Compositions and methods for activating NK cells

Abstract

To provide therapeutic compositions and methods for improved NK immunotherapy.SOLUTION: The present application provides methods of activating NK cells in vitro, ex vivo and / or in vivo by an osteoclast cell (OC) and / or a dendritic cell, and methods of treating disease using these activated NK cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 459,397, filed February 15, 2017, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Natural killer (NK) cells lyse cancer stem cells / undifferentiated tumors, differentiate, and express low levels of MHC class I, CD54, and B7H1, but high levels of CD44. Moderate to high cytotoxic activity of peripheral blood lymphocytes is associated with a decreased risk of cancer, and high tumor infiltration by NK cells is associated with a favorable prognosis, while low activity is associated with an increased risk of cancer.

[0003] NK cell suppression is mediated by downregulation of NK receptors in the tumor microenvironment. NK cell function has previously been shown to be significantly reduced in tumor patients. Several in vitro NK expansion techniques have been developed to enable higher therapeutic cell doses. Stimulating purified populations of peripheral blood mononuclear cells (PBMCs) or NK cells with feeder cells, such as K562 cells expressing interleukin (IL)-15 and 41BB ligand, EBV-TM-LCL, Wilms' tumor cells, or irradiated PBMCs, resulted in the generation of more fully functional NK cells. The generated NK cells expressed higher levels of NKG2D, the natural cytotoxicity receptor, DNAM-1, and ICAM-1. Therefore, various methods for obtaining ex vivo expanded and activated CD3+ T cell-depleted NK cells have been established for clinical use. Furthermore, the safety and efficacy of adoptive cell transfer of HLA-haploidentically transplanted NK cells in patients with advanced cancer have been established. Furthermore, clinical trials have shown that allogeneic NK cells have a therapeutic role in solid tumors and are safe to transfer into patients.

[0004] NK cell-based immunotherapy is limited by the lack of sufficient numbers of highly functional NK cells. Moreover, unlike NK cells from healthy individuals, the expansion of NK cells in patients, as well as those from tumor-bearing humanized mice, is severely limited by the proliferation of a small proportion of contaminating T cells that crowd out NK cells due to their faster proliferative capacity.

[0005] The mechanisms underlying NK cell immunoregulation are not understood. There is a great need to identify therapeutic compositions and methods for improving NK immunotherapy. Summary of the Invention

[0006] The present invention is based, at least in part, on the discovery that osteoclasts can induce NK cell proliferation and further increase the CD8+ / CD4+ T cell ratio in both healthy humans and cancer patients. While cancer patients generally have higher NK cell and CD8+ / CD4+ T cell ratios in vivo compared to healthy humans, the excess NK and CD8+ T cells are short-lived (due to the proliferation of contaminating T cells that can suppress NK cell function) and lack activity (e.g., cytotoxicity and cytokine selection). However, osteoclasts can induce NK cell proliferation and increase both the number and function of NK cells in cancer patients (e.g., as measured by cytokine secretion capacity). While dendritic cells preferentially promote T cell proliferation, osteoclasts preferentially promote NK cell proliferation, suggesting differences in the microenvironment for the selective proliferation of T and NK cells. Thus, the present invention provides methods for expanding large numbers of activated NK cells for use in immunotherapeutic strategies. Such cells can be used to inhibit or eliminate the growth of cancer stem cells and control tumors by promoting stem-like / poorly differentiated tumor differentiation.

[0007] Provided herein are methods for activating NK cells in vitro or ex vivo, comprising culturing NK cells in a medium with osteoclasts (OCs). The NKs may optionally be non-transformed primary (primary) NK cells. The activated NK cells may proliferate for approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within 4 weeks. The culture may include a plurality of osteoclasts (OCs) and a plurality of NK cells, e.g., the ratio of OCs:NK cells in the cell culture is at least 1:2. Osteoclasts are involved in, for example, the lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by NK cells or the like. 51 It can enhance NK cell cytotoxicity as measured by Cr release cytotoxicity assay.

[0008] Additionally, osteoclasts can enhance the production, secretion, and / or function of at least one cytokine or chemokine produced by NK cells. For example, osteoclasts can enhance the secretion of IFN-γ and / or IL-12 by NK cells and / or the expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by NK cells.

[0009] The NK cells may be cells purified from a cancer sample of a human subject. In certain embodiments, the cell culture further comprises T cells derived from the cancer sample. In certain such embodiments, the NK cells may be preferentially expanded relative to T cells. The NK cells may be expanded for any period of time, for example, for at least one month. The culture medium may be supplemented with at least one osteoclast to continue preferentially expanding the NK cells. The T cells may be, for example, 51The NK cells may secrete IFN-γ but not mediate cytotoxicity, as measured, for example, by lysis of OSCSCs by T cells in a Cr release cytotoxicity assay. The expanded NK cells may be capable of expanding CD8+ T cells. NK cells expanded by OC may also be capable of preferentially expanding CD8+ T cells over CD4+ T cells. In certain embodiments, the method further comprises adding an anti-CD3 antibody to the cell culture, for example, to further enhance IFN-γ secretion by NK cells. The activated NK cells may be divided into anergized cells.

[0010] In certain embodiments, the method may further include adding to the cell culture 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, where optionally the at least one bacterial strain may be live or sonicated. For example, the composition may comprise Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus. Alternatively or additionally, the composition may comprise sAJ2 bacteria. The ratio of sAJ2 bacterial concentration to NK cell and / or OC concentration in the cell culture can be, for example, i) at least 1:2 for NK cells:sAJ2; ii) at least 1:4 for OC:sAJ2; and / or iii) at least 1:2:4 for OC:NK cells:sAJ2.

[0011] In certain embodiments, the method may further comprise adding to the cell culture another agent capable of activating NK cells.

[0012] In certain preferred embodiments, the method comprises: i) providing a cell culture comprising osteoclasts (OCs), NK cells, and T cells; and ii) culturing the NK cells, T cells, and osteoclasts in the cell culture, thereby preferentially activating the NK cells relative to the T cells.

[0013] Also provided herein are methods comprising: i) providing a cell culture comprising dendritic cells (DCs), NK cells, and T cells; and ii) culturing NK cells, T cells, and dendritic cells in the cell culture, thereby preferentially activating T cells over NK cells. The NK cells may be primary NK cells, and optionally, the primary NK cells are not transformed. The culture may comprise a plurality of osteoclasts (OCs) and a plurality of NK cells, e.g., the ratio of OCs to NK cells in the cell culture is at least 1:2. The osteoclasts may enhance NK cell proliferation and / or IL-15 secretion by the NK cells. The activated NK cells may expand to approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within four weeks. Furthermore, osteoclasts can enhance the cytotoxicity of NK cells, as measured, for example, by the lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by NK cells. 51 It can be measured by a Cr release cytotoxicity assay.

[0014] In certain embodiments, osteoclasts (OCs) can enhance the production, secretion, and / or function of at least one cytokine or chemokine produced by NK cells. For example, osteoclasts can enhance the secretion of IFN-γ and / or IL-12 by NK cells. Osteoclasts can enhance the expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by NK cells. NK cells and / or T cells can be purified from a cancer sample derived from a subject, e.g., a human subject. In certain embodiments, the preferential activation of NK cells can persist for at least one month. Furthermore, after the preferential activation of NK cells has diminished or ceased, at least one osteoclast can be added to the cell culture for at least one month after the culture of the NK cells to continue the activation of the NK cells. In some embodiments, T cells can secrete IFN-γ but not mediate cytotoxicity. Cytotoxicity can be achieved, for example, by 51 Lysis of OSCSCs by T cells can be measured in a Cr release cytotoxicity assay. Expanded NK cells can expand CD8+ T cells and can preferentially expand CD8+ T cells over CD4+ T cells. In certain embodiments, NK cells expanded by DCs can preferentially expand CD4+ T cells over CD8+ T cells. In other embodiments, anti-CD3 antibodies can be added to the cell culture, for example, to further enhance IFN-γ secretion by NK cells. Furthermore, activated NK cells can be divided into anergized cells.

[0015] In certain embodiments, the method may further include adding to the cell culture 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 live or sonicated. For example, the composition may include Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus.

[0016] Alternatively, or in addition, the composition may comprise sAJ2 bacteria. In certain such embodiments, the ratio of sAJ2 bacteria concentration to NK cell and / or OC concentration in the cell culture may be, for example, i) at least 1:2 for NK cells:sAJ2; ii) at least 1:4 for OC:sAJ2; and / or iii) at least 1:2:4 for OC:NK cells:sAJ2.

[0017] Additionally, the method may further include adding another agent to the cell culture that may be capable of activating NK cells.The method may further include adding another agent to the cell culture that may be capable of activating T cells.

[0018] Also provided herein are methods of treating cancer or a cancer-related disease or disorder in a subject having or suspected of having cancer or a cancer-related disease or disorder by administering to the subject a therapeutically effective amount of osteoclasts (OC), cell cultures comprising osteoclasts (OC), and / or supernatants of cell cultures comprising osteoclasts (OC).

[0019] Osteoclasts can enhance NK cell proliferation in a subject, and optionally, osteoclasts enhance IL-15 secretion by NK cells. In certain embodiments, enhanced NK cell proliferation can be approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within 4 weeks. Furthermore, osteoclasts can enhance the cytotoxicity of NK cells, as measured, for example, by lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by NK cells. The cytotoxicity of the cells can be measured by: 51 It can be measured by a Cr release cytotoxicity assay.

[0020] In certain embodiments, osteoclasts can increase or promote the production, secretion, and / or function of at least one cytokine or chemokine produced by NK cells. For example, osteoclasts can enhance the secretion of IFN-γ and / or IL-12 by NK cells. Osteoclasts can preferentially activate NK cells relative to T cells and / or preferentially enhance the proliferation of NK cells relative to T cells. In certain embodiments, the preferential activation of NK cells can persist for at least one month. T cells can be, for example, 51 They secrete IFN-γ but do not mediate cancer cytotoxicity, as measured by lysis of OSCSCs by T cells in a Cr release cytotoxicity assay.

[0021] In certain embodiments, the activated NK cells can expand CD8+ T cells in the subject, e.g., preferentially expanding CD8+ T cells over CD4+ T cells. The subject can also be treated with an anti-CD3 antibody to further enhance IFN-γ secretion by the NK cells. The activated NK cells can be divided and anergized.

[0022] In certain embodiments, the treatment method may further include adding to the cell culture 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 live or sonicated. For example, the composition may comprise Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus. In certain embodiments, the composition may comprise sAJ2 bacteria.

[0023] In certain embodiments, the method may further comprise adding to the cell culture another agent capable of activating NK cells.

[0024] In certain embodiments, the osteoclasts, cell cultures, and / or supernatants may be administered in pharmaceutical compositions and may be administered systemically or locally to the cancer. In certain embodiments, the osteoclasts, cell cultures, and / or supernatants may be administered to the subject at least twice, for example, the osteoclasts, cell cultures, and / or supernatants may be administered to the subject at least one month after the first administration.

[0025] In some embodiments, the subject may be a human. The present invention also provides the following embodiments. Item [1] A method for activating NK cells in vitro or ex vivo, The method comprises culturing the NK cells in culture medium together with osteoclasts (OCs). Item [2] i) Providing a cell culture containing NK cells and osteoclasts; and ii) culturing the NK cells and the osteoclasts in the cell culture. thereby activating said NK cells. Item [3] The method according to Item [1] or [2], wherein the NK cells are primary NK cells, and optionally the primary NK cells are not transformed. Item [4] The method of any one of the preceding items, wherein the activated NK cells expand to at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within 4 weeks. Item [5] The method according to any one of the preceding items, wherein the culture comprises a plurality of osteoclasts (OCs) and a plurality of NK cells, and the ratio of OCs to NK cells in the cell culture is at least 1:2. Item [6] The method of any one of the preceding items, wherein the osteoclasts enhance the cytotoxicity of NK cells, and optionally the cytotoxicity of the NK cells is measured by lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by the NK cells. Item [7] The cytotoxicity of the NK cells is 51 The method according to item [6], wherein the cytotoxicity is measured by a Cr release cytotoxicity assay. Item [8] The method according to any one of the preceding items, wherein the osteoclasts enhance the secretion of IFN-γ by the NK cells, and optionally, the osteoclasts enhance the secretion of IL-12 by the NK cells. Item [9] The method of any one of the preceding items, wherein the osteoclasts enhance the expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by the NK cells. Item

[10] The method according to any one of the preceding items, wherein the NK cells are purified from a cancer sample. Item

[11] The method according to Item

[10] , wherein the cancer sample is derived from a subject having the cancer. Item

[12] The method according to Item

[11] , wherein the subject is a human. Item

[13] The method according to any one of Items

[10] to

[12] , wherein the cell culture further comprises T cells derived from the cancer sample. Item

[14] The method according to Item

[13] , wherein the NK cells are preferentially expanded relative to the T cells. Item

[15] The method according to Item

[14] , further comprising preferentially expanding the NK cells for at least one month. Item

[16] The method according to Item

[15] , further comprising supplementing the culture medium with at least one osteoclast to preferentially continue to proliferate the NK cells. Item

[17] The T cells secrete IFN-γ but do not mediate cytotoxicity, and optionally, the cytotoxicity is preferably 51 The method according to item

[14] , wherein the lysis of OSCSCs by the T cells is measured in a Cr release cytotoxicity assay. Item

[18] The method according to any one of Items

[13] to

[17] , wherein the expanded NK cells are capable of expanding CD8+ T cells. Item

[19] The method according to Item

[18] , wherein the NK cells expanded by the OC can preferentially expand CD8+ T cells over CD4+ T cells. Item

[20] The method of any one of the preceding items, further comprising adding an anti-CD3 antibody to the cell culture. Item

[21] The method according to Item

[20] , wherein the anti-CD3 antibody further enhances the secretion of IFN-γ by the NK cells. Item

[22] The method of any one of the preceding items, wherein the activated NK cells are fractionally anergized. Item

[23] The method of any one of the preceding items, further comprising adding to the cell culture 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 live or sonicated. Item

[24] The method according to Item

[23] , wherein the composition comprises Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus. Item

[25] The method according to Item

[23] , wherein the composition comprises sAJ2 bacteria. Item

[26] The ratio of the sAJ2 bacteria concentration to the NK cell and / or OC concentration in the cell culture is i) NK cells: at least 1:2 for sAJ2; ii) at least 1:4 for OC:sAJ2; and / or iii) OC:NK cells:sAJ2 ratio is at least 1:2:4 The method according to item

[25] , Item

[27] The method according to any one of the preceding items, further comprising adding to the cell culture another agent capable of activating NK cells. Item

[28] The method of any one of the preceding items, wherein the osteoclasts enhance the production, secretion, and / or function of at least one cytokine or chemokine produced by the NK cells. Item

[29] i) Providing a cell culture comprising osteoclasts (OCs), NK cells, and T cells; and ii) culturing the NK cells, the T cells, and the osteoclasts in the cell culture; thereby preferentially activating said NK cells relative to said T cells. Item

[30] i) Providing a cell culture containing dendritic cells (DCs), NK cells, and T cells; and ii) Culturing the NK cells, the T cells, and the dendritic cells in the cell culture. thereby preferentially activating said T cells relative to said NK cells. Item

[31] The method according to Item

[29] or

[30] , wherein the NK cells are primary NK cells, and optionally the primary NK cells are not transformed. Item

[32] The method according to Item

[29] or

[31] , wherein the culture comprises a plurality of osteoclasts (OCs) and a plurality of NK cells, and the concentration ratio of OCs to NK cells in the cell culture is at least 1:2. Item

[33] The method according to Item

[29] ,

[31] , or

[32] , wherein the osteoclasts enhance NK cell proliferation, and optionally, the osteoclasts enhance IL-15 secretion by the NK cells. Item

[34] The method of Item

[33] , wherein the activated NK cells expand to at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within 4 weeks. Item

[35] The method according to any one of Items

[29] and

[31] to

[34] , wherein the osteoclasts enhance the cytotoxicity of NK cells, and optionally the cytotoxicity of the NK cells is measured by lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by the NK cells. Item

[36] The cytotoxicity of the cells is51 The method according to item

[35] , wherein the cytotoxicity is measured by a Cr release cytotoxicity assay. Item

[37] The method according to any one of Items

[29] and

[31] to

[36] , wherein the osteoclasts enhance the secretion of IFN-γ by the NK cells, and optionally, the osteoclasts enhance the secretion of IL-12 by the NK cells. Item

[38] The method according to any one of Items

[29] and

[31] to

[37] , wherein the osteoclasts enhance the expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by the NK cells. Item

[39] The method according to any one of Items

[29] to

[38] , wherein the NK cells and / or T cells are purified from a cancer sample. Item

[40] The method according to Item

[39] , wherein the cancer sample is derived from a subject having the cancer. Item

[41] The method according to Item

[40] , wherein the subject is a human. Item

[42] The method according to any one of Items

[29] and

[31] to

[41] , wherein the preferential activation of NK cells persists for at least one month. Item

[43] The method of Item

[42] further comprises continuing the activation of the NK cells by adding at least one osteoclast to the cell culture after the preferential activation of the NK cells has attenuated or stopped, optionally wherein the addition of the at least one osteoclast to the cell culture is at least one month after the culture of the NK cells. Item

[44] The T cells secrete IFN-γ but do not mediate cytotoxicity, and optionally, the cytotoxicity is preferably 51 The method according to any one of items

[29] to

[43] , wherein the lysis of OSCSCs by the T cells is measured in a Cr release cytotoxicity assay. Item

[45] The method according to any one of Items

[29] and

[31] to

[44] , wherein the expanded NK cells are capable of expanding CD8+ T cells. Item

[46] The method according to Item

[45] , wherein the NK cells expanded by the OC can preferentially expand CD8+ T cells over CD4+ T cells. Item

[47] The method according to any one of Items

[30] to

[44] , wherein the NK cells expanded by the DCs can preferentially expand CD4+ T cells over CD8+ T cells. Item

[48] The method according to any one of Items

[29] and

[31] to

[46] , further comprising adding an anti-CD3 antibody to the cell culture. Item

[49] The method according to Item

[48] , wherein the anti-CD3 antibody further enhances the secretion of IFN-γ by the NK cells. Item

[50] The method according to any one of Items

[29] to

[49] , wherein the activated NK cells are divided into anergized cells. Item

[51] The method according to any one of Items

[29] and

[31] to

[50] , further comprising adding to the cell culture 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 live or sonicated. Item

[52] The method according to Item

[51] , wherein the composition comprises Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus. Item

[53] The method according to Item

[52] , wherein the composition comprises sAJ2 bacteria. Item

[54] The ratio of the sAJ2 bacteria concentration to the NK cell and / or OC concentration in the cell culture is i) NK cells: at least 1:2 for sAJ2; ii) at least 1:4 for OC:sAJ2; and / or iii) OC:NK cells:sAJ2 ratio is at least 1:2:4 The method according to item

[53] , Item

[55] The method according to any one of Items

[29] and

[31] to

[52] , further comprising adding another agent capable of activating NK cells to the cell culture. Item

[56] The method according to any one of Items

[30] to

[52] , further comprising adding another agent capable of activating T cells to the cell culture. Item

[57] The method according to any one of Items

[29] and

[31] to

[55] , wherein the osteoclasts (OCs) enhance the production, secretion, and / or function of at least one cytokine or chemokine produced by the NK cells. Item

[58] A method for treating cancer or a cancer-related disease or disorder in a subject having or suspected of having cancer or a cancer-related disease or disorder, comprising: The method comprises administering to the subject a therapeutically effective amount of osteoclasts (OC), a cell culture containing osteoclasts (OC), and / or a supernatant of a cell culture containing osteoclasts (OC). Item

[59] The method according to Item

[58] , wherein the osteoclasts activate NK cells in the subject. Item

[60] The method according to Item

[59] , wherein the NK cells are primary NK cells, and optionally the primary NK cells are not transformed. Item

[61] The method according to any one of Items

[59] to

[60] , wherein the osteoclasts enhance NK cell proliferation in the subject, and optionally, the osteoclasts enhance the secretion of IL-15 by the NK cells. Item

[62] The method of Item

[61] , wherein the enhanced NK cell proliferation is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within 4 weeks. Item

[63] The method according to any one of Items

[59] to

[62] , wherein the osteoclasts enhance the cytotoxicity of NK cells, and optionally the cytotoxicity of the NK cells is measured by lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by the NK cells. Item

[64] The cytotoxicity of the NK cells is 51 The method according to item

[63] , wherein the cytotoxicity is measured by a Cr release cytotoxicity assay. Item

[65] The method according to any one of Items

[59] to

[64] , wherein the osteoclasts enhance the secretion of IFN-γ by the NK cells, and optionally, the osteoclasts enhance the secretion of IL-12 by the NK cells. Item

[66] The method according to any one of Items

[59] to

[65] , wherein the osteoclasts preferentially activate NK cells compared to T cells, and optionally, the osteoclasts preferentially enhance the proliferation of NK cells compared to T cells. Item

[67] The method according to Item

[66] , wherein the preferential proliferation of the NK cells is sustained for at least one month. Item

[68] The T cells secrete IFN-γ but do not mediate cytotoxicity of the cancer, and optionally, the cytotoxicity is, for example, 51 The method according to item

[67] , wherein the lysis of OSCSCs by the T cells is measured in a Cr release cytotoxicity assay. Item

[69] The method according to any one of Items

[59] to

[68] , wherein the activated NK cells proliferate CD8+ T cells in the subject. Item

[70] The method according to Item

[69] , wherein the activated NK cells preferentially proliferate CD8+ T cells compared to CD4+ T cells. Item

[71] The method according to any one of Items

[58] to

[70] , further comprising adding an anti-CD3 antibody to the subject. Item

[72] The method according to Item

[71] , wherein the anti-CD3 antibody further enhances the secretion of IFN-γ by the NK cells. Item

[73] The method according to any one of Items

[59] to

[72] , wherein the activated NK cells are divided into anergized cells. Item

[74] The method according to any one of Items

[58] to

[73] , further comprising adding 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 live or sonicated. Item

[75] The method according to Item

[74] , wherein the composition comprises Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus. Item

[76] The method according to Item

[74] , wherein the composition comprises sAJ2 bacteria. Item

[77] The method according to any one of Items

[58] to

[76] , further comprising adding another agent capable of activating NK cells to the cell culture. Item

[78] The method according to any one of Items

[58] to

[77] , wherein the osteoclasts (OCs) increase or promote the production, secretion, and / or function of at least one cytokine or chemokine produced by the NK cells. Item

[79] The method according to any one of Items

[58] to

[78] , wherein the osteoclasts, the cell culture, and / or the supernatant are administered in a pharmaceutical composition. Item

[80] The method according to any one of Items

[58] to

[79] , wherein the osteoclasts, the cell culture, and / or the supernatant are administered systemically or locally to the cancer. Item

[81] The method according to any one of Items

[58] to

[80] , wherein the osteoclasts, the cell culture, and / or the supernatant are administered to the subject at least twice, and optionally the osteoclasts, the cell culture, and / or the supernatant are administered to the subject at least one month after the first administration. Item

[82] The method according to any one of Items

[58] to

[81] , wherein the subject is a human. [Brief explanation of the drawings]

