Processed osteoclasts activate NK cell function

Abstract

The present application relates to compositions and methods for activating and / or expanding NK cells.

Description

[0001] PROCESSED OSTEOCLASTS ACTIVATE NK CELL FUNCTION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 657,375, filed June 7, 2024, and U.S. Provisional Application No. 63 / 694,269, filed September 13, 2024, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Natural killer (NK) cells lyse and differentiate cancer stem cells / undifferentiated tumors with lower expression of MHC class I, CD54 and B7H1 and higher expression of CD44. Medium and high cytotoxic activity of peripheral-blood lymphocytes are associated with reduced cancer risk, and high NK-cell infiltration of the tumor is associated with a better prognosis, whereas low activity is associated with increased cancer risk.

[0006] Suppression of NK cells is mediated by downregulation of NK receptors in the tumor microenvironment. Function of NK cells was shown previously to be significantly reduced in tumor patients. Immunotherapy with NK cells has been limited due to inability to obtain sufficient numbers of highly functional NK cells. In addition, unlike NK cells from healthy individuals, expansion of cancer patient NK cells, similar to those from tumor-bearing humanized mice, is significantly limited due to the expansion of a small fraction of contaminating T cells which crowd out NK cells by their faster proliferating capability.

[0007] Accordingly, there is a great need for compositions and methods for improving NK cell immunotherapy.

[0008] SUMMARY OF THE INVENTION

[0009] The present disclosure is based, at least in part, on the discovery that a processed osteoclast (e.g., sonicated osteoclast) can induce expansion of NK cells, which further increases CD8+ / CD4+ T cell ratio in both healthy humans and cancer patients. While cancer patients generally have more NK cells and higher CD8+ / CD4+ T cell ratio in vivo relative to healthy humans, the excess NK and CD8+ T cells are short-lived (due to the expansion of contaminating T cells which may suppress NK cell function) and lack activity (e.g., the cytoxicity and cytokine selection). However, a processed osteoclast can induce NK cell expansion and increase both the cell number and the function of NK cells in cancer patients (e.g., measured by their cytokine secretion ability). Dendritic cells preferentially promote the expansion of T cells, whereas a processed osteoclast preferentially promotes the expansion of NK cells, suggesting microenvironmental differences for the selective expansion of T and NK cells. Therefore, the present disclosure provides a method to expand large numbers of activated NK cells for use in immunotherapeutic strategies. While culturing NK cells with live or intact osteoclasts activates NK cells, the use of a processed osteoclast, which can be made in bulk and stored without a need for a fresh culture, facilitates scale up and efficiency of manufacturing activated NK cells for immunotherapy.

[0010] In some embodimetns, a method of activating a NK cell in vivo, in vitro, or ex vivo, comprises contacting the NK cell with a processed osteoclast. The NK may be a primary NK cell, preferably where it has not been transformed. The activated NK cell may expand by about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, or more population doublings within 4 weeks. The compositions and methods of the present disclosure may comprise a plurality of NK cells and / or a plurality of processed osteoclasts. The osteoclast cell may enhance NK cell cytotoxicity, e.g., as measured by the lysis of oral squamous carcinoma stem-like cells (OSCSCs) by the NK cell or a51Cr release cytotoxicity assay.

[0011] Additionally, a processed osteoclast may enhance production, secretion, and / or function of at least one cytokine or chemokine produced by the NK cell. For example, the processed osteoclast cell may enhance secretion of IFN-y and / or IL-12 by the NK cell, and / or the expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by the NK cell.

[0012] The NK cell may be a cell purified from a cancer sample of a human subject. The compositions and methods that activate NK cell using a processed osteoclast may be supplemented with at least one additional processed osteoclast cell to continue preferentially expanding the NK cells. In some embodiments, the compositions and methods that activate NK cell using a processed osteoclast may be supplemented with another agent that activates and / or expands the NK cell, for example, the probiotic bacterial compositions described herein, e.g., AJ2 and / or AJ4. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. lA-Fig. IB show that processed osteoclasts are capable of expanding NK cells. 0.35xl06sorted primary NK cells treated with IL-2 (1000 U / ml) and anti-CD16 mAh (3 pg / ml) overnight were cultured with osteoclasts (OCs) and AJ2 probiotic bacteria (2: 1:4 NK:OC:AJ2), and used as the standard expansion technique. The same numbers of OCs as described in Example 2 were sonicated (sOCs) until only cell fragments were visible and added to 0.35xl06sorted primary NK cells treated as described in Example 2 in the presence of AJ2 (2:1:4 NK:OC:AJ2). To the cultures of sonicated OCs with NK cells supernatants from the cultured osteoclasts were added since osteoclasts provide all the necessary cytokines such as IL-12, IL-15, IL-18 and IFN-a to aid in the expansion of the NK cells. Fold increase in the expansion of NK cells at different days were determined using an input of 0.3xl06NK cells every three days (Fig 1A). The total numbers of expanded NK cells at days 12, 15, 18, and 23 were estimated using the input numbers of NK cells at the initial day of the culture multiplied by the fold increase within 3 days (Fig. IB).

[0014] Fig. 2A-Fig. 2B show that the percentages of NK cells and the small populations of CD8+ T cells contaminating the purified NK cells decline starting from day 18 of expansion with the Sonicated osteoclast treatment when compared to those cultured with viable osteoclasts. 0.35xl06sorted primary NK cells treated with IL-2 (1000 U / ml) and antiCD 16mAb (3 pg / ml) overnight were cultured with osteoclasts (OCs) and AJ2 probiotic bacteria (2:1:4 NK:OC:AJ2), and used as the standard expansion technique. The same numbers of OCs as described above were sonicated (sOCs) until only cell fragments were visible and added to 0.35xl06sorted primary NK cells treated as described above in the presence of AJ2 (2:1:4 NK:OC:AJ2). To the cultures of sonicated OCs with NK cells supernatants from the cultured osteoclasts were added since osteoclasts provide all the necessary cytokines such as IL-12, IL-15, IL-18 and IFN-a to aid in the expansion of the NK cells. Percentages of NK cells (Fig. 2A) and small contaminating CD8+ T cells within CD3 subset (Fig. 2B) were determined on different days of the cultures using antibody staining followed by flow cytometric analysis.

[0015] Fig. 3A-Fig. 3B show that processed osteoclasts have similar capability to induce IFN-y secretion by the expanded NK cells at the early expansion period, and the levels decrease thereafter when compared to those induced by the viable osteoclasts. 0.35xl06sorted primary NK cells treated with IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) overnight were cultured with osteoclasts (OCs) and AJ2 probiotic bacteria (2:1:4 NK:0C:AJ2), and used as the standard expansion technique. The same numbers of OCs as described above were sonicated (sOCs) until only cell fragments were visible and added to 0.35xl06sorted primary NK cells treated as described above in the presence of AJ2 (2:1:4 NK:0C:AJ2). To the cultures of sonicated OCs with NK cells supernatants from the cultured osteoclasts were added since osteoclasts provide all the necessary cytokines such as IL-12, IL-15, IL-18 and IFN-a to aid in the expansion of the NK cells. Every 3 days after culture, 0.3xl06expanding NK cells were removed and cultured for the next 3 days until day 23. At the end of three days from each period the supernatants were removed from the cultures of NK cells with osteoclasts and the levels of IFN-y were determined using ELISA (Fig. 3A). The total amount of IFN-y within the expansion period were determined by adding the amounts obtained from different time points (Fig. 3B).

[0016] Fig. 4A-Fig. 4C show that processed osteoclasts have similar capability to increase cytotoxicity by the expanded NK cells at the days 10 and 13 expansion period. 0.35xl06sorted primary NK cells treated with IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) overnight were cultured with osteoclasts (OCs) and AJ2 probiotic bacteria (2:1:4 NK:OC:AJ2), and used as the standard expansion technique. The same numbers of OCs as described above were sonicated (sOCs) until only cell fragments were visible and added to 0.35xl06sorted primary NK cells treated as described above in the presence of AJ2 (2:1:4 NK:OC:AJ2). To the cultures of sonicated OCs with NK cells supernatants from the cultured osteoclasts were added since osteoclasts provide all the necessary cytokines such as IL-12, IL-15, IL-18 and IFN-a to aid in the expansion of the NK cells. Every 3 days after culture, 0.3xl06expanding NK cells were removed and cultured for the next 3 days until day 13. On days 10 and 13 (Fig. 4A: donor 1 DaylO and Dayl3; Fig. 4B: donor 2 Day 10 and Dayl3), NK cells were counted and different NK numbers to tumor cells (E:T) were used to assess NK cytotoxicity after 4 hours of their culture.51Cr labeled oral squamous cancer stem cells (OSCSCs) were used in NK cell cytotoxicity assays. Standard 4 hour-51Cr release assay was to determine NK cell cytotoxicity. Lu30 / 106cells denotes the number of NK cells required to lyse 30% of target cells (Fig. 4C: Donor 1 and Donor2)

[0017] Fig. 5A-Fig. 5D show that while processed osteoclasts and live osteoclasts expand supercharged NK cells and increase cytotoxicity, they do not trigger the same level of IFN-y secretion.

[0018] Fig. 6 shows that the threshold of selection of CD 8+ T cells by supercharged NK cells can be determined by the level of IFN-y induction. Fig. 7 shows that the threshold of selection of CD 8+ T cells by supercharged NK cells can be determined by the level of IFN-y induction even with additional CD3+ T cells.

[0019] Fig. 8 shows that processed osteoclast induced slightly higher or similar levels of NK cell expansion as compared to live OCs. Osteoclasts (OCs) were generated and processed as described in the Materials and Methods section. NK cells (0.5X106cells / 2ml) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) for 18-20 hours before they were co-cultured with OCs or pOCs and the cultures were treated with AJ2 (1:2:4: OCs or pOCs:NK:AJ2). OC-sNK and pOC-sNK cells were counted on days 9 and 14.

[0020] Fig. 9A-Fig. 9C shows that NK cells expanded using live OCs were more highly cytotoxic against tumors than NK cells expanded using processed osteoclasts. Osteoclasts (OCs) were generated and processed as described Example 1. NK cells (0.5X106cells / 2ml) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) for 18-20 hours before they were co-cultured with OCs or pOCs and culture was treated with AJ2 (1:2:4: OCs or pOCs:NK:AJ2). On day 10 after co-culture, OC-sNK and pOC-sNK cells were used as effector cells against oral squamous cell carcinoma stem cell line (OSCSCs) to determine the cytotoxic potential of sNK cells using a standard 4-hour51Cr release assay. The lytic units 30 / 106cells were determined using the inverse number of NK cells required to lyse 30% of OSCSCs x 100 (Fig. 9A). OSCSCs were cultured on eSight plates for 20-24 hours before the OC-sNK and pOC-sNK cells were added at 2.5:1 and 6.25: 1 E:T ratios, and cocultures were continued to 48-80 hours. Microscopic images of tumor and sNK cell interactions were taken at 24 and 46 hours of incubation were captured by eSight (scale: 0- 200 pm) (Fig. 9B). The graphs for normalized cell index and % cytolysis were assessed by the eSight RTCA and RTCA pro software, OSCSC only (in grey), OSCSC+live OC (in blue) and OSCSC+pOC (in orange) (Fig. 9C).

[0021] Fig. 10 shows that NK cells expanded with live OCs secreted higher levels of IFN-y as compared to NK cells expanded using pOCs. Osteoclasts (OCs) were generated and processed as described in Example 1. NK cells (0.5X106cells / 2ml) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) for 18-20 hours before they were co-cultured with OCs or pOCs and culture was treated with AJ2 (1:2:4: OCs or pOCs:NK:AJ2). The supernatants were harvested on day 10 of culture to determine IFN-y secretion using a single ELISA.

[0022] Fig. IIA-Fig. HE show that live OC supernatant-treated pOCs induced slightly lower levels of NK cell expansion as compared to live OCs. Osteoclasts (OCs) were generated and processed as described in Example 1. NK cells (0.35X106) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) for 18-20 hours before they were co-cultured with OCs or OCs supernatant-treated pOCs and culture was treated with AJ2 (1:2:4: OCs or sup-treated pOCs:NK:AJ2). The supernatant of osteoclast culture media was collected and later used as condition media in combination with processed osteoclast to expand sNK (labeled as pOC+OC sup in the figure) OC-sNK and pOC+OC sup-sNK cells were manually counted using microscopy on days as shown in figure (Fig. 11A). Fold increases in total lymphocyte counts were determined on days as shown in the figure using the formula: final cell count / input, input was day 0: 0.35X106, day 6 and onwards: 0.3X106(Fig. 11B). The surface expression levels of CD16+CD56+ were analyzed on days shown in the figure using flow cytometry (Fig. 11C). The numbers of NK cells were determined using the percentages of CD16+CD56+ within the total cells in Fig.l lA (Fig. 11D). Fold increases in NK cell numbers were determined on days as shown in the figure using the formula: final cell count / input (Fig. HE).

[0023] Fig. 12A-Fig. 12B show that live OC supernatant-treated pOCs induced slightly lower cytotoxicity against tumors and secreted lower levels of IFN-y as compared to live OCs. Osteoclasts (OCs) were generated and processed as described in Example 1. NK cells (0.35X106) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) for 18-20 hours before they were co-cultured with OCs or OCs supernatant-treated pOCs and culture was treated with AJ2 (1:2:4: OCs or sup-treated pOCs:NK:AJ2). On day 10 after co-culture, OC-sNK and pOC+OC sup-sNK cells were used as effector cells against OSCSCs to determine the cytotoxic potential of sNK cells using a standard 4-hour51Cr release assay. The lytic units 30 / 106cells were determined using the inverse number of NK cells required to lyse 30% of OSCSCs x 100 (Fig. 12A). The supernatants were harvested on days shown in the figure to determine IFN-y secretion using a single ELISA (Fig. 12B).

[0024] Fig. 13A-Fig. 13D show that the percentages of CD3+CD4+ and CD3+CD8+ T cells in live OC supernatant-treated and live OCs sNK cell cultures in the absence or presence of autologous CD3+ T cells. Osteoclasts (OCs) were generated and processed as described in Example 1. NK cells (0.35X106) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) for 18-20 hours before they were co-cultured with OCs or OCs supernatant-treated pOCs and culture was treated with AJ2 (1:2:4: OCs or sup-treated pOCs:NK:AJ2). Additional autologous CD3+ T cells (NK:CD8+T cells: CD4+T cell, 7:1:2) were added into purified NK cells prior to the co-culture with OCs (Fig. 13C and Fig. 13D). The surface expression levels of CD3+CD4+ and CD3+CD8+ were analyzed on days shown in the figure using flow cytometry (Fig. 13A-Fig. 13D).

[0025] Fig. 14 shows growth rate of AN3CA and HEC-1B cells. AN3CA and HEC-1B cells were plated at a density of IxlO5cells per well in a 6-well culture dish. Cell counts were taken every 24 hours using microscopy to assess growth rates. The scatter plot compares the growth rate of AN3CA cells and HEC-1B cells. Error bars represent the standard deviation (SD) of triplicate samples.

[0026] Fig. 15 shows cytotoxicity of supercharged NK (sNK) cells against AN3CA and HEC- 1B cells. A 4-hour51Cr release assay was performed to evaluate the cytotoxicity of sNK cells against AN3CA and HEC-1B cells, with OSCSC serving as a control representing a stem-like cancer cell line. The lytic units 30 / 106cells were calculated using the inverse number of NK cells required to lyse 30% of OSCSCs multiplied by 100. The following symbols represent the levels of statistical significance within each analysis, ***(p-value <0.001), **(p-value 0.001- 0.01), *(p-value 0.01-0.05).

[0027] Fig. 16A-Fig. 16B show real-time cytotoxicity monitoring of supercharged NK (sNK) cells against AN3CA and HEC-1B cells using eSight. Fig. 16A shows images of AN3CA cells, and Fig. 16B shows images of HEC-1B cells, both captured at 24-hour intervals over a 72-hour co-culture period.

[0028] Fig. 17 shows comparative cytotoxicity of supercharged NK (sNK) cells and primary NK (pNK) cells treated with IL-2 or IL-2 combined with sAJ4 against AN3CA, HEC-1B, and OSCSC cells. A 4-hour51Cr release assay was performed to evaluate the cytotoxicity of these cells. The lytic units 30 / 106cells were calculated using the inverse number of NK cells required to lyse 30% of OSCSCs multiplied by 100. The following symbols represent the levels of statistical significance within each analysis, ***(p-value <0.001), **(p-value 0.001-0.01), *(p- value 0.01-0.05).

[0029] Fig. 18A-18C show real-time cytotoxicity monitoring of sNK (dark blue) cells and pNK cells treated with IL- 2 (light blue) or IL-2 combined with AJ4 (yellow) against AN3CA, HEC-1B, and OSCSC cells (red) using eSight. Fig. 18A, Fig. 18B, and Fig. 18C show the change in cell index over a 72-hour co-culture period for AN3CA, HEC-1B, and OSCSC cells, respectively.

[0030] Fig. 19A-19C show percentage cytolysis of supercharged NK (sNK) cells and primary NK (pNK) cells treated with IL-2 or IL-2 combined with AJ4 against AN3CA, HEC-1B, and OSCSC cells. Fig. 19A, Fig. 19B, and Fig. 19C show the % cytolysis over a 72-hour co-culture period for AN3CA, HEC-1B, and OSCSC cells, respectively.

[0031] Fig. 20 shows a comparison of resected endometrial tumors from huBLT mice treated with NK immunotherapy and control group.

[0032] Fig. 21A-21C show in vivo efficacy of supercharged NK (sNK) cells in targeting AN3CA tumor progression in huBLT mice. Tumor weight analysis of sNK-treated mice compared to the control group, measured in grams (Fig. 21A). Tumor volume analysis in sNK- treated mice relative to controls, measured in mm (Fig. 21B). IFN-y secretion levels (pg / mL) in PBMCs post-mortem after ex vivo IL-2 stimulation (Fig. 21C). The following symbols represent the levels of statistical significance within each analysis, ***(p-value <0.001), **(p- value 0.001-0.01), *(p-value 0.01-0.05).

