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WO2023114445A1 - Prévention de la perte osseuse - Google Patents

Prévention de la perte osseuse Download PDF

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Publication number
WO2023114445A1
WO2023114445A1 PCT/US2022/053095 US2022053095W WO2023114445A1 WO 2023114445 A1 WO2023114445 A1 WO 2023114445A1 US 2022053095 W US2022053095 W US 2022053095W WO 2023114445 A1 WO2023114445 A1 WO 2023114445A1
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cells
cancer
ifn
bone
cell
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Anahid Jewett
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • pancreatic cancer has an incidence rate nearly comparable to its mortality rate.
  • the 5-year survival rate has remained less than 6% for the past three decades, demonstrating the aggressiveness and lethal nature of this disease.
  • Cancer is a risk factor for bone loss and fracture. While the mechanism of bone loss is not well understood, the bone loss includes a decrease in both mineral content and protein matrix components of the bone, and leads to an increased fracture rate of, predominantly, femoral bones and bones in the forearm and vertebrae. These fractures, in turn, lead to an increase in general morbidity, a marked loss of stature and mobility, and, in many cases, an increase in mortality resulting from complications. Unchecked, bone loss can lead to osteoporosis and/or osteopenia. Osteopenia is reduced bone mass due to a decrease in the rate of osteoid synthesis to a level insufficient to compensate normal bone lysis. Osteoporosis is a major debilitating disease whose prominent feature is the loss of bone mass (decreased density and enlargement of bone spaces) without a reduction in bone volume, producing porosity and fragility.
  • compositions and methods for preventing and/or treating bone loss conditions including cancer-associated bone loss conditions.
  • the present invention is based, at least in part, on the discovery that administration of NK cells (e.g., super-charged NK cells), certain probiotic bacteria, sonicated osteoclasts, or any combination thereof prevents and/or treats bone loss conditions, e.g., cancer- associated bone loss conditions.
  • NK cells are also effective in killing cancer cells
  • the immunotherapy comprising NK cells provides an unexpected and surprising therapeutic benefit in preventing and/or treating either (1) bone loss conditions, or (2) both cancer proliferation and cancer-associated bone loss conditions.
  • the stage of differentiation of, e.g., pancreatic tumors has a profound effect on the function of NK cells.
  • the stem-like/poorly differentiated tumors are preferentially targeted by the NK cells, and an intact immune system is required for the elimination of tumors.
  • tumors have been shown to cause immune suppression, in particular, NK suppression.
  • NK cells from cancer patients and humanized mice implanted with tumors lose their ability to kill and differentiate tumors.
  • Inability of the NK cells from cancer patients to curtail tumor growth through cancer cell lysis and differentiation of tumors is a profound deficiency that can be remedied by administering the super-charged NK cells.
  • the role of the supercharged NK cells and interferon gamma (IFN-y) in mediating tumor dissemination and bone quality alteration related to pancreatic cancer is elucidated.
  • FIG. 1 shows a schematic diagram illustrating the regulatory interactions that maintain homeostasis of the skeletal systems.
  • FIG. 2 shows a schematic diagram illustrating the roles of natural killer cells in cancer.
  • FIG. 3 shows a schematic diagram illustrating the BLT-NSG human immune cell reconstitution.
  • FIG. 4 shows that greater than 90% of tissue infiltrated immune cells in BLT-NSG mice are of human immune cells.
  • IgG shows 99.9 % of human immune cells, m-CD45 99 % (top panels).
  • Ig G presents 99.9% of human immune cells and m-CD45 of 98 % (bottome panels).
  • FIGs. 5A-5B show the ex vivo assessment of bone architecture by micro-CT analysis.
  • FIG. 5 A shows the representative images of MP2.
  • FIG. 5B shows the representative images of MP2 + NK + AJ2.
  • FIGs. 6A-6B show the phenotypic characteristics of bone marrow, spleen, peripheral blood, pancreas in hu-BLT mice. Lack of tumor growth, metastasis and longterm survival of NSG mice after orthotopic implantation of NK-supematant differentiated MP2 tumors in pancreas. MP2 tumors were differentiated by the NK-supernatants. Patient- derived differentiated PL12 (2 /
  • OCs were generated from hu-BLT bone marrow monocytes and human peripheral blood monocytes.
  • NK cells purified from hu-BLT splenocytes were pre-treated with IL-2 (1000 U/mL) and anti-CD16mAb (3 pg/mL) for 18 hours and then either cultured alone or with hu-BLT-OCs or human OCs in the presence of sAJ2 (NK: OCs: sAJ2; 2: 1 :4) and the numbers of expanding NK cells were counted on days 6, 10, 14, 18 and 22.
  • sAJ2 sAJ2
  • sAJ2 sAJ2
  • FIG. 6A The supernatants from the NK cells and OCs cultures in the presence of sAJ2, were harvested on days 6, 10, 14, 18 and 22, and the levels of IFN-y were determined using single ELISA (FIG. 6B).
  • FIG. 8 shows that a single injection of super-charged NK-cells with/without feeding with AJ2 inhibited tumor growth due to differentiation of tumors in hu-BLT mice.
  • FIG. 10 shows the Hu-BLT mice that were implanted with MP2 tumors and injected with NK cells or implanted with NK-differentiated tumors.
  • FIGs. 11A-11C show that the NK cell cytotoxicity and ability to secrete IFN-y are severely decreased in pancreatic cancer patients.
  • FIGs. 12A-12L show that the injection of super-charged NK-cells with/without feeding with AJ2 restored and increased IFN-y secretion and/or cytotoxic function of NK cells from different tissues of tumor-bearing hu-BLT mice.
  • PBMCs were isolated from blood and treated with IL-2 (1000 U/mL) before they were used in cytotoxicity assay against OSCSCs using 4 h 51 Cr release assay. Lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the target cells x 100.
  • NK-enriched cells were isolated from splenocytes and were cultured with IL-2 (1000 U/mL) before they were used for cytotoxicity against OSCSCs using 4 h 51 Cr release assay.
  • BM cells were harvested and treated with IL-2 (1000 U/mL) for 7 days before they were used for cytotoxicity against OSCSCs using 4 h 51 Cr release assay.
  • the following symbols represent the levels of statistical significance within each analysis, *** (p-value ⁇ 0.001), **.
  • FIGs. 13A-13B show that the mice fed with AJ2 presented increased trabecular bone formation when compared to the CTRL (control) group.
  • FIGs. 14A-14B show that the MP2 tumor-bearing mice injected with NK cells and fed with AJ2, presented statistically significant higher trabecular bone volume when compared to MP2 tumor and MP2+AJ2 group, respectively.
  • FIGs. 15A-15B show that the stem-like/undifferentiated tumors implanted in hu- BLT mice injected with cultures of NK cells and fed with or without AJ2 presented similar bone formation when compared to the control group.
  • FIG. 16 shows that there was a remarkable correlation between induction and secretion of IFN-y and the bone morphology.
  • MP2 tumor-bearing mice injected with NK cells and fed with AJ2 group there was increased IFN-y in serum, cell cultures from pancreatic tumors, NK cells purified from splenocytes, PBMCs, splenocytes cell cultures, and bone marrow cells.
  • MP2 tumor-bearing hu-BLT mice showed decreased IFN-y in the same compartments.
  • FIG. 17 shows that the histological analysis of the AJ2 sample exhibited increased bone formation when compared to the Control and MP2 samples.
  • the histomorphometric values for the MP2+NK +AJ2 sample could not be confirmed due to staining failure.
  • FIG. 18 shows that the MP2 tumor sample exhibits positive TRAP staining.
  • FIG. 19 shows the decreased bone in OSCSC-implanted BLT mice and its restoration by either feeding AJ2 or injection with super-charged NK cells.
  • FIG. 20 shows the decreased bone in MP2 poorly differentiated pancreatic tumor implanted BLT mice and its restoration by either feeding AJ2 and/or injection with supercharged NK cells.
  • FIG. 21A-FIG. 21G show increased IFN-y and decreased IL-10 secretions by sAJ4 treated PBMCs in comparison to sAJ3 and sAJ2 treated PBMCs.
  • PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with sAJ3 (PBMC:sAJ3, 1 :20) or with sAJ4 (PBMC:sAJ4, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (PBMC:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (PBMC:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y (FIG.
  • PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and anti- CD3/28 antibody (25 pl/ml) or with a combination of IL-2 (1000 U/ml) and sAJ2 (PBMC:sAJ2, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (PBMC:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (PBMC:sAJ4, 1 :20) or with sAJ2 (PBMC:sAJ2, 1 :20) or with sAJ3 (PBMC:
  • PBMCs were treated as described in FIG. 2 IE for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL-10 secretion using specific single ELISAs, and ratio of IFN-y to IL-10 was determined (FIG. 21G). **(p value 0.001-0.01), *(p value 0.01-0.05).
  • FIG. 22A-FIG. 221 show increased IFN-y and decreased IL- 10 secretions by sAJ4 treated NK cells in comparison to sAJ3 and sAJ2 treated NK cells.
  • NK cells were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with sAJ3 (NK:sAJ3, 1 :20) or with sAJ4 (NK:sAJ4, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from NK cells to determine IFN-y (FIG.
  • FIG. 22A NK cells were treated as described in FIG. 22A for 18 hours, and the number of cells secreting IFN-y in the NK cells were determined as spot counts using ELISpot assay (FIG. 22C), and the supernatants were harvested to determine IFN-y using single ELISA (FIG. 22D).
  • NK cells were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and sAJ2 (NK:sAJ2, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) or with sAJ2 (NK:sAJ2, 1 :20) or with sAJ3 (NK:sAJ3, 1 :20) or with sAJ4 (NK:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from NK cells to determine IFN-y secretion using single ELISA.
  • NK were treated as described in FIG. 22E for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using specific single ELISAs, and ratio of IFN-y to IL- 10 was determined (FIG. 22H).
  • NK were treated as described in FIG.
  • FIG. 23A-FIG. 23B show increased IFN-y secretion levels in sAJ4 treated NK cells and monocytes in comparison to sAJ3 treated cells.
  • NK cells and monocytes of healthy individuals were isolated from PBMCs as described in Example 4.
  • NK cells or monocytes or co-culture of NK and monocytes (NK: monocytes; 1 : 1) treated with sAJ3 (NK:sAJ3, 1 :20) or sAJ4 (NK:sAJ4, 1 :20) or a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) or a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) and sAJ3 (NK:sAJ3, 1 :20) or or a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) and sAJ3 (NK:sAJ3, 1 :20) or or a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml)
  • FIG. 23 A NK cells and monocytes were treated as described in FIG. 23 A for 18 hours before the supernatant was harvested to determine IFN-y and IL- 10 secretion using specific single ELIS As, and ratio of IFN-y to IL- 10 was determined (FIG. 23B).
  • FIG. 24 shows increased IFN-y by sAJ4 treated CD8+ T cells in comparison to sAJ3 treated CD8+ T cells.
  • CD8+ T cells were left untreated or treated with IL-2 (100 U/ml) or IL-2 (100 U/ml) and anti-CD3/28 mAbs (25 pl/ml), or IL-2 (100 U/ml) and sAJ2 (CD8+ T:sAJ2, 1 :20) or IL-2 (100 U/ml) and sAJ3 (CD8+ T:sAJ3, 1 :20) or IL-2 (100 U/ml) and sAJ4 (CD8+ T:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from NK cells to determine IFN-y using specific ELISAs.
  • FIG. 25 shows schematic representation of AJ3 function in ALS.
  • AJ3 will be effective in alleviating auto-immunity, in particular in ALS since it will greatly regulate the levels and function of IFN-y, decreasing over activation and death of motor neurons.
  • Gene deletion or mutation may decrease MHC-class I expression on some motor neurons activating NK cells. Both cytokine and receptor mediated cross-linking will greatly sway the activation of the NK cells towards greater IFN-y secretion in the presence of no or decreased IL-10.
  • IFN-y secretion by the NK cells will not only expand CD8+ T cells but also will differentiate and increase MHC-class I expression on motor neurons, allowing the mutated motor neurons to become susceptible to CD8+ T cell activation and effector function, in which case further IFN-y secretion could exacerbate the death of not only mutated motor neurons, but also the non-mutated bystander cells, through overactivation and induction of cell death.
  • Treatment with AJ3 will regulate the increase in the secretion of IFN-y by increased induction of IL-10, decreasing the activation of NK cells and CD8+ T cells, and minimizing or even halting the death of motor neurons and slowing the progression of the disease. In the absence of disease, the default function of AJ3 formulation is towards increase in anti-inflammatory IL- 10 induction.
  • compositions and methods that are useful in preventing and/or treating bone loss conditions (e.g., cancer-associated bone loss conditions), in inducing bone formation, and/or inhibiting or preventing bone loss.
  • bone loss conditions e.g., cancer-associated bone loss conditions
  • This disclosure provides the correlation and potential impact of the immune system, specifically NK cells and interferon gamma, in bone quality alteration related to cancer, e.g., pancreatic cancer in hu-BLT mice.
  • the studies show how MP2 tumors injected in hu-BLT mice affected the bone structure.
  • the analysis show how the MP2 tumor-bearing mice that are injected with NK cells and fed with and without AJ2 influenced the bone structure.
  • IFN-y (1) induces secretion by NK cells, (2) inhibits tumor growth, and (3) decreases skeletal complications of malignancy by directly acting on host cells to inhibits osteoclast formation and function.
  • the enhanced osteolytic lesion formation in BLT tumor-bearing mice and NK cells’ ability to secrete IFN-y to significantly reduce bone loss in tumor-bearing mice indicate a direct anti-osteoclastogenic role for IFN-y in the setting of cancer-induced bone disease. Furthermore, the present study further indicates that IFN-y directly promotes bone formation.
  • NK cells into tumorbearing mice increased IFN-y secretion in hu-BLT mice.
  • the data also indicate that IFN-y has direct anti-tumor effects and can suppress tumor-induced bone loss by directly targeting host osteoclasts to inhibit osteolysis.
  • the present work provides a novel report of bone quality alteration related to pancreatic cancer in hu-BTL mice and the role of IFN-y secreted by NK cells in the suppression of tumor-induced bone loss and induction of bone formation.
  • NK and T cells make up significant percentages of lymphocytes in peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • NK cells are mainly known as the effectors of innate immunity due to their lack of antigen-specificity.
  • CD4+ and CD8+ T cells mediate the adaptive cellular immunity, which closely collaborate with the innate immune system.
  • NK cells and CD8+ T cells play significant role in cancer control, and NK and CD8+ T cellbased immunotherapies are among the leading standards in cancer therapeutics. Decreased function of these two lymphocytes result in poor prognosis in cancer patients. It has been shown that NK cells could activate and induce the proliferation of T cells, and also could kill chronically activated leukocytes.
  • CD8+ T cells Increased levels of tumor-infiltrating CD8+ T cells were found to be associated with complete responses to standard chemotherapeutic regimens, and the presence of CD8+ memory T cells is associated with patient survival. It was found that NK cells can accelerate CD8+ T cells responses against viral infections, such as those caused by cytomegaloviruses.
  • AJ2 is a combination of different strains of gram-positive probiotic bacteria selected based on their superior ability to induce optimal secretion of both pro-inflammatory and anti-inflammatory cytokines in NK cells.
  • Al-Pro AJ3
  • CA/I-Pro AJ4
  • Sonicated bacteria e.g., AJ2
  • OCs osteoclasts
  • sAJ2 sonicated AJ2
  • NK cells regulate the balance of T cell subsets, cytokine secretions, and cytotoxic activity of immune cells in various tissue compartments of mice.
  • super-charged NK cells lyse activated CD4+ T and not CD8+ T cells, thus selecting and preferentially expanding CD8+ T cells.
  • TLRs Toll-like receptors
  • NK cells were found to express TLR mRNA for TLR1-10. NK cells were found to produce higher levels of IFN-y and also increased cytotoxic activity after TLR2, TLR3, TLR4 and TLR5 stimulation.