[0026] [Figure 1-1] The four panels, identified as panels A, B, C, and D, show higher expression of NK-activating ligands by osteoclasts. To generate osteoclasts (OCs), monocytes were cultured for 21 days in medium containing macrophage colony-stimulating factor (M-CSF) (25 ng / ml) and RANKL (25 ng / ml). Highly purified NK cells (1 × 10 cells / ml) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16 mAb (3 μg / ml) for 18 hours and then cocultured with autologous OCs in the presence or absence of sAJ2 bacteria at a ratio of 1:2:4 (OC:NK:sAJ2). Surface expression of CD3, CD16, and CD56 was analyzed using flow cytometry in 1 × 10 lymphocyte samples from the coculture on days 6, 9, 12, 15, 19, 24, 29, and 34, with the culture medium refreshed and supplemented with rh-IL-2 (1000 U / ml) (Fig. 1A). [Figure 1-2]Cells were co-cultured as shown in Figure 1A, and the proliferated lymphocytes were manually counted using a microscope (Figure 1B). Monocytes were isolated from PBMCs of healthy donors. To generate dendritic cells, monocytes were cultured for 8 days in medium containing GM-CSF (150 ng / ml) and IL-4 (50 ng / ml). Osteoclasts were generated, and NK cells were purified as shown in Figure 1A, and then co-cultured with autologous cells in the presence of sAJ2 bacteria at a ratio of 1:2:4 (target cells:NK:sAJ2). On day 6 of culture, the culture medium was removed, and NK cells were treated with rh-IL-2 for 5 hours. The supernatants were harvested, and IFN-γ secretion was assessed using a simple ELISA (Figure 1C). [Figure 1-3] Monocytes were isolated, and dendritic cells and osteoclasts were generated as shown in Figure 1A and Figure 1D. OSCSCs and the K562 tumor cell line were cultured as described in Materials and Methods. 1 x 10 cells were analyzed for MHC-1, CD54, KIR2, KIR3, KLRG1, and MICA / B surface expression using PE-conjugated antibodies and flow cytometry. IgG2 isotype was used as a control (Figure 1D). [Figure 2-1] This figure contains 10 panels identified as panels A–J, and shows significant enhancement of preferential proliferation and function of NK cells and T cells by osteoclasts and dendritic cells. Monocytes were purified from human PBMCs and cultured with GM-CSF (150 ng / ml) and IL-4 (50 ng / ml) for 8 days to generate DCs. To generate osteoclasts, monocytes were cultured for 21 days in alpha-MEM medium containing M-CSF (25 ng / ml) and RANKL (25 ng / ml). For expansion, purified NK cells (1 × 10 cells / ml) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16 mAb (3 μg / ml) for 18 hours and then cocultured with autologous monocytes, DCs, or OCs in the presence of sAJ2 at a ratio of 1:2:4 (monocytes, DCs, or OCs:NKs:sAJ2). Surface expression of CD3, CD16, and CD56 was analyzed by flow cytometry at the days indicated in the figure, and the culture medium was refreshed and supplemented with rh-IL-2 (1000 U / ml) (Fig. 2A). [Figure 2-2]Cells were co-cultured as shown in Figure 2A, and the number of expanded lymphocytes was assessed using microscopic measurements (Figure 2B). The numbers of NK cells (Figure 2C) and T / NKT cells (Figure 2D) were determined using the percentage of NK and T / NKT cells (Figure 2A) among the total number of expanded cells in Figure 2B. [Figure 2-3] Cells were cocultured as shown in Figure 2A, and cytotoxicity against oral squamous cell carcinoma stem cell lines (OSCSCs) was determined using a standard 4-hour 51Cr-release assay at the days indicated. Lytic units (30 / 106 cells) were determined using the reciprocal number of lymphocytes required to lyse 30% of OSCSCs × 100 (Figure 2E). Supernatants were harvested from NK cocultures with OCs on days 6, 9, 12, and 15, as shown in Figure 1A, and IFN-γ secretion was determined using a single ELISA (Figure 2F). NK cells were cocultured with autologous osteoclasts and expanded from 10 healthy donors as shown in Figure 2A. The cumulative fold expansion of NK cells was calculated for each donor over 31 days (Figure 2G), and population doublings were calculated based on the logarithm of the ratio of the final count to the baseline count divided by the logarithm of 2 (Figure 2H). [Figure 2-4] Dendritic cells and osteoclasts were generated as shown in Figure 2A, and 1 x 10 cells were used to analyze ULBP, KIR2, KIR3, KLRG1, and MICA / B surface expression using PE-conjugated antibodies and flow cytometry analysis. An IgG2 isotype control antibody was used as a control (Figure 2I). [Figure 2-5] Freshly isolated NK cells (top row) and NK cells expanded in coculture with autologous osteoclasts (bottom row) as shown in Figure 2A were analyzed for surface expression of CD16, Nkp30, Nkp44, Nkp46, KIR2, KIR3, CD94, and NKG2D using PE-conjugated antibodies. An IgG2 isotype control antibody was used as a control (Figure 2J). [Figure 3-1]These eight panels, identified as panels A–H, demonstrate that, unlike NK cells, T cells purified from osteoclast-expanded NK cells do not mediate cytotoxicity against OSCSCs and moderately secrete IFN-γ. Freshly purified NK cells were processed and cocultured with monocyte-derived autologous osteoclasts as described in Materials and Methods. Surface expression of CD3, CD16, CD56, GL3 (TCRγ / δ), CD4, and CD8 was analyzed in lymphocyte samples from the coculture on day 9 using FITC- and PE-conjugated antibodies and flow cytometry (Figure 3A). NK cells were processed and cocultured with autologous osteoclasts as described in Figure 1A. On day 9, CD3 T-positive cells were sorted using a CD3 T-positive selection kit. The purity of CD3 T-negative (NK) cells was assessed using CD3, CD16, and CD56 FITC- and PE-conjugated antibodies and flow cytometry (Figure 3B). [Figure 3-2]CD3+ T cells and CD3+ T-negative cells (CD16+ cells) were treated with rh-IL-2 (1000 U / ml) for 18–20 hours and then tested for cytotoxicity against OSCSCs (Figure 3C) and K562 (Figure 3D) cell lines using a standard 4-hour 51Cr release assay. Lytic units of 30 / 106 cells were determined for OSCSCs and K562 cells, respectively, using the method described in Materials and Methods. Supernatants were harvested from the cultures, and IFN-γ secretion was determined using a single ELISA (Figure 3E). NK cells, CD3T, CD4T, CD8T, and γδT cells were purified from PBMCs as described in Materials and Methods and activated with rh-IL-2 for 18–20 hours, followed by testing for cytotoxicity against OSCSCs using a standard 4-hour 51Cr release assay (Figure 3F). The lytic unit (30 / 106 cells) was determined using the reciprocal number of lymphocytes required to lyse 30% of 100 OSCSCs (Figure 3F). NK and T cells were purified from PBMCs as described in Materials and Methods, and NK cells were treated as described in Materials and Methods. T cells were activated with anti-CD3 (1 μg / ml) and anti-CD28 (3 μg / ml) for 18–20 hours and then cultured with autologous OCs. Expanded lymphocytes were manually counted using a microscope after 4 days of culture (Figure 3G). NK and T cells were purified, cultured with OCs, and counted on day 4, as shown in Figure 3G. The fold expansion of lymphocytes expanded with OCs was divided by the fold expansion of lymphocytes without OCs (Figure 3H). [Figure 4-1]These 19 panels, identified as panels A–S, show the decrease in the percentage of NK cells, NK cell-mediated cytotoxicity, and IFN-γ secretion with each successive restimulation of NK cell cultures with osteoclasts and sAJ2 bacteria. Freshly purified NK cells were treated and cocultured with monocyte-derived autologous osteoclasts as shown in Figure 2A. Surface expression of CD3, CD16, and CD56 was analyzed in 1 × 10 lymphocytes from the coculture at the days indicated in the figure using flow cytometry analysis (Figure 4A). After 36 days, when NK cells ceased proliferation, they were recultured with new autologous osteoclasts as shown in Figure 2A. Surface expression of CD3, CD16, and CD56 was analyzed at the days indicated in the figure using antibody staining and flow cytometry analysis (Figure 4B). On day 63, when the cells stopped proliferating, they were recultured with OC as indicated, and surface expression of CD3, CD16, and CD56 was analyzed on the days indicated in the figure (Fig. 4C). [Figure 4-2] The number of proliferated lymphocytes was assessed using microscopic measurements (Figures 4D, 4G, 4J), and the numbers of NK (Figures 4F, 4I, 4L) and T / NKT (Figures 4E, 4H, 4K) cells were determined using the percentage of NK and T / NKT cells among the total number of proliferated cells (Figure 4D). [Figure 4-3] Cell death was determined in lymphocytes on days 36, 59, and 83 using propidium iodide staining and flow cytometry analysis (Figure 4M). Freshly purified NK cells were treated and cocultured with autologous osteoclasts as shown in Figure 2A. Lymphocytes were then tested for cytotoxicity against OSCSCs using a standard 4-hour 51Cr release assay at days 6, 17, and 34 of coculture (Figure 4N), 40 and 63 of coculture (Figure 4O), or 76 and 92 of coculture (Figure 4P). Lytic units of 30 / 106 cells were determined using the method shown in Figure 2E. Supernatants were harvested, and IFN-γ secretion was determined using a single ELISA using supernatants from days 6, 9, 12, 15, 18, and 21 (Figure 4Q); days 40, 46, 51, 55, and 59 (Figure 4R), and days 76, 83, 92, and 97 (Figure 4S). [Figure 5-1]These eight panels, identified as panels A–H, demonstrate that osteoclasts, but not K562 or OSCSCs, significantly increase NK cell proliferation and NK cell function. To generate osteoclasts, monocytes were cultured for 21 days in medium containing M-CSF (25 ng / ml) and RANKL (25 ng / ml), and the K562 tumor cell line was cultured as described in Materials and Methods. Highly purified NK cells (1 × 10 cells / ml) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16 mAb (3 μg / ml) for 18 hours and then cocultured with K562 and autologous OCs in the presence of sAJ2 bacteria at a ratio of 1:2:4 (OC:NK:sAJ2). Cultured cells were manually counted using a microscope on days 6, 10, and 13 (Figure 5A). Osteoclasts generated as shown in Figure 1A and the K562 tumor cell line were irradiated at 40 Gray (Gy) as described in Materials and Methods. NK cells were purified and treated as described in Materials and Methods, then co-cultured with irradiated K562 cells and irradiated autologous OCs in the presence of sAJ2 bacteria at a ratio of 1:2:4 (OC:NK:sAJ2), respectively. Cultured cells were manually counted using a microscope on days 6, 10, and 13 (Figure 5B). NK cells were purified and cultured with OCs and K562 cells as shown in Figure 1A. The cytotoxicity of the co-cultured lymphocytes was assessed against OSCSCs using a standard 4-hour 51Cr-release assay. Lytic units of 30 / 106 cells were determined using the method described in Figure 3F (Figure 5C). NK cells were purified and cultured with OC and K562 cells as shown in Figure 5B. The cytotoxicity of the co-cultured lymphocytes for 6 days was determined against OSCSCs using a standard 4-hour 51Cr release assay. Lytic units of 30 / 106 cells were determined using the method described in Figure 3F (Figure 5D). [Figure 5-2]NK cells were purified and cultured with OCs and OSCSCs as shown in Figure 5A. Lymphocyte cytotoxicity was assessed against OSCSCs using a standard 4-hour 51Cr-release assay after 6 days of coculture. Lytic units of 30 / 106 cells were determined using the method described in Figure 3F (Figure 5E). NK cells were purified and cultured with OCs and K562 as shown in Figure 3A. Supernatants were harvested on days 3, 6, 7, 10, and 13, and IFN-γ secretion was assessed using a single ELISA (Figure 5F). NK cells were purified and cultured with irradiated OCs and irradiated K562. Supernatants were harvested on days 3, 6, 7, 10, and 13, and IFN-γ secretion was assessed using a single ELISA (Figure 5G). NK cells were purified and cultured with OCs and OSCSCs. Supernatants were harvested on days 1, 3, 6, and 8, and IFN-γ secretion was assessed using a single ELISA (Figure 5H). [Figure 6-1] These 16 panels, identified as panels A–P, demonstrate that purified NK cells cultured with OCs from cancer patients outgrow T cells more than NK cells and mediate significantly lower cytotoxicity and cytokine secretion compared to those expanded from healthy donors. Freshly purified NK cells from healthy donors and cancer patients were treated and cocultured with monocyte-derived OCs as shown in Figure 2A. Surface expression of CD3, CD16, and CD56 was analyzed on expanded cells at days 6, 9, 12, 15, 18, 21, 24, 27, and 31 in cancer patients (Figure 6A) and healthy donors (Figure 6B) using antibody staining followed by flow cytometry analysis. [Figure 6-2] Cell death was assessed on expanded NK cells from cancer patients and healthy donors on day 19 using PI staining and flow cytometry analysis (Figure 6C). After 6, 9, 12, 15, 18, 21, 24, 27, and 31 days of coculture, expanded lymphocytes were manually counted using a microscope (Figure 6D). The numbers of NK cells (Figure 6E) and T / NKT cells (Figure 6F) were assessed using the percentage of NK and T / NKT cells among the total number of expanded cells in Figures 6A and 6B. Lymphocyte cytotoxicity against OSCSCs was assessed on days 12 and 15 using a standard 4-hour 51Cr release assay. Lytic units (30 / 106 cells) were determined using the method described in Figure 2E. [Figure 6-3] The numbers of NK cells (Figure 6E) and T / NKT cells (Figure 6F) were determined using the percentage of NK and T / NKT cells among the total number of proliferating cells in Figures 6A and 6B. Lymphocyte cytotoxicity was determined on days 12 and 15 against OSCSCs using a standard 4-hour 51Cr release assay. Lytic units of 30 / 106 cells were determined using the method described in Figure 2E. Lytic units from Figure 6F were normalized based on NK cells (Figure 6H). Supernatants were harvested from overnight, 6, 9, 12, 15, 18, 21, 24, and 27 days of co-culture, and IFN-γ (Figure 6I), IL-10 (Figure 6J), and IL-6 (Figure 6K) secretion was determined using single ELISAs. [Figure 6-4] Supernatants were harvested from overnight, 6, 9, 12, 15, 18, 21, 24, and 27 days of coculture, and IFN-γ (Figure 6I), IL-10 (Figure 6J), and IL-6 (Figure 6K) secretion was assessed using a single ELISA. Freshly purified NK cells from a healthy donor, a cancer patient with tonsillar cancer (patient #1), and a cancer patient with pancreatic cancer (patient #2) were treated and cocultured with OCs as shown in Figure 2A. Surface expression of CD3, CD16, and CD56 was analyzed on lymphocytes from the 21-day coculture of healthy donor and patient NK cells and the 87-day coculture of healthy donors (third stimulation) (Figure 6L). Surface expression of Nkp30, Nkp44, Nkp46, KIR2, KIR3, CD94, and NKG2D was analyzed in CD16-positive cells (Figure 6M). An IgG2 isotype control antibody was used as a control (FIGS. 6L and 6M). [Figure 6-5] Freshly purified NK cells from a healthy donor, a cancer patient with tonsillar cancer (patient #1), and a cancer patient with pancreatic cancer (patient #2) were treated and cocultured with OCs as shown in Figure 2A. Surface expression of CD3, CD16, and CD56 was analyzed on lymphocytes from the 21-day coculture of healthy donor and patient NK cells and the 87-day coculture of healthy donors (third stimulation) (Figure 6L). Surface expression of Nkp30, Nkp44, Nkp46, KIR2, KIR3, CD94, and NKG2D was analyzed in CD16-positive cells (Figure 6M). An IgG2 isotype control antibody was used as a control (Figures 6L and 6M). [Figure 6-6] Supernatants were harvested from the cocultures on day 13, and equal volumes of supernatant (200 μl) from each donor were used to differentiate OSCSCs overnight. The levels of MHC-I, CD54, CD44, and B7H1 surface expression were then assessed on the OSCSCs. An IgG2 isotype control antibody was used as a control (Figure 6N). Cell death was assessed in untreated and NK cell supernatant-differentiated OSCSCs using propidium iodide staining and flow cytometry analysis (Figure 6O). Highly purified NK cells were treated with IL-2 (1000 U / ml) and used to assess cytotoxicity against untreated and NK cell supernatant-differentiated OSCSCs in a 4-hour 51Cr release assay. Lytic units (30 / 106 cells) were determined using the method described in Figure 2E (Figure 6P). [Figure 7-1] These 11 panels, identified as panels A–K, demonstrate that the very small number of contaminating T cells in purified NK cells from cancer patients proliferates faster and swarms the NK cells, presumably due to impaired NK cell function. Freshly purified NK cells from healthy donors and pancreatic cancer patients were processed and cocultured with monocyte-derived allogeneic osteoclasts (from a different healthy donor) as shown in Figure 1A. Surface expression of CD3, CD16, and CD56 was analyzed in 1 x 10 lymphocyte samples from the cocultures at days 6, 10, 13, 17, 21, 24, 28, 32, and 36 using FITC- and PE-conjugated antibodies and flow cytometry in cancer patients (Figure 7A) and healthy donors (Figure 7B). [Figure 7-2] After 6, 10, 13, 17, 21, 24, 28, and 32 days of coculture, expanded lymphocytes were manually counted using a microscope (Figure 7C). Cells were counted as described in Figure 7C and adjusted based on the surface expression analyzed in Figures 7A and 7B. The number of T / NKT cells (Figure 7D) and NK cells was determined daily (Figure 7E). The cytotoxicity of lymphocytes cocultured for 18–20 h, 13, 20, and 32 days was determined against OSCSCs using a standard 4-h 51Cr release assay. The lytic units (30 / 106 cells) were determined using the method described in Figure 3F (G). [Figure 7-3]Lytic units of 30 / 10 cells were determined using the method described in Figure 3F (G). Lytic units from Figure 7F were adjusted based on the surface expression analyzed in Figures 7A and 7B to determine NK cell-mediated cytotoxicity against OSCSCs (Figure 7H). Supernatants were harvested from overnight, 6, 10, 13, 17, 21, 24, 28, and 32 days of co-culture, and IFN-γ (Figure 7I), IL-10 (Figure 7J), and IL-6 (Figure 7K) secretion was determined using a single ELISA. [Figure 8] The phenotype of CD3 T cell-depleted lymphocytes from hu-BLT mice splenocytes is shown. Humanized BLT (hu-BLT; human bone marrow / liver / thymus) mice were generated by surgically transplanting human fetal liver and thymus tissue under the kidney capsule of 6-8 week-old immunodeficient NOD.CB17-Prkdcscid / J and NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ (NSG) mice. Four to six weeks after tissue transplantation, mice were sublethally irradiated, and CD34+ cells isolated from the fetal liver were intravenously injected to support complete reconstitution of human bone marrow. Eight to 12 weeks after CD34+ cell injection, human immune system reconstitution was analyzed using blood samples. At the end of the experiment, human immune cell engraftment was confirmed by staining splenocytes and bone marrow cells with anti-human CD45, CD3, CD4, and CD8 antibodies and analyzed by flow cytometry (data not shown). Successfully reconstituted hu-BLT mice (with T cell levels and lineage comparable to healthy donors) were orthotopically injected with 1 x 10 human OSCSCs into the floor of the mouth. Disease progression and weight loss were monitored for an additional 3–4 weeks. Animals were sacrificed, spleens were harvested from sacrificed animals, and single-cell suspensions were obtained as described in Materials and Methods. CD3+ T cells were selected using a human CD3+ T-positive selection kit. Flow-through cells (CD3-negative cells) were analyzed for surface expression of human CD3, CD16, CD56, CD45, CD19, and CD14 after staining with PE-conjugated, PE-Cy5-conjugated, and FITC-conjugated antibodies, respectively. Isotype control antibodies were used as controls. [Figure 9-1]These 11 panels, identified as Panels A–K, demonstrate that T cell-depleted, in vitro-expanded lymphocytes from tumor-bearing humanized BLT mice expanded T cells, contained fewer NK cells, and mediated reduced cytotoxicity compared with those obtained from healthy hu-BLT mice. 1 × 10 human OSCSCs were orthotopically injected into the floor of the mouth of reconstituted BLT mice (with T cell levels and lineage comparable to those of healthy donors). Disease progression and weight loss were monitored for an additional 4–5 weeks. Mice were sacrificed, spleens harvested, and single-cell suspensions were obtained as described in Supplementary Materials and Methods. CD3+ T cells were sorted using a positive selection kit, and flow-through cells were analyzed for surface expression of human CD3, CD16, and CD56 after staining with the respective antibodies. Isotype control antibodies were used as controls (Figure 9A). CD3-negative cells (1 × 10 cells / ml) from hu-BLT mice were treated with a combination of IL-2 (1000 U / ml) and anti-CD16 mAb (3 μg / ml) for 18 hours, and then cultured with OCs in the presence of sAJ2 at a ratio of 1:2:4 (OC:NK:sAJ2). Surface expression of CD3, CD16, and CD56 was analyzed by flow cytometry on days 6, 10, 14, 18, and 22 (Figure 9B). [Figure 9-2] After 6, 10, 18, and 22 days of coculture, expanded lymphocytes were manually counted using a microscope (Figure 9C). The numbers of NK cells (Figure 9D) and T / NKT cells (Figure 9E) were determined using the percentage of NK and T / NKT cells among the total number of expanded cells. The cytotoxicity of NK cells cocultured for 10 and 18 days was determined against OSCSCs using a standard 4-hour 51Cr-release assay, and the lytic units (30 / 106 cells) were determined using the reciprocal number of NK cells required to lyse 30% of OSCSCs × 100 (Figure 9F). [Figure 9-3]Lytic units were normalized and adjusted for NK cell lysis against OSCSCs (Figure 9G). Supernatants were harvested from co-cultures on days 6, 10, and 13, and IFN-γ (Figure 9H), IL-10 (Figure 9I), and IL-6 (Figure 9J) secretion was determined using a single ELISA. Peripheral blood was collected from hu-BLT mice after death by cardiac puncture, and serum samples were harvested and analyzed for IFN-γ, IL-10, and IL-6 secretion using multiplexed arrays (Figure 9K). [Figure 10a] These three panels, identified as Panels A–C, show cytokines, chemokines, and growth factors and ligands secreted by primary NK cells and NK cells expanded by osteoclasts. Highly purified NK cells and monocytes were obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors. NK cells (1 × 10 cells / ml) were treated with IL-2 (1000 U / ml) for 18 hours, after which the supernatant was harvested. To generate osteoclasts, monocytes were cultured for 21 days in alpha-MEM medium containing M-CSF (25 ng / ml) and RANKL (25 ng / ml). For expansion, purified NK cells (1 × 10 cells / ml) were treated with a combination of IL-2 (1000 U / ml) and anti-CD16 mAb (3 μg / ml) for 18 hours, and then co-cultured with autologous osteoclasts in the presence of sAJ2 bacteria at a ratio of 1:2:4 (OC:NK:sAJ2). Supernatants were harvested after 6 days of co-culture, and multiplex assays were used to determine cytokine (Figure 10A), chemokine (Figure 10B), and growth factor (Figure 10C) levels secreted by primary and expanded NK cells. [Figure 10b] Supernatants were harvested after 6 days of co-culture, and multiplex assays were used to determine cytokine (Figure 10A), chemokine (Figure 10B), and growth factor (Figure 10C) levels secreted by primary and expanded NK cells. [Figure 10c] Supernatants were harvested after 6 days of co-culture, and multiplex assays were used to determine cytokine (Figure 10A), chemokine (Figure 10B), and growth factor (Figure 10C) levels secreted by primary and expanded NK cells. [Figure 11]These three panels, identified as panels A–C, demonstrate that blocking IL-12, IL-15, or a combination of both resulted in decreased NK cell proliferation, NK cell-mediated cytotoxicity, and cytokine secretion. Freshly purified NK cells from healthy donors were treated and cocultured with autologous osteoclasts at 100 ng / ml and 1 μg / ml in the presence and absence of anti-IL-12, -IL-15, or a combination of anti-IL-12 and -IL-15 mAbs, respectively, as shown in Figure 2A. IL-2 (1000 units / mL) was replenished every two days. NK cells were counted microscopically on days 6, 8, 12, 14, and 20 (Figure 11A). On days 9 and 15, 1 × 10 NK cells from each expanded sample were used for standard 4-hour 51Cr release assays against OSCSCs. Lytic units (30 / 106 cells) were determined using the reciprocal number of NK cells required to lyse 30% of OSCSCs × 100 (Figure 11B). Supernatants were harvested from co-cultures on days 8, 12, 15, and 20, and IFN-γ secretion was determined using a simple ELISA (Figure 11C). [Figure 12-1] These seven panels, identified as panels A–G, demonstrate that the addition of anti-CD3 antibody inhibits T cell proliferation and increases NK cell proliferation in OC-expanded donors. Freshly purified NK cells from healthy donors and cancer patients were expanded in OC for 27 days, after which the cultures were treated with rh-IL-2 and anti-CD3 (1 μg / ml). NK cell (FIG. 12A) and T cell (FIG. 12B) numbers were then determined by microscopic evaluation on days 29, 31, and 35 (both healthy and patient). Loss of forward and side scatter was determined in healthy cells (FIG. 12C) and patient cells (FIG. 12D) treated as shown in FIGS. 12A and 12B. [Figure 12-2] Cells were stained with PI and analyzed for DNA fragmentation (Figure 12E). Day 31 lymphocytes were used in a standard 4-hour 51Cr release assay against OSCSCs. Lytic units of 30 / 106 cells were determined using the reciprocal number of NK cells required to lyse 30% of OSCSCs x 100 (Figure 12F). Supernatants were harvested from co-cultures on day 35, and IFN-γ secretion was determined using a single ELISA (Figure 12G). [Figure 13]This figure shows that purified T cells treated with anti-CD3 mAb in the absence of NK cells did not lose forward and side scatter. Highly purified NK cells and monocytes were obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors. T cells (1 × 10 cells / ml) were treated with IL-2 (100 U / ml) and anti-CD3 (1 μg / ml) for 18 hours and then cocultured with autologous osteoclasts in the presence of sAJ2 bacteria at a ratio of 1:2:4 (OC:T cells:sAJ2). Cells were analyzed for