# US 2013/0280221 A1 · Interleukin 15 as Selectable Marker for Gene Transfer in Lymphocytes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0280221 A1 - Document ID: US20130280221A1 - Status: Published application - Kind: A1 - Application number: 13/821,474 - Filed: 2011-09-06 - Published: 2013-10-24 - Earliest claimed: 2010-09-08 - Assignees: Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus - Inventors: Kurt Schonfeld; Christiane Knopp; Winfried Wels - Web page: https://patentgrep.com/patent/US20130280221A1 ## CPC - C07K 14/5443: {IL-15} - A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells - A61K 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells - A61K 40/31: Chimeric antigen receptors [CAR] - A61K 40/35: Cytokines - A61K 40/4254: {Adhesion molecules, e.g. NRCAM, EpCAM or cadherins} - C07K 14/7051: {T-cell receptor (TcR)-CD3 complex} - C07K 14/70517: {CD8} - C07K 16/18: against material from animals or humans - C12N 15/85: for animal cells - C12N 15/86: Viral vectors - A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells} - C07K 2317/622: Single chain antibody (scFv) - C07K 2319/02: containing a signal sequence - C07K 2319/03: containing a transmembrane segment - C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies - C07K 2319/74: containing a fusion for binding to a cell surface receptor - C12N 2740/16043: viral genome or elements thereof as genetic vector - 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) ## Abstract The present invention relates to the use of interleukin-15 (IL-15) as selectable marker for gene transfer, preferably of at least one gene of therapeutic interest, into a mammalian cell or cell line, in particular a human cell or cell line. The present invention furthermore relates to transgenic mammalian cells or cell lines expressing IL-15 as selectable marker and co-expressing at least one protein of interest encoded by at least one gene of interest, which is preferably a protein of therapeutic interest. The present invention is in particular suitable for chimeric antigen receptors (CARs) as the gene or protein of interest and their expression in lymphocytes. The transgenic mammalian cells and cell lines are furthermore suitable for use as a medicament, in particular in the treatment of cancer and in immunotherapy, such as adoptive, target-cell specific immunotherapy. ## Claims ### Claim 1 A method for ex vivo or in vitro gene transfer into a mammalian cell, wherein said method comprises the use of interleukin-15 (IL-15) as a selectable marker, wherein the mammalian cell is selected from effector cells of the immune system which require cytokines for growth and survival, and wherein the effector cells of the immune system are T cells or natural killer (NK) cells, wherein the method comprises the transfer, into the mammalian cell, of an expression construct comprising a first nucleotide sequence encoding IL-15 and a second nucleotide sequence encoding a protein of interest, which is a protein other than IL-15, wherein the expression of the IL-15 as a selectable marker results in survival or growth of the mammalian cell in the absence of exogenously added IL-15, and wherein the IL-15 has the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 1, or is encoded by the nucleotide sequence of SEQ ID NO. 2 or a nucleotide sequence with at least 95% sequence identity to SEQ ID NO. 2. ### Claim 2 (depends on 1) The method according to claim 1, wherein the expression of the IL-15 as a selectable marker results in survival or growth of the mammalian cell in the absence of exogenously added IL-2. ### Claim 3 (depends on 1) The method according to claim 1, wherein the expression construct comprises a nucleotide sequence encoding human IL-15, selected from a nucleotide sequence encoding human IL-15 having the amino acid sequence of SEQ ID NO. 1, a nucleotide sequence comprising the nucleotide sequence of human IL-15 transcript variant 3 of SEQ ID NO. 2, or a complementary sequence thereof, or a codon-optimized sequence thereof, or a nucleotide sequence encoding an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 1, or a nucleotide sequence with at least 95% sequence identity to the nucleotide sequence of SEQ ID NO. 2. ### Claim 4 (depends on 1) The method according to claim 1, wherein the protein of interest is a protein of therapeutic interest. ### Claim 5 (depends on 4) The method according to claim 4, wherein the protein of therapeutic interest is a chimeric antigen receptor (CAR) that comprises the following: (i) a signal peptide; (ii) a target specific recognition domain that binds an antigen, receptor, peptide ligand or protein ligand of the target, wherein the target is a cell or a virus; (iii) a linker region, connecting domain (ii) and domain (iv); and (iv) an effector domain comprising a transmembrane region and one or more intracellular signaling domains. ### Claim 6 (depends on 5) The method according to claim 5, wherein the target specific recognition domain (ii) of the CAR comprises an antigen binding