# US 10,245,302 B2 · Fibronectin based scaffold domain proteins that bind to myostatin

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,245,302 B2
- Document ID: US10245302B2
- Status: Granted
- Kind: B2
- Application number: 15/282,277
- Filed: 2016-09-30
- Published: 2017-03-30
- Granted: 2019-04-02
- Earliest claimed: 2012-09-13
- Assignees: Bristol-Myers Squibb Company
- Applicants: Bristol-Myers Squibb Company
- Inventors: Sharon Cload; Linda Engle; Dasa Lipovsek; Malavi Madireddi; Ginger Chao Rakestraw; Joanna Swain; Wenjun Zhao; Hui Wei; Aaron P. Yamniuk; Vidhyashankar Ramamurthy; Alexander T. Kozhich; Martin J. Corbett; Stanley Richard Krystek, Jr.
- Web page: https://patentgrep.com/patent/US10245302B2

## CPC

- A61K 38/39: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
- A61K 38/17: from animals; from humans {(enzyme inhibitors A61K38/005)}
- A61K 38/1709: {from mammals}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/642: {the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 3/06: Antihyperlipidemics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 5/50: for increasing or potentiating the activity of insulin
- A61P 7/12: Antidiuretics, e.g. drugs for diabetes insipidus (ADH A61P5/10)
- 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 11/00: Drugs for disorders of the respiratory system
- A61P 13/12: of the kidneys
- A61P 19/00: Drugs for skeletal disorders
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 19/10: for osteoporosis
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/02: Muscle relaxants, e.g. for tetanus or cramps
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- C07K 14/435: from animals; from humans
- C07K 14/765: Serum albumin, e.g. HSA
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C07K 14/79: Transferrins, e.g. lactoferrins, ovotransferrins
- C07K 16/18: against material from animals or humans
- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- 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
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- 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
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates to fibronectin-based scaffold domain proteins that bind to myostatin. The invention also relates to the use of these proteins in therapeutic applications to treat muscular dystrophy, cachexia, sarcopenia, osteoarthritis, osteoporosis, diabetes, obesity, COPD, chronic kidney disease, heart failure, myocardial infarction, and fibrosis. The invention further relates to cells comprising such proteins, polynucleotides encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the proteins.

## Claims

### Claim 1

A nucleic acid encoding a polypeptide comprising a fibronectin type III tenth (10Fn3) domain which binds to myostatin and (i) comprises AB, BC, CD, DE, EF, and FG loops, wherein at least one loop of the BC, DE, and FG loops of the 10Fn3 domain has 0, 1, 2, or 3 amino acid substitutions relative to the respective BC, DE, and FG loops of SEQ ID NOs: 34, 39, and 75.

### Claim 2 (depends on 1)

The nucleic acid of claim 1, wherein at least one loop of the BC, DE, and FG loops of the 10Fn3 domain has 1 amino acid substitution relative to the respective BC, DE, and FG loops of SEQ ID NOs: 34, 39, and 75.

### Claim 3 (depends on 1)

The nucleic acid of claim 1, wherein:
(i) the serine at position 3 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, D, F, H, I, K, L, N, Q, R, T, V, W, or Y;
(ii) the leucine at position 4 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from M or V;
(iii) the proline at position 5 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, D, E, I, K, L, M, N, Q, R, S, T, V, or Y;
(iv) the histidine at position 6 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, or Y;
(v) the glutamine at position 7 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y;
(vi) the glycine at position 8 of the BC loop (SEQ ID NO: 34) is substituted with the amino acid S;
(vii) the lysine at position 9 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y;
(viii) the alanine at position 10 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of C, G, L, M, S, or T; or
(ix) the asparagine at position 11 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, F, H, P, Q, R, S, or Y.

### Claim 4 (depends on 3)

The nucleic acid of claim 3, wherein:
(i) the serine at position 3 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of C, F, I, V, W, or Y;
(ii) the histidine at position 6 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of C, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W, or Y;
(iii) the lysine at position 9 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, G, H, I, L, M, N, Q, R, S, V, W, or Y;
(iv) the alanine at position 10 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of G, L, M, or S; or
(v) the asparagine at position 11 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of C, H, Q, S, or Y.

### Claim 5 (depends on 3)

The nucleic acid of claim 3, wherein:
(i) the serine at position 3 of the BC loop (SEQ ID NO: 34) is substituted with the amino acid F or W;
(ii) the histidine at position 6 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of C, F, G, I, K, L, M, N, R, S, T, V, W, or Y;
(iii) the glutamine at position 7 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, E, F, H, I, K, L, M, P, R, S, T, V, or Y;
(iv) the lysine at position 9 of the BC loop (SEQ ID NO: 34) is substituted with an amino acid selected from the group consisting of A, C, H, L, M, N, R, V, W, or Y;
(v) the alanine at position 10 of the BC loop (SEQ ID NO: 34) is substituted with the amino acid G or L; or
(vi) the asparagine at position 11 of the BC loop (SEQ ID NO: 34) is substituted with the amino acid H or Q.