  • TLRs 2, 4, 5 and 11 on the cell surface recognize bacterial lipoproteins, lipopolysaccharide (LPS), flagellin and profilin, respectively.
  • TLRs 3, 7, 8 and 9 are expressed in endosomal compartments and recognize viral and bacterial nucleic acids.
  • HSPs heat shock proteins
  • extracellular matrix components such as fibronectin and hyaluronan
  • DNA complexes may activate TLR3, TLR7 and TLR9.
  • TLR expression has been clearly shown on the surface of innate immune cells such as monocytemacrophages and dendritic cells and therefore, TLRs have traditionally been considered to play an important role in indirectly controlling T cell responses through the activation of innate immune cells through TLRs.
  • TLRs have traditionally been considered to play an important role in indirectly controlling T cell responses through the activation of innate immune cells through TLRs.
  • compositions comprising probiotic bacteria and methods of using them.
  • probiotic bacteria e.g., AJ2, AJ3, AJ4
  • PBMC, NK and CD8+ T cells were left untreated or treated with IL-2 or IL- 2+anti-CD16mAbs or IL-2+anti-CD3/CD28mAbs in the presence and absence of sAJ2, sAJ3 and sAJ4.
  • IL-2+anti-CD16mAbs activation of PBMCs and NK cells had the highest IFN-y/IL-10 ratio whereas IL-2 combination with sAJ4 had the next highest followed by IL-2+sAJ2 and the lowest was seen with IL-2+sAJ3.
  • IL-2+anti- CD3/CD28mAbs had lower IFN-y/IL-10 in PBMCs when compared to either IL-2 alone or IL2+anti-CD16mAbs. Accordingly, the highest secretion of IFN-y was seen when the PBMCs and NK cells were treated with IL-2+sAJ4, intermediate for IL-2+sAJ2 and the lowest IL-2+sAJ3. Indeed, IFN-y secretion by IL-2+sAJ4 exceeded much higher than that of the levels of IFN-y secretion by IL-2 or IL-2+anti-CD16mAbs or IL-2+anti- CD3/CD28mAbs in PBMCs and NK cells.
  • sAJ3 probiotic bacteria had the lowest IFN-y/ILlO ratios and triggered much lower induction of IFN-y, Thus, sAJ3 probiotic bacteria was formulated to augment antiinflammatory cytokine IL- 10 to counter the aggressive nature of pro-inflammatory cytokine IFN-y induced by NK and CD8+ T cells in ALS patients.
  • sAJ3 will alleviate auto-immunity seen in ALS by regulating the levels and function of IFN-y, thereby decreasing overactivation and death of motor neurons.
  • AJ4 which is particularly effective in activating NK cells and inducing IFN-g production, will treat cancer and bone loss (including cancer-associated bone loss).
  • an element means one element or more than one element.
  • administering is intended to include routes of administration which allow an agent to perform its intended function.
  • routes of administration for treatment of a body which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes.
  • the injection can be bolus injections or can be continuous infusion.
  • the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function.
  • the agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier.
  • the agent also may be administered as a prodrug, which is converted to its active form in vivo.
  • the amount or level of IFN-y or other cytokine/chemokine is “significantly” higher or lower than the normal amount, if the amount is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least, about, or no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more than that amount.
  • the amount of the cytokines and/or chemokines can be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the cytokines and/or chemokines.
  • Such “significance” can also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.
  • the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents).
  • the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially.
  • an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.
  • control refers to any reference standard suitable to provide a comparison to the expression products in the test sample.
  • a control diseased patient e.g., those afflicted with a bone loss condition
  • can be stored sample or previous sample measurement with a known outcome
  • normal tissue or cells isolated from a subject such as a normal patient or the diseased patient (e.g., those afflicted with a bone loss condition)
  • cultured primary cells/tissues isolated from a subject such as a normal subject or the diseased patient (e.g., those afflicted with a bone loss condition)
  • adjacent normal cells/tissues obtained from the same organ or body location of the diseased patient a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository.
  • control may comprise a reference standard IFN-y level from any suitable source, including but not limited to a previously determined IFN-y level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, certain bone density) or receiving a certain treatment (for example, cancer therapy or bone loss therapy). It will be understood by those of skill in the art that such control samples and reference standard IFN-y levels can be used in combination as controls in the methods of the present invention.
  • control may comprise normal or non-diseased cell/tissue sample.
  • the control may comprise a level for a set of patients, such as a set of diseased patients (e.g., those afflicted with a bone loss condition), or for a set of patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome.
  • the control may comprise normal cells, cells from patients treated with bone loss therapy, and cells from patients having benign cancer and the associated bone loss condition.
  • the control may also comprise a measured value for example, healthy or diseased individuals who were not treated with the agents of the present disclosure, or healthy or diseased individuals who were administered with other bone loss therapy.
  • control comprises a ratio of IFN-y and other cytokine/chemokine levels, including but not limited to the level of one cytokine against the level of another cytokine, e.g., the ratio of the level of IFN-y and the level of IL-10.
  • control comprises a control sample which is of the same lineage and/or type as the test sample.
  • Immune cell refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • lymphocytes such as B cells and T cells
  • natural killer cells such as myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • immune response refers to a response mediated by any or all immune cells.
  • the “immune response” includes T cell mediated and/or B cell mediated immune responses.
  • Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity.
  • immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
  • inhibit includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction.
  • cancer is “inhibited” if at least one symptom of the bone loss condition is alleviated, terminated, slowed, or prevented.
  • a bone loss condition is also “inhibited” if recurrence of the bone loss condition is reduced, slowed, delayed, or prevented.
  • a biological function such as the function of a protein, is inhibited if it is decreased as compared to a reference state, such as a control like a wild-type state.
  • prevention of a bone loss condition or bone loss includes, for example, delaying the appearance of reduction in bone density in a population of patients receiving a prophylactic treatment relative to an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • a “therapeutically effective amount” of a substance or cells is an amount capable of producing a medically desirable result in a treated patient, e.g., increase in bone density, decrease in bone loss, with an acceptable benefit: risk ratio, preferably in a human or nonhuman mammal.
  • treating includes prophylactic and/or therapeutic treatments.
  • prophylactic or therapeutic treatment is art-recognized and includes administration to the subject one or more agents of the present disclosure. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal), then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition); whereas, if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9 and all intervening fractional values between the aforementioned integers such as, for example, 1/2, 1/3, 1/4, 1/5, 1/6, 1/8, and 1/9, and all multiples of the aforementioned values.
  • a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
  • Osteocytes are the most abundant cell type in bone (Nijweide et al. (1996) Principles of Bone Biology (Bilezikian, Riasz and Rodan, eds.), Academic Press, New York, N.Y., pp. 115-126), with approximately ten times more osteocytes than osteoblasts (Parfitt et al. (1977) Clin. Orthop. Rel. Res. 127:236-247), and with osteoblasts far more abundant than osteoclasts. Each of these different types of bone cell has a different phenotype, morphology and function.
  • Osteocytes are localized within the mineral matrix at regular intervals, and arise from osteoblasts. During their transition from osteoblasts, osteocytes maintain certain osteoblastic features, but acquire several osteocyte-specific characteristics. Mature osteocytes are stellate shaped or dendritic cells enclosed within the lacuno-canalicular network of bone. Long, slender cytoplasmic processes radiate from the central cell body, with most of the processes perpendicular to the bone surface. The processes connect the osteocyte to neighboring osteocytes and to the cells lining the bone surface.
  • osteocytes include: to respond to mechanical strain and to send signals of bone formation or bone resorption to the bone surface, to modify their microenvironment, and to regulate both local and systemic mineral homeostasis.
  • osteocytes may regulate physiological local bone remodeling, in part through their cell death and apoptosis that trigger osteoclasts formation and bone resorption, and in part by secreting sclerostin, a molecule specifically produced by osteocytes that acts as an inhibitor of bone formation (Giuliani et al. (2015) in Bone Cancer (Second Edition), Chapter 42, pp 491-500).
  • Osteoblasts are the skeletal cells responsible for bone formation, and thus synthesize and regulate the deposition and mineralization of the extracellular matrix of bone (Aubin and Liu, (1996) Principles of Bone Biology (Bilezikian, Riasz and Rodan, eds.), Academic Press, New York, N.Y., pp. 51-67).
  • Osteoclasts are multinucleated giant cells with resorbing activity of mineralized bone (Suda et al., (1996) Principles of Bone Biology (Bilezikian, Riasz and Rodan, eds.), Academic Press, New York, N.Y., pp. 87-102).
  • the term “bone loss condition” refers to a condition that occurs when the body doesn’t make new bone as quickly as it reabsorbs old bone.
  • “bone loss conditions” include bone diseases, such as osteopenia, osteolysis, osteoporosis, osteoplasia (osteomalacia), and Paget's disease of bone.
  • bone loss conditions include bone loss that is associated with other diseases, such as diabetes, chronic renal failure, hyperparathyroidism, and cancer (e.g., multiple myeloma and breast cancer), which result in abnormal or excessive bone loss.
  • the present invention is directed to methods of treating and/or preventing bone loss conditions, such as osteoporosis and osteopenia and other diseases where inhibiting bone loss may be beneficial, including Paget's disease, malignant hypercalcemia, periodontal diseasejoint loosening and metastatic bone disease, as well as reducing the risk of fractures, both vertebral and nonvertebral.
  • Osteopenia refers to bone density that is lower than normal density but not low enough to be classified as osteoporosis. Osteopenia is reduced bone mass due to a decrease in the rate of osteoid synthesis to a level insufficient to compensate normal bone lysis. Osteopenia is commonly seen in people over age 50 that have lower than average bone density but do not have osteoporosis.
  • Osteoporosis is a structural deterioration of the skeleton caused by loss of bone mass resulting from an imbalance in bone formation, bone resorption, or both, such that the resorption dominates the bone formation phase, thereby reducing the weight-bearing capacity of the affected bone.
  • the rate at which bone is formed and resorbed is tightly coordinated so as to maintain the renewal of skeletal bone.
  • an imbalance in these bone remodeling cycles develops which results in both loss of bone mass and in formation of microarchitectural defects in the continuity of the skeleton.
  • osteoporosis Although this imbalance occurs gradually in most individuals as they age (“senile osteoporosis”), it is much more severe and occurs at a rapid rate in postmenopausal women. In addition, osteoporosis also may result from nutritional and endocrine imbalances, hereditary disorders and a number of malignant transformations.
  • Bone loss is also an important consideration for treatment among cancers, particularly among multiple myeloma, breast cancer, and pancreatic cancer.
  • Osteoplasia also known as osteomalacia (“soft bones”), is a defect in bone mineralization (e.g., incomplete mineralization), and classically is related to vitamin D deficiency (1,25-dihydroxy vitamin D3).
  • the defect can cause compression fractures in bone, and a decrease in bone mass, as well as extended zones of hypertrophy and proliferative cartilage in place of bone tissue.
  • the deficiency may result from a nutritional deficiency (e.g., rickets in children), malabsorption of vitamin D or calcium, and/or impaired metabolism of the vitamin.
  • osteoporosis or osteopenia are based on inhibiting further bone resorption, e.g., by 1) inhibiting the differentiation of hemopoietic mononuclear cells into mature osteoclasts, 2) by directly preventing osteoclast-mediated bone resorption, or 3) by affecting the hormonal control of bone resorption.
  • Drug regimens used for the treatment of osteoporosis include calcium supplements, estrogen, calcitonin, estradiol, and diphosphonates. Vitamin D3 and its metabolites, known to enhance calcium and phosphate absorption, can also be used.
  • parathyroid hormone such as the 84-amino acid PTH peptide or fragments thereof, such as the teriparatide first 1-34 amino acids of human PTH
  • PTH parathyroid hormone
  • 84-amino acid PTH peptide or fragments thereof, such as the teriparatide first 1-34 amino acids of human PTH can also be used (see, for example, U.S. Pat. Publ. 2018/0028622 and U.S. Pat. 8,110,547, each of which is incorporated in their entirety herein by this reference).
  • the instant invention is drawn to a method comprising administering to the subject a composition comprising at least one probiotic bacterial strain, capable of regulating NK cell function.
  • a method may comprise contacting the NK cells in vivo, in vitro, or ex vivo with at least one probiotic bacterial strain in order to activate NK cells prior to administration to the subject.
  • probiotic bacteria induce significant production or secretion of various cytokines/chemokines, e.g., IFN-y, Gro-alpha, IL-10, and TNF-a.
  • cytokines/chemokines e.g., IFN-y, Gro-alpha, IL-10, and TNF-a.
  • probiotic bacteria induce significant activation and/or expansion of NK cells.
  • Preferred probiotic bacteria species of the present disclosure include Streptococcus (e.g., S. thermophiles'), Bifidobacterium (e.g., B. longum, B. breve, B. infantis), and/or Lactobacillus genera (e.g., L. acidophilus, L. helveticus, L. bulgaricus, L. rhamnosus, L. plantarum, and L. easel).
  • the compositions and methods of the present disclosure comprise at least one probiotic bacterial strain, preferably a combination of two or more different bacterial strains, to a subject, preferably a mammal (e.g., a human).
  • Such administration may be systemically or locally (e.g., directly to intestines, e.g., orally or rectally) performed.
  • the preferable administration route for probiotic bacteria is oral administration. Other routes (e.g., rectal) may be also used.
  • the bacteria e.g., in a wet, sonicated, ground, or dried form or formula
  • the bacterial culture medium comprising the bacteria
  • the bacterial culture medium supernatant not containing the bacteria
  • the bacteria may be alive, partially alive, or dead.
  • the bacteria may be sonicated, ground, wet, or dry (e.g., freeze-dried).
  • the composition (bacterial, pharmaceutical, and/or nutraceutical) of the present disclosure comprises at least about 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 2 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 10 x 10 8 , 100 x 10 8 , IxlO 9 , 5xl0 9 , 10xl0 9 , 100 x 10 9 , HO x 10 9 , 120 x 10 9 , 130 x 10 9 , 140 x 10 9 , 150 x 10 9 , 160 x 10 9 , 170 x 10 9 , 180 x 10 9 , 190 x 10 9 , 200 x 10 9 , 210 x 10 9 , 220 x 10 9 , 230 x 10 9 , 240 x 10 9 , 250 x 10 9 , 260 x
  • the composition comprises at least about 180 x 10 9 but no more than about 270 x 10 9 total CFU of bacteria per gram of the composition. In preferred embodiments, the composition comprises about 250 x 10 9 total CFU of bacteria per gram of the composition.
  • AJ2 is a combination of 7 strains of gram-positive probiotic bacteria with the ability to induce synergistic production of IFN-y when added to IL-2 -treated or IL-2 + anti-CD16 monoclonal antibody -treated NK cells (anti-CD16mAb).
  • the combination of strains was used to provide bacterial diversity in addition to synergistic induction of a balanced pro and anti-inflammatory cytokine and growth factor release in NK cells.
  • the quantity of each bacteria within the combination of strains was adjusted to yield a closer ratio of IFN-y to IL- 10 to that obtained when NK cells are activated with IL-2 + anti-CD16mAb in the absence of bacteria.
  • the rationale behind such selection was to obtain a ratio similar to that obtained with NK cells activated with IL-2 + anti-CD16mAb in the absence of bacteria since such treatment provided significant differentiation of the cells.
  • the composition (bacterial, pharmaceutical, and/or nutraceutical) of the present disclosure comprises at least two bacterial strains selected from: Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium inf antis.
  • one or more bacterial strains are intact.
  • one or more bacterial strains are sonicated.
  • the composition is an AJ3 composition comprising Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis.
  • the percent bacteria refers to the percentage of the colony forming unit (CFU) of said bacteria relative to the total CFU of bacteria in the composition.
