Interferon-alpha polypeptides and conjugates

Abstract

The present invention provides interferon-alpha polypeptides and conjugates, and nucleic acids encoding the polypeptides. The invention also includes compositions comprising these polypeptides, conjugates, and nucleic acids; cells containing or expressing the polypeptides, conjugates, and nucleic acids; methods of making the polypeptides, conjugates, and nucleic acids; and methods of using the polypeptides, conjugates, and nucleic acids.

1 .- 7 . (canceled)  
     
     
         8 . An isolated or recombinant polypeptide comprising a sequence which 
 (a) differs in 0 to 16 amino acid positions from SEQ ID NO:13 and    (b) comprises Ala or Leu at position 48;    which polypeptide exhibits antiviral activity.    
     
     
         9 . The polypeptide of  claim 8 , which sequence comprises Ala at position 48.  
     
     
         10 . The polypeptide of  claim 8 , comprising a sequence which differs from SEQ ID NO:13 in 0 to 8 amino acid positions.  
     
     
         11 . An isolated or recombinant polypeptide comprising the sequence SEQ ID NO:13, optionally further comprising a methionine at the N-terminus, which polypeptide exhibits antiviral activity.  
     
     
         12 .- 19 . (canceled)  
     
     
         20 . An isolated or recombinant polypeptide comprising a sequence which 
 (a) differs in 0 to 16 amino acid positions from SEQ ID NO:38 and    (b) comprises Ala or Leu at position 48;    which polypeptide exhibits antiviral activity.    
     
     
         21 . The polypeptide of  claim 20 , which sequence comprises Ala at position 48.  
     
     
         22 . The polypeptide of  claim 20 , comprising a sequence which differs from SEQ ID NO:38 in 0 to 8 amino acid positions.  
     
     
         23 . An isolated or recombinant polypeptide comprising the sequence SEQ ID NO:38 or the sequence of a variant thereof wherein the variant differs in 1 to 2 amino acid positions from SEQ ID NO:38, optionally further comprising a methionine at the N-terminus, which polypeptide exhibits antiviral activity.  
     
     
         24 . The polypeptide of  claim 8 ,  11 ,  20 ,  23 , or  77 , wherein the antiviral activity of the polypeptide is equal to or greater than the antiviral activity of huIFN-alpha 2b or huIFN-alpha 2a.  
     
     
         25 . The polypeptide of  claim 24 , wherein the antiviral activity of the polypeptide is at least two-fold greater than the antiviral activity of huIFN-alpha 2b or huIFN-alpha 2a.  
     
     
         26 . The polypeptide of  claim 8 ,  11 ,  20 ,  23 , or  77 , wherein the polypeptide exhibits a ratio of antiviral activity/antiproliferative activity at least two-fold greater than the ratio of antiviral activity/antiproliferative activity exhibited by huIFN-alpha 2b or huIFN-alpha 2a.  
     
     
         27 . A conjugate comprising 
 (a) the polypeptide of  claim 8 ,  11 ,  20 ,  23 , or  77  and    (b) a non-polypeptide moiety covalently attached to the polypeptide,    which conjugate exhibits antiviral activity.    
     
     
         28 . The conjugate of  claim 27 , comprising at least two non-polypeptide moieties covalently attached to the polypeptide.  
     
     
         29 . The conjugate of  claim 27 , comprising a non-polypeptide moiety covalently attached to a cysteine residue.  
     
     
         30 . The conjugate of  claim 27 , comprising a non-polypeptide moiety covalently attached to a lysine residue or to the N-terminal amino group.  
     
     
         31 . The conjugate of  claim 27 , comprising a non-polypeptide moiety covalently attached to a lysine residue.  
     
     
         32 . The conjugate of  claim 27 , comprising a non-polypeptide moiety attached to the N-terminal amino group.  
     
     
         33 . The conjugate of  claim 27 , wherein the non-polypeptide moiety is a polymer.  
     
     
         34 . The conjugate of  claim 33 , wherein the polymer is a polyethylene glycol.  
     
     
         35 . The conjugate of  claim 34 , wherein the polyethylene glycol is a 40 kDa mPEG2 moiety covalently attached to a lysine residue.  
     
     
         36 . The conjugate of  claim 27 , wherein the non-polypeptide moiety is a sugar.  
     
     
         37 . The conjugate of  claim 36 , wherein the sugar is attached to an N-glycosylation site of the polypeptide.  
     
