After the recipient patient signed an informed consent, the donor screening period, and cell collection performed prior to beginning the conditioning protocol, a qualified recipient patient will be assigned (stratified by prophylactic treatment and parental and non-parental transplant donors in a 1:1 ratio) to receive Intravenous (IV) injections 12-36 hours prior to HSCT transplantation or assigned:

Study group: single dose of 140 x 10 irradiated early apoptotic cells/kg body weight from multiple individual donors in Phosphate Buffered Saline (PBS)⁶20% cells/kg.

All patients will also be treated with the institutional standard of care (SOC) immunosuppressive regimen: cyclosporine/methotrexate or tacrolimus/methotrexate for complete myeloablation and mycophenolate mofetil/cyclosporine or mycophenolate/tacrolimus for strength reduction. The patient will be hospitalized according to medical instructions.

Patients will be followed up for 180 days for the secondary efficacy endpoint and for 1 year for the primary safety and tertiary efficacy endpoint. The number of visits by patients participating in this study will be comparable to the number of visits to which patients in their pathology are accustomed. For donors, study-specific visits will be made during the screening period for apheresis procedures.

Since these patients suffer from a number of potential medical conditions and may experience symptoms compatible with aGVHD, it may be difficult to absolutely determine whether toxicity is associated with apoptotic cells despite the basic data from previous phase 1-2a studies using apoptotic cells for GvHD prevention (Mevorach et al (2014) blood and bone marrow transplant biology 20(1): 58-65). Single infusion of donor mononuclear early apoptotic cells as prophylaxis against graft-versus-host disease in myeloablative HLA-matched allogeneic bone marrow transplantation: phase I/IIa clinical trial. (< BBMT > 20(1) < 58 > -65).

The data safety monitoring committee (DSMB) will meet the requirements as specified in the DSMB regulations, including at the time of planned interim analysis (180 days), provided that no safety hazards have been addressed in advance.

The study procedure was as follows:

the study will include a screening phase, a treatment phase and a follow-up phase.

1. Screening period (days-60 to-2)

Potential recipient patients will sign informed consent prior to any study-related procedure. Standard pre-approval assessments will be made by the transplant centre for donors during the screening period and typically comprise: demographic data, medical history, HLA match status validation (no match required), physical examination, height and weight, vital signs, pregnancy tests (all women), hematology, blood chemistry, infectious disease screening, ECG, and urine analysis.

The recipient (study patient) will undergo the following evaluations during the screening period: demographic data, medical history, carnofsky (Karnofsky) performance status, HLA match validation, physical examination, height and weight, vital signs, pregnancy tests (all women), ECG, pulmonary function tests, hematology, blood chemistry, coagulation markers, infectious disease screening, and urinalysis.

After the initial screening evaluation, if the recipient is eligible to participate in the study, the recipient patient will be assigned to receive 140 x 10 on the first day of the conditioning regimen ⁶Single intravenous infusion of ± 20% cells/kg of multi-donor apoptotic cells. Conditioning regimens should be completed on the day before or on the day of the apoptotic cell infusion scheduled for study day-1.

Apoptotic cell doses will be calculated for each recipient patient and the number of apheresis collections and donor numbers presumed will be decided accordingly.

For peripheral stem cell transplant donors: between day-6 and day-1, the donor will receive one or more daily injections of G-CSF to mobilize progenitor cells, and on day 0, will undergo apheresis to generate donor hematopoietic stem cells for transplantation. The preparation of hematopoietic stem cells for bone marrow transplantation will be performed by trained hospital staff according to central standard practice. Hematopoietic stem cells for HSCT will not proliferate or be depleted by T cells prior to administration.

For bone marrow transplant donors: bone marrow will be harvested and prepared according to central standard practice and will not otherwise multiply.

2. Day of treatment (day-1)

On day-1 (12-36 hours before HSCT), a qualified patient will receive 140X 10 of irradiated early apoptotic cells from multiple individual donors⁶A single intravenous infusion of ± 20% cells/kg. Vital signs should be monitored hourly during infusion, followed every 4 hours for the first 24 hours thereafter. Treatment-related AEs will be evaluated immediately after infusion. .

On day 0, the patient will undergo hematopoietic stem cell transplantation according to local institutional guidelines.

3. Short follow-up period (day 0 to day 180)

Patients will receive a follow-up visit to study day 180 to assess primary endpoint safety and tolerability as well as secondary and tertiary endpoints: cumulative incidence of grade II-IV aGVHD (based on Przepiorka et al "modified Glucksberg"); cumulative incidence of any grade and high grade aGVHD (i.e., time to develop grade II-IV aGVHD); any systemic treatment for GVHD; and the development of cGVHD.

Short follow-up visits will be made daily (typically at least day-1 to day +14 or more) during hospitalization for transplantation and once weekly during the first 7 cycles after discharge; day +7, day +14, day +21, day +28, day +35, day +42, and then day 60, day 100, day 140, and day 180. During a subsequent follow-up period of up to 180 days, the visit window will be ± 5 days for the weekly visit (the first 7 weeks), and the visit window will be ± 5 days for every two or more weekly visits.

Blood samples will be obtained on

days

1, 3, 7, +28, +42, 60, 100, 140 and 180 and examined for implantation, immune recovery, plasma and serum biomarkers ("Michigan") and documentation of cell subsets.

4. Long-term follow-up period (181 th to 365 th/1 year)

Patients will be followed a long-term follow-up for one year after HSCT to monitor secondary endpoints: non-recurring mortality and Overall Survival (OS), recurring morbidity, leukemia-free survival (LFS), and chronic GVHD. At least two long-term follow-up visits were performed, the last one being 12 ± 1 month after HSCT.

Duration of study: the duration of the study will be up to 14 months for each patient enrolled, as follows:

screening: up to 60 days (2 months)

Treatment: 1 day

Follow-up: 365 days (12 months), consisting of:

short-term: 180 days

And (3) long-term: +180 days

Study population: a total of 25 patients diagnosed with hematological malignancies who are scheduled to undergo HSCT (bone marrow transplantation or peripheral blood stem cell transplantation) with at least 15 non-relatives donors following either a myeloablative conditioning protocol or a reduced intensity conditioning protocol following central standard practice will be included in this study and will be compared to historical controls.

Inclusion/exclusion criteria:

recipient patient exclusion criteria

1. Patients eligible for allogeneic HSCT against the following malignancies, age > 18:

acute myeloid or undifferentiated or bi-epi leukemia that is in complete remission (any remission) or surpasses complete remission but morphologically < 5% blast cells in the bone marrow.

Acute Myeloid Leukemia (AML) in complete remission if it has evolved from myelodysplastic syndrome (MDS) which should be documented at least 3 months prior to diagnosis of acute myeloid leukemia. Or from polycythemia vera or essential thrombocythemia.

Acute Lymphoblastic Leukemia (ALL) in complete remission (any remission), with blast cells in the bone marrow < 5% from a morphological point of view.

Chronic Myelogenous Leukemia (CML) in chronic or accelerated phase.