  • the bacteria in the composition comprise about 50% (or about 40% to about 60%) Bifidobacterium Longum, about 10% (or about 1% to about 20%) Bifidobacterium breve, and about 40% (or about 30% to about 50%) Bifidobacterium infantis, wherein the percent bacteria refers to the percentage of the CFU of said bacteria relative to the total CFU of bacteria in the composition.
  • the composition (bacterial, pharmaceutical, and/or nutraceutical) of the present disclosure comprises at least two bacterial strains selected from: Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei.
  • one or more bacterial strains are intact.
  • one or more bacterial strains are sonicated.
  • the composition is an AJ4 composition comprising Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei.
  • the percent bacteria refers to the percentage of the colony forming unit (CFU) of said bacteria relative to the total CFU of bacteria in the composition.
  • the bacteria in the composition comprise about 30% (or about 20% to about 40%) Streptococcus thermophiles, about 20% (or about 10% to about 30%) Lactobacillus acidophilus, about 40% (or about 30% to about 50%) Lactobacillus plantarum, and about 10% (or about 1% to about 20%) Lactobacillus paracasei, wherein the percent bacteria refers to the percentage of the CFU of said bacteria relative to the total CFU of bacteria in the composition.
  • Osteoclasts are multinuclear cells, have the unique ability to degrade bone to initiate normal bone remodeling and mediate bone loss in pathologic conditions by increasing their resorptive activity. Osteoclasts are derived from hematopoietic stem cells, precursors in the myeloid/ monocyte lineage that circulate in the blood after their formation in the bone marrow, and are tartrate-resistant acid phosphatase (TRAP)-positive cells.
  • TRIP tartrate-resistant acid phosphatase
  • Osteoclasts differentiation is controlled by interactions between osteoblasts and/or stromal cells and pre-osteoclasts.
  • M-CSF and RANKL are required for osteoclasts formation.
  • M-CSF is a cytokine released from osteoblasts as a result of endocrine stimulation from parathyroid hormone. It binds to receptors on osteoclast precursor cells (OPC) and induces differentiation into osteoclasts.
  • OPC osteoclast precursor cells
  • M-CSF is required for both the proliferative and differentiation phase of osteoclast development.
  • RANKL is critical for osteoclastogenesis and bone resorption.
  • RANKL interacts with its receptor RANK (receptor activator of NF- kB), a transmembrane receptor that is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on the surface of pre-osteoclasts and mature osteoclasts.
  • RANK receptor activator of NF- kB
  • TNF tumor necrosis factor
  • Osteoprotegerin (OPG) is a soluble decoy receptor which is produced by osteoblasts and can block osteoclast formation in vitro and bone resorption in vivo by binding to RANKL and reducing its ability to bind to RANK.
  • SZ sealing zone
  • Bone resorption is necessary for many skeletal processes. It is an obligatory event during bone growth, tooth eruption and fracture healing, and is also necessary for the maintenance of an appropriate level of blood calcium. Bone resorption is tightly coupled to bone formation in the healthy skeleton, however several diseases manifest as a result of an imbalance between resorption and formation. Osteopetrosis is a disease caused by a lack of osteoclast activity, leading to an increase in bone mass, whereas osteoporosis is a disease caused by osteoclast over activity, therefore leading to reduced bone mass and an increased risk of fracture.
  • Osteoclasts also express many ligands for receptors present on activated NK cells. They reported that osteoclasts express ULBP-1, ULBP-2/5/6 and ULBP-3, but little or no MIC-A, MIC-B, or MHC class Llike ligands for NKG2D, the activating receptor of NK cells.
  • Osteoclasts in comparison to dendritic cells (DCs) and monocytes, are significant activators of NK cell expansion and function (Tseng et al. (2015) Oncotarget 6(24):20002-25). Additionally, osteoclasts secrete significant amounts of IL-12, IL-15, IFN- y and IL- 18, which are known to activate NK cells; osteoclasts also express important NK- activating ligands. Accordingly, osteoclasts expand and activate NK cells to levels that are higher than those established by other methodologies.
  • IFN-y is a multifunctional cytokine produced mainly by NK cells and activated T cells that plays a critical role in host immune responses against pathogens and cancer.
  • IFN-y can inhibit the critical osteoclast regulator, receptor activator of NFkB ligand (RANKL), by activating ubiquitin-mediated degradation of its signaling pathway adaptor protein TRAF-6.
  • RTKL receptor activator of NFkB ligand
  • IFN-y could also be used to inhibit experimental tooth movement and bone erosion by decreasing osteoclastic activity. It has been shown that IFN- y participates in the regulation of RANKL signaling and bone destruction. Bone and immune system are functionally interconnected. Immune and bone cells derive from same progenitors in the bone marrow, they share a common microenvironment and are being influenced by similar mediators, different immune cells such as macrophages, T and B lymphocytes, mast cells, natural killer cells (NK), etc. have been shown to influence bone cells as well (FIG. 1).
  • Immune cells and their products play an important role in the regulation of skeletal development and function, particularly of the osteoclast, which implies that immune cell dysfunction may be involved in the pathogenesis of certain skeletal disorders.
  • IFN-y produced by both NK cells and Thl lymphocytes, has been shown to inhibit osteoclastogenesis in vitro.
  • IFN-y the in vivo effects of IFN-y on bone tissue are less clear since often provide a contrasting effect when compared to in vitro studies.
  • Reduced functioning of osteoclast and NK cell function coexist in osteopetrotic mutant rat.
  • OC progenitor activity is positively regulated by TNF-a and negatively regulated by IFN-y.
  • IFN-y binds to its receptor on osteoclasts, degrades RANKL signaling and thus inhibits the activation of osteoclasts and protects our bones from being resorbed.
  • This cytokine is produced predominantly by NK and natural killer T (NKT) cells involved in the innate immune response, and by CD4+ Thl and CD8+ cytotoxic T lymphocyte (CTL) effector T cells, once antigen-specific immunity develops.
  • ITIM-bearing NK receptor positively regulates osteoclasts differentiation
  • immunoreceptor tyrosine-based activation motif (ITAM)- mediated signaling is critical for osteoclast differentiation.
  • Crosstalk between the skeletal system and T cells is termed as osteoimmunology.
  • RANKL expressed by CD4+ and CD8+ T-cells can induce osteoclast genesis, providing a link between immune and skeletal system. Osteoclasts produce chemokines that recruit CD8 positive T cells.
  • Osteoclasts induced the secretion of IL-2, IL-6, IFN-y and induced the proliferation of CD8 positive T cells.
  • CD8 positive T cells activated by osteoclasts expressed FoxP3, CTLA4, and receptor activator of NF-kB ligand.
  • Anti- CD3/CD28- stimulated y6 T cells or CD4+ T cells inhibit human osteoclast formation and resorptive activity in vitro. Cytokine production by CD3/CD28-stimulated y6 T cells and observed a lack of IL- 17 production, with activated y6 T cells producing abundant interferon (IFN)-y.
  • IFN interferon
  • osteoimmunology a close relationship between immune system and osteoclasts, which is termed osteoimmunology.
  • RANKL is secreted by activated T-cells, and dysregulation of RANKL leads to defective formation of lymph nodes and lymphocyte differentiation as well as impaired osteoclastic bone resorption.
  • IFN-y also known as immune interferon, is the only type II IFN and was discovered in 1965. It is secreted predominantly by T-cells, natural killer cells, and some other cells such as macrophages, dendritic cells and B cells. IFN-y signal transduction is mediated by binding with IFNGR1 and IFNGR2 resulting in activation of intracellular molecular signaling networks such as JAK-STAT pathway and STAT-independent pathways such as the MAP kinease, NF-kB, and PI3K pathways.
  • IFN-y activated factor GAF
  • IFN-y uniquely regulates a variety of autoinflammatory and autoimmune diseases, such as systemic lupus erythematosus, hemophagocytic lymphohistiocytosis (HLH), and macrophage activation syndrome (MAS).
  • HHL hemophagocytic lymphohistiocytosis
  • MAS macrophage activation syndrome
  • IFN-y has long been used as an immunosuppressive. Further studies showed that IFN-y could also be used to inhibit experimental tooth movement and bone erosion by decreasing osteoclastic activity. It has been shown that IFN-y participates in the regulation of RANKL signaling and bone destruction.
  • IFN-y has also recently been found to participate in a number of signaling pathways in osteoclastogenesis. Li et al. revealed that IFN-y mediates a previously unknown feedback loop that exits between osteoclasts and activated T-cells by inducing indoleamine 2,3- dioxygenase (IDO) expression in osteoclasts. Ji et al. identified another mechanism through which IFN-y regulates osteoclastogenesis. In contrast to previous studies, they found that IFN-y alone did not affect TRAF6 expression in human osteoclast precursors, whereas, IFN-y cooperated with TLRs to suppress RANK expression by inhibiting colony stimulating factor 1 receptor (c-Fms), a potent stimulator of RANK expression.
  • c-Fms colony stimulating factor 1 receptor
  • IFN-y In addition to directly inhibiting osteoclast formation. IFN-y was recently reported to promote osteoblastogenesis and bone formation both in vitro and in vivo. The anabolic effect of IFN-y is mediated by increasing both osteoblastogenesis and osteoclastogenesis with a predominant stimulatory effect on the osteoblast lineage, thus increasing bone mass and rescuing oophorectomized (OVX) mice from osteoporosis. In addition, IFN-y significantly increases the expression of osteogenic markers in differentiating mesenchymal stem cell (MSC) into osteoblasts in vitro, including runt-related transcription factor 2 and osteopontin.
  • MSC mesenchymal stem cell
  • IFN-y the effects of IFN-y on bone remain complex with some investigators reporting contrasting findings about its effect on osteoclastogenesis and bone resorption.
  • High doses of IFN-y have been used as treatment in patients with osteopetrosis to induce bone resorption and reduce bone mass.
  • Other studies report that IFN-y directly inhibits osteoclast differentiation and induce osteoclast apoptosis.
  • the effect of IFN-y on osteoclast differentiation and function could be affected, among others, by estrogen deficiency, inflammation, and bacterial toxins.
  • IFN-y Interfereon-gamma
  • IFN-y INDUCES APOPTOSIS OF CANCER CELLS It has been demonstrated that high doses of IFN-y could induce apoptosis in NSCLC cell-lines, namely A549 and H460, by activating JAK-STAT1 -caspase signaling.
  • Western blot analyses showed that STAT1 forced transcription and synthesis of caspase 3 and caspase 7, which further initiated apoptotic processes in cancer cells (Song et al. (2019) Cancer Res, 81771781 : 1-29. Additionally, it was shown that IFN-y can increase the motility of antigen-specific CD8+ T-cells to the antigen-expressing (target) cells and enhance the killing capacity of target cells.
  • IFN-y+/+ and IFN-y-/- CD8 T-cells were incubated with the target cells, significantly higher effectiveness of IFN-y competent cells was observed.
  • Addition of anti-IFN-y-antibody to the co-culture system markedly reduced target cell killing.
  • IFN-y can selectively induce apoptosis in stem-like colon cancer cells through JAK-STAT1-IRF1 signaling in a dose-dependent manner.
  • Kundu et al. reported that precise neutralization of cytokine from IL-12 family, namely p40 monomer, induces IL-12-IFN-y signaling cascade in prostate cancer both in vitro and in vivo, which subsequently leads to cancer cells death and tumor regression.
  • IFN-y can directly affect the viability of tumor cells, increasing evidence points to interactions with surrounding stromal cells for effective rejection of solid tumors.
  • immunohistology analyses of large tumor sections revealed that IFN-y could reduce the number of endothelial cells and induce blood vessel destruction and later promote tumor tissue necrosis.
  • Kammertoens et al. showed responsiveness of cancer endothelial cells by highlighting the necessary role that IFN-y plays in the regression of solid tumors. By using electron microscopy they observed that IFN-y-exposed endothelial cells became round, condensed, and more occluded, which reduced blood flow in tumor tissues and subsequently, prompted tumor ischemia (Kammertoen et al.
  • IFN-y downregulated the expression of vascular endothelial growth factor A, a growth factor critical for tumor neovascularization. Therefore, it is equally important to investigate IFN- y-mediated effects on tumor stromal cells, especially in solid, well-established tumors.
  • TME tumor microenvironment
  • IFN-y interacts with distinct cytokines from the TME to induce cancer growth arrest. Synergistically with TNF, IFN-y stimulates the senescence of tumor cell growth through stabilization of pl6INK4a - Rb pathway. This effect is mediated by activation of STAT1 and TNF receptor 1 and is maintained permanently in vitro and in vivo. Together with inducing apoptosis or senescence, IFN-y can shift tumors to a dormant state. As recently shown IFN-y - mediated upregulation of IDO 1 increased the intracellular concentration of kynurenine (kyn, IDO1 - catalyzed tryptophan metabolite), which activated aryl hydrocarbon receptor (AhR).
  • IDOl-Kyn-AhR- p27 pathway was proposed as a mechanism which explains how high concentration of IFN- y induces tumor dormancy.
  • the existence of IL-12-IFN-y relationship has also been described.
  • DCs dendritic cells
  • IFN-y produced by NK cells altered tumor structure and limited the number of metastasis by increasing the expression of the extracellular matrix protein, fibronectin 1 (Glasner et al. (2016) Immunity 48: 107-19).
  • IFN-y is believed to be one of the critical factors determining the success of immunotherapy.
  • Ayers et al. reported that metastatic melanoma, head and neck squamous cell carcinoma, and gastric cancer patients who responded to anti-PD-1 therapy had higher expression scores for IFN- y-related genes when compared to non-responders.
  • IFN-y signature IDO1, CXCL10, CXCL9, HLA-DRA, STAT1, and IFNG
  • IDO1, CXCL10, CXCL9, HLA-DRA, STAT1, and IFNG IFN-y signature
  • IFNG four-gene IFN-y signature
  • this antibody stimulates CD8+ T-cells to secrete IFN-y, which activates its receptor on DCs, thus increasing the production of IL- 12 in the TME.
  • the newly generated interleukin acts back on CD8+ T cells to further stimulate IFN-y production and enhance cytotoxic tumor cell function. Therefore, a combination of anti-PD-1 antibody and induction of INF -y via the compositions of the present disclosure would be particularly useful in preventing and/or treating cancer.
  • Alternative mechanism by which IFN-y contributes to efficiency of cancer immunotherapy was described by Wang et al. In that model, tumor-infiltrating CD8+ T-cells secreted IFN-y in response to nivolumab, an anti-PD-Ll antibody.
  • the released IFN-y mediated lipid peroxidation and ferroptosis in tumor cells by reducing the uptake of cystine and excretion of glutamate, resulting in tumor cells death both in vitro and in vivo.
  • type II interferon activated the JAK1-STAT1 signaling pathway, which further downregulated the transcription of SLC7A11 and SLC3 A2 proteins of the glutamate-cystine antiporter system.
  • the clinical benefits of cancer immunotherapy were reduced in nivolumab -treated mice bearing INFGR-/- tumors.
  • IFN-y may be a good therapeutic option for improving the efficacy of PD-1 blockade therapy for pancreatic cancer by preventing the trafficking of CXCR2+ CD68+ immunosuppressive macrophages to the TME by blocking the CXCL8-CXCR2 axis (Zhang et al. (2020) J Immunother Cancer 8(1): 1-15).
  • the efficiency of anti-CTLA-4 therapy was also IFN-y dependent.
  • Whole exome sequencing data showed that melanoma tumors resistant to immunotherapy had defects in IFN-y signaling, namely loss of IFNGR1, IRF-1, JAK2 and IFNGR2 genes, as well as amplification of SOCS1 and PIAS4 inhibitory genes. Therefore, the combination of immune checkpoint inhibitors and IFN-y can be a good strategy to increase the overall efficiency of cancer immunotherapy. Indeed, two such clinical trials have already been initiated testing the combination of nivolumab or pembrolizumab with IFN-y (NCT02614456 and NCT03063632, respectively).
  • IFN-y signaling in tumor cells could boost tumor growth and impact the efficiency of given immune checkpoint inhibitor therapy.
