Compositions and methods for activating NK cells
阅读说明：本技术 用于使nk细胞活化的组合物和方法 (Compositions and methods for activating NK cells ) 是由 A·朱厄特 于 2018-02-15 设计创作，主要内容包括：本申请涉及在体外、离体和/或在体内由破骨细胞(OC)和/或树突细胞使NK细胞活化的方法,以及使用这些经活化NK细胞来治疗疾病的方法。(The present application relates to methods of activating NK cells by Osteoclasts (OCs) and/or dendritic cells in vitro, ex vivo and/or in vivo, and methods of using these activated NK cells to treat diseases.)
1. A method of activating NK cells in vitro or ex vivo, comprising culturing the NK cells in a culture medium together with Osteoclasts (OCs).
2. A method, comprising:
i) providing a cell culture comprising NK cells and osteoclasts; and
ii) culturing the NK cell and the osteoclast in the cell culture,
thereby activating the NK cells.
3. The method of claim 1 or 2, wherein the NK cells are primary NK cells, optionally wherein the primary NK cells have not been transformed.
4. The method of any preceding claim, wherein activated NK cells expand to achieve at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more population doublings within 4 weeks.
5. The method of any preceding claim, wherein the culture comprises a plurality of Osteoclasts (OCs) and a plurality of NK cells, and the ratio of OC: NK cells in the cell culture is at least 1: 2.
6. The method of any preceding claim, wherein the osteoclast enhances NK cell cytotoxicity, optionally wherein the NK cell cytotoxicity is measured by lysis of oral squamous carcinoma stem cell like cells (osccs) by the NK cell.
7. The method of claim 6, wherein the NK cell cytotoxicity is through51Cr release cytotoxicity assay.
8. The method of any preceding claim, wherein the osteoclast enhances secretion of IFN- γ by the NK cell, optionally wherein the osteoclast enhances secretion of IL-12 by the NK cell.
9. The method of any preceding claim, wherein the osteoclast enhances expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by the NK cell.
10. The method of any preceding claim, wherein the NK cells are purified from a cancer sample.
11. The method of claim 10, wherein the cancer sample is from a subject having cancer.
12. The method of claim 11, wherein the subject is a human.
13. The method of any one of claims 10-12, wherein the cell culture further comprises T cells also derived from the cancer sample.
14. The method of claim 13, whereby the NK cells are preferentially expanded relative to the T cells.
15. The method of claim 14, further comprising preferentially expanding the NK cells for at least one month.
16. The method of claim 15, further comprising supplementing the culture medium with at least one osteoclast to continue preferentially expanding the NK cells.
17. The method of claim 14, wherein the T cell secretes IFN- γ but does not mediate cytotoxicity, optionally wherein the cytotoxicity is by preferential51Cr release cytotoxicity assay is measured by the dissolution of OSCSC by the T cells.
18. The method of any one of claims 13-17, wherein the expanded NK cells are capable of expanding CD8+ T cells.
19. The method of claim 18, wherein said NK cells expanded by said OC are capable of preferentially expanding CD8+ T cells over CD4+ T cells.
20. The method of any preceding claim, further comprising adding an anti-CD 3 antibody to the cell culture.
21. The method of claim 20, wherein the anti-CD 3 antibody further enhances secretion of IFN- γ by the NK cell.
22. The method of any preceding claim, wherein the activated NK cells are segmentation-unresponsive.
23. The method of any preceding claim, further comprising adding to the cell culture a composition comprising at least one bacterial strain selected from the group consisting of: streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99 and lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is live or sonicated.
24. The method of claim 23, wherein the composition comprises streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99, and lactobacillus bulgaricus.
25. The method of claim 23, wherein the composition comprises sAJ2 bacteria.
26. The method of claim 25, wherein the ratio of the sAJ2 bacteria concentration to the NK cells and/or the OC concentration in the cell culture
i) sAJ2 is at least 1:2 for NK cells;
ii) at least 1:4 for OC: sAJ 2; and/or
iii) for OC: NK cells: sAJ2 is at least 1:2: 4.
27. The method of any preceding claim, further comprising adding to the cell culture another agent capable of activating NK cells.
28. The method of any preceding claim, wherein the osteoclast enhances production, secretion and/or function of at least one cytokine or chemokine produced by the NK cell.
29. A method, comprising:
i) providing a cell culture comprising Osteoclasts (OCs), NK cells and T cells; and
ii) culturing the NK cell, the T cell and the osteoclast in the cell culture,
thereby preferentially activating the NK cells over the T cells.
30. A method, comprising:
i) providing a cell culture comprising Dendritic Cells (DCs), NK cells, and T cells; and
ii) culturing said NK cells, said T cells and said dendritic cells in said cell culture,
thereby preferentially activating the T cells over the NK cells.
31. The method of claim 29 or 30, wherein the NK cells are primary NK cells, optionally wherein the primary NK cells have not been transformed.
32. The method of claim 29 or 31, wherein the culture comprises a plurality of Osteoclasts (OCs) and a plurality of NK cells, and the ratio of the concentration of OC: NK cells in the cell culture is at least 1: 2.
33. The method of any one of claims 29, 31, or 32, wherein the osteoclast enhances NK cell expansion, optionally wherein the osteoclast enhances IL-15 secretion by the NK cell.
34. The method of claim 33, wherein activated NK cells expand to achieve at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more population doublings within 4 weeks.
35. The method of any one of claims 29 and 31-34, wherein the osteoclast enhances NK cell cytotoxicity, optionally wherein the NK cell cytotoxicity is measured by lysis of oral squamous carcinoma stem cell like cells (osccs) by the NK cell.
36. The method of claim 35, wherein the cell is a humanCytotoxic passage through51Cr release cytotoxicity assay.
37. The method of any one of claims 29 and 31-36, wherein the osteoclast enhances secretion of IFN- γ by the NK cell, optionally wherein the osteoclast enhances secretion of IL-12 by the NK cell.
38. The method of any one of claims 29 and 31-37, wherein the osteoclasts enhance expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by the NK cells.
39. The method of any one of claims 29-38, wherein the NK cells and/or the T cells are purified from a cancer sample.
40. The method of claim 39, wherein the cancer sample is from a subject having cancer.
41. The method of claim 40, wherein the subject is a human.
42. The method of any one of claims 29 and 31-41, wherein said preferential activation of said NK cells persists for at least one month.
43. The method of claim 42, further comprising adding at least one osteoclast to the cell culture after the preferential activation of the NK cell is attenuated or halted, thereby continuing to activate the NK cell, optionally wherein at least one osteoclast is added to the cell culture at least one month after culturing the NK cell.
44. The method of any one of claims 29-43, wherein the T cell secretes IFN- γ but does not mediate cytotoxicity, optionally whereinThe cytotoxicity is preferably in51Cr release cytotoxicity assay is measured by the dissolution of OSCSC by the T cells.
45. The method of any one of claims 29 and 31-44, wherein the expanded NK cells are capable of expanding CD8+ T cells.
46. The method of claim 45, wherein said NK cells expanded by said OC are capable of preferentially expanding CD8+ T cells over CD4+ T cells.
47. The method of any one of claims 30-44, wherein the NK cells expanded by the DCs are capable of preferentially expanding CD4+ T cells over CD8+ T cells.
48. The method of any one of claims 29 and 31-46, further comprising adding an anti-CD 3 antibody to the cell culture.
49. The method of claim 48, wherein the anti-CD 3 antibody further enhances secretion of IFN- γ by the NK cell.
50. The method of any one of claims 29-49, wherein activated NK cells are segmentally non-reactive.
51. The method of any one of claims 29 and 31-50, further comprising adding to the cell culture a composition comprising at least one bacterial strain selected from the group consisting of: streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99 and lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is live or sonicated.
52. The method of claim 51, wherein the composition comprises Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus.
53. The method of claim 52, wherein said composition comprises sAJ2 bacteria.
54. The method of claim 53, wherein the ratio of the sAJ2 bacteria concentration to the NK cells and/or the OC concentration in the cell culture
i) sAJ2 is at least 1:2 for NK cells;
ii) at least 1:4 for OC: sAJ 2; and/or
iii) for OC: NK cells: sAJ2 is at least 1:2: 4.
55. The method of any one of claims 29 and 31-52, further comprising adding to the cell culture another agent capable of activating NK cells.
56. The method of any one of claims 30-52, further comprising adding to the cell culture another agent capable of activating T cells.
57. The method of any one of claims 29 and 31-55, wherein the Osteoclast (OC) enhances production, secretion and/or function of at least one cytokine or chemokine produced by the NK cell.
58. A method of treating cancer or a cancer-related disease or disorder in a subject having or suspected of having cancer or a cancer-related disease or disorder, comprising administering to the subject a therapeutically effective amount of Osteoclasts (OC), a cell culture comprising Osteoclasts (OC), and/or a supernatant of a cell culture comprising Osteoclasts (OC).
59. The method of claim 58, wherein the osteoclast activates an NK cell in the subject.
60. The method of claim 59, wherein the NK cells are primary NK cells, optionally wherein the primary NK cells have not been transformed.
61. The method of any one of claims 59-60, wherein the osteoclast enhances NK cell expansion in the subject, optionally wherein the osteoclast enhances IL-15 secretion by the NK cell.
62. The method of claim 61, wherein enhanced NK cell expansion is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more population doublings within 4 weeks.
63. The method of any one of claims 59-62, wherein the osteoclast enhances NK cell cytotoxicity, optionally wherein the NK cell cytotoxicity is measured by lysis of oral squamous carcinoma stem cell like cell (OSCSC) by the NK cell.
64. The method of claim 63, wherein the NK cell cytotoxicity is through51Cr release cytotoxicity assay.
65. The method of any one of claims 59-64, wherein the osteoclast enhances secretion of IFN- γ by the NK cell, optionally wherein the osteoclast enhances secretion of IL-12 by the NK cell.
66. The method of any one of claims 59-65, wherein the osteoclast preferentially activates NK cells relative to T cells, optionally wherein the osteoclast preferentially enhances NK cell expansion relative to T cells.
67. The method of claim 66, wherein preferential expansion of NK cells persists for at least one month.
68. The method of claim 67, wherein the T cell secretes IFN- γ, but does not mediate cytotoxicity to the cancer, optionally wherein the cytotoxicity is by, e.g., in51Cr release cytotoxicity assay is measured by the dissolution of OSCSC by the T cells.
69. The method of any one of claims 59-68, wherein activated NK cells expand CD8+ T cells in the subject.
70. The method of claim 69, wherein activated NK cells preferentially expand CD8+ T cells over CD4+ T cells.
71. The method of any one of claims 58-70, further comprising administering to the subject an anti-CD 3 antibody.
72. The method of claim 71, wherein the anti-CD 3 antibody further enhances secretion of IFN- γ by the NK cell.
73. The method of any one of claims 59-72, wherein the activated NK cells are segmentation-unresponsive.
74. The method of any one of claims 58-73, further comprising administering to the subject a composition comprising at least one bacterial strain selected from the group consisting of: streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99 and lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is live or sonicated.
75. The method of claim 74, wherein the composition comprises Streptococcus thermophilus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, KE99, and Lactobacillus bulgaricus.
76. The method of claim 74, wherein said composition comprises sAJ2 bacteria.
77. The method of any one of claims 58-76, further comprising adding to the cell culture another agent capable of activating NK cells.
78. The method of any one of claims 58-77, wherein the Osteoclast (OC) increases or promotes the production, secretion and/or function of at least one cytokine or chemokine produced by the NK cell.
79. The method of any one of claims 58-78, wherein the osteoclast, the cell culture and/or the supernatant are administered in a pharmaceutical composition.
80. The method of any one of claims 58-79, wherein the osteoclasts, the cell culture and/or the supernatant are administered to the cancer systemically or locally.
81. The method of any one of claims 58-80, wherein the osteoclasts, the cell culture and/or the supernatant are administered to the subject at least twice, optionally wherein the osteoclasts, the cell culture and/or the supernatant are administered to the subject at least one month after the first administration.