CD3, CD16, and CD56 after 9 days of culture. Anti-CD3-treated T cells did not lose forward and side scatter in the absence of NK cells. [Figure 14-1] This figure includes eight panels identified as panels A–H, and shows that osteoclast-activated NK cells substantially increase CD8+ T cell numbers. PBMCs from healthy donors and cancer patients were analyzed for surface expression of CD3, CD4, and CD8 using PE- and FITC-conjugated antibody staining, followed by flow cytometry analysis (Figure 14A). Freshly purified NK cells from healthy donors and cancer patients were treated and cocultured with OCs as shown in Figure 2A. T cells were purified from PBMCs of healthy donors and cancer patients using a CD3-positive selection kit. T cells were activated with a combination of rh-IL2 (100 U / ml) and anti-CD3 (1 μg / ml) and anti-CD28 mAbs (1 μg / ml) for 18–20 hours, then cocultured with OCs in the presence of sAJ2 at a ratio of 1:2:4 (OC:T cells:sAJ2). Surface expression of CD3, CD4, and CD8 was analyzed on lymphocytes (Figure 14B). Monocytes were purified from human PBMCs to generate OCs and DCs, and purified NK cells were cocultured as shown in Figure 2A. The number of expanded lymphocytes was assessed using microscopic measurements (Figure 14C). The numbers of T cells (Figure 14D) and NK cells (Figure 14E) were determined using the percentages of NK and T cells, as shown in Figure 2A, within the total number of expanded cells in Figure 14C. [Figure 14-2]Lymphocytes were analyzed for surface expression of CD3+CD4+ and CD3+CD8+ cells, and the numbers of CD3+CD4+ T cells (Figure 14F) and CD3+CD8+ T cells (Figure 14G) were determined using the percentages of CD4 and CD8 cells among the total number of T cells in panel D. NK cells activated by OCs expanded T cells, NK cells activated by DCs expanded T cells, and T cells expanded / activated by OCs and T cells expanded / activated by DCs were stained with antibodies against CD45RO, CD62L, CD28, CD44, CCR7, and CD127 and analyzed by flow cytometry. The numbers in the second quadrant represent the percentage of cells positive for each antibody within CD3+ T cells (Figure 14H). [Figure 15] This figure includes two panels, identified as Panels A and B, and shows that NK cells expanded by osteoclasts retained their cytokine secretion and cytotoxic function after freezing. Freshly purified NK cells were processed and co-cultured with monocyte-derived autologous osteoclasts after 9 days of culture, and the expanded NK cells were frozen as shown in Figure 1A. NK cells were thawed and treated with rh-IL-2 (1000 U / ml) after 6 and 9 days of culture. Supernatants were harvested, and IFN-γ secretion was determined using a single ELISA (Figure 15A). NK cells were cultured as shown in Figure 1A, and lymphocyte cytotoxicity after 6 and 9 days of culture was determined against OSCSCs using a standard 4-hour 51Cr-release assay. Lytic units of 30 / 106 cells were determined using the method described in Figure 3F (Figure 15B). [Figure 16] The five panels identified as panels A-E show the reduction in the number of PBMCs obtained from the peripheral blood of pancreatic (FIG. 16B), colon (FIG. 16C), oral (FIG. 16D), and prostate (FIG. 16E) cancer patients. FIG. 16A shows the reduction in patients versus healthy controls. [Figure 17-1] The five panels identified as panels A-E show that the percentages of NK and CD14 monocytes were increased, whereas T cells and B cells were significantly decreased, from PBMCs obtained from peripheral blood of healthy subjects (FIG. 17A), pancreatic (FIG. 17B), colon (FIG. 17C), oral (FIG. 17D), and prostate (FIG. 17E) cancer patients. [Figure 17-2] The five panels identified as panels A-E show that the percentages of NK and CD14 monocytes were increased, whereas T cells and B cells were significantly decreased, from PBMCs obtained from peripheral blood of healthy subjects (FIG. 17A), pancreatic (FIG. 17B), colon (FIG. 17C), oral (FIG. 17D), and prostate (FIG. 17E) cancer patients. [Figure 18] It includes four panels identified as panels A-D, showing the reduced NK cell cytotoxicity of patient NK cells compared to healthy NK cells. [Figure 19-1] It includes eight panels identified as panels AH, showing NK cells expanded by patient-derived osteoclasts and their cytotoxicity and secretion of IFN-γ. [Figure 19-2] It includes eight panels identified as panels AH, showing NK cells expanded by patient-derived osteoclasts and their cytotoxicity and secretion of IFN-γ. [Figure 20] 1 shows cytokine secretion in non-osteoclast-expanded NK cells from pancreatic cancer patients. [Figure 21] The five panels identified as panels A-E show IFN-γ secretion by osteoclast-expanded T cells from healthy subjects (FIG. 21A), pancreatic (FIG. 21B), colon (FIG. 21C), oral (FIG. 21D), and prostate (FIG. 21E) cancer patients. [Figure 22] Includes six panels identified as panels A-F, showing IFN-γ secretion from NK cells versus T cells (FIG. 22A), healthy subjects (FIG. 22B), pancreatic (FIG. 22C), colon (FIG. 22D), prostate (FIG. 22E), and oral (FIG. 22F) cancer patients, and from NK cells, T cells, and osteoclast-expanded NK cells and T cells. [Figure 23] It includes four panels identified as panels A to D, showing T cell secretion of IFN-γ from healthy subjects (FIG. 23A), pancreatic (FIG. 23B), colon (FIG. 23C), and prostate (FIG. 23D) cancer patients. [Figure 24]Includes six panels identified as panels A-F, showing cumulative NK cells vs. T cells (Figure 24A), NK cells from healthy subjects (Figure 24B), and total numbers of NK cells activated through proliferating T cells vs. T cell effects activated through surface receptor crosslinking determined within 0-15 days in pancreatic (Figure 24C), colon (Figure 24D), oral (Figure 24E), and prostate (Figure 24F) cancer patients. [Figure 25] Includes five panels identified as Panels A-E, showing the ability to proliferate T cells from pancreatic (FIG. 25B), colon (FIG. 25C), and prostate (FIG. 25D) cancer patients compared to the ability of healthy individuals (FIG. 25A) assessed within 0-15 days. [Figure 26] 1 shows the reduction of cytokines and chemokines in the serum of patients versus healthy individuals. [Figure 27] Two panels, A and B, are included, showing the percentages of CD4 and CD8 T cells in PBMCs from pancreatic (FIG. 27A) and colon (FIG. 27B) cancer patients compared to healthy individuals. [Figure 28] The figure includes five panels, A-E, showing the ratio of CD4 / CD8 T cells in PBMCs of pancreatic (Figure 28B), colon (Figure 28C), oral (Figure 28D), and prostate (Figure 28E) cancer patients versus healthy individuals (Figure 28A). [Figure 29-1] Contains five panels showing the ratio of CD4 to CD8 T cells in healthy subjects (Figure 29A) versus pancreatic (Figure 29B), colon (Figure 29C), oral (Figure 29D), and prostate (Figure 29E) cancer patients. [Figure 29-2] Contains five panels showing the ratio of CD4 to CD8 T cells in healthy subjects (Figure 29A) versus pancreatic (Figure 29B), colon (Figure 29C), oral (Figure 29D), and prostate (Figure 29E) cancer patients. [Figure 30] Two panels, A and B, are included, showing the effect of culturing CD8 and CD4 T cells without (FIG. 30A) and with (FIG. 30B) osteoclasts. [Figure 31] IFN-γ secretion from NK and CD8 T cells is shown. [Figure 32]1 shows CD8 and CD4 T cell proliferation promoted by osteoclasts and NK cells, respectively. [Figure 33] Cytotoxicity of NK cells expanded by osteoclasts against cancer stem cells / undifferentiated tumors. [Figure 34] 1 shows the effect of osteoclast-expanded NK cells on NK cell proliferation and the cytotoxic effect of osteoclast-expanded NK cells. [Figure 35] Figure 1 shows T cells with an effector memory phenotype among NK cells expanded by OC compared to NK cells expanded by DC. [Figure 36-1] The number of T cells with an exhausted phenotype among NK cells expanded by OC versus NK cells expanded by DC is shown. [Figure 36-2] The number of T cells with an exhausted phenotype among NK cells expanded by OC versus NK cells expanded by DC is shown. [Figure 37] IFN-γ-expressing NK cells among OC-expanded NK cells from a pancreatic cancer patient are shown. [Figure 38] Table showing CD8 and NK-specific cytokines, costimulatory ligands, granzymes, perforin, and soluble Fas and Fas ligand secreted by OC-expanded T cells. [Figure 39] Table showing CD8-associated cytokines, chemokines, costimulatory ligands, sFas and Fas ligand, granzymes, and perforin secreted by CD8+ T cells from OC-expanded NK cell cultures. [Figure 40] Shown are levels of GM-CSF, IFN-g, IL-10, TNF-a, the low costimulatory ligand sCD137, granzymes, perforin, soluble Fas, and Fas ligand secreted from CD8 cells expanded by NK or OC. [Figure 41] 1 shows the cytotoxic activity of NK cells from oral tumor-implanted BLT mice compared to tumor-free mice. [Figure 42]CD8+ T cells in the BM, spleen, and blood after immunotherapy with supercharged NK cells following tumor implantation are shown. [Figure 43] Serum IFN-γ, IL-6, ITAC, GM-CSF, and IL-8 after immunotherapy with supercharged NK cells in BLT mice after tumor implantation are shown. [Figure 44] 1 shows the cytotoxicity of patient NK cells expanded with osteoclasts at various days of expansion. [Figure 45-1] The number of NK cells in the NK cell population expanded by osteoclasts in patients at various days of expansion is shown. [Figure 45-2] The number of NK cells in the NK cell population expanded by osteoclasts in patients at various days of expansion is shown. [Figure 46] The number of osteoclast-expanded NK cells compared with DC-expanded NK cells on days 15-25 is shown. [Figure 47] The number of osteoclast-expanded NK cells compared with DC-expanded NK cells at various days of expansion is shown. [Figure 48] The number of DC-expanded T cells compared with osteoclast-expanded T cells at various days of expansion is shown. [Figure 49] Cytotoxicity of osteoclast-expanded NK cells compared to DC-expanded NK cells at various days of expansion is shown. [Figure 50] IFN-γ secretion by primary, non-osteoclast-expanded and osteoclast-expanded patient NK cells compared to healthy donor NK cells at various days of expansion is shown. [Figure 51] IFN-γ secretion by primary, non-osteoclast-expanded and osteoclast-expanded patient T cells compared to healthy donor T cells at various days of expansion is shown. [Figure 52-1] IFN-γ secretion by osteoclast-expanded patient NK cells compared with that obtained from healthy donor NK cells at various days of expansion is shown. [Figure 52-2] IFN-γ secretion by osteoclast-expanded patient NK cells compared with that obtained from healthy donor NK cells at various days of expansion is shown. [Figure 53-1] Figure 1 shows IFN-γ secretion by primary, non-osteoclast-expanded and osteoclast-expanded patient T cells compared with that obtained from healthy donor T cells in several patients at various days of expansion. [Figure 53-2] Figure 1 shows IFN-γ secretion by primary, non-osteoclast-expanded and osteoclast-expanded patient T cells compared with that obtained from healthy donor T cells in several patients at various days of expansion. [Figure 54] IFN-γ secretion (combined secretion from all expansion days) by primary, non-osteoclast-expanded, and osteoclast-expanded patient T cells (T cells were positively selected) compared to that obtained from healthy donor T cells at various expansion days is shown. [Figure 55] IFN-γ secretion by positively selected primary, non-osteoclast-expanded, and osteoclast-expanded patient T cells (combined secretion from all expansion days) compared to negatively selected T cells from healthy donors is shown. [Figure 56] Figure 1 shows IFN-γ secretion / cell by osteoclast-expanded T cells (positively selected T cells) compared with NK cells obtained from healthy donors. Primary positively selected T cells activated with IL-2 secreted more IFN-γ when compared with NK cells. [Figure 57] IFN-γ secretion / cell by primary, non-osteoclast and osteoclast-expanded patient T cells (T cells were positively selected) compared to T cells obtained from healthy donors. [Figure 58]IFN-γ secretion / cell by osteoclast-expanded patient T cells (T cells were positively selected) and NK cells. IL-2-activated primary, non-osteoclast-expanded T cells secreted more IFN-γ when compared to IL-2-treated primary NK cells. [Figure 59] 1 shows the increased number of expanded cells by positively selected primary, non-osteoclast-expanded and osteoclast-expanded T cells compared to negatively selected NK cells or negatively selected T cells from healthy donors. [Figure 60] 1 shows the reduced number of expanded cells by positively selected primary, non-osteoclast-expanded and osteoclast-expanded patient T cells compared to those obtained from healthy donors. [Figure 61-1] 1 shows serum cytokine and chemokine levels from the blood of pancreatic patients. [Figure 61-2] 1 shows serum cytokine and chemokine levels from the blood of pancreatic patients. [Figure 62-1] This figure includes 11 panels, identified as panels A–C, and shows that a single injection of supercharged NK cells inhibited tumor growth in hu-BLT mice, regardless of whether AJ2 was administered. Hu-BLT mice were generated as described in Materials and Methods and are shown in the figure (Figure 62A). Hu-BLT and NSG mice were orthotopically implanted with 1×10 human OSCSCs into the floor of their mouths. Seven to 10 days later, a group of hu-BLT mice was injected with 1.5×10 supercharged NK cells via the tail vein, and the mice were monitored for disease progression. Another group of hu-BLT mice was fed AJ2 probiotic bacteria (5 million cells / day) every 48 hours for 2 weeks before OSCSC implantation and after tumor implantation, with or without NK injection, and the experiment was terminated (Figure 62B). Weight loss was monitored by weighing the mice weekly. One representative experiment of three is shown in this figure (Fig. 62C). [Figure 62-2]At the end of the experiment, mice were sacrificed, and tumors were photographed after excision (Figure 62D) and weighed (n = 4) (Figure 62E). As shown in Figure 62B, mice were transplanted with human OSCSCs, injected with NK cells, and fed AJ2. Tumors were excised and weighed postmortem (n = 4) (Figure 62F). PBMCs were isolated from hu-BLT mice and humans, and surface expression of human CD3 (n = 5) (Figure 62G), CD4 (n = 5) (Figure 62H), CD8 (n = 5) (Figure 62I), CD19 (n = 3) (Figure 62J), and CD16 (n = 5) (Figure 62K) was determined in CD45+ immune cells using antibody staining and then analyzed by flow cytometry. [Figure 63-1] The nine panels, labeled A–I, show that infusion of supercharged NK cells restored and increased IFN-γ secretion and cytotoxic function of NK cells in the blood, spleen, BM, enriched NK cells, and purified CD3+ T cells in tumor-bearing hu-BLT mice, regardless of whether they were fed AJ2. As shown in Figure 62B, Hu-BLT mice were transplanted with human OSCSCs, infused with NK cells, and fed AJ2. One week after NK cell infusion, the mice were injected with anti-PD1 (50 μg / mouse) via tail vein injection. After sacrifice, spleens (n=5) (Figure 63A), BM (n=5) (Figure 63B), and peripheral blood (n=5) (Figure 63C) were collected, and single-cell suspensions were prepared from each tissue (1x106 cells / ml for spleen and BM, and 0.7x106 cells / ml for PBMCs) and treated with IL-2 (1000 U / ml) for 7 days. NK-enriched cells were isolated from splenocytes (1x106 cells / ml) and treated with IL-2 (1000 U / ml) for 7 days (n=3) (Figure 63D). [Figure 63-2]Cytotoxicity assays were performed on OSCSCs using a standard 4-hour 51Cr-release assay, and LU30 / 106 cells was determined using the reciprocal number of cells required to lyse 30% of OSCSCs × 100. Splenocytes (n = 5) (Figure 63E), BM cells (n = 5) (Figure 63F), and PBMCs (n = 5) (Figure 63G) at 1 × 106 cells / ml for spleen and BM, and 0.7 × 106 cells / ml for PBMCs were treated with IL-2 (1000 U / ml), respectively. Positively selected CD3+ T cells (n = 4) from splenocytes at 1 × 106 cells / ml were treated with IL-2 (100 U / ml) (Figure 63H) for 7 days, after which supernatants were harvested and IFN-γ levels were determined using a specific ELISA. The fold change in IFN-γ secretion in each tissue from each group of mice was determined from that obtained from mice injected with OSCSCs alone (FIGS. 63E-63H). [Figure 64] Figure 62B includes two panels identified as A and B, showing that a single infusion of supercharged NK cells increased the number of CD8+ T cells in hu-BLT mice, regardless of whether they were fed AJ2. As shown in Figure 62B, Hu-BLT mice were transplanted with OSCSCs, infused with NK cells, and fed AJ2. The percentage of human CD8+ T cells within BM cells (n=3) (Figure 64A) and splenocytes (n=3) (Figure 64B) was determined using antibody staining and then analyzed by flow cytometry. [Figure 65-1]This panel includes 10 panels identified as A–J, and shows that a single injection of supercharged NK cells mediated in vivo tumor differentiation, increased IFN-γ secretion, and mobilized a greater number of human immune cells into tumors, resulting in reduced ex vivo tumor growth, regardless of whether BLT mice were fed AJ2. Hu-BLT and NSG mice were transplanted with OSCSCs and injected with NK cells, as shown in Figure 62B. After sacrifice, oral tumors were harvested, single-cell suspensions were prepared, and equal numbers of cells from each group (1 × 10 cells / ml, for a total of 3 × 10 cells) were cultured on day 0. On day 10, the supernatant was removed, and the attached tumor cells were counted. In subsequent cultures, the numbers in each group were adjusted to the number obtained from NK-injected mice, as they grew the fewest tumors. On days 10, 14, 19, and 20, the total number of ex vivo-growing tumor cells was determined for each group. One of several representative experiments is shown in this figure (Figure 65A). As shown in Figure 62B, Hu-BLT mice were transplanted with OSCSCs, in vitro NK-differentiated OSCSCs (differentiated OSCSCs), or NK-differentiated OSCSCs treated with antibodies against IFN-γ and TNF-α, and differentiation was blocked. After sacrifice, oral tumors were dissociated, single cells were prepared, and cultured (at 1 x 106 cells / ml for a total of 3 x 106 cells). The number of proliferating tumor cells was determined, as shown in Figure 65A. One of several representative experiments is shown in this figure (Figure 65B). As shown in Figure 62B, Hu-BLT mice were transplanted with OSCSCs, differentiated OSCSCs, or differentiated OSCSCs treated with antibodies against IFN-γ and TNF-α, and NK cells were injected and / or fed AJ2. After sacrifice, oral tumors were harvested and cultured, and the number of proliferating tumor cells was determined (n=7) (Figure 65C), as shown in Figure 65A. As shown in Figure 62A, OSCSCs were transplanted into Hu-BLT and NSG mice, and then NK cells were injected into the hu-BLT mice. Oral tumors were harvested, and single-cell suspensions were prepared. The percentage of infiltrating hu-CD45C immune cells within nonadherent cells on day 12 of culture was determined using antibody staining and then analyzed by flow cytometry. One representative image of three is shown in this figure (Figure 65D).Oral tumors from hu-BLT and NSG mice were cultured as shown in Figure 4A and treated with IL-2 (1000 U / ml). Their supernatants were harvested on the days indicated, and IFN-g levels were determined using ELISA. One representative image is shown in this figure (Figure 65E). [Figure 65-2] Expression of human CD54 and MHC-I was assessed on day 10 in oral tumor cultures from hu-BLT and NSG mice using flow cytometry analysis after staining with the respective antibodies. One representative experiment is shown in this figure (Figure 65F). Purified NK cells (1 × 10 cells / ml) from peripheral blood of healthy human donors were either untreated or treated with IL-2 (1000 U / ml) for 18 hours before addition to 51Cr-labeled OSCSCs cultured from resected tumors of different experimental groups of hu-BLT mice. This was compared with cultures of OSCSCs maintained in the laboratory at various effector-to-target ratios. NK cell-mediated cytotoxicity was assessed using a standard 4-hour 51Cr-release assay. LU30 / 106 cells was determined (n = 4) as described in Materials and Methods (Figures 65G and 65H). As shown in Figure 65A, oral tumor cells from hu-BLT mice were treated with IL-2 (1000 U / ml). Supernatants were harvested after 3 and 7 days, and the level of VEGF secretion was determined using a specific ELISA. The reduction in VEGF secretion by tumors obtained from NK-injected animals (n = 6) was calculated based on the amount obtained from mice injected with OSCSCs alone (Figure 65I). The percentage of infiltrating hu-CD45C immune cells within oral tumors dissociated from different experimental groups of hu-BLT mice was determined using flow cytometry analysis after antibody staining. One representative experiment is shown in this figure (Figure 65J). [Figure 66]A single infusion of supercharged NK cells, including three panels identified as A–C, restored and increased the secretion of cytokines, chemokines, and growth factors in serum obtained from peripheral blood of hu-BLT mice, regardless of whether tumor-bearing mice were fed AJ2. Serum from peripheral blood was obtained as described in Example 4, and a multiplexed array was performed to determine IFN-γ secretion; one of four representative figures is shown here (Figure 66A). IFN-γ fold changes were determined based on values ​​obtained from control hu-BLT mice (n=5) (Figure 66B). A multiplexed array was also used to determine the secretion of cytokines, chemokines, and growth factors in serum obtained from peripheral blood (Figure 66C). [Figure 67] This figure includes two panels labeled A and B, showing that CDDP or paclitaxel induces significant cell death in OSCSCs differentiated with NK supernatant but not in poorly differentiated tumors, regardless of the presence or absence of NAC. Highly purified NK cells were treated with a combination of IL-2 (1000 U / mL) and anti-CD16 mAb (3 mg / mL) for 18 hours, after which NK supernatant was added to OSCSCs in the presence of anti-TNF-α (1:100) and anti-IFN-γ (1:100) for 5 days. OSCSCs were then detached and treated with or without cisplatin for 18–24 hours. OSCSC viability was determined using PI staining and flow cytometry analysis. One representative experiment from three is shown in this figure (Figure 67A). OSCSCs were treated with supernatant from NK cells as shown in Figure 67A. Tumors were then removed and treated with or without NAC (20 nM) for 24 hours, followed by paclitaxel treatment for 18–24 hours. OSCSC viability was assessed by PI staining and flow cytometry analysis. One representative experiment from three was shown in this figure (Figure 67B). [Figure 68]The five panels, identified as A–E, show that monocytes or osteoclasts from tumor-bearing mice injected with NK cells or transplanted with NK-differentiated OSCSC tumors alone induced significantly more IFN-γ from autologous or allogeneic NK tumor cocultures compared with NK cell-bearing mice transplanted with tumors alone. As shown in Figure 62B, Hu-BLT mice were transplanted with OSCSCs, injected with NK cells, and fed AJ2. After sacrifice, NK cells from splenocytes and monocytes from BM cells were isolated as described herein. Autologous NK cells were left untreated or treated with IL-2 (1000 U / ml) in combination with monocytes (NK:monocytes, 2:1). After 7 days of coculture, NK cells were used as effector cells in a standard 4-hour chromium release assay against OSCSCs. LU30 / 106 cells was determined using the reciprocal number of NK cells required to lyse 30% of 100 target cells (Figure 68A). Autologous NK cells were left untreated or treated with IL-2 (1000 U / ml) or a combination of IL-2 (1000 U / ml) and LPS (100 ng / ml) in the absence and presence of monocytes (NK:monocytes, 2:1) for 7 days. Supernatants were harvested, and IFN-γ secretion was assessed using a single ELISA (Figure 68B). OCs were generated from purified hu-BLT monocytes as described in Example 4. Purified allogeneic NK cells from healthy human donors were pretreated with IL-2 (1000 U / ml) and anti-CD16 mAb (3 mg / ml) for 18 hours and then cultured with hu-BLT-OCs in the presence of sAJ2 (NK:OC:sAJ2, 2:1:4). After culture, the number of NK cells in the cultures was counted microscopically on days 5, 8, 12, and 15 (Figure 68C). Supernatants were harvested from cultures on days 5, 8, 12, and 15, and IFN-g secretion was determined using a single ELISA (Figure 68E). IFN-g levels obtained from the ELISA were determined at 1 x 10 cells using the cell count from Figure 68C. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates, in part, to methods for activating NK cells in vitro or ex vivo, comprising culturing NK cells in a medium containing osteoclasts (OCs). Similarly, provided herein are methods for activating T cells in vitro or ex vivo, comprising culturing T cells in a medium containing dendritic cells (DCs). The present invention further provides methods for activating NK cells relative to T cells in vitro or ex vivo, comprising culturing NK cells and T cells in a medium containing osteoclasts (OCs). The present invention further provides methods for activating T cells relative to NK cells in vitro or ex vivo, comprising culturing NK cells and T cells in a medium containing dendritic cells (DCs). Such activated NK cells may be used to improve a host immune response or to treat a disease (e.g., cancer). In some aspects, the present invention provides methods for activating NK cells in vivo, optionally activating NK cells relative to T cells, by osteoclasts (OCs). In some embodiments, OCs or OC culture supernatants may be administered to a subject to treat a disease (e.g., cancer). In some embodiments, probiotic bacteria (e.g., sAJ2) may be added to improve the function of OCs and activate NK cells. In addition to osteoclasts (OCs) and dendritic cells (DCs), other agents that can activate NK cells or T cells, such as any gene, protein, metabolite, etc., may be added to the cell culture or administered to a subject.

