Kv1.3 Antagonists and Methods of Use

Abstract

The present invention relates to Kv1.3 antagonists, and polynucleotides encoding them, and methods of making and using the foregoing.

1 ) An isolated fusion protein comprising a peptide antagonist of Kv1.3 conjugated to a half-life extending moiety, wherein the peptide antagonist of Kv1.3 comprises
 a) the sequence shown in SEQ ID NO: 1 having a substitution of glycine to isoleucine at position 10 (G10I), and optionally having 1, 2, 3, 4, 5, 6 or 7 additional substitutions; or   b) an amino acid sequence which is at least 80% identical to SEQ ID NO: 1, further comprising a G10I substitution; and   c) the peptide antagonist of Kv1.3 optionally comprises a C-terminal extension of four amino acids.   
     
     
         2 ) The fusion protein of  claim 1 , wherein the peptide antagonist of Kv1.3 comprises the sequence 
       
         
           
                 
               
                   (SEQ ID NO: 426) 
                 
                   GVPXaa 1 Xaa 2 VKCXaa 3 ISRQCXaa 4 Xaa 5 PCKDAGMRFGKCMNGKCHCT  
                 
                   PK; 
                 
             
                
                
                
               
            
           
         
       
       wherein
 a) Xaa 1  is I or T, Q or E; 
 b) Xaa 2  is N or D; 
 c) Xaa 3  is K, R, E, A or Q; 
 d) Xaa 4  is I, E, L, D, Q, H, V, K or A; and 
 e) Xaa 5  is E, K, L, Q, D, V or H. 
 
     
     
         3 ) The fusion protein of  claim 2 , wherein the peptide antagonist of Kv1.3 comprises the sequence 
       
         
           
                 
               
                   (SEQ ID NO: 427) 
                 
                   GVPXaa 1 Xaa 2 VKCXaa 3 ISRQCXaa 4 Xaa 5 PCKDAGMRFGKCMNGKCHCT  
                 
                   PK; 
                 
             
                
                
                
               
            
           
         
       
       wherein
 a) Xaa 1  is I or T; 
 b) Xaa 2  is N or D; 
 c) Xaa 3  is K or H; 
 d) Xaa 4  is I or E; and 
 e) Xaa 5  is E or K. 
 
     
     
         4 ) The fusion protein of  claim 2 , wherein the peptide antagonist of Kv1.3 comprises the amino acid sequence of SEQ ID NOs: 3, 13, 21, 22, 24, 26, 29, 30, 32, 34, 38, 39, 42-46, 49, 51, 59, 63, 65, 69, 71, 73, 76, 78, 81-83, 85, 87, 89, 92, 96, 101, 103, 104, and 108. 
     
     
         5 ) The fusion protein of  claim 3 , wherein the peptide antagonist of Kv1.3 comprises the amino acid sequence of SEQ ID NOs: 3, 22, 34 or 42. 
     
     
         6 ) The fusion protein of  claim 1 , wherein the C-terminal extension comprises the amino acid sequence of SEQ ID NOs: 123-268. 
     
     
         7 ) The fusion protein of  claim 6 , wherein the C-terminal extension comprises the amino acid sequence of SEQ ID NOs: 128, 143, 155, 188, 206-210, 212, 214, 216, 219, 223, 224, 227, 230, 232-235, 237, 239, 240, 243, 252, 261-263, or 268. 
     
     
         8 ) The fusion protein of  claim 1 , wherein the half-life extending moiety is human serum albumin (HSA), albumin binding domain (ADB), or polyethylene glycol (PEG). 
     
     
         9 ) The fusion protein of  claim 8 , wherein the half-life extending moiety is human serum albumin. 
     
     
         10 ) The fusion protein of  claim 1 , wherein the half-life extending moiety is conjugated to the peptide antagonist of Kv1.3 via a linker. 
     
     
         11 ) The fusion protein of  claim 10 , wherein the linker comprises the amino sequence of SEQ ID NOs: 112-122 or 428. 
     