### Claim 6 (depends on 1)

The nucleic acid of claim 1, wherein the valine at position 5 of the DE loop (SEQ ID NO: 39) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, I, K, L, M, N, Q, S, or T.

### Claim 7 (depends on 6)

The nucleic acid of claim 6, wherein the valine at position 5 of the DE loop (SEQ ID NO: 39) is substituted with an amino acid selected from the group consisting of C, E, I, L, M, Q, or T.

### Claim 8 (depends on 6)

The nucleic acid of claim 6, wherein the valine at position 5 of the DE loop (SEQ ID NO: 39) is substituted with an amino acid selected from the group consisting of C, E, I, L, or M.

### Claim 9 (depends on 1)

The nucleic acid of claim 1, wherein:
(i) the valine at position 2 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, F, I, L, M, Q, T, W, or Y;
(ii) the threonine at position 3 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, F, G, H, I, K, L, M, N, Q, R, S, V, W, or Y;
(iii) the aspartic acid at position 4 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(iv) the threonine at position 5 of the FG loop (SEQ ID NO: 75) is substituted to with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y;
(v) the glycine at position 6 of the FG loop (SEQ ID NO: 75) is substituted to with an amino acid selected from the group consisting of A, C, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;
(vi) the tyrosine at position 7 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, F, H, I, L, M, N, P, S, T, V, or W;
(vii) the leucine at position 8 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, E, F, H, I, K, M, N, Q, R, S, T, V, W, or Y;
(viii) the lysine at position 9 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
(ix) the tyrosine at position 10 of the FG loop (SEQ ID NO: 75) is substituted with the amino acid F or W; or
(x) the lysine at position 11 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y.

### Claim 10 (depends on 9)

The nucleic acid of claim 9, wherein:
(i) the valine at position 2 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, I, L, or M;
(ii) the threonine at position 3 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of C, F, H, I, L, M, Q, R, S, V, W, or Y;
(iii) the aspartic acid at position 4 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, E, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y;
(iv) the threonine at position 5 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, V, W, or Y;
(v) the glycine at position 6 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, D, E, F, H, I, L, M, N, Q, S, T, V, W, or Y;
(vi) the tyrosine at position 7 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of C, F, I, L, M, P, T, V, or W;
(vii) the leucine at position 8 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of C, F, H, I, K, M, N, Q, R, T, V, W, or Y;
(viii) the lysine at position 9 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, E, F, G, I, L, M, N, P, Q, R, S, T, V, W, or Y;
(ix) the tyrosine at position 10 of the FG loop (SEQ ID NO: 75) is substituted with the amino acid W; or
(x) the lysine at position 11 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, D, E, G, H, L, M, N, P, Q, R, S, T, or V.

### Claim 11 (depends on 9)

The nucleic acid of claim 9, wherein:
(i) the valine at position 2 of the FG loop (SEQ ID NO: 75) is substituted with the amino acid I;
(ii) the threonine at position 3 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of C, F, I, L, M, V, W, or Y;
(iii) the aspartic acid at position 4 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, E, F, G, H, I, L, M, N, Q, S, T, or V;
(iv) the threonine at position 5 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, D, F, G, I, L, M, N, Q, S, V, W, or Y;
(v) the glycine at position 6 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, S, T, or W;
(vi) the tyrosine at position 7 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of F, I, V, or W;
(vii) the leucine at position 8 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of F, H, I, M, V, W, or Y;
(viii) the lysine at position 9 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, C, F, G, I, L, M, T, V, or W; or
(ix) the lysine at position 11 of the FG loop (SEQ ID NO: 75) is substituted with an amino acid selected from the group consisting of A, G, L, M, P, Q, or R.

### Claim 12 (depends on 1)

The nucleic acid of claim 1, wherein the 10Fn3 domain comprises the amino acid sequence of SEQ ID NO: 118.

### Claim 13 (depends on 1)

The nucleic acid of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 281.

### Claim 14 (depends on 1)

The nucleic acid of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 331.

### Claim 15 (depends on 1)

The nucleic acid of claim 1, wherein the polypeptide further comprises one or more pharmacokinetic (PK) moieties selected from the group consisting of Fc, Fc fragment, transferrin, serum albumin, a serum albumin binding protein, and a serum immunoglobulin binding protein.

### Claim 16 (depends on 15)

The nucleic acid of claim 15, wherein the one or more PK moieties is an Fc.

### Claim 17 (depends on 1)

The nucleic acid of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 273.