# US 11,896,616 B2 · Stimulatory cell lines for ex vivo expansion and activation of natural killer cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,896,616 B2 - Document ID: US11896616B2 - Status: Granted - Kind: B2 - Application number: 16/497,682 - Filed: 2018-03-27 - Published: 2020-01-16 - Granted: 2024-02-13 - Earliest claimed: 2017-03-27 - Assignees: National University of Singapore - Applicants: National University of Singapore - Inventors: Takahiro Kamiya; Dario Campana - Web page: https://patentgrep.com/patent/US11896616B2 ## CPC - A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics - C07K 14/5443: {IL-15} - A61K 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells - A61K 40/42: Cancer antigens - A61P 35/00: Antineoplastic agents - C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex} - C12N 5/0646: {Natural killers cells [NK], NKT cells} - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C07K 14/54: Interleukins [IL] - C07K 2317/622: Single chain antibody (scFv) - C12N 2502/30: tumour cells - C12N 2510/00: Genetically modified cells - C12N 2510/04: Immortalised cells - C12N 2740/10043: viral genome or elements thereof as genetic vector - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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) - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The present invention relates to genetically engineered cell populations derived from an immortalised/cancerous cell that do not express MHC class I molecules but that are modified to express membrane-bound IL-15, membrane-bound 4-1 BBL ligand, and at least one other membrane bound molecule, such as an interleukin or anti-CD3 antibody. The co-culture of said cells with a population of immune cells results in the activation and expansion of at least one subpopulation of immune cells. Expanded populations of NK cells derived from the co-culture of a mixed cell culture with the stimulatory cell lines may be used in methods of treating cancer or an infectious disease. In a separate embodiment, a plurality of nucleic acids for use in preparing the engineered cell population are provided. ## Claims ### Claim 1 A genetically engineered cell population that does not express major histocompatibility complex (MHC) I molecules, wherein said genetically engineered cell population is derived from K562 cells, wherein said genetically engineered cell population is modified to express membrane-bound interleukin-15 (mbIL15), wherein the mbIL15 comprises the amino acid sequence of SEQ ID NO: 2, wherein said genetically engineered cell population is modified to express membrane-bound 4-1BB ligand (mb4-1BBL) comprising the amino acid sequence of SEQ ID NO: 14, wherein said genetically engineered cell population is modified to express membrane-bound interleukin-18 (mbIL18), wherein the mbIL18 comprises the amino acid sequence of SEQ ID NO: 8, wherein said genetically engineered cell population is modified to express membrane-bound interleukin-12A (mbIL12A), wherein the mbIL12A comprises the amino acid sequence of SEQ ID NO: 4, wherein said genetically engineered cell population is modified to express membrane-bound interleukin-12B (mbIL12B), wherein the mbIL12B comprises the amino acid sequence of SEQ ID NO: 6, wherein said genetically engineered cell population does not express major histocompatibility complex (MHC) I molecules, and wherein co-culture of said genetically engineered cell population with a population of natural killer (NK) cells results in the activation and expansion of the NK cells. ### Claim 2 (depends on 1) The genetically engineered cell population of claim 1, wherein the genetically engineered cell population lacks expression of MHC II molecules. ### Claim 3 (depends on 1) The genetically engineered cell population of claim 1, wherein the genetically engineered cell population is further modified to express: membrane-bound interleukin-21 (mbIL21) comprising the sequence of SEQ ID NO: 10; or membrane-bound interleukin-22 (IL22) comprising the sequence of SEQ ID NO: 12. ### Claim 4 (depends on 1) The genetically engineered cell population of claim 1, wherein the cells further comprise a membrane-bound anti-CD3 antibody (mbα-CD3), an antibody fragment thereof, or an anti-CD3 scFv. ### Claim 5 (depends on 4) The genetically engineered cell population of claim 4, wherein the mbα-CD3 is a monoclonal antibody. ### Claim 6 (depends on 5) The genetically engineered cell population of claim 5, wherein the mbα-CD3 targets an epitope within the epsilon subunit of the CD3 receptor. ### Claim 7 A genetically engineered cell population that does not express major histocompatibility complex (MHC) I molecules, wherein said genetically engineered cell population is derived from K562 cells, wherein said genetically engineered cell population is modified to express membrane-bound interleukin-15 (mbIL15), wherein the mbIL15 is encoded by a nucleic acid sequence that comprises SEQ ID NO: 1, wherein said genetically engineered cell population is modified to express membrane-bound 4-1BB ligand (mb4-1BBL) comprising the amino acid sequence of SEQ ID NO: 14, wherein said genetically engineered cell population is modified to express membrane-bound interleukin-18 (mbIL18), wherein the mbIL18 comprises the amino acid sequence of SEQ ID NO: 8, wherein said genetically engineered cell population is modified to express membrane-bound interleukin-12A (IL12A), wherein the mbIL12A comprises the amino acid sequence of SEQ ID NO: 4, wherein said genetically engineered cell population is modified to express membrane-bound interleukin-12B (IL12B), wherein the mbIL12B comprises the amino acid sequence of SEQ ID NO: 6, wherein said genetically engineered cell population does not express major histocompatibility complex (MHC) I molecules, and wherein co-culture of said genetically engineered cell population with a population of natural killer (NK) cells results in the activation and expansion of the NK cells. ### Claim 8 (depends on 7) The genetically engineered cell population of claim 7, wherein the amino acid sequence of SEQ ID NO:14 is encoded by a nucleic acid sequence that comprises SEQ ID NO: 13. ### Claim 9 (depends on 7) The genetically engineered cell population of claim 7, wherein the engineered cell population is further modified to express membrane-bound interleukin-21 or membrane-bound interleukin-22.