# US 7,612,040 B2 · Lefty polypeptides and derivatives thereof

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- Number: US 7,612,040 B2
- Document ID: US7612040B2
- Status: Granted
- Kind: B2
- Application number: 11/479,181
- Filed: 2006-06-30
- Published: 2007-02-22
- Granted: 2009-11-03
- Earliest claimed: 2005-07-01
- Assignees: Acceleron Pharma Inc.
- Inventors: John Knopf; Jasbir Seehra; Ravindra Kumar
- Web page: https://patentgrep.com/patent/US7612040B2

## CPC

- C07K 14/47: from mammals
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/04: Anorexiants; Antiobesity agents
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/12: Antihypertensives
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/495: Transforming growth factor [TGF]
- 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: 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)

## Abstract

The disclosure relates to Lefty derivatives and the uses of Lefty polypeptides as antagonists of the function of certain ligands such as Nodal, GDF-8 (Myostatin), and GDF-11. These derivatives may be fused to other functional heterologous proteins such as IgG, especially the Fc portion of IgG. According to the disclosure, Lefty polypeptides are useful in the treatment of a variety of disorders, including, for example, neuronal diseases, muscle and bone conditions, and metabolic disorders.

## Claims

### Claim 1

A recombinant Lefty derivative polypeptide comprising an amino acid sequence as set forth in the formula: W-A-X-B-Y,
wherein A consists essentially of an amino acid sequence at least 85% identical to the sequence of Region 2 of SEQ ID NO: 1;
wherein B consists essentially of an amino acid sequence at least 85% identical to the sequence of Region 4 of SEQ ID NO: 1;
wherein X consists of zero, one or more than one amino acid;
wherein W consists of zero, one or more than one amino acid;
wherein Y consists of zero, one or more than one amino acid;
wherein the recombinant Lefty derivative polypeptide binds to one or more of Nodal, myostatin and GDF-11;
wherein the recombinant Lefty derivative polypeptide is not a wild-type Lefty polypeptide;
wherein the recombinant Lefty derivative polypeptide comprises an amino acid sequence that is at least 95% identical to a human Lefty polypeptide sequence selected from the group consisting of amino acids 78-353 of SEQ ID NO: 1 and amino acids 78-353 of SEQ ID NO:2; and
wherein the recombinant Lefty derivative polypeptide comprises one or more mutations within the RXXR cleavage sequence corresponding to amino acid residues 132-135 of SEQ ID NO: 1 or 2 so as to prevent cleavage at the mutated sequence.

### Claim 2 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein the recombinant Lefty derivative polypeptide comprises an amino acid sequence that is at least 85% identical to the cysteine knot portion of a human Lefty polypeptide.

### Claim 3 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein A consists of an amino acid sequence at least 95% identical to the sequence of Region 2 of SEQ ID NO: 1 and wherein B consists of an amino acid sequence at least 95% identical to the sequence of Region 4 of SEQ ID NO:1.

### Claim 4 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein A is selected from the group consisting of: CRQEMYIDLQGMKWAKNWVLEPPG FLAYECVGT (SEQ ID NO: 5) and CRQEMYIDLQGMKWAENWVLEPPGFLAYECVGT (SEQ ID NO: 7), and wherein B is selected from the group consisting of: CIASETASLPMIVSIKEGGRTRPQVVSLPNMRVQKC (SEQ ID NO: 6) and CIASETDSLPMIVSIKEGGRTRPQVVSLPNMRVQKC (SEQ ID NO: 8).

### Claim 5 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein X comprises an amino acid sequence that has low immunogenicity.

### Claim 6 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein X comprises a glycosylation site.

### Claim 7 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein the length of X is between 0-50 amino acids.

### Claim 8 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein X comprises a dimerization domain.

### Claim 9 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein the polypeptide is fused to an additional domain.

### Claim 10 (depends on 9)

The recombinant Lefty derivative polypeptide of claim 9, wherein the additional domain is a dimerization domain.

### Claim 11 (depends on 9)

The recombinant Lefty derivative polypeptide of claim 9, wherein the additional domain is fused to the carboxyl or amino terminus of the Lefty polypeptide.

### Claim 12 (depends on 10)

The recombinant Lefty derivative polypeptide of claim 10, wherein the dimerization domain comprises a Lefty propeptide sequence.

### Claim 13 (depends on 11)

The recombinant Lefty derivative polypeptide of claim 11, wherein the dimerization domain is selected from an immunoglobulin heavy chain constant region and an immunoglobulin light chain constant region.

### Claim 14 (depends on 10)

The recombinant Lefty derivative polypeptide of claim 10, wherein the dimerization domain is a leucine zipper domain.

### Claim 15 (depends on 14)

The recombinant Lefty derivative polypeptide of claim 14, wherein said leucine zipper domain comprises at least four leucine heptads.

### Claim 16 (depends on 15)

The recombinant Lefty derivative polypeptide of claim 15, wherein said leucine zipper domain is selected from the group consisting of a Fos and a Jun leucine zipper domain.

### Claim 17 (depends on 10)

The recombinant Lefty derivative polypeptide of claim 10, further comprising a linker sequence interposed between and covalently joining the Lefty polypeptide and the dimerization domain.

### Claim 18 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein X comprises a domain that binds Nodal, myostatin and/or GDF-11.