domain derived from an antibody against an antigen of the target, or a peptide that binds an antigen of the target, or a peptide or protein that binds an antibody that binds an antigen of the target, or a peptide or protein ligand that binds a receptor on the target, or a domain derived from a receptor that binds a peptide or protein ligand on the target, and/or, where domain (ii) of the CAR binds an antigen of the target, wherein the antigen is a tumor-associated surface antigen, a lineage-specific or tissue-specific surface antigen or a virus-specific surface antigen; and/or, where domain (ii) of the CAR comprises an antigen binding domain, which is derived from an antibody or an antibody fragment. ### Claim 7 (depends on 5) The method according to claim 5, wherein the linker region (iii) of the CAR comprises a hinge region derived from the human CD8 alpha-chain and/or wherein the effector domain (iv) of the CAR comprises the zeta-chain of the human CD3 complex of the T-cell receptor or a fragment thereof or a functional equivalent thereof. ### Claim 8 A transgenic mammalian cell expressing IL 15 as a selectable marker for gene transfer and co-expressing at least one protein of interest, wherein the mammalian cell is selected from effector cells of the immune system which require cytokines for growth and survival, and wherein the effector cells of the immune system are T cells or natural killer (NK) cells, wherein the protein of interest is a protein other than IL-15, wherein the IL-15 and a nucleotide sequence encoding the protein of interest were transferred into the cell using one expression construct or using different expression constructs, wherein the expression of the IL-15 as a selectable marker results in survival or growth of the mammalian cell in the absence of exogenously added IL-15 and/or IL-2, and wherein the IL-15 has the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 1, or is encoded by the nucleotide sequence of SEQ ID NO. 2 or a nucleotide sequence with at least 95% sequence identity to SEQ ID NO. 2. ### Claim 9 (depends on 8) The transgenic mammalian cell according to claim 8, wherein the protein of interest is a protein of therapeutic interest. ### Claim 10 (depends on 9) The transgenic mammalian cell according to claim 9, wherein the protein of therapeutic interest is a chimeric antigen receptor (CAR), wherein the CAR comprises the following: (i) a signal peptide; (ii) a target specific recognition domain that binds an antigen, receptor, peptide ligand or protein ligand of the target, wherein the target is a cell or a virus; (iii) a linker region, connecting domain (ii) and domain (iv); and (iv) an effector domain comprising a transmembrane region and one or more intracellular signaling domains. ### Claim 11 (depends on 8) The transgenic mammalian cell according to claim 8, wherein the IL-15 is encoded by a nucleotide sequence encoding human IL-15 having the amino acid sequence of SEQ ID NO. 1, a nucleotide sequence comprising the nucleotide sequence of human IL-15 transcript variant 3 of SEQ ID NO. 2, or a complementary sequence thereof, or a codon-optimized sequence thereof, or a nucleotide sequence encoding an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 1, or a nucleotide sequence with at least 95% sequence identity to the nucleotide sequence of SEQ ID NO. 2. ### Claim 12 (canceled) ### Claim 13 (depends on 8) A method for treating cancer or for use in immunotherapy, wherein said method comprises administering, to a subject in need of such treatment or immunotherapy, the cell of claim 8. ### Claim 14 (depends on 8) A pharmaceutical composition comprising the cell of claim 8, and a pharmaceutical carrier. ### Claim 15 (depends on 4) The method according to claim 4, wherein the protein of therapeutic interest is a chimeric antigen receptor (CAR). ### Claim 16 (depends on 10) The transgenic mammalian cell, according to claim 10, wherein the CAR comprises an antigen binding domain derived from an antibody against an antigen of the target, or a peptide that binds an antigen of the target, or a peptide or protein that binds an antibody that binds an antigen of the target, or a peptide or protein ligand that binds a receptor on the target, or a domain derived from a receptor that binds a peptide or protein ligand on the target, and/or, where domain (ii) of the CAR binds an antigen of the target, wherein the antigen is a tumor-associated surface antigen, a lineage-specific or tissue-specific surface antigen or a virus-specific surface antigen; and/or, where domain (ii) of the CAR comprises an antigen binding domain, which is derived from an antibody or an antibody fragment. ### Claim 17 (depends on 10) The transgenic mammalina cell, according to claim 10, wherein the linker region (iii) of the CAR comprises a hinge region derived from the human CD8 alpha-chain and/or wherein the effector domain (iv) of the CAR comprises the zeta-chain of the human CD3 complex of the T-cell receptor or a fragment thereof or a functional equivalent thereof.