Myelodysplastic syndrome-refractory cytopenia with multiple-lineage dysplasia (RCMD), RA (refractory anemia), RA with ringed sideroblasts (RARS; all < 5% blasts), RA with blast (RAEB; 5% to 20% blasts).

Transplant donor and recipient patients must have at least 8/8HLA matches at the HLAA, B, C, DQ and DR loci, and no antigen or allele mismatches. However, the donor or donors of leukocytes for apoptotic cell formation are not limited to HLA matching.

The performance status at screening visit was scored at least 70% (carnofsky for adults and Lansky (Lansky) for < 16-year-old recipients).

Left ventricular ejection fraction of heart in adults within 4 weeks of starting conditioning>40 percent; if previously exposed to anthracyclines or a history of cardiac disease, a MUGA scan or cardiac ECHO needs to be performed.

For DLCO¹FEV1 (forced expiratory volume) and FVC (forced vital capacity) pulmonary function testing>Predicted 60%.

¹Diffusion capacity of lung to carbon monoxide

The oxygen saturation in the room air is at least 90%.

The patient must have sufficient organ function as defined below:

AST (SGOT)/ALT (SGPT) <3 × Upper Normal Limit (ULN).

Serum creatinine <2.0mg/dL (adult, >16 years old), or <0.8(1-2 years old), <1(3-4 years old), <1.2(5-9 years old), <1.6(10-13 years old), and 1.8(14-15 years old).

Serum bilirubin <3mg/dL unless due to Gilbert's disease or hemolysis.

Written informed consent for independent study participation was signed by the patient or guardian (in the case of minor).

Capability of meeting research requirements.

During the duration of 4 weeks (from day-1), both women and men must agree to:

surgical sterilization is performed within the first month or longer using acceptable contraceptive methods, or in the presence of BMT related limitations.

Pregnancy tests were negative regardless of fertility.

Recipient patient exclusion criteria

All diseases eligible for HSCT not specified in the criteria were included.

Interventional study trials were enrolled within 30 days of the screening visit.

Malignant tumors with progressive or poorly controlled malignancy.

If the BMT program comprises a T cell depleting allogeneic transplant.

If the BMT program contains anti-thymocyte globulin (ATG) or alemtuzumab as part of an immunosuppressive regimen or high dose cyclophosphamide therapy for prevention of post-transplant GVHD.

Uncontrolled infections, including sepsis, pneumonia with hypoxemia, persistent bacteremia, or meningitis, occurred within two weeks of the screening visit.

Active acute or chronic HBV or HCV infection is currently known.

Known as Human Immunodeficiency Virus (HIV) infection.

Patients with severe or symptomatic restrictive or obstructive pulmonary disease or respiratory failure who require ventilator support.

Patients with other complicated serious and/or uncontrolled medical conditions that may compromise the participation of the study (i.e., active infection, uncontrolled diabetes, uncontrolled hypertension, congestive heart failure, unstable angina, ventricular arrhythmia, active ischemic heart disease, myocardial infarction within six months, chronic liver or kidney disease, active upper gastrointestinal ulcer).

Any chronic or acute condition that would interfere with the efficacy assessment of the study product.

The investigator's opinion is believed to be susceptible to any form of substance abuse (including drug or alcohol abuse), mental disorder or any chronic condition that would interfere with the progress of the study.

Prior history of allogeneic organ transplantation or stem cell transplantation (allogenic only).

Breastfeeding of women with fertility.

Patients who may not be compliant or not functioning during the study.

Study of product pathways and dosage forms

Will be 140X 10 intravenous infusion 12-36 hours prior to HSCT⁶+ 20% cells/kg irradiated multi-donor apoptotic cell product.

Apoptotic cells are cell-based therapeutics consisting of apoptotic cells from multiple individual donors. The product contains enriched mononuclear cells of the allogeneic donor in the form of a liquid suspension, of which at least 40% are early apoptotic cells. Suspensions were prepared from multiple individual donors with PBS solution according to GMP regulations and should be stored at 2-8 ℃ until infusion. The total volume of the final product in the opaque transfer bag was 300-600mL and it was irradiated to 25Gy after preparation. The study product should be administered to the patient within 48 hours of completion of the manufacturing process.

Safety results/efficacy endpoints/results measures

Mainly:

safety and tolerability endpoints included the time of transplant and physical examination to determine adverse events, concomitant medication, and safety laboratories at day 180 and day 360 (1 year). Further, it is expected that irradiated pooled apoptotic cell preparations will show a lack of cell proliferation in vitro and in vivo and a lack of activation in vivo. This shows that the pooled apoptotic cell preparation was identified as safe to use.

And (2) secondarily:

cumulative incidence of grade II-IV aGVHD at day 180 was based on a retrospective comparison with the grade of Gluckberg (IBMTR Severity Index for grading acute graft versus host disease-subjects-disease: retrospective comparison with the grade of Gluckberg) using the "modified Gluckberg" consensus (Br J Haematol. 1997, 6.97; 4): 855-64).

1 year non-recurrent mortality and Overall Survival (OS)

Incidence of 1 year recurrence

Survival time of leukemia-free 1 year (LFS)

Highest grade of aGVHD in the first 180 days

Cumulative incidence of grade III-IV aGVHD

The incidence of chronic GVHD on days 180 and 360 (1 year) was based on (Jagasia et al, 2015).

Any "systemic treatment" (used and cumulative dose) containing corticosteroids for treatment of aGvHD from day 20 to day 180

Immune reconstitution and function on days +28, 100, 180 and 360 (1 year) associated with T, B, NK and monocytes

Major infection rates (including lung infiltration, CMV reactivation and any other infections requiring hospitalization) within day 180 and 1 year.

Third/exploratory:

the number of hospitalizations as a percentage of total days at risk, total days alive and days discharged. Or total days of hospitalization until first discharge after transplantation.

Organ-specific GVHD

T reg, CD4 Tcon, CD8, NK and B cell levels at day 180

Statistical analysis:

where individuals have comparable baseline characteristics, the study results are compared to historical controls.

Descriptive statistics will be used to aggregate the result metrics and baseline characteristics. In this analysis, all available data will be presented without interpolating any missing data. Subjects will contribute data available until withdrawal or study completion or death. Descriptive statistics (such as mean, median, standard deviation, minimum and maximum) will be used to summarize the continuous variables. All subjects receiving apoptotic cell infusions will be included in the safety analysis. Subjects who also received HSCT will be included in the efficacy analysis. Since this study is exploratory in nature, a specialized analysis is planned.

Sample size considerations

A total of 25 patients will be enrolled and at least 15 matched non-relatives will be enrolled. Apoptotic cell activity will be given to all (stratified according to GVHD prevention protocol) as well as both parental and non-parental transplant donors.

Definition of group analysis

All efficacy analyses will be performed on intent-to-treat (ITT) populations and compared to adequate historical controls. The safety population will be defined as all patients who received a dose of study drug.

Statistical method

The frequency and percentage or median (range) will be used as appropriate to characterize the patient, disease and transplant.

