# US 11,078,249 B2 · Heterodimeric Fc-fused cytokine and pharmaceutical composition comprising the same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,249 B2 - Document ID: US11078249B2 - Status: Granted - Kind: B2 - Application number: 16/886,177 - Filed: 2020-05-28 - Published: 2020-11-19 - Granted: 2021-08-03 - Earliest claimed: 2016-08-10 - Assignees: Ajou University Industry-Academic Cooperation Foundation - Applicants: Ajou University Industry-Academic Cooperation Foundation - Inventors: Yong Sung Kim; Keunok Jung; Ji Hee Ha; Dong Ki Choi; Hye Ji Choi; Ye Jin Kim - Web page: https://patentgrep.com/patent/US11078249B2 ## CPC - C07K 14/5434: {IL-12} - A61K 38/208: {IL-12} - A61P 35/00: Antineoplastic agents - C07K 14/54: Interleukins [IL] - C07K 14/59: Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH] - 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 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto - Y02E 60/10: Energy storage using batteries - 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 - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract The present invention relates to a heterodimeric Fc-fused protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc pair and a physiologically active protein composed of two or more different subunits, wherein one or more subunits of the physiologically active protein are linked separately to one or more ends of the N-terminus or C-terminus of the first Fc region and/or the second Fc region, and CH3 domains of the first Fc region and the second Fc region are mutated so as to promote the heterodimeric Fc formation. Moreover, the present invention relates to a pharmaceutical composition comprising the heterodimeric Fc-fused protein. The heterodimeric Fc-fused protein according to the present invention has an advantage in that it can retain the activity of a naturally occurring physiologically active protein whose two or more different subunits exhibit physiological activity by forming a protein complex, because the physiologically active protein can be linked to an immunoglobulin heterodimeric Fc such that the naturally occurring form and structure of the fused protein thereof can be maintained. When the heterodimeric Fc-fused protein according to the present invention is used, there is an advantage in that the in vivo half-life of the physiologically active protein contained in the heterodimeric Fc-fused protein can be significantly increased due to the Fc-mediated long half-life such that various physiological activities thereof in vivo can be long-lasting. ## Claims ### Claim 1 A heterodimeric Fc-fused protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are linked separately to the first Fc region and the second Fc region, or to the second Fc region and the first Fc region, respectively, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc regions, and wherein CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations promoting heterodimerization. ### Claim 2 (depends on 1) The heterodimeric Fc-fused protein according to claim 1, wherein the p40 and p35 subunits are each linked to the N-terminus of the Fc regions. ### Claim 3 (depends on 2) The heterodimeric Fc-fused protein according to claim 2, wherein the p40 or p35 subunit is linked to its corresponding Fc region by a linker. ### Claim 4 (depends on 3) The heterodimeric Fc-fused protein according to claim 3, wherein the p35 subunit is linked to its corresponding Fc region by a linker. ### Claim 5 (depends on 4) The heterodimeric Fc-fused protein according to claim 4, wherein the linker comprises a (G4S)3 linker. ### Claim 6 (depends on 1) The heterodimeric Fc-fused protein according to claim 1, wherein each of the first Fc region and the second Fc region is from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4. ### Claim 7 (depends on 1) The heterodimeric Fc-fused protein according to claim 1, wherein the first Fc region and the second Fc region are comprised in a whole antibody form selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4. ### Claim 8 (depends on 1) The heterodimeric Fc-fused protein according to claim 1, wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations, wherein the mutation at the CH3 domain of the first Fc region or the second Fc region comprises one or more mutations selected from the group consisting of: (a) glutamic acid (E), arginine (R), methionine (M), aspartic acid (D), or histidine (H) substitution of the amino acid residue at position 370 in the CH3 domain of the first Fc region; (b) glutamic acid (E) substitution of the amino acid residue at position 360 in the CH3 domain of the first Fc region; (c) tryptophan (W) substitution of the amino acid residue at position 409 in the CH3 domain of the first Fc region; (d) asparagine (N), aspartic acid (D), alanine (A), isoleucine (I), glycine (G), or methionine (M) substitution of the amino acid residue at position 357 in the CH3 domain of the second Fc region, and threonine (T) or tryptophan (W) substitution of the amino acid reside