Engineered polypeptides having enhanced duration of action

Abstract

Compounds are provided having inter alia good duration of action, high potency and/or convenient dosing regimens including once weekly administration. The compounds are engineered polypeptides which incorporate an albumin binding domain in combination with one or more biologically active polypeptides. Also provided are pharmaceutical compositions and methods of treatment for diseases and disorders including lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease or diabetes (including type I and type II). Additional diseases and disorders which can be treated by the compounds and methods described herein include nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X and Huntington's Disease.

What is claimed is: 
     
         1 . An engineered polypeptide comprising: an albumin binding domain polypeptide (ABD) and a first peptide hormone domain (HD1) selected from a leptin, a leptin analog or an active fragment thereof. 
     
     
         2 . The engineered polypeptide according to  claim 1 , further comprising a first linker (L1) covalently linked to said HD1. 
     
     
         3 . The engineered polypeptide according to  claim 1 , wherein said engineered polypeptide comprises said ABD as an N-terminal moiety and said HD1 as a C-terminal moiety. 
     
     
         4 . The engineered polypeptide according to  claim 1 , wherein said engineered polypeptide comprises said ABD as a C-terminal moiety and said HD1 as an N-terminal moiety. 
     
     
         5 . The engineered polypeptide according to  claim 1 , wherein said HD1 has at least 50% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33. 
     
     
         6 . The engineered polypeptide according to  claim 1 , wherein said ABD comprises an albumin binding motif (ABM) that consists of the amino acid sequence: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 34) 
                 
                     
                   GVSD X 5  YK X 8  X 9  I X 11  X 12  A X 14  TVEGV X 20  AL X 23   
                 
                     
                     
                 
                     
                   X 24  X 25  I 
                 
             
                
                
                
                
               
            
           
         
         wherein, independently of each other,
 X 5  is selected from Y and F; 
 X 8  is selected from N, R and S; 
 X 9  is selected from V, I, L, M, F and Y; 
 X 1i  is selected from N, S, E and D; 
 X 12  is selected from R, K and N; 
 X 14  is selected from K and R; 
 X 20  is selected from D, N, Q, E, H, S, R and K; 
 X 23  is selected from K, I and T; 
 X 24  is selected from A, S, T, G, H, L and D; and 
 X 25  is selected from H, E and D. 
 
       
     
     
         7 . The engineered polypeptide according to  claim 6 , wherein, independently of each other,
 X 5  is Y;   X 8  is N;   X 23  is T or I;   X 24  is S or L; and   X 25  is E or H.   
     
     
         8 . The engineered polypeptide according to  claim 7 , wherein the albumin binding motif comprises an amino acid sequence that is selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 114) 
                 
                     
                   GVSDYYKNLINKAKTVEGVEALTLHI 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 115) 
                 
                     
                   GVSDYYKNLINKAKTVEGVEALISEI. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         9 . The engineered polypeptide according to  claim 1 , wherein said ABD comprises an albumin binding motif (ABM) that is not GVSDYYKNLINNAKTVEGVKALIDEI (SEQ ID NO:35). 
     
     
         10 . The engineered polypeptide according to  claim 2 , wherein the albumin binding domain polypeptide (ABD) comprises an amino acid sequence that is selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO: 50) 
                 
                   LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP; 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO: 51) 
                 
                   LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALISEILAALP. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         11 . The engineered polypeptide according to  claim 1 , wherein said ABD comprises the amino acid sequence: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 36) 
                 
                     
                   LAEAK X a  X b  A X c  X d  EL X e  KY-[ABM]-LAALP 
                 
             
                
                
               
            
           
         
         wherein
 [ABM] is an albumin binding motif, and, 
 independently of each other, 
 X a  is selected from V and E; 
 X b  is selected from L, E and D; 
 X e  is selected from N, L and I; 
 X d  is selected from R and K; 
 X e  is selected from D and K; 
 the leucine at position 45 is present or absent; 
 the proline at position 46 is present or absent; and 
 
         wherein ABM consists of the amino acid sequence: 
       
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 34) 
                 
                     
                   GVSD X 5  YK X 8  X 9  I X 11  X 12  A X 14  TVEGV X 20  AL X 23   
                 
                     
                     
                 
                     
                   X 24  X 25  I 
                 
             
                
                
                
                
               
            
           
         
         wherein, independently of each other,
 X 5  is selected from Y and F; 
 X 8  is selected from N, R and S; 
 X 9  is selected from V, I, L, M, F and Y; 
 X 11  is selected from N, S, E and D; 
 X 12  is selected from R, K and N; 
 X 14  is selected from K and R; 
 X 20  is selected from D, N, Q, E, H, S, R and K; 
 X 23  is selected from K, I and T; 
 X 24  is selected from A, S, T, G, H, L and D; and 
 X 25  is selected from H, E and D. 
 
