Myostatin binding agents

Abstract

The present invention provides binding agents comprising peptides capable of binding myostatin and inhibiting its activity. In one embodiment the binding agent comprises at least one myostatin-binding peptide attached directly or indirectly to at least one vehicle such as a polymer or an Fc domain. The binding agents of the present invention produced increased lean muscle mass when administered to animals and decreased fat to muscle ratios. Therapeutic compositions containing the binding agents of the present invention are useful for treating muscle-wasting disorders and metabolic disorders including diabetes and obesity.

1 . A binding agent comprising at least one peptide capable of binding myostatin, wherein the peptide comprises the amino acid sequence  C a 1 a 2   W a 3   WMCPP  (SEQ ID NO: 352), wherein a 1 , a 2  and a 3  are selected from a neutral hydrophobic, neutral polar, or basic amino acid, and wherein the peptide is between 10 and 50 amino acids in length, and physiologically acceptable salts thereof. 
     
     
         2 . A binding agent comprising at least one peptide capable of binding myostatin, wherein the peptide comprises the sequence  C b 1 b 2   W b 3   WMCPP  (SEQ ID NO: 353), wherein
 b 1  is selected from any one of the amino acids T, I, or R; 
 b 2  is selected from any one of R, S, Q; 
 b 3  is selected from any one of P, R and Q, 
 
       and wherein the peptide is between 10 and 50 amino acids in length, and physiologically acceptable salts thereof. 
     
     
         3 . A binding agent comprising at least one peptide capable of binding myostatin, wherein the peptide is selected from the group consisting of:
 (a) a peptide comprising the amino acid sequence  WY e 1 e 2   Y e 3   G , (SEQ ID NO: 356), wherein e 1  is P, S or Y, e 2  is C or Q, and e 3  is G or H;   (b) a peptide comprising the amino acid sequence f 1   EMLf   2   SL f 3 f 4   LL , (SEQ ID NO: 455), wherein f 1  is M or I, f 2  is any amino acid, f 3  is L or F, and f 4  is E, Q or D;   (c) a peptide comprising the amino acid sequence  L g 1 g 2   LL g 3 g 4   L , (SEQ ID NO: 456), wherein g 1  is Q, D or E, g 2  is S, Q, D or E, g 3  is any amino acid, and g 4  is L, W, F, or Y; and   (d) a peptide comprising the amino acid sequence h 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h 6  (SEQ ID NO: 457), wherein h 1  is R or D, h 2  is any amino acid, h 3  is A, T S or Q, h 4  is L or M, h 5  is L or S, h 6  is any amino acid, h 7  is F or E, h 8  is W, F or C, and h 9  is L, F, M or K,   
       and physiologically acceptable salts thereof. 
     
     
         4 . A binding agent wherein said agent has the structure:
 (X 1 ) a —F 1 —(X 2 ) b , or multimers thereof;   
       wherein F 1  is a vehicle; and X 1  and X 2  are each independently selected from
 -(L 1 ) c -P 1 ; 
 -(L 1 ) c -P 1 -(L 2 ) d -P 2 ; 
 -(L 1 ) c -P 1 -(L 2 ) d -P 2 -(L 3 ) e -P 3 ; 
 and -(L 1 ) c -P 1 -(L 2 ) d -P 2 -(L 3 ) e -P 3 -(L 4 ) f -P 4 ; 
 
       wherein P 1 , P 2 , P 3 , and P 4  are peptides capable of binding myostatin, wherein L 1 , L 2 , L 3 , and L 4  are each linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1, and physiologically acceptable salts thereof. 
     
     
         5 . The binding agent of  claim 4  wherein one or more of the myostatin binding peptides comprise the amino acid sequence  C a 1 a 2   W a 3   WMCPP  (SEQ ID NO: 352), wherein a 1 , a 2  and a 3  are selected from a neutral hydrophobic, neutral polar, or basic amino acid, and wherein the peptide is between 10 and 50 amino acids in length, and physiologically acceptable salts thereof. 
     
     
         6 . The binding agent of  claim 4 , wherein one or more of the myostatin binding peptides comprise the amino acid sequence  C b 1 b 2   W b 3   WMCPP  (SEQ ID NO: 353), wherein
 b 1  is selected from any one of the amino acids T, I, or R; 
 b 2  is selected from any one of R, S, Q; 
 b 3  is selected from any one of P, R and Q, 
 
       and wherein the peptide is between 10 and 50 amino acids in length, and physiologically acceptable salts thereof. 
     