  • Amplification of IFN-y-pathway inhibitory molecules or downregulation and loss of its receptor and downstream signaling mediators are common mechanisms for tumor cells to avoid generated immune response. It was recently shown that aging can also consistently attenuate IFN-y signaling in triple-negative breast cancer patients and limit the efficiency of immune checkpoint blockade (ICB) therapy.
  • ICB immune checkpoint blockade
  • Another hypothesis is that enhanced intratumoral production of IFN-y can improve the potency of ICB therapy in patients with cancer.
  • CBM complex CARMAl-BCLlO-MALTl
  • CARMAl-BCLlO-MALTl CARMAl-BCLlO-MALTl
  • Tregs reprogram them to secrete IFN-y which results in tumor regression.
  • CBM inhibition and anti-PD-1 antibodies enabled tumor control in MC38 colon carcinoma-bearing mice who were resistant to anti-PD-1 monotherapy.
  • tumor regression has been observed only in melanoma-bearing mice treated with PD-1 targeted therapy together with antibodies against neuropilin- 1.
  • Neuropilin- 1 is a protein found on most of the tumor-infiltrating Tregs, important for their suppressive function.
  • Treg-secreted IFN-y drove intratumoral fragility of the remaining immune- suppressive Tregs via hypoxia-inducible factor 1 -alpha (HIFla) which stimulated host immunity to eliminate cancer cells.
  • HIFla hypoxia-inducible factor 1 -alpha
  • metastatic potential of tumor cells after receiving immunotherapy was due to reduction of IFN-y in the TME and augmented activity of integrin avP3 signaling axis.
  • NK cells Natural Killer (NK) cells are granular lymphocytes that function at the interference of innate and adaptive immunity. Discovered in the early 1970’s by accident when investigators were studying specific cytotoxic effects of lymphocytes, it was not until the 1980’s that they became generally accepted despite the accumulated evidence. NK cells are a subset of 8 cytotoxic lymphocytes able to recognize and lyse tumor cells and virus infected cells without prior sensitization. Traditionally they have been classified as effectors of innate immunity due to the lack of antigen specific cell surface receptors. NK cells are known to mediate direct and antibody dependent cellular cytotoxicity (ADCC) against tumors as well as to regulate the function of other cells through the secretion of cytokines and chemokines.
  • ADCC antibody dependent cellular cytotoxicity
  • NK cells derive from CD34+ hematopoietic stem cells (HSC’s) found in the bone marrow. They can be found throughout the body in the spleen, liver, placenta, and peripheral blood.
  • HSC hematopoietic stem cells
  • Human NK cells are defined phenotypically by the surface expression of CD56 and CD 16, and by their lack of CD3 surface expression.
  • CD56 is a human neural-cell adhesion molecule, but its function on human NK cells is yet to be understood. Although the function of CD56 is unknown, its expression correlates with the expression of other surface markers that confer important functional properties to NK cells. Two subsets of NK cells have been identified based on surface expression of CD56 and CD 16.
  • the major subset of NK cells is defined by low expression of CD56 (CD56dim) and high expression of CD16 (CD16 bright).
  • the minor subset makes up approximately 10% of human NK cells and is defined by high expression of CD56 (CD56 bright) and low or lack of CD 16 (CD 16 dim) expression.
  • the CD56dim CD 16 bright cells were found to be the more cytotoxic subset of human NK cells.
  • CD56bright CD16dim/- NK cells were found to secrete more cytokines such as interferon-y (IFN-y), tumor necrosis factor-a (TNF-a), TNF-P, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin- 10 (IL- 10), and IL 13 after being stimulated with pro-inflammatory cytokines.
  • IFN-y interferon-y
  • TNF-a tumor necrosis factor-a
  • TNF-P tumor necrosis factor-a
  • GM-CSF granulocyte macrophage colony stimulating factor
  • IL- 10 interleukin- 10
  • IL 13 interleukin- 10
  • NK cells develop in the bone marrow and constitute about 5-10% of total lymphocytes in the peripheral circulation and secondary lymphoid organs. Effector function of NK cells include direct natural cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), as well as secretion of inflammatory cytokines and chemokines that indirectly regulate the functions of other immune cells.
  • ADCC antibody-dependent cellular cytotoxicity
  • NK cells mediate cytotoxicity against transformed tumor cells, as well as healthy cells, by releasing pre-formed granules of proteins, known as perforin and granzyme B, which can induce apoptosis, or programmed cell death in target cell.
  • NK cells have also been identified within inflamed synovial fluid and express RANKL and M-CSF which during their interaction with monocytes can trigger the formation of osteoclasts in a process that is RANKL and M-CSF dependent.
  • NK cell cytotoxic activity in cancer patients is severely reduced, correlating with the decreased expression of NK cell activating receptors even at the early stages of disease, and are further diminished in advanced cancers.
  • Patients’ NK cells are significantly defective in their function, and the defect is seen at both preneoplastic and neoplastic stages of pancreatic cancer.
  • Patients’ NK cells do not recover their full functional potential even when the best activating conditions are provided for their expansion and function.
  • NK cells under the expansion conditions give rise to T cell expansion at a much faster rate, with a subsequent decrease in the percentages of NK cells when compared to those from healthy individuals.
  • allogeneic NK cells from healthy individuals is preferable than autologous NK cells for therapeutic purposes.
  • the use of allogeneic NK cells in hematologic malignancies following HLA-haploidentical HSCT in clinical trials has previously been reported.
  • allogeneic NK cells exert less collateral damage due to graft versus host disease (GVHD) when used therapeutically in solid tumors.
  • GVHD graft versus host disease
  • NK cells In search of a more potent therapeutic dose of NK cells, a novel strategy to expand highly functional NK cells has been established, coined as super-charged NK cells, by employing osteoclasts as feeder cells in the presence of a combination of sonicated probiotic bacteria (sAJ2).
  • the potency and effectiveness of super-charged NK cells are significantly superior to those established by other methodologies or when compared to primary activated NK cells.
  • the term super-charged was used to describe the magnitude and superiority of their functional potential in lysing and differentiating CSCs/poorly differentiated tumors.
  • NK cells have the following roles: 1) Selection and differentiation of healthy stem cells. 2) Regulate inflammation, 3) activate CAR NK, CAR T, and T cells, 4) control the tumors through tumor differentiation, 5) kill cancer stem cells, 6) kill through oncologic viruses. 7) act synergistically with radiation and chemotherapy, 8) kill through antibodies and checkpoint inhibition, 9) kill suppressor cells, 10) exert preferential expansion of CD8+ T cells, and 11) gene knockout recognition (FIG. 2).
  • NK cells have very diverse biological functions including significant roles in defense against tumor cells. Based on knowledge of NK cell function and evidence that they become nonfunctional in cancer patients, several approaches have been proposed for the use of NK cells in immunotherapy:
  • Cytokines can be used to boost NK cells for immunotherapeutic means, both in vitro and in vivo.
  • Antibodies can be applied to NK cell immunotherapy based on different approaches.
  • NK cells can target tumor cells coated with IgG antibodies through the ADCC.
  • There are several monoclonal antibody treatments available specific to different tumor antigens such as the use of anti-CD20 for the treatment of B cell lymphoma or anti-Her2 for the treatment of Her2-overexpressing invasive breast cancer.
  • Antibodies can be also being used to block NK cells’ checkpoint inhibitors. Studies have shown that NK cells express checkpoint molecules such as PD-1, CTLA-4, TIM-3 and TIGIT.
  • NK cells can be harvested from different sources, and their functional competencies may vary depending on the strategies used to separate, activate or expand them.
  • Sources of natural killer cells for adoptive transfer a) NK cells isolated from Peripheral Blood Mononuclear Cells (PBMCs)
  • NK cells can be isolated from autologous and allogeneic PBMCs.
  • the strategies to expand and activate NK cells from PBMCs are different.
  • Investigators have tried to Expand NK cells directly from PBMCs without isolating the NK cells population, or by depleting CD3+ cells, or selecting CD16+, or CD 16+ and CD56+ cells.
  • Different cells have been used as feeder cells to improve NK cells expansion.
  • Irradiated K562 and OK432 are two of the most popular feeder cells for NK expansions.
  • Different Cytokines and other activators also have been applied to expand NK cells in vitro.
  • IL-2, IL-21, IL- 15 and IL- 18 are some of the cytokines used for this means.
  • NK cells are treated with IL-2 and CD 16 antibody and probiotic bacteria and Osteoclasts are used as feeder cells.
  • the Expanded NK cells called “Super-Charged NK cell” have high cytotoxicity and IFN- y secretion.
  • NK cells as a source of therapy has not been very effective. It has been shown that NK cells from cancer patients are less functional both in terms of cytotoxicity and of IFN- y secretion in vitro. In an in vivo study adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression.
  • NK cells have also demonstrated a limited effect on tumor suppression in malignant glioma. Allogenic NK cells can be a better resource for NK cell therapy since NK cells from healthy donors have better functionality than NK cells in cancer patient, besides the KIR receptor from the donor mismatch the MHC class I of the recipient letting NK cells to skip some of the inhibitory processes.
  • NK cells Due to the pluripotency of stem cells, using them as a precursor of NK cells has become one of the interesting sources of NK cells.
  • Generation of NK cells from ESCs and iPSCs enerally requires two steps. First, CD34+ hematopoietic precursors must be generated. The CD34+ cells are then sorted and differentiated into NK cells with cytokines and feeder cells (usually murine stromal cells). When NK cells were generated from hESCs, they were mostly CD56+CD45+ NK cells, which also expressed inhibitory and activating receptors typically found on adult NK cells.
  • NK cells were also able to mediate cytotoxicity against of leukemic cells, K562 (erythroleukemia), and several solid tumors, including breast cancer (MCF7), testicular embryonal carcinoma (NTERA2), prostate cancer (PC3), and glioma (U87) cell lines.
  • MCF7 breast cancer
  • NTERA2 testicular embryonal carcinoma
  • PC3 prostate cancer
  • U87 glioma
  • the Kaufman research group established a feeder-free, sorting-free approach to generate NK cells from human ESCs. These authors used a spin-embryoid body system with BMP4 and VEGF to derive hematopoietic progenitors. After 11 days of culture, the spin-EBs were transferred to the NK cell culture containing the cytokines IL-3, IL-15, IL-7, SCF and Flt3L for 28 days. This feeder-free system can generate NK cells that have no difference from those derived from the murine embryonic liver cell line EL08-1D2 as feeder cell. Meanwhile, this group established a clinical-scale derivation of NK cells from ESCs and iPSCs without cell sorting and in the absence of feeder cells. c) NK cells isolated from Umbilical Cord blood.
  • Umbilical cord blood has become a known source for NK cells.
  • the NK cells from UCB and peripheral blood (PB) have some differences.
  • UCB NK cells express similar levels of CD56, NKp46, NK30 and NKG2D as PB NK cells but lower levels of CD 16 CD2, CD 11 a, CD 18, CD62L), KIRs, DNAM-1, NKG2C, IL-2R, and CD57 and CD8.
  • UCB has a higher percentage of NK cells but these cells have lower cytotoxicity in comparison to NK cells from PB which could be due to lower levels of Granzyme B and perforin in CB NK cells. Studies have shown that with proper signaling NK cells from UCB can be expanded to create many cells with proper function. d) Genetically modified NK cells
  • NK cells can be genetically modified to secrete cytokines in favor of their survival and activation.
  • Engineering Chimeric antigen receptor (CAR) NK cells has currently become the topic of interest. Currently, several tumor antigen-binding domains have been designed as CAR extracellular domains and tested.
  • the activity or level of NK cells can be detected by any means known in the art.
  • the NK cell level in PBMC can be determined by detecting the presence/absence of surface markers that phenotypically define the NK cells (e.g., the surface expression of CD56 and CD 16, and by their lack of CD3 surface expression - see the section “NK Cells” above).
  • Such detection of NK cells can utilize any methods known in the art, including those described below for detecting a cytokine/chemokine.
  • the NK cell level and/or activity can also be detected by the amount of IFN-y or other cytokines/chemokines secreted by NK cells.
  • NK cells can be isolated from PBMC using e.g., using isolation kits from Stem Cell Technologies (Vancouver, BC, Canada), and their cytotoxicity activity can be measured using e.g., 51 Cr release assay (see below).
  • NK cell’s ADCC activity can also be used as a measure of the NK cell amount and/or activity.
  • the activity or level of a cytokine or chemokine can be detected and/or quantified by detecting or quantifying the expressed polypeptide.
  • the polypeptide can be detected and quantified by any of a number of means well-known to those of skill in the art. Accordingly, any method known in the art for detecting polypeptides can be used.
  • Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference).
  • binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.
  • ELISA and RIA procedures may be conducted such that a desired cytokine/chemokine standard is labeled (with a radioisotope such as 125 I or 35 S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabeled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay).
  • a radioisotope such as 125 I or 35 S
  • an assayable enzyme such as horseradish peroxidase or alkaline phosphatase
  • the cytokine/chemokine in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme- labeled anti-biomarker protein antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay).
  • radioactivity or the enzyme assayed ELISA-sandwich assay.
  • Other conventional methods may also be employed as suitable.
  • a “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody.
  • a “two-step” assay involves washing before contacting, the mixture with labeled antibody.
  • Other conventional methods may also be employed as suitable.
  • a method for measuring the cytokine/chemokine levels comprises the steps of: contacting a biological specimen with an antibody or variant (e.g., fragment) thereof which selectively binds the cytokine/chemokine, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of the fFN-y or cytokine/chemokine.
  • an antibody or variant e.g., fragment
  • Enzymatic and radiolabeling of a cytokine/chemokine and/or the antibodies may be effected by conventional means.
  • Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected.
  • some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.
  • Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.
  • cytokine/chemokine may be used to detect a cytokine/chemokine according to a practitioner's preference based upon the present disclosure.
  • One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter.
  • Anti-cytokine/chemokine antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including 125 I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
  • Immunohistochemistry may be used to detect expression of IFN-y or cytokine/chemokine, e.g., in a biopsy sample.
  • a suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody.
  • Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabeling. The assay is scored visually, using microscopy.
  • Antibodies that may be used to detect a cytokine/chemokine include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker protein to be detected.
  • An antibody may have a Kd of at most about 10' 6 M, 10' 7 M, 10' 8 M, 10' 9 M, 10' 10 M, 1O' U M, 10" 12 M.
  • the phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant.
  • An antibody may bind preferentially to the biomarker protein relative to other proteins, such as related proteins.
  • Antibodies may be commercially available or may be prepared according to methods known in the art.
  • Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered or single-chain antibodies as well as functional fragments, /. ⁇ ?., cytokine/chemokine-binding fragments, of antibodies.
  • antibody fragments capable of binding to a biomarker protein or portions thereof including, but not limited to, Fv, Fab, Fab' and F(ab')2 fragments can be used.
  • Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively.
  • Fab or F(ab')2 fragments can also be used to generate Fab or F(ab')2 fragments.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain.
  • agents that specifically bind to IFN-y or a cytokine/chemokine other than antibodies are used, such as peptides.
  • Peptides that specifically bind to a cytokine/chemokine is well known in the art (e.g., receptor fragment for the cytokine/chemokine), and can also be identified by any means known in the art.
  • specific peptide binders of a biomarker protein can be screened for using peptide phage display libraries.
  • NK cell or cytokine/chemokine (e.g., IFN-y) amount and/or activity measurement(s) in a sample from a subject is compared to a pre-determined control (standard) sample.
  • the sample from the subject is typically from blood or tissue.
  • the control sample can be from the same subject or from a different subject.
  • the control sample is typically a normal, non-diseased sample. However, in some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, the control sample can be from a diseased subject.
  • the control sample can be a combination of samples from several different subjects.
  • the NK cell or cytokine/chemokine amount and/or activity measurement(s) from a subject is compared to a pre-determined level. This pre-determined level is typically obtained from normal samples.