[0028] I. Definition The articles "a" and "an" are used herein to refer to one or to more than one (i.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.

[0029] The term "administering" is intended to include routes of administration by which an agent (e.g., at least one osteoclast (OC) or dendritic cell (DC), a cell culture comprising at least one osteoclast (OC) or dendritic cell (DC), the supernatant of such a cell culture, any composition comprising such OC(s) or DC(s), at least one probiotic bacterium, any composition comprising such probiotic bacteria, other agents capable of activating NK cells and / or T cells or promoting the function of such OC(s) or DC(s) and / or probiotic bacteria, etc., including processed (i.e., isolated, purified, concentrated, or after other processing for therapeutic or other use) forms of the various agents described herein) can perform its intended function. Examples of routes of administration of bodily treatments that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection may be a bolus injection or a continuous infusion. Depending on the route of administration, the drug may be coated with or encased in a selected material to protect the drug from natural conditions that may interfere with the drug's ability to perform its intended function. The drug may be administered alone or in combination with a pharmaceutically acceptable carrier. The drug may also be administered as a prodrug that is converted to its active form in vivo.

[0030] The terms "activating" or "activation" refer to the enhancement of a target function. For example, the present disclosure provides methods of activating NK cells or T cells in vitro, ex vivo, and / or in vivo, optionally where such activation is preferential relative to T cells or NK cells, respectively. In the present disclosure, activation of cells refers to the enhancement of the function of such cells, e.g., at least enhancing the activity and / or at least one cell function (e.g., cytotoxicity, cell division and / or growth rate, etc.) of each cell of a cell type (e.g., NK cells or T cells), enhancing the number of cells of a cell type (e.g., cell proliferation), or both. In some embodiments, an agent used herein activates at least one cell, e.g., NK cell(s) or T cell(s). In other embodiments, an agent used herein preferentially activates one cell type (e.g., NK cells or T cells) relative to another (e.g., T cells or NK cells).

[0031] The term "enhancement" is used interchangeably with terms such as "increase," "upregulation," "improvement," etc. in this disclosure, which refer to any meaningful increase in the function of an agent and / or target. For example, enhancement of the activity and / or cell number of a cell type (e.g., NK cells or T cells) can be "significant" if the increase in amount is greater than the original amount in a control and / or normal amount (e.g., the activity and / or cell number of the cell type in a normal subject or a subject without a disease or disorder (e.g., cancer)) by an amount greater than the standard error of the assay used to assess the amount, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. Alternatively, an enhancement of a cell type's activity and / or cell number may be considered "significant" if such enhancement is at least about 2, preferably at least about 3, 4, or 5 times, or more, over the original and / or normal activity / amount. Such "significance" may also apply to any other measured parameter described herein, e.g., expression, inhibition, cytotoxicity, cell growth, etc. In some embodiments, an enhancement of a cell type's activity and / or cell number (e.g., NK cells or T cells) may not be "significant" as described herein, but may be an increase sufficient for one of skill in the art to understand its biological relevance.

[0032] Unless otherwise specified herein, the terms "antibody(s)" broadly include naturally occurring antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, and fragments and derivatives of all of the foregoing that contain at least one antigen-binding site. Antibody derivatives may include proteins or chemical moieties that are conjugated to the antibody.

[0033] The term "antibody," as used herein, also includes an "antigen-binding portion" (or simply "antibody portion") of an antibody. The term "antigen-binding portion," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or a fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows these domains to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a specific scFv can also be joined to human immunoglobulin constant region cDNA or genomic sequences to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL may also be used in the production of Fab, Fv or other fragments of immunoglobulins, using either protein chemistry or recombinant DNA techniques. Other forms of single chain antibodies, such as diabodies, are also encompassed.Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the domains are forced to pair with the complementary domains on another chain and form two antigen-binding sites, using a linker that is too short to allow pairing between the two domains on the same chain (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0034] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified versions thereof (e.g., humanized, chimeric, etc.). Antibodies may be fully human. Preferably, the antibodies of the invention bind specifically or substantially specifically to a biomarker polypeptide or a fragment thereof. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one antigen-binding site capable of immunoreacting with a particular epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

[0035] The terms "cancer" or "tumor" or "hyperproliferative" refer to the presence of cells that have characteristics typical of cancer-causing cells, such as unregulated proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological properties. Unless otherwise specified, the term includes metaplasia. In some embodiments, such characteristics include at least one of silencing, reducing, and / or evading a host immune response, and / or resistance to host cell (e.g., NK cell) lysis and / or differentiation. In some embodiments, such cancer-causing cells are cancer stem cells (e.g., oral squamous cell carcinoma stem cells (OSCSCs)). In some embodiments, such cells exhibit some or all of these characteristics due to at least one genetic mutation. While cancer cells are often in the form of tumors, such cells may exist alone in an animal or may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers. Cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, blood tissue cancer, and the like.Other non-limiting examples of cancer types applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, colon 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 adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, liver carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, Included are testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngeal duct tumor, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrocyte glioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is epithelial in nature, including, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, 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 oral cancer, breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers can be characterized in a variety of other ways, including, but not limited to, serous, endometrial, mucinous, clear cell, Brenner, or undifferentiated.

[0036] In some embodiments, the cancer is "triple-negative breast cancer" or "TNBC," which refers to breast cancer that is strogen receptor (ER)-negative, progesterone receptor (PR)-negative, and human epidermal growth factor receptor 2 (HER-2)-negative (Pegram et al. (1998) J. Clin. Oncol. 16:2659-2671; Wiggans et al. (1979) Cancer Chemother. Pharmacol. 3:45-48; Carey et al. (2007) Clin. Cancer Res. 13:2329-2334).

[0037] In certain embodiments, cancer is "PI3K beta-dependent cancer", which may refer to cancer that is functionally dependent on PI3K beta.For example, even if the expression level of PI3K beta (for example, PI3K beta mRNA, PI3K beta protein, newly synthesized PI3K beta protein, etc.) in tumor tissue is comparable to that in normal tissue, if the direct or indirect inhibition of PI3K beta mRNA and / or protein, for example, by using RNAi or any other means, or the deletion of PI3K beta gene (for example, knockout or clustered regularly interspaced short palindromic repeats (CRISPR) technology) leads to the inhibition of carcinogenesis, tumor cell proliferation, tumor metastasis, or induces tumor cell differentiation, then the cancer is PI3K beta-dependent. The term "PI3K beta-dependent cancer" also refers to cancer in which PI3K beta is expressed at levels significantly higher than the normal amount of PI3K beta (e.g., PI3K beta mRNA, PI3K beta protein, newly synthesized PI3K beta protein, etc.) expressed in non-cancerous cells of the same cell type as the PI3K beta-dependent cancer.

[0038] The term "micrometastasis," as used herein, is preferably defined as a confluent group of cancer cells greater than 0.2 mm in size and / or having more than 200 cells up to 2 mm in width. More preferably, a "micrometastasis" is defined as a confluent group of cancer cells measuring from 0.2 mm to 2 mm in width. (See Edge et al. (2010) AJCC Cancer Staging Manual and Handbook (7th ed.)). An alternative, preferred definition of "micrometastasis" is a confluent group of at least 1,000 cancer cells measuring at least 0.1 mm in its widest dimension up to 1 mm in its widest dimension. Micrometastases are generally not visible with standard contrast MRI imaging or other clinical imaging techniques. However, in certain cancers, radioactive antibodies directed against tumor-selective antigens (e.g., Her2 for breast cancer metastases) allow visualization of micrometastases. Other indirect detection methods include contrast agent leakage at brain micrometastasis sites due to VEGF-induced vascular leakage (Yano et al. (2000) Cancer Res. 60:4959-49067; U.S. Patent Publication No. 2015 / 0352113). More sensitive imaging techniques can also be applied to detect micrometastases. For example, blood volume can be imaged by MRI using the alternative contrast agent USPIO (Molday Iron, Biopal, Worcester, Mass.) to detect micrometastases (Yin et al. (2009) Clin. Exp. Metastasis. 26:403-414).

[0039] The term "control" refers to any reference standard suitable to provide a comparison with the expression product, cell number, and / or cell function in a test sample. In certain embodiments, control involves obtaining a "control sample" from which the level of expression product, cell number, and / or cell function from the test sample is detected and compared. Such a control sample may include any suitable sample, including, but not limited to, a sample from a control cancer patient with a known result (which may be an archived sample or a previous sample measurement), normal tissue or cells isolated from a subject such as a normal patient or cancer patient, cultured primary cells / tissues isolated from a subject such as a normal subject or cancer patient, adjacent normal cells / tissues obtained from the same organ or body site of a cancer patient, a tissue or cell sample isolated from a normal subject, or primary cells / tissues obtained from an archive. In other preferred embodiments, the control may comprise a reference standard expression product level from any suitable source, including, but not limited to, a housekeeping gene, a range of expression product levels from normal tissue (or other previously analyzed control sample), a range of expression product levels previously determined in test samples from a group of patients, or a set of patients with a particular outcome (e.g., 1-, 2-, 3-, 4-year survival rate, etc.) or who have received a particular treatment (e.g., standard of care for cancer treatment). It will be understood by those of skill in the art that a combination of such control sample and reference standard expression product, cell number, and / or cell function levels can be used as controls in the methods of the invention. In certain embodiments, the control may comprise a normal or non-cancerous cell / tissue sample. In other preferred embodiments, the control may comprise expression levels, numbers of a particular cell type (e.g., NK cells or T cells), and / or cell function of a particular cell type from a set of patients, such as a set of cancer patients, or a set of cancer patients who have received a particular treatment, or a set of patients with one outcome versus another. In the former case, each patient's level of a particular expression product, cell number, and / or cell function can be assigned a percentile expression, cell number, and / or cell function level, or expressed as being higher or lower than the mean value of a reference standard expression, cell number, and / or cell function level.In other preferred embodiments, the control may include normal cells, cells from patients treated with combination chemotherapy, and cells from patients with benign tumors. In other embodiments, the control may also include a measurement, e.g., the average expression level of a particular gene, cell number, and / or cell function of a particular cell type (e.g., NK cells or T cells) in a population compared to the expression level of a housekeeping gene or another cell type in the same population. Such populations may include normal subjects, cancer patients not receiving any treatment (i.e., treatment-naive), cancer patients receiving standard treatment, or patients with benign tumors. As demonstrated by the data below, the methods of the present invention are not limited to the use of a particular cutpoint when comparing the level of expression product, cell number, and / or cell function in a test sample with a control.

[0040] The term "determining an appropriate treatment plan for a subject" refers to the determination of a treatment plan (i.e., a monotherapy or a combination of various therapies used to prevent and / or treat cancer in a subject) for a subject that is initiated, modified, and / or terminated based on, or based essentially on, or at least in part on, the results of an analysis according to the present invention. One example would be to initiate adjuvant therapy after surgery, the purpose of which is to reduce the risk of recurrence, and another example would be to modify the dosage of a particular chemotherapy. The determination can be based on the results of an analysis according to the present invention as well as the personal characteristics of the subject being treated. In most cases, the actual determination of an appropriate treatment plan for a subject will be made by the attending physician or doctor.

[0041] The term "cancer diagnosis" includes determining the presence or absence of cancer or its subtypes in an individual using the methods, systems, and code of the present invention. The term also includes methods, systems, and code for assessing the level of disease activity in an individual.

[0042] The term "immune cell" refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, e.g., B cells or T cells; natural killer cells; myeloid cells, e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0043] The term "cytokine" refers to a broad and loose category of small proteins (~5-20 kDa) important in cell signaling. Their release influences the behavior of surrounding cells, and cytokines participate in autocrine, paracrine, and endocrine signaling as immunomodulators. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, and may further include hormones or growth factors of the present disclosure. Cytokines are produced by a wide range of cells, including immune cells such as 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 figures of the present disclosure, e.g., Table 1.

[0044] The term "cytokine / chemokine activity" includes the ability of a cytokine or chemokine to regulate at least one cellular function. Generally, cytokines or chemokines regulate the balance between humoral and cell-based immune responses and regulate the maturation, growth, and responsiveness of specific cell populations. Thus, the term "cytokine / chemokine activity" includes the ability of a cytokine or chemokine to bind to its natural cell receptor(s), regulate cell signaling, and regulate the immune response.

[0045] The term "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 cell-mediated cytotoxicity. The term immune response also includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral response) and activation of cytokine-responsive cells, e.g., macrophages.

[0046] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate the host immune system to generate an immune response against a tumor or cancer in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0047] The term "inhibit" includes, for example, reducing, limiting, or blocking 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 the recurrence or metastasis of the cancer is alleviated, slowed, delayed, or prevented. Similarly, a biological function, e.g., a protein function, is inhibited if it is reduced compared to a control, such as a baseline state, e.g., a wild-type state. For example, the kinase activity of a mutant PI3 kinase or PI3 kinase contacted with a PI3 kinase inhibitor is inhibited if the kinase activity is reduced due to the mutation and / or contact with the inhibitor, compared to a wild-type PI3 kinase and / or PI3 kinase not contacted with the inhibitor. Such inhibition can be induced, for example, by application of an agent at a particular time and / or location, or can be constituted, for example, by an inherited mutation. Such inhibition can be partial or complete (e.g., essentially no measurable activity compared to a control, such as a baseline state, e.g., a wild-type state). Essentially complete inhibition is referred to as blocking.

[0048] The term "interaction," when referring to an interaction between two molecules, refers to physical contact (e.g., binding) between the molecules. Generally, such an interaction results in an activity (producing a biological effect) in one or both of the molecules.

[0049] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent, e.g., a probe or small molecule, for specifically detecting and / or affecting the expression of a marker of the invention. The kit can be advertised, distributed, or sold as a unit for performing the methods of the invention. The kit may contain one or more reagents necessary to express a composition useful in the methods of the invention. In certain embodiments, the kit may further include a reference substance, e.g., a nucleic acid encoding a protein that does not affect or regulate signaling pathways controlling cell growth, division, migration, survival, or apoptosis. One of skill in the art can envision many such control proteins, including, but not limited to, common molecular tags (e.g., green fluorescent protein and beta-galactosidase), proteins that are not classified by gene ontology standards into any of the pathways encompassing cell growth, division, migration, survival, or apoptosis, or universal housekeeping proteins. The reagents in the kit can be provided in individual containers or as a mixture of two or more reagents in a single container. Additionally, instructional materials describing the use of the compositions in the kit may also be included.

[0050] The term "neoadjuvant therapy" refers to treatment given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy. For example, in the treatment of breast cancer, neoadjuvant therapy can allow patients with large breast tumors to undergo breast-conserving surgery.

[0051] A "normal" level of expression and / or activity of a biomarker is the level of expression and / or activity of the biomarker in the cells of a subject, e.g., a human patient, not afflicted with cancer. "Overexpression" or "significantly high expression level" of a biomarker refers to an expression level in a test sample that is greater than the standard error of the assay used to assess expression, and is preferably at least 10%, more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times greater than the expression activity or level of the biomarker in a control sample (e.g., a sample from a healthy subject without a biomarker-associated disease), and preferably the average expression level of the biomarker in several control samples. A "significantly lower expression level" of a biomarker refers to a level in a test sample that is at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times lower than the expression level of the biomarker in a control sample (e.g., a sample from a healthy subject free of a biomarker-associated disease), and preferably the average expression level of the biomarker in several control samples. The same determination can be made to determine overexpression or underexpression.

[0052] NK cells Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte crucial to the innate immune system. Their role is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells (acting approximately three days after infection) and respond to tumor formation. Typically, immune cells detect major histocompatibility complex (MHC) molecules displayed on the surface of infected cells, triggering cytokine release and causing lysis or apoptosis. NK cells are unique, however, in their ability to recognize stressed cells in the absence of antibodies and MHC molecules, allowing for a much more rapid immune response. NK cells were named "natural killer" due to the initial concept that they do not need to be activated to kill cells lacking the "self" marker MHC class I. This role is particularly important because harmful cells lacking MHC class I markers cannot be detected and destroyed by other immune cells, such as T lymphocytes.

[0053] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute the third cell type differentiated from a common lymphoid progenitor cell that gives rise to B and T lymphocytes. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, from which they then enter the circulatory system. NK cells are phenotypically distinct from natural killer T cells (NKT) depending on their origin and respective effector functions; NKT cell activity often promotes NK cell activity by secreting IFNγ. In contrast to NKT cells, NK cells do not express the T cell antigen receptor (TCR) or pan-T marker CD3 or surface immunoglobulin (Ig) B cell receptor. However, they generally express the surface markers CD16 (FcγRIII) and CD56 in humans and NK1.1 or NK1.2 in C57BL / 6 mice. The NKp46 cell surface marker constitutes another NK cell marker of choice, being expressed in both humans, some mouse strains (e.g., BALB / c mice), and three common monkey species.

[0054] NK cells are negatively regulated by major histocompatibility complex (MHC) class I-specific inhibitory receptors (Karre et al., 1986; Ohlen et al., 1989). These specific receptors bind to polymorphic determinants on MHC class I molecules or HLA present on other cells and inhibit NK cell lysis. In humans, some members of a family of receptors called killer Ig-like receptors (KIRs) recognize groups of HLA class I alleles.

[0055] KIRs are a large family of receptors present on specific subsets of lymphocytes, including NK cells. KIR nomenclature is based on the number of extracellular domains (KIR2D or KIR3D) and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). Within humans, the presence or absence of a particular KIR can vary from NK cell to NK cell within a single individual's NK population. Within the human population, there is also a relatively high level of polymorphism in KIR molecules, with certain KIR molecules present in some, but not all, individuals. Certain KIR gene products, when bound to the appropriate ligand, trigger the stimulation of lymphocyte activity. All identified stimulatory KIRs possess short cytoplasmic tails with charged transmembrane residues that associate with adaptor molecules containing immunostimulatory motifs (ITAMs). Other KIR gene products are inhibitory in nature.