     
         12 ) The fusion protein of  claim 11 , wherein
 a) the peptide antagonist of Kv1.3 comprises the amino acid sequence of SEQ ID NOs: 3, 22, 34 or 42;   b) optionally the C-terminal extension comprises the amino acid sequence of SEQ ID NOs: 128, 143, 155, 188, 206-210, 212, 214, 216, 219, 223, 224, 227, 230, 232-235, 237, 239, 240, 243, 252, 261-263, or 268;   c) the linker comprises the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO:119; and   d) the half-life extending moiety is human serum albumin.   
     
     
         13 ) The fusion protein of  claim 11 , wherein
 a) the peptide antagonist of Kv1.3 comprises the amino acid sequence of SEQ ID NO: 42;   b) the linker comprises the amino acid sequence of SEQ ID NO: 116; and   c) the half-life extending moiety is human serum albumin.   
     
     
         14 ) The fusion protein of  claim 11 , wherein
 a) The peptide antagonist of Kv1.3 comprises the amino acid sequence of SEQ ID NO: 42;   b) the C-terminal extension comprises the amino acid sequence of SEQ ID NO: 209;   c) the linker comprises the amino acid sequence of SEQ ID NO: 116; and   d) the half-life extending moiety is human serum albumin.   
     
     
         15 ) The fusion protein of  claim 11 , wherein
 a) the peptide antagonist of Kv1. 3 comprises the amino acid sequence of SEQ ID NO: 3;   b) the C-terminal extension comprises the amino acid sequence of SEQ ID NO: 235;   c) the linker comprises the amino acid sequence of SEQ ID NO: 116; and   d) the half-life extending moiety is human serum albumin.   
     
     
         16 ) The fusion protein of  claim 11 , wherein
 a) the peptide antagonist of Kv1.3 comprises the amino acid sequence of SEQ ID NO: 42;   b) the C-terminal extension comprises the amino acid sequence of SEQ ID NO: 235;   c) the linker comprises the amino acid sequence of SEQ ID NO: 116; and   d) the half-life extending moiety is human serum albumin.   
     
     
         17 ) The fusion protein of  claim 1 , wherein the fusion protein is at least 100 fold more selective towards human Kv1.3 than towards human Kv1.1, when selectivity is measured as a ratio of an IC 50  value of the isolated fusion protein for Kv1.1 to an IC 50  value of the isolated fusion protein for Kv1.3 in a patch clamp assay in cells transfected with Kv1.1 and Kv1.3, respectively. 
     
     
         18 ) The fusion protein of  claim 1 , wherein the antagonist inhibits potassium currents with an IC 50  value at least about 10 fold less than an IC 50  value for a parent KV1C2 fusion protein of SEQ ID NO: 425 in a patch clamp assay in cells transfected with human Kv1.3. 
     
     
         19 ) The fusion protein of  claim 1 , wherein the fusion protein inhibits currents with an IC 50  value of about 1.5×10 −8  M or less in a patch clamp assay in cells transfected with human Kv1.3. 
     
     
         20 ) The fusion protein of  claim 1 , wherein the fusion protein inhibits in vitro thallium flux with an IC 50  value of about 2.2×10 −8  M or less in cells transfected with human Kv1.3. 
     
     
         21 ) An isolated fusion protein comprising a peptide antagonist of Ev1.3 conjugated to a half-life extending moiety via a linker, the peptide antagonist of Kv1.3 having an optional C-terminal extension of four amino acids, wherein
 a) the peptide antagonist of Kv1.3 comprises the amino acid sequence of SEQ ID NOs: 3-110;   b) the C-terminal extension comprises the amino acid sequence of SEQ ID NOs: 123-268;   c) the linker comprises the amino acid sequence of SEQ ID NOs: 112-122 or 428; and   d) the half-life extending moiety is human serum albumin.   
     
     
         22 ) An isolated polynucleotide encoding the fusion protein of  claim 1  or  21 . 
     
     
         23 ) A vector comprising the isolated polynucleotide of  claim 22 . 
     
     
         24 ) A host cell comprising the vector of  claim 23 . 
     
     
         25 ) A method of producing the isolated fusion protein of  claim 1  or  21 , comprising culturing the host cell of  claim 25  and recovering the fusion protein expressed by the host cell. 
     