  • a pre-determined NK cell or cytokine/chemokine amount and/or activity measurement(s) may be determined in populations of patients with or without a disease (e.g., bone loss condition and/or cancer).
  • the pre-determined NK cell or cytokine/chemokine amount and/or activity measurement(s) can be a single number, equally applicable to every patient, or the pre-determined NK cell or cytokine/chemokine amount and/or activity measurement(s) can vary according to specific subpopulations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined NK cell or cytokine/chemokine amount and/or activity measurement s) of the individual. Furthermore, the pre-determined cytokine/chemokine amount and/or activity can be determined for each subject individually. In some embodiments, the amounts determined and/or compared in a method described herein are based on absolute measurements.
  • the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios e.g., NK cell or cytokine/chemokine level, and/or activity before a treatment vs. after a treatment, and the like).
  • the relative analysis can be based on the ratio of pre-treatment cytokine/chemokine measurement as compared to post-treatment cytokine/chemokine measurement.
  • Pre-treatment NK cell or cytokine/chemokine measurement can be made at any time prior to initiation of anti-cancer therapy or an anti-inflammation therpay.
  • Posttreatment cytokine/chemokine measurement can be made at any time after initiation of anticancer therapy or an bone loss therapy.
  • post-treatment NK cell or cytokine/chemokine measurements are made 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after initiation of the administration of the compositions of the present disclosure.
  • the change of NK cell or cytokine/chemokine amount and/or activity measurement(s) from the pre-determined level is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 fold or greater, or any range in between, inclusive.
  • Such cutoff values apply equally when the measurement is based on relative changes, such as based on the ratio of pre-treatment NK cell or cytokine/chemokine measurement as compared to post-treatment NK cell or cytokine/chemokine measurement.
  • Body fluids refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
  • the subject and/or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.
  • the sample is serum, plasma, or urine. In other embodiments, the sample is serum.
  • the samples can be collected from individuals repeatedly over a longitudinal period of time (e.g., once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an individual over a period of time can be used to verify results from earlier detections and/or to identify an alteration in biological pattern as a result of, for example, disease progression, drug treatment, etc. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three month intervals according to the present invention.
  • cytokine/chemokine amount and/or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.
  • Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and/or analysis of cytokine/chemokine measurement(s).
  • Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.
  • compositions of the compositions disclosed herein may be formulated for administration in solid or liquid form, including those adapted for the following exemplary routes: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; or (2) parenteral administration, for example, by subcutaneous, intratumoral, intramuscular or intravenous injection as, for example, a sterile solution or suspension.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes
  • parenteral administration for example, by subcutaneous, intratumoral, intramuscular or intravenous injection as, for example, a sterile solution or suspension.
  • Cells e.g., NK cells, super-charged NK cells, osteoclasts
  • NK cells can be administered at 1, 10, 1000, 10,000, 0.1 x 10 6 , 0.2 x 10 6 , 0.3 x 10 6 , 0.4 x 10 6 , 0.5 x 10 6 , 0.6 x 10 6 , 0.7 x 10 6 , 0.8 x 10 6 , 0.9 x 10 6 , 1.0 x 10 6 , 5.0 x 10 6 , 1.0 x 10 7 , 5.0 x 10 7 , 1.0 x 10 8 , 5.0 x 10 8 , 1.0 x 10 9 or more, or any range in between or any value in between, cells per kilogram of subject body weight.
  • the number of cells injected may be adjusted based on the desired level of administration in a given amount of time. Generally, 1 * 10 5 to about 1 x 10 9 cells/kg of body weight, from about 1 * 10 6 to about 1 x 10 8 cells/kg of body weight, or about 1 x 10 7 cells/kg of body weight, or more cells, as necessary, may be injected. In some embodiments, injection of at least about 100, 1000, 10,000, O.lxlO 6 , 0.5xl0 6 , l.Ox lO 6 , 2.0x l0 6 , 3.0x l0 6 , 4.0x l0 6 , or 5.0x l0 6 total cells relative to an average size mouse is effective.
  • Cells e.g., NK cells, super-charged NK cells, osteoclasts
  • NK cells e.g., NK cells, super-charged NK cells, osteoclasts
  • Cells can also be administered before, concurrently with, or after, other bone loss therapy and/or anticancer agents.
  • Agents, including cells may be introduced to the desired site by direct injection, or by any other means used in the art including, but are not limited to, intravascular, intracerebral, parenteral, intraperitoneal, intravenous, epidural, intraspinal, intrastemal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, intratumoral, or intramuscular administration.
  • agents of interest may be administered with cells by various routes.
  • Such routes include, but are not limited to, intravenous administration, subcutaneous administration, administration to a specific tissue (e.g, focal transplantation), injection into the femur bone marrow cavity, injection into the spleen, injection into the tumor, administration under the renal capsule of fetal liver, and the like.
  • the NK cells e.g., super-charged NK cells
  • Cells may be administered in one infusion, or through successive infusions over a defined time period sufficient to generate a desired effect.
  • NK cells e.g., super-charged NK cells
  • osteoclasts e.g., a cell types that can be combined and administered, such as NK cells (e.g., super-charged NK cells) and osteoclasts, and the like.
  • the ratio of NK cells (e.g., supercharged NK cells) to other cell types (e.g., osteoclasts) can be 1 : 1, but can modulated in any amount desired (e.g, 1 : 1, 1.1 : 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4:1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7:1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5:1, 10: 1, or greater).
  • Engraftment of transplanted cells may be assessed by any of various methods, such as, but not limited to, bone density, tumor volume, cytokine levels, time of administration, flow cytometric analysis of cells of interest obtained from the subject at one or more time points following transplantation, and the like. For example, a time-based analysis of waiting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 days or more can signal the time for sample harvesting. Any such metrics are variables that can be adjusted according to well-known parameters in order to determine the effect of the variable on a response to the agents of the present disclosure.
  • the transplanted cells can be co-transplanted with other agents, such as cytokines, extracellular matrices, cell culture supports, and the like.
  • An agent including cells, e.g., NK cells, super-charged NK cells, osteoclasts
  • an agent can be administered to an individual in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol.
  • Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).
  • compositions described herein may be used for oral administration to the gastrointestinal tract, directed at the objective of introducing the probiotic bacteria to tissues of the gastrointestinal tract.
  • the formulation for a therapeutic composition of the present invention may also include other probiotic agents or nutrients which promote spore germination and/or bacterial growth.
  • An exemplary material is a bifidogenic oligosaccharide, which promotes the growth of beneficial probiotic bacteria.
  • the probiotic bacterial strain is combined with a therapeutically-effective dose of an (preferably, broad spectrum) antibiotic, or an antifungal agent.
  • the compositions described herein are encapsulated into an enterically-coated, time-released capsule or tablet.
  • the enteric coating allows the capsule/tablet to remain intact (i.e., undisolved) as it passes through the gastrointestinal tract, until after a certain time and/or until it reaches a certain part of the GI tract (e.g., the small intestine).
  • the time-released component prevents the “release” of the probiotic bacterial strain in the compositions described herein for a pre-determined time period.
  • the therapeutic compositions of the present invention may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins or minerals.
  • the therapeutic compositions of the present invention are combined with a carrier which is physiologically compatible with the gastrointestinal tissue of the species to which it is administered.
  • Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule or powdered form; or the carrier can be comprised of liquid or gel-based materials for formulations into liquid or gel forms.
  • the specific type of carrier, as well as the final formulation depends, in part, upon the selected route(s) of administration.
  • the therapeutic composition of the present invention may also include a variety of carriers and/or binders.
  • a preferred carrier is micro-crystalline cellulose (MCC) added in an amount sufficient to complete the one gram dosage total weight.
  • Carriers can be solid-based dry materials for formulations in tablet, capsule or powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes of administration.
  • Typical carriers for dry formulations include, but are not limited to: trehalose, malto-dextrin, rice flour, microcrystalline cellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and like carriers.
  • Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like).
  • water-based carriers possess a neutral pH value (i.e., pH 7.0).
  • Other carriers or agents for administering the compositions described herein are known in the art, e.g., in U.S.Patent No. 6,461,607.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydro
  • Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of one or more bacterial strains as disclosed herein.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid or as an oil-in-water or water-in-oil liquid emul
  • the composition (e.g., bacterial composition, pharmaceutical composition, nutraceutical composition) comprises at least one carbohydrate.
  • a “carbohydrate” refers to a sugar or polymer of sugars.
  • saccharide e.g., bacterial composition, pharmaceutical composition, nutraceutical composition
  • oligosaccharide e.g., oligosaccharide
  • Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnHznOn.
  • a carbohydrate may be a monosaccharide, a disaccharide, tri saccharide, oligosaccharide, or polysaccharide.
  • the most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose.
  • Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose.
  • an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units.
  • Exemplary polysaccharides include starch, glycogen, and cellulose.
  • Carbohydrates may contain modified saccharide units such as 2’-deoxyribose wherein a hydroxyl group is removed, 2’ -fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2’- fluororibose, deoxyribose, and hexose).
  • Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
  • the composition comprises at least one lipid.
  • a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans).
  • the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16: 1), margaric acid (17:0), heptadecenoic acid (17: 1), stearic acid (18:0), oleic acid (18: 1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20: 1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EP A), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and
  • the composition comprises at least one supplemental mineral or mineral source.
  • supplemental mineral or mineral source examples include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium.
  • Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
  • the composition comprises at least one supplemental vitamin.
  • the at least one vitamin can be fat-soluble or water soluble vitamins.
  • Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin Bl 2, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
  • Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
  • the composition comprises an excipient.
  • suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • the excipient comprises a buffering agent.
  • suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • the excipient comprises a preservative.
  • suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • the composition comprises a binder as an excipient.
  • Nonlimiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
  • the composition comprises a lubricant as an excipient.
  • suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
  • the composition comprises a dispersion enhancer as an excipient.
  • suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.
  • the composition comprises a disintegrant as an excipient.
  • the disintegrant is a non-effervescent disintegrant.
  • suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth.
  • the disintegrant is an effervescent disintegrant.
  • suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
  • the composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food product e.g., a food or beverage
  • a food or beverage such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, carb
  • a nutraceutical composition is a pharmaceutical alternative which may have physiological benefits.
  • a nutraceutical composition is a food (or part of a food) that provides medical or health benefits, including the prevention and/or treatment of a disease. See, e.g., Brower (1998) Nat. Biotechnol. 16:728-731; Kalra (2003) AAPS Pharm Sci. 5(3):25.
  • a nutraceutical composition is a dietary or nutritional supplement.
  • a nutraceutical composition of the invention can be a food product, foodstuff, functional food, or a supplement composition for a food product or a foodstuff.
  • the term food product refers to any food or feed which provides a nutritional source and is suitable for oral consumption by humans or animals.
  • the food product may be a prepared and packaged food (e.g., mayonnaise, salad dressing, bread, or cheese food) or an animal feed (e.g., extruded and pelleted animal feed, coarse mixed feed or pet food composition).
  • the term foodstuff refers to a nutritional source for human or animal oral consumption.
  • Functional foods refer to foods being consumed as part of a usual diet but are demonstrated to have physiological benefits and/or reduce the risk of chronic disease beyond basic nutritional functions.
  • Food products, foodstuffs, functional foods, or dietary supplements may be beverages such as non-alcoholic and alcoholic drinks as well as liquid preparations to be added to drinking water and liquid food.
  • Non-alcoholic drinks are for instance soft drinks; sport drinks; fruit juices, such as orange juice, apple juice and grapefruit juice; lemonades; teas; near-water drinks; and milk and other dairy drinks such as yogurt drinks, and diet drinks.
  • food products, foodstuffs, functional foods, or dietary supplements refer to solid or semi-solid foods.
  • These forms can include, but are not limited to, baked goods such as cakes and cookies; puddings; dairy products; confections; snack foods (e.g., chips); or frozen confections or novelties (e.g., ice cream, milk shakes); prepared frozen meals; candy; liquid food such as soups; spreads; sauces; salad dressings; prepared meat products; cheese; yogurt and any other fat or oil containing foods; and food ingredients (e.g., wheat flour).
  • the food products, foodstuffs, functional foods, or dietary supplements may be in the form of tablets, boluses, powders, granules, pastes, pills or capsules for the ease of ingestion.
  • compositions of the present disclosure and other ingredients can be added to food products, foodstuffs, or functional foods described herein, for example, fillers, emulsifiers, preservatives, etc. for the processing or manufacture of the same.
  • flavors, coloring agents, spices, nuts and the like may be incorporated into the nutraceutical composition.
  • Flavorings can be in the form of flavored extracts, volatile oils, chocolate flavorings, peanut butter flavoring, cookie crumbs, crisp rice, vanilla or any commercially available flavoring.
  • Emulsifiers can also be added for stability of the nutraceutical compositions.
  • suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soy), and/or mono- and di-glycerides.
  • Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product.
  • Preservatives can also be added to the nutritional supplement to extend product shelf life.
  • preservatives such as potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate or calcium disodium EDTA are used.
  • the nutraceutical composition can contain natural or artificial (preferably low calorie) sweeteners, e.g., saccharides, cyclamates, aspartamine, aspartame, acesulfame K, and/or sorbitol.
  • natural or artificial sweeteners e.g., saccharides, cyclamates, aspartamine, aspartame, acesulfame K, and/or sorbitol.
  • artificial sweeteners can be desirable if the nutraceutical composition is intended to be consumed by an overweight or obese individual, or an individual with type II diabetes who is prone to hyperglycemia.
  • a multi-vitamin and mineral supplement can be added to the nutraceutical compositions of the present invention to obtain an adequate amount of an essential nutrient, which is missing in some diets.
  • the multi-vitamin and mineral supplement can also be useful for disease prevention and protection against nutritional losses and deficiencies due to lifestyle patterns.
  • modulation of commensal bacterial populations can provide additional benefit against the development and progression of inflammatory diseases, autoimmune diseases, and cancer. Accordingly, particular embodiments of the invention provide for the nutritional source of the nutraceutical to modulate endogenous commensal bacterial populations.
  • Such modulation can be achieved by modification of gut pH, consumption of beneficial bacteria (e.g., as in yogurt), by providing nutritional sources (e.g., prebiotics) that select for particular populations of bacteria, or by providing antibacterial compounds.
  • Such modulation can mean an increase or decrease in the gut microbiota populations or ratios.
  • the absolute or relative numbers of desirable gut microorganisms is increased and/or the absolute or relative numbers of undesirable gut microorganisms is decreased.
  • nutritional sources exhibiting antibacterial activity that can be used to modulate gut microbiota populations.
  • garlic has been shown to produce the compound allicin (allyl 2-propenethiosulfinate), which exhibits antibacterial activity toward E.
  • the food products, foodstuffs, functional foods, or dietary supplements may be combined with antibiotics to control the gut microbiota populations.
  • the nutraceutical composition of the present invention can be provided in a commercial package, alone, or with additional components, e.g., other food products, food stuffs, functional foods, dietary supplement.
  • the commercial package has instructions for consumption of the instant nutraceutical, including preparation and frequency of consumption, and use in the prevention or treatment of inflammatory diseases, autoimmune diseases and cancer.
  • the commercial package further includes a natural product (e.g., the food, extracts, antibiotics, and oils) that modulates endogenous commensal bacterial populations.
  • a package containing both a nutraceutical of the present disclosure in combination with said natural product can contain instructions for consuming the natural product, e.g., in advance (e.g., 2, 4, 6 or 8 or more hours) of consuming the nutraceutical in order to enhance the activity of the nutraceutical composition.
  • the subject suitable for the compositions and methods disclosed herein is a mammal e.g., mouse, rat, primate, non-human mammal, domestic animal, such as a dog, cat, cow, horse, and the like), and is preferably a human.
  • the subject is healthy (e.g., as a prevention of a bone loss condition).