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] Osteoclasts (OCs) are multinuclear bone-resorbing cells1,2necessitating two essential factors M-CSF and RANKL for their formation3. Natural killer (NK) cells are known for their effector functions, the most important of which are cytotoxicity against tumors and, secretion of inflammatory cytokines and chemokines that indirectly regulate the functions of other immune cells4,5. Several studies have shown that increased NK cell number and functional activity in peripheral blood and / or NK cell infiltration of tumor tissue were found to be associated with a better prognosis in cancer patients6-9. Immune and bone cells derived from the progenitors in the bone marrow, share a common microenvironment, and are influenced by similar mediators. NK cells have been identified to express RANKL which during their interaction with monocytes can trigger the formation of osteoclasts10. IFN- y binds to its receptor on osteoclasts, degrades RANKL signaling and thus inhibits the activation of osteoclasts and protects our bones from being resorbed.

[0035] OCs were found to secrete a wide range of cytokines and chemokines including IL- 12, IL-15, IFN-a, and IL-18 by OCs. These factors play a crucial role in NK cells’ functional activation. In addition, OCs were found to express important NK-activating ligands11,12. These OC-expressed ligands and secreted factors contribute to OC-mediated expansion in NK cells. Large numbers of NK cells can be expanded using specific strains of processed probiotic bacteria (e.g., AJ2 or AJ4) in combination with OCs. The probiotic strains selected for NK cell expansion methodology were shown to increase cytokine secretion levels in NK cells. OC-expanded supercharged NK cells (sNK) were demonstrated to exhibit significantly increased proliferative, effector, and cytotoxic function in comparison to primary NK cells.2 18sNK cells were found to be highly effective against several tumors in preclinical models (humanized-BLT mice).2 14 16 18-38

[0036] It is demonstrated herein that processed OCs are surprisingly effective in activating and expanding NK cells to generate superchaged NK cells.

[0037] The present disclsoure relates, in part, to compositions and methods that activate and / or expand an NK cell in vitro or ex vivo, comprising contacting the NK cell with a processed osteoclast cell (pOC). Further presented herein are compositions (e.g., processed osteoclasts) and methods that activate and / or expand an NK cell in vivo, comprising administering the composition to a patient in need thereof, e.g., cancer patients. While culturing NK cells with live or intact osteoclasts activates NK cells, the use of a processed osteoclast (e.g., sonicated osteoclast, ground osteoclast), which can be made in bulk and stored without a need for a fresh culture, facilitates scale up and convenience and efficiency of manufacturing activated NK cells for immunotherapy. Accordingly, the use of pOCs is preferable for the expansion of NK cells (and hence generation of supercharged NK cells) for the shorter NK expansion period and higher cost effectiveness and faster availability for patient infusion.

[0038] Definitions

[0039] The articles “a” and “an” are used herein to refer to one or to more than one (z.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0040] The term “administering” is intended to include routes of administration which allow an agent to perform its intended function. Examples of routes of administration for treatment of a body which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.

[0041] The amount of a biomarker in a cell is “significantly” higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000% or than that amount. Alternately, the amount of the biomarker in the cell can be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker. Such “significance” can also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.

[0042] The term “conjoint”, with respect to administration of two or more agents, refers to the simultaneous, sequential, or separate dosing of the individual agents provided that some overlap occurs in the simultaneous presence of the agents or compositions in a cell or a subject. Accordingly, the term “conjoint therapy”, as used herein, refers to the administration of two or more therapeutic substances. The different agents comprising the conjoint therapy may be administered concomitant with, prior to, or following the administration of one or more therapeutic agents, such that some overlap occurs in the simultaneous presence of the agents in a cell or a subject.

[0043] The term “control” refers to any reference standard suitable to provide a comparison to the expression products in the test sample. Such a control may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells / tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells / tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells / tissues obtained from a depository. In some embodiments, the control may comprise differentiated cancer cells, CSCs, or heterogeneous cancer cells at various stages of differentiation. In other embodiments, the control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy). It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present disclosure. In some embodiments, the control may comprise normal or non- cancerous cell / tissue sample. In other embodiments, the control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In other preferred embodiments, the control may comprise normal cells, cells from patients treated with combination chemotherapy, and cells from patients having benign cancer. In still other embodiments, the control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population. Such a population may comprise normal subjects, cancer patients who have not undergone any treatment (i.e., treatment naive), cancer patients undergoing standard of care therapy, or patients having benign cancer. In other embodiments, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control. In particularly preferred embodiments, the control comprises a control sample which is of the same lineage and / or type as the test sample.

[0044] The term “immune cell” refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0045] The term “immune response” refers to a response mediated by any or all immune cells. The “immune response” includes T cell mediated and / or B cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. The term “immunotherapeutic agent” can include any molecule, peptide, antibody or other agent which can stimulate a host immune system to generate an immune response to a tumor or cancer in the subject.

[0046] The term “inhibit” includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. In some embodiments, cancer is “inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented. Similarly, a biological function, such as the function of a protein, is inhibited if it is decreased as compared to a reference state, such as a control like a wild-type state.

[0047] A “kit” is any manufacture (e.g. a package or container) comprising at least one reagent described herein, e.g. a composition (e.g., a pharmaceutical composition) comprising at least one immune cell, at least one cytokine / chemokine that support survival of the at least one immune cell, at least one reagent that aids the viscosity of sNK cells, and / or at least one cancer therapy described herein or known in the art. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present disclosure. The kit may comprise one or more reagents necessary to produce a composition useful in the methods of the present disclosure. In certain embodiments, the kit may also include instructional materials disclosing or describing the use of the kit. A kit may also include additional components to facilitate the particular application for which the kit is designed. A packaged pharmaceutical or nutraceutical composition may also be referred to as a kit.

[0048] The “level” or “amount” of a biomarker (e.g., a cell surface marker) in a sample is “significantly” higher or lower than the level of a biomarker in a control, if the amount of the biomarker is greater or less, respectively, than the level in a control by an amount greater than the standard error of the assay employed to assess amount.

[0049] In some embodiments, the amount or level of a biomarker in a sample can be considered “significantly” higher or lower than the normal and / or control amount if the amount is at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about 160%, at least or about 170%, at least or about

[0050] 180%, at least or about 190%, at least or about 200%, at least or about 250%, at least or about

[0051] 300%, at least or about 350%, at least or about 400%, at least or about 450%, at least or about

[0052] 500%, at least or about 550%, at least or about 600%, at least or about 650%, at least or about

[0053] 700%, at least or about 750%, at least or about 800%, at least or about 850%, at least or about

[0054] 900%, at least or about 950%, at least or about 1000%, at least or about 1500%, at least or about 2000%, at least or about 2500%, at least or about 3000%, or more, or any range in between, such as 5%-100%, higher or lower, respectively, than the normal and / or control amount of the biomarker. Such significant modulation values can be applied to any metric described herein, such as the level of a cell surface marker or a level of a cytokine / chemokine.

[0055] The term “processed osteoclast” encompasses any form of an osteoclast that is different from the native state of the osteoclast. In some embodiments, a processed osteoclast is not viable. In some embodiments, a processed osteoclast is not capable of proliferation. In some embodiments, processed osteoclasts comprise fragmented osteoclasts. In some embodiments, a composition comprising processed osteoclasts comprise at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about

[0056] 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about

[0057] 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about

[0058] 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about

[0059] 90%, at least or about 95%, at least or about 100% of the fragmented osteoclasts.

[0060] In some embodiments, processed osteoclasts comprise sonicated osteoclasts. In some embodiments, processed osteoclasts comprise osteoclasts that have undergone a freeze-thaw cycle. In some embodiments, processed osteoclasts comprise osteoclasts that have been treated with an extreme temperature (extremely cold temperature or extremely high temperature) (e.g., frozen or heat-inactivated). In some embodiments, processed osteoclasts comprise ground osteoclasts. In some embodiments, processed osteoclasts comprise osteoclasts that have been blended using a blender. In some embodiments, processed osteoclasts comprise dried or lyophilized osteoclasts. In some embodiments, processed osteoclasts comprise wet processed osteoclasts. In some embodiments, processed osteoclasts may have been treated with any combination of the processings described herein.

[0061] In some embodiments, processed osteoclasts are anchored to a solid support (e.g., solid phase). In some embodiments, the solid support comprises beads (e.g., magnetic beads, sepharose beads, agarose beads, etc.). In some embodiments, the solid support comprises a plate (e.g, a glass plate, a plastic plate). In some embodiments, the solid support comprises a petri dish. In some such embodiments, the petri dish support the growth of a cell culture. Anchoring processed osteoclasts to a solid support may increase the local concentration, which may improve the efficiency of activation and / or proliferation of an NK cell. Anchoring processed osteoclasts may be effected using any suitable method known in the art. Processed osteoclasts may be anchored to a solid support via covalent coupling (e.g., chemical conjugation, e.g., using reactive groups (e.g., carboxyl groups, NHS groups)), affinity binding (e.g., via an antibody that recognizes a cell surface protein of an osteoclast), and / or physical adsorption (facilitated by electrostatic forces, hydrophobic interactions, etc.). In some embodiments, a processed osteoclast comprises one or more features selected from: non- viable, fragmented, sonicated, freeze-thawed, ground, blended, heat-inactivated, wet, dried, lyophilized, and frozen.

[0062] The term “preventing” is art-recognized, and when used in relation to a disease such as cancer, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0063] The term “subject” or “patient” refers to any healthy or diseased animal, e.g., any human or non-human animal. The non-human animal can be a vertebrate, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses and non-mammals such as reptiles, amphibians, chickens, and turkeys.

[0064] In some embodiments, the subject is afflicted with cancer. In various embodiments, the subject is in need of and / or benefit from the compositions and methods of the present disclosure. In various embodiments of the methods of the present disclosure, the subject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and / or immunotherapies. In other embodiments, the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and / or immunotherapies. In certain embodiments, the subject has had surgery to remove cancerous or precancerous tissue. In other embodiments, the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient.

[0065] A “therapeutically effective amount” of a substance or cells is an amount capable of producing a medically desirable result in a treated patient, e.g., decrease tumor burden, decrease the growth of tumor cells, or alleviate any symptom associated with cancer, with an acceptable benefit: risk ratio, preferably in a human or non-human mammal.

[0066] The term “treating” includes prophylactic and / or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal), then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition); whereas, if it is administered after manifestation of the unwanted condition, the treatment is therapeutic i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0067] All numerical ranges provided herein are understood to be shorthand for all of the decimal and fractional values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9 and all intervening fractional values between the aforementioned integers such as, for example, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 8, and 1 / 9, and all multiples of the aforementioned values. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0068] Probiotic bacteria

[0069] In some embodiments, the compositions and / or methods of the present disclosure comprises the at least one probiotic bacterial strain, capable of regulating NK cell function. Such probiotic bacteria induce significant production or secretion of various cytokines / chemokines, e.g., IFN-y, Gro-alpha, IL-10, and TNF-a. In addition, such probiotic bacteria induce significant activation and / or expansion of NK cells. Preferred probiotic bacteria species of the present disclosure include Streptococcus (e.g., S. thermophiles), Bifidobacterium (e.g., B. longum, B. breve, B. infantis, B. breve, B. infantis), and / or Lactobacillus genera (e.g., L. acidophilus, L. helveticus, L. bulgaricus, L. rhamnosus, L. plantarum, and L. casei). The compositions and methods of the present disclosure comprise at least one probiotic bacterial strain, preferably a combination of two or more different bacterial strains. Such probiotic bacteria can be used to generate supercharged NK cells. Alternatively, the probiotic bacteria can be administered to a subject, preferably a mammal (e.g., a human) to enhance NK cell function in the subject (e.g., as a cancer therapy). Such administration may be systemically or locally (e.g., directly to intestines, e.g., orally or rectally) performed. The preferable administration route is oral administration. Other routes (e.g., rectal) may be also used.

[0070] For administration, either the bacteria (e.g., in a wet, sonicated, ground, or dried form or formula), the bacterial culture medium comprising the bacteria, or the bacterial culture medium supernatant (not containing the bacteria), may be administered. The bacteria may be alive, partially alive, or dead. The bacteria may be sonicated, ground, wet, or dry (e.g., freeze- dried).

[0071] In some embodiments, the composition (bacterial, pharmaceutical, and / or nutraceutical) of the present disclosure comprises at least about 1 x 104, at least about 1 x 105, at least about 1 x 106, at least about 2 x 106, at least about 5 x 106, at least about 1 x 107, at least about 5 x 107, at least about 1 x 108, at least about 5 x 108, at least about 10 x 108, at least about 100 x 108, at least about IxlO9, at least about 5xl09, at least about 10xl09, at least about 100 x 109, at least about 110 x 109, at least about 120 x 109, at least about 130 x 109, at least about 140 x 109, at least about 150 x 109, at least about 160 x 109, at least about 170 x 109, at least about 180 x 109, at least about 190 x 109, at least about 200 x 109, at least about 210 x 109, at least about 220 x 109, at least about 230 x 109, at least about 240 x 109, at least about 250 x 109, at least about 260 x 109, at least about 270 x 109, at least about 280 x 109, at least about 290 x 109, at least about 300 x 109, at least about 310 x 109, at least about 320 x 109, at least about 330 x 109, at least about 340 x 109, at least about 350 x 109, at least about 360 x 109, at least about 370 x 109, at least about 380 x 109, at least about 390 x 109, at least about 400 x 109, at least about 410 x 109, at least about 420 x 109, at least about 430 x 109, at least about 440 x 109, at least about 450 x 109, at least about 460 x 109, at least about 470 x 109, at least about 480 x 109, at least about 490 x 109, or at least about 500 x 109. In some embodiments, the composition (bacterial, pharmaceutical, and / or nutraceutical) of the present disclosure comprises no more than 510 x 109, no more than 520 x 109, no more than 530 x 109, no more than 540 x 109, no more than 550 x 109, no more than 600 x 109, no more than 650 x 109, no more than 700 x 109, no more than 750 x 109, no more than 800 x 109, no more than 850 x 109, no more than 900 x 109, no more than 950 x 109, or no more than 1000 x 109total CFU of bacteria per gram of the composition.

[0072] In some embodiments, the composition comprises at least about 180 x 109but no more than about 270 x 109total CFU of bacteria per gram of the composition. In some embodiments, the composition comprises about 250 x 109total CFU of bacteria per gram of the composition. AJ2 BACTERIA

[0073] The present disclosure encompasses AJ2 probiotic bacteria. AJ2 is a combination of at least 7 different strains of gram positive probiotic bacteria (Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, and optionally further comprising KE99 and Lactobacillus bulgaricus). In some embodiments, AJ2 comprises Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei. In some embodiments, AJ2 comprises Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus bulgaricus).

[0074] AJ2 has the ability to induce synergistic production of IFN-y when added to IL-2- treated or IL-2 + anti-CD16 monoclonal antibody-treated NK cells (anti-CD16mAb). The combination of strains was used to provide bacterial diversity in addition to synergistic induction of a balanced pro and anti-inflammatory cytokine and growth factor release in NK cells. Moreover, the quantity of each bacteria within the combination of strains was adjusted to yield a closer ratio of IFN-y to IL-10 to that obtained when NK cells are activated with IL- 2 + anti-CD16mAb in the absence of bacteria. The rationale behind such selection was to obtain a ratio similar to that obtained with NK cells activated with IL-2 + anti-CD16mAb in the absence of bacteria since such treatment provided significant differentiation of the cells. AJ4 BACTERIA

[0075] In some embodiments, the composition (bacterial, pharmaceutical, and / or nutraceutical) of the present disclosure comprises at least two bacterial strains selected from: Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei. In some embodiments, one or more bacterial strains are intact. In some embodiments, one or more bacterial strains are sonicated. In preferred embodiments, the composition is an AJ4 composition comprising Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei.

[0076] In some embodiments, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, or at least about 60%, of the bacteria in the composition are Streptococcus thermophiles. In some embodiments, no more than about 61%, no more than about 62%, no more than about 63%, no more than about 64%, no more than about 65%, no more than about 66%, no more than about 67%, no more than about 68%, no more than about 69%, no more than about 70%, no more than about 71%, no more than about 72%, no more than about 73%, no more than about 74%, no more than about 75%, no more than about 76%, no more than about 77%, no more than about 78%, no more than about 79%, no more than about 80%, no more than about 81%, no more than about 82%, no more than about 83%, no more than about 84%, no more than about 85%, no more than about 86%, no more than about 87%, no more than about 88%, no more than about 89%, no more than about 90%, no more than about 91%, no more than about 92%, no more than about 93%, no more than about 94%, no more than about 95%, no more than about 96%, no more than about 97%, no more than about 98%, or no more than about 99% of the bacteria in the composition are Streptococcus thermophiles.

[0077] In some embodiments, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, or at least about 50% of the bacteria in the composition are Lactobacillus acidophilus. In some embodiments, no more than about 51%, no more than about 52%, no more than about 53%, no more than about 54%, no more than about 55%, no more than about 56%, no more than about 57%, no more than about 58%, no more than about 59%, no more than about 60%, no more than about 61%, no more than about 62%, no more than about 63%, no more than about 64%, no more than about 65%, no more than about 66%, no more than about 67%, no more than about 68%, no more than about 69%, no more than about 70%, no more than about 71%, no more than about 72%, no more than about 73%, no more than about 74%, no more than about 75%, no more than about 76%, no more than about 77%, no more than about 78%, no more than about 79%, no more than about 80%, no more than about 81%, no more than about 82%, no more than about 83%, no more than about 84%, no more than about 85%, no more than about 86%, no more than about 87%, no more than about 88%, no more than about 89%, no more than about 90%, no more than about 91%, no more than about 92%, no more than about 93%, no more than about 94%, no more than about 95%, no more than about 96%, no more than about 97%, no more than about 98%, or no more than about 99% of the bacteria in the composition are Lactobacillus acidophilus.