82. The method of any one of claims 58-81, wherein the subject is a human.
Background
Natural Killer (NK) cells lyse and differentiate cancer stem cells/undifferentiated tumors with lower expression of MHC class I, CD54 and B7H1, and higher expression of CD 44. Moderate and high cytotoxic activity of peripheral blood lymphocytes is associated with a reduced risk of cancer, and high infiltration of tumors by NK cells is associated with a better prognosis, while low activity is associated with an increased risk of cancer.
Inhibition of NK cells is mediated by down-regulation of NK receptors in the tumor microenvironment. It was previously shown that NK cell function was significantly reduced in tumor patients. Several in vitro NK expansion techniques have been developed to allow higher therapeutic cell doses to be achieved. Stimulation of Peripheral Blood Mononuclear Cells (PBMC) or purification of the NK cell population with feeder cells such as K562 cells expressing Interleukin (IL) -15 and 41BB ligands, EBV-TM-LCL, Wilms's tumor (Wilm's tumor) or irradiated PBMC has produced a larger number of NK cells with sufficient function. The resulting NK cells expressed higher levels of NKG2D, natural cytotoxicity receptor, DNAM-1 and ICAM-1. Thus, various methods to obtain ex vivo expanded, activated and CD3+ T cell depleted NK cells have been established for clinical use. Furthermore, it has been determined that adoptive cell transfer with HLA haploidentical NK cells is safe and effective in patients with advanced cancer. In addition, clinical trials have shown that allogeneic NK cells play a therapeutic role in the context of solid tumors and are safe for transfer into patients.
Immunotherapy with NK cells has been limited by the inability to obtain a sufficient number of highly functional NK cells. Furthermore, unlike NK cells from healthy individuals, the expansion of patient NK cells is significantly limited by the expansion of a small fraction of contaminating T cells that, due to their faster proliferative capacity, deplete NK cells, similar to those from tumor-bearing humanized mice.
The underlying mechanism of NK cell immunomodulation is not understood. There is a great need to identify therapeutic compositions and methods for achieving improved NK immunotherapy.
Disclosure of Invention
The present invention is based, at least in part, on the discovery that osteoclasts can induce expansion of NK cells in both healthy humans and cancer patients, which further increases the CD8+/CD4+ T cell ratio. Although cancer patients typically have more NK cells and a greater CD8+/CD4+ T cell ratio in vivo relative to healthy humans, excess NK cells and CD8+ T cells are short lived (NK cell function may be inhibited due to expansion of contaminating T cells) and lack activity (e.g., cytotoxicity and cytokine selection). However, osteoclasts can induce NK cell expansion in cancer patients and increase both cell number and function of NK cells (e.g., as measured by their cytokine secretion capacity). Dendritic cells preferentially promote T cell expansion, while osteoclasts preferentially promote NK cell expansion, indicating a difference in the microenvironment of selectively expanding T cells and NK cells. Accordingly, the present invention provides a method to expand large numbers of activated NK cells for use in immunotherapeutic strategies. The cells can be used to inhibit or eliminate cancer stem cells by promoting differentiation of stem cell-like/poorly differentiated tumors, and to control tumor growth.
Provided herein is a method of activating NK cells in vitro or ex vivo, comprising culturing the NK cells in a culture medium together with Osteoclasts (OCs). The NK can be a primary NK cell, optionally, wherein it has not been transformed. Activated NK cells can expand about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more population doublings within 4 weeks. The culture can comprise a plurality of Osteoclasts (OCs) and a plurality of NK cells, for example wherein the ratio of OC to NK cells in the cell culture is at least 1: 2. Osteoclasts may potentiate NK cell cytotoxicity, e.g., as measured by lysis of oral squamous carcinoma stem-like cells (osccs) by NK cells or by51Cr release cytotoxicity assay.
In addition, osteoclasts may enhance the production, secretion and/or function of at least one cytokine or chemokine produced by NK cells. For example, osteoclasts may enhance secretion of IFN- γ and/or IL-12 by NK cells, and/or expression of one or more of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2, and KIR3 by NK cells.
The NK cells can be cells purified from a cancer sample of a human subject. In certain embodiments, the cell culture further comprises T cells also derived from the cancer sample. In certain such embodiments, NK cells may be preferentially expanded relative to T cells. NK cells can be expanded for any length of time, for example at least one month. The medium may be supplemented with at least one osteoclast to continue to preferentially expand NK cells. T cells may secrete IFN- γ, but may not mediate cytotoxicity, e.g., as by, e.g., in51Cr release cytotoxicity assay was measured by T cell dissolution of OSCSC. The expanded NK cells may be capable of expanding CD8+ T cells. NK cells expanded by OC may also be able to preferentially expand CD8+ T cells over CD4+ T cells. In certain embodiments, the methods further comprise adding an anti-CD 3 antibody to the cell culture, e.g., to further enhance secretion of IFN- γ by NK cells. Activated NK cells may be division-unresponsive.
In certain embodiments, the method may further comprise adding to the cell culture a composition comprising at least one bacterial strain selected from the group consisting of: streptococcus thermophilus (Streptococcus thermophilus), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium infantis (Bifidobacterium infantis), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus paracasei (Lactobacillus paracasei), KE99 and Lactobacillus bulgaricus (Lactobacillus bulgaricus), optionally wherein the at least one bacterial strain may be live or sonicated. For example, the composition may comprise streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99, and lactobacillus bulgaricus. Alternatively or additionally, the composition may comprise sAJ2 bacteria. The ratio of sAJ2 bacteria concentration to NK cell and/or OC concentration in the cell culture can be, for example, i) at least 1:2 for NK cell:
In certain embodiments, the method may further comprise adding to the cell culture another agent capable of activating NK cells.
In certain preferred embodiments, the method comprises: i) providing a cell culture comprising Osteoclasts (OCs), NK cells and T cells; and ii) culturing the NK cell, the T cell and the osteoclast in the cell culture, thereby preferentially activating the NK cell over the T cell.
Similarly, provided herein is a method comprising: i) providing a cell culture comprising Dendritic Cells (DCs), NK cells, and T cells; and ii) culturing said NK cells, said T cells and said dendritic cells in said cell culture, thereby preferentially activating said T cells over said NK cells. In an embodiment, the NK cell may be a primary NK cell, optionally wherein said primary NK cell has not been transformed. The culture can comprise a plurality of Osteoclasts (OCs) and a plurality of NK cells, for example wherein the ratio of OC to NK cells in the cell culture is at least 1: 2. Osteoclasts may expand NK cells, and/or secretion of IL-15 by NK cells may be enhanced. Activated NK cells can expand to achieve at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more population doublings within 4 weeks. In addition, osteoclasts may potentiate NK cell cytotoxicity, e.g., as measured by the lysis of oral squamous carcinoma stem-like cells (osccs) by NK cells. Cytotoxicity can be induced by51Cr release cytotoxicity assay.
In certain embodiments, the Osteoclast (OC) may enhance the production, secretion, and/or function of at least one cytokine or chemokine produced by NK cells. For example, osteoclasts may enhance secretion of IFN- γ and/or IL-12 by NK cells. The osteoclast can be one of NKG2D, NKp46, NKp44, NKp30, CD94, KIR2 and KIR3 treated by NK cellOne or more of them is enhanced. NK cells and/or T cells can be purified from a cancer sample from a subject, e.g., a human subject. In certain embodiments, preferential activation of NK cells may last for at least one month. In addition, after preferential activation of NK cells is attenuated or stopped, at least one osteoclast may be added to the cell culture that persists for at least one month after culturing NK cells, thereby continuing to activate NK cells. In some embodiments, the T cell may secrete IFN- γ, but may not mediate cytotoxicity. Cytotoxicity can be measured by the lysis of OSCSC by T cells, e.g., by51Cr release cytotoxicity assay. Expanded NK cells may be capable of expanding CD8+ T cells, and may be capable of preferentially expanding CD8+ T cells over CD4+ T cells. In certain embodiments, NK cells expanded by DCs may be capable of preferentially expanding CD4+ T cells over CD8+ T cells. In other embodiments, an anti-CD 3 antibody can be added to the cell culture, for example, to further enhance secretion of IFN- γ by NK cells. In addition, activated NK cells may be division-unresponsive.
In certain embodiments, the method may further comprise adding to the cell culture a composition comprising at least one bacterial strain selected from the group consisting of: streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99 and lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is live or sonicated. For example, the composition may comprise streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99, and lactobacillus bulgaricus. Alternatively or additionally, the composition may comprise sAJ2 bacteria. In certain such embodiments, the ratio of sAJ2 bacteria concentration to NK cells and/or OC concentration in the cell culture can be i) at least 1:2 for NK cell:
In addition, the method may further comprise adding to the cell culture another agent that may be capable of activating NK cells. The method may further comprise adding to the cell culture another agent capable of activating T cells.
Similarly, provided herein is a method of treating cancer or a cancer-related disease or disorder in a subject having or suspected of having cancer or a cancer-related disease or disorder by: administering to the subject a therapeutically effective amount of an Osteoclast (OC), a cell culture comprising an Osteoclast (OC), and/or a supernatant of a cell culture comprising an Osteoclast (OC).
The osteoclast may enhance NK cell expansion in the subject, optionally wherein the osteoclast enhances IL-15 secretion by NK cells. In certain embodiments, the enhanced NK cell expansion may be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more population doublings within 4 weeks. In addition, osteoclasts may potentiate NK cell cytotoxicity, e.g., as measured by the lysis of oral squamous carcinoma stem-like cells (osccs) by NK cells. Cytotoxicity can be induced by51Cr release cytotoxicity assay.
In certain embodiments, the osteoclast may increase or promote the production, secretion and/or function of at least one cytokine or chemokine produced by NK cells. For example, osteoclasts may enhance secretion of IFN- γ and/or IL-12 by NK cells. Osteoclasts may preferentially activate NK cells relative to T cells, and/or preferentially enhance NK cell expansion relative to T cells. In certain embodiments, preferential expansion of NK cells may last for at least one month. T cells can secrete IFN-gamma but do not mediate cytotoxicity against cancer, e.g., as by administration of a peptide in51Cr release cytotoxicity assay was measured by T cell dissolution of OSCSC.
In certain embodiments, the activated NK cells can expand CD8+ T cells in the subject, e.g., CD8+ T cells can be preferentially expanded relative to CD4+ T cells. The subject may also be treated with an anti-CD 3 antibody to further enhance secretion of IFN- γ by NK cells. Activated NK cells may be division-unresponsive.
In certain embodiments, the method of treatment may further comprise administering to the subject a composition comprising at least one bacterial strain selected from the group consisting of: streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99 and lactobacillus bulgaricus, optionally wherein the at least one bacterial strain is live or sonicated. For example, the composition may comprise streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei, KE99, and lactobacillus bulgaricus. In certain embodiments, the composition may comprise sAJ2 bacteria.
In certain embodiments, the method may further comprise adding to the cell culture another agent capable of activating NK cells.
In certain embodiments, the osteoclasts, cell culture, and/or supernatant may be administered in the form of a pharmaceutical composition that can be administered to the cancer systemically or locally. In certain embodiments, the osteoclast, cell culture, and/or supernatant may be administered to the subject at least twice, e.g., the osteoclast, cell culture, and/or supernatant may be administered to the subject at least one month after the first administration.
In some embodiments, the subject may be a human.
