# US 9,434,783 B2 · Shortened CD95-Fc variant fusion proteins, nucleic acids and host cells thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,434,783 B2 - Document ID: US9434783B2 - Status: Granted - Kind: B2 - Application number: 14/415,871 - Filed: 2013-07-18 - Published: 2015-06-18 - Granted: 2016-09-06 - Earliest claimed: 2012-07-18 - Assignees: Apogenix AG - Applicants: Apogenix GmbH - Inventors: Oliver Hill; Christian Gieffers; Meinolf Thiemann - Web page: https://patentgrep.com/patent/US9434783B2 ## CPC - C07K 16/18: against material from animals or humans - A61K 38/1793: {for cytokines; for lymphokines; for interferons} - A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines} - A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics - A61P 7/00: Drugs for disorders of the blood or the extracellular fluid - A61P 9/00: Drugs for disorders of the cardiovascular system - A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis - A61P 13/12: of the kidneys - A61P 25/00: Drugs for disorders of the nervous system - A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] - A61P 31/04: Antibacterial agents - A61P 31/18: for HIV - A61P 35/00: Antineoplastic agents - A61P 35/02: specific for leukemia - A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) - C07K 14/525: Tumour necrosis factor [TNF] - C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)} - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) - C07K 2317/52: Constant or Fc region; Isotype - C07K 2317/524: CH2 domain - C07K 2317/526: CH3 domain - C07K 2319/00: Fusion polypeptide - C07K 2319/02: containing a signal sequence - C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto - C07K 2319/74: containing a fusion for binding to a cell surface receptor - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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 an isolated fusion protein comprising an extracellular CD95 domain or a functional fragment thereof and an Fc domain or functional fragment thereof, formulations providing such fusion protein in a stable form as well as a method for producing such a fusion protein. ## Claims ### Claim 1 A fusion protein comprising (i) at least a functional fragment of an N-terminally truncated extracellular cluster of differentiation 95 (CD95) domain, wherein the functional fragment of the N-terminally truncated extracellular CD95 domain consists of the amino acid sequence of SEQ ID NO: 11 or 12, or a sequence having at least 97% identity to SEQ ID NO: 11 or 12, and (ii) at least an Fc domain or a functional fragment thereof, wherein the Fc domain is a human Fc domain consisting of the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 97% identity to SEQ ID NO: 8. ### Claim 2 (depends on 1) The fusion protein according to claim 1, wherein the functional fragment of the N-terminally truncated extracellular CD95 domain consists of the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. ### Claim 3 (depends on 1) The fusion protein according to claim 1, said fusion protein being free of a signal peptide. ### Claim 4 (depends on 1) The fusion protein according to claim 1, comprising an N-terminally located signal peptide. ### Claim 5 (depends on 4) The fusion protein according to claim 4, wherein the N-terminally located signal peptide is a naturally occurring CD95 signal peptide. ### Claim 6 (depends on 5) The fusion protein according to claim 5, wherein the N-terminally located signal peptide is an artificial signal peptide. ### Claim 7 (depends on 6) The fusion protein according to claim 6, wherein the N-terminally located signal peptide comprises SEQ ID NO:2 or a sequence having at least 90%, or at least 95% identity to SEQ ID NO:2. ### Claim 8 (depends on 1) The fusion protein according to claim 1, wherein the functional fragment of the N-terminally truncated extracellular CD95 domain is located N-terminally to the at least an Fc domain or the functional fragment thereof. ### Claim 9 (depends on 6) The fusion protein according to claim 6, wherein the signal peptide is directly fused to the functional fragment of the N-terminally truncated extracellular CD95 domain. ### Claim 10 (depends on 1) The fusion protein according to claim 1, wherein the functional fragment of the N-terminally truncated extracellular CD95 domain is directly fused to the N-terminal of the Fc domain or to the functional fragment thereof, wherein the two domains have an overlapping amino acid of serine. ### Claim 11 (depends on 10) The fusion protein according to claim 10, comprising a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and a sequence having at least 97% identity to SEQ ID NO:5, or SEQ ID NO:6. ### Claim 12 (depends on 1) The fusion protein of claim 1, wherein the at least an Fc domain or the functional fragment thereof provides a pl in the range of 4.0-8.5. ### Claim 13 (depends on 12) The fusion protein according to claim 12, wherein the pl is in the range of 4.5-7.8. ### Claim 14 (depends on 1) The fusion protein according to claim 1, comprising sialic acids. ### Claim 15 (depends on 1) The fusion protein according to claim 1, which is N-terminally blocked. ### Claim 16 (depends on 1) The fusion protein according to claim 1, which is not glycosylated in the CH2 domain of the Fc domain or of the functional fragment thereof. ### Claim 17 (depends on 1) A formulation comprising the fusion protein according to claim 1. ### Claim 18 (depends on 17) The formulation of claim 17 further comprising: (a) a phosphate, (b) a viscosity enhancing agent, and/or (c) having a pH value in the range of 4-8. ### Claim 19 (depends on 15) The fusion protein according to claim 15, wherein the N-terminally blocked fusion proteins are blocked by pyro-Glu modification. ### Claim 20 (depends on 18) The formulation of claim 18, wherein the phosphate is in an amount of 20 mM to 100 mM, and the viscosity enhancing agent is sorbitol in an amount of about 0.1-10 weight %. ### Claim 21 (depends on 1) A nucleic acid molecule encoding the fusion protein according to claim 1. ### Claim 22 (depends on 21) An isolated host cell comprising a nucleic acid molecule of claim 21. ### Claim 23 (depends on 1) The method for producing the fusion protein according to claim 1, comprising recombinantly expressing a nucleic acid molecule encoding the fusion protein. ### Claim 24 (depends on 1, 23) The method of claim 23, comprising the steps of: (a) producing the fusion protein according to claim 1 by a fed-batch production process providing a cell harvest, and (b) isolating the fusion protein from the cell harvest. ### Claim 25 (depends on 23) The method according to claim 23, wherein step (a) comprises a series of cultivation steps of a given master cell batch until relevant harvest parameters are reached, followed by cell sedimentation and filtration of fusion protein containing supernatant, and/or step (b) comprises capture chromatography, virus inactivation, a series of anion and/or cation chromatography, virus filtration and/or adjustment to a desired final protein concentration. ### Claim 26 (depends on 21) A formulation comprising the nucleic acid molecule according to claim 21.