[0078] In some embodiments, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, or at least about 70% of the bacteria in the composition are Lactobacillus plantarum. In some embodiments, no more than about 71%, no more than about 72%, no more than about 73%, no more than about 74%, no more than about 75%, no more than about 76%, no more than about 77%, no more than about 78%, no more than about 79%, no more than about 80%, no more than about 81%, no more than about 82%, no more than about 83%, no more than about 84%, no more than about 85%, no more than about 86%, no more than about 87%, no more than about 88%, no more than about 89%, no more than about 90%, no more than about 91%, no more than about 92%, no more than about 93%, no more than about 94%, no more than about 95%, no more than about 96%, no more than about 97%, no more than about 98%, or no more than about 99% of the bacteria in the composition are Lactobacillus plantarum.

[0079] In some embodiments, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, or at least about 40% of the bacteria in the composition are Lactobacillus paracasei. In some embodiments, no more than about 41%, no more than about 42%, no more than about 43%, no more than about 44%, no more than about 45%, no more than about 46%, no more than about 47%, no more than about 48%, no more than about 49%, no more than about 50%, no more than about 51%, no more than about 52%, no more than about 53%, no more than about 54%, no more than about 55%, no more than about 56%, no more than about 57%, no more than about 58%, no more than about 59%, no more than about 60%, no more than about 61%, no more than about 62%, no more than about 63%, no more than about 64%, no more than about 65%, no more than about 66%, no more than about 67%, no more than about 68%, no more than about 69%, no more than about 70%, no more than about 71%, no more than about 72%, no more than about 73%, no more than about 74%, no more than about 75%, no more than about 76%, no more than about 77%, no more than about 78%, no more than about 79%, no more than about 80%, no more than about 81%, no more than about 82%, no more than about 83%, no more than about 84%, no more than about 85%, no more than about 86%, no more than about 87%, no more than about 88%, no more than about 89%, no more than about 90%, no more than about 91%, no more than about 92%, no more than about 93%, no more than about 94%, no more than about 95%, no more than about 96%, no more than about 97%, no more than about 98%, or no more than about 99% of the bacteria in the composition are Lactobacillus paracasei.

[0080] In some embodiments, the percent bacteria refers to the percentage of the colony forming unit (CFU) of said bacteria relative to the total CFU of bacteria in the composition.

[0081] In some embodiments, the bacteria in the composition comprise about 30% (or about 20% to about 40%) Streptococcus thermophiles, about 20% (or about 10% to about 30%) Lactobacillus acidophilus, about 40% (or about 30% to about 50%) Lactobacillus plantarum, and about 10% (or about 1% to about 20%) Lactobacillus paracasei, wherein the percent bacteria refers to the percentage of the CFU of said bacteria relative to the total CFU of bacteria in the composition.

[0082] Cytokines that support activation and survival of NK cells

[0083] Cytokines include a broad and loose category of small proteins (-5-20 kDa) that are important in cell signaling. Their release has an effect on the behaviour of cells around them, cytokines are involved in autocrine signalling, paracrine signaling and endocrine signalling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors, and may additionally include hormones or growth factors in the instant disclosure. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. Preferred cytokines are exemplified in the specification and the Tables of the instant disclosure.

[0084] Cytokines that support activation and survival of NK cells include: interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, and type I interferons (IFNs) (see Zwirner and Domaica (2010) Biofactors 36(4):274-88 which is incorporated herein by reference). The type I IFN family is a multi- gene cytokine family that encodes 13 partially homologous IFNa subtypes in humans (14 in mice), a single IFNP and several poorly defined single gene products (IFNe, IFNr, IFNK, IFNCO, IFN5 and IFNQ (see McNab et al. (2015) Nature Reviews Immunology, 15:87- 103, which is incorporated herein by reference).

[0085] Supercharged NK cells

[0086] Supercharged NK cells (sNK cells) are NK cells that are generated by contacting NK cells with osteoclasts (OCs) or a processed (e.g., sonicated) lysate thereof. An exemplary method to generate sNK cells using live and intact osteoclasts was described by Kaur et al. (2017) Front Immunol 8:297, which is incorporated herein by reference. In some embodiments, purified human NK cells are activated with rh-IL-2 (1000 lU / ml) and antiCD 16 mAbs (3 ug / ml) for 18-20 hours prior to contacting or co-culturing with OCs. In some embodiments, the NK cells are contacted or co-cultured with OCs in the presence of AJ2. In some embodiments, the ratio of NK: OCs: AJ2 is 2:1:4. The medium may be renewed with RPMI supplemented with (1500 lU / mL) rhIL-2 every three days.

[0087] Osteoclasts

[0088] Osteoclasts are a type of bone cell, derived from hematopoietic stem cells. Their function, resorbing bone tissue, is critical for the maintenance, repair, and remodeling of bones. Bone homeostasis is achieved when there is a balance between osteoblast bone formation and osteoclast bone resorption. Osteoclasts mature through stimulation from osteoblasts expressing RANKL, and their interaction, mediated by firm adhesion via ICAM-1. Osteoclasts also express many ligands for receptors present on activated NK cells. They reported that osteoclasts express ULBP-1, ULBP-2 / 5 / 6 and ULBP-3, but little or no MIC-A, MIC-B, or MHC class I-like ligands for NKG2D, the activating receptor of NK cells.

[0089] Osteoclasts (OCs), in comparison to dendritic cells (DCs) and monocytes, are significant activators of NK cell expansion and function (Tseng et al. (2015) Oncotarget 6(24):20002-25). Additionally, osteoclasts secrete significant amounts of IL-12, IL-15, IFN-y and IL- 18, which are known to activate NK cells; osteoclasts also express important NK- activating ligands. Accordingly, osteoclasts expand and activate NK cells to levels that are higher than those established by other methodologies.

[0090] Method of Detection

[0091] A biomarker (e.g., cell surface protein, a cytokine, etc.) can be detected and quantified by any of a number of means well-known to those of skill in the art. For example, cell surface proteins can be detected using Fluorescence-activated Cell Sorting (FACS), immunohistochemistry, next-generation sequencing, or the like. Additionally, a biomarker can be detected by methods including, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference), NMR, MALDLTOF, or LC-MS / MS. Preferred are binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.

[0092] For example, ELISA and RIA procedures may be conducted such that a desired standard is labeled (with a radioisotope such as125I or35S, or an assay able enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabeled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, the biomarker protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarker protein antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.

[0093] The above techniques may be conducted essentially as a “one-step” or “two-step” assay. A “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A “two-step” assay involves washing before contacting, the mixture with labeled antibody. Other conventional methods may also be employed as suitable.

[0094] In certain embodiments, a method for measuring the biomarker levels comprises the steps of: contacting a biological specimen with an antibody or variant e.g., fragment) thereof which selectively binds the biomarker protein, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of the biomarker protein.

[0095] Enzymatic and radiolabeling of biomarker protein and / or the antibodies may be effected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.

[0096] It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.

[0097] It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.

[0098] Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

[0099] Other techniques may be used to detect biomarker protein according to a practitioner’s preference based upon the present disclosure. One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-biomarker protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or antiimmunoglobulin (suitable labels including125I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.

[0100] Immunohistochemistry may be used to detect expression of biomarker protein, e.g., in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabeling. The assay is scored visually, using microscopy.

[0101] Antibodies that may be used to detect biomarker protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker protein to be detected. An antibody may have a Kd of at most about 10"6M, at most about 10"7M, at most about 10"8M, at most about 10-9M, at most about 1010M, at most about 10-11M, at most about 1012M. The phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to the biomarker protein relative to other proteins, such as related proteins.

[0102] Antibodies may be commercially available or may be prepared according to methods known in the art.

[0103] Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered or single-chain antibodies as well as functional fragments, i.e., biomarker protein binding fragments, of antibodies. For example, antibody fragments capable of binding to a biomarker protein or portions thereof, including, but not limited to, Fv, Fab, Fab’ and F(ab’)2 fragments can be used. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab’)2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab’)2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab’)2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain.

[0104] In some embodiments, agents that specifically bind to a biomarker other than antibodies are used, such as peptides. Peptides that specifically bind to a biomarker may be well known in the art (e.g., receptor fragment for a ligand), or can be identified by any means known in the art. For example, specific peptide binders of a biomarker protein can be screened for using peptide phage display libraries.

[0105] Controls

[0106] Often, a sample from the subject is typically from blood, cells, or tissue. The control sample can be from the same subject or from a different subject. The control sample is typically a normal, non-diseased sample. However, in some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, the control sample can be from a diseased subject. The control sample can be a combination of samples from several different subjects.

[0107] In some embodiments, the biomarker amount and / or activity measurement(s) from a subject is compared to a pre-determined level. This pre-determined level is typically obtained from normal samples. As described herein, a “pre-determined” biomarker amount and / or activity measurement(s) may be a biomarker amount and / or activity measurement(s) used to, by way of example only, evaluate a subject that may be selected for treatment, evaluate a response to a composition as disclosed herein, alone or in combination with other immunotherapies and with one or more additional anti-cancer therapies. A pre-determined biomarker amount and / or activity measurement(s) may be determined in populations of patients with or without a disease (e.g., cancer). The pre-determined biomarker amount and / or activity measurement(s) can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and / or activity measurement(s) can vary according to specific subpopulations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and / or activity measurement(s) of the individual. Furthermore, the pre-determined biomarker amount and / or activity can be determined for each subject individually. In some embodiments, the amounts determined and / or compared in a method described herein are based on absolute measurements.

[0108] In other embodiments, the amounts determined and / or compared in a method described herein are based on relative measurements, such as ratios (e.g., biomarker level, and / or activity before a treatment vs. after a treatment, and the like). For example, the relative analysis can be based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement. Pre-treatment biomarker measurement can be made at any time prior to initiation of anti-cancer therapy. Post-treatment biomarker measurement can be made at any time after initiation of anti-cancer therapy. In some embodiments, post-treatment biomarker measurements are made 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after initiation of the administration of the compositions of the present disclosure.

[0109] In some embodiments, the change of biomarker amount and / or activity measurement(s) from the pre-determined level is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0 fold or greater, or any range in between, inclusive. Such cutoff values apply equally when the measurement is based on relative changes, such as based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement.

[0110] Accordingly, in some embodiments, a control is a biomarker level in a healthy subject, in a diseased subject (e.g., afflicted with a cancer), in a subject prior to a certain therapy (e.g., a cancer therapy), or in a subject after being treated with a certain therapy. Diagnostic Methods

[0111] The present disclosure provides, in part, methods, systems, and code for accurately classifying whether a biological sample according to the presence and / or level of a biomarker (e.g., a cell surface marker of the present disclosure), thereby indicative of the state of a disorder of interest, such as cancer (e.g., Grade 1 cancer or cancer with a differentiated cancer cell). In some embodiments, the present disclosure is useful for classifying a sample (e.g., from a subject) as associated with or at risk for cancer or a subtype thereof, which can be treated with a composition of the present disclsoure (e.g., a composition comprising a supercharged NK cell). Such classification uses a statistical algorithm and / or empirical data e.g., the presence, absence, and / or level of a biomarker described herein).

[0112] An exemplary method for detecting the level of a biomarker of the present disclosure, and thus useful for classifying whether a sample is associated with a cancer or a clinical subtype thereof or different stages of a cancer involves obtaining a biological sample from a test subject and contacting the biological sample with an antibody or antigen-binding fragment thereof capable of detecting a biomarker of the present disclosure such that the level of the biomarker is detected in the biological sample.

[0113] Any method or a combination of two or more known in the art or those described herein can be used to detect the biomarker. In some embodiments, FACS analysis and / or immunohistochemistry are used to detect the biomarker. In some embodiments, at least one antibody or antigen-binding fragment thereof is used, wherein two, three, four, five, six, seven, eight, nine, ten, or more such antibodies or antibody fragments can be used in combination (e.g., in sandwich ELISAs) or in serial. Alternatively, certain grade of cancer (e.g, Grade I, II, etc.) can be determined using histology or any technique known to a pathologist or a clinician, without detecting the biomarker of the present disclosure. It is well known in the art that different grade or stage of cancer is associated with specific cellular morphology.

[0114] In certain instances, the statistical algorithm is a single learning statistical classifier system. For example, a single learning statistical classifier system can be used to classify a sample as a cancer sample based upon a prediction or probability value and the presence or level of ganglioside. The use of a single learning statistical classifier system typically classifies the sample as a cancer sample with a sensitivity, specificity, positive predictive value, negative predictive value, and / or overall accuracy of at least or about 75%, at least or about 76%, at least or about 77%, at least or about 78%, at least or about 79%, at least or about 80%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99%.

[0115] Other suitable statistical algorithms are well-known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets. In some embodiments, a single learning statistical classifier system such as a classification tree e.g., random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning e.g., decision / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg- Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In some embodiments, the method of the present disclosure further comprises sending the sample classification results to a clinician (a non-specialist, e.g., primary care physician; and / or a specialist, e.g., a histopathologist or an oncologist).

[0116] In some embodiments, the method of the present disclosure further provides a diagnosis in the form of a probability that the individual has a cancer or a certain stage of a cancer (e.g., Grade 1 cancer). For example, the individual can have about a 0%, about a 5%, about a 10%, about a 15%, about a 20%, about a 25%, about a 30%, about a 35%, about a 40%, about a 45%, about a 50%, about a 55%, about a 60%, about a 65%, about a 70%, about a 75%, about a 80%, about a 85%, about a 90%, about a 95%, or greater probability of having the cancer. In yet another embodiment, a method of the present disclosure further provides a prognosis of the cancer in the individual. In some instances, the method of classifying a sample as a cancer sample may be further based on the symptoms (e.g., clinical factors) of the individual from which the sample is obtained. The symptoms or group of symptoms can be, for example, lymphocyte count, white cell count, erythrocyte sedimentation rate, diarrhea, abdominal pain, bloating, pelvic pain, lower back pain, cramping, fever, anemia, weight loss, anxiety, depression, and combinations thereof. In some instances, the method of classifying a sample as a cancer sample may be further based on genetic mutations and / or predisposition to cancer, irrespective of the symptoms. In some embodiments, the diagnosis of an individual as having a cancer is followed by administering to the individual a therapeutically effective amount of a cancer therapy e.g., chemotherapeutic agents).

[0117] An exemplary method for detecting the presence or absence of a biomarker comprises using an antibody of the present disclosure, or fragment thereof, capable of binding to a biomarker, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. Such agents can be labeled. The term “labeled”, with regard to the antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody. The term “biological sample” is intended to include tissues, cells, and biological fluids isolated from a subject, such as serum, blood, as well as tissues, cells, and fluids present within a subject. That is, the detection method of the present disclosure can be used to detect a biomarker in a biological sample in vitro, ex vivo, as well as in vivo. In vitro techniques for detection of a biomarker include enzyme linked immunosorbent assays (ELIS As), immunoprecipitations, immunohistochemistry (IHC), flow cytometry and related techniques, and immunofluorescence. Furthermore, in vivo techniques for detection of a biomarker include introducing into a subject a labeled anti- biomarker antibody. For example, the antibody can be labeled with a radioactive, luminescent, fluorescent, or other similar marker whose presence and location in a subject can be detected by standard imaging techniques, either alone or in combination with imaging for other molecules, such as markers of cell type (e.g., CD8+ T cell markers).

[0118] In some embodiments, the methods further involve obtaining a control biological sample (e.g., biological sample from a subject who does not have a cancer), a biological sample from the subject during remission or before developing a cancer, or a biological sample from the subject during treatment for developing a cancer.

[0119] In some embodiments, the methods comprise contacting the control sample with a compound or agent capable of detecting a biomarker such that the presence and / or the level of a biomarker is detected in the biological sample, and comparing the presence or the level of a biomarker in the control sample with the presence or the level of a biomarker in the test sample.

[0120] A preferred biological sample is a cell, tissue, serum, blood, saliva, tumor microenvironment, peritumoral, or intratumoral, isolated by conventional means from a subject.

[0121] In still other embodiments, the antibodies can be associated with a component or device for the use of the antibodies in an ELISA or RIA. Non-limiting examples include antibodies immobilized on solid surfaces for use in these assays (e.g., linked and / or conjugated to a detectable label based on light or radiation emission as described above). In other embodiments, the antibodies are associated with a device or strip for detection of a biomarker by use of an immunochromatographic or immunochemical assay, such as in a “sandwich” or competitive assay, immunohistochemistry, immunofluorescence microscopy, and the like. Additional examples of such devices or strips are those designed for home testing or rapid point of care testing. Further examples include those that are designed for the simultaneous analysis of multiple analytes in a single sample. For example, an unlabeled antibody may be applied to a “capture” a biomarker in a biological sample and the captured (or immobilized) biomarker may be bound to a labeled form of an anti-biomarker antibody of the present disclosure for detection. Other embodiments of immunoassays are well-known the skilled artisan, including assays based on, for example, immunodiffusion, immunoelectrophoresis, immunohistopathology, immunohistochemistry, and histopathology. In some embodiments, the compositions and methods of the present disclosure can be used to determine a grade of a cancer. A cancer’s grade describes how abnormal the cancer cells and tissue look under a microscope when compared to healthy cells. Cancer cells that look and organize most like healthy cells and tissue are low grade tumors. Doctors describe these cancers as being well differentiated. Lower grade cancers are typically less aggressive and have a better prognosis. The more abnormal the cells look and organize themselves, the higher the cancer’s grade. Cancer cells with a high grades tend to be more aggressive. They are called poorly differentiated or undifferentiated. Some cancers have their own system for grading tumors. Many others use a standard 1-4 grading scale.