### Claim 19 (depends on 9)

The recombinant Lefty derivative polypeptide of claim 9, wherein the additional domain is a domain that binds Nodal, myostatin and/or GDF-11.

### Claim 20 (depends on 19)

The recombinant Lefty derivative polypeptide of claim 19, wherein the domain that binds Nodal, myostatin and/or GDF-11 inhibits the binding of Nodal, myostatin and/or GDF-11 to a Type I receptor.

### Claim 21 (depends on 20)

The recombinant Lefty derivative polypeptide of claim 20 wherein the domain that binds Nodal, myostatin and/or GDF-11 competitively inhibits the binding of Nodal, myostatin and/or GDF-11 to a Type I receptor selected from the group consisting of ALK4 and ALK7.

### Claim 22 (depends on 20)

The recombinant Lefty derivative polypeptide of claim 20, wherein the domain that binds Nodal, myostatin and/or GDF-11 is selected from the group consisting of:
(a) an extracellular portion of ALK4;
(b) an extracellular portion of ALK7;
(c) an antigen-binding portion of an antibody that binds Nodal, myostatin and/or GDF-11; and
(d) a randomized polypeptide that has been selected for binding to Nodal, myostatin and/or GDF-11.

### Claim 23 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein said modified Lefty polypeptide inhibits signaling mediated by a protein selected from an ActRII receptor, myostatin, Nodal, and GDF-11 in a cell.

### Claim 24 (depends on 1)

The recombinant Lefty derivative polypeptide of claim 1, wherein said modified Lefty polypeptide comprises a heterogenous sequence that mediates secretion of the recombinant Lefty derivative polypeptide.

### Claim 25 (depends on 24)

The recombinant Lefty derivative polypeptide of claim 24, wherein said heterogenous sequence that mediates secretion of the recombinant Lefty derivative polypeptide is a honey bee melatin leader sequence.

### Claim 26

An isolated Lefty polypeptide complex comprising:
a) a first Lefty polypeptide; and
b) a second Lefty polypeptide,
wherein the first and second Lefty polypeptides are associated to form a complex, wherein the complex binds to a TGF-β family member selected from the group consisting of: myostatin, Nodal and GDF-11, and wherein at least one of the Lefty polypeptides of the complex comprises one or more mutations within the RXXR cleavage sequence corresponding to amino acid residues 132-135 of SEQ ID NO:1 or 2 so as to prevent cleavage at the mutated sequence.

### Claim 27 (depends on 26)

The Lefty polypeptide complex of claim 26, wherein the polypeptide complex is a homodimer.

### Claim 28 (depends on 1)

A pharmaceutical preparation comprising a modified Lefty polypeptide of claim 1.

### Claim 29

A recombinant Lefty derivative polypeptide comprising an amino acid sequence as set forth in the formula: W-A-X-B-Y;
wherein the Lefty derivative polypeptide comprises an amino acid sequence that is at least 85% identical to a human Lefty polypeptide sequence selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:2;
wherein A is selected from the group consisting of: CRQEMYIDLQGMKWAKNWVLEPPGFLAYECVGT (SEQ ID NO: 5) and CRQEMYIDLQGMKWAENWVLEPPGFLAYECVGT (SEQ ID NO: 7);
wherein B is selected from the group consisting of: CIASETASLPMIVSIKEGGRTRPQVVSLPNMRVQKC (SEQ ID NO: 6) and CIASETDSLPMIVSIKEGGRTRPQVVSLPNMRVQKC (SEQ ID NO: 8);
wherein X consists of zero, one or more than one amino acid;
wherein W consists of zero, one or more than one amino acid;
wherein Y consists of zero, one or more than one amino acid;
wherein the recombinant, Lefty derivative polypeptide binds to one or more of Nodal, myostatin and GDF-11;
wherein the recombinant Lefty derivative polypeptide is not a wild-type Lefty polypeptide; and
wherein the recombinant Lefty derivative polypeptide comprises one or more mutations within the RXXR cleavage sequence corresponding to amino acid residues 132-135 of SEQ ID NO: 1 or 2 so as to prevent cleavage at the mutated sequence.

### Claim 30 (depends on 29)

The recombinant Lefty derivative polypeptide of claim 29, wherein the recombinant Lefty derivative polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, and wherein A is SEQ ID NO: 5 and B is SEQ ID NO: 6.

### Claim 31 (depends on 29)

The recombinant Lefty derivative polypeptide of claim 29, wherein the recombinant Lefty derivative polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 2, and wherein A is SEQ ID NO: 7 and B is SEQ ID NO: 8.

### Claim 32 (depends on 4)

The recombinant Lefty derivative polypeptide of claim 4, wherein the recombinant Lefty derivative polypeptide comprises an amino acid that is at least 95% identical to amino acids 78-353 of SEQ ID NO: 1, and wherein A is SEQ ID NO: 5 and B is SEQ ID NO: 6.

### Claim 33 (depends on 29)

The recombinant Lefty derivative polypeptide of claim 29, wherein the recombinant Lefty derivative polypeptide comprises an amino acid that is at least 95% identical to amino acids 78-353 of SEQ ID NO: 2, and wherein A is SEQ ID NO: 7 and B is SEQ ID NO: 8.