Security analysis

Descriptive statistics will be used to summarize safety results with emphasis on the AEs reported between study treatment infusion and HSCT procedure (24-30 hour window). No changes to the study performance were caused by DSMB review, including sample sizing. Therefore, as a result of the interim analysis, no penalty adjustment for overall type I errors will be required.

Minor endpoint analysis

Grade II-IV aGVHD will be described using an accumulative morbidity estimator, with pre-aGVHD mortality as a competitive event.

Cumulative morbidity will be used to describe neutrophil and platelet recovery, aGVHD grade III-IV, chronic GVHD, infection, relapse and graft-related mortality, with relapse as a competitive event for TRM and death as all other competitive events. Overall survival and leukemia-free survival will be described using a kaplan-meier estimator. The highest grade of aGVHD in the first 180 days and the demand for steroids at 180 days will be described using the man-wheaten U test (Mann Whitney U test) and chi-square test (chi-square test), respectively, frequency of use and percentage. Immune recovery of each cell subpopulation and TREG will be described at each time point using the median and range mann-whitney assay.

Example 15: comparison of pooled apoptotic cell preparations in GVHD leukemia/lymphoma model to Single Donor apoptotic cell preparations

The purpose is as follows: the beneficial clinical effects of human early apoptotic cells obtained from a single donor on the severity of GvHD in the murine model of GvHD were compared to the clinical effects of human early apoptotic cells obtained from multiple individual donors, if any, on the severity of GvHD in the murine model of GvHD, wherein the multiple individual donors represent HLA-mismatched heterologous donors.

Example 12 above illustrates the beneficial effects of irradiated apoptotic cells pooled from multiple individual donors. The results shown in fig. 28 and 29 are surprising, as the skilled person would recognise that mismatched cells from multiple sources may have increased diversity in antigenicity of the cells, and would therefore be expected to have significantly reduced clinical effects. Unexpectedly, pooled unmatched early apoptotic cells also reduced the known beneficial effect of early apoptotic cell pairs in reducing GvHD severity and thus prolonging the days to death (fig. 28), which is said to increase antigenicity due to pooled multiple unmatched source cells.

An additional objective was to see if there was a difference between using irradiated early apoptotic cells and using non-irradiated apoptotic cells.

The skilled person will appreciate that mismatched, irradiated cells retain their antigen profile as recognized by the APC mechanism and the T cells of the host into which they have been infused. Thus, when pooling heterogeneous, mismatched cell populations, concerns include cross-reactivity between individual populations being pooled, mixed cell lymphomas of pooled populations, or T cell immune responses between pooled populations that may reduce or eliminate cells, or any combination thereof.

Method

Mouse model: female 7-9 week old BALB/c mice (H-2) in the mismatched GVHD model^d) Used as recipients, and female 8-9 week old C57BL/6 mice (H-2)^b) Used as donor. Recipients were irradiated systemically at 850cGy 24 hours prior to bone marrow and spleen cell transplantation. Bone marrow reconstitution was performed using donor bone marrow cells. Bone marrow cells were extracted from femur and tibia with RPMI 1640. The red blood cells were lysed, then the cells were washed and resuspended in PBS. Evaluation of viability using trypan blue dye exclusion method (>90% viability). GVHD induction was performed using donor splenocytes. Spleens were removed and homogenized and single cell suspensions were obtained. Erythrocytes were lysed and splenocytes resuspended with PBS. At least 90% of the viable cells were evaluated using trypan blue dye.

Early apoptotic cells: apoptotic cells were generated by apheresis of an enriched mononuclear cell fraction from a healthy donor similar to that of example 1. Briefly:

enriched mononuclear cell (MNC) fractions were obtained from healthy, qualified donors by leukapheresis. In addition to 400-

The apheresis system collects cells from 12 liters of blood. The estimated yield of enriched monocyte fractions from donors is expected to be about 1.2-1.5X 10¹⁰And (4) cells. Prior to leukopheresis procedures, donors were tested and confirmed to be negative for the following viral vectors:

human Immunodeficiency Virus (HIV)

types

1 and 2;

2. hepatitis B Virus (HBV);

3. hepatitis C Virus (HCV);

4. cytomegalovirus (CMV);

5. treponema pallidum (syphilis);

human T Lymphotropic Virus (HTLV) type I and type II.

After cell collection, cells were washed with RPMI and frozen as follows. The frozen formulation consisted of plasma lyte a for injection at pH 7.4, 10% DMSO, 5% human serum albumin and 5% anticoagulant citrate dextrose solution seeded with 10U/ml heparin.

The freezing medium was prepared in bags and the freezing procedure was performed in a closed system under cGMP conditions.

After completion of the leukapheresis procedure, the enriched MNC fraction was washed with PlasmaLyte a and resuspended to 50-65 x 10 with ice cold freezing medium⁶Concentration of individual cells/ml. The cells were then transferred to a freezing bag, the bag was transferred to a pre-cooled aluminum cassette, and the cassette was immediately transferred to-18- (-25) deg.C for two hours.

After two hours, the cassettes were transferred to-80 ℃ for an additional 2 hours, and then transferred to liquid nitrogen (> -135 ℃) for long-term storage.

Autologous plasma was divided into 50gr aliquots. Plasma aliquots were transferred to-80 ℃ for 2 hours and then to-18- (-25) ℃ for long term storage.

For apoptosis induction, cells were thawed and washed with pre-warmed RPMI1640 containing 10mM Hepes buffer, 2mM L-glutamine and 5% anticoagulant citrate glucose solution seeded with 10U/ml heparin. After supernatant extraction, cells were plated at 5X 10⁶The concentration of/ml was resuspended in RPMI1640 supplemented with 10mM Hepes, 2 mML-glutamine, and 10% autologous plasma and 50. mu.g/ml methylprednisolone and 5% anticoagulant citrate glucose solution inoculated with 10U/ml heparin were added. The cells were then transferred to a cell culture bag and incubated at 37 ℃ with 5% CO ₂For 6 hours to stabilize apoptosis.

After incubation, cells were harvested, washed with PBS and resuspended in PBS.

Early apoptotic cell products are produced from a single donor or from 10 different individual donors combined, in which case the cells are combined just prior to irradiation. Because of the potential for interference between components in the multi-donor product (e.g., between live non-apoptotic cells), the early apoptotic cell product was subdivided and a sample of early apoptotic cells to be tested in vivo was irradiated at 2500cGy prior to administration (sample F below) and stored at 2-8 ℃ until administration. Table 3 below of example 6 presents details of annexin V positive/propidium iodide negative ratios and cell surface markers for early apoptotic cell products, establishing the consistency of maintaining apoptotic cells administered to mice. The final product was stable at 2-8 ℃ for 48 hours.