[0122] • Grade 1 : Tumor cells and tissue looks most like healthy cells and tissue. These are, by definition, well-differentiated tumors. Grade 1 cancer can be identified by histology / histopathology. Alternatively, Grade 1 cancer or a well-differentiated cancer cell can be identified by the expression level of at least one cell surface markers selected from CD44, CD26, CD166, CD326, CD338, CD133, CD54, PD-L1, and MHC-class I. In preferred embodiments, the at least one cell surface markers is selected from CD44, CD54, PD-L1, and MHC-class I markers.

[0123] • Grade 2: The cells and tissue are somewhat abnormal and are called moderately differentiated. These are intermediate grade tumors.

[0124] • Grade 3: Cancer cells and tissue look very abnormal. These cancers are considered poorly differentiated, since they no longer have an architectural structure or pattern. Grade 3 tumors are considered high grade.

[0125] • Grade 4: These undifferentiated cancers have the most abnormal looking cells. These are the highest grade and typically grow and spread faster than lower grade tumors.

[0126] As used herein, low grade cancer refers to Grade 1 cancer; and high grade cancer refers to cancer of Grades 2-4.

[0127] A cancer’s stage explains how large the primary tumor is and how far the cancer has spread in the patient’s body. There are several different staging systems. Many of these have been created for specific kinds of cancers. Others can be used to describe several types of cancer. One common system that many people are aware of puts cancer on a scale of 0 to IV.

[0128] • Stage 0 is for abnormal cells that haven’t spread and are not considered cancer, though they could become cancerous in the future. This stage is also called “in-situ.” • Stage I through Stage III are for cancers that haven’t spread beyond the primary tumor site or have only spread to nearby tissue. The higher the stage number, the larger the tumor and the more it has spread.

[0129] • Stage IV cancer has spread to distant areas of the body.

[0130] As used herein, cancer at the early / low stage refers to cancer at Stage I; and cancer at the late / high / advanced stage includes cancer at Stage II to Stage IV.

[0131] NK cells activated using processed osteoclasts can be used to treat cancer of any grade (e.g., Grade 1, 2, 3, or 4) or stage (e.g., Stage I, II, III, or IV).

[0132] Cancer cell differentiation can be determined using morphology, proliferation rate (e.g., doubling time), as well as the presence and / or abundance of certain cell surface marker(s). The table below shows exemplary cell surface markers that indicate cancer cell proliferation.

[0133] Table

[0134] Samples

[0135] Biological samples can be collected from a variety of sources from a patient including a body fluid sample, cell sample, or a tissue sample. “Body fluids” refer to fluids that are excreted or secreted from the body as well as fluids that are normally not e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In some embodiments, the subject and / or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. In some embodiments, the sample is serum, plasma, or urine. In other embodiments, the sample is serum.

[0136] The samples can be collected from individuals repeatedly over a longitudinal period of time e.g., once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an individual over a period of time can be used to verify results from earlier detections and / or to identify an alteration in biological pattern as a result of, for example, disease progression, drug treatment, etc. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three month intervals. In addition, the cytokine / chemokine amount and / or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.

[0137] Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and / or analysis of biomarker measurement(s). Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.

[0138] Administration

[0139] The cells (e.g., sNK cells) of the present disclosure can be administered at a dose of 1, 10, 1000, 10,000, 0.1 x 106, 0.2 x 106, 0.3 x 106, 0.4 x 106, 0.5 x 106, 0.6 x 106, 0.7 x 106, 0.8 x 106, 0.9 x 106, 1.0 x 106, 5.0 x 106, 1.0 x 107, 5.0 x 107, 1.0 x 108, 5.0 x 108, 1.0 x 109or more, or any range in between or any value in between, cells per kilogram of subject body weight. The number of cells transplanted may be adjusted based on the desired level of engraftment in a given amount of time. Generally, about IxlO5to about IxlO9cells / kg of body weight, from about IxlO6to about IxlO8cells / kg of body weight, or about IxlO7cells / kg of body weight, or more cells, as necessary, may be transplanted. In some embodiment, transplantation of at least about 100, at least about 1000, at least about 10,000, at least about O.lxlO6, at least about 0.5xl06, at least about l.OxlO6, at least about 2.0xl06, at least about 3.0xl06, at least about 4.0xl06, or at least about 5.0xl06total cells to a subject is effective.

[0140] In some embodiments, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 billion cells (e.g., sNK cells) are administered to a subject via systemic administration. In preferred embodiments, at least about 10 billion cells (e.g., sNK cells) are administered to a subject via systemic administration. In another preferred embodiments, about 10 billion cells (e.g., sNK cells) are administered to a subject via systemic administration.

[0141] The cells can be administered in any suitable route as described herein, such as by infusion or image-guided needle or transcatheter delivery. The cells can also be administered before, concurrently with, or after, other anti-cancer agents or locoregional therapies.

[0142] Administration can be accomplished using methods generally known in the art. Agents, including cells or at least one cancer therapy of the present disclosure, may be introduced to the desired site by direct injection, or by any other means used in the art including, but are not limited to, intravascular, intracerebral, parenteral, intraperitoneal, intravenous, epidural, intraspinal, intrasternal, intra-articular, intra-synovial, intrathecal, intraarterial, intracardiac, or intramuscular administration. For example, subjects of interest may be engrafted with the transplanted cells by various routes. Such routes include, but are not limited to, intravenous administration, subcutaneous administration, administration to a specific tissue e.g., focal transplantation), injection into the femur bone marrow cavity, injection into the spleen, administration under the renal capsule of fetal liver, and the like. In some embodiments, the cancer vaccine is injected to the subject intratumorally or subcutaneously. Cells may be administered in once, or multiple times over a defined time period sufficient to generate a desired effect. Exemplary methods for transplantation, engraftment assessment, and marker phenotyping analysis of transplanted cells are well- known in the art (see, for example, Pearson et al. (2008) Curr. Protoc. Immunol. 81:15.21.1- 15.21.21; Ito et al. (2002) Blood 100:3175-3182; Traggiai et al. (2004) Science 304: 104-107; Ishikawa et al. Blood (2005) 106:1565-1573; Shultz et al. (2005) 7. Immunol. 174:6477- 6489; and Holyoake et al. (1999) Exp. Hematol. 27:1418-1427).

[0143] The cells (e.g., supercharged NK cells) can be administered to a subject conjointly with at least one cytokine / chemokine that supports activation and / or survival of NK cells. The cells can be administered before, concurrently, or after the administration of the at least one cytokine / chemokine.

[0144] The cells (e.g., supercharged NK cells) can be administered to a subject conjointly with at least one cancer therapy described herein or those known in the art. The cells can be administered before, concurrently, or after the administration of the at least one cancer therapy.

[0145] Pharmaceutical Compositions

[0146] The present disclosure provides pharmaceutically acceptable compositions of the compositions disclosed herein.

[0147] The phrase “pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0148] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0149] An agent can be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. '.TT).

[0150] The agent may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0151] Pharmaceutical compositions of agents suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the composition will preferably be sterile and must be fluid to the extent that easy syringeability exists. It will preferably be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0152] Sterile injectable solutions can be prepared by incorporating an agent of the disclosure in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the agent plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0153] It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form “, as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by, and directly dependent on, (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0154] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of one or more bacterial strains as disclosed herein.

[0155] In some embodiments, the composition comprises at least one carbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnFbnOn. A carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates may contain modified saccharide units such as 2 ’-deoxyribose wherein a hydroxyl group is removed, 2 ’-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N- acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2 ’-fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

[0156] In some embodiments, the composition comprises at least one lipid. As used herein a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans). In some embodiments the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0). In some embodiments the composition comprises at least one modified lipid, for example a lipid that has been modified by cooking.

[0157] In some embodiments, the composition comprises at least one supplemental mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.

[0158] In some embodiments, the composition comprises at least one supplemental vitamin. The at least one vitamin can be fat-soluble or water soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.

[0159] In some embodiments, the composition comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent. In some embodiments, the excipient comprises a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.

[0160] In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.

[0161] In some embodiments, the composition comprises a binder as an excipient. Nonlimiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.

[0162] In some embodiments, the composition comprises a lubricant as an excipient. Nonlimiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.

[0163] In some embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.

[0164] In some embodiments, the composition comprises a disintegrant as an excipient. In some embodiments the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth. In some embodiments the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.

[0165] The compositions of the present disclosure may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins or minerals. Accordingly, a composition of the present disclosure can be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0166] Cancer Therapies

[0167] The therapeutic agents of the present disclosure can be used alone or can be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radiolabelled compounds, or with surgery, cryotherapy, and / or radiotherapy. The preceding treatment methods can be administered in conjunction with other forms of conventional therapy (e.g., standard-of-care treatments for cancer well-known to the skilled artisan), either consecutively with, pre- or post-conventional therapy. For example, agents of the present disclsoure can be administered with a therapeutically effective dose of chemotherapeutic agent. In other embodiments, agents of the present disclosure are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physicians’ Desk Reference (PDR) discloses dosages of chemotherapeutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art, and can be determined by the physician.

[0168] Immunotherapy is a targeted therapy that may comprise, for example, the use of cancer vaccines and / or sensitized antigen presenting cells. For example, an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site. The immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). For example, anti-VEGF is known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and / or pathology of a tumor or cancer.

[0169] Immunotherapy also encompasses immune checkpoint modulators. Immune checkpoints are a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well-known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD 160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG- 3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, for example, WO 2012 / 177624). Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer. In some embodiments, the composition of the present disclosure is administered in combination with one or more inhibitors of immune checkpoints, such as PD1, PD-L1, and / or CD47 inhibitors.

[0170] Adoptive cell -based immunotherapies can be combined with the therapies of the present disclosure. Well-known adoptive cell-based immunotherapeutic modalities, including, without limitation, irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines, and / or antigen presenting cells. Such cellbased immunotherapies can be further modified to express one or more gene products to further modulate immune responses, such as expressing cytokines like GM-CSF, and / or to express tumor-associated antigen (TAA) antigens, such as Mage-1, gp-100, and the like.

[0171] In other embodiments, immunotherapy comprises non-cell-based immunotherapies. In some embodiments, compositions comprising antigens with or without vaccine-enhancing adjuvants are used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigen comprising fusion proteins, and the like. In some embodiments, immunomodulatory cytokines, such as interferons, G-CSF, imiquimod, TNFalpha, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory interleukins, such as IL-2, IL-6, IL-7, IL-12, IL-17, IL-23, and the like, as well as modulators thereof e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory chemokines, such as CCL3, CCL26, and CXCL7, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory molecules targeting immunosuppression, such as STAT3 signaling modulators, NFkappaB signaling modulators, and immune checkpoint modulators, are used. The terms “immune checkpoint” and “anti- immune checkpoint therapy” are described above.

[0172] In still other embodiments, immunomodulatory drugs, such as immunocytostatic drugs, glucocorticoids, cytostatics, immunophilins and modulators thereof e.g., rapamycin, a calcineurin inhibitor, tacrolimus, ciclosporin (cyclosporin), pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, a non-glucocorticoid steroid, a pyrimidine synthesis inhibitor, leflunomide, teriflunomide, a folic acid analog, methotrexate, anti-thymocyte globulin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, an opioid, an IMPDH inhibitor, mycophenolic acid, myriocin, fingolimod, an NF- xB inhibitor, raloxifene, drotrecogin alfa, denosumab, an NF-xB signaling cascade inhibitor, disulfiram, olmesartan, dithiocarbamate, a proteasome inhibitor, bortezomib, MG132, Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, non-steroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxyquinomycin (DHMEQ), I3C(indole-3-carbinol) / DIM(di- indolmethane) (13C / DIM), Bay 11-7082, luteolin, cell permeable peptide SN-50, IKBa.- super repressor overexpression, NFKB decoy oligodeoxynucleotide (ODN), or a derivative or analog of any thereo, are used. In yet other embodiments, immunomodulatory antibodies or protein are used. For example, antibodies that bind to CD40, Toll-like receptor (TLR), 0X40, GITR, CD27, or to 4-1BB, T-cell bispecific antibodies, an anti-IL-2 receptor antibody, an anti-CD3 antibody, 0KT3 (muromonab), otelixizumab, teplizumab, visilizumab, an anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, an anti-CDll a antibody, efalizumab, an anti-CD18 antibody, erlizumab, rovelizumab, an anti-CD20 antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, an anti-CD23 antibody, lumiliximab, an anti-CD40 antibody, teneliximab, toralizumab, an anti-CD40L antibody, ruplizumab, an anti-CD62L antibody, aselizumab, an anti-CD80 antibody, galiximab, an anti- CD147 antibody, gavilimomab, a B-Lymphocyte stimulator (BLyS) inhibiting antibody, belimumab, an CTLA4-Ig fusion protein, abatacept, belatacept, an anti-CTLA4 antibody, ipilimumab, tremelimumab, an anti-eotaxin 1 antibody, bertilimumab, an anti-a4-integrin antibody, natalizumab, an anti-IL-6R antibody, tocilizumab, an anti-LFA-1 antibody, odulimomab, an anti-CD25 antibody, basiliximab, daclizumab, inolimomab, an anti-CD5 antibody, zolimomab, an anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atorolimumab, cedelizumab, dorlimomab aritox, dorlixizumab, fontolizumab, gantenerumab, gomiliximab, lebrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, aflibercept, alefacept, rilonacept, an IL-1 receptor antagonist, anakinra, an anti-IL-5 antibody, mepolizumab, an IgE inhibitor, omalizumab, talizumab, an IL 12 inhibitor, an IL23 inhibitor, ustekinumab, and the like.

[0173] Nutritional supplements that enhance immune responses, such as vitamin A, vitamin E, vitamin C, and the like, are well-known in the art (see, for example, U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publ. No. WO 2004 / 004483) can be used in the methods described herein.

[0174] Similarly, agents and therapies other than immunotherapy or in combination thereof can be used with in combination with the composition of present disclosure to treat a condition that would benefit therefrom. For example, chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HD AC) modifiers, methylation modifiers, phosphorylation modifiers, and the like), targeted therapy, and the like are well-known in the art.

[0175] In some embodiments, chemotherapy is used. Chemotherapy includes the administration of a chemotherapeutic agent. Such a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5 -fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2’ -deoxy-5 -fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, sorafenib, doxorubicin, and rhizoxin. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, PARP e.g., PARP-1 and / or PARP-2) inhibitors are used and such inhibitors are well-known in the art e.g., Olaparib, ABT-888, BSI-201, BGP- 15 (N-Gene Research Laboratories, Inc.); INO-lOOl (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide (Trevigen); 4-amino-l,8- naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); and NU1025 (Bowman et al.). The mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity. PARP catalyzes the conversion of .beta.-nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly- ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J. et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 Jun. 2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA single-strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame J C, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang Z Q, et al. (1997) Genes Dev 11 :2347-2358). Knockout of SSB repair by inhibition of PARP1 function induces DNA double-strand breaks (DSBs) that can bigger synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant H E, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The foregoing examples of chemotherapeutic agents are illustrative, and are not intended to be limiting.

[0176] In other embodiments, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J. B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy- hypocrellin A; and 2BA-2-DMHA.

[0177] In other embodiments, hormone therapy is used. Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0178] In other embodiments, photodynamic therapy (also called PDT, photoradiation therapy, phototherapy, or photochemotherapy) is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light.

[0179] In yet other embodiments, laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors.

[0180] The immunotherapy and / or cancer therapy may be administered before, after, or concurrently with the compositions described herein. The duration and / or dose of treatment with the compositions may vary according to the particular composition, or the particular combinatory therapy. An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan. The instant disclosure contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by the methods of the disclosure is a factor in determining optimal treatment doses and schedules.

[0181] Clinical Efficacy

[0182] Clinical efficacy can be measured by any method known in the art. For example, the response to a therapy (e.g., a composition of the present disclosure, e.g, a supercharged NK cells), relates to e.g., any response of the cancer, e.g., a tumor, to the therapy, preferably to a change in tumor mass and / or volume after initiation of neoadjuvant or adjuvant chemotherapy. Tumor response may be assessed in a neoadjuvant or adjuvant situation where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation and the cellularity of a tumor can be estimated histologically and compared to the cellularity of a tumor biopsy taken before initiation of treatment. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or cellularity or using a semi-quantitative scoring system such as residual cancer burden (Symmans et al. (2007) J. Clin. Oncol. 25:4414-4422) or Miller-Payne score (Ogston et al. (2003) Breast (Edinburgh, Scotland) 12:320-327) in a qualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria. Assessment of tumor response may be performed early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferably after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and / or the tumor bed.

[0183] In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR for a particular regimen is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or more.

[0184] Additional criteria for evaluating the response to a therapy e.g., a composition of the present disclosure) are related to “survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0185] For example, in order to determine appropriate threshold values, a particular agent encompassed by the present disclosure can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of a therapy e.g., a composition of the present disclosure). The outcome measurement may be pathologic response to therapy given in the neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival can be monitored over a period of time for subjects following a therapy (e.g., a composition of the present disclosure). In certain embodiments, the same doses of the agent are administered to each subject. In related embodiments, the doses administered are standard doses known in the art for the agent. The period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 months.

[0186] Cancer

[0187] As described herein, the methods and compositions provided herein can be used for preventing or treating cancer.