       
     
     
         12 . The engineered polypeptide according to  claim 11 , wherein said ABD comprises an amino acid sequence having at least 85% identity with an amino acid sequence that is selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52. 
     
     
         13 . The engineered polypeptide according to  claim 12 , wherein said ABD comprises any one of the peptides selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO: 38) 
                 
                   LAEAKVLANRELDKYGVSDFYKSYINRAKTVEGVHTLIGHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 39) 
                 
                   LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVNALTHHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 40) 
                 
                   LAEAKVLANRELDKYGVSDYYKNLINRARTVEGVHALIDHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 41) 
                 
                   LAEAKVLANRELDKYGVSDYYKNIINRAKTVEGVRALKLHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 42) 
                 
                   LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVSSLKGHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 43) 
                 
                   LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 44) 
                 
                   LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVDALIAHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 45) 
                 
                   LAEAKVLANRELDKYGVSDFYKSLINRAKTVEGVDALTSHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 46) 
                 
                   LAEAKVLANRELDKYGVSDFYKNLINRAKTVEGVNSLTSHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 47) 
                 
                   LAEAKVLANRELDKYGVSDFYKNVINKAKTVEGVEALIADILAALP, 
                 
                     
                 
                   (SEQ ID NO: 48) 
                 
                   LAEAKVLANRELDKYGVSDYYKNLINKAKTVEGVQALIAHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 49) 
                 
                   LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 50) 
                 
                   LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALTLHILAALP, 
                 
                     
                 
                   (SEQ ID NO: 51) 
                 
                   LAEAKEDAIKELDKYGVSDYYKNLINKAKTVEGVEALiseILAALP, 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO: 52) 
                 
                   LAEAKEDAIKELDKYGVSDYYKRLISKAKTVEGVKALISEILAALP. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         14 . The engineered polypeptide according to  claim 2 , wherein said linker L1 is a peptide of from 1 to 30 amino acids. 
     
     
         15 . The engineered polypeptide according to  claim 14 , wherein said linker L1 comprises a N-terminal TG dipeptide. 
     
     
         16 . The engineered polypeptide according to  claim 14 , wherein said linker L1 comprises a C-terminal AS dipeptide. 
     
     
         17 . The engineered polypeptide according to  claim 14 , wherein said linker L1 comprises an N-terminal TG dipeptide and a C-terminal AS dipeptide. 
     
     
         18 . The engineered polypeptide according to  claim 14 , wherein said linker L1 comprises an amino acids sequence that is selected from the group consisting of TG-(GGGS) 1  (SEQ ID NO: 215), TG-(GGGS) 2  (SEQ ID NO: 216), TG-(GGGS) 3  (SEQ ID NO: 217), TG-(GGGS) 4  (SEQ ID NO: 218), TG-(GGGS) 5  (SEQ ID NO: 219), (GGGS) 1 -AS (SEQ ID NO: 220), (GGGS) 2 -AS (SEQ ID NO: 221), (GGGS) 3 -AS (SEQ ID NO: 222), (GGGS) 4 -AS (SEQ ID NO: 223), (GGGS) 5 -AS (SEQ ID NO: 224), TG-(GGGS) 1 -AS (SEQ ID NO: 225), TG-(GGGS) 2 -AS (SEQ ID NO: 226), TG-(GGGS) 3 -AS (SEQ ID NO: 227), TG-(GGGS) 4 -AS (SEQ ID NO: 228), and TG-(GGGS) 5 -AS (SEQ ID NO: 229). 
     
     
         19 . The engineered polypeptide according to  claim 1 , wherein said polypeptide further comprises one or more additional linkers. 
     
     
         20 . The engineered polypeptide according to  claim 1 , wherein said engineered polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, and SEQ ID NO:107. 
     
     
         21 . A method for treating a disease or disorder in a subject, comprising administering a engineered polypeptide according to  claim 1  to a subject in need thereof in an amount effective to treat said disease or disorder. 
     
     
         22 . The method according to  claim 21 , wherein said disease or disorder is disease or disorder can be lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes, type I diabetes, type II diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), metabolic syndrome X and Huntington's Disease. 
     
     
         23 . The method according to  claim 22 , wherein said disease or disorder is lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease or diabetes. 
     
     
         24 . A pharmaceutical composition comprising an engineered polypeptide according to  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         25 . The pharmaceutical composition according to  claim 24 , wherein said pharmaceutical composition is formulated as an injectable pharmaceutical composition. 
     