Drawings
Figure 1 includes four panels, identified as panels A, B, C and D, which show higher expression of NK activating ligands by osteoclasts. To generate Osteoclasts (OC), monocytes were cultured for 21 days in medium containing macrophage colony stimulating factor (M-CSF) (25ng/ml) and RANKL (25 ng/ml). Treatment of highly purified NK cells (1X 10) with a combination of IL-2(1000U/ml) and anti-CD 16mAb (3. mu.g/ml)6Individual cells/ml) for 18 hours, then co-cultured with autologous OCs in the presence of sAJ2 bacteria at a 1:2:4 ratio (OC: NK: sAJ2) or in the absence of sAJ2 bacteria, respectively. Analysis of 1x 10-containing samples from co-cultures on
FIG. 2 includes ten panels, identified as panels A through J, showing preferential expansion and significant functional gain of NK cells by osteoclasts and T cells by dendritic cells monocyte monocytes purified from human PBMC and cultured with GM-CSF (150ng/ml) and IL-4(50ng/ml) for 8 days to produce DC, to produce osteoclasts, monocytes cultured in α -MEM medium containing M-CSF (25ng/ml) and RANKL (25ng/ml) for 21 days, to expand purified NK cells (1 x 10) treated with a combination of IL-2(1000U/ml) and anti-CD 16mAb (3 μ g/ml)6Individual cells/ml) for 18 hours, and then co-cultured with autologous monocytes, DCs or OCs in the presence of sAJ2 at a 1:2:4 ratio (monocytes, DCs or OC: NK: sAJ 2). On the days indicated in the figure, surface expression of CD3, CD16 and CD56 was analyzed using flow cytometry, and the media was refreshed and supplemented with rh-IL-2(1000U/ml) (fig. 2A). Cells were co-cultured as described in fig. 2A, and the number of expanded lymphocytes was assessed using a microscopic assay (fig. 2B). Percentage of intracellular NK cells and T/NKT cells was expanded using the population in FIG. 2B (FIG. 2A)To determine the number of NK cells (FIG. 2C) and T/NKT cells (FIG. 2D). Cells were co-cultured as described in fig. 2A, and standard 4 hours for oral squamous cell carcinoma stem cell line (OSCSC) were used51Cr release assay to determine cytotoxicity on the days shown in the figure.
Figure 3 includes eight panels, identified as panels a through H, which show that unlike NK cells, T cells purified from osteoclast-expanded NK cells do not mediate cytotoxicity against OSCSC and secrete IFN- γ moderately. Freshly purified NK cells were treated as described in materials and methods and co-cultured with monocyte-derived autologous osteoclasts. Samples of lymphocytes from co-cultures at
Figure 4 includes 19 panels, identified as panels a through S, showing a reduction in NK cell proportion, NK cell-mediated cytotoxicity, and IFN- γ secretion with each successive re-stimulation of NK cell cultures with osteoclasts and sAJ2 bacteria. Freshly purified NK cells were treated as described in figure 2A and co-cultured with monocyte-derived autologous osteoclasts. On days indicated in the figure,
Figure 5 includes eight panels, identified as panels a through H, showing that osteoclasts, but not K562 or OSCSC, substantially expand NK cells and increase NK cell function. To generate osteoclasts, monocytes were cultured for 21 days in medium containing M-CSF (25ng/ml) and RANKL (25ng/ml), and the K562 tumor cell line was cultured as described in materials and methods. Treatment of highly purified NK cells (1X 10) with a combination of IL-2(1000U/ml) and anti-CD 16mAb (3. mu.g/ml)6Individual cells/ml) for 18 hours, then they were co-cultured with K562 and autologous OCs, respectively, in the presence of sAJ2 bacteria at a 1:2:4 ratio (OC: NK: sAJ 2). On
Figure 6 includes 16 panels, identified as panels a through P, which show that purified NK cells from cancer patients cultured with OCs expanded T cells to a greater extent than NK cells, mediating much lower cytotoxicity and cytokine secretion, compared to expanded NK cells from healthy donors. Freshly purified NK cells from healthy donors and cancer patients were treated as described in figure 2A and co-cultured with monocyte-derived OCs. Analysis of cancer patients (FIG. 6A) and healthy donors (FIG. 6B) using antibody staining and flow cytometry analysis in sequence at 6, 9, 12,15. Surface expression of CD3, CD16, and CD56 on expanded cells at
Figure 7 includes 11 panels, identified as panels a through K, which show that a small fraction of contaminating T cells within purified NK cells from cancer patients expand faster and may deplete NK cells due to reduced NK cell function. Freshly purified NK cells from healthy donors and pancreatic cancer patients were treated as described in figure 1A and co-cultured with monocyte-derived allogeneic (from different healthy donors) osteoclasts. Analysis of co-cultures from cancer patients (fig. 7A) and healthy donors (fig. 7B) at
FIG. 8 shows the phenotype of CD3T cells depleted of lymphocytes from splenocytes from hu-BLT mice. Humanized BLT (hu-BLT; human bone marrow/liver/thymus) mice were generated by surgical implantation of human fetal liver and thymus tissues under the kidney capsule of 6-8 week old immunocompromised nod.cb17-Prkdcscid/J and nod.cg-Prkdcscid Il2rgtm1Wjl/szj (nsg) mice. 4-6 weeks after tissue transplantation, mice were sublethally irradiated and injected intravenously with CD34+ cells isolated from fetal liver to support complete reconstitution of human bone marrow. In situ injection with CD34+ cellsBlood samples were used to analyze the reconstitution of the human immune system 8-12 weeks later. At the end of this experiment, engraftment of human immune cells was confirmed by staining spleen and bone marrow cells with anti-human CD45, CD3, CD4 and CD8 antibodies and analyzed by flow cytometry (data not shown). Successfully reconstituted hu-BLT mice (levels and lineage of T cells similar to healthy donors) were treated with
Figure 9 includes 11 panels, identified as panels a through K, which show that T cell depleted in vitro expanded lymphocytes from tumor bearing humanized BLT mice expanded T cells and contained fewer NK cells and mediated lower cytotoxicity when compared to those obtained from healthy hu-BLT mice. Reconstituted BLT (levels and lineage of T cells similar to healthy donors) with
Fig. 10 includes three panels, identified as panels a through C, showing cytokines, chemokines, and growth factors secreted by primary NK cells and by osteoclast-expanded NK cells. Highly purified NK cells and monocytes were obtained from Peripheral Blood Mononuclear Cells (PBMC) of healthy donors and NK cells (1X 10) were treated with IL-2(1000U/ml)6Individual cells/ml) for 18 hours, followed by collection of supernatant to generate osteoclasts, monocytes were cultured in α -MEM medium containing M-CSF (25ng/ml) and RANKL (25ng/ml) for 21 days for expansion, purified NK cells (1X 10) were treated with a combination of IL-2(1000U/ml) and anti-CD 16mAb (3. mu.g/ml)6Individual cells/ml) for 18 hours, and then separately cocultured with autologous osteoclasts in the presence of sAJ2 bacteria at a ratio of 1:2:4 (OC: NK: sAJ 2). Supernatants were collected after 6 days of co-culture and multiplexed assays were used to determine cytokine (fig. 10A), chemokine (fig. 10B), and growth factor (fig. 10C) levels secreted by primary NK cells and expanded NK cells.
Figure 11 includes three panels, identified as panels a through C, showing that blocking IL-12, IL-15, or a combination of both results in reduced NK cell expansion, NK cell-mediated cytotoxicity, and cytokine secretion. Freshly purified NK cells from healthy donors were treated as described in figure 2A and in the presence and absence, respectively, of autologous osteoclastsanti-IL 12mAb, anti-IL-15 mAb, or a combination of anti-IL-12 mAb and anti-IL-15 mAb at 100ng/ml and 1. mu.g/ml. Co-cultures were supplemented every 2 days with IL-2(1000 units/mL). On
Figure 12 includes seven panels, identified as panels a through G, showing that addition of anti-CD 3 antibody inhibits T cell expansion and increases OC-expanded NK cells. Freshly purified NK cells from healthy donors and cancer patients were expanded with OC for 27 days, followed by treatment of the cultures with rh-IL-2 and CD3 antibody (1 μ g/ml), followed by determination of NK cell (fig. 12A) and T cell (fig. 12B) numbers (both healthy donors and patients) at
Figure 13 shows that purified T cells treated with anti-CD 3 mAb in the absence of NK cells did not lose forward scatter and side scatter. Highly purified T cells and monocytes were obtained from Peripheral Blood Mononuclear Cells (PBMC) of healthy donors and T cells (1X 10) were treated with IL-2(100U/ml) and CD3 antibody (1. mu.g/ml)6Individual cells/ml) for 18 hours, and then co-cultured with autologous osteoclasts in the presence of sAJ2 bacteria at a ratio of 1:2:4 (OC: T cells: sAJ2), respectively. On
Figure 14 includes eight panels, identified as panels a through H, which show a substantial increase in the number of CD8+ T cells by osteoclast activation of NK cells. PBMCs from healthy donors and cancer patients were analyzed for CD3, CD4, and CD8 surface expression using PE and FITC conjugated antibody staining and flow cytometry analysis in sequence (fig. 14A). Freshly purified NK cells from healthy donors and cancer patients were treated as described in figure 2A and co-cultured with OCs. The CD3 positive selection kit was used to purify T cells from PBMCs of healthy donors and cancer patients and activate T cells with rh-IL2(100u/ml) and a combination of anti-CD 3 mAb (1 μ g/ml) and anti-CD 28 mAb (1 μ g/ml) for 18-20 hours before co-culturing them with OC in the presence of sAJ2 at a 1:2:4 ratio (OC: T cells: sAJ 2). Lymphocytes were analyzed for CD3, CD4, and CD8 surface expression (fig. 14B). Monocytes were purified from human PBMCs and OC and DC were produced as described in figure 2A and NK cells were purified by co-culture and the number of expanded lymphocytes was assessed using microscopy (figure 14C) and the number of T cells (figure 14D) and NK cells (figure 14E) were determined using the percentage of NK cells and T cells as described in figure 2A within the overall expanded cells in figure 14C. Lymphocytes were analyzed for surface expression of CD3+ CD4+ cells and CD3+ CD8+ cells, and the number of CD3+ CD4+ T cells (fig. 14F) and CD3+ CD8+ T cells (fig. 14G) was determined using the percentage of CD4 cells and CD8 cells within the total T cells in panel D. OC-activated NK-expanded T cells, DC-activated NK-expanded T cells, OC-expanded/activated T cells and DC-expanded/activated T cells were stained with antibodies against CD45RO, CD62L, CD28, CD44, CCR7 and CD127 and analyzed by flow cytometry. The values in
Figure 15 includes two panels, identified as a and B, showing that osteoclastically expanded NK cells retain their cytokine secretion and cytotoxic function after freezing. Freshly purified NK cells were treated and co-cultured with monocyte-derived autologous osteoclasts as described in figure 1A, and allowed to pass through on
Figure 16 includes five panels, identified as panels a-E, showing a reduction in the number of PBMCs obtained from peripheral blood of pancreatic (figure 16B), colon (figure 16C), oral (figure 16D), and prostate (figure 16E) cancer patients. Figure 16A shows the reduction in the condition of healthy subjects relative to patients.
Figure 17 includes five panels, identified as panels a-E, showing that the percentage of NK and CD14 monocytes was increased, but T and B cells were significantly decreased in cancer patients according to PBMCs obtained from peripheral blood of healthy subjects (figure 17A), pancreas (figure 17B), colon (figure 17C), oral (figure 17D), and prostate (figure 17E) cancer patients.
Fig. 18 includes four panels, identified as panels a-D, showing a reduction in NK cell cytotoxicity achieved by NK cells of the patient compared to healthy NK cells.
Figure 19 includes eight panels, identified as panels a-H, showing osteoclast-expanded NK cells from patients as well as cytotoxicity and IFN- γ secretion.
Fig. 20 shows cytokine secretion in the case of non-osteoclastically expanded NK cells from pancreatic cancer patients.
Figure 21 includes five panels, identified as panels a-E, showing secretion of IFN- γ by osteoclast-expanded T cells from healthy subjects (figure 21A), pancreas (figure 21B), colon (figure 21C), oral (figure 21D), and prostate (figure 21E) cancer patients.
Figure 22 includes six panels, identified as panels a-F, showing IFN- γ secretion from NK cells, T cells, and osteoclastically expanded NK cells and osteoclastically expanded T cells, relative to T cells (figure 22A), healthy subjects (figure 22B), pancreas (figure 22C), colon (figure 22D), prostate (figure 22E), and oral (figure 22F) cancer patients.
Figure 23 includes four panels, identified as panels a-D, showing T cell IFN- γ secretion from healthy subjects (figure 23A), pancreatic (figure 23B), colon (figure 23C), and prostate (figure 23D) cancer patients.