[0188] Cancer, tumor, or hyperproliferative disease refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. In some embodiments, cancer cells are highly differentiated. In other embodiments, cancer cells are poorly differentiated. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to, B cell cancer, (e.g., multiple myeloma, Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphomas, Burkitt lymphoma, Waldenstrom’s macroglobulinemia, Hairy cell leukemia, Primary central nervous system (CNS) lymphoma, Primary intraocular lymphoma, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis), T cell cancer (e.g., T-lymphoblastic lymphoma / leukemia, non-Hodgkin lymphomas, Peripheral T-cell lymphomas, Cutaneous T-cell lymphomas (e.g., mycosis fungoides, Sezary syndrome), Adult T-cell leukemia / lymphoma, Angioimmunoblastic T-cell lymphoma, Extranodal natural killer / T-cell lymphoma, Enteropathy-associated intestinal T- cell lymphoma (EATL), Anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma), melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, hepatocellular carcinoma (HCC), bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma (SCLC), bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin’s disease and non-Hodgkin’s disease), multiple myeloma, Waldenstrom’s macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0189] Exemplary Embodiments

[0190] 1. A composition comprising processed osteoclasts, optionally (a) wherein the processed osteoclasts comprise an osteoclast that is non-viable, fragmented, sonicated, ground, heat- inactivated, dried, lyophilized, and / or frozen; (b) wherein the composition comprises at least or about 70% of fragmented osteoclasts; and / or (c) wherein the processed osteoclasts are anchored to a solid support (e.g., beads).

[0191] 2. The composition of embodiment 1, further comprising

[0192] (a) an NK cell, optionally wherein the NK cell has been contacted with IL-2 and / or an anti-CD16 antibody; or

[0193] (b) an NK cell in combination with IL-2 and / or an anti-CD16 antibody, optionally wherein the NK cell is autologous or allogeneic to the processed osteoclasts.

[0194] 3. The composition of embodiment 1 or 2, further comprising at least one bacterial strain selected from: Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is either alive or sonicated.

[0195] 4. The composition of embodiment 3, wherien the at least one bacterial strain comprises

[0196] (a) Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei',

[0197] (b) AJ2 bacteria; or

[0198] (c) AJ4 bacteria.

[0199] 5. The composition of any one of embodiments 1-4, further comprising at least one cytokine or chemokine that activates and / or supports the survival of the NK cell. 6. The method of embodiment 6, wherein the at least one cytokine is selected from interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, type I interferons (e.g., IFN-a), and any combination of two or more thereof, optionally wherein the at least one cytokine comprises IL-12, IL-15, IL-18, and IFN-a.

[0200] 7. The composition of any one of embodiments 1-6, wherein the composition is a pharmaceutical composition, which optionally comprises a pharmaceutically acceptable excipient.

[0201] 8. A method of activating and / or expanding an NK cell in vitro or ex vivo, the method comprising contacting the NK cell with the composition of any one of embodiments 1-7.

[0202] 9. A method of activating and / or expanding an NK cell in vitro or ex vivo, the method comprising:

[0203] (a) contacting the NK cell with IL-2 and / or an anti-CD16 antibody; and

[0204] (b) contacting the NK cell of (a) with the composition comprising the processed osteoclasts of embodiment 1 , optionally wherein the NK cell is autologous or allogeneic to the processed osteoclasts.

[0205] 10. The method of embodiment 9, further comprising contacting the NK cell with a composition comprising at least one bacterial strain selected from: Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is either alive or sonicated.

[0206] 11. The method of embodiment 10, wherein the at least one bacterial strain comprises

[0207] (a) Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei',

[0208] (b) AJ2 bacteria; or

[0209] (c) AJ4 bacteria.

[0210] 12. The method of any one of embodiments 9-11, further comprising contacting the NK cell with at least one cytokine or chemokine that activates the NK cell.

[0211] 13. The method of embodiment 12, wherein the at least one cytokine is selected from interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, and type I interferons (e.g., IFN-a), optionally wherein the at least one cytokine comprises IL-12, IL-15, IL-18, and IFN-a. 14. A method of activating and / or expanding an NK cell in a subject, the method comprising administering to the subject the composition of any one of embodiments 1-7.

[0212] 15. A method of treating a disease in a subject in need thereof, comprising administering to the subject (a) the composition of any one of embodiments 1-7, and / or (b) the NK cell activated and / or expanded according to the method of any one of embodiments 8-13.

[0213] 16. The method of embodiment 15, wherein the disease is a cancer or an infection (e.g., viral or bacterial infection).

[0214] 17. A method of killing a cancer cell in a subject, comprising administering to the subject

[0215] (a) the composition of any one of embodiments 1-7, and / or (b) the NK cell activated and / or expanded according to the method of any one of embodiments 8-13.

[0216] 18. The method of any one of embodiments 14-17, further comprising administering to the subject a composition comprising at least one bacterial strain selected from: Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is administered orally.

[0217] 19. The method of embodiment 18, wherein the at least one bacterial strain comprises

[0218] (a) Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei',

[0219] (b) AJ2 bacteria; or

[0220] (c) AJ4 bacteria.

[0221] 20. The method of any one of embodiments 14-19, further comprising administering to the subject at least one cytokine or chemokine that activates and / or supports the survival of the NK cell.

[0222] 21. The method of embodiment 20, wherein the at least one cytokine is selected from interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, type I interferons (e.g., IFN-a), and any combination of two or more thereof, optionally wherein the at least one cytokine comprises IL-12, IL-15, IL-18, and IFN-a.

[0223] 22. The method of any one of embodiments 16-21, further comprising conjointly treating the subject with at least one cancer therapy, optionally wherein the subject is treated with the at least one cancer therapy before, after, or concurrently with (a) the composition of any one of embodiments 1-7, and / or (b) the NK cell prepared according to the method of any one of embodiments 8-13.

[0224] 23. The method of embodiment 22, wherein the at least one cancer therapy is selected from a surgery, radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

[0225] 24. The method of embodiment 23, wherein the at least one cancer therapy is chemotherapy.

[0226] 25. The method of embodiment 23, wherein the at least one cancer therapy is immunotherapy.

[0227] 26. The method of embodiment 23 or 25, wherein the immunotherapy inhibits an immune checkpoint.

[0228] 27. The method of embodiment 26, wherein the immune checkpoint is selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.

[0229] 28. The method of any one of embodiments 16-21, wherein the cancer is an oral cancer, an adenocarcinoma, a uterine cancer, or an endometrial adenocarcinoma.

[0230] 29. The method of any one of embodiments 14-28, wherein the NK cell is autologous or allogeneic to the subject, preferably allogeneic to the subject.

[0231] 30. The method of any one of embodiments 14-28, wherein the subject is a mammal, preferably a human.

[0232] EXAMPLES

[0233] Example 1: Materials and Methods

[0234] Cell lines, reagents, and antibodies

[0235] Oral squamous carcinoma stem cells (OSCSCs) were isolated from patients with tongue tumors at UCLA39-42, and were grown in RPMI 1640 medium (Gibco, ThermoFisher, CA) with 10% FBS (Gemini Bio-Products, CA, USA), 2% Antibiotic / Antimycotic Solution (Cytiva, MA), 1.4% Sodium Pyruvate (Gibco, CA, USA), 1.4% MEM Non-Essential Amino Acids (Gibco, CA, USA) and 0.15% sodium bicarbonate. Recombinant IL-2 was obtained from NIH-BRB. Antibodies used in flow cytometry - IgG2, CD45, CD16, CD56, CD3, and CD14 were purchased from Biolegend (San Diego, CA, USA). Human NK cells and monocyte purification kits were obtained from Stem Cell Technologies (Vancouver, BC, Canada).

[0236] Bacteria sonication

[0237] Gram-positive probiotic bacteria (e.g., AJ2 or AJ4) were thoroughly vortexed, then processed using a sonicator with the amptitue of 60% pulse on ice for 15 seconds with 30 second interval for approximately 20 rounds until 80% of the bacteria were lysed microscopely. Finally, the processed AJ2 or AJ4 were weighed and resuspended in RPMI 1640 containing 10% FBS at a concentration of 10 mg / lmL and stored in a -80°C freezer. Sonication of bacteria is not required or necessary to render its activities presented herein. Purification of NK cells and monocytes from the peripheral blood

[0238] Written informed consents, approved by the UCLA Institutional Review Board (IRB#11-000781), were obtained from healthy individuals, and all procedures were approved by the UCLA-IRB. Peripheral blood was separated using Ficoll-hypaque centrifugation, after which the white, cloudy layer, containing peripheral blood mononuclear cells (PBMCs) was harvested. NK cells and monocytes were negatively selected from PBMCs using the EasySep® Human NK cell enrichment and EasySep® Human Monocytes enrichment kits, respectively. To assess the purity of NK cells and monocytes, were stained them with anti- CD45 and anti-CD3 / CD16 / CD56 antibodies for NK cells, and anti-CD45 and anti-CD14 antibodies for monocytes. Then, flow cytometric analysis was performed to quantify the percentage of positive cells in each population. Samples showing greater than 95% purity were used for the study.

[0239] Generation of osteoclasts

[0240] To generate osteoclasts (OCs), monocytes were cultured in alpha-MEM media supplemented with M-CSF (25 ng / mL) for 21 days and RANKL (25 ng / mL) from day 6 to 21 days. The media were replenished every three days.

[0241] Live osteoclasts were counted and suspended in alpha-MEM medium supplemented with 10% FBS, reaching a final concentration of 106cells / ml. Osteoclasts were then processed by sonication and the freeze-thaw methods. The sample was then sonicated for 20 seconds with a 30-second interval for 15 rounds to ensure complete disruption of osteoclasts. After processing osteoclasts, a sample was taken and examined under a microscope until at least 80% of the cell walls were disrupted.

[0242] Generation of supercharged NK cells

[0243] Purified NK cells were activated with rh-IL-2 (5000 U / ml) and anti-CD16 mAh (3 pg / ml) for 18-20 hours before they were co-cultured with OCs or processed OCs (pOCs) and AJ2 (0Cs:NK:AJ2; 1:2:4) in RPMI 1640 medium containing 10% human serum AB (Gemini Bio-Products, CA, USA). The media were refreshed every three days with RPMI complete medium containing rh-IL-2 (5000 U / ml). The supercharged NK cells were used on day 15 for cytotoxicity assay. eSight xCELLigence RTCA eSight (Agilient, USA) was purchased and cell behavior and cell function were studied using real-time biosensor impedance-based and image-based measurements. The impedance-based xCELLigence technology utilizes proprietary microplates (E-Plates View 96) embedded with gold biosensors at the bottom of each well, which serve to non-invasively quantify cell behavior. Over the course of an experiment, the biosensors monitor cell metrics such as proliferation, adhesion strength, changes in morphology, migration, and differentiation. On day 1, 50 pl of the respective media was added to each well, and the machine was run once to measure the background. Subsequently, IxlO4(OSCSCs) target cells were seeded per well and the machine was run overnight for adhesion. The impedance of each well was monitored every 15 minutes, and the images of the cells were acquired every hour. After incubating for 18-24 hours, different concentrations of effector cells were added to each well, with a twofold dilution for each target cell type. To perform the serial dilution, we used different E:T ratios. Impedance readings were recorded at 15-minute intervals, and images were at 24 and 48 hours.

[0244] Surface staining analysis

[0245] Staining was performed by labeling the cells with antibodies as described previously43-45. Flow cytometric analysis was performed using Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA), and FlowJo vl0.4 (BD, Oregon, USA) were used for analysis. Beckman Coulter Epics XL cytometer (Brea, CA), and results were analyzed in the FlowJo vX software (Ashland, OR).

[0246] Enzyme-Linked Immunosorbent Assays (ELISAs)

[0247] Single ELISAs were performed as previously described45. To analyze and obtain the cytokine and chemokine concentration, a standard curve was generated by either two- or three-fold dilutions of recombinant cytokines provided by the manufacturer.

[0248] 51Cr release cytotoxicity assay

[0249] The51Cr release cytotoxicity assay was performed as previously described46. Briefly, different ratios of PBMCs or NK cells and51Cr-labeled OSCSCs or patient-derived ovarian cancers were incubated for four hours. After this, the supernatants were harvested from each sample, and the released radioactivity was counted using the gamma counter. The percentagespecific cytotoxicity was calculated as follows:

[0250] LU 30 / 106is calculated by using the inverse of the number of PBMCs or NK cells needed to lyse 30% of tumors xlOO.

[0251] Statistical analyses

[0252] An unpaired or paired, two-tailed Student’s t-test was performed for experiments with two groups. Duplicate or triplicate samples were used for assessment. The following symbols represent the levels of statistical significance within each analysis: ****(p-value<0.0001), ***(p-value <0.001), **(p-value 0.001-0.01), *(p-value 0.01-0.05).

[0253] Example 2: Processed osteoclasts are capable of expanding NK cells

[0254] Sonicated osteoclasts can expand NK cells

[0255] 0.35xl06sorted primary NK cells treated with IL-2 (1000 U / ml) and anti-CD16 mAh (3 pg / ml) overnight were cultured with osteoclasts (OCs) and AJ2 probiotic bacteria (2:1:4 NK:OC:AJ2) as described previously (1-3), and used as the standard expansion technique. The same numbers of OCs as described above were sonicated (sOCs) until only cell fragments were visible and added to 0.35xl06sorted primary NK cells treated as described above in the presence of AJ2 (2:1:4 NK:OC:AJ2). To the cultures of sonicated OCs with NK cells supernatants from the cultured osteoclasts were added since osteoclasts provide all the necessary cytokines such as IL-12, IL-15, IL-18 and IFN-a to aid in the expansion of the NK cells. Every 3 days after culture, 0.3xl06expanding NK cells were removed and re-cultured for the next 3 days until day 23. When compared to the standard NK expansion by live OCs, sOCs+sup had approximately 50% lower in the fold expansion rate (Fig. 1A) and lower ability to expand NK cells (Fig. IB). At the initial 12-15 days sOC had about 25%-67% lower capability (donors 1-3) to expand NK cells when compared to those expanded by live OCs, however, the expansion was usually terminated in the samples which received sOC at around 13-15 days, whereas those cultured with live OCs continued expansion and resulted in the greater expansion of NK cells (Fig. IB).

[0256] The percentages ofNK cells and the small populations of CD8+ T cells contaminating the purified NK cells decline starting from day 18 of expansion with the Sonicated osteoclast treatment when compared to those cultured with viable osteoclasts Treatments were carried out as described in the description of Figure 1A and IB. Briefly, 0.35xl06sorted primary NK cells treated with IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) overnight were cultured with osteoclasts (OCs) and AJ2 probiotic bacteria (2:1:4 NK:OC:AJ2) as described above and used as the standard expansion technique. The same numbers of OCs as described above were sonicated (sOCs (sonicated osteoclasts, exemplary processed osteoclasts)) until only cell fragments were visible and added to 0.35xl06sorted primary NK cells treated as described above in the presence of AJ2 (2:1:4 NK:OC:AJ2). To the cultures of sonicated OCs with NK cells supernatants from the cultured osteoclasts were added since osteoclasts provide all the necessary cytokines such as IL-12, IL-15, IL-18 and IFN-a to aid in the expansion of the NK cells. The purity of NK cells were high at the start of the culture and after culturing with OCs or sOCs declined on day 7 but gradually started rising from day 7 onwards as the NK cells expanded and increased their function (please see below). Although the purity remains high in NK cultures that had been activated with viable OCs until day 23, the purity of those cultured with sOCs+sup started declining from day 18 cultures (Fig. 2B). Super-charged NK cells cultured with OCs preferentially can expand CD8+ T cells by targeting the CD4+ T cells. Therefore, the percentages of CD8+ T cells contaminating the cultures of NK cells were determined (Fig. 2B). When compared to the standard NK expansion by viable OCs, sOCs+sup had high levels of CD8+ T cells at the start of the culture but decreased the levels of CD8+ T cells from day 18 onwards (Fig. 2B). NK cells cultured with the viable OCs maintained high levels of NK purity and the purity of CD8+ T cells contaminating the NK cultures remained high throughout the expansion period (Fig. 2A-2B).

[0257] Sonicated osteoclasts have similar capability to induce IFN-y secretion by the expanded NK cells at the early expansion period, and the levels decrease thereafter when compared to those induced by the viable osteoclasts

[0258] 0.35xl06sorted primary NK cells treated with IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) overnight were cultured with osteoclasts (OCs) and AJ2 probiotic bacteria (2:1:4 NK:OC:AJ2) as described above, and used as the standard expansion technique. The same numbers of OCs as described above were sonicated (sOCs) until only cell fragments were visible and added to 0.35xl06sorted primary NK cells treated as described above in the presence of AJ2 (2:1:4 NK:OC:AJ2). To the cultures of sonicated OCs with NK cells supernatants from the cultured osteoclasts were added since osteoclasts provide all the necessary cytokines such as IL-12, IL-15, IL-18 and IFN-a to aid in the expansion of the NK cells. Every 3 days after culture, 0.3xl06expanding NK cells were removed and cultured for the next 3 days until day 23. At the end of three days the supernatants were removed and the levels of IFN-y were determined using ELISA (Fig. 3A). The total amount of IFN-y within the expansion period were determined by adding the amounts obtained from different time points (Fig. 3B). When compared to the standard NK expansion by live OCs, sOCs+sup had similar capability to induce IFN-y at day 7 of expansion, however, after day 7 the levels of secreted IFN-y were less in the sOC expanded NK cells when compared to those obtained in the presence of live OCs (Fig. 3A). Overall sOC had lower ability to induce IFN-y within the 23 days of expansion when compared to those obtained in the presence of viable OCs (Fig. 3A and 3B).