On the day of transplantation, mice received 5 × 10 mice according to the following experimental design⁶Bone marrow cell, 3X 10⁶Spleen cells and 30X 10⁶Single donor or multi-donor early apoptotic cell products:

irradiation contrast

Reconstitution control-irradiation + bone marrow transplantation (BM)

GVHD control-irradiation + bone marrow and splenocyte transplantation

Single donor, irradiated-irradiated + bone marrow and spleen cell transplantation + irradiated early apoptotic cell product from single donor

Single donor, unirradiated-irradiated + bone marrow and spleen cell transplantation + unirradiated early apoptotic cell product from a single donor

Multiple donors, irradiated-irradiation + bone marrow and spleen cell transplantation + irradiated early apoptotic cell products from multiple donors

Multiple donors, unirradiated-irradiated + bone marrow and spleen cell transplantation + unirradiated early apoptotic cell products from multiple individual donors.

Monitoring-labeling of transplanted mice and monitoring of their survival rates. Body weight was assessed every two days for the first two weeks of the experiment, and then once daily. 35% loss from initial body weight was determined as the primary endpoint and mice were sacrificed and the survival curves were updated accordingly. The body weight results were comparable to those observed in example 12, fig. 29.

The severity of GVHD was assessed using a known scoring system (Cooke KR et al, Experimental model of idiopathic pneumonia syndrome after bone marrow transplantation, I. Effect of minor H antigen and endotoxin (An experimental model of immunological pulmonary tuberculosis bone marrow transplantation. I. the roles of minor H antigens and endotoxin), "blood" 1996; 8: 3230-3239), which involves five clinical parameters: weight loss, posture (stoop), activity, fur texture and skin integrity. Mice were evaluated for each standard and ranked from 0 to 2. By aggregating the five clinical scores, clinical index values were generated (the number of indices increased with the severity of GVHD).

Results

The percent survival of the different mouse populations is presented graphically in fig. 30. As expected from mice that did not receive bone marrow reconstitution, only irradiated control mice died between day 8 and day 12 (n-13). Most GVHD control mice (receiving bone marrow and spleen) died between day 6 and day 27. One mouse did not die (n-18). In the bone marrow reconstitution control group (BM), 3 of 7 mice died between

day

6 and 8. In the remaining mice, bone marrow was reconstituted from donor bone marrow and mice remained viable (>50 days).

Individual donor, unirradiated mice died between day 15 and day 36. Thus, as previously shown, single donor, unirradiated, early apoptotic cells have beneficial effects and prolonged survival (p < 0.01).

Single donor, irradiated mice died between day 7 and day 35, with one mouse remaining disease-free to survive (>50 days). This demonstrates that single donor, irradiated apoptotic cells also provide beneficial effects relative to GVHD. Thus, irradiation does not impair the immunomodulatory effects of early apoptotic cells. All had a beneficial effect on the survival of the murine model of GVHD compared to GVHD controls (p < 0.01).

Non-irradiated multi-donor treatment provided no beneficial effect compared to the GVHD control (n-11). Survival pattern was similar to GVHD control and mice died between day 6 and day 28 (p ═ NS-not significant). Surprisingly and in contrast to non-irradiated apoptotic cells, irradiated multiple individual donor apoptotic cells (treatment F) (n ═ 10) had beneficial effects similar to single donor treatment compared to GVHD controls. GVHD symptoms appeared significantly later and mice died between day 18 and day 34 (p < 0.01).

Irradiated multiple individual donors (n-10), irradiated single donors (n-10) and non-irradiated single donor treatment (n-10) had similar survival patterns and no significant differences in the effects on survival were observed between the three treatment groups.

Experiments indicate that in GVHD-induced mice, infusion of apoptotic cells had a significant effect. Treatment of irradiated multiple individual donor apoptotic cells and irradiated and unirradiated donor apoptotic cells has a significant life-prolonging effect.

Multi-donor treatment did not prolong the survival of mice without irradiation, but irradiation of apoptotic cell products prior to administration to mice improved the results and treatment had a survival pattern close to that of single donor treatment.

As described above, fig. 30 shows the following comparisons: (1) unirradiated apoptotic cells obtained from a single donor that does not match; (2) irradiated apoptotic cells obtained from a single donor that does not match; and (3) irradiated apoptotic cells obtained from a plurality of mismatched donors, which have significantly reduced positive clinical effects on GvHD and reduction in mortality (% survival). In addition, all three (non-irradiated early apoptotic cells, single donor; and irradiated early apoptotic cells, multiple individual donors) had similar effects.

This data is surprising because the antigenicity of unirradiated apoptotic cells obtained from multiple individual donors is expected to be similar to that of irradiated apoptotic cells obtained from multiple individual donors, why neither would produce a similar hostile antigen response to implanted bone marrow, and why neither would reduce GvHD and mortality?

If antigenicity is a major problem here, it is expected to see the difference between the clinical effects of non-irradiated apoptotic cells obtained from a single donor versus irradiated apoptotic cells obtained from a single donor. However, the data does not show such differences.

One possibility is the lack of efficacy of unirradiated pooled apoptotic cell preparations prepared from multiple individual donors due to cross-interactions between individual mononuclear populations present in the pooled preparation. These interactions do not appear to be directly attributable to antigenicity to the host, as irradiated cells maintain their antigenicity, but differ significantly in efficacy from non-irradiated cells. Thus, it appears that the cross-interactions in the irradiated pooled early apoptotic cell preparation are unexpectedly eliminated and the host response to the administration of the cells is good.

As shown, the irradiated pooled donor had essentially the same effect as the unirradiated single donor.

Example 16: effect of irradiation on the final apoptotic cell product

Apoptotic cells are increasingly being used in new therapeutic strategies due to their inherent immunomodulatory and anti-inflammatory properties. Early apoptotic cell preparations may contain up to 20-40% live cells (as measured by lack of PS exposure and no PI entry; annexin V negative and propidium iodide negative), some of which may undergo apoptosis after use in infusion, but some will remain viable. In the case of bone marrow transplantation from matched donors, viable cells do not represent a clinical problem, as the recipient has already received more viable cells in the actual transplant. However, in the case of a third party infusion (or a fourth or more as may be indicated in a pooled mononuclear apoptotic cell preparation), the use of an apoptotic cell population comprising live cells may introduce a second GvHD inducing agent. Furthermore, the effect of irradiation on the immunomodulatory potential of early apoptotic cells has not been evaluated to date. The skilled person may consider that additional irradiation of the early apoptotic cell population may result in the cells progressing to a late stage of apoptosis or necrosis. Since this seems to be a particularly relevant problem with respect to clinical applications, the experiments presented below are aimed at solving this problem, at least one of whose objectives is to improve the biosafety of functionally apoptotic cells.

Thus, the aim is to facilitate the clinical use of apoptotic cells in a number of indications where the efficacy of apoptotic cells may depend on bystander effects rather than the engraftment of transplanted cells.

The purpose is as follows: the effect of irradiation on early apoptotic cells was examined, where irradiation occurred after induction of apoptosis.

Method (in brief): cells were harvested and early apoptotic cells were prepared essentially as described in example 5.

Three separate batches of early apoptotic cells were prepared on different days (collections 404-1, 0044-1 and 0043-1).