at position 364 in the CH3 domain of the second Fc region; and (e) threonine (T) substitution of the amino acid residue at position 405 in the CH3 domain of the second Fc region, and valine (V) substitution of the amino acid residue at position 399 in the CH3 domain of the second Fc region, wherein mutation positions are numbered according to the EU index. ### Claim 9 (depends on 8) The heterodimeric Fc-fused protein according to claim 8, wherein the CH3 domain of the second Fc region comprises: arginine (R) substitution of the amino acid residue at position 347, wherein the positions are numbered according to the EU index. ### Claim 10 (depends on 9) The heterodimeric Fc-fused protein according to claim 9, wherein the CH3 domains in the first Fc region and the second Fc region further include comprise the following residues: (i) cysteine (C) substitution at position 349 in the CH3 domain of the first Fc region; and (ii) cysteine (C) substitution at position 354 in the CH3 domain of the second Fc region, wherein the positions are numbered according to the EU index. ### Claim 11 (depends on 1) The heterodimeric Fc-fused protein according to claim 1, wherein the IL-12 is human IL-12. ### Claim 12 A pharmaceutical composition comprising a heterodimeric Fc-fused protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, and a pharmaceutically acceptable carrier, wherein the p40 and p35 subunits of IL-12 are linked separately to the first Fc region and the second Fc region, or to the second Fc region and the first Fc region, respectively, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc regions, and wherein CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations promoting heterodimerization. ### Claim 13 (depends on 12) The pharmaceutical composition according to claim 12, wherein the p40 and p35 subunits are each linked to the N-terminus of the Fc regions. ### Claim 14 (depends on 13) The pharmaceutical composition according to claim 13, wherein the p40 or p35 subunit is linked to its corresponding Fc region by a linker. ### Claim 15 (depends on 14) The pharmaceutical composition according to claim 14, wherein the p35 subunit is linked to its corresponding Fc region by a linker. ### Claim 16 (depends on 15) The pharmaceutical composition according to claim 15, wherein the linker comprises a (G4S)3 linker. ### Claim 17 (depends on 12) The pharmaceutical composition according to claim 12, wherein each of the first Fc region and the second Fc region is from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4. ### Claim 18 (depends on 12) The pharmaceutical composition according to claim 12, wherein the first Fc region and the second Fc region are comprised in a whole antibody form selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4. ### Claim 19 (depends on 12) The pharmaceutical composition according to claim 12, wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations, wherein the mutation at the CH3 domain of the first Fc region or the second Fc region includes comprises one or more mutations selected from the group consisting of: (a) glutamic acid (E), arginine (R), methionine (M), aspartic acid (D), or histidine (H) substitution of the amino acid residue at position 370 in the CH3 domain of the first Fc region; (b) glutamic acid (E) substitution of the amino acid residue at position 360 in the CH3 domain of the first Fc region; (c) tryptophan (W) substitution of the amino acid residue at position 409 in the CH3 domain of the first Fc region; (d) asparagine (N), aspartic acid (D), alanine (A), isoleucine (I), glycine (G), or methionine (M) substitution of the amino acid residue at position 357 in the CH3 domain of the second Fc region, and threonine (T) or tryptophan (W) substitution of the amino acid reside at position 364 in the CH3 domain of the second Fc region; and (e) threonine (T) substitution of the amino acid residue at position 405 in the CH3 domain of the second Fc region, and valine (V) substitution of the amino acid residue at position 399 in the CH3 domain of the second Fc region, wherein mutation positions are numbered according to the EU index. ### Claim 20 (depends on 19) The pharmaceutical composition according to claim 19, wherein the CH3 domain of the second Fc region comprises: arginine (R) substitution of the amino acid residue at position 347, wherein the positions are numbered according to the EU index. ### Claim 21 (depends on 20) The pharmaceutical composition according to claim 20, wherein the CH3 domains in the first Fc region and the second Fc region further include comprise the following residues (wherein the positions are numbered according to the EU index): (i) cysteine (C) substitution at position 349 in the CH3 domain of the first Fc region; and (ii) cysteine (C) substitution at position 354 in the CH3 domain of the second Fc region, wherein the positions are numbered according to the EU index. ### Claim 22 (depends on 12) The pharmaceutical composition according to claim 12, wherein the IL-12 is human IL-12.