[0259] Sonicated osteoclasts have similar capability to increase cytotoxicity by the expanded NK cells at the days 10 and 13 expansion period

[0260] 0.35xl06sorted primary NK cells treated with IL-2 (1000 U / ml) and anti-CD16mAb (3 pg / ml) overnight were cultured with osteoclasts (OCs) and AJ2 probiotic bacteria (2:1:4 NK:OC:AJ2) as described above, and used as the standard expansion technique. The same numbers of OCs as described above were sonicated (sOCs) until only cell fragments were visible and added to 0.35xl06sorted primary NK cells treated as described above in the presence of AJ2 (2:1:4 NK:OC:AJ2). To the cultures of sonicated OCs with NK cells supernatants from the cultured osteoclasts were added since osteoclasts provide all the necessary cytokines such as IL-12, IL-15, IL-18 and IFN-a to aid in the expansion of the NK cells. Every 3 days after culture, 0.3xl06expanding NK cells were removed and cultured for the next 3 days until day 13. On days 10 and 13 (Fig. 4A donor 1 Day 10 and Day 13, Fig. 4B donor 2 Day 10 and Day 13) NK cells were counted and their cytotoxicity was determined using standard 4 hour51Cr release assay. As shown in Figure 4A-4C, NK cells expanded by sOC had similar levels of cytotoxicity on both days to those cultured by viable osteoclasts. In addition, the cytotoxicity of NK cells increased from day 10 to 13 in both donors with either viable OCs or those cultured with sOCs (Fig. 4A and 4B). Cytotoxicity were also compared with adding supernatants from osteoclasts in the absence of sOC. In comparison to viable OCs and sOC+sup, addition of only supernatants to NK cells mediated lower cytotoxicity when compared to those cultured with either viable OCs or sOCs+sup. Example 3: Processed OCs induced similar or slightly higher levels of cell expansion in NK cells compared to live OCs

[0261] Here, the NK cell expansion efficiency was compared between live and processed OC (pOC) for IL-2+anti-CD16mAbs treated NK in the presence of probiotic bacteria AJ2 (Fig. 8). Both live OC and pOC were capable of inducing significant levels of cell expansion in NK cells, however, pOCs in comparison to live OCsr induced slightly higher cell expansion on day 14 (Fig. 8).

[0262] Example 4: pOC-generated sNK cells exhibited lower lysis of OSCSC tumors in comparison to live OC-generated sNK cells

[0263] Short-term (4-hour51chromium killing assay), and long-term (killing assay using esight) killing assays were conducted to compare the cytotoxic potential of pOC-generated sNK cells (pOC-sNK cells) and live OC-generated sNK cells (OC-sNK cells). In the shortterm killing assay, OC-sNK cells induced higher cytotoxicity against oral squamous cancer stem-like cells (OSCSCs) (Fig. 9A). For the long-term killing assay, OSCSCs were cultured on eSight plates for 20-24 hours before pOC-sNK cells or OC-sNK cells were added to the tumors and co-cultures were continued for 48-90 hours (Figs. 9B-9C). Microscopic images were captured by eSight until 48 hours of incubation. As seen in the microscopic analysis, OC-sNK cells lysed greater number of tumors compared to pOC-sNK cells (Fig. 9B). Also, OC-sNK cells compared to pOC-sNK cells, had the lowest cell index with the highest percentages of cytolysis of OSCSCs (Fig. 9C). As shown in Fig. 8, there was a clear correlation between the data obtained in two different killing assays, both showing that OC- sNK cells slightly lysed tumors compared to pOC-sNK cells at higher E:T ratio (Effector:Target ratio) of 2.5:1. At lower E:T ratio of 0.625:1 OC-sNKs had higher killing than pOC-sNK cells.

[0264] Example 5: Lower levels of IFN-y secretion in pOC-sNK cells than those of live OC-sNK cells

[0265] The levels of IFN-y secretion levels in both OC-sNK cells or pOC-sNK cells were determined using ELISA. pOC-sNK cells secreted significantly lower amounts of IFN-y in comparison to OC-sNK cells (Fig. 10).

[0266] Example 6: NK cell expansion by pOC Cell expansion rates were compared between OC-sNK cells and pOC-sNK cells. Similar numbers of NK cells were co-cultured with OC-sNK cells or pOC-sNK cells. As shown in Fig. 8, very similar or slightly higher cell expansion using pOC vs live OC was observed. Here, pOC-sNK cells co-cultures were treated with the supernatants harvested from live OC in order to supply OC-secreted factors to pOC expanded NK cells. Even though, the supernatants from live OC were added, cell expansion was still significantly low in pOC-sNK in comparison to OC-sNK cells (Fig. 11 A-l IB). Also, the percentages of NK cells in the expanded lymphocytes were lower in pOC-sNK compared to OC-sNK cells post day 18 of cultures (Fig. 11C). When the number of NK cells in the co-culture was determined, they were found to be lower in pOC-sNK as compared to OC-sNK cells (Figs. 1 ID-1 IE).

[0267] Example 7: Cytotoxic function and secretion levels of IFN-y remained slightly lower in pOC-sNK compared to OC-sNK cells even after adding the supernatants harvested from live OC to pOC-sNK culture

[0268] As shown in Figs 8 and 9A-9B, pOC-sNK cells mediated lower cytotoxicity against tumors and secreted lower levels of IFN-y. Next, pOC-sNK cells co-cultures were treated with the supernatants harvested from live OC, and the cells were cultured for 13 days before they were used as effectors against OSCSCs in chromium-51 killing assay. pOC-sNK cells (treated with the supernatant of live OCs) induced lower cytotoxicity against tumors as compared to OC-sNK cells (Fig. 12A). pOC-sNK cells co-cultures were treated with the supernatants harvested from live OC, and the levels of IFN-y secretion in both OC-sNK cells or pOC-sNK cells were determined using EEISA on different days of culture. pOC-sNK cells (treated with the supernatant of live OCs) secreted lower levels of IFN-y compared to OC- sNK cells (Fig. 12B).

[0269] Example 8: Selection of CD8+ T cells in both OC-sNK and pOC-sNK cells

[0270] Here, NK cells were expanded using live and pOCs with NK cells harvested from three different individuals and observed that in both OC-sNK and pOC+OC sup-sNK cells, the majority of CD3+ T cells were CD8+ T cells (Figs. 13A-13B). To mimic the possible expansion scenario in vivo, a mixture of autologous NK and T cells (NK:CD8+T cells: CD4+ T cells, 7:1:2) was used for the NK expansion. Similar to purified NK expansion culture, even with extra T cells in the OC co-cultures, CD8+ T cells remained to be the major T cell population in both OC-sNK and pOC+OC sup-sNK cells (Figs. 13C-13D). Osteoclasts stimulate the expansion and functional activation of NK cells through the secretion of cytokines necessary for NK cell activation such as IL-12, IL-15, IL-18 and IFN- a, and provide activating ligands to increase the functional activation of NK cells2. However, these studies were performed using live, PBMC-derived monocyte-differentiated osteoclasts. Here, it is demonstrated that the fragmented osteoclasts are surprisingly very good activators of the NK cells in comparison to live osteoclasts. To understand how to condition processed (e.g., fragmented) osteoclasts to supercharge NK cells similarly to live osteoclasts, a series of studies were performed to compare the function of live osteoclasts to processed osteoclasts. The same numbers of osteoclasts were used to activate NK cells from either live or processed osteoclasts. The rationale for performing these experiments is to simplify the supercharging process of NK cells using processed osteoclasts in comparison to live osteoclasts which is labor intensive and time-consuming. For example, live osteoclasts require several weeks of differentiation with RankL and MCS-F before their use with NK cells, which makes it more challenging for NK cell expansion regimen. More importantly, it requires constant blood donations from healthy individuals for supplying fresh PBMC-derived monocytes. With the use of processed osteoclasts, it can be used as a supplement to the culture by batch preparation and could benefit the sNK expansion for convenience and effectiveness, especially in a large-scale manufacturing process.

[0271] Though pOC exhibited slightly higher ability to expand NK cells than live OCs, live OC-expanded sNK cells were slighlty more capable of lysing OSCSC in both short- and long-term killing assays, using51Cr release assay and eSight respectively (Fig. 9A-9B). At higher effector to target ratios there were comparable tumor-killing ability between two methods, whereas at lower E:T ratios, live OC-expanded sNK cells induced slightly better killing than pOC-sNK cells in long-term killing assay (Fig. 9C). In addition, live OC expanded sNK cells also secreted slightly higher levels of IFN-y secretion when compared to pOCs. Overall, these studies indicated that while live OCs may be slightly better in expanding NK cells than pOCs, pOCs still had a good expansion and functional activation of NK cells (thereby generation of supercharged NK cells). In addition, the pOC-expanded NK cells expand the same levels of CD8+ T cells as by live OC-expanded NK cells (Fig. 13A- 13D). Therefore, pOCs are still great feeders to expand sNK cells, even though they have slightly lower capability to expand functional sNK cells than live OCs. In this case the numbers of pOCs in expansion of NK cells can be increased to achieve the same level of expansion as live OCs. pOCs on solid support may also be used to enhance the expansion of NK cells.

[0272] To understand how to enhance the function of processed osteoclasts to obtain the same levels of expansion and functional activation of NK cells per osteoclasts used, the pOCs were cultured with supernatants from the live osteoclasts in order to provide the cytokines that would not have been secreted by the pOCs in cultures with the NK cells. However, similar trends were observed as indicated above. Therefore, the supernatants from the live OCs did not change the trend seen with processed OC-expanded NK cells when compared to live OCs.

[0273] Processed OCs have many advantages over the live OCs in the expansion of NK cell to generate sNK cells. As indicated above, it shortens the preparation time for generating sNK cells by several weeks for clinical use. In addition, large batch preparations of pOC for sNK culture is beneficial for sNK quality control as live OCs from different healthy individuals might show variations in expanding sNK cells. Additionally, there is a concern of having contaminating live OCs when using them for generating sNK cells. In such a case, the sNK infusion for patient treatment may lead to infusion of the contaminating live OCs. With pOC expansion approach, one can make sure that there are no longer any live cells other than sNK in the infusion for cell therapy. Lastly, using pOC as a primary approach for sNK expansion can be cost-effective and will provide a speedy sNK generation turnaround time as a cancer cell therapeutic option to a broader patient population.

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[0294] 21 Ko, M.-W. et al. in NK Cells in Cancer Immunotherapy: Successes and Challenges (eds Anahid Jewett & Yuman Fong) 281-297 (Academic Press, 2023).

[0295] 22 Kaur, K., Ko, M.-W., Chen, F. & Jewett, A. Defective NK cell expansion, cytotoxicity, and lack of ability to differentiate tumors from a pancreatic cancer patient in a long term follow-up: implication in the progression of cancer. Cancer Immunology, Immunotherapy 71, 1033-1047, doi:10.1007 / s00262-021-03044-w (2022).

[0296] 23 Kaur, K., Safaie, T., Ko, M.-W., Wang, Y. & Jewett, A. ADCC against MICA / B Is Mediated against Differentiated Oral and Pancreatic and Not Stem-Like / Poorly Differentiated Tumors by the NK Cells; Loss in Cancer Patients due to Down-Modulation of CD 16 Receptor. Cancers 13, 239 (2021).

[0297] 24 Jewett, A. et al. in Successes and Challenges ofNK Immunotherapy (eds Benjamin Bonavida & Anahid Jewett) 101-119 (Academic Press, 2021).

[0298] 25 Kaur, K. et al. Probiotics in Health and Disease: Distinct Roles of Different Strains in

[0299] Natural Killer Cell Activation and Regulation. 41, 1-19, doi: 10.1615 / CritRevImmunol.2021037163 (2021 ). 26 Kaur, K., Ko, M.-W., Ohanian, N., Cook, J. & Jewett, A. Osteoclast-expanded supercharged NK-cells preferentially select and expand CD8+ T cells. Scientific Reports 10, 20363, doi:10.1038 / s41598-020-76702-l (2020).

[0300] 27 Jewett, A. et al. Natural Killer Cells: Diverse Functions in Tumor Immunity and Defects in Pre-neoplastic and Neoplastic Stages of Tumorigenesis. Mol Ther Oncolytics 16, 41-52, doi:10.1016 / j.omto.2019.11.002 (2020).

[0301] 28 Jewett, A. et al. Multiple Defects of Natural Killer Cells in Cancer Patients: Anarchy, Dysregulated Systemic Immunity, and Immunosuppression in Metastatic Cancer. 40, 93-133, doi: 10.1615 / CritRevImmunol.2020033391 (2020).

[0302] 29 Kaur, K. et al. Probiotic-Treated Super-Charged NK Cells Efficiently Clear Poorly Differentiated Pancreatic Tumors in Hu-BLT Mice. Cancers 12, 63 (2020).

[0303] 30 Jewett, A. et al. NK cells shape pancreatic and oral tumor microenvironments; role in inhibition of tumor growth and metastasis. Seminars in Cancer Biology 53, 178-188, doi.org / 10.1016 / j.semcancer.2018.08.001 (2018).

[0304] 31 Kaur, K. et al. Super-charged NK cells inhibit growth and progression of stem- like / poorly differentiated oral tumors in vivo in humanized BLT mice; effect on tumor differentiation and response to chemotherapeutic drugs. Oncolmmunology 7, M426518, doi: 10.1080 / 2162402X.2018.1426518 (2018).

[0305] 32 Kaur, K. et al. Natural killer cells target and differentiate cancer stem-like cells / undifferentiated tumors: strategies to optimize their growth and expansion for effective cancer immunotherapy. Current Opinion in Immunology 51, 170-180, doi.org / 10.1016 / j.coi.2018.03.022 (2018).

[0306] 33 Kaur, K., Chang, H.-H., Cook, J., Eibl, G. & Jewett, A. Suppression of Gingival NK Cells in Precancerous and Cancerous Stages of Pancreatic Cancer in KC and BLT- Humanized Mice. Frontiers in Immunology 8, doi: 10.3389 / fimmu.2017.01606 (2017).

[0307] 34 Kozlowska, A. K., Kaur, K., Topchyan, P. & Jewett, A. Novel strategies to target cancer stem cells by NK cells; studies in humanized mice. Front Biosci (Landmark Ed) 22, 370-384, doi: 10.2741 / 4489 (2017).

[0308] 35 Kozlowska, A. K., Kaur, K., Topchyan, P. & Jewett, A. Adoptive transfer of osteoclast-expanded natural killer cells for immunotherapy targeting cancer stem-like cells in humanized mice. Cancer Immunology, Immunotherapy 65, 835-845, doi:10.1007 / s00262- 016-1822-9 (2016). 36 Breznik, B., Novak, M., Majc, B., Habic, A. & Jewett, A. in NK Cells in Cancer Immunotherapy: Successes and Challenges (eds Anahid Jewett & Yuman Fong) 335-367 (Academic Press, 2023).

[0309] 37 Breznik, B. et al. P06.07.A Natural killer cells lyse glioblastoma stem cells and increase their sensitivity to chemotherapy. (Neuro Oncol. 2022 Sep 5;24(Suppl 2):ii39. doi: 10.1093 / neuonc / noacl74.131. eCollection 2022 Sep.).

[0310] 38 Breznik, B. et al. Infiltrating natural killer cells bind, lyse and increase chemotherapy efficacy in glioblastoma stem-like tumorospheres. Communications Biology 5, 436, doi: 10.1038 / s42003-022-03402-z (2022).

[0311] 39 Tseng, H. C. et al. Increased lysis of stem cells but not their differentiated cells by natural killer cells; de-differentiation or reprogramming activates NK cells. PLoS One 5, el 1590, doi:10.1371 / journal.pone.0011590 (2010).

[0312] 40 Tseng, H. C., Bui, V., Man, Y. G., Cacalano, N. & Jewett, A. Induction of Split Anergy Conditions Natural Killer Cells to Promote Differentiation of Stem Cells through Cell-Cell Contact and Secreted Factors. Front Immunol 5, 269, doi: 10.3389 / fimmu.2014.00269 (2014).

[0313] 41 Tseng, H. C. et al. Differential Targeting of Stem Cells and Differentiated Glioblastomas by NK Cells. Journal of Cancer 6, 866-876, doi: 10.7150 / jca.11527 (2015).

[0314] 42 Bui, V. T. et al. Augmented IFN-y and TNF-a Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL- 10. Frontiers in immunology 6, doi: 10.3389 / fimmu.2015.00576 (2015).

[0315] 43 Jewett, A., Cavalcanti, M. & Bonavida, B. Pivotal role of endogenous TNF-alpha in the induction of functional inactivation and apoptosis in NK cells. J Immunol 159, 4815-4822 (1997).

[0316] 44 Jewett, A. & Bonavida, B. Interferon-alpha activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-alpha secretion by immature human natural killer cells. J Clin Immunol 15, 35-44 (1995).

[0317] 45 Jewett, A. & Bonavida, B. Target-induced inactivation and cell death by apoptosis in a subset of human NK cells. J Immunol 156, 907-915 (1996).

[0318] 46 Jewett, A. et al. Cytokine dependent inverse regulation of CD54 (ICAM1) and major histocompatibility complex class I antigens by nuclear factor kappaB in HEp2 tumor cell line: effect on the function of natural killer cells. Hum Immunol 64, 505-520 (2003). 47 Bui, V. T. et al. Augmented IFN-gamma and TNF-alpha Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10. Front Immunol 6, 576, doi:10.3389 / fimmu.2015.00576 (2015).

[0319] 48 Kaur, K., Ko, M. W., Ohanian, N., Cook, J. & Jewett, A. Osteoclast-expanded supercharged NK-cells preferentially select and expand CD8+ T cells. Sci Rep 10, 20363, doi: 10.1038 / s41598-020-76702-l (2020).

[0320] 49 Knochelmann, H. M. et al. CAR T Cells in Solid Tumors: Blueprints for Building Effective Therapies. Front Immunol 9, 1740, doi:10.3389 / fimmu.2018.01740 (2018).

[0321] Example 9: Investigation of efficacy of supercharged NK cells against uterine cancer cells in vitro and in vivo

[0322] The aims of this study were 1) to investigate and compare the lysing capacity of supercharged NK (sNK) cells and primary NK cells against Uterine Cancer Stem-like and Differentiated Cell Lines AN3CA and HEC-1B and 2) to investitate the in vivo efficacy of sNK Cells in Targeting AN3CA Tumor Progression in huBLT mice.