[0056] Probiotic bacteria In some embodiments, the present invention relates to compositions comprising at least one probiotic bacterial strain capable of modulating NK cell function. Such probiotic bacteria induce significant split anergy in activated NK cells, resulting in significant induction of IFN-γ and TNF-α. Furthermore, such probiotic bacteria induce significant proliferation of NK cells.

[0057] Many commercially available probiotics are available with various effects, such as reducing gastrointestinal discomfort or strengthening the immune system. Preferred probiotic bacterial species for use in the compositions and methods described herein include those derived from commercially available probiotic bacterial strains (e.g., sAJ2 bacteria), particularly those derived from Streptococcus (e.g., S. thermophiles), Bifidobacterium (e.g., B. longum, B. breve, B. infantis, B. breve, B. infantis), and Lactobacillus genera (e.g., L. acidophilus, L. helveticus, L. bulgaricus, L. rhamnosus, L. plantarum, and L. casei). The present disclosure includes methods of administering at least one probiotic bacterial strain, preferably a combination of two or more different bacterial strains, to a subject, preferably a mammal (e.g., a human). Such administration may be systemic or local (e.g., directly to the intestine). Oral administration is preferred. Other routes (e.g., rectal) may also be used. For administration, either the bacteria (e.g., in wet, sonicated, ground, or dried form or format), bacterial culture medium containing the bacteria, or bacterial culture medium supernatant (free of bacteria) may be administered.

[0058] osteoclasts Osteoclasts are a type of bone cell derived from hematopoietic stem cells. Their function, bone tissue resorption, is crucial for bone maintenance, repair, and remodeling. Bone homeostasis is achieved when bone formation by osteoblasts and bone resorption by osteoclasts are balanced. Osteoclasts mature through stimulation by RANKL-expressing osteoblasts, and their interaction is mediated by firm adhesion via ICAM-1. Osteoclasts also express many ligands for receptors present on activated NK cells. It has been reported that osteoclasts express ULBP-1, ULBP-2 / 5 / 6, and ULBP-3, but express little or no MIC-A, MIC-B, or MHC class I-like ligands for NKG2D, an activating receptor for NK cells.

[0059] Compared with dendritic cells (DCs) and monocytes, osteoclasts (OCs) are key activators of NK cell proliferation and function (Tseng et al. (2015) Oncotarget 6(24):20002-25). Furthermore, osteoclasts secrete significant amounts of IL-12, IL-15, IFN-γ, and IL-18, which are known to activate NK cells, and osteoclasts also express important NK-activating ligands. This disclosure provides a novel strategy for expanding highly functional, supercharged, osteoclast-expanded NK cells to levels significantly higher than those established by other methodologies. Several in vitro NK expansion techniques have been developed to enhance NK cell activity and in vivo expansion capacity while establishing higher therapeutic cell doses. Some of these techniques involve the use of peripheral blood mononuclear cells (PBMCs), stimulation of PBMC-purified populations of NK cells, or human umbilical cord blood, sometimes in combination with various feeder cells, such as K562 cells expressing membrane-bound IL-15 and 41BB ligand (K562-mb15-41BBL), EBV-TM-LCL, Wilms' tumor, or irradiated PBMCs. These studies have generated clinically relevant numbers of NK cells with good function.

[0060] dendritic cells Dendritic cells (DCs) are antigen-presenting cells (also known as accessory cells) of the mammalian immune system. Their main function is to process antigenic material and present it on their surface to T cells of the immune system. Dendritic cells act as messengers between the innate and adaptive immune systems.

[0061] Dendritic cells are present in tissues that come into contact with the external environment, such as the skin (where a specialized dendritic cell type called Langerhans cells reside) and the lining of the nose, lungs, stomach, and intestine. They can also be found in an immature state in the blood. After activation, they migrate to lymph nodes, where they interact with T cells and B cells to initiate and shape adaptive immune responses. At a certain developmental stage, they generate branched processes, the dendrites that give the cells their name. Although similar in appearance, these are distinct structures from the dendrites of nerve cells. Immature dendritic cells are also called veil cells because they possess a large cytoplasmic "veil" rather than a dendrite.

[0062] The present disclosure provides a novel method for activating NK cells using osteoclasts, resulting in NK cell-mediated apoptosis of tumor target cells and enhanced sensitization to cytokine production. The terms "activate," "activation," or "activating" refer to enhancing NK cell proliferation and / or activation of NK cell function, alone or in combination. The term "NK cell function(s)" refers to any function of NK cells, for example, cytotoxicity and / or cytokine / chemokine production / secretion activity.

[0063] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the chance of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition but is at risk of or susceptible to developing the disease, disorder, or condition.

[0064] The terms "response to anti-cancer treatment" or "response to therapy with a composition comprising at least one probiotic bacterium alone or in combination with other NK immunotherapies" refer to any response of a hyperproliferative disorder (e.g., cancer) to treatment with an anti-cancer agent(s), e.g., a composition comprising at least one probiotic bacterium alone or in combination with other NK immunotherapies, preferably the change in tumor mass and / or volume after the initiation of neoadjuvant or adjuvant therapy. For example, hyperproliferative disease response can be assessed for efficacy or in the neoadjuvant or adjuvant setting, and tumor size after systemic intervention may be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound, or palpation. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded quantitatively, such as by percentage change in tumor volume, or qualitatively, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. Assessment of hyperproliferative disease response may be performed early after the initiation of neoadjuvant or adjuvant therapy, e.g., hours, days, weeks, or preferably months. A typical endpoint for response assessment is after the end of neoadjuvant chemotherapy or after surgical removal of residual tumor cells and / or tumor bed. This is typically 3 months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the percentage of patients with a complete response (CR), the number of patients with a partial response (PR), and the number of patients with stable disease (SD) at least 6 months after the end of treatment. An abbreviation for this formula is CBR = CR + PR + SD over 6 months. In some embodiments, the CBR for a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. An additional criterion for assessing response to cancer treatment relates to "survival," which includes all of the following:These include survival until death, also known as overall survival (which death may be cause-unrelated or tumor-related), "recurrence-free survival" (the term recurrence is intended to include both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease is intended to include cancer and related diseases). The length of survival may be calculated by reference to a defined starting point (e.g., time of diagnosis or initiation of treatment) and an end point (e.g., death, recurrence, or metastasis). Criteria for treatment efficacy may also be expanded to include response to chemotherapy, probability of survival, probability of outcome within a given time period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a particular cancer treatment regimen can be applied to a population of subjects, and results can be correlated to biomarker measurements determined before any cancer treatment is administered. The outcome measurement may be a pathological response to therapy given in a neoadjuvant setting. Alternatively, outcome measures such as overall survival and disease-free survival can be monitored over a period of time after cancer treatment for subjects with known measurements. In certain embodiments, the dosage is a standard dose known in the art for cancer therapeutics. The length of time a subject is monitored can vary. For example, a subject may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds that correlate with cancer treatment outcome can be determined using methods well known in the art, such as those described in the Examples section.

[0065] The term "resistance" refers to the acquired or innate resistance of a cancer sample or mammal to cancer therapy (i.e., non-responsiveness, reduced response, or limited response to therapeutic treatment), e.g., a reduced response of 25% or more to therapeutic treatment, e.g., 30%, 40%, 50%, 60%, 70%, 80%, or 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or more. The reduced response can be measured by comparing the same cancer sample or mammal before resistance was acquired or by comparing it to another cancer sample or mammal known not to be resistant to therapeutic treatment. A typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance can be mediated by P-glycoprotein or other mechanisms, or can occur when a mammal is infected with a multidrug-resistant microorganism or a mixture of microorganisms. Determining resistance to therapeutic treatment is routine in the art and within the skill of one of ordinary skill in the art and can be measured, for example, by cell proliferation and cell death assays, referred to herein as "sensitization." In some embodiments, the term "reverse resistance" refers to the use of a second agent in combination with a first cancer treatment (e.g., chemotherapy or radiation therapy) to significantly reduce tumor volume at a statistically significant level (e.g., p<0.05) compared to the tumor volume of an untreated tumor, in situations where the first cancer treatment (e.g., chemotherapy or radiation therapy) alone fails to achieve a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor. This typically applies to measurements of tumor volume when the untreated tumor is growing logarithmically.

[0066] The term "response" or "responsiveness" refers to an anti-cancer response, for example, in the sense of a reduction in tumor size or inhibition of tumor growth. The term can also refer to an improved prognosis, reflected, for example, by an increase in time to recurrence, which is the time to first recurrence, censoring a second primary cancer as the first event without evidence of recurrence, or death, or an increase in overall survival, which is the time from treatment to death from any cause. Responding or having a response means that there is a beneficial endpoint that is reached when exposed to a stimulus. Alternatively, upon exposure to a stimulus, negative or adverse symptoms are minimized, reduced, or alleviated. It will be understood that assessing the likelihood that a tumor or subject will exhibit a favorable response is equivalent to assessing the likelihood that the tumor or subject will not exhibit a favorable response (i.e., exhibit a lack of response or be non-responsive).

[0067] The term "sample" as used to detect or determine the presence or level of at least one biomarker typically refers to brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, feces (e.g., stool), tears, and any other bodily fluid (e.g., as defined above in the definition of "bodily fluid"), or a tissue sample (e.g., biopsy material), such as a small intestine, large intestine sample, or surgically resected tissue. In certain cases, the methods of the present invention further comprise obtaining a sample from an individual prior to detecting or determining the presence or level of at least one marker in the sample.

[0068] The term "sensitize" refers to altering cancer or tumor cells in a way that allows for more effective treatment of the associated cancer with cancer therapy (e.g., treatment with a composition described herein). In some embodiments, normal cells are not affected to an extent that would unduly harm the normal cells. Increased or decreased sensitivity to therapeutic treatment is measured by methods known in the art for the particular treatment and described herein below, including, but not limited to, cell proliferation assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164), cell death assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal et al. (1985) Cancer Treat. Rep. 69:615-632; Weisenthal LM, In: Kaspers et al. eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Publishers, 1993:415-432; Weisenthal et al. (1994) Contrib. Gynecol. Obstet. 19:82-90). Sensitivity or resistance may also be measured in animals by measuring tumor size reduction over a period of time, e.g., six months in humans and four to six weeks in mice. A composition or method is said to sensitize a response to a therapeutic treatment if the increase in therapeutic sensitivity or decrease in resistance, compared to the sensitivity or resistance to treatment in the absence of the composition or method, is 25% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or more. Determining sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of one of ordinary skill in the art. It should be understood that any method described herein for improving the effectiveness of cancer treatment is equally applicable to methods for sensitizing hyperproliferative or other cancer cells (e.g., resistant cells) to cancer treatment.

[0069] The term "specific binding" refers to an agent, such as an antibody, that binds to a predetermined target, such as an antigen. Typically, an antibody binds to a specific target, e.g., approximately 10, as determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using the antigen of interest as the analyte and the antibody as the ligand. -8 M, 10 -9 M or 10 -10 Less than M, or even lower, approximately 10 -7 An antibody binds to a predetermined antigen with an affinity (KD) of less than M and that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen." Selective binding is a relative term that refers to the ability of an antibody to distinguish binding of one antigen from another.

[0070] The term "synergistic effect" refers to the fact that the combined effect of two or more anti-cancer agents (e.g., treatment with a combination of a composition comprising at least one of the probiotic bacteria alone or in combination with other NK immunotherapies) can be greater than the sum of the individual effects of the anti-cancer agents alone.

[0071] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human afflicted with cancer, such as brain metastasis, lung, ovarian, pancreatic, liver, breast, prostate, colon cancer, melanoma, multiple myeloma, etc. The term "subject" is interchangeable with "patient."

[0072] The term "survival" includes all of the following: survival until death, also known as overall survival (the death may be cause-unrelated or tumor-related), "recurrence-free survival" (the term recurrence is intended to include both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease is intended to include cancer and related diseases). The length of survival may be calculated by reference to a defined starting point (e.g., time of diagnosis or initiation of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for efficacy of treatment may be expanded to include response to chemotherapy, probability of survival, probability of outcome within a given time period, and probability of tumor recurrence.

[0073] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or the enhancement of desired physical or mental development and conditions in animals or humans. The phrase "therapeutically effective amount" refers to that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. In certain embodiments, a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like. For example, a particular compound discovered by the methods of the present invention can be administered in an amount sufficient to provide a reasonable benefit / risk ratio applicable to such treatment.

[0074] As used herein, the term "unresponsiveness" includes the unresponsiveness of cancer cells to a treatment or the unresponsiveness of therapeutic cells, e.g., immune cells, to stimuli, such as stimulation via an activating receptor or cytokine. Unresponsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigen. As used herein, the terms "anergy" or "tolerance" include unresponsiveness to activating receptor-mediated stimuli. Such unresponsiveness is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (unlike unresponsiveness) is characterized by the lack of cytokine production, e.g., IL-2. T cell anergy occurs when a T cell is exposed to an antigen and receives a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, re-exposure of the cell to the same antigen (even if re-exposure occurs in the presence of a costimulatory polypeptide) results in a failure of cytokine production and therefore a failure of proliferation. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can be observed by proliferation assays using indicator cell lines or by the lack of IL-2 production by T lymphocytes as measured by ELISA. Alternatively, a reporter gene construct may be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by multimers of AP1 sequences that can be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0075] II. Subject In certain embodiments, a subject suitable for the compositions and methods disclosed herein is a mammal (e.g., a mouse, a rat, a primate, a non-human mammal, a domestic animal such as a dog, a cat, a cow, a horse, etc.), preferably a human. In other embodiments, the subject is an animal model of cancer. For example, the animal model can be a human oral squamous cell carcinoma or an orthotopic xenograft animal model containing cancer stem cell (CSC) / anaplastic tumors.

[0076] In other embodiments of the methods of the invention, the subject has not received a treatment such as chemotherapy, radiation therapy, targeted therapy, and / or anti-immunotherapy (e.g., NK cell-related immunotherapy). In yet other embodiments, the subject has received a treatment such as chemotherapy, radiation therapy, targeted therapy, and / or anti-immunotherapy (e.g., NK cell-related immunotherapy).

[0077] In certain embodiments, the subject has undergone surgery to remove cancerous or pre-cancerous tissue, hi other embodiments, the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in an inoperable area of ​​the body, e.g., vital tissue, or an area where surgical procedures pose a significant risk of harm to the patient.

[0078] The methods of the present invention can be used to treat and / or assess responsiveness to many different cancers in a subject, including those described herein, using a composition comprising at least one of the probiotic bacteria, alone or in combination with other NK immunotherapies.

[0079] III. Anti-cancer treatment In one aspect, combinations of other anti-cancer therapies and / or immunotherapies, or combinations of therapies (e.g., one or more PI3K beta selective inhibitors, e.g., KIN193, in combination with one or more immune checkpoint inhibitors, e.g., anti-PD-1 antibodies, alone or in combination with additional anti-cancer therapies, e.g., targeted therapies) can be administered, particularly if the subject is initially shown to be a likely responder to the compositions disclosed herein. In other embodiments, such therapies can be avoided if the subject is not shown to be a likely responder to such therapies, and alternative treatment regimens, e.g., targeted and / or non-targeted anti-cancer therapies, can be administered in conjunction with the compositions disclosed herein.

[0080] Combination therapy can also be contemplated and can include, for example, one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation, and chemotherapy, each of which combinations can be a therapy disclosed herein. As described below, agents can be administered in combination with, for example, chemotherapeutic agents, hormones, antiangiogenic agents, radiolabeled compounds, or with surgery, cryotherapy, and / or radiation therapy. The aforementioned therapeutic methods can be administered in conjunction with other forms of conventional therapy (e.g., standard of care for cancer known to those skilled in the art), either sequentially with, prior to, or after the conventional therapy. For example, these modulating agents can be administered together with a therapeutically effective dose of a chemotherapeutic agent. In other embodiments, these modulating agents are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The U.S. Pharmaceutical Register (PDR) discloses dosages of chemotherapeutic agents used in the treatment of various cancers. The dosage regimens and dosages of these aforementioned chemotherapeutic agents that are therapeutically effective will depend on the particular melanoma being treated, the extent of the disease, and other factors familiar to physicians of skill in the art, and can be determined by a physician.

[0081] The term "targeted therapy" refers to the administration of drugs that treat cancer by selectively interacting with selected biomolecules, such as breast or ovarian cancer antigens.

[0082] Alternatively, immunotherapy is a form of targeted therapy that may involve, for example, the use of cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are viruses that can infect and lyse cancer cells while leaving normal cells unharmed, making them potentially useful in cancer treatment. The replication of oncolytic viruses promotes tumor cell destruction and results in dose amplification at the tumor site. They may also act as vectors for anti-cancer genes, allowing them to be delivered specifically to the tumor site. Immunotherapy may involve passive immunotherapy for short-term host defense, achieved by administering preformed antibodies directed against cancer or disease antigens (e.g., monoclonal antibodies, optionally chemotherapeutic agents or toxins, monoclonal antibodies bound to tumor antigens). Immunotherapy may focus on the use of 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 contribute to tumor or cancer initiation, progression, and / or pathology.

[0083] The term "non-targeted therapy" refers to the administration of an agent that does not selectively interact with a selected biomolecule but still treats cancer. Representative examples of non-targeted therapy include, but are not limited to, chemotherapy, gene therapy, and radiation therapy.

[0084] In certain embodiments, chemotherapy is used. Chemotherapy involves the administration of a chemotherapeutic agent. Such chemotherapeutic agents may be selected from, but are not limited to, the following groups of compounds: cytotoxic antibiotics, antimetabolites, antimitotics, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant alkaloids, and toxins, and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trophosphamide; plant alkaloids: vinblastine, paclitaxel, docetaxel; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; antifolates: 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, and rhizoxin. Compositions containing one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG includes fludarabine, cytosine arabinoside (Ara-C), and G-CSF. CHOP includes cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiment, a PARP (e.g., PARP-1 and / or PARP-2) inhibitor is 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-1001 (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide (Trevigen); 4-amino-1,8-naphthalimide (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Reissue Patent No. 36,397); and NU1025 (Bowman et al.).This mechanism of action is generally related to the ability of PARP inhibitors to bind to and reduce the activity of PARP. PARP catalyzes the conversion of β-nicotinamide adenine dinucleotide (NAD+) to nicotinamide and poly-ADP-ribose (PAR). Both poly(ADP-ribose) and PARP are involved in the regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard VJ 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 June 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 JC, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang ZQ, et al. (1997) Genes Dev 11:2347-2358). Knockout of SSB repair by inhibiting PARP1 function induces DNA double-strand breaks (DSBs), which can induce synthetic lethality in cancer cells due to defective homology-induced DSB repair (Bryant HE, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The above examples of chemotherapeutic agents are illustrative and not limiting.

[0085] 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, intracellular implants of radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or whole abdominal and pelvic radiation therapy. For a review 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. Radiation therapy can be administered as external beam radiation therapy or teletherapy, in which radiation is directed from a distant source. Radiation therapy can also be administered as internal radiation therapy or brachytherapy, in which a radiation source is placed inside the body in close proximity to the cancer cells or tumor mass. Also encompassed is the use of photodynamic therapy, which involves the administration of photosensitizers such as hematoporphyrin or its derivatives, vertoporphine (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxy-hypocrelin A, and 2BA-2-DMHA.

[0086] In yet other embodiments, surgical intervention can physically remove cancerous cells and / or tissue.

[0087] In yet other embodiments, hormone therapy is used. Hormonal therapeutic treatments can include, for example, hormone agonists, hormone antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), hormone biosynthesis and processing inhibitors, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogens, 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).

[0088] Yet other embodiments use hyperthermia, a technique in which body tissues are exposed to high temperatures (up to 106°F). Heat can help shrink tumors by damaging cells or starving them of substances necessary for survival. Hyperthermia can be local, regional, or whole-body hyperthermia using external and internal heating devices. Hyperthermia is almost always used in conjunction with other forms of treatment (radiation therapy, chemotherapy, and biological therapy) in an attempt to enhance their effectiveness. Local hyperthermia refers to heat applied to a very small area, such as a tumor. An area can be heated externally with radiofrequency waves, targeting the tumor from a device outside the body. To achieve internal heating, one of several types of sterile probes can be used, including thin heated wires or hollow tubes filled with warm water; implanted microwave antennas; and radiofrequency electrodes. Regional hyperthermia involves heating an organ or limb. High-energy generating magnets and devices are placed over the area to be heated. Another approach, called perfusion, involves removing a portion of the patient's blood, heating it, and then pumping (perfusing) it into the area to be internally heated. Whole-body heating is used to treat metastatic cancer that has spread throughout the body. This can be achieved using warm water blankets, hot wax, induction coils (like those in electric blankets), or thermal chambers (similar to large incubators). Hyperthermia does not cause a significant increase in radiation side effects or complications. Heat applied directly to the skin, however, can cause uncomfortable or even significant local pain in about half of treated patients. It can also cause blisters, which generally heal quickly.

[0089] In yet another embodiment, photodynamic therapy (PDT, also called photoradiotherapy, phototherapy, or photochemotherapy) is used to treat some types of cancer.

[0090] In yet other embodiments, laser therapy involves the use of high-intensity light to destroy cancer cells. This technique is often used to relieve cancer symptoms such as bleeding or blockage, especially when the cancer is resistant to other treatments. It may also be used to treat cancer by shrinking or destroying tumors.

[0091] The duration and / or dosage of therapy may vary depending on the specific therapeutic agent or combination thereof. The appropriate treatment time for a particular cancer therapeutic agent will be understood by one of ordinary skill in the art. The present invention contemplates ongoing evaluation of the optimal treatment schedule for each cancer therapeutic agent, and the subject's cancer phenotype, as determined by the methods of the present invention, is a factor in determining the optimal treatment dosage and schedule.

[0092] In other embodiments, recombinant biomarker polypeptides and fragments thereof can be administered to a subject. In some embodiments, fusion proteins with enhanced biological properties can be constructed and administered. Additionally, biomarker polypeptides and fragments thereof can be modified according to pharmacological methods well known in the art (e.g., pegylation, glycosylation, oligomerization, etc.) to further enhance desirable biological activity, such as increasing bioavailability and reducing proteolysis.

[0093] Clinical efficacy can be measured by any method known in the art. For example, response to a treatment, e.g., a composition disclosed herein, relates to any response to cancer, e.g., tumor, treatment, preferably a change in tumor mass and / or volume after the initiation of neoadjuvant or adjuvant chemotherapy. Tumor response may be assessed in the neoadjuvant or adjuvant setting, where tumor size after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound, or palpation, and tumor cellularity can be estimated histologically and compared to the cellularity of a tumor biopsy taken before the 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 quantitatively, such as by percentage change in tumor volume or cellularity, or qualitatively, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria, using semiquantitative scoring systems such as residual tumor burden (Symmans et al., J. Clin. Oncol. (2007) 25:4414-4422) or the Miller-Payne score (Ogston et al., (2003) Breast (Edinburgh, Scotland) 12:320-327). Tumor response assessment may be performed early after the initiation of neoadjuvant or adjuvant therapy, for example, hours, days, weeks, or preferably months. A typical endpoint for response assessment is after the end of neoadjuvant chemotherapy or after surgical removal of residual tumor cells and / or the tumor bed.