Each final product was divided into three groups:

untreated

2500rad

4000rad。

Immediately after irradiation (t)₀) Early apoptotic cells were tested for cell count, annexin V positive-PI negative staining, cell surface markers (% of different cell types) and potency (dendritic cells (DCs)). At t₀After examination, early apoptotic cells were stored at 2-8 ℃ for 24 hours and the same test panel was used the following day (t)_{24 hours}) (cell count, annexin V positive-PI negative staining and cell surface markers and potency).

Previously, a post-release efficacy assay was developed that assesses the ability of donor mononuclear early apoptotic cells (early apoptotic cells) to induce tolerance (Mevorach et al BBMT 2014 supra). The assay is based on assessing class II MHC molecule (HLA-DR) and co-stimulatory molecule (CD86) expression on iDC membranes following exposure to LPS using flow cytometry. As previously and repeatedly shown, tolerant DCs can be produced upon interaction with apoptotic cells (Verbovetsky et al, journal of experimental medicine (J Exp Med) 2002, Krispin et al, blood 2006), and inhibition of maturation (inhibition of DR and CD86 expression) of LPS-treated DCs occurs in a dose-dependent manner.

During the 1/2a phase of the early apoptotic cell clinical study, a post-release efficacy assay (overall result n-13) was performed on each early apoptotic cell batch in order to assess the ability of each batch to induce tolerance (results shown in figure 1, Mevorach et al (2014) blood and bone marrow transplantation biology 20(1): 58-65).

DCs were generated for each batch of early apoptotic cells from fresh buffy coats collected from unknown and non-relatives healthy donors and combined with early apoptotic cells in different ratios (1: 2, 1:4 and 1:8 DCs: early apoptotic cells, respectively). Following incubation with early apoptotic cells and exposure to LPS, potency was determined based on DC membrane expression downregulation of HLA DR or CD86 at one or more DC: early apoptotic cell ratios. In all 13 assays, early apoptotic cells showed a tolerogenic effect, which was seen in the case of preparations with maximal DC: early apoptotic cell ratio, and for both markers in a dose-dependent manner.

Immature DCs (iDCs) obtained from monocytes were generated from peripheral blood PBMCs of healthy donors and cultured in the presence of 1% autologous plasma, G-CSF and IL-4. The iDCs were then preincubated for 2 hours at 1:2, 1:4, and 1:8 ratios with either freshly prepared apoptotic cell final products or apoptotic cell final products stored for 24 hours at 2-8 ℃. Both end products were examined simultaneously to determine if storage affected the potency of apoptotic cells. After incubation, LPS was added to the indicated wells and left for another 24 hours. At the end of the incubation, the idcs were collected, washed and stained with both DC-sign and HLA-DR or CD86 in order to determine changes in expression. Cells were analyzed using flow cytometry and FCS-express software from the DC-sign positive cell gate to ensure that only DCs were analyzed.

Fig. 31A and 31B and fig. 32A and 32B show potency testing of irradiated pooled apoptotic cells compared to unirradiated donor cells.

As a result:

single donor formulation

Table 21 presents the results of comparing non-irradiated apoptotic cells to irradiated apoptotic cells; mean cell loss (%) at 24 hours;annexin positivity at 0 and 24 hours (II)⁺) Propidium Iodide (PI) negative: (^-) % of early apoptotic cells (%); annexin positivity at 0 and 24 hours (II)⁺) Propidium Iodide (PI) positive (A), (B)⁺) % of late apoptotic cells (%); cell surface antigens at 0 and 24 hours CD3(T cells), CD19(B cells), CD56(NK cells), CD14 (monocytes) and CD15^{Height of}(granulocytes) are present.

Table 21:

the results in table 21 show that both non-irradiated apoptotic cells and irradiated apoptotic cells have a significant percentage of early (lines 2 and 3) apoptotic cells and late (lines 4 and 5) apoptotic cells. Thus, prior to this high level of gamma irradiation, 25Gy or 40Gy irradiation did not accelerate the apoptotic or necrotic process. Further, with respect to cell type, there was agreement between the irradiated cell population and the control non-irradiated population.

The results of the potency assays presented in fig. 31A-31B (HLA-DR expression) and fig. 32A-32B (CD86 expression) show that the immunomodulatory capacity of fresh (fig. 32A, 32A) and irradiated (fig. 31B and 32B) apoptotic cells stored for 24 hours was not changed when compared to non-irradiated apoptotic cells.

In both FIGS. 31A-31B and FIGS. 32A-32B, there was a clear upregulation of both HLA-DR and CD86 expression after exposure to the maturation agent LPS. In the presence of freshly prepared apoptotic cells from either single donors or irradiated pooled donors, significant (p <0.01) dose-dependent downregulation of both co-stimulatory markers was observed. In addition, in the presence of apoptotic cells stored at 2-8 ℃ for 24 hours, a dose-dependent down-regulation was maintained in both markers, indicating stability and efficacy of the final product after 24 hours of storage.

Effect on dendritic cells. To test the immunomodulatory capacity of apoptotic cells, a post-release potency assay was used (Mevorach et al, (2014) BBMT, supra). No changes in the immunomodulatory assay were observed in dendritic cells. (data not shown).

Effect on Mixed Lymphocyte Response (MLR). To further test the immunomodulatory effects, standardized MLR assays were established. Here, co-culture of the stimulatory cells and the responsive cells (i.e., MLR) results in robust and reliable proliferation. After the addition of unirradiated apoptotic cells to the MLR, the proliferation of lymphocytes decreased significantly > 5-fold, clearly demonstrating the inhibition of proliferation by the cells. The inhibition of lymphocyte proliferation in MLR mediated by irradiated apoptotic cells was entirely comparable. (data not shown).

The next step is the in vivo evaluation of irradiated and non-irradiated apoptotic cells in a complete mismatched mouse model. As shown in figures 28 and 29, irradiated and non-irradiated early apoptotic cell preparations had comparable beneficial effects in vivo.

Single donor formulation conclusion:

taken together, irradiation of 25Gy or 40Gy did not significantly accelerate apoptosis or induction of necrosis of apoptotic cell populations. Notably, these populations maintain the immunomodulatory effects of apoptotic cells both in vitro and in vivo.

Multi-donor formulations

Next, experiments were performed to verify that the phenomena observed with a single donor, third party formulation, were also true for multiple third party donors. Unexpectedly, the beneficial effects of mismatched single donors were lost when pooled individual donor apoptotic cell preparations were used (fig. 30). This is not due to GvHD, as the beneficial effects of each donor were maintained separately (test results not shown). One possibility is that the beneficial effects of early apoptotic cell preparations are lost due to interactions between individual donor cells in them. It was further examined whether this possible interaction of different donors could be avoided by gamma irradiation.

As shown in figure 30, the beneficial effects of a single donor were fully recovered after gamma irradiation, with irradiated multi-donor formulations and single donor formulations (irradiated or non-irradiated) having similar survival patterns.

And (4) conclusion:

it is shown here for the first time that, surprisingly, irradiation (and any method that may lead to inhibition of the T cell receptor) not only avoids unwanted proliferation and activation of T cells, but also allows for the beneficial effects of immunomodulation when using preparations of multi-donor third-party apoptotic cells.