[0323] Natural Killer Cell

[0324] Natural Killer (NK) cells, essential constituents of the immune system, are lymphocytes originating from the bone marrow, embodying characteristics that allow them to act as the body’s frontline defense against tumors and virulent pathogens. These cells, notable for their large granules filled with potent cytotoxic molecules, represent approximately 5 to 15% of the lymphocytes in human blood [1]. Their identification is facilitated by distinct surface markers, notably CD16 and CD56, with the absence of CD3 that sets them apart from T cells [2]. NK cells thrive within peripheral blood mononuclear cells (PBMCs), playing a crucial role in the innate immune system, where they directly engage and neutralize threats and shape the adaptive immune response through their production of cytokines [3].

[0325] NK cell-mediated cytotoxicity is largely dependent on the action of perforin, which is a protein known to pierce through cell membranes, and granzyme B, an enzyme that acts as a serine protease [4]. The functionality of NK cells is delineated into two main subsets, characterized by their surface marker expression and inherent roles. The CD 16+ CD56dim subset, constituting the majority of NK cells in circulation, is primarily associated with direct cytotoxic activities against compromised cells [5]. Conversely, the CD56bright subset, though less prevalent, is essential for cytokine secretion, influencing both innate and adaptive immune mechanisms [6]. The balance between activating and inhibitory signals received through receptors such as NKG2D, NKp44, and KIR2 dictates the cytotoxic response of NK cells, ensuring targeted action against malignancies while sparing healthy tissues [7]. Beyond their innate cytolytic functions, NK cells possess the ability to induce differentiation in cancer stem cells, particularly those within poorly differentiated tumors, through the secretion of cytokines like IFN-y and TNF-a [8]. This capacity highlights the versatility of NK cells in tumor surveillance and underscores their potential in advancing cancer immunotherapy strategies.

[0326] Endometrial Cancer

[0327] Endometrial cancer (EC), the most common form of uterine cancer, originates from the endometrium, the lining of the uterus. Globally, EC ranks as the fourth most common cancer among women, with its incidence rising by approximately 20%, affecting about 1 in 37 women in their lifetime [9]. Traditionally seen in postmenopausal women, there’s a noticeable increase in younger women, attributed to early-onset obesity and hyperinsulinemia

[0010] .

[0328] Despite being often detected at an early stage due to symptoms like irregular vaginal bleeding, EC remains a significant health burden for several reasons. Firstly, the incidence and mortality rates of this cancer are on the rise. Type II endometrial cancer, which is highgrade and often detected at an advanced stage, accounts for a substantial proportion of all deaths from endometrial cancer

[0011] . This type’s aggressive nature and late detection contribute significantly to the overall health burden of the disease, with an estimated 13,030 deaths anticipated in 2023 out of 66,200 new cases

[0012] . This places a spotlight on the necessity for effective diagnostic and therapeutic strategies. Among the established cell lines AN3CA, ECC-1, HEC1A, HEC1B, and Ishikawa, we specifically chose AN3CA and HEC- 1B to examine their interactions with NK cells.

[0329] Differentiation of Tumors

[0330] Stem-like / poorly differentiated and well-differentiated tumors represent contrasting states of cancer cell maturity, with significant implications for treatment and prognosis. Stem- like / poorly differentiated tumors are characterized by a lack of mature cellular features and often exhibit aggressive growth patterns, making them less responsive to conventional chemotherapies. These tumors tend to grow rapidly, forming large masses, and show lower expression of differentiation markers like MHC-class I, CD54, and PD-L1

[0013] . In contrast, well-differentiated tumors resemble their tissue of origin more closely, displaying higher expression of differentiation antigens and are generally more susceptible to chemotherapeutic agents.

[0331] Research has shown that the differentiation stage of cancer cells influences their interaction with the immune system, particularly Natural Killer (NK) cells

[0013] . Poorly differentiated tumors are preferentially targeted by NK cells, which can also induce differentiation in these tumors, thereby increasing their susceptibility to drug-mediated cell death. This highlights the potential of NK cell-based therapies to not only directly lyse cancer cells but also to enhance the effectiveness of conventional treatments by promoting tumor differentiation. For instance, supercharged NK cells, when used in combination with chemotherapy or immunotherapy, have shown promise in reducing tumor size and enhancing NK cell function in experimental models, underscoring the importance of considering tumor differentiation in designing treatment strategies.

[0332] AN3CA

[0333] The AN3CA cell line, utilized in EC research, is classified as poorly differentiated

[0014] . Poor differentiation indicates that the cells exhibit low similarity to normal endometrial cells, reflecting a high grade of malignancy and aggressive tumor behavior

[0015] . AN3CA exhibits migratory properties and is tumorigenic in nude mice

[0016] . Such characteristics make AN3CA a valuable model for studying advanced disease stages and testing therapeutic strategies targeting aggressive cancer forms. While NK cells have been extensively studied for their cytotoxic capabilities against various cancers, specific research on NK cells’ effectiveness against the AN3CA EC cell line is not directly highlighted in the available literature.

[0334] HEC-1B

[0335] The HEC-1B cell line is a specific substrain of HEC-l-A, isolated for its distinct characteristics including a stationary growth phase observed between the 135th and 190th days in culture

[0017] . This substrain exhibits morphological changes upon recovery, appearing flatter and adopting a more pavement-like pattern compared to the parent line. HEC-1B forms moderately well-differentiated adenocarcinomas in nude mice and steroid-treated hamsters, consistent with grade II endometrial carcinoma

[0015] . Distinct from AN3CA, known for its aggressive and poorly differentiated characteristics, HEC-1B is often utilized in studying hormone responses and gene expression.

[0336] Osteoclasts

[0337] Osteoclasts are derived from hematopoietic stem cells and they play a crucial role in bone tissue maintenance, repair, and remodeling by resorbing bone tissue. This process, along with osteoblast bone formation, is essential for bone homeostasis [18, 19, 20]. Osteoclast maturation is stimulated by osteoblasts expressing RANKL, with their interaction facilitated by firm adhesion through ICAM-1

[0019] . Previous studies demonstrated that osteoclasts express ligands for receptors present on activated NK cells, such as ULBP-1, ULBP-2 / 5 / 6, and ULBP-3, while lacking expression of MIC-A, MIC-B, or MHC class I-like ligands for NKG2D

[0021] ,

[0338] Previous research has highlighted osteoclasts as significant activators of NK cell expansion and function compared to dendritic cells and monocytes

[0022] . Osteoclasts secrete IL-12, IL-15, IFN-y, and IL-18, known to activate NK cells, and express important NK- activating ligands

[0023] .

[0339] Super-Charged NK Cells (Osteoclast-expanded NK Cells)

[0340] Super-charged NK cells (sNK) are NK cells that have undergone an expansion process facilitated by osteoclasts, significantly enhancing their functional capabilities

[0024] . This expansion leads to a marked increase in their proliferation rate, cytotoxicity against cancer cells, and an increase in the secretion of IFN-y, a critical cytokine in immune response modulation

[0025] . Unlike primary NK cells (pNK) from PBMCs, sNK cells maintain their enhanced functions even when targeting challenging targets like oral squamous carcinoma stem-like cells (OSCSCs) or poorly differentiated tumors

[0026] . sNK cells exhibit a higher expression of activating receptors such as NKG2D, NKp44, NKp46, and granzyme B, which contribute to their increased cytotoxicity and enhanced IFN-y secretion

[0024] . Compared to primary NK (pNK) cells, sNK cells are more polyfunctional, showcasing superior multifaceted immune responses. This makes sNK cells a promising field for research and application in cancer immunotherapy, offering a potential for more effective targeting of cancer cells. OSCSC (Oral Squamous Carcinoma Stem Cells)

[0341] Oral Squamous Carcinoma Stem Cells (OSCSCs) are a specialized subset of cells found within oral squamous cell carcinomas, known for their lower differentiation and higher susceptibility to NK cell-mediated cytotoxicity. Unlike differentiated carcinoma cells, OSCSC exhibits slight B7H1 and EGF-R expression and none of MHC-Class II or CD90

[0027] . Yet, OSCSC expresses high levels of CD133, CD44, EpCAM, CD26, and CD338, making them prone to NK cell cytotoxicity and able to stimulate IFN-y secretion.

[0342] Humanized Mouse Model

[0343] The Humanized Mouse Model, utilized to study Cancer Stem Cells (CSCs) and their ability to initiate human tumors, exhibits varied NK cell impairment across strains like nude, NOD-scid, and NSG

[0028] . Questions persist about immune subsets’ roles in cancer control, motivating the use of humanized mice with restored immune systems for better assessment

[0029] .

[0344] Efforts to develop mice with fully reconstituted human immune systems are ongoing, primarily using NSG or NRG strains due to their severe immunodeficiency [30, 31]. Various methods exist for humanizing mice, such as injecting them with human PBMCs or isolating CD34+ progenitor cells for transplantation into NSG mice [28, 32].

[0345] Among these methods, the BLT humanized mouse model (hu-BLT) is the most notable

[0033] . It involves surgically implanting human fetal liver and thymus tissue under the renal capsule of NSG mice, followed by CD34+ cell injection [28, 33]. This model allows for human T cell development in the presence of a human thymus, resulting in functional CD4+ and CD8+ T cells with human MHC restriction [34, 35]. BLT mice also exhibit mucosal immunity and robust human leukocyte reconstitution, making them a promising tool for studying human immunity and cancer

[0036] .

[0346] Materials and Methods

[0347] Cell lines, reagents, and antibodies

[0348] Human immune cells were cultured in RPMI 1640, supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, CA). Oral Squamous Cancer Stem Cells (OSCSCs) were isolated from oral cancer patient tongue tumors at UCLA School of Medicine and cultured in RPMI 1640 supplemented 10% Fetal Bovine Serum (FBS) (Gemini Bio-Products, CA), 1.4% antibiotic antimycotic, 1% sodium pyruvate, 1.4% non-essential amino acids, 1% L-glutamine, 0.2% gentamicin (Gemini Bio-Products, CA, USA), and 0.15% sodium bicarbonate (Fisher Scientific, PA, USA). Endometrial cancer cell lines AN3CA and HEC-1B were purchased (ATCC, Manassas, VA) and cultured in EMEM medium with 10% FBS and 1% penicillin and streptomycin (Gemini Bio-Products, CA, USA). Osteoclasts were cultured with Alpha-MEM medium (Life Technologies, CA) supplemented with 10% FBS (Gemini Bio-Products, CA). Recombinant IL-2 was obtained from NIH-BRB. Antibodies used for flow cytometry- isotype control, CD45 (human), CD3, CD4, CD16, CD56, CD8, CD14, and CD19 - were purchased from Biolegend (San Diego, CA). Human NK purification kits were obtained from Stem Cell Technologies (Vancouver, BC, Canada).

[0349] Purification ofNK Cells from Human Peripheral Blood

[0350] Written informed consents approved by UCLA Institutional Review Board (IRB) were obtained from healthy blood donors and all the procedures were approved by the UCLA-IRB. The Ficoll-Hypaque technique was used to fractionate the red blood cells and the white cloudy layer, also known as the huffy coat. The huffy coat contains Peripheral Blood Mononuclear Cells (PBMC), which were harvested, washed, and re-suspended in RPMI 1640 (Invitrogen by Life Technologies, CA) supplemented with 10% FBS. NK cells were negatively selected and isolated from PBLs using the EasySep® Human NK cell enrichment kit purchased from Stem Cell Technologies (Vancouver, BC, Canada). The isolated NK cells were stained with anti-CD45, anti- CD16, anti-CD56, and anti-CD3 antibodies to ensure at least 90% cell purity through flow cytometry analysis. Purified NK cells were cultured in RPMI Medium 1640 supplemented with 10% FBS (Gemini BioProducts, CA), 1% antibiotic antimycotic, 1% sodium pyruvate, and 1% MEM non-essential amino acids (Invitrogen, Life Technologies, CA).

[0351] Generation of Osteoclasts from Human Peripheral Blood

[0352] Written informed consents, approved by UCLA Institutional Review Board (IRB) were obtained from healthy blood donors, and all procedures were approved by the UCLA- IRB. After 1-2 hours of incubation of PBMC in culture dishes, the adherent subpopulation of PBMCs was detached from the tissue culture plates. Monocytes were purified using the EasySep® Human monocyte cell enrichment kit obtained from Stem Cell Technologies (Vancouver, BC, Canada). Based on flow cytometric analysis of CD14 the antibody-stained, enriched monocyte cells, the monocyte population was found to have at least 95% purity. Monocytes were cultured using alpha- MEM medium containing M-CSF (25 ng / mL) and RANKL (25 ng / mL) for 21 days, and the medium was refreshed every 3 days.

[0353] Sonication

[0354] Osteoclasts and probiotics were sonicated to disrupt cellular membranes and release the cell contents, effectively inducing cell death. A homogeneous population of sonicated osteoclasts and probiotics were thus obtained.

[0355] Live osteoclasts were counted and suspended in alpha-MEM medium supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products), reaching a final concentration of 106 cells / ml. After every five pulses, a sample was taken and examined under a microscope until at least 80% of the cell wall was dissolved. The sample was then sonicated for 20 seconds while kept on ice, followed by a 30-second incubation on ice. This sonication process was repeated 15 times to ensure complete sonication.

[0356] AJ2 or AJ4 was weighed and resuspended in RPMI Medium 1640 containing 10% FBS at a concentration of lOmg per 1 mL. The bacteria was thoroughly vortexed, then sonicated on ice for 15 seconds, at 6 to 8 amplitude. Sonicated samples were then incubated for 30 seconds on ice. After every five pulses, a sample was taken to observe under the microscope until at least 80 percent of cell walls were lysed. It was determined that approximately 20 rounds of sonication / incubation on ice, were conducted to achieve complete sonication. Finally, the sonicated samples (sAJ2, sAJ4, or osteoclasts) were aliquoted and stored in a -80 °C freezer. Sonication of bacteria is not required or necessary to render its activities presented herein.

[0357] Surface staining and Flow cytometry analysis

[0358] Staining was performed by staining the cells with antibodies as described previously. Briefly, all samples (4 x 104) were stained with lOOpL of 1% BSA-PBS (Gemini BioProducts, CA) and pre-determined optimal concentration of desired fluorochrome (PE, FITC or PEcy5) conjugated antibodies. The samples were then incubated at 4°C for 30 min. The sample was washed and resuspended with 1% BSA-PBS. For the cell death assay, 3 x 104cells in 100 pl of cold 1% BSA-PBS were stained with 8 mg / ml propidium iodide and reconstituted to a final volume of 200 pl with BSA-PBS. Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA) and FlowJo vl0.4 (BD, Oregon, USA) were used for data analysis. Expansion ofNK Cells human NK cells were purified and activated using rhIL-2 (1000 U / ml) and anti-CD16 monoclonal antibody (3 ug / ml) for 18-20 hours. Subsequently, the activated NK cells were co- cultured with osteoclasts and AJ2 in a specific ratio of 2:1:4 (NK cells:osteoclasts:AJ2). The culture medium was replenished with rhIL-2 every three days to maintain optimal conditions.

[0359] 51Chromium Release Cytotoxicity Assay

[0360] Chromium-51 (51Cr) was obtained from Perkin Elmer (Santa Clara, California) for use in the standard51Cr release cytotoxicity assay.51Cr release cytotoxicity assay was employed to assess the cytotoxic function of NK cells in the experimental cultures. The assay was performed by incubating effector cells (IxlO5NK cells / well) and51Cr-labeled target cells (5xl05OSCSC / HEC- 1B / AN3CA) for four hours at four (5:1; 2.5:1; 1.25:1; 0.625:1) to six serial dilutions (5:1; 2.5:1; 1.25:1; 0.625:1; 0.3125:1; 0.15625:1). Following incubation, target cells were washed twice to remove excess unbound51Cr.51Cr-labeled target cells were aliquoted into the 96-well round bottom microwell plates containing effector cells at a concentration of IxlO4cells / well at a top effector: target (E:T) ratio of 5:1. After a 4-hour incubation period, the supernatant of each sample was harvested and the released radioactivity was measured using a gamma counter. The total release (containing51Cr-labeled target cells) and spontaneous release (supernatants of target cells alone) values were recorded to calculate the percentage of specific cytotoxicity. The percentage- specific cytotoxicity was calculated using the following formula:

[0361] Experimental cpm — Spontaneous cpm % Cytotoxicity = - - -

[0362] Total cpm — Spontaneous cpm

[0363] Lytic unit (LU) 30 / 106is calculated by using the inverse of the number of effector cells needed to lyse 30% of tumor target cells x 100.

[0364] Enzyme-Linked Immunosorbent Assays (ELIS As)

[0365] Human ELISA kit for IFN-y was purchased from Biolegend (San Diego, CA) to measure the IFN-y levels in cell culture. The assay was conducted as described in the manufacturer’s protocol. Briefly, 96-well EIA / RIA plates were coated with diluted capture antibody corresponding to target cytokine and incubated overnight at 4°C. After 16-18 hours of incubation, the plates were washed 3 times with wash buffer (0.05% Tween in IxPBS) and blocked with assay diluent (1%BSA in IxPBS). The plates were incubated for 1 hour at room temperature, on a plate shaker at 200rpm; plates were washed 3 times following incubation. Then, lOOuL of standards and samples collected from each culture were added to the wells and incubated for 2 hours at room temperature, on the plate shaker at 200rpm. After incubation, plates were washed 4 times, loaded with detection antibody, and incubated for 1 hour at room temperature, on the plate shaker at 200rpm. After 1 hour of incubation, the plates were washed 3 times; wells were loaded with Avidin- HRP solution and incubated for 30 minutes at room temperature, on the plate shaker at 200rpm. After washing the plates 5 times with wash buffer; lOOuL of TMB substrate solution was added to the wells and plates were incubated in the dark until they developed a desired blue color (or up to 30 minutes). Then, 50uL of stop solution (2NH2SO4) was added per well to stop the reaction. Finally, plates were read in a microplate reader, at 450nm to obtain absorbance values (Biolegend, ELISA manual).