     
         38 . A conjugate comprising 
 (a) a polypeptide comprising the sequence SEQ ID NO:13, optionally further comprising a methionine at the N-terminus; and    (b) a PEG moiety covalently attached to a lysine residue of the polypeptide, which conjugate exhibits antiviral activity.    
     
     
         39 . The conjugate of  claim 38 , wherein the PEG moiety is a 40 kDa mPEG2 moiety.  
     
     
         40 . The conjugate of  claim 39 , wherein the 40 kDa mPEG2 moiety is covalently attached to a lysine residue selected from Lys122 and Lys135.  
     
     
         41 . A conjugate comprising 
 (a) a polypeptide comprising the sequence SEQ ID NO:38 or the sequence of a variant thereof wherein the variant differs in 1 to 2 amino acid positions from SEQ ID NO:38, optionally further comprising a methionine at the N-terminus; and    (b) a PEG moiety covalently attached to a lysine residue of the polypeptide, which conjugate exhibits antiviral activity.    
     
     
         42 . The conjugate of  claim 41  or  79 , wherein the PEG moiety is a 40 kDa mPEG2 moiety.  
     
     
         43 . The conjugate of  claim 42 , wherein the 40 kDa mPEG2 moiety is covalently attached to a lysine residue selected from Lys31, Lys122, and Lys135.  
     
     
         44 . A composition comprising 
 (a) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:13, optionally further comprising a methionine at the N-terminus, and a 40 kDa mPEG2 moiety covalently attached to Lys122; and    (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:13, optionally further comprising a methionine at the N-terminus, and a 40 kDa mPEG2 moiety covalently attached to Lys135;    which composition exhibits antiviral activity.    
     
     
         45 . A composition comprising 
 (a) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:38, optionally further comprising a methionine at the N-terminus, and a 40 kDa mPEG2 moiety covalently attached to Lys31;    (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:38, optionally further comprising a methionine at the N-terminus, and a 40 kDa mPEG2 moiety covalently attached to Lys122; and    (b) a conjugate comprising a polypeptide comprising the sequence SEQ ID NO:38, optionally further comprising a methionine at the N-terminus, and a 40 kDa mPEG2 moiety covalently attached to Lys135;    which composition exhibits antiviral activity.    
     
     
         46 . A composition comprising the polypeptide of  claim 8 ,  11 ,  20 ,  23  or  77 , and a pharmaceutically acceptable excipient.  
     
     
         47 . A composition comprising the conjugate of  claim 38 ,  41 , or  79 , and a pharmaceutically acceptable excipient.  
     
     
         48 . The composition of  claim 44  or  45  further comprising a pharmaceutically acceptable excipient.  
     
     
         49 . An isolated or recombinant polynucleotide comprising a nucleic acid sequence which encodes the polypeptide of  claim 8 ,  11 ,  20 ,  23 , or  77 .  
     
     
         50 . The polynucleotide of  claim 49 , comprising a nucleic acid sequence which encodes a polypeptide comprising the sequence SEQ ID NO:13.  
     
     
         51 . The polynucleotide of  claim 50 , comprising a nucleic acid sequence selected from SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:89.  
     
     
         52 . The polynucleotide of  claim 49 , comprising a nucleic acid sequence which encodes a polypeptide comprising the sequence SEQ ID NO:38.  
     
     
         53 . The polynucleotide of  claim 52 , comprising a nucleic acid sequence selected from SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:90.  
     
     
         54 . A host cell comprising the polynucleotide of  claim 49 .  
     
     
         55 . A vector comprising the polynucleotide of  claim 49 .  
     
     
         56 . The vector of  claim 55 , which is an expression vector comprising the polynucleotide operably linked to a promoter.  
     
     
         57 . A host cell comprising the vector of  claim 56 .  
     
     
         58 . A composition comprising the polynucleotide of  claim 49  and an excipient.  
     
     
         59 . A method for preparing the polypeptide of  claim 8 ,  11 ,  20 ,  23 , or  77  the method comprising: 
 providing a culture comprising a host cell, the host cell comprising an expression vector comprising a promoter operably linked to a polynucleotide, the polynucleotide comprising a nucleic acid sequence which encodes the polypeptide,    culturing the culture under conditions which permit expression of the polypeptide, and    recovering the polypeptide.    
     
     
         60 . The method of  claim 59 , wherein the host cell is a glycosylating host cell.  
     
     
         61 . The method of  claim 59 , wherein the host cell is a bacterial host cell.  
     