     
         26 . The pharmaceutical composition according to  claim 24 , wherein said pharmaceutical composition is formulated as a sustained release or long lasting pharmaceutical composition. 
     
     
         27 . The pharmaceutical composition according to  claim 24  formulated for use in the treatment of lipodystrophy, dyslipidemia, hyperlipidemia, overweight, obesity, hypothalamic amenorrhea, Alzheimer's disease, leptin deficiency, fatty liver disease, diabetes (including type I and type II), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome X. 
     
     
         28 . The engineered polypeptide according to  claim 1 , wherein said HD1 is selected from the group consisting of:
 (a) the amino acid sequence 1-146 of a leptin selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; in which a different amino acid is substituted in one or more of the following positions and retaining the same numbering (even in the absence of a glutaminyl residue at position 28): 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145;   (b) the amino acid sequence of subpart (a) in which the glutaminyl residue at position 28 is absent;   (c) the amino acid sequence of subparts (a) or (b) in which a methionyl residue is added at the N-terminus;   (d) a leptin consisting of a fragment of the amino acid sequence of (a), (b), or (c) selected from the group consisting of:
 (i) amino acids 98-146; 
 (ii) amino acids 1-32; 
 (iii) amino acids 40-116; 
 (iv) amino acids 1-99 and 112-146; 
 (v) amino acids 1-99 and 112-146 in which one or more of amino acids 100-111 is placed between amino acids 99 and 112; 
 (vi) the amino acid sequence of subpart (i) wherein one or more of amino acids 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145 is substituted with another amino acid; 
 (vii) the amino acid sequence of subpart (ii) wherein one or more of amino acids 4, 8 and 32 is substituted with another amino acid; 
 (viii) the amino acid sequence of subpart (iii) wherein one or more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111 and 112 is replaced with another amino acid; 
 (ix) the amino acid sequence of subpart (iv) wherein one or more of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142, and 145 is replaced with another amino acid; and 
 (x) the amino acid sequence of subpart (v) wherein one or more of amino acids 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145 is replaced with another amino acid; 
 (xi) the amino acid sequence of any of subparts (i)-(x) wherein a methionine has been added at the N-terminus; 
   (e) the amino acid sequence of any of subparts (a) through (d) wherein said amino acid sequence is attached to a chemical moiety;   (f) the amino acid sequence of subpart (e) wherein said chemical moiety is a water soluble polymer moiety;   (g) the amino acid sequence of subpart (f) wherein said water soluble polymer moiety is selected from the group consisting of: polyethylene glycol, an ethylene glycol/propylene glycol copolymer, a carboxymethylcellulose, a dextran, a polyvinyl alcohol, a polyvinyl pyrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid homopolymer, a polyaminoacid random copolymer, an albumin, an Fc protein, a poly(n-vinyl pyrolidone)polyethylene glycol, a propylene glycol homopolymer, a polypropylene oxide/ethylene oxide copolymer, a polyoxyethylated polyol, a polyvinyl alcohol, a polyethylene glycol propionadehyde, a succinate, and a styrene;   (h) the amino acid sequence of subpart (g) wherein said water soluble polymer moiety is a polyethylene glycol; and   (i) the amino acid sequence of subpart (g) wherein said water soluble polymer is a polyamino acid-selected from the group consisting of: an albumin, an antibody, an Fc protein, and a polylysine moiety.   
     
     
         29 . The engineered polypeptide according to  claim 28 , wherein said HD1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; wherein one or more amino acid substitutions have been made. 
     
     
         30 . The engineered polypeptide according to  claim 1 , wherein said HD1 is SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145 or SEQ ID NO:146. 
     
     
         31 . A method of treating obesity in a subject comprising peripherally administering therapeutically effective amounts of at least two different anti-obesity agents, wherein at least one anti-obesity agent is an amylin, an amylin analog, an amylin agonist, or an amylin derivative (i.e. an amylin agent) and at least one anti-obesity agent is an engineered polypeptide comprising: an albumin binding domain polypeptide (ABD); and a first peptide hormone domain (HD1) selected from a leptin, a leptin analog or an active fragment thereof. 
     
     
         32 . A method of reducing body weight in a subject comprising peripherally administering therapeutically effective amounts of at least two different anti-obesity agents, wherein at least one anti-obesity agent is an amylin, an amylin analog, an amylin agonist, or an amylin derivative (i.e. an amylin agent) and at least one anti-obesity agent is an engineered polypeptide comprising: an albumin binding domain polypeptide (ABD); and a first peptide hormone domain (HD1) selected from a leptin, a leptin analog or an active fragment thereof.