[0366] Analysis of human AN3CA cell growth in humanized mice

[0367] Animal research was performed under the written approval of the UCLA Animal Research Committee (ARC) in accordance to all federal, state, and local guidelines. Humanized- BLT (hu-BLT; human bone marrow / liver / thymus) mice were prepared and maintained in the animal facilities at UCLA under protocols approved by the UCLA as previously described [37, 38].

[0368] In vivo growth of human endometrial carcinoma stem cells (AN3CA) was determined by orthotopic cell implantation of tumor cells into hu-BLT mice. To establish orthotopic tumors, mice were first anesthetized using an isoflurane setup, and AN3CA were then transferred by direct injection of 1x106 cells mixed with 10 pL HC Matrigel (Corning, NY, USA) directly into the uterus. Immediately before tumor cell injection, 5.0-mg / kg carprofen was injected subcutaneously, and this injection was repeated every 24 hours for 48 hours.

[0369] Following injection of tumor cells, all mice were continuously monitored for disease progression every other day. Mice were observed for overall signs of morbidity, such as loss of weight, ruffled fur, hunched posture, and immobility. Seven days after tumor implantation selected hu-BLT mice received 1.5x106 human expanded NK cells via tail vein (IV) injection. Mice were euthanized in 6 weeks when signs of morbidity were evident. Tumor volumes were determined using the formula:

[0370] Length x Width2n volume = - x —

[0371] 2 4

[0372] Cell dissociation and cell culture from tissues of tumor bearing hu-BLT mice

[0373] At the end of the experiment, mice were euthanized and the tumor, liver, bone marrow, spleen and blood were obtained from the hu-BLT mice. Single cell suspensions were obtained by digesting tissues using DMEM medium supplemented with collagenase II (Img / mL) (oral tumor) (Invitrogen, CA) and DNAse (lOu / mL) (Sigma-Aldrich, CA) and 1%BSA. The digested tissues were passed through 70 pM filters (Fisher Scientific, CA) to obtain single-cell suspensions. To obtain single-cell suspensions from BM, femurs were flushed using media, and filtered through a 40 pm cell strainer. Spleens were removed and single-cell suspensions were prepared and filtered through a 40 pm cell strainer and centrifuged at 1500 rpm for 5 min at 4 °C. The pellets were then re-suspended in ACK buffer to remove the red blood cells. Murine peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll-Hypaque centrifugation of heparinized blood specimens. The white, cloudy layer containing PBMCs was harvested, washed, and re-suspended in the RPMI 1640 media (Life Technologies, CA), supplemented with 10% FBS. Single-cell suspensions of each tissue were cultured in the presence and / or absence of IL-2 (1000 units / mL) treatment, using RPMI 1640 media (Life Technologies, CA), supplemented with 10% FBS.

[0374] Impedance Assay ( eSight)

[0375] The xCELLigence RTCA eSight system (Agilent, USA) utilizes advanced impedance-based and image-based biosensor technology to dynamically analyze cell behavior and functions. Equipped with specialized E-Plates containing gold biosensors at the bottom of each well, the system offers a non-invasive means to continuously monitor cellular parameters such as proliferation, adhesion, morphology, migration, and differentiation. The process began by establishing a background impedance measurement after adding the media. Target cells were then introduced at a concentration of 5xl03cells per well. The system operated overnight for target cell attachment, capturing impedance measurements every 15 minutes and images hourly. After 24 hours, effector cells were added in a sequence of serial dilutions starting at a 2.5:1 effector-to- target (E:T) ratio, reducing through several stages down to a 0.625:1. The system then continued to monitor these interactions through impedance and visual data for an extended period of 72 hours, providing detailed insights into cell dynamics.

[0376] Statistical analysis

[0377] A one-tailed, unpaired Student t-test was conducted for statistical analysis. To compare different groups, one-way ANOVA with a Bonferroni post-test was employed. Cytotoxicity and cytokine analysis were performed using duplicate or triplicate samples for assessment. The levels of statistical significance are represented by the following symbols: ***(p-value < 0.001), **(p-value 0.001-0.01), *(p-value 0.01-0.05).

[0378] Results

[0379] Investigation and comparison of the lysing capacity of supercharged NK (sNK) cells and primary NK cells against Uterine Cancer Stem-like and Differentiated Cell Lines AN3CA and HEC-1B

[0380] AN3CA cells exhibited a higher growth rate in comparison to HEC-1B cells

[0381] The growth of AN3CA and HEC-1B cells was assessed by plating 1x10^ cells per well in a 6-well culture dish. One well was trypsinized and counted every 24 hours using microscopy. AN3CA cells exhibited a higher growth rate compared to HEC-1B cells (fig. 14), consistent with their poorly differentiated and more aggressive nature. This observation aligns with the expected behavior of poorly differentiated cancer cells, which typically demonstrate rapid proliferation. sNK cells exhibit higher cytotoxicity against AN3CA cells sNK cells exhibit higher cytotoxicity against poorly differentiated cancer cells. To further investigate, a 4-hour 'Cr release assay was performed to evaluate sNK cell performance in lysing AN3CA and HEC-1B cells, with OSCSC serving as a control, representing a stem-like cancer cell line. sNK cells exhibited high cytotoxicity against AN3CA cells, similar to their performance against OSCSC (fig. 15). This indicated that sNK cells are particularly effective against poorly differentiated cancer cells. sNK cells showed lower cytotoxicity against HEC-1B cells, which are more differentiated. This aligns with the hypothesis that sNK cells are more potent against less differentiated, more aggressive cancer cells.

[0382] These results were further corroborated by eSight real-time cytotoxicity monitoring. Over a 72-hour co-culture period, images captured at 24-hour intervals demonstrated that sNK cells were able to significantly reduce the viability of AN3CA cells, while the impact on HEC-1B cells was markedly less pronounced (figs. 16A, 16B). The real-time monitoring provided dynamic evidence of the cytotoxic process, with sNK cells visibly reducing the number of viable AN3CA cells over time. sNK cells (supercharged NK cells) exhibit higher cytotoxicity than treated pNK cells (primary NK cells) pNK cells treated with IL-2 or IL-2 combined with sAJ4 exhibited higher cytotoxicity than untreated NK cells. To evaluate the cytotoxicity of these treated pNK cells in comparison to sNK cells, performed herein are a51Cr release assay and eSight real-time cytotoxicity monitoring with AN3, HEC-1B, and OSCSC as target cells. sNK cells demonstrated superior killing ability compared to pNK cells, regardless of the treatment with IL-2 or IL-2+sAJ4 (fig. 17). sNK cells showed a greater reduction in cell index over time when co-cultured with AN3CA and OSCSC cells compared to pNK cells treated with IL-2 or IL-2+sAJ4 (fig. 18A-C). The cell index for HEC-1B cells showed a less pronounced reduction, consistent with the lower cytotoxicity observed. sNK cells achieved higher % cytolysis across all three cell lines, with the most significant impact on AN3CA and OSCSC cells (fig. 19A-C). IL-2+sAJ4 treated pNK cells showed improved cytolysis compared to IL- 2 treated pNK cells, but did not match the efficacy of sNK cells. This reinforces the enhanced efficacy of sNK cells in targeting and lysing cancer cells.

[0383] The findings highlight the cytotoxic capabilities of sNK cells against poorly differentiated cancer cells like AN3CA, surpassing the performance of treated pNK cells. The data indicate that osteoclast expansion and subsequent activation of sNK cells significantly enhance their therapeutic potential.

[0384] The cytotoxic effects of supercharged NK (sNK) cells on two uterine cancer cell lines, AN3CA and HEC-1B, as well as a stem-like cancer cell line, OSCSC were explored. The findings provided valuable insights into the differential effectiveness of sNK cells against cancer cells with varying degrees of differentiation.

[0385] The initial growth rate assessment of AN3CA and HEC-1B cells revealed that AN3CA cells proliferate at a faster rate compared to HEC-1B cells. This is consistent with the poorly differentiated and aggressive nature of AN3CA cells. The rapid proliferation of AN3CA cells highlights their malignant potential and underscores the need for effective therapeutic strategies to target such aggressive cancer types.

[0386] The 4-hour51Cr release assay demonstrated that sNK cells exhibit significantly higher cytotoxicity against AN3CA cells compared to HEC-1B cells. This higher cytotoxicity against AN3CA cells was on par with the cytotoxicity observed against OSCSC, a stem-like cancer cell line. These results suggest that sNK cells are particularly effective against poorly differentiated and stem-like cancer cells, which are typically more challenging to treat with conventional therapies.

[0387] The real-time cytotoxicity monitoring using eSight provided dynamic evidence supporting these findings. Over the 72-hour co-culture period, sNK cells significantly reduced the viability of AN3CA and OSCSC cells, while the impact on HEC-1B cells was less pronounced. The cell index and % cytolysis data further corroborated these observations, with sNK cells achieving higher % cytolysis across all three cell lines. The greatest cytolytic effect was observed against AN3CA and OSCSC cells, indicating the enhanced capability of sNK cells to target and eliminate poorly differentiated and stem-like cancer cells. pNK cells treated with IL-2 or IL-2 combined with sAJ4 exhibited higher cytotoxicity than untreated NK cells. The51Cr release assay and eSight results confirmed these findings, with treated pNK cells showing improved cytotoxicity compared to untreated pNK cells. However, even with these treatments, pNK cells did not match the efficacy of sNK cells.

[0388] The superior performance of sNK cells compared to treated pNK cells highlights the significant impact of osteoclast expansion on NK cell functionality. sNK cells, expanded through osteoclast co-culture, demonstrate enhanced cytotoxicity and the ability to target aggressive cancer cells.

[0389] In Vivo Efficacy ofsNK Cells in Targeting AN3CA Tumor Progression in huBLT mice Decreased tumor burden huBLT mice treated with sNK cells

[0390] After the hu-BLT mice were sacrificed, endometrial tumors were resected. A comparative analysis showed that the tumors from mice treated with NK immunotherapy were visibly smaller (fig. 20). Furthermore, these tumor’s weight (fig. 21 A) and volume (fig. 21B) decreased compared to those from the control group. Increased IFN-y secretion level after IL-2 treatment on PBMC of sNK treated group

[0391] Further analysis involved assessing the immune response via IFNy production by PBMCs post-mortem after ex vivo IL-2 stimulation. The results showed an increase in IFNy levels in sNK- treated mice compared to controls (fig. 21C). This finding indicated that sNK cell treatment not only suppresses tumor growth but may also reinvigorate the immune system’s capacity to combat cancer, potentially reversing tumor-induced immunosuppression.

[0392] The substantial decrease in tumor volume among sNK-treated mice confirms the cells’ potent cytotoxic capabilities. This supports the hypothesis that a well-maintained physiological environment during sNK cell cultivation is crucial for developing highly effective therapeutic agents. The observed efficacy suggests that sNK cells retain their functional integrity and aggressive anti-tumor properties in a live setting.

[0393] The increase in IFNy production after ex vivo IL-2 stimulation of PBMCs is particularly significant. This indicates that sNK cell treatment extends beyond direct tumor cell cytotoxicity, modulating the systemic immune environment to counteract tumor-induced immunosuppression. IFNy plays a vital role in enhancing both innate and adaptive immune responses, crucial for effective anti-tumor activity and potentially reducing recurrence and metastasis.

[0394] The dual action of sNK cells — direct cytotoxic effects against tumor cells and immunomodulatory effects enhancing systemic immunity — suggests their potential as a versatile and robust therapeutic option. Their applicability may extend across various cancer types, offering a broad therapeutic impact. The study’s findings advocate for clinical trials exploring sNK cell therapies, focusing on optimizing therapeutic protocols to maximize benefits and minimize potential side effects.

[0395] In the context of cytotoxic efficacy against cancer cell lines, sNK cells exhibited significantly higher cytotoxicity against the poorly differentiated AN3CA cell line, akin to their effect on stem-like cancer cells (OSCSC). Conversely, sNK cells showed lower cytotoxicity against the more differentiated HEC-1B cell line. The performance of sNK cells was superior to that of pNK cells treated with IL-2 or IL-2+sAJ4, highlighting the enhanced effectiveness of osteoclast-expanded sNK cells.

[0396] In vivo studies using huBLT mouse models demonstrated that sNK cell treatment led to a substantial reduction in tumor burden, with significant decreases in tumor weight and volume compared to controls. Additionally, increased IFN-y secretion levels in sNK-treated mice indicated a strengthened immune response, suggesting that sNK cells can potentially reverse tumor-induced immunosuppression and improve anti-tumor immunity.

[0397] These findings shed light on the therapeutic potential of sNK cells in targeting aggressive and poorly differentiated cancer cells.

[0398] References

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[0439] Incorporation by Reference

[0440] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0441] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.

[0442] Equivalents

[0443] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A composition comprising processed osteoclasts, optionally (a) wherein the processed osteoclasts comprise an osteoclast that is non-viable, fragmented, sonicated, ground, heat- inactivated, dried, lyophilized, and / or frozen; (b) wherein the composition comprises at least or about 70% of fragmented osteoclasts; and / or (c) wherein the processed osteoclasts are anchored to a solid support (e.g., beads).

2. The composition of claim 1, further comprising(a) an NK cell, optionally wherein the NK cell has been contacted with IL-2 and / or an anti-CD16 antibody; or(b) an NK cell in combination with IL-2 and / or an anti-CD16 antibody, optionally wherein the NK cell is autologous or allogeneic to the processed osteoclasts.

3. The composition of claim 1 or 2, further comprising at least one bacterial strain selected from: Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is either alive or sonicated.

4. The composition of claim 3, wherien the at least one bacterial strain comprises(a) Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei',(b) AJ2 bacteria; or(c) AJ4 bacteria.

5. The composition of any one of claims 1-4, further comprising at least one cytokine or chemokine that activates and / or supports the survival of the NK cell.

6. The method of claim 6, wherein the at least one cytokine is selected from interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, type I interferons (e.g., IFN-a), and any combination oftwo or more thereof, optionally wherein the at least one cytokine comprises IL-12, IL-15, IL- 18, and IFN-a.

7. The composition of any one of claims 1-6, wherein the composition is a pharmaceutical composition, which optionally comprises a pharmaceutically acceptable excipient.

8. A method of activating and / or expanding an NK cell in vitro or ex vivo, the method comprising contacting the NK cell with the composition of any one of claims 1-7.

9. A method of activating and / or expanding an NK cell in vitro or ex vivo, the method comprising:(c) contacting the NK cell with IL-2 and / or an anti-CD16 antibody; and(d) contacting the NK cell of (a) with the composition comprising the processed osteoclasts of claim 1 , optionally wherein the NK cell is autologous or allogeneic to the processed osteoclasts.

10. The method of claim 9, further comprising contacting the NK cell with a composition comprising at least one bacterial strain selected from: Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is either alive or sonicated.

11. The method of claim 10, wherein the at least one bacterial strain comprises(a) Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei',(b) AJ2 bacteria; or(c) AJ4 bacteria.

12. The method of any one of claims 9-11, further comprising contacting the NK cell with at least one cytokine or chemokine that activates the NK cell.

13. The method of claim 12, wherein the at least one cytokine is selected from interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, and type I interferons (e.g., IFN-a), optionally wherein the at least one cytokine comprises IL-12, IL-15, IL-18, and IFN-a.

14. A method of activating and / or expanding an NK cell in a subject, the method comprising administering to the subject the composition of any one of claims 1-7.

15. A method of treating a disease in a subject in need thereof, comprising administering to the subject (a) the composition of any one of claims 1-7, and / or (b) the NK cell activated and / or expanded according to the method of any one of claims 8-13.

16. The method of claim 15, wherein the disease is a cancer or an infection (e.g., viral or bacterial infection).

17. A method of killing a cancer cell in a subject, comprising administering to the subject (a) the composition of any one of claims 1-7, and / or (b) the NK cell activated and / or expanded according to the method of any one of claims 8-13.

18. The method of any one of claims 14-17, further comprising administering to the subject a composition comprising at least one bacterial strain selected from: Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is administered orally.

19. The method of claim 18, wherein the at least one bacterial strain comprises(a) Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasev,(b) AJ2 bacteria; or(c) AJ4 bacteria.

20. The method of any one of claims 14-19, further comprising administering to the subject at least one cytokine or chemokine that activates and / or supports the survival of the NK cell.

21. The method of claim 20, wherein the at least one cytokine is selected from interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, type I interferons (e.g., IFN-a), and any combination of two or more thereof, optionally wherein the at least one cytokine comprises IL-12, IL-15, IL- 18, and IFN-a.

22. The method of any one of claims 16-21, further comprising conjointly treating the subject with at least one cancer therapy, optionally wherein the subject is treated with the at least one cancer therapy before, after, or concurrently with (a) the composition of any one of claims 1-7, and / or (b) the NK cell prepared according to the method of any one of claims 8- 13.

23. The method of claim 22, wherein the at least one cancer therapy is selected from a surgery, radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

24. The method of claim 23, wherein the at least one cancer therapy is chemotherapy.

25. The method of claim 23, wherein the at least one cancer therapy is immunotherapy.

26. The method of claim 23 or 25, wherein the immunotherapy inhibits an immune checkpoint.

27. The method of claim 26, wherein the immune checkpoint is selected from CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.

28. The method of any one of claims 16-21, wherein the cancer is an oral cancer, an adenocarcinoma, a uterine cancer, or an endometrial adenocarcinoma.

29. The method of any one of claims 14-28, wherein the NK cell is autologous or allogeneic to the subject, preferably allogeneic to the subject.

30. The method of any one of claims 14-28, wherein the subject is a mammal, preferably a human.

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