     
         62 . The method of  claim 59 , wherein the bacterial host cell is  E. coli.    
     
     
         63 . The method of  claim 62 , wherein the step of recovering the polypeptide comprises: 
 (a) isolating inclusion bodies (IB) comprising the polypeptide;    (b) solubilizing the IB, thereby obtaining unfolded polypeptide;    (c) refolding the unfolded polypeptide, thereby obtaining refolded polypeptide; and    (d) purifying the refolded polypeptide.    
     
     
         64 . A method for preparing a conjugate, the method comprising 
 (i) providing the polypeptide of  claim 8 ,  11 ,  20 ,  23 , or  77 , and    (ii) attaching at least one non-polypeptide moiety to the polypeptide, wherein the resulting conjugate exhibits antiviral activity.    
     
     
         65 . The method of  claim 64 , wherein the step of providing the polypeptide comprises: 
 providing a culture comprising a host cell, the host cell comprising an expression vector comprising a promoter operably linked to a polynucleotide, the polynucleotide comprising a nucleic acid sequence which encodes the polypeptide,    culturing the culture under conditions which permit expression of the polypeptide, and    recovering the polypeptide.    
     
     
         66 . The method of  claim 65 , wherein the host cell is a glycosylating host cell or a bacterial host cell.  
     
     
         67 . The method of  claim 66 , wherein the bacterial host cell is  E. coli.    
     
     
         68 . The method of  claim 67 , wherein the step of recovering the polypeptide comprises: 
 (a) isolating inclusion bodies (IB) comprising the polypeptide;    (b) solubilizing the IB, thereby obtaining unfolded polypeptide;    (c) refolding the unfolded polypeptide, thereby obtaining refolded polypeptide; and    (d) purifying the refolded polypeptide.    
     
     
         69 . The method of  claim 64 , wherein the step of attaching the at least one non-polypeptide moiety to the polypeptide comprises: 
 reacting the polypeptide with 40 kDa mPEG2-NHS at a molar ratio of mPEG2-NHS:polypeptide of about 3:1 to 5:1 at pH 9 and 4° C. for 1 hour to overnight.    
     
     
         70 . A method for reducing the number of copies of a virus in cells infected with the virus, the method comprising: administering the conjugate of  claim 27  to the cells in an amount effective to reduce the number of copies of the virus in the cells, thereby reducing the number of copies of the virus in said cells.  
     
     
         71 . A method for reducing the level of HCV RNA in the serum of a patient infected with HCV, comprising administering to the patient the conjugate of  claim 27  in an amount effective to reduce the level of HCV RNA compared to the HCV RNA level present prior to the start of treatment.  
     
     
         72 . A method for reducing the level of HBV DNA in serum of a patient infected with HBV, comprising administering to the patient the conjugate of  claim 27  in an amount effective to reduce the level of IIBV DNA compared to the HBV DNA level present prior to the start of treatment.  
     
     
         73 . A method for reducing the level of HIV RNA in serum of a patient infected with HIV, comprising administering to the patient the conjugate of  claim 27  in an amount effective to reduce the level of HIV RNA compared to the HIV RNA level present prior to the start of treatment.  
     
     
         74 . The polypeptide of  claim 8 , comprising a sequence which differs from SEQ ID NO:13 in 0 to 4 amino acid positions.  
     
     
         75 . The polypeptide of  claim 22 , comprising a sequence which differs from SEQ ID NO:38 in 0 to 4 amino acid positions.  
     
     
         76 . The polypeptide of  claim 23 , comprising the sequence SEQ ID NO:38.  
     
     
         77 . An isolated or recombinant polypeptide comprising the sequence SEQ ID NO:39 or the sequence of a variant thereof wherein the variant differs in 1 to 3 amino acid positions from SEQ ID NO:39, optionally further comprising a methionine at the N-terminus, which polypeptide exhibits antiviral activity.  
     
     
         78 . The conjugate of  claim 41 , wherein the polypeptide comprises the sequence SEQ ID NO:38.  
     
     
         79 . A conjugate comprising 
 (a) a polypeptide comprising the sequence SEQ ID NO:39 or the sequence of a variant thereof wherein the variant differs in 1 to 3 amino acid positions from SEQ ID NO:39, optionally further comprising a methionine at the N-terminus; and    (b) a PEG moiety covalently attached to a lysine residue of the polypeptide, which conjugate exhibits antiviral activity.