  • the subject is afflicted with a disease (e.g., a cancer and/or bone loss condition). It is important to point out that the agents and methods of the present disclosure are effective and beneficial to any subject (healthy or diseased) who is in need of prevention and/or treatment of a bone loss condition, irrespective of whether the subject is afflicted with a cancer.
  • the subject is an animal model of a cancer and/or bone loss condition.
  • the animal model can be an orthotopic xenograft animal model of human oral squamous carcinoma, or comprising cancer stem cells (CSCs)/undifferentiated tumors.
  • CSCs cancer stem cells
  • the subject is an animal model of an inflammatory disease or an autoimmune disease.
  • the subject has not undergone treatment, such as bone loss therapy or a cancer therapy (chemotherapy, radiation therapy, targeted therapy, and/or anti- immune therapy - such as NK cell-related immunotherapies).
  • the subject has undergone treatment, such as bone loss therapy or a cancer therapy (chemotherapy, radiation therapy, targeted therapy, and/or anti-immune therapy - such as NK cell-related immunotherapies).
  • the subject has had surgery to remove cancerous or pre- cancerous tissue.
  • the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient.
  • the subject is in need of an NK cell activation.
  • the subject is in need of an increased level of certain cytokines (e.g., cytokines or chemokines described herein).
  • the subject would benefit from the compositions or methods of the present disclosure, irrespective of whether they would need e.g., an NK cell activation.
  • the methods of the present invention can be used to treat and/or determine the responsiveness to a composition comprising at least one of probiotic bacteria, alone or in combination with other NK immunotherapies, of many different cancers in subjects such as those described herein.
  • ALS Amyotrophic Lateral Sclerosis
  • Amyotrophic lateral sclerosis also known as motor neurone disease, is considered the most common form of a motoneuron disease with an onset in adult age of, in average, about 50-60 years and an incidence of 1 :50,000 per year.
  • ALS results in the death of neurons controlling voluntary muscles. ALS is characterized by stiff muscles, muscle twitching, and gradually worsening weakness due to muscles decreasing in size. It may begin with weakness in the arms or legs, or with difficulty speaking or swallowing. About half of the people affected develop at least mild difficulties with thinking and behavior and most people experience pain. Most eventually lose the ability to walk, use their hands, speak, swallow, and breathe. ALS is a progressive disease with a fatal outcome due to gradual paralysis of all voluntary muscles throughout the body, whereby the breathing and swallowing muscles become affected early on already.
  • the cause is not known in 90% to 95% of cases, but is believed to involve both genetic and environmental factors. The remaining 5-10% of cases are inherited from a person's parents. The most common familial forms of ALS in adults are caused by mutations of the superoxide dismutase gene, or SOD1, located on chromosome 21. The underlying mechanism involves damage to both upper and lower motor neurons.
  • ALS Amyotrophic Lateral Sclerosis
  • riluzole prolongs ALS survival; it increases survival rates at 12 months by 10% and prolongs survival by 6 months.
  • Edaravone is effective in treating ALS.
  • Masitinib is a tyrosine kinase inhibitor used to treat cancer in dogs. It was proven that mastinib inhibited glial cell activation in the appropriate rat model and increased survival.
  • Retigabine is an approved drug for epilepsy, and acts by binding to the voltagegated potassium channels and increasing the M-current, thus leading to membrane hyperpolarization. Retigabine is able to prolong motor neuron survival and decrease excitability, which is advantageous in the treatment of ALS, since it is believed that, in this disease, neurons are hyper-excitable, firing more than normal and ultimately leading to cell death. This drug is still under clinical trial for the treatment of ALS.
  • Tamoxifen is an antioestrogen drug, approved for the chemotherapy and chemoprevention of breast cancer.
  • Its neuroprotective properties appear to be related to inhibition of protein kinase C, which is overexpressed in the spinal cord of ALS patients.
  • tamoxifen was found to be able to modulate a proteinopathy present in ALS, through its capacity to be an autophagy modulator.
  • the methods and compositions provided herein can be used for preventing or treating a cancer-associated bone loss.
  • eradication/treatment of cancer may halt further bone loss resulting from cancer
  • the eradication/treatment of cancer alone does not restore the bone density already lost due to cancer.
  • the compositions and methods provided herein are necessary to restore the lost bone density. Accordingly, the compositions and methods provided herein carry out multiple functions in a subject afflicted with cancer: (1) prevent/treat cancer, which aids in preventing future bone loss due to cancer, (2) prevent future bone loss (i.e., cancer-independent loss) by regulating the bone homeostasis, and (3) actively restore the lost bone density.
  • Cancer, tumor, or hyperproliferative disease refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell.
  • Cancers include, but are not limited to, B cell cancer, (e.g., multiple myeloma, Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphomas, Burkitt lymphoma, Waldenstrom's macroglobulinemia, Hairy cell leukemia, Primary central nervous system (CNS) lymphoma, Primary intraocular lymphoma, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis), T cell cancer (e.g., T-lymphoblastic lymphoma/leukemia, non-Hodgkin lymphomas, Peripheral T-cell lymphomas, Cutaneous T-cell lymphomas (e
  • cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
  • the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma.
  • the epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.
  • the therapeutic agents of the present invention can be used alone or can be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, and/or radiotherapy.
  • chemotherapeutic agents e.g., hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, and/or radiotherapy.
  • the preceding treatment methods can be administered in conjunction with other forms of conventional therapy (e.g, standard-of-care treatments for cancer well-known to the skilled artisan), either consecutively with, pre- or post-conventional therapy.
  • agents of the present invention can be administered with a therapeutically effective dose of chemotherapeutic agent.
  • agents of the present invention are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent.
  • the Physicians’ Desk Reference discloses dosages of chemotherapeutic agents that have been used in the treatment of various cancers.
  • the dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art, and can be determined by the physician.
  • Immunotherapy is a targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells.
  • an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site.
  • the immunotherapy can involve passive immunity for shortterm protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen).
  • a cancer antigen or disease antigen e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen.
  • anti-VEGF is known to be effective in treating renal cell carcinoma.
  • Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.
  • antisense polynucleotides can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.
  • Immunotherapy also encompasses immune checkpoint modulators.
  • Immune checkpoints are a group of molecules on the cell surface of CD4+ and/or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response.
  • Immune checkpoint proteins are well-known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, for example, WO 2012/177624).
  • Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer.
  • the composition of the present disclosure is administered in combination with one or more inhibitors of immune checkpoints, such as PD1, PD-L1, and/or CD47 inhibitors.
  • Adoptive cell-based immunotherapies can be combined with the therapies of the present invention.
  • Well-known adoptive cell-based immunotherapeutic modalities including, without limitation, irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines, and/or antigen presenting cells.
  • Such cellbased immunotherapies can be further modified to express one or more gene products to further modulate immune responses, such as expressing cytokines like GM-CSF, and/or to express tumor-associated antigen (TAA) antigens, such as Mage-1, gp-100, and the like.
  • TAA tumor-associated antigen
  • immunotherapy comprises non-cell-based immunotherapies.
  • compositions comprising antigens with or without vaccineenhancing adjuvants are used.
  • Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigen comprising fusion proteins, and the like.
  • immunomodulatory cytokines such as interferons, G- CSF, imiquimod, TNF alpha, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used.
  • immunomodulatory interleukins such as IL-2, IL-6, IL-7, IL- 12, IL- 17, IL-23, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used.
  • immunomodulatory chemokines such as CCL3, CCL26, and CXCL7, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used.
  • immunomodulatory molecules targeting immunosuppression such as STAT3 signaling modulators, NFkappaB signaling modulators, and immune checkpoint modulators, are used.
  • the terms “immune checkpoint” and “anti-immune checkpoint therapy” are described above.
  • immunomodulatory drugs such as immunocytostatic drugs, glucocorticoids, cytostatics, immunophilins and modulators thereof (e.g., rapamycin, a calcineurin inhibitor, tacrolimus, ciclosporin (cyclosporin), pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, a non-glucocorticoid steroid, a pyrimidine synthesis inhibitor, leflunomide, teriflunomide, a
  • immunomodulatory antibodies or protein are used.
  • Nutritional supplements that enhance immune responses such as vitamin A, vitamin E, vitamin C, and the like, are well-known in the art (see, for example, U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publ. No. WO 2004/004483) can be used in the methods described herein.
  • agents and therapies other than immunotherapy or in combination thereof can be used with in combination with an agent that increases the activity/amount of NK cells and/or IFN-y to treat a condition that would benefit therefrom.
  • agents and therapies other than immunotherapy or in combination thereof can be used with in combination with an agent that increases the activity/amount of NK cells and/or IFN-y to treat a condition that would benefit therefrom.
  • chemotherapy, radiation, epigenetic modifiers e.g., histone deacetylase (HD AC) modifiers, methylation modifiers, phosphorylation modifiers, and the like
  • targeted therapy and the like are well-known in the art.
  • Chemotherapy includes the administration of a chemotherapeutic agent.
  • a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof.
  • Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphi dicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin.
  • alkylating agents cisplatin, treosulfan, and trofosfamide
  • compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used.
  • FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF.
  • CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.
  • PARP e.g., PARP-1 and/or PARP-2
  • 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. Pat. Re.
  • the mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity.
  • PARP catalyzes the conversion of .beta. -nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP -ribose (PAR).
  • NAD+ nicotinamide adenine dinucleotide
  • PARP poly-ADP -ribose
  • Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J. et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q.
  • PARP1 Poly(ADP-ribose) polymerase 1
  • SSBs DNA single- strand breaks
  • DSBs DNA double-strand breaks
  • chemotherapeutic agents are illustrative, and are not intended to be limiting.
  • radiation therapy is used.
  • the radiation used in radiation therapy can be ionizing radiation.
  • Radiation therapy can also be gamma rays, X-rays, or proton beams.
  • Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy.
  • the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source.
  • the radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass.
  • photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfm (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and 2B A-2-DMHA.
  • hormone therapy is used.
  • Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).
  • hormonal antagonists e.g., flutamide, bicalu
  • photodynamic therapy also called PDT, photoradiation therapy, phototherapy, or photochemotherapy
  • PDT photoradiation therapy
  • phototherapy phototherapy
  • photochemotherapy is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light.
  • laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors.
  • the immunotherapy and/or cancer therapy may be administered before, after, or concurrently with the agents/compositions described herein.
  • the duration and/or dose of treatment with the compositions may vary according to the particular composition, or the particular combinatory therapy.
  • An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan.
  • the invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by the methods of the invention is a factor in determining optimal treatment doses and schedules.
  • a method of preventing and/or treating a bone loss condition in a subject comprising administering to the subject an agent that increases (a) the amount and/or activity of the NK cells; and/or (b) the amount of IFN-y.
  • a method of preventing and/or treating a bone loss condition and a cancer in a subject comprising administering to the subject an agent that increases (a) the amount and/or activity of the NK cells; and/or (b) the amount of IFN-y.
  • a method of inducing bone formation and/or preventing bone loss in a subject comprising administering to the subject an agent that increases (a) the amount and/or activity of the NK cells; and/or (b) the amount of IFN-y.
  • the agent comprises NK cells, super-charged NK cells, at least one probiotic bacteria, osteoclasts, IFN-y, IL-2, anti-CD16 antibody, or any combination thereof.
  • NK cells are generated by co-culturing the NK cells with osteoclasts, optionally in the presence of at least one probiotic bacteria.
  • NK cells are further activated by IL-2, anti-CD16 antibody, at least one probiotic bacteria, or any combination thereof.
  • the agent comprises (a) at least one probiotic bacteria and NK cells; or (b) at least one probiotic bacteria and super-charged NK cells.
  • the agent comprises (a) at least one probiotic bacteria, NK cells, and osteoclasts; or (b) at least one probiotic bacteria, super-charged NK cells, and osteoclasts.
  • the at least one probiotic bacteria comprises the bacteria of the genus Streptococcus (e.g., S. thermophiles'), Bifidobacterium (e.g., B. longum, B. breve, B. infantis), and/ or Lactobacillus (e.g., L. acidophilus, L. helveticus, L. bulgaricus, L. rhamnosus, L. plantarum, and L. easel).
  • Streptococcus e.g., S. thermophiles'
  • Bifidobacterium e.g., B. longum, B. breve, B. infantis
  • Lactobacillus e.g., L. acidophilus, L. helveticus, L. bulgaricus, L. rhamnosus, L. plantarum, and L. easel.
  • the at least one probiotic bacteria comprises one or more bacteria selected from Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei, optionally further comprising Lactobacillus bulgaricus.
  • the at least one probiotic bacteria comprises AJ2 or a composition comprising Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei, optionally further comprising Lactobacillus bulgaricus.
  • the at least one probiotic bacteria comprises AJ4 or a composition comprising Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei.
  • the at least one probiotic bacteria comprises a composition comprising about 30% (or about 20% to about 40%) Streptococcus thermophiles, about 20% (or about 10% to about 30%) Lactobacillus acidophilus, about 40% (or about 30% to about 50%) Lactobacillus plantarum, and about 10% (or about 1% to about 20%) Lactobacillus paracasei.
  • the bone loss therapy comprises calcium supplements, estrogen, calcitonin, estradiol, diphosphonates, vitamin D3, parathyroid hormone, or any combination thereof.
  • bone loss condition is selected from osteopenia, osteoporosis, osteolysis, and cancer-associated bone loss.
  • the immunotherapy inhibits an immune checkpoint
  • the immune checkpoint is selected from CTLA-4, PD-1, VISTA, B7- H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.
  • the immunotherapy is selected from: atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, BGB-A317, STI-Al l 10, TSR-042, RG-7446, BMS- 936559, MEDI-4736, MSB-0010718C, AUR-012, and STI-A1010.
  • any one of 2 and 4-28 wherein the cancer is selected from multiple myeloma, prostate cancer, stomach cancer, bladder cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bone cancer, brain cancer, leukemia, head and neck cancer, oral cancer, pancreatic cancer, lung cancer, colon cancer, melanoma, breast cancer, ovarian cancer, and glioblastoma.
  • Example 1 Materials and Methods for Examples 2 and 3
  • human CD34+ progenitor cells When human CD34+ progenitor cells are provided with an appropriate thymic microenvironment, they can mature into naive single-positive human T cells. Thus, it was first determined whether CD34+ cells introduced by bone marrow transplantation could systemically repopulate the mouse and sustain thymopoiesis in the implanted human thymic tissue.
  • the NOD/SCID mice were used because they support significantly higher percentages of reconstitution following transplantation with human CD34+ cells. In essence, NOD/SCID mice were first implanted with human fetal liver and thymic tissues, and the mice were allowed to recover from surgery.
  • mice were preconditioned with a sublethal dose of gamma radiation and were transplanted with autologous CD34+cells obtained from fetal liver.
  • Human T cells, B cells, monocytes and macrophages, and DCs were present in all tissues.
  • the differentiation state of the human T cells in the peripheral blood of BLT mice was also determined. BLT mice kept under sterile conditions had a higher percentage of naive T cells (CD45RA+CD27+) than did healthy human controls.
  • BLT mice Liver and lung from BLT mice contained substantial numbers of human T cells, B cells, monocytes and macrophages, and both CDl lc+ and CD123+ DCs. Multilineage reconstitution in the gut of BLT mice analyzed by immunohistology was also observed. These data show that BLT mice can generate an extensive state of sustained systemic multilineage reconstitution with human hematopoietic cells (FIG. 4).
  • Recombinant human IL-2 was obtained from NIH-BRB.
  • Human TNF-a and IFN-y was obtained from Biolegend (San Diego, CA, USA).
  • Antibody to CD 16 was purchased from Biolegend (San Diego, CA, USA).
  • Fluorochrome-conjugated human and mouse antibodies for flow cytometry were obtained from Biolegend (San Diego, CA, USA).
  • Monoclonal antibodies to TNF-a and IFN-y were prepared in our laboratory and used at 1 : 100 dilutions to block rhTNF-a and rhIFN-y functions.
  • the human NK cell and monocyte purification kits were obtained from Stem Cell Technologies (Vancouver, BC, Canada).