     
         26 ) A pharmaceutical composition comprising the fusion protein of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         27 ) A method of suppressing T cell activation in a subject having a condition associated with undesired T cell activation, comprising administering to the subject an effective amount of the isolated fusion protein of  claim 1  to suppress T cell activation. 
     
     
         28 ) The method of  claim 27 , wherein the condition associated with undesired T cell activation is an inflammatory condition, an immune and proliferative disorder, rheumatoid arthritis (PA), ankylosing spondylitis, psoriatic arthritis, osteoarthritis, osteoporosis, uveitis, inflammatory fibrosis, scleroderma, lung fibrosis, cirrhosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, asthma, allergic asthma, allergies, Chronic Obstructive Pulmonary Disease (COPD), multiple sclerosis, psoriasis, contact-mediated dermatitis, systemic lupus erythematosus (SLE) and other forms of lupus, diabetes, type I diabetes, obesity, cancer, lupus, restenosis, systemic sclerosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, or graft-versus-host disease. 
     
     
         29 ) An isolated peptide antagonist of Kv1.3 comprising
 a) the sequence shown in SEQ ID NO: 1 having a substitution of glycine to isoleucine at position 10 (G10I), and optionally having 1, 2, 3, 4, 5, 6 or 7 additional substitutions; or   b) an amino acid sequence which is at least 80% identical to SEQ ID NO: 1, further comprising a G10I substitution; and   c) the peptide antagonist of Kv1.3 optionally comprises a C-terminal extension of four amino acids.   
     
     
         30 ) The peptide antagonist of Kv1.3 of  claim 29  comprising the sequence 
       
         
           
                 
               
                   (SEQ ID NO: 426) 
                 
                   GVPXaa 1 Xaa 2 VKCXaa 3 ISRQCXaa 4 Xaa 5 PCKDAGMRFGKCMNGKCHCT  
                 
                   PK; 
                 
             
                
                
                
               
            
           
         
       
       wherein
 a) Xaa 1  is I or T, Q or E; 
 b) Xaa 2  is N or D; 
 c) Xaa 3  is K, R, E, A or Q; 
 d) Xaa 4  is I, E, L, D, Q, H, V, K or A; and 
 e) Xaa 5  is E, K, L, Q, D, V or H. 
 
     
     
         31 ) The peptide antagonist of Kv1.3 of  claim 29  comprising the sequence 
       
         
           
                 
               
                   (SEQ ID NO: 427) 
                 
                   GVPXaa 1 Xaa 2 VKCXaa 3 ISRQCXaa 4 Xaa 5 PCKDAGMRFGKCMNGKCHCT  
                 
                   PK; 
                 
             
                
                
                
               
            
           
         
       
       wherein
 a) Xaa 1  is I or T; 
 b) Xaa 2  is N or D; 
 c) Xaa 3  is K or R; 
 d) Xaa4 is I or E; and 
 e) Xaa 5  is E or K. 
 
     
     
         32 ) The peptide antagonist of Kv1.3 of  claim 29  comprising the amino acid sequence of SEQ ID NOs: 3, 13, 21, 22, 24, 26, 29, 30, 32, 34, 38, 39, 42-46, 49, 51, 59, 63, 65, 69, 71, 73, 76, 78, 81-83, 85, 87, 89, 92, 96, 101, 103, 104, 108 or 269-414. 
     
     
         33 ) The peptide antagonist of Kv1.3 of  claim 29  comprising the sequence of SEQ ID NOs: 3-110 or 269-414. 
     
     
         34 ) An isolated polynucleotide encoding the peptide antagonist of Kv1.3 of  claim 29 . 
     
     
         35 ) A vector comprising the isolated polynucleotide of  claim 34 . 
     
     
         36 ) A host cell comprising a vector of  claim 35 . 
     
     
         37 ) A method of producing the isolated peptide antagonist of Kv1.3 of  claim 29 , comprising culturing the host cell of  claim 36  and recovering the peptide antagonist of KV1.3 expressed by the host cell.