# US 11,442,069 B2 · Method of diagnosing cancer and diagnosis kit using measurement of NK cell activity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,442,069 B2 - Document ID: US11442069B2 - Status: Granted - Kind: B2 - Application number: 13/985,301 - Filed: 2012-02-10 - Published: 2014-01-16 - Granted: 2022-09-13 - Earliest claimed: 2011-02-14 - Assignees: Atgen Co., Ltd. - Applicants: Jae Myun Lee; Joo Chun Yoon; Sang Woo Park; Jong Sun Kim - Inventors: Jae Myun Lee; Joo Chun Yoon; Sang Woo Park; Jong Sun Kim - Web page: https://patentgrep.com/patent/US11442069B2 ## CPC - G01N 33/6866: {Interferon} - C07K 14/47: from mammals - C07K 14/54: Interleukins [IL] - C07K 14/5434: {IL-12} - C07K 14/5443: {IL-15} - C07K 14/55: IL-2 - G01N 33/575: for cancer - C07K 2319/00: Fusion polypeptide - C07K 2319/21: containing a His-tag - G01N 2800/54: Determining the risk of relapse - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) The method according to claim 1, wherein the composition further comprises interleukin 2. ### Claim 3 (depends on 1) 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). ### Claim 4 (depends on 1) The method according to claim 1, wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ). ### Claim 5 (depends on 1) The method according to claim 1, wherein the NK cell-secreting cytokine is tumor necrosis factor-alpha (TNF-α). ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 1) 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. ### Claim 8 (depends on 7) The method according to claim 7, wherein the stabilizing peptide is a C-terminal acidic tail domain peptide of a synuclein family. ### Claim 9 (depends on 8) 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). ### Claim 10 (depends on 1) 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. ### Claim 11 (depends on 1) 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. ### Claim 12 (depends on 2) The method according to claim 2, wherein interleukin 2 is in the form of a fusion protein with a stabilizing peptide. ### Claim 13 (depends on 1) The method according to claim 1, wherein the interleukin 15 is in the form of a fusion protein with a stabilizing peptide. ### Claim 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. ### Claim 15 (depends on 14) The method of claim 14, wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), or both. ### Claim 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. ### Claim 17 (depends on 16) 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. ### Claim 18 (depends on 16) 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. ### Claim 19 (depends on 16) 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. ### Claim 20 (depends on 16) 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. ### Claim 21 (depends on 16) 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). ### Claim 22 (depends on 16) The method according to claim 16, wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ). ### Claim 23 (depends on 16) The method according to claim 16, wherein the NK cell-secreting cytokine is tumor necrosis factor-alpha (TNF-α). ### Claim 24 (depends on 16) The method according to claim 16, wherein the stabilizing peptide is a C-terminal acidic tail domain peptide of a synuclein family. ### Claim 25 (depends on 16) 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). ### Claim 26 (depends on 16) 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. ### Claim 27 (depends on 16) 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. ### Claim 28 (depends on 16) 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. ### Claim 29 (depends on 27) The method according to claim 27, wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ). ### Claim 30 (depends on 28) The method according to claim 28, wherein the NK cell-secreting cytokine is interferon-gamma (IFN-γ). ### Claim 31 (depends on 1) 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). ### Claim 32 (depends on 31) The method of claim 31, wherein the stabilizing peptide comprises the amino acid sequence of SEQ ID NO: 24. ### Claim 33 (depends on 32) 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.