Figure 24 includes six panels, identified as panels a-F, showing the total number of expanded T cells versus the total number of expanded NK cells determined within
Figure 25 includes five panels, identified as panels a-E, showing the ability of pancreatic (figure 25B), colon (figure 25C), and prostate (figure 25D) cancer patients to expand T cells compared to healthy individual (figure 25A) ability measured within
Figure 26 shows a reduction in cytokines and chemokines in the serum of patients relative to healthy individuals.
Figure 27 includes two panels a-B showing the percentage of CD4T cells and CD8T cells in PBMCs from pancreatic (figure 27A) and colon (figure 27B) cancer patients compared to healthy individuals.
Fig. 28 includes five panels a-E showing the ratio of CD4T cells/CD 8T cells in PBMCs of cancer patients of the pancreas (fig. 28B), colon (fig. 28C), oral cavity (fig. 28D), and prostate (fig. 28E) relative to healthy individuals (fig. 28A).
Fig. 29 includes five panels showing the ratio of CD4T cells to CD8T cells in healthy subjects (fig. 29A) relative to pancreatic (fig. 29B), colon (fig. 29C), oral (fig. 29D), and prostate (fig. 29E) cancer patients.
Figure 30 includes two panels a and B showing the effect of culturing CD8T cells and CD4T cells without (figure 30A) and with (figure 30B) osteoclasts.
FIG. 31 shows IFN- γ secretion by NK cells and CD8T cells.
Figure 32 shows CD8T cell expansion and CD4T cell expansion promoted by osteoclasts and NK cells, respectively.
Fig. 33 shows cytotoxicity of osteoclastically expanded NK cells against cancer stem cells/undifferentiated tumors.
Figure 34 shows the effect of using osteoclasts to expand NK cells on NK cell expansion and the NK cell cytotoxic effect achieved by osteoclast-expanding cells.
Figure 35 shows that T cells in OC expanded NK cells have effector memory phenotype compared to DC expanded NK cells.
Figure 36 shows the number of T cells with depleted phenotype in OC-expanded NK cells relative to DC-expanded NK cells.
FIG. 37 shows IFN- γ expressing NK cells in OC expanded NK cells from pancreatic cancer patients.
Fig. 38 shows a table of CD8 and NK-specific cytokines, costimulatory ligands, granzymes, perforins, and soluble Fas and Fas ligand secreted by OC-expanded T cells.
FIG. 39 shows a table of CD 8-related cytokines, chemokines, co-stimulatory ligands, sFas and Fas ligands, and granzymes and perforins secreted by CD8+ T cells from OC expanded NK cell cultures.
FIG. 40 shows the levels of GM-CSF, IFN-g, IL-10, TNF-a, the less co-stimulatory ligand sCD137, granzyme, perforin, soluble Fas and Fas ligand secreted from NK-expanded or OC-expanded CD8 cells.
Figure 41 shows cytotoxic activity of NK cells from mice implanted with oral tumors into BLT compared to mice without tumors.
Figure 42 shows CD8+ T cells in BM, spleen and blood following immunotherapy with hyperactive NK cells following tumor implantation.
FIG. 43 shows serum IFN-. gamma.IL-6, ITAC, GM-CSF and IL-8 after tumor implantation following immunotherapy with hyperdynamic NK cells in BLT mice.
FIG. 44 shows NK cell cytotoxicity of patients expanded by osteoclasts at different days of expansion.
Fig. 45 shows the number of NK cells in the NK cell population expanded by osteoclasts in the case of patients on different expansion days.
Figure 46 shows the number of osteoclast expanded NK cells compared to DC expanded cells from
Figure 47 shows the number of osteoclast expanded NK cells compared to DC expanded NK cells at different expansion days.
Figure 48 shows the number of DC expanded T cells compared to osteoclast expanded T cells at different expansion days.
Figure 49 shows cytotoxicity of osteoclast expanded NK cells compared to DC expanded NK cells at different expansion days.
Figure 50 shows IFN- γ secretion achieved by primary non-osteoclast expanded patient NK cells and osteoclast expanded patient NK cells compared to healthy donor NK cells at different expansion days.
Figure 51 shows IFN- γ secretion by primary non-osteoclast expanded patient T cells and osteoclast expanded patient T cells compared to healthy donor T cells on different expansion days.
Figure 52 shows IFN- γ secretion achieved by osteoclastically expanded patient NK cells over different expansion days when compared to those obtained from healthy donor NK cells.
Figure 53 shows IFN- γ secretion achieved by primary non-osteoclastically expanded patient T cells and osteoclastically expanded patient T cells in the case of some patients, when compared to those obtained from healthy donor T cells, at different expansion days.
Figure 54 shows IFN- γ secretion (pooled secretion from all expansion days) achieved by primary non-osteoclastically expanded patient T cells and osteoclastically expanded patient T cells when compared to those obtained from healthy donor T cells over different expansion days (T cells were selected in a positive manner).
Figure 55 shows IFN- γ secretion achieved by positively selected primary non-osteoclastically expanded patient T cells and osteoclastically expanded patient T cells (pooled secretion from all expansion days) when compared to negatively selected T cells from healthy donors.
Figure 56 shows per cell-based IFN- γ secretion achieved by osteoclastically expanded T cells (T cells selected in a positive manner) when compared to NK cells obtained from healthy donors. Primary positively selected T cells activated with IL-2 secrete higher IFN- γ when compared to NK cells.
Figure 57 shows per-cell based IFN-g secretion achieved by primary non-osteoclastically expanded patient T cells and osteoclastically expanded patient T cells (T cells selected in a positive manner) when compared to T cells obtained from healthy donors.
FIG. 58 shows per-cell based secretion of IFN-g by osteoclast-expanded patient T cells (T cells were selected in a positive manner) and NK cells. Primary non-osteoclast-expanded T cells have higher IFN-g secretion in the case of IL-2 when compared to primary NK cells treated with IL-2.
Figure 59 shows an increase in the number of expanded cells achieved by positively selected primary non-osteoclast expanded T cells and osteoclast expanded T cells when compared to negatively selected NK cells or negatively selected T cells from healthy donors.
Figure 60 shows the reduction in expanded cell number achieved by positively selected primary non-osteoclastically expanded patient T cells and osteoclastically expanded patient T cells when compared to those obtained from healthy donors.
FIG. 61 shows the levels of cytokines and chemokines in serum from blood of pancreatic patients.
FIG. 62 includes 11 panels, identified as panels A-C, showing that single injections of hyperinsulinemic NK cells inhibit tumor growth in hu-BLT mice fed with/without
FIG. 63 includes nine panels, identified as A-I, showing restoration and increase of IFN-y secretion and cytotoxic function of NK cells, enriched NK cells and purified CD3+ T cells in blood, spleen, BM with injection of hyperactive NK cells in tumor-bearing hu-BLT mice with/without AJ2 feeding. Hu-BLT mice were implanted with human osccs and injected with NK cells and fed with AJ2 as described in fig. 62B, and mice were injected with PD1 antibody (50 μ g/mouse) by tail vein injection one week after NK cell injection. After sacrifice, spleens (n-5) (fig. 63A), BMs (n-5) (fig. 63B) and peripheral blood (n-5) (fig. 63C) were collected, single cell suspensions were prepared from each tissue and treated with IL-2(1000U/ml) for 7 days (1 x10 for spleens and BMs)6Individual cells/ml and 0.7 x10 for PBMC6Individual cells/ml). NK-rich cells were isolated from splenocytes and treated with IL-2(1000U/ml) for 7 days (1X 10)6One cell/ml) (n-3) (fig. 63D). Standard 4 hours for OSCSC was used51Cr Release assay cytotoxicity assays were performed and LU30/10 was determined using the reciprocal of the number of cells required to dissolve 30% of OSCSCs, x1006And (4) cells. Will be provided withSplenocytes (n ═ 5) (fig. 63E), BM cells (n ═ 5) (fig. 63F), PBMC (n ═ 5) (fig. 63G) (at
FIG. 64 includes two panels, identified as A and B, showing that a single injection of hyperinsulinemic NK cells with/without AJ2 increased the number of CD8+ T cells in hu-BLT mice. Hu-BLT mice were implanted with osccs and injected with NK cells as described in figure 62B and fed with AJ2 and the percentage of human CD8+ T cells within BM cells (n-3) (figure 64A) and splenocytes (n-3) (figure 64B) was determined using antibody staining and flow cytometric analysis, in sequence.
Fig. 65 includes ten panels, identified as a-J, showing that a single injection of hyperactive NK cells mediated in vivo tumor differentiation in BLT mice fed with/without AJ2, increased IFN- γ secretion and mobilized an increased number of human immune cells to the tumor, and resulted in decreased ex vivo tumor growth. Hu-BLT and NSG mice were implanted with osccs and injected with NK cells as described in figure 62B. After sacrifice, oral tumors were collected and single cell suspensions were prepared and the same number of cells from each group (at 1x 10) were cultured on
Oral tumor cells from hu-BLT mice are described in fig. 65A, and supernatants were collected after 3 and 7 days and VEGF secretion levels were determined using a specific ELISA. The reduction in VEGF secretion achieved by tumors obtained from NK-injected animals (n-6) was calculated based on the amount obtained from OSCSC-injected mice alone (fig. 65I). The percent infiltration of hu-CD45C immune cells within oral tumors dissociated from different experimental groups of hu-BLT mice was determined using flow cytometry analysis after staining with antibody. One of several representative experiments is shown in this figure (fig. 65J).
FIG. 66 includes three panels, identified as A-C, showing that single injections of hyperactive NK cells restored and increased secretion of cytokines, chemokines and growth factors in serum obtained from peripheral blood of hu-BLT mice in tumor-bearing mice with/without feeding of
Figure 67 includes two panels, identified as a and B, showing that CDDP or paclitaxel with and without NAC induces significant cell death in osccs differentiated with NK supernatants rather than poorly differentiated tumors. Highly purified NK cells were treated with a combination of IL-2(1000U/mL) and anti-CD 16mAb (3mg/mL) for 18 hours, after which NK supernatant was added to OSCSC in the presence of TNF-a antibody (1:100) and IFN-g antibody (1:100) for a period of 5 days. Thereafter, OSCSC were detached and treated with/without cisplatin for 18-24 hours. Viability of OSCSC was then determined using PI staining and flow cytometric analysis. One of 3 representative experiments is shown in this figure (fig. 67A). OSCSC was treated with supernatant from NK cells as described in figure 67A. Thereafter, the tumors were detached and treated with/without NAC (20nM) for 24 hours followed by paclitaxel for 18-24 hours. OSCSC viability was determined by PI staining and flow cytometric analysis. One of 3 representative experiments is shown in this figure (fig. 67B).
Figure 68 includes five panels, identified as a-E, which show that monocytes or osteoclasts from tumor-bearing mice injected with NK cells or implanted with NK differentiated OSCSC tumors alone induce significantly more IFN-g from autologous or allogeneic NK-tumor co-cultures when compared to those implanted with NK cells alone. Hu-BLT mice were implanted with osccs and injected with NK cells as described in figure 62B and fed with
Detailed Description
The present invention relates in part to a method of activating NK cells in vitro or ex vivo, comprising culturing said NK cells in a medium comprising Osteoclasts (OCs). Similarly, provided herein is a method to activate T cells in vitro or ex vivo, comprising culturing the T cells in a culture medium comprising Dendritic Cells (DCs). The present invention further provides a method of activating NK cells in vitro or ex vivo relative to T cells, comprising culturing said NK cells and said T cells in a medium comprising Osteoclasts (OCs). The invention further provides a method of activating T cells in vitro or ex vivo relative to NK cells, comprising culturing said NK cells and said T cells in a culture medium comprising Dendritic Cells (DCs). The activated NK cells are useful for improving host immune responses, and for treating diseases (e.g., cancer). In some aspects, the invention provides a method of activating NK cells in vivo by Osteoclasts (OCs), optionally, relative to T cells. In some embodiments, the OC or OC culture supernatant can be administered to a subject to treat a disease (e.g., cancer). In some embodiments, probiotic bacteria (e.g., sAJ2) may be added to improve the function of OCs to activate NK cells. In addition to Osteoclasts (OCs) and Dendritic Cells (DCs), other agents capable of activating NK or T cells may also be added to the cell culture or administered to the subject, including any genes, proteins, metabolites, etc.
