# US 8,540,982 B2 · Methods for inducing a natural killer (NK) cell-mediated immune response and for increasing 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 8,540,982 B2 - Document ID: US8540982B2 - Status: Granted - Kind: B2 - Application number: 13/443,703 - Filed: 2012-04-10 - Published: 2012-10-11 - Granted: 2013-09-24 - Earliest claimed: 2007-03-22 - Assignees: Dendreon Corporation - Applicants: Nadeem Sheikh; Lori Jones - Inventors: Nadeem Sheikh; Lori Jones - Web page: https://patentgrep.com/patent/US8540982B2 ## CPC - G01N 33/5047: {Cells of the immune system} - A61K 38/193: {Colony stimulating factors [CSF]} - A61K 38/57: from animals; from humans {(A61K38/553, A61K38/556 take precedence)} - A61P 35/00: Antineoplastic agents - A61P 37/02: Immunomodulators - G01N 2333/535: Granulocyte CSF; Granulocyte-macrophage CSF - G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis - 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) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS ## Abstract This disclosure relates to methods of inducing a natural killer (NK) cell-mediated immune response and increasing NK activity in a mammal for the treatment of tumors and virus infections, comprising isolating peripheral blood mononuclear cells (PBMCs) from a subject, exposing them in vitro to a protein conjugate comprising granulocyte macrophage colony stimulating factor (GM-CSF) covalently linked to a soluble peptide antigen to activate the PBMCs, and administering the activated PBMCs to the subject. The method also relates to assessing NK cell activity of activated PBMCs to determine whether the subject has had a therapeutically effective response to the protein conjugate. ## Claims ### Claim 1 A method for detecting a cytotoxic NK cell-mediated immune response in a subject, comprising: (a) isolating peripheral blood mononuclear cells (PBMCs) comprising dendritic cells from the subject; (b) exposing said PBMCs in vitro to a protein conjugate comprising granulocyte macrophage colony stimulating factor (GM-CSF) covalently linked to a soluble peptide antigen, said protein conjugate comprising SEQ ID NO: 5 or SEQ ID NO: 7, under conditions effective to activate said PBMCs; (c) assessing the level of an NK cell response in the activated PBMCs; (d) administering said activated PBMCs to the subject; (e) repeating steps (a) and (b) at least 10 days after the previous step (d) has occurred, resulting in a second isolation of PBMCs; and (f) assessing the level of an NK cell response in the activated PBMCs from the second isolation, wherein said activated PBMCs are effective in activating NK cells to produce a cytotoxic cellular response at a level higher than that produced by the PBMCs which have not been activated by the protein conjugate. ### Claim 2 (depends on 1) The method of claim 1, wherein said protein conjugate comprises a fusion protein having the sequence depicted as SEQ ID NO: 5. ### Claim 3 (depends on 1) The method of claim 1, wherein said protein conjugate comprises a fusion protein having the sequence depicted as SEQ ID NO: 7. ### Claim 4 (depends on 1) The method of claim 1, wherein said mammalian subject is a human. ### Claim 5 (depends on 1) The method of claim 1, wherein said protein conjugate is produced in a baculovirus expression system. ### Claim 6 (depends on 1) The method of claim 1, wherein said method further comprises repeating steps (a), (b) and (c) at least once with each cycle beginning at least 14 days after step (c) has occurred. ### Claim 7 (depends on 6) The method of claim 6, wherein said step (d) is performed in vitro by CD336 surface expression. ### Claim 8 (depends on 6) The method of claim 6, wherein said step (d) is performed in vitro lysis of the K562 tumor line. ### Claim 9 A method for determining whether a subject has had a therapeutically effective response to a protein conjugate, comprising, (a) isolating PBMCs comprising dendritic cells from the subject; (b) exposing said PBMCs in vitro to a protein conjugate comprising granulocyte macrophage colony stimulating factor (GM-CSF) covalently linked to a soluble peptide antigen, said protein conjugate comprising SEQ ID NO: 5 or SEQ ID NO: 7, under conditions effective to activate said PBMCs; (c) administering said activated PBMCs to the subject; (d) repeating steps (a) and (b) at least 10 days after step (c) has occurred; and (e) determining a change in NK cell activity of the activated PBMCs. ### Claim 10 (depends on 9) The method of claim 9, wherein determining step (e) includes assessing the NK activity of the activated PBMCs from the previous isolation; and determining the change in the NK activity over the level of NK activity of the activated PBMCs prior to the first administration. ### Claim 11 (depends on 9) The method of claim 9, wherein said step (e) is performed in vitro by CD336 surface expression. ### Claim 12 (depends on 9) The method of claim 9, wherein said step (e) is performed in vitro lysis of the K562 tumor line. ### Claim 13 (depends on 12) The method of claim 12, wherein said steps (a) through (c) are performed a total of two times and wherein 14 days has elapsed since the previous step (c) has occurred. ### Claim 14 (depends on 9) The method of claim 9, wherein said steps (a) through (c) are performed a total of three times and wherein 14 days has elapsed since the previous step (c) has occurred.