Example 17 Effect of Combined apoptotic cell and CAR-T cell therapy on solid tumor models

The purpose is as follows: to test the effect of CAR-T cell therapy on a model of human peritoneal solid tumors and to evaluate any possible synergistic effect of apoptotic cell infusion on the anticancer effect of CAR-T cell therapy.

Method

hela-CD 19 cell line: hela cells expressing CD19 were purchased from pomi (pomi biotechnology limited, usa under catalog number PM-hela-CD 19). To follow tumor growth in vivo, hela-CD 19 was stably transduced with pllenti-PGK-V5-Luc-Neo (Addgene, usa #21471) and single clones were isolated by flow cytometry cell sorter and cultured with 500 μ G/ml G418 (sigma aldrich, usa, catalog No. a1720-1G) as selection reagent. The cell line stably and continuously expresses the firefly luc gene under the PGK promoter. Genomic integration was verified by PCR and expression was verified by flow cytometry and bioluminescence imaging (BLI) positive clones.

Cells were cultured in RP1640 MI (Gibco, Sammerfeill technologies, USA, Cat. No. 31870-025) supplemented with 10% FBS (Gibco, Sammerfeill technologies, south America, Cat. No. 12657-029), 2mM GlutaMAX (Gibco, Sammerfeill technologies, USA, Cat. No. 35050-038), and 100U/ml penicillin +100U/ml streptomycin (Gibco, Sammerfeill technologies, Cat. No. 15140-122), referred to as "complete medium". The hela-CD 19 medium was further supplemented with 1 μ g/ml puromycin (sigma aldrich, usa, catalog No. P9620) as a selective antibiotic during standard culture.

For bioluminescence imaging, mice were injected intraperitoneally with 150mg/kg Xenolight D-fluorescein (PerkinElmer, U.S.A., cat. No. 122799) and anesthetized with 4% isoflurane. Bioluminescence was taken 10 minutes after injection using the Caliper IVIS-Kinetics (perkin elmer, usa) and the Living Image software v4.2 (perkin elmer, usa).

CD19-CAR-T cells: a Ficoll gradient was used to isolate a fresh Monocyte (MNC) fraction from peripheral blood of healthy donors. The cells were then gradually frozen and stored in liquid nitrogen until use. After thawing, cells were washed and introduced into beads at 1: 2: cell-scale loaded CD28+ CD3+ beads (american whirlpool biotechnology limited). The cells were then incubated for 3 days in the presence of 300U/ml recombinant human IL-2(rhIL-2, Peprotech). 293Lenti-X cells (Clontech-Takara,632180) were transfected with a third generation CD19-CAR plasmid (Innovation laboratory) 48 hours prior to MNC infection with JetPrime reagent (Polyplus,114-01) at a JetPrime: DNA ratio of 2: 1. 24 hours before infection, according to the manufacturer's instructions, using 7.5ug/cm ²Fibronectin (Takara-Clontech, T100A) coated untreated 24-well plates (SPL, BN 30006). On the day of infection, virus-containing supernatants collected from transfected 293Lenti-X cells were filtered using a 0.45um filter (Millipore) according to manufacturer's instructions and transferred to fibronectin-coated plates (0.75 ml of virus supernatant per well) and centrifuged. Activated T cells (0.5 x10 per well) were then added to the plates⁶Individual cells) and also centrifuged at 290 g. The plates were then incubated in a humidified incubator for 60 hours. After incubation, cells were collected and the remaining adherent cells were detached using trypsin. Cells were washed twice with hank's balanced salt solution (HBSS, bio-industry limited) supplemented with 2.5% Hepes buffer (Lonza). The cells were re-seeded in the presence of 300U/ml rhIL-2 and diluted every 48 hours to 1.0X 10 with fresh medium containing 300U/ml rhIL-2⁶The concentration of (c). On day 12 post-infection, the doses were measured in 1:2 beads: cell ratio cells were reactivated with CD28+ CD3+ beads. Cells were incubated for two additional days, then animals were injected (in RPMI) on day 14 post-infection, for a total of 10X 10 injections per animal⁶And (4) cells. Preparation of pseudo-T cells (pseudo-T) as CAR-T cells, the plasmid of which did not contain the anti-CD 19 region, were injected as a control. PCR and EGFR (erbitux, merck) expression were used for transduction efficacy. In vitro cytotoxicity assays were used for functional testing of CAR T cells.

Apoptotic cells: an enriched mononuclear cell fraction was collected from healthy, qualified human donors by leukapheresis procedure and prepared as provided in example 1 herein and as produced by gamma irradiated apoptotic cells (2000-. The irradiated apoptotic cells used in this example are considered "Over The Shelf (OTS), where the donor and recipient are not necessarily HLA matched. In some embodiments, for methods of use herein, the donor and recipient are not HLA matched. In some embodiments, for the methods of use herein, the donor and recipient are HLA matched. [ apoptosis and viability of apoptotic cells were determined using annexin V and PI staining (MBL, MA, USA) as described herein. Staining with carboxyfluorescein succinimidyl ester (CFSE) and bead stimulation showed lack of proliferation.

CFSE staining: cells were brought to 10 × 10 with RPMI1640⁶Final concentration of individual cells/mL and staining with 5 μ M CFSE for 10 min at Room Temperature (RT) and light shielding with slight tilt. After the end time of incubation, the staining reaction was stopped with complete FBS for 1 min. The cells were then washed twice to remove excess CFSE dye.

Cell inoculation: cells were brought to 10X10 with medium (RPMI1640 and Biotarget, supplemented with 2mM L-glutamine, 10mM Hepes, 10% heat-activated FBS and 100U/mL recombinant human IL-2)⁶Concentration of individual cells/mL. Cells were then seeded in 24-well plates, 1mL per well, with or without CD3+ CD28+ loaded beads at a cell to bead ratio of 1: 3. In an incubator (Enlevex Equipment number ENX01051) at 5% CO²The plate is incubated in the presence of (a).

And (3) sample analysis: every 48 hours, each preparation was sampled, counted and analyzed by flow cytometry to obtain cell number and CFSE staining. Using culture medium to make concentration more than 1.5X10⁶Dilution of individual cells/mL formulation to 1X10⁶Individual cells/mL. Not more than 1.5x10⁶Individual cells/mLThe preparation was renewed with medium (0.3 mL of medium was discarded and fresh medium was added). A total of 4 test points were run for the entire 8 days.

FACS analysis of subpopulations: peritoneal cells were assessed by flow cytometry (LSR-II, BD) for the following markers and isotype controls: mMHC-II-PE (12-5321), mCD19-FITC (11-0193), mCD11c-PE-Cy5.5(35-0114), mF4/80-eFlour450(48-4801), mCD11b-APC-eFlour780(47-0112), rIgG2b-PE (12-4031), rIgG2a-FITC (11-4321), Armenian-hamster IgG-PE-Cy5.5(35-4888), rIgG2a-eFlour450(48-4801), rIgG2b-APC-eFlour780(47-4031), hCD19-APC (17-0198) and mIgG1-APC (17-4714) (bioscience, Inc.).