I.Definition of
The article "a" or "an" is used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an" element means one element or more than one element.
The term "administering" is intended to include routes of administration that allow an agent (e.g., at least one Osteoclast (OC) or Dendritic Cell (DC), a cell culture comprising at least one Osteoclast (OC) or Dendritic Cell (DC), a supernatant of the cell culture, any composition comprising the OC or DC, at least one probiotic bacterium, any composition comprising the probiotic bacterium, other agents capable of activating NK cells and/or T cells or contributing to the function of the OC or DC and/or probiotic bacterium, etc.), and also include treated (i.e., isolated, purified, concentrated, or after other procedures for therapeutic or other use) forms of the various agents described herein, to perform its intended function. Examples of administration routes for physical therapy that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection may be bolus injection or may be continuous infusion. Depending on the route of administration, the agent may be coated with or disposed within the selected substance to protect it from natural conditions that may adversely affect its ability to perform its intended function. The agents may be administered alone or in combination with a pharmaceutically acceptable carrier. The agent may also be administered in the form of a prodrug which is converted in vivo to its active form.
The term "activating" refers to enhancing the function of a target. For example, the present disclosure provides a method of activating NK cells or T cells in vitro, ex vivo, and/or in vivo, optionally wherein the activation is preferential relative to T cells or NK cells, respectively. In the present disclosure, activation of a cell refers to enhancement of the function of the cell, including at least enhancement of the activity and/or at least one cell function (e.g., cytotoxicity, cell division and/or growth rate, etc.) of each cell of a certain cell type (e.g., NK cells or T cells), enhancement of the number of cells of a certain cell type (e.g., cell expansion), or both. In some embodiments, an agent used herein activates at least one cell, such as an NK cell or a T cell. In other embodiments, an agent used herein preferentially activates one cell type (e.g., NK cell or T cell) over another cell type (e.g., T cell or NK cell).
In the present disclosure, the term "enhance" is used interchangeably with the terms "increase", "upregulation", "improve", and the like, to refer to any increase in number that is meaningful to the function of an agent and/or target. For example, an increase in the activity and/or number of cells of a certain cell type (e.g., NK cells or T cells) can be "significant" when the increase in the quantity is increased by a quantity greater than the standard error of the assay used to assess the quantity, as compared to the original quantity and/or normal quantity in the context of a control (e.g., the activity and/or number of cells of the certain cell type in a normal subject or a subject not having a disease or disorder (e.g., cancer), and preferably by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more as compared to that original quantity and/or normal quantity. Alternatively, an enhancement in the activity and/or number of cells of a certain cell type may be considered "significant" if the enhancement is at least about two-fold, and preferably at least about three-fold, four-fold, or five-fold or more, compared to the original activity/amount and/or normal activity/amount. The "significance" may also apply to any other measured parameter described herein, such as expression, inhibition, cytotoxicity, cell growth parameters, and the like. In some embodiments, the enhancement in activity and/or cell number of a certain cell type (e.g., NK cells or T cells) may not be "significant" as described herein, but still sufficient for the skilled artisan to understand its biologically relevant increase.
Unless otherwise specified herein, the term "antibody" broadly encompasses naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies such as single chain antibodies, chimeric and humanized antibodies and multispecific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigen binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
The term "antibody" as used herein also includes the "antigen-binding portion" of an antibody (or simply "antibody portion"). The term "antigen-binding portion" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab')2 fragment, i.e. a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546) consisting of the VH domain; and (vi) isolating the Complementarity Determining Regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that enable them to be a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science242:423 + 426; and Huston et al (1988) Proc. Natl. Acad. Sci. USA85:5879 + 5883; and Osbourn et al 1998, Nature Biotechnology 16: 778). The single chain antibody is also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of a particular scFv can be linked to a human immunoglobulin constant region cDNA or genomic sequence to generate an expression vector encoding the complete IgG polypeptide or other isotype. VH and VL can also be used to produce Fab, Fv or other immunoglobulin fragments using protein chemistry or recombinant DNA techniques. Other forms of single chain antibodies, such as minibifunctional antibodies, are also contemplated. The minibifunctional antibody is a bivalent bispecific antibody in which the VH domain and the VL domain are expressed on a single polypeptide chain, but a linker is used which is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and generating two antigen binding sites (see e.g.Holliger, P. et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-.
The antibody may be a polyclonal or monoclonal antibody; xenogenic, allogeneic or allogeneic antibodies; or modified forms thereof (e.g., humanized antibodies, chimeric antibodies, etc.). The antibody may also be a fully human antibody. Preferably, the antibodies of the invention specifically or substantially specifically bind to a biomarker polypeptide or fragment thereof. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one species of antigen binding site that is capable of immunoreacting with a particular epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen binding sites that are capable of interacting with a particular antigen. Monoclonal antibody compositions typically exhibit a single binding affinity for the particular antigen to which it is immunoreactive.
The term "cancer" or "tumor" or "hyperproliferative" refers to the presence of cells having characteristics typical of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological characteristics. Unless stated otherwise, the term includes metaplasia. In some embodiments, the features include at least one of: silence, reduce and/or avoid a host immune response, and/or be resistant to host cell (e.g., NK cell) lysis and/or differentiation. In some embodiments, the oncogenic cell is a cancer stem cell (e.g., an Oral Squamous Carcinoma Stem Cell (OSCSC)). In some embodiments, the cell exhibits the characteristic due, in part or in whole, to at least one genetic mutation. Cancer cells are often in the form of tumors, but the cells may be present alone in the animal, or may be non-tumorigenic cancer cells such as leukemia cells. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers. Cancers include, but are not limited to, B cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia (II)
In some embodiments, the Cancer is "triple negative breast Cancer" or "TNBC," which refers to breast Cancer that is Estrogen Receptor (ER) negative, Progesterone Receptor (PR) negative, and human epidermal growth factor receptor 2(HER-2) negative (Pegram et al (1998) J. Clin. Oncol.16: 2659-2671; Wiggans et al (1979) Cancer Chemothers. Pharmacol.3: 45-48; Carey et al (2007) Clin. Cancer Res.13: 2329-2334).
For example, even if PI3K β 0 (e.g., PI3K β 1mRNA, PI3K β protein, newly synthesized PI3K β protein, etc.) in tumor tissue is expressed at a level similar to its expression in normal tissue, the cancer is also 3K β dependent if inhibition of PI3K β mRNA and/or protein, such as by using RNAi or any other means, or deletion of PI3K β gene (e.g., by knocking out or Clustering Regularly Interspaced Short Palindromic Repeats (CRISPR) technology) results in inhibition of tumorigenesis, tumor cell proliferation, tumor metastasis, or induction of tumor cell differentiation, the term "PI 3K β dependent cancer" also refers to cancers in which PI3K β is expressed at a significantly higher level than PI3K β expressed in noncancerous cells of the same cell type as PI3K β dependent cancer (PI 3K β, PI3 protein synthesis at PI 8945, e.g., PI3, 82K β protein).
The term "micrometastases" as used herein is preferably defined as a group of confluent cancer cells measured in terms of maximum width of more than 0.2mm and/or having more than 200 cells up to 2 mm. More preferably, "micrometastases" are defined as a group of confluent Cancer cells that are 0.2mm to 2mm in maximum width (see Edge et al (2010) AJCC Cancer Staging Manual and handbook (7 th edition)). An alternative preferred definition of "micrometastases" is a group of at least 1000 confluent cancer cells and is at least 0.1mm in its widest dimension up to 1mm in its widest dimension. In the case of standard contrast MRI imaging or other clinical imaging techniques, micrometastases are generally not visible. However, in the case of certain cancers, radioactive antibodies directed against tumor-selective antigens (e.g., Her2 for breast cancer metastasis) allow imaging of micrometastases. Other indirect detection methods include contrast media leakage at brain micrometastasis sites due to VEGF-induced vascular leakage (Yano et al (2000) cancer Res.60: 4959-49067; U.S. patent publication 2015/0352113). More sensitive imaging techniques can also be applied to detect micrometastases. For example, blood volume can be imaged by MRI using the alternative contrast agent USPIO (Molday Iron, Biopal, Worcester, Mass.) to detect micrometastases (Yin et al (2009) clin. exp. metastasis.26: 403-.
The term "control" refers to any reference standard suitable for providing a comparison to the expression product, cell number, and/or cell function in a test sample. In certain embodiments, a control comprises obtaining a "control sample" from which the expression product level, cell number, and/or cell function is detected and compared to the expression product level, cell number, and/or cell function from the test sample. Such control samples may include any suitable sample, including but not limited to samples from control cancer patients with known results (which may be stored samples or previous sample measurements); a normal tissue or cell isolated from a subject, such as a normal patient or a cancer patient, a cultured primary cell/tissue isolated from a subject, such as a normal subject or a cancer patient, an adjacent normal cell/tissue obtained from the same organ or body location of a cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cell/tissue obtained from a depository. In other preferred embodiments, controls may include reference standard expression product levels from any suitable source including, but not limited to, housekeeping genes, ranges of expression product levels from normal tissue (or other previously analyzed control samples), ranges of expression product levels previously determined within a test sample from a group of patients or a group of patients having a certain outcome (e.g., survival for 1 year, 2 years, 3 years, 4 years, etc.) or receiving a certain treatment (e.g., standard of care cancer therapy). It will be appreciated by those skilled in the art that the control sample and reference standard expression product levels, cell numbers and/or cell functions may be used in combination as controls in the methods of the invention. In certain embodiments, a control can include a normal or non-cancerous cell/tissue sample. In other preferred embodiments, a control may comprise a group of patients, such as a group of cancer patients, or a group of cancer patients receiving a certain treatment, or a group of patients having a certain outcome of expression level, number of a certain cell type (e.g., NK cells or T cells), and/or cell function of a certain cell type relative to another outcome. In the foregoing cases, the specific expression product level, cell number, and/or cell function of each patient may be assigned as a percentile expression level, cell number, and/or cell function, or expressed as an average or mean value that is higher or lower than a reference standard expression level, cell number, and/or cell function. In other preferred embodiments, the control can include normal cells, cells from a patient treated with combination chemotherapy, and cells from a patient with benign cancer. In other embodiments, the control may also include measurements such as the average expression level of a particular gene in the same population, the average cell number and/or cell function of a particular cell type (e.g., NK cells or T cells) as compared to the expression level of a housekeeping gene or another cell type in the population. Such a population may comprise normal subjects, cancer patients who have not undergone any treatment (i.e., treatment naive), cancer patients who have undergone standard of care therapy, or patients with benign cancer. As shown by the data below, the methods of the invention are not limited to the use of specific truncation points when comparing expression product levels, cell numbers, and/or cell function in a test sample to a control.
The term "determining a treatment regimen appropriate for a subject" is used to mean determining a treatment regimen for a subject (i.e. a monotherapy or a combination of different therapies for preventing and/or treating cancer in a subject) that is initiated, modified and/or terminated based, or substantially based, or at least partially based, on the results of an analysis according to the invention. One example is the initiation of adjuvant therapy after surgery with the aim of reducing the risk of relapse, and another example would be to modify the dosage of a particular chemotherapy. In addition to the results of the analysis according to the invention, the determination may be based on the personal characteristics of the subject to be treated. In most cases, the actual determination of a treatment regimen appropriate for a subject will be made by the attending physician or doctor.
The term "diagnosing cancer" includes the use of the methods, systems and procedures of the present invention to determine the presence or absence of cancer or a subtype thereof in an individual. The term also includes methods, systems, and procedures for assessing the level of disease activity in an individual.
The term "immune cell" refers to a cell that plays a role in an immune response. Immune cells have hematopoietic origin and include lymphocytes, such as B cells and T cells; a natural killer cell; bone marrow cells such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.
The term "cytokine" refers to a broad and loose class of small proteins (about 5-20 kDa) important in cell signaling. Their release has an effect on the behaviour of the cells around them. Cytokines are involved in autocrine signaling, paracrine signaling, and endocrine signaling as immunomodulators. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, and may additionally include hormones or growth factors of the present disclosure. Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. Preferred cytokines are exemplified in the specification and drawings of the present disclosure, for example, in table 1.