     
         7 . The binding agent of  claim 4 , wherein the myostatin binding peptides P 1 , P 2 , P 3 , and P 4  are selected from the group consisting of:
 (a) a peptide comprising the amino acid sequence  WY e 1 e 2   Y e 3   G , (SEQ ID NO: 356), wherein e 1  is P, S or Y, e 2  is C or Q, and e 3  is G or H;   (b) a peptide comprising the amino acid sequence f 1   EMLf   2   SL f 3 f 4   LL , (SEQ ID NO: 455), wherein f 1  is M or I, f 2  is any amino acid, f 3  is L or F, and f 4  is E, Q or D;   (c) a peptide comprising the amino acid sequence  L g 1 g 2   LL g 3 g 4   L , (SEQ ID NO: 456), wherein g 1  is Q, D or E, g 2  is S, Q, D or E, g 3  is any amino acid, and g 4  is L, W, F, or Y; and   (d) a peptide comprising the amino acid sequence h 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h 9  (SEQ ID NO: 457), wherein h 1  is R or D, h 2  is any amino acid, h 3  is A, T S or Q, h 4  is L or M, h 5  is L or S, h 6  is any amino acid, h 7  is F or E, h 8  is W, F or C, and h 9  is L, F, M or K;   
       wherein the peptide is between 9 and 50 amino acids in length, 
       wherein L 1 , L 2 , L 3 , and L 4  are each linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1, and physiologically acceptable salts thereof 
     
     
         8 . A binding agent wherein said agent has the structure:
 (X 1 ) a —F 1 —(X 2 ) b , or multimers thereof   
       wherein F 1  is a vehicle; and X 1  and X 2  are each independently selected from
 -(L 1 ) c -P 1 ; 
 -(L 1 ) c -P 1 -(L 2 ) d -P 2 ; 
 -(L 1 ) c -P 1 -(L 2 ) d -P 2 -(L 3 ) e -P 3 ; 
 and -(L 1 ) c -P 1 -(L 2 ) d -P 2 -(L 3 ) e -P 3 -(L 4 ) f -P 4 ; 
 
       wherein P 1 , P 2 , P 3 , and P 4  are peptides capable of binding myostatin, and are independently selected from SEQ ID NO: 305 through 351 and SEQ ID NO: 357 through 454; 
       wherein L 1 , L 2 , L 3 , and L 4  are each linkers; 
       and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1, and physiologically acceptable salts thereof. 
     
     
         9 . The binding agent of  claim 8 , wherein the binding agent has the structure F 1 -(L 1 ) c -P 1  or F 1 -(L 1 ) c -P 1 -(L 2 ) d -P 2 . 
     
     
         10 . The binding agent of  claim 9 , wherein the peptide is selected from any one of SEQ ID NO: 305-308, 310-313, 315-331, 333, 335-346, wherein F 1  is an Fc domain. 
     
     
         11 . A binding agent capable of binding myostatin comprising the structure F 1 -(L 1 )-P 1 , wherein F 1  is a human IgG Fc, wherein L 1  is (Gly) 5 , wherein the binding agent further comprises AQ and wherein P 1  is selected from SEQ ID NO: 311, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 336, SEQ ID NO: 337, and SEQ ID NO: 345. 
     
     
         12 . A pharmaceutical composition comprising a therapeutically effective amount of the binding agent of  claim 6  in admixture with a pharmaceutically acceptable carrier thereof. 
     
     
         13 . An isolated nucleic acid molecule comprising a polynucleotide encoding the binding agent of any one of  claim 6  or  8 . 
     
     
         14 . The nucleic acid molecule of  claim 13 , wherein the polynucleotide selected from any one of SEQ ID NO: 573 through 614, and 616, 618, 620, 622, 624, 626, 628, 630, 632. 
     
     
         15 . An expression vector comprising the polynucleotide of  claim 13 . 
     
     
         16 . A host cell comprising the expression vector of  claim 15 . 
     
     
         17 . A method of making a myostatin binding agent comprising culturing the host cell of  claim 16  under conditions suitable for expressing the binding agent, and purifying the binding agent. 
     
     
         18 . A method of inhibiting myostatin activity in a subject comprising administering an effective amount of the composition of  claim 12  to the subject. 
     
     
         19 . A method of increasing lean muscle mass in a subject comprising administering the composition of  claim 12  to the subject. 
     
     
         20 . A method of increasing the ratio of lean muscle mass to fat in a subject comprising administering a therapeutically effective amount of the composition of  claim 12  to the subject. 
     
     
         21 . A method of treating a muscle-wasting disease in a subject comprising administering a therapeutically effective amount of the composition of  claim 12  to the subject. 
     
     
         22 . The method of  claim 21 , wherein the disease is selected from muscular dystrophy, amyotrophic lateral sclerosis, congestive obstructive pulmonary disease, chronic heart failure, cancer, AIDs, renal failure, uremia, rheumatoid arthritis, age-related sarcopenia, and muscle-wasting due to prolonged bedrest, spinal chord injury, stroke, bone fracture, and aging. 
     
     
         23 . A method of treating a myostatin-related metabolic disorder in a subject comprising administering a therapeutically effective amount of the composition of  claim 12  to the subject. 
     
     
         24 . The method of  claim 23  wherein the metabolic disorder is selected from diabetes, obesity, hyperglycemia, and bone loss. 
     
     
         25 . A method of detecting or measuring myostatin in a sample comprising contacting the sample with a binding agent of  claim 1  and detecting or measuring the bound complex.