  • RPMI 1640 Human immune cells were cultured in RPMI 1640, supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, CA).
  • FBS fetal bovine serum
  • OSCSCs Oral squamous carcinoma stem cells (OSCSCs) were isolated from oral cancer patient tongue tumors at UCLA School of Medicine and cultured in RPMI 1640, supplemented 10% FBS (Gemini Bio-Products, CA), 1.4% antibiotic antimycotic, 1% sodium pyruvate, 1.4% MEM non-essential amino acids, 1% L-glutamine, 0.2% gentimicin (Gemini Bio-products, CA) and 0.15% sodium bicarbonate (Fisher Scientific, PA).
  • Human monocytes/osteoclasts were cultured in alpha-MEM medium (Life Technologies, CA), supplemented with 10% FBS, and penicillin-streptomycin (Gemini Bio-Products, CA).
  • Human M-CSF Biolegend, CA
  • soluble RANKL PeproTech, NJ
  • NK cells and monocytes were negatively selected from PBMCs using isolation kits from Stem Cell Technologies (Vancouver, BC, Canada). Greater than 96% purity was obtained both for purified NK cells and monocytes based on flow cytometric analysis.
  • Pancreatic tumors were harvested from hu-BLT mice and cut into 1 mm 3 pieces and placed into a digestion buffer containing 1 mg/mL collagenase IV, 10 U/mL DNAse I, and 1% bovine serum albumin (BSA) in DMEM media for 20 min at 37 °C. The samples were then filtered through a 40 mm cell strainer and centrifuged at 1500 rpm for 10 min at 4 °C. To obtain singlecell suspensions from BM, femurs were flushed using media, and filtered through a 40 pm cell strainer. Spleens were removed and single cell suspensions were prepared and filtered through a 40 pm cell strainer and centrifuged at 1500 rpm for 5 min at 4 °C. The pellets were re-suspended in ACK buffer to remove the red blood cells. Peripheral blood mononuclear cells (PBMCs) were isolated using ficoll-hypaque centrifugation.
  • PBMCs Peripheral blood
  • NK cells and T cells from hu-BLT splenocytes were obtained as described previously by using the human CD56+ and CD3+ selection kits respectively (Stem Cells Technologies, Vancouver, BC, Canada). Monocytes from hu-BLT bone marrow were isolated using human CD14 isolation kit (eBioscience, San Diego, CA, U.S.A.). Generation of Osteoclasts and Expansion of Human and hu-BLT NK Cells — Generation of Super-charged NK cells
  • Monocytes were purified form human peripheral blood or hu-BLT BM and cultured using alpha-MEM medium containing M-CSF (25 ng/mL) and RANKL (25 ng/mL) for 21 days (medium was refreshed every 3 days).
  • NK cells were activated with rh-IL-2 (1000 U/mL) and anti-CD16 mAh (3pg/mL) for 18-20 h before they were cultured with osteoclasts and sonicated-AJ2 to generate super-charged NK cells.
  • the medium was refreshed every 3 days with RMPI containing rh-IL-2 (1000 U/mL).
  • Human ELISA kits for IFN-y and IL-6 were purchased from Biolegend (San Diego, CA, USA). The assays were conducted as recommended by the manufacturer. For certain experiments multiplex arrays were used to determine the levels of secreted cytokines and chemokines. Analysis was performed using MAGPIX (Millipore, Danvers, MA, USA) and data was analyzed using xPONENT 4.2 (Luminex, Austin, TX, USA).
  • Staining was performed by staining the cells with antibodies as described previously, briefly, antibodies were added to 1 x 104 cells in 50 pL of cold-PBS+ 1% BSA and cells were incubated on ice for 30 min. Thereafter cells were washed in cold PBS+ 1% BSA and flow cytometric analysis was performed using Beckman Coulter Epics XL cytometer (Brea, CA, USA) and results were analyzed in FlowJo vX software (Flowjo, Ashland, OR, USA).
  • AJ2 is a combination of 7 different strains of gram positive probiotic bacteria which has been cloned to withstand high temperature and also low pH (Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei) were used to induce NK activation which in turn increases IFN-y and promotes differentiation of stem cells.
  • AJ2 was weighed and resuspended in RPMI Medium 1640 containing 10% FBS at a concentration of 10 mg per 1 mL. The bacteria were thoroughly vortexed, then sonicated on ice for 15 seconds, at 6 to 8 amplitude. Sonicated samples were then incubated for 30 seconds on ice. After every five pulses, a sample was taken to observe under the microscope until at least 80 percent of cell walls were lysed. It was determined that approximately 20 rounds of sonication/incubation on ice, were conducted to achieve complete sonication. Finally, the sonicated samples (sAJ2) were aliquoted and stored in a - 80 degrees Celsius freezer. Sonication of bacteria is not required or necessary to render its activities presented herein. Analysis of Human Pancreatic Cancer Cell Growth in Humanized-BLT Mice
  • mice Humanized-BLT (hu-BLT; human bone marrow/liver/thymus) mice were generated as previously described. In vivo growth of pancreatic tumors was performed by orthotopic tumor implantation in the pancreas hu-BLT mice. To establish orthotopic tumors, mice were anesthetized using isoflurane, and tumors in a mixture with Matrigel (10 pL) (Corning, NY, USA) were injected in the pancreas using insulin syringe. Mice received 1.5 x 10 6 super-charged NK cells via tail vein injection 7 to 10 days after the tumor implantation. They were also fed AJ2 (5 billion/dose) orally.
  • AJ2 5 billion/dose
  • the first dose of AJ2 was given one or two weeks before tumor implantation, and feeding was continued throughout the experiment at an interval of every 48 h. Mice were euthanized when signs of morbidity were evident. Lumbar vertebrae, pancreas, pancreatic tumors, bone marrow, spleen, and peripheral blood were harvested, and single cell suspensions were prepared from each tissue as described previously and below.
  • micro computed tomography micro computed tomography
  • Samples were harvested, formalin-fixed and imaged using high-resolution microCT (Skyscan 1275, Skyscan, Belgium) at an image resolution of 10 u pixels and analyzed using Data Viewer, Recon, CTAn and CTVol software provided by the manufacturer.
  • mice will be euthanized in a CO2 chamber with the appropriate CO2 concentrations and exposure times.
  • Lumbar vertebrae (L3) were dissected and fixed in 70% ethanol.
  • vertebras were scanned with Skyscan 1275 (Bruker microCT N. V., Belgium), equipped with a 5-pm focal spot micro-focus x-ray tube at the resolution of 10pm (60 kVp, 166 mA, and 1mm Al Filter).
  • Specimens were aligned with the vertical axis of the scanner, and low-density foam (a non-attenuating material) was used to stabilize the specimens firmly into a 0.25-diameter-tube.
  • Phantom calibration was performed to relate the micro-CT values to a mineral-equivalent value (g/cm3) of calcium hydroxyapatite.
  • scanned images were reconstructed with NRecon (Bruker microCT N.V., Belgium) for attenuation correction, ring artifact reduction, and beam hardening. After data acquisition, images were aligned in 3D view for vertical orientation with Data Viewer software for accuracy.
  • NRecon Bruker microCT N.V., Belgium
  • Segmentation of the images will be completed manually by comparing the binarized image with the unsegmented image, and a single global threshold of 60 will be applied. An irregular ROI selection will be manually drawn parallel and close to the endocortical surface.
  • Length of the ROI was adjusted in proportion to the total vertebral height.
  • transverse micro-CT slices were acquired for the entire vertebral body, and trabecular bone was evaluated within the region of 0.5 mm away from the growth plate.
  • each ROI were drawn manually in a sequential manner for each trans- axial micro-CT slice.
  • Morphometric parameters were computed from the binarized images using a direct threedimensional approach that does not rely on any prior assumptions about the underlying structure.
  • trabecular morphology assessment of bone volume fraction (BV/TV %), trabecular thickness (Tb. Th, mm), trabecular number (Tb. N, mm), and trabecular separation (Tb.Sp, mm) were used.
  • Static histomorphometry was carried out on hu-BLT mice.
  • Third lumbar vertebras (L3) were dissected, fixed in 70% ethanol, dehydrated and embedded undecalcified in methyl methacrylate. Frontal sections, 5 pm thick and stained with 0.1% toluidine blue, pH 6.4.
  • Static parameters of bone formation (OB) and resorption (OC) were measured in a defined area between 0.25mm from both growth plates and endochondral bone surfaces.
  • Additional histochemical stain tartrate-resistant acid phosphatase (TRAP) was performed to identify osteoclasts.
  • 51 Cr was purchased from Perkin Elmer (Santa Clara, CA). Standard 51 Cr release cytotoxicity assays were used to determine NK cell cytotoxic function in the experimental cultures and the sensitivity of target cells to NK cell mediated lysis.
  • the effector cells (1 x 10 5 NK cells/well) were aliquoted into 96-well round-bottom microwell plates (Fisher Scientific, Pittsburgh, PA) and titrated at four to six serial dilutions.
  • Patient-derived OSCSCs were used as a specific and sensitive NK target to assess NK cell-mediated cytotoxicity.
  • the target cells (5 x 10 5 OSCSCs) were labeled with 50 pCi 51 Cr (Perkin Elmer, Santa Clara, CA) and chromated for 1 hour.
  • 51 Cr-labeled target cells were aliquoted into the 96-well round bottom microwell plates containing effector cells at a concentration of 1 x 10 4 cells/well at a top effectortarget (E:T) ratio of 5: 1. Plates were centrifuged and incubated for a period of 4 hours. After a 4-hour incubation period, the supernatants were harvested from each sample and counted for released radioactivity using the gamma counter. Total (containing 51 Cr-labeled target cells) and spontaneous (supernatants of target cells alone) release values were measured and used to calculate the percentage specific cytotoxicity. The percentage specific cytotoxicity was calculated using the following formula:
  • LU 30/10 6 is calculated by using the inverse of the number of effector cells needed to lyse 30% of target cells xlOO.
  • Example 2 The impact of MP2 tumors, super-charged NK cells, and AJ2 on the secretion of IFN Gamma and its association with bone loss.
  • NK cells have a profound effect on NK cells' function and that stemlike / poorly differentiated tumors were preferentially targeted by the NK cells. Also, we determined the role of super-charged NK cells in immune mobilization, lysis, and differentiation of stemlike/ undifferentiated tumors implanted in the pancreas of humanized-BLT (hu-BLT) mice fed with or without AJ2 probiotics.
  • hu-BLT humanized-BLT
  • NK differentiated MP2 MoaPaCa-2
  • NK differentiated MP2 MiaPaCa-2
  • the differentiation of stemlike tumors by the NK cells was prevented by the addition of antibodies to IFN-y and TNF-a, tumors grew rapidly and metastasized, and they remained resistant to chemotherapeutic drugs.
  • NK cells prevent the growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stem-like/undifferentiated-tumors.
  • Hu-BLT NK cells cultured with osteoclasts expanded greatly and increased secretion of IFN-y
  • Hu-BLT NK cells purified from the spleen of mice responded to the activation signals provided by the IL-2 and anti-CD16 mAb treatment and expanded greatly and demonstrated increased secretion of IFN-y when cultured with both autologous and allogeneic osteoclasts in the presence of sAJ2 treatment (FIG. 6A and FIG. 6B), indicating close similarity between hu-BLT and human donor derived NK cell expansion and function by osteoclasts.
  • a J2 and super-charged NK cells increased the levels of IFN-y in the serum of the hu- BLT mice
  • NK cells Injection of NK cells into tumor-bearing mice restored IFN-y secretion in pancreatic cell cultures and the levels exceeded those seen in the control mice with no tumors.
  • Blocking MP2 differentiation with anti-IFN-y and anti-TNF-a antibodies resulted in the inhibition of tumor differentiation and generation of tumors with higher tumor weights. (FIG. 10).
  • PBMCs from tumor-bearing mice which were similar to PBMCs and NK cells from pancreatic cancer patients, had significantly lower NK cell-mediated cytotoxicity and exhibited decreased IFN-y secretion, when compared to those from healthy mice or humans, respectively.
  • FIG. 11A-FIG. 11C Tumor-bearing mice had much lower cytotoxicity and/or secretion of IFN-y in cells obtained from all tissue compartments, in comparison to those obtained from control mice without tumor, or tumor-bearing mice injected with NK cells, or those implanted with NK- differentiated tumors (FIG. 12).
  • NK cells prevent pancreatic tumors' growth through lysis and differentiation, thereby curtailing the growth and metastatic potential of stemlike/undifferentiated-tumors. Also, we demonstrated that NK cells induced differentiation of MP2 tumors through the functions of IFN-y and TNF-a.
  • NK cells in the presence and absence of feeding AJ2 increased IFN-y secretion in different compartments of hu-BLT mice; like the serum, cell cultures from pancreatic tumors, NK cells purified from hu-BLT splenocytes, PBMCs, splenocytes cell cultures, and bone marrow.
  • MP2 tumor-bearing hu-BLT mice showed decreased IFN-y in the same compartments.
  • Example 3 Changes in the bone structure of MP2 tumor-bearing mice injected with cultures of NK cells and fed with and without AJ2.
  • mice fed with AJ2 presented increased bone formation when compared to the CTRL group.
  • the AJ2 group presented increased bone formation with increased bone volume and trabecular number when compared to the CTRL group. (FIG. 13A-FIG. 13B).
  • MP2 tumor-bearing mice injected with cultures of NK cells and fed with AJ2 presented increased bone formation with statistically significant higher trabecular bone volume when compared to MP2 tumor and MP2+AJ2 group, respectively.
  • MP2 tumor-bearing mice injected with NK cells and fed with AJ2 group showed a statistically significant increase in bone volume fraction (BV/TV), augmented trabecular thickness (Tb.Th), higher trabecular number (Tb.n), and decreased trabecular spacing (Tb.Sp) when compared to the MP2 tumor group.
  • BV/TV bone volume fraction
  • Tb.Th augmented trabecular thickness
  • Tb.n higher trabecular number
  • Tb.Sp decreased trabecular spacing
  • FIG. 14A-FIG. 14B Stem-like/undifferentiated tumors implanted in hu-BLT mice injected with cultures of NK cells and fed with or without A J2 presented similar bone formation when compared to the control group.
  • FIG. 15A-FIG. 15B Stem-like/undifferentiated tumors implanted in hu- BLT mice injected with NK cells' cultures and fed with or without AJ2 presented similar bone formation compared to the control group.
  • FIG. 15A-FIG. 15B the increase in bone formation was more evident in the lumber microCT than bone parameters (FIG. 15 A), and FIG. 15B clearly showed that there was more bone formation for treatment group than control. It is also evident that hu-BLT mice with MP2 without any treatment showed more significant bone loss.
  • Histological, immunohistochemical analysis, and static indices assessments at the third lumbar vertebra exhibited an increase of trabecular bone formation in the AJ2 treated group whencompared with the Control and MP2 groups.
  • histological analysis and static indices assessment at the third lumbar vertebra were performed. Consistent with the micro-CT findings, histological analysis of the AJ2 treatment group exhibited an increase in bone volume fraction (B V/TV), augmented trabecular thickness (Tb.Th), higher trabecular number (Tb.n), and decreased trabecular spacing (Tb.Sp) when compared to the Control and MP2 tumor groups. The histomorphometric values for the MP2+NK +AJ2 sample could not be confirmed due to staining failure (FIG. 17 and Table 2).
  • MP2 tumor-bearing mice showed reduced bone formation and decreased IFN-y secretion in the same compartments. (FIG. 16 and Table 1). Accordingly, MP2 tumor-bearing mice group presented statistically significant reduced trabecular bone volume compared to the treatment groups.
  • IFN-y can either inhibit osteoclast formation or enhance osteoclastogenesis according to the conditions to which the osteoclast precursors are exposed. While IFN-y has been shown to be involved in bone cell differentiation and function with complex effects on skeletal helath, the role of IFN-y in pathological bone diseases has been controversial.
