Method of diagnosing cancer and diagnosis kit using measurement of NK cell activity
Provided are a method for diagnosing cancer, a diagnosis kit and compositions useful for measurement of NK cell activity. The incidence of cancer may be diagnosed by monitoring changes in the in vivo immune system through measurement of NK cell activity in blood. Thus, the incidence of cancer may be readily predicted as described herein using a blood sample from a subject.
1. A method of measuring natural killer (NK) cell activity, comprising:
stimulating NK cells in a whole blood sample by incubating the whole blood sample with a composition comprising interleukin 15 and interleukin 12, thereby artificially activating the NK cells to generate and secrete NK cell-secreting cytokines; and
measuring an amount of the NK cell-secreting cytokines secreted into the whole blood sample and using the amount as a measure of NK cell activity;
wherein the whole blood sample is a human whole blood sample.
2. The method according to claim 1 , wherein the composition further comprises interleukin 2.
3. The method according to claim 1 , wherein the NK cell-secreting cytokines are selected from the group consisting of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1β(MIP-1β).
4. The method according to claim 1 , wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ).
5. The method according to claim 1 , wherein the NK cell-secreting cytokine is tumor necrosis factor-alpha (TNF-α).
6. The method according to claim 1 , wherein the measuring of the amount of the NK cell-secreting cytokines is performed by enzyme-linked immunosorbent assay (ELISA) or other immunoassay.
7. The method according to claim 1 , wherein the IL-15 and IL-12 are in the form of a fusion protein with a stabilizing peptide.
8. The method according to claim 7 , wherein the stabilizing peptide is a C-terminal acidic tail domain peptide of a synuclein family.
9. The method according to claim 8 , wherein the stabilizing peptide comprises amino acid residues 103-115 (SEQ ID NO: 22), amino acid residues 114-126 (SEQ ID NO: 23), amino acid residues 119-140 (SEQ ID NO: 24) or amino acid residues 130-140 (SEQ ID NO: 25) of the C-terminal acidic tail domain of α-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of β-synuclein (SEQ ID NO: 27), amino acid residues 1-127 of γ-synuclein (SEQ ID NO: 28), or amino acid residues 96-127 of the C-terminal acidic tail domain of γ-synuclein (SEQ ID NO: 29).
10. The method according to claim 1 , wherein the step of stimulating NK cells in a whole blood sample thereby artificially activating the NK cells to generate and secrete NK cell-secreting cytokines is performed in solution containing a carrier protein.
11. The method according to claim 1 , wherein the IL-12 is in the form of a fusion protein with a stabilizing peptide, wherein the fusion protein comprises an amino acid sequence of SEQ ID NO: 4 or 6, or the IL-15 is in the form of a fusion protein with a stabilizing peptide, wherein the fusion protein comprises an amino acid sequence of SEQ ID NO: 8.
12. The method according to claim 2 , wherein interleukin 2 is in the form of a fusion protein with a stabilizing peptide.
13. The method according to claim 1 , wherein the interleukin 15 is in the form of a fusion protein with a stabilizing peptide.
14. A method of measuring natural killer (NK) cell activity, comprising:
stimulating NK cells in a whole blood sample by incubating the whole blood sample with an agent comprising the isolated stimulating cytokine interleukin 2 in the form of a fusion protein with a stabilizing peptide, thereby artificially activating the NK cells to generate and secrete NK cell-secreting cytokines; and
measuring an amount of the NK cell-secreting cytokines secreted into the whole blood sample and using the amount as a measure to evaluate NK cell activity;
wherein the whole blood sample is a human whole blood sample.
15. The method of claim 14 , wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), or both.
16. A method of measuring natural killer (NK) cell activity, comprising:
stimulating NK cells in a whole blood sample by incubating the whole blood sample with an agent comprising at least one isolated stimulating cytokine selected from the group consisting of interleukin 2 and interleukin 15, thereby artificially activating the NK cells to generate and secrete NK cell-secreting cytokines; and
measuring an amount of the NK cell-secreting cytokines secreted into the whole blood sample and using the amount as a measure to evaluate NK cell activity;
wherein the at least one stimulating cytokine is in the form of a fusion protein with a stabilizing peptide.
17. The method according to claim 16 , wherein the stimulation of the NK cells is performed by incubating the whole blood sample with at least interleukin 2 in the form of a fusion protein with a stabilizing peptide.
18. The method according to claim 16 , wherein the stimulation of the NK cells is performed by incubating the whole blood sample with at least interleukin 15 in the form of a fusion protein with a stabilizing peptide.
19. The method according to claim 16 , wherein the stimulation of the NK cells is performed by incubating the whole blood sample with interleukin 2 and interleukin 12.
20. The method according to claim 16 , wherein the stimulation of the NK cells is performed by incubating the whole blood sample with interleukin 15 and interleukin 12.
21. The method according to claim 16 , wherein the NK cell-secreting cytokines are selected from the group consisting of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1β(MIP-1β).
22. The method according to claim 16 , wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ).
23. The method according to claim 16 , wherein the NK cell-secreting cytokine is tumor necrosis factor-alpha (TNF-α).
24. The method according to claim 16 , wherein the stabilizing peptide is a C-terminal acidic tail domain peptide of a synuclein family.
25. The method according to claim 16 , wherein the stabilizing peptide comprises amino acid residues 103-115 (SEQ ID NO: 22), amino acid residues 114-126 (SEQ ID NO: 23), amino acid residues 119-140 (SEQ ID NO: 24) or amino acid residues 130-140 (SEQ ID NO: 25) of the C-terminal acidic tail domain of α-synuclein, amino acid residues 85-134 of the C-terminal acidic tail domain of β-synuclein (SEQ ID NO: 27), amino acid residues 1-127 of γ-synuclein (SEQ ID NO: 28), or amino acid residues 96-127 of the C-terminal acidic tail domain of γ-synuclein (SEQ ID NO: 29).
26. The method according to claim 16 , wherein the fusion protein comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 8.
27. The method according to claim 16 , wherein the stimulation of the NK cells is performed by incubating the whole blood sample with interleukin 2 in the form of a fusion protein with a stabilizing peptide.
28. The method according to claim 16 , wherein the stimulation of the NK cells is performed by incubating the whole blood sample with interleukin 15 in the form of a fusion protein with a stabilizing peptide.
29. The method according to claim 27 , wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ).
30. The method according to claim 28 , wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ).
31. The method of claim 1 , wherein the stimulation of the NK cells is performed by incubating the whole blood sample with interleukin 2 in the form of a fusion protein with a stabilizing peptide, and wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ).
32. The method of claim 31 , wherein the stabilizing peptide comprises the amino acid sequence of SEQ ID NO: 24.
33. The method of claim 32 , wherein the interleukin 2 in the form of a fusion protein with a stabilizing peptide comprises the amino acid sequence of SEQ ID NO: 2.