[0094] In some embodiments, the clinical effectiveness 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 percentage of patients in complete response (CR), the number of patients in partial response (PR), and the number of patients with stable disease (SD) at least 6 months after the end of treatment. An abbreviation for this formula is CBR = CR + PR + SD over 6 months. In some embodiments, the CBR for a particular treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.

[0095] An additional criterion for assessing response to the treatments disclosed herein relates to "survival," which includes all of the following: survival until death, also known as overall survival (the death may be cause-unrelated or tumor-related), "recurrence-free survival" (the term recurrence is intended to include both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease is intended to include cancer and related diseases). The length of survival may be calculated by reference to a defined starting point (e.g., time of diagnosis or initiation of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of outcome within a given time period, and probability of tumor recurrence.

[0096] For example, to determine an appropriate threshold, a particular cancer treatment regimen can be applied to a population of subjects, and the results can be correlated with biomarker measurements determined before administration of any of the compositions disclosed herein. The outcome measurement can be a pathological response to therapy administered in a neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival, can be monitored over a period of time for subjects with known measurements after treatment. In certain embodiments, the same dose of a therapeutic composition is administered to each subject. The length of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds that correlate with the outcomes of the treatments disclosed herein can be determined using methods well known in the art, such as those described in the Examples section.

[0097] 3. Pharmaceutical Compositions The present invention provides pharmaceutically acceptable compositions of the compositions disclosed herein. As described in detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including forms adapted for (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes, (2) parenteral administration, e.g., as a sterile solution or suspension, e.g., subcutaneous, intramuscular, or intravenous injection, (3) topical application, e.g., as a cream, ointment, or spray applied to the skin, (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam, or (5) aerosol, e.g., as an aqueous aerosol, liposomal formulation, or solid particles comprising the compound.

[0098] The compositions described herein, e.g., probiotic bacterial compositions, may be used for oral administration to the gastrointestinal tract with the goal of introducing the probiotic bacteria into the tissues of the gastrointestinal tract. The therapeutic composition formulation of the present invention may also include other probiotic agents or nutrients that promote spore germination and / or bacterial growth. Exemplary substances include bifidogenic oligosaccharides, which promote the growth of beneficial probiotic bacteria. In certain embodiments, the probiotic bacterial strains are combined with a therapeutically effective dose of a (preferably broad-spectrum) antibiotic or antifungal agent. In some embodiments, the compositions described herein are encapsulated in an enteric-coated, sustained-release capsule or tablet. The enteric coating allows the capsule / tablet to remain intact (i.e., undissolved) during transit through the gastrointestinal tract until a certain time has elapsed and / or until a specific portion of the GI tract (e.g., the small intestine) is reached. The sustained-release component prevents the "release" of the probiotic bacterial strains in the compositions described herein for a predetermined period of time.

[0099] Therapeutic compositions of the present invention may also contain known antioxidants, buffers, and other agents such as colorants, flavorings, vitamins, or minerals.

[0100] In some embodiments, the therapeutic compositions of the present invention, e.g., osteoclasts, osteoclast cell cultures, and / or osteoclast cell culture supernatants, can be administered alone or in combination with a carrier that is physiologically compatible with the species to which they are administered. The carrier can be a solid-based dry material for tablet, capsule, or powder formulations, or it can be a liquid or gel-like material for formulation into a liquid or gel-like formulation. The particular type of carrier, as well as the final formulation, will depend, in part, on the route(s) of administration selected. The therapeutic compositions of the present invention may also include various carriers and / or binders. A preferred carrier is microcrystalline cellulose (MCC), added in an amount sufficient to complete the dosage to 1 g total weight. The carrier can be a solid-based dry material for tablet, capsule, or powder formulations, or a liquid or gel-like material for liquid or gel formulations, the form of which will depend, in part, on the route of administration. Typical carriers for dry formulations include, but are not limited to, trehalose, maltodextrin, rice flour, microcrystalline cellulose (MCC), magnesium stearate, inositol, FOS, GOS, dextrose, sucrose, and similar carriers. Suitable liquid or gel-like carriers include, but are not limited to, water and saline, urea, alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol), glycols (e.g., ethylene glycol, propylene glycol, etc.). Preferably, aqueous carriers have a neutral pH (i.e., pH 7.0). Other carriers or agents for administering the compositions described herein are known in the art, for example, U.S. Pat. No. 6,461,607. The osteoclasts, osteoclast cell cultures, and / or osteoclast cell culture supernatants of the present invention can be administered using local and / or systemic administration routes known in the art and described herein.

[0101] The osteoclasts (OCs) or dendritic cells (DCs) described herein may be used for administration to a subject in any pharmaceutically acceptable composition via any route of administration known in the art. For example, the OCs or DCs, cell cultures containing such OCs or DCs, or supernatants of such cell cultures, optionally together with additional agent(s), may be administered in a pharmaceutical composition via injection (e.g., intravenously) systemically and / or locally (e.g., at or near cancer or tumor tissue).

[0102] The phrase "pharmaceutically acceptable" is used herein to refer to those agents, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, and for a reasonable benefit / risk ratio.

[0103] The phrase "pharmaceutically acceptable carrier," as used herein, refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which is involved in carrying or transporting the subject chemical substance from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) sorbents, such as cocoa butter and suppository wax; and (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) buffers 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, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0104] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), powder, granules, or as a solution or 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 a troche (with an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or mouthwash, etc., each containing a predetermined amount of one or more of the bacterial strains disclosed herein.

[0105] The present invention also encompasses kits for detecting and / or modulating the biomarkers described herein. Kits of the present invention also include instructions disclosing or describing the use of the disclosed kits or antibodies in the disclosed methods provided herein. Kits may also include additional components to facilitate the particular application for which the kit is intended. For example, the kit may further include a means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as sheep anti-mouse HRP, etc.), and reagents needed for controls (e.g., a control biological sample or a reference material). The kit may further include buffers and other reagents approved for use in the disclosed methods. Non-limiting examples include agents for reducing nonspecific binding, such as carrier proteins or detergents. [Example]

[0106] Example 1: Materials and Methods for Examples 2 and 3 Cell lines, reagents, and antibodies RPMI 1640 complete medium (Gemini Bio-Product) containing 10% fetal bovine serum (FBS) was used for cell culture. Oral squamous cell carcinoma cells (OSCC) and oral squamous cell carcinoma stem cells (OSCSC) were isolated from cancer patients with tongue tumors at UCLA [see references 2 and 33–35; all citations below refer to the same reference list]. Alpha-MEM (Life Technologies, CA) containing 10% FBS was used for the culture of osteoclasts and DCs. M-CSF (Biolegend, CA), RANKL, GM-CSF, and IL-4 were purchased from PeproTech (NJ), and rh-IL-2 was obtained from NIH-BRB. Human CD3 / CD28 T cell activating factor was purchased from Stem Cell Technologies.

[0107] Antibodies against MHC-I, KIR2, KIR3, CD44, CD54, B7H1, CD16, NKG2D, MICA / B, KLGR1, CD45, CD3 / 16 / 56, CD8, CD3, CD28, CD4, GL3, NKp40, NKp30, NKp44, NKp46, and CD94 were purchased from Biolegend (San Diego, CA). ULBP1-6 antibody was purchased from R&D Systems. Propidium iodide (PI) was purchased from Sigma (St. Louis, MO). sAJ2 was prepared as previously described

[36] .

[0108] Purification of NK cells and T cells from human PBMCs and hu-BLT splenocytes NK cells and T cells were purified as previously described

[37] . T cells from hu-BLT splenocytes were actively purified using an isolation kit from Stem Cell Technologies (Stem Cell Technologies, Vancouver, Canada).

[0109] Purification of monocytes and generation of dendritic cells and osteoclasts from hu-BLT mice and human PBMCs Written informed consent, approved by the UCLA Institutional Review Board (IRB), was obtained from healthy blood donors, and all procedures were approved by the UCLA IRB. Monocytes were purified as previously described

[37] . Monocytes from hu-BLT mice were actively isolated from bone marrow using a human CD14 isolation kit (eBioscience, San Diego, CA). Purity of >95% was achieved for each subset based on flow cytometry analysis. Monocytes were differentiated into osteoclasts by treatment with M-CSF (25 ng / mL) and RANKL (25 ng / mL) for 21 days. To obtain DCs, monocytes were treated with GM-CSF (150 ng / mL) and IL-4 (50 ng / mL) for 7 days.

[0110] NK cell proliferation Human purified NK cells and hu-BLT-enriched NK cells were activated with rh-IL-2 (1000 U / ml) and anti-CD16 mAb (3 μg / ml) for 18–20 h and then cocultured with feeder cells and sAJ2. The culture medium with IL-2 was refreshed every 3 days.

[0111] Tumor implantation and tissue preparation from hu-BLT mice The animal studies described herein were conducted in accordance with all federal, state, and local regulations and with written approval from the UCLA Animal Research Committee (ARC). Combined immunodeficient NOD.CB17-Prkdcscid / J and NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ mice (deficient in NSG T, B, and natural killer cells) were purchased from the Jackson Laboratory. Humanized BLT (hu-BLT; human bone marrow / liver / thymus) mice were prepared on an NSG background as described above and shown in Figure 8 [38, 39]. To establish orthotopic tumors, mice were first anesthetized with isoflurane in combination with oxygen, after which tumor cells were implanted in 10 μl of HC Matrigel (Corning, NY, USA) (1 × 10 6 The tumor cells were injected directly into the floor of the mouth in a suspension containing 1000 cells (1000 cells). Four to five weeks after tumor injection, the mice were euthanized, and the bone marrow, spleen, and blood were harvested and single-cell suspensions were prepared

[40] .

[0112] ELISA and multiplex cytokine array kits Single ELISA and multiplex assays were performed as previously described

[37] .

[0113] Cancer stem cell differentiation by NK cell supernatant Supernatant from NK cells was prepared and used for differentiation of OSCSCs as described above

[36] . On day 13, supernatant from NK cells expanded by OC was used for differentiation.

[0114] Surface staining and cell death assay Cells were stained by antibody or propidium iodide labeling as previously described [37, 41, 42]. Flow cytometry analysis was performed using a Beckman Coulter 20 Epics XL cytometer (Brea, CA), and results were analyzed using FlowJo vX software (Ashland, OR).

[0115] 51 Cr release cytotoxicity assay 51 Cr release assays were performed as previously described

[43] .

[0116] statistical analysis Statistical analysis was performed using unpaired or paired two-tailed Student's t-test. Different groups were compared using one-way ANOVA with Bonferroni post-hoc test. *** (p < 0.001), ** (p 0.001-0.01), * (p 0.01-0.05).

[0117] Example 2: Osteoclasts preferentially activate supercharged NK cells, rapidly expand CD8+ T cells, and lead to a reduction in natural killer cell numbers from cancer patients and BLT-humanized mice. Preferential proliferation of NK cells by osteoclasts and T cells by dendritic cells and significant enhancement of their functions. The activation effects of osteoclasts, monocytes, and DCs on NK cell proliferation and function were compared. NK cells were activated with IL-2 and anti-CD16 mAb for 18–20 h and then cocultured with OC and / or sAJ2. The combination of OC and sAJ2 preferentially expanded NK cells while maintaining a low proportion of T cells (Figure 1). The proliferation rate and level of contaminating T cells in NK cultures were then compared between cocultures with OC, DC, and monocytes treated with sAJ2. NK cells cocultured with OC preferentially expanded NK cells, while the proliferation rate of contaminating T cells remained very low over the first 1–2 months of culture (Figures 2 and 4A). In contrast, DCs preferentially expanded T cells, while the proportion of NK cells remained low. Monocytes could also expand T cells, but T cell proliferation remained lower than NK cell proliferation. The proportion of T cells initially increased in all three cultures; however, in subsequent cultures, the rate of T cell proliferation decreased in cocultures with OCs, whereas T cells continued to proliferate and increased substantially in cultures with DCs. A steady state of T cell proliferation was also observed in cocultures with monocytes (Figure 2A-D).

[0118] NK cells cocultured with OCs were significantly more able to lyse OSCSCs than NK cells cocultured with monocytes or DCs, with a significant increase from days 9 to 15. This correlated with faster proliferation of NK cells in cocultures with OCs on day 15 (Figure 2E). IL-2 and anti-CD16 mAb-activated NK cells cultured with OCs secreted significantly higher amounts of IFN-γ compared with NK cells cocultured with monocytes or DCs (Figure 2F). Analysis of the proliferation rate and population doublings (defined as the logarithm of the ratio of the final count to the baseline count divided by the logarithm of 2) of NK cells revealed that OCs expanded NK cells 21,000–132,000-fold on day 20 and 300,000–5,100,000-fold on day 31, resulting in 17–21 population doublings within 4 weeks (Figure 2G–H).

[0119] Freshly isolated monocytes were compared with mature DCs and OCs, which express key surface receptors. CD54 was upregulated on DCs and OCs, whereas MHC-I expression was reduced on DCs and OCs compared with monocytes (Figure 1D). Killer cell immunoglobulin-like receptors (KIRs), KLRG1, and MICA / B, were high on OCs, intermediate on monocytes, and very low on DCs (Figure 2I). ULBP1-6 was high on monocytes, intermediate on OCs, and low on DCs (Figure 2I). NK cell receptors, including CD94 and NKG2D, were higher on OC-expanded NK cells (Figure 2J, bottom row) compared with untreated primary NK cells (Figure 2J, top row). KIR2 and KIR3 expression was intermediate on expanded NK cells (Figure 2J, bottom row).

[0120] The residual population of T cells purified from OC-expanded NK cells does not mediate cytotoxicity but does secrete IFN-γ. The majority of T cell contamination from OC-expanded NK cells was CD8+ T cells (Figure 3A). On day 9, the OC-expanded NK cells were sorted to obtain purified T and NK cells. The purity of NK cells was then tested using CD16 and CD3 / 56 antibodies (Figure 3B). NK and T cells were then treated with IL-2 for 18–20 hours, after which they were cultured against OSCSCs and K562. 51 CD3+ T cells isolated from OC-expanded NK cells were used in a Cr release assay. CD3+ T cells isolated from OC-expanded NK cells were unable to lyse OSCSCs (Figure 3C) or K562 (Figure 3D). Supernatants from NK cells secreted significantly higher levels of IFN-γ compared with T cells (Figure 3E).

[0121] Osteoclast-induced NK cell proliferation remained high at 1 month, gradually decreased at 2 months, and significantly decreased at 3 months. NK cells cultured with OC expanded for 31–36 days, while the rate of contaminating T cells remained low (Figure 4A). NK cells expanded on day 36 were recultured with OC for a second round of expansion, and NK proliferation continued for 27 days (Figure 4B). Similarly, the rate of T cell proliferation remained very low during the second round of OC-induced NK cell expansion (Figure 4B). NK cells expanded on day 67 were recultured with OC for a third round of expansion; however, NK cells were gradually lost due to T cell proliferation (Figure 4C). Little or no cell death was observed in expanded NK cells during the third round of OC-induced expansion (Figure 4M). The ability of NK cells to lyse cancer stem cells and secrete IFN-γ gradually declined during the first and second rounds of expansion, and these functions were minimized during the third round, when a larger percentage of T cells expanded (Figures 4N–4S).

[0122] Osteoclasts, but not K562 or OSCSCs, proliferate NK cells and increase NK cell function. Activated NK cells were cultured with OSCSCs, K562, OC, irradiated K562, or irradiated OC in the presence of sAJ2 to determine NK proliferation and their functional levels (Figures 5A-5H). NK cell proliferation and function (cytotoxicity and IFN-γ secretion) induced by either non-irradiated, irradiated K562, or OSCSCs were significantly lower than those induced by non-irradiated or irradiated OC (Figure 5).

[0123] The reduced cytotoxicity and low secretion of IFN-γ by NK cells from patients is consistent with increased T cell proliferation. When cultured with OC, purified NK cells from cancer patients were unable to sustain NK cell proliferation; in fact, by day 12, more than half of the expanded cells were T cells. Furthermore, by day 31, only 10% of NK cells remained in the culture (Figures 6A and 7A). Furthermore, the total number of expanded NK and T cells was determined within 31–36 days of expansion in cancer patients. Fewer expanded cells were observed from cancer patients compared with healthy controls (Figures 6D and 7C), and the level of T cell proliferation was significantly higher than that of NK cells (Figures 6E–F and 7D–F). In contrast, NK cells isolated from healthy donors sustained NK cell proliferation, with the level of NK proliferation being significantly higher than that of T cells (Figures 6B, E, F and 7B, 7E, and 7F). No or very little NK cell proliferation was observed in patient NK cells compared with healthy NK cells at various days of culture (Figure 7F). No significant cell death could be observed in expanded cells from either healthy donors or patients, although the rate of cell death was slightly higher in cells from patients than in healthy donors (Fig. 6C).

[0124] Patient NK cells cultured with OC lysed significantly fewer OSCSCs compared with healthy NK cells cultured with OC (Figures 6G and 7G). When normalized based on NK cell number, the cytotoxicity induced per NK cell by patients was lower than that of NK cells from healthy controls (Figures 6H and 7H). Patient NK cells expanded with OC secreted significantly less IFN-γ compared with healthy NK cells expanded with OC (Figures 6I and 7I). NK cells from oral cancer patients expanded with OC secreted significantly less IL-10 compared with healthy NK cells (Figure 6J), whereas those from pancreatic cancer patients secreted higher IL-10 compared with healthy NK cells (Figure 7J). No significant differences were observed in the levels of IL-6 secretion by healthy or cancer patient NK cells (Figures 6K and 7K). Compared with healthy control NK cells expanded by osteoclasts, NKG2D surface expression levels were similar on the surface of patient NK cells, whereas CD94 expression was high and KIR2, NKp30, NKp44, and NKp46 expression was low (Figures 6L and 6M).

[0125] Supernatant from patients' expanded NK cells has very low capacity to differentiate into OSCSCs. After treating OSCSCs with equal volumes of day 13 supernatants from patients and healthy donors for 18–20 hours, the expression levels of CD44, B7H1, CD54, and MHC-I were analyzed (Figure 6N). Healthy NK supernatants increased MHC-I expression by 7.1-fold, whereas patient NK supernatants only increased it by 2.56-fold (Figure 6N). A 13-fold increase in CD54 expression was observed with healthy NK supernatants compared with a 2.1-fold increase with patient NK supernatants. A 3.75-fold increase in B7H1 expression was observed with healthy NK supernatants compared with a 1.5-fold increase with patient NK supernatants. CD44 expression was decreased by healthy NK supernatants, whereas no decrease was observed with patient NK supernatants (Figure 6N). No significant cell death was observed after treating OSCSCs with NK supernatants (Figure 6O). As shown in Figure 6P , a 74% reduction in NK cell-mediated cytotoxicity was observed when treated with healthy NK supernatant, whereas only a 33% reduction was observed with patient NK supernatant ( Figure 6P ).

[0126] Oral tumors in humanized mice preferentially expand T cells, resulting in loss of NK cytotoxicity while retaining IFN-γ secretion. Oral tumors were implanted into humanized BLT mice, which were sacrificed 4 weeks after tumor implantation. Spleens were harvested from hu-BLT mice and T cells were sorted. Flow-through cells containing B cells (Figure 8) were then treated with IL-2 and anti-CD16 mAb for 18–20 hours and then cultured with BLT-OC. Tumor-bearing hu-BLT mice contained a greater proportion of NK cells (Figure 9A), but proliferation gradually led to significant T cell proliferation. This began on day 6 and continued through day 22, at which point 96% of cells were T cells and only 1.1% were NK cells. In contrast, flow-through cells from hu-BLT tumor-free mice, which initially contained fewer NK cells, expanded NK cells, with levels rising from 28.6% on day 6 to 69% on day 22 (Figure 9B). When cultured with autologous OC, NK cell levels increased in both animals from the first day of culture, but tumor-free mice increased 10.59-fold from day 0 to day 6, while tumor-bearing mice increased 4.56-fold (Fig. 9B). The total number of proliferated lymphocytes was lower in tumor-bearing mice compared with tumor-free ones (Fig. 9C), and the majority of these were T cells, not NK cells (Fig. 9D and E).

[0127] When cocultured with OCs, NK-enriched cells from tumor-bearing mice were able to lyse significantly fewer OSCSCs than those from tumor-free control mice (Figure 9F). Furthermore, when cytotoxicity per NK cell was assessed, NK cells from tumor-bearing mice exhibited lower cytotoxicity compared with NK cells from tumor-free control mice (Figure 9G). NK-enriched cells from tumor-bearing mice secreted significantly higher IFN-γ (Figure 9H), lower IL-10 (Figure 9I), and slightly lower IL-6 (Figure 9J) compared with tumor-free control mice. Serum from the peripheral blood of tumor-bearing hu-BLT mice showed increased secretion of IFN-γ, IL-10, and IL-6 compared with control tumor-free mice (Figure 9K).

[0128] IL-15 mediates, in part, osteoclast-induced proliferation of NK cells, while IL-12 is responsible for increasing IFN-γ secretion by NK cells. The levels of cytokines, chemokines, growth factors, and ligands secreted by primary NK cells and NK cells expanded by OC on day 6 were determined (Figure 10). Most of the secreted cytokines, chemokines, growth factors, and ligands were higher in OC-expanded NK cells compared with those secreted by primary NK cells activated with IL-2 and anti-CD16 mAb (Figure 10). A 50- to 60-fold higher induction of IL-12 and a 20- to 26-fold higher secretion of IL-15 were observed in OC-expanded NK cells compared with primary NK cells (Figure 10). The addition of anti-IL-12 and / or anti-IL-15 mAb significantly reduced cell proliferation, with 1 μg / ml anti-IL-15 being the most effective (Figure 11A). Treatment with anti-IL-12 and / or anti-IL-15 did not affect the cytotoxic function of NK cells on day 9 (Fig. 11B), but significantly inhibited it on day 15 (Fig. 11B). The level of IFN-γ secretion by OC-expanded NK cells was reduced more with anti-IL-12 than with anti-IL-15 treatment (Fig. 11C).

[0129] The addition of anti-CD3 antibody suppresses T cell proliferation and increases NK cells expanded by OC. Lymphocytes were treated with anti-CD3, and NK or T cell proliferation was assessed at various days (Figures 12A and 12B). The levels of cells that lost both forward and side scatter increased (Figures 12C and 12D), and elevated DNA fragmentation was obtained in anti-CD3-treated cells, indicating T cell loss, as evidenced by an increased sub-G0 / G1 peak in cell cycle analysis (Figure 12E). Accordingly, NK cell levels were elevated in both patients and healthy donors (Figures 12A-12B). The population that lost forward and side scatter was CD3+ T cells, as judged by CD3 and CD16 mAb staining (data not shown). In the absence of NK cells, anti-CD3 mAb-treated T cells did not lose forward and side scatter (Figure 13) or exhibit cell death (data not shown). When NK cell cytotoxicity was assessed before and after the addition of anti-CD3 mAb, NK cells from both healthy and patient donors had significantly lower cytotoxicity, although levels were higher in healthy NK cells (Figure 12G). In contrast, the level of IFN-γ secretion was significantly elevated in both healthy and patient NK samples, indicating the induction of fractional anergy in NK cells by anti-CD3 mAb bound to T cells (Figure 12F).