The term "cytokine/chemokine activity" includes the ability of a cytokine or chemokine to modulate at least one cellular function. In general, cytokines or chemokines regulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of specific cell populations. Thus, the term "cytokine/chemokine activity" includes the ability of a cytokine or chemokine to bind to its native cellular receptor, to modulate cellular signaling, and to modulate an immune response.
The term "immune response" includes T cell-mediated and/or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are affected by T cell activation, such as antibody production (humoral responses) and activation of cytokine-responsive cells such as macrophages.
The term "immunotherapeutic agent" may include any molecule, peptide, antibody, or other agent that can stimulate the host immune system to generate an immune response against a tumor or cancer in a subject. Various immunotherapeutic agents are suitable for use in the compositions and methods described herein.
The term "inhibiting" includes reducing, limiting or blocking, for example, a particular effect, function or interaction. In some embodiments, a cancer is "inhibited" if at least one symptom of the cancer is alleviated, stopped, slowed, or prevented. As used herein, a cancer is also "inhibited" if the recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented. Similarly, if a biological function, such as the function of a protein, is reduced compared to a reference state, such as a control, e.g., wild-type state, it is inhibited. For example, if kinase activity is reduced due to mutation and/or contact with an inhibitor compared to wild-type PI3 kinase and/or PI3 kinase not contacted with the inhibitor, the kinase activity of the mutant PI3 kinase or PI3 kinase contacted with the PI3 kinase inhibitor is inhibited. The inhibition may be induced, such as by application of an agent at a particular time and/or location, or may be constitutive, such as due to a heritable mutation. The inhibition may also be partial or complete (e.g., substantially no measurable activity as compared to a reference state, such as a control, e.g., wild-type state). Substantially complete inhibition is referred to as blocking.
The term "interaction" when referring to an interaction between two molecules refers to the physical contact (e.g., binding) of the molecules to each other. Typically, such interaction results in one or both of the molecules being active (which produces a biological effect).
A "kit" is any article of manufacture (e.g., a package or container) that includes at least one agent, e.g., a probe or small molecule, for specifically detecting and/or affecting the expression of a marker of the invention, the kit can be marketed, sold or marketed in a unit format for performing the methods of the invention.
The term "neoadjuvant therapy" refers to a treatment administered prior to primary treatment. Examples of neoadjuvant therapy may include chemotherapy, radiation therapy, and hormone therapy. For example, in treating breast cancer, neoadjuvant therapy may allow patients with large breast cancer to undergo breast conservation surgery.
A "normal" level of expression and/or activity of a biomarker is a level of expression and/or activity of the biomarker in cells of a subject, e.g., a human patient, that is not afflicted with cancer. By "overexpression" or "significantly higher level expression" of a biomarker is meant that the expression level in a test sample is increased over the standard error of the assay used to assess expression compared to a control sample (e.g., a sample from a healthy subject not suffering from a biomarker-related disease), and preferably is increased by at least 10% compared to the average expression level of the biomarker in several control samples, and more preferably is increased by 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.5, 9, 10.5, 10, 11, 12.9, 13, 13.9, 1.9, 2.0, 2.1, 2.2.2, 2, 2.3, 2, 3,5, 2.5, 5, 6, 14. 15, 16, 17, 18, 19, 20 times higher or more. By "significantly lower level expression" of a biomarker is meant that the expression level in the test sample is reduced by at least 10% compared to the expression level of said biomarker in a control sample (e.g. a sample from a healthy subject not suffering from a biomarker-related disease), and preferably compared to the average expression level of said biomarker in several control samples, and more preferably is 1/1.2, 1/1.3, 1/1.4, 1/1.5, 1/1.6, 1/1.7, 1/1.8, 1/1.9, 1/2.0, 1/2.1, 1/2.2, 1/2.3, 1/2.4, 1/2.5, 1/2.6, 1/2.7, 1/2.8, 1/2.9, 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, 1/10.5, 1/11, 1/12, 1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20. The same assay can be performed to determine over-activity or under-activity.
NK cells
Natural killer cells or NK cells are one type of cytotoxic lymphocyte cell that is critical to the innate immune system. NK cells play a role similar to that of cytotoxic T cells in vertebrate adaptive immune responses. NK cells provide a rapid response to virus-infected cells, act at about 3 days after infection, and respond to tumor formation. Typically, immune cells detect the presence of Major Histocompatibility Complex (MHC) on the surface of infected cells, triggering cytokine release, leading to lysis or apoptosis. However, NK cells are unique in that they are able to recognize excitable cells in the absence of antibodies and MHC, allowing much faster immune responses to be achieved. They were named "natural killers" due to the following initial insights: they do not require activation to kill cells that lack "self"
NK cells, belonging to the group of congenital lymphoid cells, are defined as Large Granular Lymphocytes (LGLs) and constitute a third class of cells differentiated from common lymphoid progenitors that give rise to B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in bone marrow, lymph nodes, spleen, tonsils and thymus, where they then enter the circulation. NK cells differ from natural killer T cells (NKTs) in phenotype, origin and corresponding effector function; often, NKT cell activity promotes NK cell activity by secreting IFN γ. In contrast to NKT cells, NK cells do not express the T cell antigen receptor (TCR) or the full T marker CD3 or the surface immunoglobulin (Ig) B cell receptor, but they typically express the surface markers CD16(Fc γ RIII) and CD56 in humans and NK1.1 or NK1.2 in C57BL/6 mice. Currently, NKp46 cell surface markers constitute another preferential NK cell marker expressed in humans, several mouse strains (including BALB/c mice) and three common monkey species.
NK cells are negatively regulated by class I Major Histocompatibility Complex (MHC) -specific inhibitory receptors (Karre et al, 1986; Ohlen et al, 1989). These specific receptors bind to MHC class I molecules or polymorphic determinants of HLA present on other cells and inhibit NK cell lysis. In humans, certain members of a family of receptors known as killer Ig-like receptors (KIRs) recognize groups of HLA class I alleles.
KIR is a large family of receptors present on certain subsets of lymphocytes, including NK cells. The nomenclature for KIRs is based on the number of extracellular domains (KIR2D or KIR3D), and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3 DS). Within humans, the presence or absence of a given KIR is variable from one NK cell to another within a population of NK cells present in a single individual. In the human population, there are also relatively high levels of polymorphism of KIR molecules, some of which are present in some but not all individuals. Certain KIR gene products, when bound to appropriate ligands, result in stimulation of lymphocyte activity. It was confirmed that stimulatory KIRs all have short cytoplasmic tails with charged transmembrane residues associated with adaptor molecules with immunostimulatory motifs (ITAMs). Other KIR gene products are inhibitory in nature.
Probiotic bacteria
In some embodiments, the present invention relates to a composition capable of modulating NK cell function comprising at least one strain of probiotic bacteria that induces significant fragmentation anergy in activated NK cells, resulting in significant induction of IFN- γ and TNF- α.
Many commercial probiotics are available, having various effects of alleviating gastrointestinal discomfort or strengthening the immune system. Preferred probiotic bacterial species for use in the compositions and methods described herein include those commercially available probiotic bacterial strains such as sAJ2 bacteria, in particular those from the genera Streptococcus (Streptococcus) (e.g. Streptococcus thermophilus (s)), Bifidobacterium (Bifidobacterium) (e.g. Bifidobacterium longum (b.longum), Bifidobacterium breve (b.breve), Bifidobacterium infantis (b.infarnatis), Bifidobacterium breve (b.infantis), Bifidobacterium infantis (b.infantis), and Lactobacillus (Lactobacillus) (e.g. Lactobacillus acidophilus (l.acidophilus), Lactobacillus helveticus (l.helveticus), Lactobacillus bulgaricus (l.bulgaricus), Lactobacillus rhamnosus (l.rhamnonosus), Lactobacillus plantarum (l.plantarum) and Lactobacillus casei). The present disclosure includes methods of administering to a subject, preferably a mammal (e.g., a human), at least one probiotic bacterial strain, preferably a combination of two or more different bacterial strains. The administration may be systemic or local (e.g., directly to the intestine). A preferred route of administration is oral administration. Other routes (e.g., transrectal) may also be used. For application, bacteria (e.g., in moist, sonicated, ground or dried form, or in a formulation), bacterial culture containing the bacteria, or bacterial culture supernatant (without the bacteria) can be applied.
Osteoclast
Osteoclasts are a type of bone cell derived from hematopoietic stem cells. Their function of resorbing bone tissue is critical to the maintenance, repair and remodeling of bone. Intrabody balance is achieved when there is a balance between osteoblast bone formation and osteoclast bone resorption. Osteoclasts mature through stimulation from osteoblasts expressing RANKL and their interactions mediated by robust adhesion by ICAM-1. Osteoclasts also express many ligands for receptors present on activated NK cells. They reported that osteoclasts expressed ULBP-1, ULBP-2/5/6 and ULBP-3, but expressed little or no MIC-A, MIC-B or class I MHC-like ligands of NK cell activating receptor NKG 2D.
Osteoclasts (OCs) are prominent activators of NK cell expansion and function compared to Dendritic Cells (DCs) and monocytes (Tseng et al (2015) Oncotarget 6(24): 20002-25). In addition, osteoclasts secrete a large number of IL-12, IL-15, IFN-. gamma.and IL-18, which are known to activate NK cells; osteoclasts also express important NK activating ligands. The present disclosure provides a novel strategy as to how to expand highly functional hyperactive NK cells via osteoclast expansion to levels significantly higher than those established by other methods. Several in vitro NK expansion techniques have been developed to establish higher therapeutic cell doses while boosting NK cell activity and in vivo proliferative potential. Some of these techniques include stimulation of Peripheral Blood Mononuclear Cells (PBMC), purification of NK cell populations from PBMC, or the use of human cord blood, sometimes in combination with various feeder cells such as K562 cells expressing membrane-bound IL-15 and 41BB ligands (K562-mb15-41BBL), EBV-TM-LCL, Wilms' tumor, or irradiated PBMC. These studies have resulted in clinically relevant NK cell numbers with good function.
Dendritic cells
Dendritic Cells (DCs) are antigen presenting cells (also called helper cells) of the mammalian immune system. Their main function is to process the antigenic material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and adaptive immune systems.
Dendritic cells are present in those tissues that are in contact with the external environment such as the skin in which specialized dendritic cell types, known as Langerhans cells, are present, and the lining of the nose, lungs, stomach, and intestines. They may also be found in the blood in an immature state. Once activated, they migrate to the lymph nodes where they interact with T and B cells to initiate and shape adaptive immune responses. At some developmental stage they produce branched overhangs, i.e. dendrites which give the cell its name. Although similar in appearance, these are structures that differ from the dendrites of neurons. Immature dendritic cells are also called veil cells because they have large cytoplasmic "veil" rather than dendrites.
The present disclosure provides a novel method to activate NK cells using osteoclasts, resulting in enhanced sensitivity of tumor target cells to NK cell-mediated apoptosis and enhanced cytokine production. The term "activation" refers to enhancement of NK cell expansion and/or activation of NK cell function, either alone or in combination. The term "NK cell function" refers to any function of NK cells, such as cytotoxicity and/or cytokine/chemokine production/secretion activity.
The terms "preventing/presenting", "prophylactic treatment", and the like refer to a reduction in the probability of developing a disease, disorder or condition in a subject who does not have the disease, disorder or condition, but who is at risk of developing the disease, disorder or condition or who is susceptible to developing the disease, disorder or condition.
