# US 2019/0169252 A1 · Cytokine fused to immunoglobulin fc heterodimer and pharmaceutical composition comprising same

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- Number: US 2019/0169252 A1
- Document ID: US20190169252A1
- Status: Published application
- Kind: A1
- Application number: 16/323,839
- Filed: 2017-08-10
- Published: 2019-06-06
- 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/US20190169252A1

## 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 a physiologically active protein,
wherein the physiologically active protein is composed of two or more different subunits, wherein the two or more different subunits exhibit a physiological activity by forming a protein complex,
wherein the two or more different subunits of the physiologically active protein are linked to one or more ends of N-terminus or C-terminus of the first Fc region and/or the second Fc region, and
wherein CH3 domains of the first Fc region and the second Fc region are mutated so as to promote heterodimeric Fc formation.

### Claim 2 (depends on 1)

The heterodimeric Fc-fused protein according to claim 1, wherein one of the two or more different subunits of the physiologically active protein is linked only to any one end of the N-terminus or C-terminus of the first Fc region or the second Fc region, and the remaining subunit(s) of the physiologically active protein is(are) linked to one end of the N-terminus and C-terminus of the other of said first and the second Fc region.

### Claim 3 (depends on 1)

The heterodimeric Fc-fused protein according to claim 1, wherein the two or more different subunits of a physiologically active protein are linked separately to each of the N-terminus and/or C-terminus of each of the first Fc region and the second Fc region.

### Claim 4 (depends on 1)

The heterodimeric Fc-fused protein according to claim 1, wherein the physiologically active protein is selected from the group consisting of interleukin-12 (IL-12), interleukin-23 (IL-23), interleukin-27 (L-27), interleukin-35 (IL-35), and follicle stimulating hormone (FSH).

### Claim 5 (depends on 4)

The heterodimeric Fc-fused protein according to claim 4, wherein the physiologically active protein is IL-12.

### Claim 6 (depends on 5)

The heterodimeric Fc-fused protein according to claim 5, wherein either p35 or p40 subunit of IL-12 is linked only to any one end of the N-terminus or C-terminus of the first Fc region or the second Fc region, and the other subunit is linked to by a linker to any one end of the N-terminus or C-terminus of the other of said first Fc region and the second Fc region.

### Claim 7 (depends on 5)

The heterodimeric Fc-fused protein according to claim 5, wherein the p35 and p40 subunits of IL-12 are linked separately to each of the N-terminus and/or C-terminus of each of the first Fc region and the second Fc region.

### Claim 8 (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 derived from an Fc region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE.

### Claim 9 (depends on 1)

The heterodimeric Fc-fused protein according to claim 1, wherein the first Fe region and the second Fc region are included in a whole antibody form consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE.

### Claim 10 (depends on 1)

The heterodimeric Fc-fused protein according to claim 1, wherein the mutation of the CH3 domain of the first Fc region or the second Fc region includes one or more mutations selected from the following group (wherein mutation positions are numbered according to the EU index):
(1) a substitution K370E, K370R, K370M, K370D or K370H of the amino acid residue at position K370 in the CH3 domain of the first Fc region;
(2) a substitution E357N, E357D, E357A, E357I, E357G or E357M of the amino acid residue at position E357 in the CH3 domain of the second Fc region, and substitution S364T or S364W of the amino acid residue at position S364 in the CH3 domain of the second Fe region;
(3) a substitution K409W of the amino acid residue at position K409 in the CH3 domain of the first Fc region; and
(4) a substitution F405T of the amino acid residue at position F405 in the CH3 domain of the second Fc region, and substitution D399V of the amino acid residue at position D399 in the CH3 domain of the second Fc region.

### Claim 11 (depends on 1)

The heterodimeric Fc-fused protein according to claim 1, wherein the mutation of the CH3 domain of the first Fc region or the second Fc region includes one or more mutations selected from the following group (wherein mutation positions are numbered according to the EU index):
(1) a substitution K360E of the amino acid residue at position K360 in the CH3 domain of the first Fc region;
(2) a substitution E347R of the amino acid residue at position E347 in the CH3 domain of the second Fc region;
(3) a substitution K409W of the amino acid residue at position K409 in the CH3 domain of the first Fc region; and
(4) a substitution F405T of the amino acid residue at position F405 in the CH3 domain of the second Fc region, and a substitution D399V of the amino acid residue at position D399 in the CH3 domain of the second Fc region.

### Claim 12 (depends on 10, 11)

The heterodimeric Fc-fused protein according to claim 10 or 11, wherein the CH3 domains in the first Fc region and the second Fc region further include the following residue (wherein the positions are numbered according to the EU index):
(i) cysteine (C) substituted at position Y349 in the CH3 domain of the first Fc region; and
(ii) cysteine (C) substituted at position S354 in the CH3 domain of the second Fc region.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

A pharmaceutical composition comprising the heterodimeric Fc-fused protein of any one of claims 1 to 12.

### Claim 14 (depends on 13)

The pharmaceutical composition according to claim 13, wherein the physiologically active protein contained in the heterodimeric Fc-fused protein is IL-12 (IL-12).

### Claim 15 (depends on 14)

The pharmaceutical composition according to claim 14, which is used to treat cancer.

### Claim 16 (depends on 15)

The pharmaceutical composition according to claim 15, wherein the cancer is selected from the group consisting of colorectal cancer, melanoma, breast cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, small intestine cancer, esophageal cancer, cervical cancer, lung cancer, lymphoma, and blood cancer.

### Claim 17 (depends on 15)

The pharmaceutical composition according to claim 15, which is used for combination therapy with other anticancer drugs.