[0130] Osteoclast-activated NK cells substantially increase CD8+ T cell numbers. Cancer patients, on average, have a moderately higher percentage of CD8+ T cells and a lower percentage of CD4+ T cells compared with healthy controls (Figure 14A). When cultured with osteoclasts, T cells in the absence of NK cells failed to expand CD8+ T cells; however, purified NK cells activated with OCs, which contained very small amounts of contaminating T cells, expanded CD8+ T cells from both healthy and patient cultures, even though patient NK cell cultures expanded T cells faster than healthy NK cells (Figure 14B). When T cells expanded by OC-activated NK cells were compared with T cells expanded by OC-activated NK cells, OCs preferentially expanded NK cell-mediated CD8+ T cells, whereas DCs expanded NK cell-mediated CD4+ T cells (Figures 14C-G). Furthermore, CD8+ T cells expanded by OC-activated NK cells were all of the activated phenotype, being CD45RO+CD44+CD62L- / low CCR7- / low-expressing memory / effector phenotype (Figure 14H).

[0131] In vivo and in vitro studies have recently demonstrated that osteoclasts are the primary activator of NK cells. More importantly, a single month of stimulation with osteoclasts was able to sustain the proliferation of supercharged NK cells from healthy donors for more than two months. Furthermore, the cytotoxic function of NK cells remained significantly elevated at one month and declined at the second month of expansion. It is unclear why NK cell function continues to decline at the second month of stimulation despite the continued proliferation of large numbers of NK cells. It may be that additional signals are required to maintain NK cell cytotoxicity at the second month and / or that OCs need to be replenished more frequently.

[0132] Compared to previous methods, using OCs as feeder cells was the best strategy for expanding large numbers of NK cells (Table 1). First, we obtained 21,000-132,000-fold expansion by day 20 and 0.3-5.1 million cells by day 31, resulting in 17-21 population doublings within 4 weeks of expansion, a rate much faster than any previously reported method (Table 1). While the cytotoxic function of the NK cells expanded throughout the study is difficult to compare due to the variety of targets used, the strategy disclosed herein provides large numbers of NK cells with the significant ability to target and lyse cancer stem cells and secrete IFN-γ (Table 1). Furthermore, primary NK cells were expanded with little or no transformation potential. It is known that transformation affects the killing potential of NK cells, as most NK lines (e.g., YT, NK92, or NK-L) or transfected NK cells are prone to losing most or part of their cytotoxic and IFN-γ secretion capabilities, respectively. Furthermore, anti-CD3 antibody treatment to maintain NK cell proliferation is an effective strategy for limiting T cell proliferation, but the cytotoxic activity of such NK cells is low due to the significant NK cell-induced division anergy. This strategy may be excellent for inducing tumor differentiation by greatly increasing IFN-γ secretion; however, it is highly suboptimal for tumor elimination. This may be one of the reasons why the use of NK cells in immunotherapy of solid tumors has not resulted in tumor regression in a minority of patients tested.

[0133] [Table 1] TIFF2025183317000002.tif250165

[0134] Unlike OCs, DCs stimulated preferential proliferation of a small number of contaminating T cells among purified NK cells, reducing NK proliferation because T cell levels remained elevated for 1 month. Therefore, low levels of cytotoxicity against OSCSCs were observed. Interestingly, when NK cell cytotoxicity was assessed at earlier time points, when similar proportions of NK cells were observed in OC and DC cultures, elevated NK cytotoxicity could be observed by NK cells cultured with OCs (data not shown). Similarly, IFN-γ secretion by NK cells cultured with OCs was increased compared with those cultured with DCs, indicating that, on a per-cell basis, NK cells cultured with OCs secreted higher IFN-γ than those cultured with DCs (Figure 1C). T cells sorted from OC-expanded NK cells were very low in cytotoxicity, and the minimal cytotoxicity observed by T cells was likely due to contaminating NK cells (Figures 3C and 3B). On the other hand, T cells could release IFN-γ, but at significantly lower levels than NK cells (Figure 3E). When T cell subsets, CD8, CD4, and gdT cells, were sorted and used in cytotoxicity assays, only NK cells were able to lyse these cells, and the residual cytotoxicity observed in the T cell subsets was due to contaminating NK cells (Figure 3F). OCs expressed more activating NK ligands than DCs, while both showed similar levels of the differentiation antigen CD54. Interestingly, both showed lower levels of MHC class I expression compared to monocytes (Figure 1D). Furthermore, IL-15 secretion appeared to be important for NK cell proliferation (Figure 11A), whereas IL-12 was important for IFN-γ secretion (Figure 11C). Both cytokines were important for NK cell cytotoxic function when assessed at later times compared to earlier time points of proliferation (Figure 11B).

[0135] Compared with OCs, all tumor cell lines tested, whether non-irradiated or irradiated, did not support NK cell proliferation over the long term. Even though IFN-γ secretion could be observed in cultures with K562 and OSCSCs in the short term, this effect was short-lived (Figure 5). Furthermore, OC-induced proliferation was significantly inferior to that of irradiated PBMCs (data not shown). OC-expanded NK cells, when compared with primary NK cells, expressed significantly higher levels of activating receptors (including NKG2D, NKp46, NKp44, NKp30, and CD94), increased inhibitory receptors KIR2 and KIR3, and very low levels of the CD16 receptor (Figure 2J, bottom). When compared with OC-expanded NK cells from healthy donors, cancer patients generally had significantly lower receptor expression, and the levels were even lower than those seen in healthy NK cells stimulated with OCs, which exhibited significant cytotoxicity and loss of cytokine secretion capacity, and were capable of expanding T cells. Similar to patient NK cells, NK cells expanded by K562 and OSCSCs were short-lived and had very low cytotoxicity and cytokine secretion capabilities. Unlike OCs, K562 or OSCSCs expressed low levels of NK-activating ligands (Figure 1D), and increasing these signals by OCs can enhance NK cell proliferation and function; therefore, they lacked the secretion of key cytokines responsible for NK cell proliferation (Figure 10A). Because the proliferation rate of NK cells expanded by engineered K562 cells was 100-fold lower than that of NK cells expanded by OCs (Table 1 and Figures 5A and 5B), it remains to be determined whether the signals delivered by engineered K562 (Table 1) are inferior to those delivered by OCs. Furthermore, while sustained stimulation by engineered K562 was necessary to maintain NK proliferation, a single stimulation by OCs was sufficient to expand supercharged NK cells for more than a month. Furthermore, OC-expanded NK cells, unlike primary NK cells, tolerate freezing temperatures well and retain their supercharged properties and proliferation rate without loss of viability or function (Figure 15).

[0136] A completely different profile was observed when NK cells from cancer patients were expanded using autologous or allogeneic OCs. OCs from cancer patients also expanded T cells early in culture, reducing the overall proliferation of NK cells over various days. When the function of patient NK cells was assessed after OC culture, a significant loss of NK cell cytotoxicity and reduced IFN-γ secretion could be observed for each NK cell (Figure 6H and Figure 15G). This observation is significant because the rapid expansion of very small amounts of contaminating T cells in purified NK cultures from cancer patients correlates with the loss of NK cell cytotoxic function. Thus, the loss of NK cells may also provide fertile ground for the growth and metastasis of cancer stem cells.

[0137] To test whether OCs obtained from tumor-implanted humanized mice, like those from cancer patients, expanded minimally contaminating T cells in purified NK cultures more rapidly than their non-tumor-bearing counterparts, we implanted and expanded OSCSCs in the floor of the mouth. After 5 weeks of expansion, mice were euthanized, T cells were removed, and the cells were cultured with autologous and allogeneic OCs to determine the rate of NK cell expansion. Similar to NK cells from healthy donors, NK cells from tumor-free hu-BLT mice expanded NK cells for a long period of time, while those from tumor-bearing mice expanded minimally contaminating T cells more rapidly in NK cultures, favoring T cell expansion over NK cells. Interestingly, similar to the loss of NK cell cytotoxicity observed in cancer patients, a significant loss of NK cell cytotoxicity was also observed in tumor-implanted hu-BLT mice, which may be the underlying mechanism behind T cell expansion. However, OC-expanded NK+ T cells from tumor-bearing mice secreted higher levels of IFN-γ compared with tumor-free ones, suggesting that potential induction of division anergy in NK cells drives the differentiation of cancer stem cells. This was confirmed by the fact that single-cell preparations of tumors in NK-injected tumor-bearing hu-BLT mice displayed higher differentiation antigens and were resistant to NK cell-mediated cytotoxicity.

[0138] Because the addition of human IL-15 promoted NK cell expansion, we hypothesize that the microenvironment in humanized mice is not conducive to NK cell expansion due to the lack of cross-reactivity between murine and human IL-15. Unlike NK cells, which require signals from osteoclasts for their proliferation, T cells proliferate rapidly in the absence of osteoclasts, and osteoclasts modestly stimulate T cell proliferation (Figures 3G and 3H). While signals received from the mouse tissue microenvironment are sufficient to maintain T and B cell proliferation, NK cell proliferation likely requires signals from myeloid subsets. Interestingly, the frequencies of both NK and myeloid subsets decrease in peripheral blood and tissues, whereas the levels of T and NK cells remain similar in the myeloid cell-rich bone marrow. Whether DCs favor T cell expansion in peripheral blood and osteoclasts favor NK cell expansion in bone marrow requires further study. In both models, low percentages and impaired function of NK cells are known to potentially contribute to cancer progression, so the humanized mouse is the best and closest model to a human cancer model.

[0139] The most exciting finding is the ability of NK cells to expand CD8+ T cells when cultured in the presence of OC. This data suggests that NK cells, as important effectors in CD8+ T cell expansion, can increase the targeting and lysis of tumor cells expressing higher levels of MHC class I. Whether NK cells increase tumor cell lysis in an antigen-specific manner by expanding antigen-specific CD8+ T cells requires further study. The rapid proliferation of T cells and reduced numbers of NK cells in the peripheral blood / tissues of cancer patients and humanized mice may be detrimental to NK cell targeting of MHC class I-low targets, including cancer stem cells / undifferentiated tumors, in order to minimize tumor cell burden. Furthermore, NK cells promote optimal differentiation of cancer stem cells and high MHC class I expression, and also provide large amounts of IFN-γ for targeting CD8+ T cells. Therefore, restoring NK cell numbers and function in cancer patients may be important for establishing effective tumor control.

[0140] Example 3: Osteoclast-activated supercharged NK cells preferentially and rapidly expand supercharged CD8+ T cells: Increased kinetics of CD8+ T cell expansion by OC-expanded NK cells in cancer patients and BLT-humanized mice. Further studies were conducted to test Example 2, and the results are summarized below.

[0141] The residual population of T cells purified from OC-expanded NK cells does not mediate cytotoxicity but does secrete IFN-γ. The majority of T cell contamination from OC-expanded NK cells was CD8+ T cells (Supplementary Figure S2A). On day 9, the OC-expanded NK cells were sorted to obtain purified T and NK cells. The purity of NK cells was then tested using CD16 and CD3 / 56 antibodies (Supplementary Figure S2B). NK and T cells were then treated with IL-2 for 18–20 hours, after which they were cultured against OSCSCs and K562. 51 CD3+ T cells isolated from OC-expanded NK cells were used in Cr release assays. CD3+ T cells isolated from OC-expanded NK cells failed to lyse OSCSCs (Supplementary Figure S2C) or K562 (Supplementary Figure S2D). Supernatants from NK cells secreted significantly higher levels of IFN-γ compared with T cells (Supplementary Figure S2E).

[0142] The reduced cytotoxicity and low secretion of IFN-γ by NK cells from patients is consistent with increased T cell proliferation. When cultured with OC, purified NK cells from cancer patients were unable to sustain NK cell proliferation; in fact, by day 12, more than half of the expanded cells were T cells. Furthermore, by day 31, only 10% of NK cells remained in culture (Figure 3A and Supplementary Figure S4A). Furthermore, the total number of expanded NK and T cells was determined within 31–36 days of expansion in cancer patients. Compared to healthy controls, fewer expanded cells were found in cancer patients (Figure 3D and Supplementary Figure S4C), and the level of T cell proliferation was significantly higher than that of NK cells (Figure 3E–F and Supplementary Figure S4D–E). In contrast, NK cells isolated from healthy donors sustained NK cell proliferation, with the level of NK proliferation being significantly higher than that of T cells (Figure 3B, Figure 3E, Figure 3F and Supplementary Figures S4B, S4D, S4E). No or very little NK cell proliferation was observed in patient NK cells compared with healthy NK cells at various days of culture (Figs. 3E and S4D). No significant cell death was observed in expanded cells from either healthy donors or patients, although the rate of cell death was slightly higher in cells from patients than in healthy donors (Fig. 3C).

[0143] Patient NK cells cultured with OC lysed significantly fewer OSCSCs compared with healthy NK cells cultured with OC (Figure 3G and Supplementary Figure S4F). When normalized based on NK cell number, the cytotoxicity induced per NK cell by patients was lower than that of NK cells from healthy controls (Figure 3H and Supplementary Figure S4G). Patient NK cells expanded with OC secreted significantly less IFN-γ compared with healthy NK cells expanded with OC (Figure 3I and Supplementary Figure S4H). NK cells from oral cancer patients expanded with OC secreted significantly less IL-10 compared with healthy NK cells (Figure 3J), whereas those from pancreatic cancer patients secreted higher IL-10 compared with healthy NK cells (Supplementary Figure S4I). No significant differences were observed in the levels of IL-6 secretion by healthy or cancer patient NK cells (Figure 3K and Supplementary Figure S4J). NKG2D surface expression levels were similar to those of healthy NK cells compared with patient NK cells expanded with osteoclasts (Figure 3L). The intensity of CD94 expression was higher on the surface of patient NK cells compared with healthy controls (Figure 3L). KIR2, NKp30, NKp44, and NKp46 expression was lower on the surface of patient NK cells expanded with OC compared with healthy NK cells (Figure 3L), whereas KIR3 expression was either the same or lower on the surface of patient NK cells expanded with OC compared with healthy NK cells (Figure 3L).

[0144] Osteoclast-activated NK cells substantially increase CD8+ T cell numbers. Cancer patients have, on average, a higher percentage of CD8+ T cells and a lower percentage of CD4+ T cells compared with healthy controls (Figure 7A). When cultured with osteoclasts, T cells in the absence of NK cells failed to expand CD8+ T cells; however, purified NK cells activated with OCs containing undetectable or very low levels of contaminating T cells expanded CD8+ T cells from both healthy and patient cultures, although patient NK cell cultures expanded T cells faster than healthy NK cells (Figure 7B). T cells isolated from patients had higher levels of CD45RO and lower levels of CD45RA, CD62L, CD28, CCR7, and CD127 compared with T cells isolated from healthy controls (Figure 7C).

[0145] NK cells activated by OCs preferentially expanded CD8+ T cells, whereas NK cells activated by DCs preferentially expanded CD4+ T cells. To determine whether there were differences between T cell subpopulations expanded by OC-activated versus OC-activated NK cells, we analyzed CD4 and CD8 subpopulations in healthy donors. OC-activated NK cells preferentially expanded CD8+ T cells, whereas OC-activated NK cells expanded CD4+ T cells (Figures 7D–7I). CD8+ T cells expanded by OC-activated NK cells exhibited high CD45RO and CD44, very low levels of CD62L, CCR7, and CD127, and intermediate levels of CD28, whereas CD4+ T cells expanded by OC-activated NK cells exhibited low levels of CD45RO, intermediate levels of CD44, and high levels of CD62L and CCR7, with little change in CD127 and low levels of CD28 (Figure 7J). T cells activated with either OC or DC in the absence of NK cells displayed a surface profile similar to that obtained with NK activated with DC, except for CD28 expression, which was similar to that obtained with NK activated with OC (Figure 7J). The proportions of CD4 and CD8 T cells within CD3+ T cells were similar between PBMCs and those activated with either OC or DC in the absence of NK cells, and no significant levels of PD-1, Tim3, or KLRG-1 could be observed on T cells activated with either OC or DC in the presence or absence of NK cells (Figures 7K-M).

[0146] Example 4: Materials and Methods for Example 5 Cell lines, reagents, and antibodies Immune cells were cultured using RPMI 1640 (Gemini Bio-Products, CA) supplemented with 10% fetal bovine serum (FBS). OSCSCs and OSCCs were dissociated and propagated from patient tongue tumors at UCLA and cultured in RPMI 1640 supplemented with 10% FBS. Recombinant IL-2 was obtained from NIH-BRB. Flow cytometry antibodies used in this study were obtained from Biolegend (San Diego, CA). Monoclonal anti-TNF-α and monoclonal anti-IFN-γ antibodies were obtained from commercial sources or prepared in our laboratory; a dilution of 1:100 was found to be the optimal concentration used for blocking experiments, as described above.

[0147] Purification of human NK cells and monocytes Written consent was obtained and all procedures were approved by the UCLA Institutional Review Board (IRB). PBMCs from 380 healthy human donors were isolated, and NK cells and monocytes were purified using isolation kits obtained from Stem Cell Technologies, as previously described. The purity of the NK cell and monocyte populations, respectively, was found to be 95% or greater based on flow cytometry analysis.

[0148] Probiotic bacteria AJ2 is a combination of eight different strains of Gram-positive probiotic bacteria (Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium 10 infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus bulgaricus) used to induce stem cell differentiation. These strains were selected for their superior ability to induce optimal secretion of both pro- and anti-inflammatory cytokines in NK cells. Furthermore, specific colonies were selected after three rounds of subcloning based on their ability to grow and tolerate environmental pressures such as temperature and acidity.

[0149] Osteoclast generation and proliferation of supercharged human and hu-BLT-derived NK cells Purified monocytes were cultured in alpha-MEM medium 400 containing M-CSF (25 ng / mL) and RANKL (25 ng / mL) for 21 days or without other conditions. The medium was refreshed every 3 days with fresh alpha-MEM containing M-CSF and RANKL. Human purified and hu-BLT-enriched NK cells were activated with rh-IL-2 (1000 U / mL) and anti-CD16 mAb (3 μg / mL) for 18–20 hours, then co-cultured with osteoclasts and sonicated AJ2. As described above, the culture medium was refreshed every 3 days with rh-IL-2. NK cells expanded ex vivo by osteoclasts and sAJ2 cells were termed supercharged NK cells because they possessed superior cytotoxicity and IFN-γ secretion and survived for a long period of time compared to other expansion methodologies. For sonication, AJ2 was weighed and resuspended in RPMI 1640 medium containing 10% FBS at a concentration of 10 mg / ml. The bacteria were thoroughly vortexed and then sonicated on ice for 15 seconds at amplitudes of 6–8. The sonicated sample was then incubated on ice for 30 seconds. After every 5 pulses, a sample was taken and observed under a microscope until at least 80% of the cell wall was lysed. Approximately 20 rounds of sonication / incubation on ice were performed to determine complete sonication. Finally, the sonicated sample (sAJ2) was aliquoted and stored in a -80°C freezer until use.

[0150] Analysis of human oral cancer cell growth in immunodeficient and humanized mice Animal studies were conducted under written approval from the UCLA Animal Research Committee (ARC) (2012-101-13A). Humanized BLT (hu-BLT; human bone marrow / liver / thymus) mice were prepared in our core facility as previously described.

[0151] In vivo growth of oral tumors was performed by orthotopically implanting tumor cells into the floor of the mouth of 8- to 10-week-old NSG or hu-BLT mice. After anesthetizing the mice with isoflurane, tumor cells were transferred together with 10 pL of HC Matrigel (Corning, NY, USA) by direct injection into the floor of the mouth. Seven to 10 days after surgery, the mice were inoculated with 1.5 × 10 6 Mice received 1000 supercharged NK cells. They were fed AJ2 (5 million bacteria / dose) starting 1 or 2 weeks before tumor implantation and every 48 hours throughout the experiment. Mice were euthanized when signs of morbidity became evident. Oral tumors, BM, spleens, and peripheral blood were harvested.

[0152] Dissociation and culture of cells from tissues of hu-BLT and NSG mice Animals were sacrificed and BM, spleen, peripheral blood, and oral tumors were obtained to prepare single cell suspensions of mouse tissues for subsequent analysis. 3 The tissue was minced into small pieces and placed in a digestion buffer containing 1 mg / ml (adipose tissue) collagenase II, 10 U / ml DNAse I, and 1% bovine serum albumin in DMEM. The mixture was then incubated at 37°C for 20 minutes in an oven equipped with a 150 rpm agitator. After digestion, the sample was filtered through a 40-μm cell strainer and centrifuged at 1500 rpm for 10 minutes at 4°C. The pellet was resuspended in DMEM, and the cells were counted. Single-cell suspensions from BM and spleen were obtained by digesting the tissues as described above. PBMCs were obtained using Ficoll-Hypaque centrifugation of heparinized blood samples. The buffy coat containing PBMCs was harvested, washed, and resuspended in RPMI 1640 medium.

[0153] Purification of NK cells, CD3+ T cells, and monocytes from hu-BLT mice CD3C T cells were isolated from hu-BLT splenocytes using a T cell selection kit (Stem-Cell Technologies), and T cell-depleted cells were used as NK-enriched cells; NK cells from hu-BLT mice were enriched for human CD56 cMonocytes from hu-BLT mice were isolated from BM cells using a human CD14 positive selection kit (Stem-Cell Technologies, Canada).

[0154] ELISA A single ELISA was performed as described above. To obtain assay cytokine and chemokine concentrations, standard curves were generated by either 2- or 3-fold dilutions of the recombinant cytokines provided by the manufacturer.

[0155] Surface staining and cell death assay Staining was performed by labeling cells with antibodies or propidium iodide as described above. Cells were washed twice with ice-cold PBS containing 1% BSA. Specific human monoclonal antibodies at the predetermined optimal concentration were added to 1 × 10 cells in 50 μl of chilled BSA. 4 1 × 10 cells in 50 μl of cold BSA. 4 Cells were stained with 8 pg / ml propidium iodide, incubated on ice for 10 min, and made up to 500 μl with PBS-BSA. Flow cytometry analysis was performed using a Beckman 485 Coulter Epics XL cytometer (Brea, CA), and results were analyzed using FlowJo vX software (Ashland, OR).

[0156] 51 Cr release cytotoxicity assay 51 Cr release assays were performed as described above. Briefly, different numbers of effector cells were added to the Cr release assay. 51 Cr-labeled target cells. After a 4-hour incubation period, supernatants were harvested from each sample and the released radioactivity was counted using a gamma counter. The percentage of specific cytotoxicity was calculated as follows: % Cytotoxicity Experiment cpm-Spontaneous cpm Total cpm-Spontaneous cpm LU30 / 10 6 is calculated using the reciprocal of the number of effector cells required to lyse 30% of the tumor target cells.

[0157] Stem cell differentiation by NK cell supernatant NK cells were treated with a combination of anti-CD16 monoclonal antibody (3 pg / mL) and IL-2 (1,000 U / mL) for 18 hours, after which the supernatant was removed and used for differentiation experiments. The amount of IFN-γ produced by activated NK cells was measured using an IFN-γ ELISA (Biolegend, CA, USA). OSCSCs were differentiated by gradually adding increasing amounts of NK cell supernatant daily. On average, a total of 3,500 pg of IFN-γ-containing supernatant was added for 4 days to induce differentiation, inducing OSCSC differentiation and resistance to NK cell-mediated cytotoxicity. Target cells were then washed with 1x PBS, removed, and used in experiments as described above.