Macrophage subpopulation characterization was performed according to Ghosn et al (PNAS 2010) February 9,2010.107(6)2568-2573 and Casado et al (Ploss one 2011) (published in 7/22/2011). Briefly, non-CD 19, non-CD 11c peritoneal cells were classified by their CD11b and F4/80 expression into Large Peritoneal Macrophages (LPM) -F4/80 high CD11bpos, Small Peritoneal Macrophages (SPM) -F4/80 low CD11bpos, and granulocytes (Gra) -F4/80 low CD11bneg (Ghosn et al (2010 supra); Casado et al (2011 supra) described the same macrophage subpopulation somewhat differently-classifying non-CD 19, non-CD 11c, F4/80 positive cells into LPM-F4/80 high MHC-IIneg, SPM-F4/80 low MHC-IIpos, Gra-F4/80 low-IIneg by their MHC-II and F4/80 expression.

Single cell analysis of Tim4, MerTK, F4/80, CD11b, CCR2, Ly6C, CD206, CD64, C169 and CD74 were confirmed by FACS analysis. Peritoneal macrophages were identified based on the expression levels of cell surface F4/80, CD11b, Tim4 and MerTK. Permeable macrophages were identified based on the expression of cell surface CCR2, Ly6c, CD206, CD64, CD169, and CD 74.

Cytokine analysis: human or mouse cytokines were tested by Luminex cytokine analysis. Custom Multiplex kits were purchased from e biosciences (vienna, austria) or R &D Systems (Minneapolis, USA).

For intraperitoneal entitiesTumor model SCID-Bg mice were injected intraperitoneally (i.p) with 2 consecutive doses of 0.25X10 on

days

1 and 2 of the experiment⁶Human hela-CD 19-luciferase cells. On day 9, mice also received 10x10⁶Apoptotic cells in humans (irradiation as described above, 2000-6000rad irradiation), or vectors; and received 10x10 on day 10⁶Individual CD19-CAR-T (passage 3) cells or pseudo-T cells. Mice were weighed twice a week and monitored daily for clinical symptoms and peritoneal fluid accumulation. In some experiments, pre-arranged sacrifice was performed to characterize the cell and macrophage subpopulation profiles. The remaining mice were retained for survival analysis. Survival endpoints were defined by scores based on severe peritoneal effusion, manifested as abdominal enlargement and tone, decreased mobility or increased respiratory effort. These clinical findings are associated with the accumulation of large numbers of hela cells in the peritoneum. Survival analysis was performed according to kaplan-meier log rank statistic test.

And (3) treatment: fig. 33 presents the experimental protocol.

Results

In this model, mice survive approximately 30 ± 5 days (27-37) and die as a result of the formation of solid tumors in the peritoneal cavity followed by accumulation of hematologic peritoneal fluid and clinical deterioration. Sham treatment showed no significant improvement in solid tumors or changes in mouse survival (34. + -.4 days; range 30-38). CAR T cell therapy alone significantly improved mouse survival to 55 ± 11 (range 34-76, p <0.05) (fig. 34A). However, when mice received co-administration of apoptotic cells and CAR T cells, a further significant increase in survival was seen compared to CAR T cells alone, with survival reaching 70 ± 20 days (range 48-90, P < 0.05). In addition, 2/10 mice had no disease for 150 days (end of experiment). Fig. 34B (representing 5 separate experiments) shows the survival curve of the peritoneal solid tumor hela-CD 19 under control and treatment conditions. Figure 34C presents a survival curve showing that apoptotic cells (Allocetra-OTS) greatly enhance CAR T anti-cancer effects. Fig. 34C shows tumor progression visualized by In Vivo Imaging System (IVIS), showing the results for the survival curve of fig. 34B. Tumor spread was initially observed on day 15 (hela-CD 19-Luc), whereas mice treated with CAR did not show any tumor spread until day 43. When mice received apoptotic cells, most mice did not show tumor spread until day 50 and tumor size (see correlation ratios) was significantly smaller as reflected in survival curves (fig. 34B).

Figures 35A and 35B provide additional evidence that the effect of CAR T cells was enhanced by co-administration with early apoptotic cells (allocenta-OTS), where survival of mice bearing tumor burden was prolonged by the addition of apoptotic cell administration. SCID-Bg mice were injected intraperitoneally with human hela-CD 19-luciferase cells, followed by CD19-CAR T cells, where administration of CAR-T cells increased percent survival and extended the life expectancy of the mice (fig. 35A). Similar experiments in which SCID-Bg mice were injected intraperitoneally with human hela-CD 19-luciferase cells, followed by CD19-CAR T cells (with or without apoptotic cells (allocenta-OTS)) showed an even greater percentage increase in survival over time, with some mice (hela-CD 19+ CAR-T + apoptotic cells) remaining alive at day 140 (fig. 35B).

Analysis of macrophage subpopulations during treatment revealed surprising results. FIG. 36A shows, based on the expression of the colonizing peritoneal macrophage features of markers F4/80, CD11b, Tim4 and MerTK, the colonizing macrophages disappeared during tumor progression (compare ascending (tumor-free) and neutral (Hela-CD 19 tumor)). Surprisingly, treatment with CAR-T cells resulted in the re-appearance of resident macrophages (descending + CAR T). At the same time, measurement of the expression of the tumor-associated macrophage (TAM) signature CCR2, Ly6c, CD206, CD64, CD169, and CD74 (fig. 36B) showed that TAMs appeared during tumor progression (compare ascending (no tumor) and neutral (hela-CD 19 tumor)). Treatment with CAR-T cells resulted in the reduction and disappearance of these TAMs (descending + CAR T). Resident macrophages in SCID mice were mostly identified as Large Peritoneal Macrophages (LPM) (fig. 36C). The results shown in FIGS. 36A-36C show that all F4/80 high cells were CD11b positive, while only 13.5% of F4/80 high cells were MHCII positive. Thus, the resident macrophage population in SCID mice was F4/80 high/TIM 4+, MHCII low, MerTK +/CD11b +, and during tumor progression this population disappeared while TAM, monocytes and dendritic cells appeared. Treatment with CAR T cells reversed this effect, with resident macrophages reappearing and TAMs disappearing.

To address the effects of resident macrophages present after CAR T cells, SCID-Bg mice were injected intraperitoneally with human hela-CD 19-luciferase cells followed by CD19-CAR T cells (with or without apoptotic cells (Allocetra-OTS)) or opsonized apoptotic cells (D89E _ Allocetra-OTS) that avoided apoptotic cell clearance by resident macrophages. The results unexpectedly showed that the percent survival of mice bearing tumor burden was reduced to the level seen by CAR T cell administration alone (hela-CD 19+ CAR-T) in the absence of apoptotic cell (hela-CD 19+ CAR-T + D89E _ Allocetra-OTS) clearance administered (figure 37). This result indicates that, in some embodiments, resident macrophages can be an effective tool during cancer therapy, where after CAR T cell administration, resident macrophage markers can be targeted to trigger resident macrophage activation. Furthermore, one skilled in the art will recognize that cancer therapies may include cancer therapies other than CAR T cell therapy. For example, immunotherapy, radiation therapy, chemotherapy, transplantation, targeted therapy, hormonal therapy, photodynamic therapy, or surgery, or a combination thereof, where stabilization of resident macrophages would be an effective tool for cancer therapy.