The term "response to an anti-cancer therapy" or "response to a therapy with a composition comprising at least one probiotic bacterium, alone or in combination with other NK immunotherapies" relates to any response of a hyperproliferative disorder (e.g. cancer) to the treatment with an anti-cancer agent, such as a composition comprising at least one probiotic bacterium, alone or in combination with other NK immunotherapies, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant therapy. Hyperproliferative disorder response can be assessed, for example, for efficacy, or hyperproliferative disorder response in neoadjuvant or adjuvant situations, where tumor size after systemic intervention can be compared to initial size and size as measured by CT, PET, mammography, ultrasound, or palpation. Response can also be assessed by caliper measurements or pathological examination of the tumor after biopsy or surgical resection. Responses may be recorded in a quantitative manner, such as percent change in tumor volume, or in a qualitative manner, such as "pathologic complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinically stable disease" (cSD), "clinically progressive disease" (cPD), or other qualitative criteria. The hyperproliferative disorder response assessment may be performed early, e.g. after hours, days, weeks or preferably months, after the initiation of neoadjuvant or adjuvant therapy. A typical endpoint for response assessment is after termination of neoadjuvant chemotherapy or after surgical removal of residual tumor cells and/or tumor bed. This is usually 3 months after initiating neoadjuvant therapy. In some embodiments, the clinical efficacy of a therapeutic treatment described herein can be determined by measuring the Clinical Benefit Rate (CBR). Clinical benefit rate was measured by determining the sum of the percentage of patients in Complete Remission (CR), the number percentage of patients in Partial Remission (PR), and the number percentage of patients with Stable Disease (SD) at a time point of at least 6 months after the end of therapy. A shorthand form of this formula is CBR ═ CR + PR + SD over 6 months. In some embodiments, the CBR of a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or greater. Additional criteria for assessing response to cancer therapy are associated with "survival," which includes all of the following: survival through death, also known as overall survival (where the death may be regardless of cause or associated with the tumor); "relapse-free survival" (where the term relapse shall include both localized relapse and distant relapse); survival without transfer; disease-free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of the survival period can be calculated by reference to determining a starting point (e.g., time to diagnose or initiate treatment) and an end point (e.g., death, recurrence, or metastasis). In addition, the criteria for determining treatment efficacy can be extended to include response to chemotherapy, survival probability, probability of metastasis over a given period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a particular cancer treatment regimen may be administered to a population of subjects, and the results may be correlated with biomarker measurements determined prior to administration of any cancer therapy. Outcome measures may be pathological responses to therapy given in a neoadjuvant setting. Alternatively, outcome measures such as overall survival and disease-free survival may be monitored for a subject over a period of time following a cancer therapy for which biomarker measurements are known. In certain embodiments, the administered dose is a standard dose known in the art for cancer therapeutics. The period over which the subject is monitored may vary. For example, a subject may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds associated with the outcome of cancer therapy can be determined using methods well known in the art, such as those described in the examples section.
The term "resistance" refers to acquired or natural resistance of a cancer sample or mammal to cancer therapy (i.e., non-responsive to, or having a reduced or limited response to, therapeutic treatment), such as having a response to therapeutic treatment that is reduced by 25% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80% or more, to 1/2, 1/3, 1/4, 1/5, 1/10, 1/15, 1/20 or greater. The decrease in response can be measured by comparison to the same cancer sample or mammal prior to acquiring resistance, or by comparison to a different cancer sample or mammal known to not have resistance to a therapeutic treatment. The typical acquired resistance to chemotherapy is referred to as "multi-drug resistance". Multidrug resistance can be mediated by P-glycoprotein, or can be mediated by other mechanisms, or it can occur when a mammal is infected with a multidrug resistant microorganism or combination of microorganisms. Determining resistance to therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician, and can be measured, for example, as "sensitization" by a cell proliferation assay and a cell death assay as described herein. In some embodiments, the term "reversing resistance" means that the use of a second agent in combination with a primary cancer therapy (e.g., chemotherapy or radiation therapy) is capable of producing a statistically significant reduction in tumor volume at a statistically significant level (e.g., p <0.05) when compared to the tumor volume of an untreated tumor in cases where the primary cancer therapy (e.g., chemotherapy or radiation therapy) alone is not capable of producing a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor. This is generally applicable to tumor volume measurements performed when untreated tumors grow at logarithmic rhythm.
The term "response" or "responsiveness" refers to an anti-cancer response, such as in the sense of reducing tumor size or inhibiting tumor growth. The term may also refer to improvement in prognosis, e.g., as reflected by an increase in the time to achieve a relapse, which is the period to achieve a first relapse, abrogation of a second primary cancer as a first event, or death with no evidence of relapse; or increased overall survival, which is the period from treatment to death for any reason. Responsive or having a response means that there is a beneficial endpoint obtained when exposed to a stimulus. Alternatively, negative or harmful symptoms are minimized, alleviated, or reduced upon exposure to the stimulus. It will be appreciated that assessing the likelihood that a tumor or subject will exhibit a favorable response is equivalent to assessing the likelihood that the tumor or subject will not exhibit a favorable response (i.e., will exhibit a lack of response or be non-responsive).
The term "sample" for detecting or determining the presence or level of at least one biomarker is typically brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, feces (e.g. feces), tears and any other bodily fluid (e.g. as described above under the definition of "bodily fluid"), or a tissue sample (e.g. a living specimen) such as a small intestine, colon sample or surgically excised tissue. In certain instances, the methods of the invention further comprise obtaining a sample from the individual prior to detecting or determining the presence or level of the at least one marker in the sample.
The term "sensitizing" means altering a cancer cell or tumor cell in a manner that allows for more effective treatment of the associated cancer with a cancer therapy (e.g., by treatment with a composition described herein). In some embodiments, the normal cells are not affected to the extent that they are caused to be unduly damaged. Increased or decreased sensitivity to therapeutic treatment is measured according to methods known in the art for the particular treatment and described herein below, including, but not limited to, cell proliferation assays (Tanigawa et al (1982) Cancer Res.42: 2159-. Sensitivity or resistance can also be measured in animals by measuring the reduction in tumor size over a period of time, e.g., 6 months for humans and 4-6 weeks for mice. The composition or method sensitizes the response to the therapeutic treatment if the increase in treatment sensitivity or decrease in resistance is 25% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80% or more, the increase in treatment sensitivity is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, and the decrease in resistance is 1/2, 1/3, 1/4, 1/5, 1/10, 1/15, 1/20 or more, as compared to the sensitivity or resistance of the treatment in the absence of the composition or method. Determining sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any of the methods described herein for enhancing the efficacy of a cancer therapy are equally applicable to methods of sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to a cancer therapy.
The term "specifically binds" refers to an agent, such as an antibody, that binds to a predetermined target, such as an antigen. Generally, when the target antigen is used as the analyte and the antibody is used as the ligand, the method is described in
The term "synergistic effect" means that the combined effect of two or more anticancer agents (e.g., treatment with a combination of compositions comprising at least one probiotic bacterium, alone or in combination with other NK immunotherapies) may be greater than the sum of the individual effects of the individual anticancer agents.
The term "subject" refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a cancer, such as brain metastases, lung cancer, ovarian cancer, pancreatic cancer, liver cancer, breast cancer, prostate cancer, colon cancer, melanoma, multiple myeloma, and the like. The term "subject" is interchangeable with "patient".
The term "survival" includes all of the following: survival through death, also known as overall survival (where the death may be regardless of cause or associated with the tumor); "relapse-free survival" (where the term relapse shall include both localized relapse and distant relapse); survival without transfer; disease-free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of the survival period can be calculated by reference to determining a starting point (e.g., time to diagnose or initiate treatment) and an end point (e.g., death, recurrence, or metastasis). In addition, the criteria for determining treatment efficacy can be extended to include response to chemotherapy, survival probability, probability of metastasis over a given period, and probability of tumor recurrence.
The term "therapeutic effect" refers to a local or systemic effect in an animal, particularly a mammal, and more particularly a human, caused by a pharmacologically active substance. Thus, the term means any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease, or the enhancement of desirable physical or psychological development and condition in an animal or human. The phrase "therapeutically effective amount" means that amount of such a substance that produces a certain desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. In certain embodiments, a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like. For example, certain compounds found by the methods of the invention may be administered in amounts sufficient to produce a reasonable benefit/risk ratio applicable to the treatment.
As used herein, the term "non-responsive" includes resistance of a cancer cell to a therapy or resistance of a therapeutic cell, such as an immune cell, to stimulation, e.g., by an activating receptor or cytokine. The non-response may occur, for example, due to exposure to an immunosuppressive agent or to exposure to a high dose of antigen. As used herein, the term "anergy" or "tolerance" includes resistance to stimulation mediated by an activating receptor. The resistance is typically antigen-specific and persists after exposure to the tolerance antigen has ceased. For example, anergy in the case of T cells (as opposed to non-response) is characterized by a lack of cytokine production, such as IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3 mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if the re-exposure occurs in the presence of the co-stimulatory polypeptide) results in the inability to produce cytokines, and thus the inability to proliferate. However, if cultured with cytokines (e.g., IL-2), anergic T cells may proliferate. For example, T cell anergy can also be observed as a lack of IL-2 production by T lymphocytes, as measured by ELISA or by proliferation assays using indicator cell lines. Alternatively, reporter gene constructs may be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of a 5' IL-2 gene enhancer, or by multimers of the AP1 sequence found in enhancers (Kang et al (1992) Science257: 1134).
II.Test subject
In certain embodiments, a subject suitable for the compositions and methods disclosed herein is a mammal (e.g., a mouse, rat, primate, non-human mammal, domestic animal such as a dog, cat, cow, horse, etc.), and preferably a human. In other embodiments, the subject is an animal model of cancer. For example, the animal model may be an in situ xenograft animal model of human oral squamous carcinoma, or comprise Cancer Stem Cells (CSCs)/undifferentiated tumors.
In other embodiments of the methods of the invention, the subject has not been subjected to a treatment, such as chemotherapy, radiation therapy, targeted therapy and/or anti-immunotherapy (such as NK cell-related immunotherapy). In other embodiments, the subject has been treated, such as chemotherapy, radiation therapy, targeted therapy, and/or anti-immunotherapy (such as NK cell-related immunotherapy).
In certain embodiments, the subject has undergone surgery to remove cancerous or precancerous tissue. In other embodiments, the cancerous tissue has not been removed, for example the cancerous tissue may be located in an inoperable region of the body, such as in tissue that is essential to life, or in an area where a surgical procedure would result in a considerable risk of patient injury.
The methods of the invention may be used to treat and/or determine the responsiveness of a number of different cancers in a subject, such as those described herein, to a composition comprising at least one probiotic bacterium, alone or in combination with other NK immunotherapies.
III.Anti-cancer therapy
In one aspect, other anti-cancer therapies and/or combinations of immunotherapy or combinations of therapies (e.g., one or more PI3K β selective inhibitors such as KIN193 in combination with one or more immune checkpoint inhibitors such as anti-PD-1 antibodies, alone or in combination with additional anti-cancer therapies such as targeted therapies) can be administered, particularly if the subject has first been indicated as a likely responder to a composition as disclosed herein.
Combination therapies are also contemplated and may include, for example, one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation, and chemotherapy, each combination may be with a therapy as disclosed herein. As described below, the agent may be administered in combination therapy with, for example, a chemotherapeutic agent, a hormone, an anti-angiogenic agent, a radiolabeled compound, or with surgery, cryotherapy, and/or radiation therapy. The prior treatment methods can be administered in combination with other forms of conventional therapy (e.g., standard of care treatment for cancer, which is well known to the skilled artisan), either sequentially with, prior to, or after the conventional therapy. For example, these modulators may be administered with a therapeutically effective dose of a chemotherapeutic agent. In other embodiments, these modulators are administered in combination with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. Physician's Desk Reference (PDR) discloses dosages of chemotherapeutic agents that have been used to treat various cancers. The dosage regimen and dosage of these above-mentioned chemotherapeutic agents which are therapeutically effective will depend on the particular melanoma being treated, the extent of the disease and other factors familiar to the skilled practitioner and can be determined by the practitioner.
The term "targeted therapy" refers to the administration of an agent that selectively interacts with a selected biomolecule to thereby treat cancer. One example includes breast or ovarian cancer antigens.
Alternatively, immunotherapy is a form of targeted therapy that may include, for example, the use of cancer vaccines and/or sensitized antigen presenting cells. For example, oncolytic viruses are viruses that are capable of infecting and lysing cancer cells while sparing normal cells, thereby making them potentially useful in cancer therapy. Replication of oncolytic viruses contributes to tumor cell destruction and also produces dose expansion at the tumor site. They can also serve as carriers for anticancer genes, allowing them to be delivered specifically to the tumor site. Immunotherapy may involve passive immunity to short-term protection of the host by administration of pre-formed antibodies against cancer or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens optionally linked to chemotherapeutic agents or toxins). Immunotherapy may also focus on the use of cancer cell line epitopes recognized by cytotoxic lymphocytes. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, and the like can be used to selectively modulate a biomolecule associated with the initiation, progression, and/or pathology of a tumor or cancer.