  • NK cells and AJ2 This is a novel report of the role of NK cells and AJ2 in the treatment and prevention of tumor growth and their role in increasing the levels of IFN-y in the hu-BLT mice suppressing tumorinduced bone loss.
  • NK cells prevented the growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stemlike/undifferentiated-tumors through the functions of IFN-y and TNF-a.
  • Increased IFN-y secretion in the presence of decreased IL-6 was seen in tumors resected and cultured from NK-injected, and AJ2 fed mice.
  • IFN-y is a multifunctional cytokine produced mainly by NK cells and activated T cells that play a critical role in host immune responses against pathogens and cancer.
  • Hu- BLT NK cells cultured with osteoclasts expanded greatly and increased secretion of IFN-y.
  • Hu-BLT NK cells were purified from the spleen of mice, responded to the IL-2 and antiCD 16 mAh treatment's activation signals, expanded greatly and demonstrated increased secretion IFN-y when cultured with both autologous and allogeneic osteoclasts in the presence of sAJ2 treatment.
  • AJ2 and super-charged NK cells increased the levels of IFN-y in the serum of the hu-BLT mice.
  • an increase in IFN-y secretion from the pancreatic cell cultures could be observed in mice implanted with MP2 tumors, injected with NK and fed with AJ2 when compared to mice implanted with MP2 tumors and control mice.
  • IFN-y seems to be a critical regulator of bone resorption.
  • IFN-y decreased tumor growth and prevented tumor-associated bone loss by inhibiting tumor cell growth and osteolysis.
  • RPMI 1640 (Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, CA) was used to culture peripheral blood mononuclear cells (PBMCs), NK cells, and CD8+ T cells.
  • FBS fetal bovine serum
  • Recombinant IL-2 was obtained from NIH- BRB.
  • Anti-CD16 mAbs, and anti-CD3/CD28 mAbs were obtained from Biolegend (San Diego, CA).
  • Oral squamous carcinoma stem cells (OSCSCs) were isolated from patients with tongue tumors at UCLA.
  • Human ELISA kits for IFN-y were purchased from Biolegend (San Diego, C A).
  • Chroimum-51 radionucleotide was purchased from PerkinElmer, CA, USA.
  • PBMCs were isolated from peripheral blood as described before. PBMCs were used to isolate NK cells, and CD8+T cells using the EasySep® Human NK cell and EasySep® Human CD8+ T cells, respectively, purchased from stem cell technologies (Vancouver, BC, Canada). Isolated NK cells and CD8+ T cells were stained with anti-CD16, anti-CD3/CD8 antibodies, respectively, to measure the cell purity using flow cytometric analysis.
  • Enzyme-linked immunosorbent assays ELISAs
  • Enzyme-linked immunospot ELISpot
  • multiplex cytokine arrays ELISAs
  • ELISAs Enzyme-linked immunosorbent assays
  • ELISpot Enzyme-linked immunospot
  • Single ELISAs were performed as previously described. To analyze and obtain the cytokine and chemokine concentrations, a standard curve was generated by either two- or three-fold dilution of recombinant cytokines provided by the manufacturer. The ELISpot was conducted according to manufacturer’s instructions. The number of IFN-y secreting cells was determined by using human IFN-y single-color enzymatic ELISpot assay, and analyzed by the ImmunoSpot® S6 UNIVERSAL analyzer and ImmunoSpot® software (all CTL Europe GmbH, Bohn, Germany). For multiple cytokine arrays, the levels of cytokines and chemokines were also determined by multiplex cytokine arrays as recommended by the manufacturer. Analysis was performed using a luminex instrument (MAGPIX, Millipore, Billerica, MA), and data were analyzed using the proprietary software (xPONENT 4.2, Millipore, Billerica, MA).
  • the 51 Cr release assay was performed as described previously. Briefly, different numbers of effector cells were incubated with 51 Cr-labeled target cells. After a 4-hour incubation period, the supernatants were harvested from each sample and the released radioactivity was counted using the gamma counter. The percentage specific cytotoxicity was calculated as follows:
  • Lytic units (LU) 30/10 6 is calculated by using the inverse of the number of effector cells needed to lyse 30% of tumor target cells 400.
  • Gram-positive probiotic bacteria strains for AJ2, AJ3, and AJ4 were weighed and re-suspended in RPMI 1640 medium containing 10% FBS at a concentration of 10 mg/ml. The bacteria were thoroughly vortexed and sonicated on ice for 15 seconds at 6 to 8 amplitudes. Sonicated samples were then incubated for 30 seconds on ice, and the cycle was repeated for five rounds. After every five rounds of sonication, the samples were examined under the microscope until at least 80% of bacterial walls were lysed. It was determined that approximately 20 rounds of sonication/incubation on ice were necessary to achieve complete sonication. Finally, the sonicated probiotic bacteria, sAJ2, sAJ3, and sAJ4 were aliquoted and stored at -80°C until use. Sonication of bacteria is not required or necessary to render its activities presented herein.
  • Example 5 Decreased IFN-Y and increased IL-10 secretions by sAJ3 treated PBMCs in comparison to sAJ4 and sAJ2 treated PBMCs
  • Examples 5-8 demonstrate how the combination of different strains, sAJ2, sAJ3, and sAJ4 differ in their potential to activate PBMCs, NK cells and CD8+ T cells. In addition, we compared the functional activation of NK cells by sAJ3 and sAJ4 in the presence of monocytes.
  • Example 6 Decreased IFN-Y and increased IL-10 secretion by sAJ3 treated NK cells in comparison to sAJ4 and sAJ2 treated NK cells
  • NK cells were sorted from the PBMCs and used in the treatments as described above for PBMCs. Similar to PBMCs, we observed significantly increased levels of IFN-y in NK cells when treated with IL-2 and sAJ4 in comparison to IL-2+sAJ3 or IL-2+sAJ2 as shown in figure (Figs. 22A, 22C, 22D, 22E, 22F, and 22G). The ratio of IFN-y to IL- 10 was the highest in IL-2+sAJ4 and the lowest in IL-2+sAJ3 (Figs. 22H and 221, and Table 8).
  • Example 7 Decreased IFN-Y secretion in sAJ3 treated NK cells cultured with monocytes in comparison to sAJ4 treated cells
  • NK and monocytes co-cultures either with IL-2 alone or IL-2 with sAJ3 or sAJ4, or sAJ3 or sAJ4 alone and determined IFN-y and IL- 10 secretion (Fig. 23).
  • NK cells exhibited higher IFN-y secretion when treated with sAJ4 in comparison to sAJ3 (Fig. 23 A).
  • NK cells exhibited higher IFN-y to IL- 10 ratio when treated with sAJ4 in comparison to sAJ3 (Fig. 23B).
  • CD8+ T cells were sorted from the peripheral blood and treated with IL-2 or IL- 2+anti-CD3/CD28 mAbS or IL-2 with each of sAJ4 and sAJ3.
  • Treatment with IL-2+anti- CD3/CD28 mAbs induced the highest secretion of IFN-y, whereas combination of IL- 2+sAJ4 was considerably lower, but still higher than IL2+sAJ3 in comparison to IL-2+anti- CD3/CD28 mAbs (Fig. 24).
  • AJ3 probiotic was formulated to augment anti-inflammatory cytokine to counter the aggressive nature of pro-inflammatory cytokine such as IFN-g which is primarily secreted by NK cells and T cells.
  • ALS patients have significantly higher functions of NK and CD8+ T cells and they secrete large amounts of IFN-y upon activation. Indeed, the serum levels of IFN-y in patients is higher in comparison to the healthy controls, and even upon treatment with NAC which blocks most of the other pro-inflammatory cytokines secreted from the immune cells, it is not capable of decreasing IFN-y and TNF-a and IL- 17a.
  • IFN-y secretion mainly occurs when cells are activated with IL-2 or other activators such as anti-CD16 mAbs (Figs. 21 and 22).
  • IL-2+anti-CD16 mAbs activated PBMCs and NK cells resulted in a much greater increase in IFN-y/IL-10 ratio, indicating that NK cells when receive activation signals through CD 16 receptor significantly augment the levels of IFN-y in the presence of no or low levels of IL-10 induction (Figs. 21G, 22H and 221).
  • sAJ4 unlike sAJ3, synergistically augment IFN-y in the presence of decreased induction of IL- 10.
  • sAJ3 acts on the opposite, increasing IL-10 in the presence of less IFN-y, a scenario which is desirable in auto-immune diseases such as ALS.
  • T cells when T cells are triggered by the sAJ2, sAJ3 and sAJ4 they secrete relatively much less IFN-y in comparison to NK cells which significant levels of IFN-y is released, and the levels exceed those triggered by IL-2+anti-CD16 mAbs. Whereas, the levels of IL-2 and probiotic in CD8+ T cells is much less than trigger by IL- 2+anti-CD3/CD28 mAbs.
  • AJ3 is effective in increasing IL- 10 and regulating the levels and function of IFN-y
  • AJ4 triggers higher levels of IFN-y without the increase in IL-10 and therefore, this probiotic will be effective in the treatment of cancer and infections where the increased levels and functions of IFN-y is required for differentiation of tumor cells and prevention of the replication of virus, respectively.
  • AJ3 will be effective in alleviating auto-immunity, in particular in ALS since it will greatly regulate the levels and function of IFN-y, decreasing over activation and death of motor neurons (Fig. 25).
  • AJ2 on the other hand can be used in healthy individuals as a maintenance since it produces IFN-y in the presence of intermediate levels of IL-10, to regulate the IFN-y, and therefore, a balance between the secretion of IFN-y and IL-10 is established.
  • Table 2 Histological analysis of the AJ2 sample exhibited increased bone formation when compared to the Control and MP2 samples.
  • Table 3 Decreased BV/TV in OSCSC implanted BLT mice and its restoration by either feeding AJ2 or injection with super-charged NK cells and/or sonicated osteoclasts.
  • Table 4 Decreased bone in MP2 poorly differentiated pancreatic tumor implanted BLT mice and its restoration by either feeding AJ2 and/or injection with super-charged NK cells.
  • Table 5 Decreased BV/TV in MP2 poorly differentiated pancreatic tumor implanted BLT mice and its restoration by either feeding AJ2 and/or injection with super-charged NK cells.
  • Table 6 Increased IFN-y by sAJ4 treated PBMCs in comparison to sAJ3 and sAJ2 treated PBMCs.
  • PBMCs were isolated from healthy individuals’ peripheral blood as described in Materials and Methods section in Example 4.
  • PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD3/28 antibody (25 pl/ml) or with a combination of IL-2 (1000 U/ml) and sAJ2 (PBMC:sAJ2, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (PBMC:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (PBMC:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y secretion using multiplex assay.
  • Tables 7A-7B Increased IFN-y and decreased IL- 10 secretions by sAJ4 treated PBMCs in comparison to sAJ3 and sAJ2 treated PBMCs.
  • PBMCs were isolated from healthy individuals’ peripheral blood as described in Materials and Methods section in Example 4. PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD3/28 antibody (25 pl/ml) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using specific single ELIS As, and ratio of IFN-y to IL- 10 was determined (Table 7A).
  • PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with sAJ3 (PBMC:sAJ3, 1 :20) or with sAJ4 (PBMC:sAJ4, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (PBMC:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (PBMC:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using multiplex assay, and ratio of IFN-y to IL- 10 was determined (Table 7B).
  • Tables 8A-8B Increased IFN-y and decreased IL- 10 secretions by sAJ4 treated NK cells in comparison to sAJ3 and sAJ2 treated NK cells.
  • NK cells were isolated from healthy individuals’ PBMCs as described in Materials and Methods section in Example 4. NK cells were treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using specific single ELIS As, and ratio of IFN-y to IL- 10 was determined (Table 8 A).
  • NK cells were left untreated or treated with IL-2 (1000 U/ml) or with sAJ3 (NK:sAJ3, 1 :20) or with sAJ4 (NK:sAJ4, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using multiplex assay, and ratio of IFN-y to IL- 10 was determined (Table 8B).
  • Example 9 Exemplary formulations of bacterial compositions
  • the AJ3 bacterial composition comprising Bifidobacterium Longum, Bifidobacterium breve, and Bifidobacterium infantis was effective in inducing secretion of a high level of IL- 10 and G-CSF that are useful for downregulating inflammation in patients afflicted with autoimmune diseases. It is notable that the AJ3 bacterial compositions comprising either intact or sonicated bacteria were equally effective.
  • the effective AJ3 composition comprised: (i) about 40-60% of Bifidobacterium Longum, (ii) about 5-20% of Bifidobacterium breve, and (iii) about 30-50% of Bifidobacterium infantis.
  • An exemplary formulation for AJ3 is shown in Table 9.
  • the AJ4 bacterial composition comprising Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei was effective in inducing secretion of IFN-g and MCP-1, proinflammatory cytokines that increase the immune response to cancer. It is notable that the AJ4 bacterial compositions comprising either intact or sonicated bacteria were equally effective.
  • the effective AJ4 composition comprised: (i) about 20-40% of Streptococcus thermophiles, (ii) about 10-30% of Lactobacillus acidophilus, (iii) about 30-50% Lactobacillus plantarum, and (iv) about 5- 20% Lactobacillus paracasei.
  • An exemplary formulation for AJ4 is shown in Table 10.
  • M-CSF Macrophage colony stimulating factor
  • RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis, in Biochem Biophys Res Commun. 1998. p. 253(2): p. 395-400.
  • Li, J., et al., RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. , in Proc Natl AcadSci USA. 2000. p. 97(4): p. 1566-71.
  • IFN-gamma stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation., in The Journal of clinical investigation. 2007. p. 117(1): p. 122-32.
  • heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol.
  • Jewett A Bonavida B. Target-induced inactivation and cell death by apoptosis in a subset of human NK cells. J Immunol. 1996;156(3):907-15.
  • any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and/or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.
  • TIGR The Institute for Genomic Research
  • NCBI National Center for Biotechnology Information

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Abstract

La présente invention concerne des compositions et des procédés qui sont utiles pour induire la formation osseuse, prévenir la perte osseuse, et/ou prévenir/traiter une affection avec perte osseuse, telle que, par exemple, une affection avec perte osseuse associée au cancer, par administration, à un sujet qui en a besoin, d'un agent qui augmente (a) la quantité et/ou l'activité des cellules NK; et/ou (b) la quantité d'IFN-y
PCT/US2022/053095 2021-12-16 2022-12-16 Prévention de la perte osseuse Ceased WO2023114445A1 (fr)

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CN117180495A (zh) * 2023-08-16 2023-12-08 广东省科学院生物与医学工程研究所 具有免疫调控功能的多孔钽支架及其制备方法、应用
CN117305188A (zh) * 2023-11-27 2023-12-29 山东中微众康生物科技有限公司 促进儿童骨生长的嗜酸乳杆菌RZKLa0701及其应用
CN119258045A (zh) * 2024-12-10 2025-01-07 北京大学口腔医学院 双硫仑在制备治疗糖尿病股骨骨丢失的药物中的应用

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WO2018152340A1 (fr) * 2017-02-15 2018-08-23 The Regents Of The University Of California Compositions et méthodes d'activation de cellules nk

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117180495A (zh) * 2023-08-16 2023-12-08 广东省科学院生物与医学工程研究所 具有免疫调控功能的多孔钽支架及其制备方法、应用
CN117305188A (zh) * 2023-11-27 2023-12-29 山东中微众康生物科技有限公司 促进儿童骨生长的嗜酸乳杆菌RZKLa0701及其应用
CN117305188B (zh) * 2023-11-27 2024-02-13 山东中微众康生物科技有限公司 促进儿童骨生长的嗜酸乳杆菌RZKLa0701及其应用
CN119258045A (zh) * 2024-12-10 2025-01-07 北京大学口腔医学院 双硫仑在制备治疗糖尿病股骨骨丢失的药物中的应用

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