To summarize: as discussed, Chimeric Antigen Receptor (CAR) T cell therapy is a novel and inventive immunotherapy. CAR-T cells are genetically engineered T cells, carrying MHC independent specific antigen receptors and co-stimulatory molecules that can activate the immune response to cancer specific antigens. This therapy has shown good efficacy in hematologic malignancies, but fails to achieve similar efficacy in both hematologic and non-hematologic solid tumors. Possible causes of this failure may be a lack of antigen, a poorly transported and hostile tumor microenvironment. Here, the effect of CAR T cell therapy on the model of human peritoneal solid tumors in SCID mice was demonstrated, where apoptotic cell infusion was significantly synergistic to the anti-cancer effect of CAR T cell therapy.

The results provided herein show that mice survived for 30 ± 5 days, with the sham treatment not significantly improving their survival to 34 ± 4 days. CAR T cell therapy significantly improved survival to 55 ± 11 days. Single cell analysis confirmed by flow cytometry revealed that resident macrophages associated with anti-tumor activity disappeared completely during tumor progression and reappeared during successful CAR T therapy. Apoptotic cells injected during tumor progression were able to stabilize the presence of macrophages, as confirmed by single cell and flow cytometry analysis, and synergized with the anti-tumor CAR-T cell effect, resulting in a significant increase in the anti-tumor macrophage population and increased survival to 75 ± 10 days (p < 0.05).

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

Claims

1. A method of slowing, reducing, inhibiting, or eliminating metastatic spread of a cancer or tumor, or any combination thereof, in a subject, the method comprising the step of administering to the subject a combination therapy comprising an early apoptotic cell population and one or more cancer therapeutic agents, wherein the method slows, reduces, inhibits, or eliminates metastatic spread of a cancer or tumor, or any combination thereof, in the subject.

2. The method of claim 1, wherein the cancer therapeutic comprises a chimeric antigen receptor-expressing T cell (CAR T cell).

3. The method of claim 1, wherein the cancer therapeutic comprises an antibody or antigen-binding fragment thereof.

4. The method of claim 1, wherein the survival of the subject is increased.

5. The method of claim 1, wherein said early apoptotic cell population comprises:

(a) An enriched monocyte population;

(b) stabilizing the apoptotic population for more than 24 hours;

(c) an apoptotic population that is free of cell aggregates;

(d) an early apoptotic cell population irradiated after the induction of apoptotic cells;

(e) a pooled population of early apoptotic cells; or

(f) A mononuclear apoptotic cell population comprising reduced non-quiescent non-apoptotic cells, suppressed cellular activation of any living non-apoptotic cells, or reduced proliferation of any living non-apoptotic cells, or any combination thereof; or

Any combination thereof.

6. The method of claim 1, wherein the subject is a human subject.

7. The method of claim 1, wherein the cancer or tumor comprises a solid tumor or a non-solid tumor.

8. The method of claim 7, wherein the non-solid cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, multiple myeloma (plasma cell myeloma), acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or plasma cell leukemia.

9. The method of claim 7, wherein the solid tumor comprises sarcoma or carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma angiosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer or tumor, breast cancer or tumor, ovarian cancer or tumor, prostate cancer or tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer or tumor, uterine cancer or tumor, testicular cancer or tumor, lung cancer, small cell lung cancer, bladder cancer, choriocarcinoma, seminoma, embryonal carcinoma, angioma, cervical cancer, or tumor, and combinations thereof, Epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

10. The method of claim 1, wherein the cancer therapeutic and the early apoptotic cells are included in separate compositions.

11. The method of claim 10, wherein the composition comprising the cancer therapeutic is administered prior to, concurrently with, or after the administration of the early apoptotic cells.

12. A method of slowing, reducing, inhibiting, or eliminating metastatic spread of a cancer or tumor, or any combination thereof, in a subject undergoing a cancer therapy, the method comprising the step of administering to the subject a population of early apoptotic cells, wherein the method slows, reduces, inhibits, or eliminates metastatic spread of a cancer or tumor, or any combination thereof, in the subject as compared to a subject undergoing a cancer therapy and not being administered a population of early apoptotic cells.

13. The method of claim 12, wherein the cancer therapy comprises radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormonal therapy, photodynamic therapy, or surgery, or a combination thereof.

14. The method of claim 12, wherein the cancer therapy comprises chimeric antigen receptor T cell (CAR T-cell) therapy.

15. The method of claim 12, wherein the survival of the subject is increased.

16. The method of claim 12, wherein said early apoptotic cell population comprises:

(a) an enriched monocyte population;

(b) stable apoptotic populations for more than 24 hours;

(c) an apoptotic population that is free of cell aggregates;

(e) a pooled population of early apoptotic cells; or

Any combination thereof.

17. The method of claim 12, wherein the subject is a human subject.

18. The method of claim 12, wherein the cancer or tumor comprises a solid tumor or a non-solid tumor.

19. The method of claim 18, wherein the non-solid cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, multiple myeloma (plasma cell myeloma), acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, or plasma cell leukemia.

20. The method of claim 18, wherein the solid tumor comprises a sarcoma or carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer or tumor, breast cancer or tumor, ovarian cancer or tumor, prostate cancer or tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer or tumor, uterine cancer or tumor, testicular cancer or tumor, lung cancer, small cell lung cancer, bladder cancer, choriocarcinoma, seminoma, embryonal carcinoma, carcinoma of the like, Epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma or retinoblastoma.

21. The method of claim 12, wherein administering the early apoptotic cells comprises administering prior to, concurrently with, or after the cancer therapy, or a combination thereof.

22. A method of improving a cancer therapy in a subject, the method comprising the step of administering an early apoptotic cell population to the subject, wherein improving a cancer therapy comprises increasing the survival time of the subject, and wherein the method improves the cancer therapy compared to a subject undergoing a cancer therapy and not being administered an early apoptotic cell population.

23. The method of claim 22, wherein the cancer therapy comprises radiation therapy, chemotherapy, transplantation, immunotherapy, targeted therapy, hormonal therapy, photodynamic therapy, or surgery, or a combination thereof.

24. The method of claim 22, wherein the cancer therapy comprises chimeric antigen receptor expressing T cell (CAR T-cell) therapy.

25. The method of claim 22, wherein the early apoptotic cell population comprises:

(a) an enriched monocyte population;

(b) stabilizing the apoptotic population for more than 24 hours;

(c) an apoptotic population that is free of cell aggregates;

(e) a pooled population of early apoptotic cells; or

Any combination thereof.

26. The method of claim 22, wherein the subject is a human subject.

27. The method of claim 22, wherein administering the early apoptotic cells comprises administering prior to, concurrently with, or after the cancer therapy, or a combination thereof.