[0158] statistical analysis Statistical analysis was performed using an unpaired two-tailed Student's t-test. Different groups were compared using one-way analysis of variance with Bonferroni post-hoc test, and (n) indicates the number of mice used in the experiment. For cytotoxicity and cytokine analyses, either duplicate or triplicate samples were used for evaluation. The following symbols represent the level of statistical significance within each analysis: *** (p value < 0.001), ** (p value 0.001-0.01), * (p value 0.01-0.05).

[0159] Example 5: Supercharged NK cells inhibit in vivo growth and progression of stem-like / poorly differentiated oral tumors in humanized BLT mice. A single infusion of supercharged NK cells inhibited OSCSC tumor growth and significantly improved the health of mice. Hu-BLT mice were generated, and human OSCSCs were transplanted into the floor of the mouth of NSG and hu-BLT mice (Figures 62A and 62B). Weight loss was monitored weekly (Figure 62C). A single injection of supercharged NK cells resulted in minimal weight loss in OSCSC-transplanted mice (Figure 62C). While OSCSC-transplanted and NK cell-injected mice showed no morbidity and were able to consume food, mice bearing oral tumors in the absence of NK injection became morbid, had food intake complications due to tumor growth (data not shown), and exhibited rapid weight loss (Figure 62C). Interestingly, tumor-bearing hu-BLT mice without NK injection lost less weight than tumor-bearing NSG mice, indicating that reconstituted human immune cells can slightly, but not efficiently, limit tumor growth (Figure 62C). The therapeutic effect of NK injection in hu-BLT mice was also observed when tumor size was compared after tumor resection. Tumors from tumor-bearing hu-BLT mice not injected with NK were much larger than those from tumor-bearing hu-BLT mice injected with NK (Figures 62D and 62E). Tumor weight remained substantially smaller in NK- or NK-injected / AJ2-fed mice (Figure 62F) compared with the large tumors formed in tumor-bearing mice that did not receive NK treatment (Figures 62D-F). Furthermore, consistent with the weight loss data, tumor-bearing hu-BLT mice not injected with NK had slightly smaller tumors compared with tumor-bearing NSG mice, indicating that reconstituted human immune cells can limit tumor growth, but not efficiently (data not shown).

[0160] Hu-BLT mice were transfected with contaminating mouse CD45 C Regarding immune cells, hCD45 in BM, spleen, and peripheral blood C The CD3+ cells in the peripheral blood of hu-BLT mice were reconstituted with immune cells to over 96%–99%. c T cells (Figure 16G), CD3 C CD4 C T cells (Figure 16H), and CD3 C CD8 CThe profile of T cells (Figure 62I) resembled that of human peripheral blood; however, compared to humans, the percentage of B cells was slightly higher (Figure 62J) while the percentage of NK cells was lower (Figure 62K) in hu-BLT mice.

[0161] Loss of NK cytotoxicity and IFN-γ secretion in tumor-bearing mice in all tissue compartments and restoration by NK infusion and / or feeding AJ2 and anti-PD1 infusion Tumor-bearing mice were cultured in spleen cells (Fig. 63A), BM-derived immune cells (Fig. 63B), PBMCs (Fig. 63C), and CD3 + Depleted splenocytes (Fig. 63D) show low NK-mediated cytotoxicity. Tumor-bearing mice injected with NK, both alone and in combination with AJ2 feeding, showed increased NK cytotoxicity in all tissue compartments, with the greatest increase observed when mice were fed AJ2 and injected with NK cells and anti-PD1 antibody (Fig. 63A-D). CD3 + When T cells were depleted from splenocytes by a positive selection kit, a similar NK cytotoxicity profile was observed in the CD3 + This was observed in the depleted splenocytes (Fig. 63D). The CD3 expression levels were also observed in the splenocytes (Fig. 63E and Table 2), BM-derived immune cells (Fig. 63F), PBMCs (Fig. 63G), and splenocyte-depleted cells of 125 tumor-bearing hu-BLT mice. + T cells (Figure 63H) secreted low IFN-γ. Injection of anti-PD1 antibody in combination with NK cells and AJ2 had either no effect or a reduced effect on IFN-γ secretion in all tissue compartments when compared to the NK and AJ2 groups (Figures 63E-63G). Tumor-bearing mice injected with NK showed increased IFN-γ secretion in all tissue compartments, with the greatest increase observed when mice were fed AJ2 (Figures 63E-63H). CD3 + When T cells were depleted from splenocytes by a positive selection kit, tumor-bearing mice that received NK infusion had the greatest increase in IFN-γ secretion compared to tumor-bearing mice in the absence of NK infusion or non-tumor-bearing control mice infused with NK (Figure 63I).

[0162] [Table 2]

[0163] When hu-BLT mice were infused with NK cells and fed AJ2, CD3 + CD8 + The proportion of T cells increased. CD3 + or CD3 + CD8 + Increased percentages of T cells were observed in the BM (Fig. 64A), spleen (Fig. 64B), and blood (data not shown) of mice injected with NK cells alone or in combination with AJ2 feeding (Tables 3 and 4). Considering the percentage of T cells at the time of sacrifice, PBMC, spleen, and BM contained a high percentage of CD3 + T cells and BM showed HLA-DR1 expression in tumor-bearing mice injected with NK cells alone or in combination with AJ2 feeding. + CD11B + The immune subsets were elevated (Table 3). + T cells and CD3 + CD8 + The percentage of T cells was increased in tumor-bearing mice injected with NK cells alone or in combination with AJ2 feeding when BM and splenocytes were cultured (Table 3). Similarly, tumors dissociated from tumor-bearing mice injected with NK and / or AJ2 feeding showed increased CD3 T cell counts at the time of sacrifice. + showed elevated T cell levels (Table 4).

[0164] [Table 3]

[0165] [Table 4]

[0166] NK infusion and / or AJ2 feeding inhibits the growth and progression of stem-like oral tumors and differentiates CSCs in vivo in hu-BLT mice. Tumor cells from NSG (Figure 65A) and hu-BLT mice without NK injections grew rapidly, whereas those with NK cells either did not grow or grew very slowly (Figures 65A-65C). Similarly, tumors from hu-BLT mice implanted with tumors differentiated with NK supernatants did not grow (Figure 65C), and blocking tumor differentiation with a combination of anti-IFN-γ and anti-TNF-α antibodies prior to implantation restored tumor growth in vivo (Figures 65B and 65C). Tumor growth was less in NK-injected mice fed AJ2 compared with mice injected with NK alone (Figure 65C), and both were substantially less than those receiving oral tumor implants alone (Figure 65C). 9-11 times more hCD45 C Immune cells infiltrated tumors in tumor-bearing mice injected with NK cells compared with tumor-only mice (Fig. 65D). The percentage of epithelial cells expressing surface EpCAM in tumors was approximately 5-fold higher in tumor-bearing mice not injected with NK cells compared with tumor-bearing mice injected with NK cells.

[0167] Significantly greater IFN-γ secretion was obtained from tumors of tumor-bearing mice injected with NK cells at various culture days compared with those from tumor-bearing hu-BLT or NSG mice (Figure 65E). Expression of CD54 and MHC-I was higher on tumors obtained from tumor-bearing mice injected with NK cells compared with tumor-bearing hu-BLT and NSG mice that received tumor alone (Figure 65F). Tumors from tumor-bearing mice injected with NK cells in the absence and presence of AJ2 feeding were highly resistant to NK cell-mediated cytotoxicity compared with tumors from tumor-bearing mice not injected with NK cells, demonstrating their differentiated phenotype (Figures 65G and 65H). When NK-mediated differentiation of tumor cells was blocked with anti-IFN-γ and anti-TNF-α antibodies before transplantation, the sensitivity of tumors to NK cell-mediated cytotoxicity was restored (Figure 65H). Oral tumors from NK cell-injected hu-BLT mice secreted relatively little VEGF when normalized based on VEGF secretion from tumor-bearing mice (Fig. 65I). Tumors from NK-injected hu-BLT mice secreted high levels of hCD45 C The highest increase in infiltrating lymphocytes was seen in those injected with NK cells and fed AJ2 (Fig. 65J). C The majority of immune cells are CD3 + T cells, CD4 + T cell subsets are CD8 + Moderately higher proportions than T cells, CD3 + CD56 + CD16 C We demonstrated that the NKT subset exhibited high-intensity CD4, CD8, and CD16 / CD56 surface expression, which was downregulated during tumor interaction, when tumors were treated with IL-2.

[0168] Serum from the peripheral blood of NK-injected tumor-bearing hu-BLT mice showed increased IFN-γ secretion and other cytokines / chemokines / growth factors and ligands compared with that from tumor-bearing hu-BLT mice lacking NK cells, and notably, IFN-γ secretion was further enhanced by feeding the mice AJ2 (Figures 66A and 66C).

[0169] CDDP or paclitaxel, with or without NAC, induced significant cell death in OSCSCs differentiated with NK supernatants treated with IL-2 plus anti-CD16 mAb. Differentiation of OSCSCs with NK supernatant resulted in significant tumor sensitivity to CDDP (Figure 67A). Similarly, paclitaxel mediated higher cell death in OSCSCs differentiated with NK supernatant, and NAC significantly increased paclitaxel-mediated cell death (Figure 67B). Blocking NK-mediated differentiation of OSCSCs with anti-IFN-γ and anti-TNF-α antibodies substantially reduced cell death induced by CDDP or paclitaxel, regardless of the presence or absence of NAC (Figure 67B). Treatment of OSCC (patient-derived differentiated oral tumors) with CDDP or paclitaxel and NAC resulted in higher cell death.

[0170] Monocytes and osteoclasts from NK-infused tumor-bearing mice had a greater ability to activate NK cells when compared with those from tumor-bearing mice in the absence of NK infusion. NK cells from tumor-bearing mice injected with NK cells and differentiated with NK supernatant when cultured with autologous monocytes showed significantly increased cytotoxicity (Figure 68A) and IFN-γ secretion (Figure 68B) compared with mice transplanted with undifferentiated tumors in the absence of NK cell infusion. Blocking NK-mediated differentiation of tumors through the addition of antibodies against TNF-α and IFN-γ before transplantation reduced NK cell cytotoxicity (Figure 68A) and IFN-γ secretion (Figure 68B). OCs were then generated from monocytes derived from the BM of hu-BLT mice and cultured with allogeneic NK cells from healthy human donors to test the extent of NK cell proliferation. NK cell proliferation (Figure 68C) and IFN-γ secretion (Figures 68D and 68E) were increased in tumor-bearing mice injected with NK cells or differentiated with NK supernatant compared with those obtained from tumor-bearing mice not injected with NK cells (Figures 68C-68E). Thus, monocytes and osteoclasts from NK-infused tumor-bearing mice had a greater capacity to activate NK cells than those from tumor-bearing mice in the absence of NK infusion.

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[0172] Incorporation by Reference All publications, patents, and patent applications mentioned herein are herein incorporated by reference in their entirety to the same extent 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 definitions herein, will control.

[0173] Also incorporated by reference in its entirety are any polynucleotide and polypeptide sequences that cite an accession number that correlates to an entry in a public database such as, for example, those maintained by The Institute for Genomic Research (TIGR) at the World Wide Web at tigr.org and / or the National Center for Biotechnology Information (NCBI) at the World Wide Web at ncbi.nlm.nih.gov.

[0174] equivalent 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 which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A method for activating NK cells in vitro or ex vivo, comprising: The method comprising co-culturing the NK cells in culture medium with osteoclasts (OCs).

2. i) providing a cell culture comprising NK cells and osteoclasts; and ii) culturing the NK cells and the osteoclasts in the cell culture. thereby activating said NK cells.

3. 3. The method of claim 1 or 2, wherein the NK cells are primary NK cells, optionally wherein the primary NK cells are not transformed.

4. 10. The method of any one of the preceding claims, wherein the activated NK cells expand to at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within 4 weeks.

5. 10. The method of any one of the preceding claims, wherein the culture comprises a plurality of osteoclasts (OC) and a plurality of NK cells, and the ratio of OC:NK cells in the cell culture is at least 1:

2.

6. 10. The method of any one of the preceding claims, wherein the osteoclasts enhance the cytotoxicity of NK cells, and optionally the cytotoxicity of the NK cells is measured by lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by the NK cells.

7. The cytotoxicity of the NK cells ５１ 7. The method of claim 6, wherein the cytotoxicity is measured by a Cr release cytotoxicity assay.

8. 10. The method of any one of the preceding claims, wherein said osteoclasts enhance the secretion of IFN-γ by said NK cells, and optionally said osteoclasts enhance the secretion of IL-12 by said NK cells.

9. 10. The method of any one of the preceding claims, wherein the osteoclasts enhance expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by the NK cells.

10. 10. The method of any one of the preceding claims, wherein the NK cells are purified from a cancer sample.

11. The method of claim 10, wherein the cancer sample is from a subject with the cancer.

12. The method of claim 11 , wherein the subject is a human.

13. The method of any one of claims 10 to 12, wherein the cell culture further comprises T cells also arising from the cancer sample.

14. 14. The method of claim 13, wherein the NK cells are preferentially expanded relative to the T cells.

15. 15. The method of claim 14, further comprising preferentially expanding the NK cells for at least one month.

16. 16. The method of claim 15, further comprising supplementing the culture medium with at least one osteoclast cell to preferentially continue to expand the NK cells.

17. The T cells secrete IFN-γ but do not mediate cytotoxicity, and optionally the cytotoxicity is preferably ５１ 15. The method of claim 14, wherein the lysis of OSCSCs by the T cells is measured in a Cr release cytotoxicity assay.

18. The method of any one of claims 13 to 17, wherein the expanded NK cells are capable of expanding CD8+ T cells.

19. 19. The method of claim 18, wherein the NK cells expanded by the OC are capable of preferentially expanding CD8+ T cells over CD4+ T cells.

20. 10. The method of any one of the preceding claims, further comprising adding an anti-CD3 antibody to the cell culture.

21. The method of claim 20, wherein the anti-CD3 antibody further enhances the secretion of IFN-γ by the NK cells.

22. 10. The method of any one of the preceding claims, wherein the activated NK cells are fractionally anergized.

23. 10. The method of any one of the preceding claims, further comprising adding to the cell culture 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 live or sonicated.

24. 24. The method of claim 23, wherein the composition comprises Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus.

25. 24. The method of claim 23, wherein the composition comprises sAJ2 bacteria.

26. the ratio of the sAJ2 bacteria concentration to the NK cell and / or OC concentration in the cell culture is i) NK cells:sAJ2 at least 1:2; ii) at least 1:4 for OC:sAJ2; and / or iii) OC:NK cells:sAJ2 ratio of at least 1:2:4 26. The method of claim 25, wherein:

27. 10. The method of any one of the preceding claims, further comprising adding to the cell culture another agent capable of activating NK cells.

28. 10. The method of any one of the preceding claims, wherein the osteoclasts enhance the production, secretion, and / or function of at least one cytokine or chemokine produced by the NK cells.

29. i) providing a cell culture comprising osteoclasts (OCs), NK cells, and T cells; and ii) culturing the NK cells, the T cells, and the osteoclasts in the cell culture; thereby preferentially activating said NK cells relative to said T cells.

30. i) providing a cell culture comprising dendritic cells (DCs), NK cells, and T cells; and ii) culturing the NK cells, the T cells, and the dendritic cells in the cell culture. thereby preferentially activating said T cells relative to said NK cells.

31. 31. The method of claim 29 or 30, wherein the NK cells are primary NK cells, optionally wherein the primary NK cells are not transformed.

32. 32. The method of claim 29 or 31, wherein the culture comprises a plurality of osteoclasts (OCs) and a plurality of NK cells, and the concentration ratio of OCs:NK cells in the cell culture is at least 1:

2.

33. 33. The method of claim 29, 31, or 32, wherein the osteoclasts enhance NK cell proliferation, and optionally, the osteoclasts enhance the secretion of IL-15 by the NK cells.

34. 34. The method of claim 33, wherein the activated NK cells expand to at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within 4 weeks.

35. 35. The method of any one of claims 29 and 31-34, wherein the osteoclasts enhance the cytotoxicity of NK cells, and optionally the cytotoxicity of the NK cells is measured by lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by the NK cells.

36. the cytotoxicity of the cells ５１ 36. The method of claim 35, as measured by a Cr release cytotoxicity assay.

37. 37. The method of any one of claims 29 and 31-36, wherein the osteoclasts enhance secretion of IFN-γ by the NK cells, and optionally, the osteoclasts enhance secretion of IL-12 by the NK cells.

38. 38. The method of any one of claims 29 and 31-37, wherein the osteoclasts enhance expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by the NK cells.

39. The method of any one of claims 29 to 38, wherein the NK cells and / or the T cells are purified from a cancer sample.

40. 40. The method of claim 39, wherein the cancer sample is from a subject with the cancer.

41. 41. The method of claim 40, wherein the subject is a human.

42. 42. The method of any one of claims 29 and 31 to 41, wherein the preferential activation of NK cells persists for at least one month.

43. 43. The method of claim 42, further comprising continuing the activation of the NK cells by adding at least one osteoclast to the cell culture after the preferential activation of the NK cells has diminished or ceased, optionally wherein the addition of at least one osteoclast to the cell culture is at least one month after culturing the NK cells.

44. The T cells secrete IFN-γ but do not mediate cytotoxicity, and optionally, the cytotoxicity is preferably ５１ The method of any one of claims 29 to 43, wherein the lysis of OSCSCs by the T cells is measured in a Cr release cytotoxicity assay.

45. The method of any one of claims 29 and 31 to 44, wherein the expanded NK cells are capable of expanding CD8+ T cells.

46. 46. ​​The method of claim 45, wherein the NK cells expanded by the OC are capable of preferentially expanding CD8+ T cells relative to CD4+ T cells.

47. The method according to any one of claims 30 to 44, wherein the NK cells expanded by the DCs are capable of preferentially expanding CD4+ T cells over CD8+ T cells.

48. 47. The method of any one of claims 29 and 31 to 46, further comprising adding an anti-CD3 antibody to the cell culture.

49. 49. The method of claim 48, wherein the anti-CD3 antibody further enhances the secretion of IFN-γ by the NK cells.

50. 50. The method of any one of claims 29 to 49, wherein the activated NK cells are fractionally anergized.

51. 51. The method of any one of claims 29 and 31-50, further comprising adding to the cell culture 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 live or sonicated.

52. 52. The method of claim 51, wherein the composition comprises Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus.

53. 53. The method of claim 52, wherein the composition comprises sAJ2 bacteria.

54. the ratio of the sAJ2 bacteria concentration to the NK cell and / or OC concentration in the cell culture is i) NK cells:sAJ2 at least 1:2; ii) at least 1:4 for OC:sAJ2; and / or iii) OC:NK cells:sAJ2 ratio of at least 1:2:4 54. The method of claim 53, wherein:

55. 53. The method of any one of claims 29 and 31 to 52, further comprising adding to the cell culture another agent capable of activating NK cells.

56. 53. The method of any one of claims 30 to 52, further comprising adding to the cell culture another agent capable of activating T cells.

57. 56. The method of any one of claims 29 and 31 to 55, wherein the osteoclasts (OCs) enhance the production, secretion, and / or function of at least one cytokine or chemokine produced by the NK cells.

58. 1. A method for treating cancer or a cancer-related disease or disorder in a subject having or suspected of having cancer or a cancer-related disease or disorder, comprising: The method comprises administering to the subject a therapeutically effective amount of osteoclasts (OCs), a cell culture comprising osteoclasts (OCs), and / or a supernatant of a cell culture comprising osteoclasts (OCs).

59. 59. The method of claim 58, wherein the osteoclasts activate NK cells in the subject.

60. 60. The method of claim 59, wherein the NK cells are primary NK cells, optionally wherein the primary NK cells are not transformed.

61. 61. The method of any one of claims 59-60, wherein said osteoclasts enhance NK cell proliferation in said subject, and optionally, said osteoclasts enhance secretion of IL-15 by said NK cells.

62. 62. The method of claim 61 , wherein said enhanced NK cell expansion is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more population doublings within 4 weeks.

63. 63. The method of any one of claims 59 to 62, wherein the osteoclasts enhance the cytotoxicity of NK cells, and optionally the cytotoxicity of the NK cells is measured by lysis of oral squamous cell carcinoma stem-like cells (OSCSCs) by the NK cells.

64. The cytotoxicity of the NK cells ５１ 64. The method of claim 63, wherein the cytotoxicity is measured by a Cr release cytotoxicity assay.

65. 65. The method of any one of claims 59 to 64, wherein the osteoclasts enhance secretion of IFN-γ by the NK cells, and optionally, the osteoclasts enhance secretion of IL-12 by the NK cells.

66. 66. The method of any one of claims 59 to 65, wherein the osteoclasts preferentially activate NK cells relative to T cells, and optionally, the osteoclasts preferentially enhance the proliferation of NK cells relative to T cells.

67. 67. The method of claim 66, wherein the preferential expansion of NK cells persists for at least one month.

68. The T cells secrete IFN-γ but do not mediate cytotoxicity of the cancer, and optionally, the cytotoxicity is, for example, ５１ 68. The method of claim 67, wherein the lysis of OSCSCs by the T cells is measured in a Cr release cytotoxicity assay.

69. 69. The method of any one of claims 59 to 68, wherein the activated NK cells expand CD8+ T cells in the subject.

70. 70. The method of claim 69, wherein the activated NK cells preferentially expand CD8+ T cells relative to CD4+ T cells.

71. 71. The method of any one of claims 58 to 70, further comprising administering to the subject an anti-CD3 antibody.

72. 72. The method of claim 71, wherein the anti-CD3 antibody further enhances the secretion of IFN-γ by the NK cells.

73. 73. The method of any one of claims 59 to 72, wherein the activated NK cells are fractionally anergized.

74. 74. The method of any one of claims 58-73, 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 live or sonicated.

75. 75. The method of claim 74, wherein the composition comprises Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus.

76. 75. The method of claim 74, wherein the composition comprises sAJ2 bacteria.

77. 77. The method of any one of claims 58 to 76, further comprising adding to the cell culture another agent capable of activating NK cells.

78. 78. The method of any one of claims 58 to 77, wherein the osteoclasts (OCs) increase or promote the production, secretion, and / or function of at least one cytokine or chemokine produced by the NK cells.

79. 79. The method of any one of claims 58 to 78, wherein the osteoclasts, the cell culture, and / or the supernatant are administered in a pharmaceutical composition.

80. 80. The method of any one of claims 58 to 79, wherein the osteoclasts, the cell culture, and / or the supernatant are administered systemically or locally to the cancer.

81. 81. The method of any one of claims 58 to 80, wherein the osteoclasts, the cell culture, and / or the supernatant are administered to the subject at least twice, and optionally the osteoclasts, the cell culture, and / or the supernatant are administered to the subject at least one month after the first administration.

82. 82. The method of any one of claims 58 to 81, wherein the subject is a human.

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