Human alpha-galactosidase variants

Abstract

The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides have been optimized to provide improved stability under both acidic (pH <4.5) and basic (pH >7) conditions. The invention also relates to the use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.

1 . A recombinant alpha galactosidase A and/or biologically active recombinant alpha galactosidase A fragment comprising an amino acid sequence comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:5. 
     
     
         2 . The recombinant alpha galactosidase A of  claim 1 , wherein said alpha galactosidase A comprises at least one mutation in at least one position as provided in Tables 2.1, 2.2, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and/or 7.1, wherein the positions are numbered with reference to SEQ ID NO:5. 
     
     
         3 . The recombinant alpha galactosidase A of  claim 2 , wherein said alpha galactosidase A comprises at least one mutation in at least one position as provided in Table 2.3, wherein the positions are numbered with reference to SEQ ID NO:10. 
     
     
         4 . The recombinant alpha galactosidase A of  claim 1 , wherein said recombinant alpha galactosidase A is derived from a human alpha galactosidase A. 
     
     
         5 . A recombinant alpha galactosidase A comprising the polypeptide sequence of SEQ ID NO:15, 13, 10, 18, 40, 42, 44, or 46. 
     
     
         6 . The recombinant alpha galactosidase A of  claim 1 , wherein said recombinant alpha galactosidase A is more thermostable than the alpha galactosidase A of SEQ ID NO:5. 
     
     
         7 . The recombinant alpha galactosidase A of  claim 1 , wherein said recombinant alpha galactosidase A is more stable at pH 7.4 than the alpha galactosidase A of SEQ ID NO:5. 
     
     
         8 . The recombinant alpha galactosidase A of  claim 7 , wherein said recombinant alpha galactosidase A is more stable at pH 4.3 than the alpha galactosidase A of SEQ ID NO:5. 
     
     
         9 . The recombinant alpha galactosidase A of  claim 7 , wherein said recombinant alpha galactosidase A is more stable to exposure to serum than the alpha galactosidase A of SEQ ID NO:5. 
     
     
         10 . The recombinant alpha galactosidase A of  claim 1 , wherein said recombinant alpha galactosidase A is a deimmunized alpha galactosidase A. 
     
     
         11 . The recombinant alpha galactosidase A of  claim 1 , wherein said recombinant alpha galactosidase A is a deimmunized alpha galactosidase A provided in Table 7.1. 
     
     
         12 . The recombinant alpha galactosidase A of  claim 1 , wherein said recombinant alpha galactosidase A is purified. 
     
     
         13 . The recombinant alpha galactosidase A of  claim 1 , wherein said recombinant alpha galactosidase A exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to pH 7.4; iii) increased tolerance to pH 4.3; iv) increased tolerance to serum; or v) reduced immunogenicity; or a combination of any of i), ii), iii), iv), or v), as compared to a reference sequence. 
     
     
         14 . The recombinant alpha galactosidase A of  claim 13 , wherein said reference sequence is SEQ ID NO:5 or SEQ ID NO:10. 
     
     
         15 . A composition comprising at least one recombinant alpha galactosidase A of  claim 1 . 
     
     
         16 . A recombinant polynucleotide sequence encoding at least one recombinant alpha galactosidase A set forth in  claim 1 . 
     
     
         17 . The recombinant polynucleotide sequence of  claim 16 , wherein said polynucleotide sequence is codon-optimized. 
     
     
         18 . An expression vector comprising the recombinant polynucleotide sequence of  claim 16 . 
     
     
         19 . The expression vector of  claim 18 , wherein said recombinant polynucleotide sequence is operably linked to a control sequence. 
     
     
         20 . The expression vector of  claim 19 , wherein said control sequence is a promoter. 
     
     
         21 . The expression vector of  claim 20 , wherein said promoter is a heterologous promoter. 
     
     
         22 . A host cell comprising the expression vector of  claim 18 . 
     
     
         23 . The host cell of  claim 22 , wherein said host cell is eukaryotic. 
     
     
         24 . A method of producing an alpha galactosidase A variant, comprising culturing said host cell of  claim 22 , under conditions that said alpha galactosidase A encoded by said recombinant polynucleotide is produced. 
     
     
         25 . The method of  claim 24 , further comprising the step of recovering said alpha galactosidase A. 
     
     
         26 . The method of  claim 25 , further comprising the step of purifying said alpha galactosidase A. 
     
     
         27 . A pharmaceutical composition for the treatment of Fabry disease, comprising the enzyme composition of  claim 15 . 
     
     
         28 . The pharmaceutical composition of  claim 27 , further comprising a pharmaceutically acceptable carrier and/or excipient. 
     
     
         29 . The pharmaceutical composition of  claim 27 , wherein said composition is suitable for parenteral injection or infusion to a human. 
     
     
         30 . A method for treating and/or preventing the symptoms of Fabry disease in a subject, comprising providing a subject having Fabry disease, and providing the pharmaceutical composition of  claim 27 , to said subject. 
     
     
         31 . The method of  claim 30 , wherein said symptoms of Fabry disease are ameliorated. 
     
     
         32 . The method of  claim 30 , wherein said subject is able to eat a diet that is less restricted in its fat content than diets required by subjects exhibiting the symptoms of Fabry disease. 
     
     
         33 . The method of  claim 30 , wherein said subject is an infant or child. 
     
     
         34 . The method of  claim 30 , wherein said subject is an adult or young adult. 
     
     
         35 . Use of the compositions provided in  claim 15 .