The term "untargeted therapy" refers to the administration of an agent that does not selectively interact with a selected biomolecule, but treats cancer. Representative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.
In certain embodiments, chemotherapy includes administration of chemotherapeutic agents selected from the group consisting of platinum compounds, cytotoxic antibiotics, antimetabolites, antimitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogs, plant bases and toxins, and synthetic derivatives thereof, including but not limited to cisplatin (cissplatin), trosufam (treosulfan) and chloroacetphosphoramide (trofosfamide), phytoalkaloids vinblastine (vinblastine), paclitaxel (paclitaxel), teniposide (teniposide), clinatropine (crisnato) and mitomycin (mitomycin), anti-folate (methotrexate), phenolic acid (copephycin), and cysteine (cysteine), as described in the aforementioned examples, such as the introduction of the factor I), the factor II (adenine D-7), the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the factor III, the factor II, the.
In other embodiments, radiation therapy is used. The radiation used in radiotherapy may be ionizing radiation. The radiation therapy may also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external beam radiation therapy, interstitial implanted radioisotopes (I-125, palladium, iridium), radioisotopes such as strontium-89, chest radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of Radiation Therapy, see Hellman,
In other embodiments, surgical intervention can physically remove cancerous cells and/or tissue.
In other embodiments, hormone therapy is used. Hormonal therapeutic treatments may include, for example, hormone agonists, hormone antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate, Lipprolide (LUPRON)), LH-RH antagonists, inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, desotropides, deltolides, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoid corticotropin, glucocorticoid, mineral corticoids, estrogens (estrogen), testosterone, progestogen (progestogen), progestogen A, trans-vitamin A derivatives (e.g., vitamin D, anti-retinoids, e.g., 3, vitamin D, and anti-progestogen analogs, Onapristone) or an antiandrogen agent (e.g., cyproterone acetate).
In other embodiments, hyperthermia is used, i.e., a procedure in which body tissue is exposed to hyperthermia (up to 106 ° f). Heat can help shrink tumors by damaging cells or depriving them of substances they need to survive. Hyperthermia can be localized, regional, and systemic hyperthermia achieved using external and internal heating devices. Hyperthermia is almost always used with other forms of therapy (e.g., radiation therapy, chemotherapy, and biological therapy) in an attempt to increase their effectiveness. Localized hyperthermia refers to the application of heat to a very small area such as a tumor. The region may be heated externally with high frequency waves from a device external to the body aimed at the tumor. To achieve internal heating, one of several types of sterile probes may be used, including a fine heated filament or a hollow tube filled with warm water; implanting a microwave antenna; and a radio frequency electrode. In regional hyperthermia, heat is applied to an organ or limb. Magnets and devices that generate high energy are placed on the area to be heated. In another method, known as perfusion, some of the patient's blood is removed, heated, and then pumped (perfused) into the area to be internally heated. Systemic heating is used to treat metastatic cancer that has spread throughout the body. This can be achieved using a warm water blanket, hot wax, induction coils (such as those in an electric blanket), or a thermal chamber (similar to a large incubator). The high temperature does not result in any significant increase in radiation side effects or complications. However, heat applied directly to the skin can cause discomfort or even significant local pain in about half of the treated patients. This can also lead to blisters, which typically heal quickly.
In other embodiments, photodynamic therapy (also known as PDT, light radiation therapy, phototherapy or photochemotherapy) is used to treat some types of cancer.
In other embodiments, laser therapy is used to destroy cancer cells using high intensity illumination. This technique is often used to alleviate symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It can also be used to treat cancer by causing the tumor to shrink or destroy.
The duration and/or dosage of treatment with a therapy may vary depending on the particular therapeutic agent or combination thereof. The appropriate treatment time for a particular cancer therapeutic will be appreciated by the skilled artisan. The invention encompasses the continuous assessment of the optimal treatment time course for each cancer therapeutic, where the cancer phenotype of the subject as determined by the methods of the invention is one factor in determining the optimal therapeutic dose and time course.
In other embodiments, recombinant biomarker polypeptides and fragments thereof may be administered to a subject. In some embodiments, fusion proteins with enhanced biological properties can be constructed and administered. In addition, biomarker polypeptides and fragments thereof may be modified (e.g., pegylated, glycosylated, oligomerized, etc.) according to pharmacological methods well known in the art to further enhance desirable biological activities, such as increased bioavailability and decreased proteolytic degradation.
Clinical efficacy can be measured by any method known in the art. For example, the response to a therapy, such as a composition as disclosed herein, relates to any response of a cancer, e.g. a tumor, to the therapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant chemotherapy or adjuvant chemotherapy. Tumor response in neoadjuvant or adjuvant situations can be assessed where the tumor size after systemic intervention can be compared to the initial size and size as measured by CT, PET, mammography, ultrasound or palpation, and the cytology of the tumor can be histologically estimated and compared to that of a live tumor specimen taken prior to initiation of treatment. Response can also be assessed by caliper measurements or pathological examination of the tumor after biopsy or surgical resection. Responses can be recorded in quantitative ways such as percent change in tumor volume or cellular, or using semi-quantitative scoring systems such as residual cancer burden (Symmans et al, j. clin. oncol. (2007)25: 4414-. Tumor response assessment may be performed early, e.g., hours, days, weeks, or preferably months, after the initiation of neoadjuvant or adjuvant therapy. A typical endpoint for response assessment is after termination of neoadjuvant chemotherapy or after surgical removal of residual tumor cells and/or tumor bed.
In some embodiments, the clinical efficacy of a therapeutic treatment described herein can be determined by measuring the Clinical Benefit Rate (CBR). Clinical benefit rate was measured by determining the sum of the percentage of patients in Complete Remission (CR), the number percentage of patients in Partial Remission (PR), and the number percentage of patients with Stable Disease (SD) at a time point of at least 6 months after the end of therapy. A shorthand form of this formula is CBR ═ CR + PR + SD over 6 months. In some embodiments, the CBR of a particular treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or greater.
Additional criteria for assessing response to a therapy as disclosed herein are associated with "survival," which includes all of the following: survival through death, also known as overall survival (where the death may be regardless of cause or associated with the tumor); "relapse-free survival" (where the term relapse shall include both localized relapse and distant relapse); survival without transfer; disease-free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of the survival period can be calculated by reference to determining a starting point (e.g., time to diagnose or initiate treatment) and an end point (e.g., death, recurrence, or metastasis). In addition, the criteria for determining treatment efficacy can be extended to include response to chemotherapy, survival probability, probability of metastasis over a given period, and probability of tumor recurrence.
For example, to determine an appropriate threshold, a particular anti-cancer treatment regimen may be administered to a population of subjects, and the results may be correlated with biomarker measurements determined prior to administration of any composition as disclosed herein. Outcome measures may be pathological responses to therapy given in a neoadjuvant setting. Alternatively, outcome measures such as overall survival and disease-free survival may be monitored for a subject over a period of time following therapy for which biomarker measurements are known. In certain embodiments, the same dose of the therapeutic composition is administered to each subject. The period over which the subject is monitored may vary. For example, a subject may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds associated with the results of a therapy as disclosed herein can be determined using various methods, such as those described in the examples section.
3.Pharmaceutical composition
The present invention provides pharmaceutically acceptable compositions of the compositions disclosed herein. As detailed below, the pharmaceutical compositions of the present invention may be specifically formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, e.g., drench (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste; (2) parenteral administration, for example by subcutaneous, intramuscular or intravenous injection, for example in the form of a sterile solution or suspension; (3) topical application, e.g. to the skin in the form of a cream, ointment or spray; (4) intravaginally or intrarectally, e.g. in the form of pessaries, creams or foams; or (5) spraying, for example in the form of an aqueous aerosol, liposome formulation or solid particles.
The compositions described herein, e.g., compositions of probiotic bacteria, may be used for oral administration to reach the gastrointestinal tract, targeting the introduction of probiotic bacteria into the tissues of the gastrointestinal tract. The formulation of the therapeutic composition of the present invention may also include other probiotics or nutrients that promote spore germination and/or bacterial growth. An exemplary substance is a bifidogenic oligosaccharide, which promotes the growth of beneficial probiotic bacteria. In a certain embodiment, the probiotic bacterial strain is combined with a therapeutically effective dose of an (preferably broad spectrum) antibiotic or antifungal agent. In some embodiments, the compositions described herein are encapsulated into an enterically coated time release capsule or tablet. The enteric coating allows the capsule/tablet to remain intact (i.e., undissolved) as it passes through the gastrointestinal tract until after a certain time and/or until it reaches a certain portion of the gastrointestinal tract (e.g., the small intestine). The timed release component prevents "release" of the probiotic bacterial strain in the compositions described herein for a predetermined period of time.
The therapeutic compositions of the present invention may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavoring agents, vitamins, or minerals.
In some embodiments, a therapeutic composition of the invention, e.g., an osteoclast, a cell culture of an osteoclast, and/or a supernatant of a cell culture of an osteoclast, may be administered alone or in combination with a carrier that is physiologically compatible with the species to which it is administered. The carrier may comprise a solid-based dry substance for formulation into a tablet, capsule or powder form; or the carrier may comprise a liquid or gel based substance for formulation into a liquid or gel form. The particular type of carrier and final formulation will depend, in part, on the route of administration chosen. The therapeutic compositions of the present invention may also include various carriers and/or binders. A preferred carrier is microcrystalline cellulose (MCC) added in an amount sufficient to complete a total weight of 1 gram dose. The carrier may be a solid-based dry material for formulation in tablet, capsule, or powder form, and may be a liquid or gel-based material for formulation in liquid or gel form, depending in part on the route of administration. Typical carriers for dry formulations include, but are not limited to: trehalose, maltodextrin, rice flour, microcrystalline cellulose (MCC), magnesium stearate, inositol, FOS, GOS, dextrose, sucrose, and similar carriers. Suitable liquid or gel based carriers include, but are not limited to: water and physiological saline solution; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, etc.). Preferably, the water-based carrier has a neutral pH value (i.e., pH 7.0). Other carriers or agents for administering the compositions described herein are known in the art, for example, in U.S. patent No. 6,461,607. Osteoclasts, cell cultures of osteoclasts, and/or supernatants of cell cultures of osteoclasts of the present disclosure may be administered using local and/or systemic routes of administration known in the art and described herein.
The Osteoclasts (OCs) or Dendritic Cells (DCs) described herein may be used to be administered to a subject in any pharmaceutically acceptable composition by any route of administration known in the art. For example, an OC or DC, a cell culture comprising the OC or DC, or a supernatant of the cell culture, optionally together with an additional agent, can be administered in the form of a pharmaceutical composition by systemic and/or local (e.g., to or near a cancer or tumor tissue) injection (e.g., intravenously).
The phrase "pharmaceutically acceptable" is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulated substance, involved in carrying or transporting a subject chemical substance from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered gum 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 glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline water; (18) ringer's solution (Ringer's solution); (19) ethanol; (20) a phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations.
Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and acacia or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as pastilles (using an inert base such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes and the like, each containing a predetermined amount of one or more bacterial strains as disclosed herein.
The invention also encompasses kits for detecting and/or modulating the biomarkers described herein. Kits of the invention may also include instructional materials disclosing or describing the use of the kit or the antibodies of the disclosed invention in the methods of the disclosed invention as provided herein. Kits may also include additional components to aid in the particular application for which the kit is designed. For example, the kit may additionally contain means for detecting the label (e.g., an enzyme-labeled enzyme substrate, a filter kit to detect fluorescent labels, an appropriate secondary label such as sheep anti-mouse HRP antibody, etc.) and reagents necessary for a control (e.g., a control biological sample or standard). The kit may additionally include buffers and other reagents recognized for use in the methods of the disclosed invention. Non-limiting examples include agents to reduce non-specific binding such as carrier proteins or detergents.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：降低LDL-胆固醇的细胞提取物和食品补充剂