Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof

Abstract

The present invention is directed to phenylalanine ammonia-lyase (PAL) variants produced by prokaryotes, wherein such prokaryotic PAL variant has a greater phenylalanine-converting activity and/or a reduced immunogenicity as compared to a wild-type PAL. The invention provides compositions of prokaryotic PAL and biologically active fragments, mutants, variants or analogs thereof, as well as methods for the production, purification, and use of such compositions for therapeutic purposes, including the treatment of cancer.

1 . A pharmaceutical composition comprising comprising a prokaryotic phenylalanine ammonia-lyase (PAL) variant and a pharmaceutically acceptable carrier, wherein said PAL variant has a greater phenylalanine-converting activity and/or a reduced immunogenicity as compared to a wild-type PAL, wherein the pharmaceutically acceptable carrier comprises a stabilizer. 
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein one or more amino acid residues of said PAL variant have been substituted by another amino acid residue wherein the substitution increases said activity and/or reduces said immunogenicity as compared to the wild-type PAL. 
     
     
         3 . The pharmaceutical composition of  claim 2 , wherein the one or more cysteine residues of said PAL variant have been substituted by a serine residue. 
     
     
         4 . The pharmaceutical composition of  claim 3 , wherein said PAL variant is  Anabaena variabilis  PAL (AvPAL). 
     
     
         5 . The pharmaceutical composition of  claim 4 , wherein the one or more cysteine residues of said AvPAL variant have been substituted by a serine residue is selected from the group consisting of cysteine residues at positions 64, 318, 503 and 565. 
     
     
         6 . The pharmaceutical composition of  claim 5 , wherein the cysteine residue at position 565 of said AvPAL variant has been substituted by a serine residue. 
     
     
         7 . The pharmaceutical composition of  claim 5 , wherein the cysteine residues at positions 503 and 565 of said AvPAL variant have been substituted by serine residues. 
     
     
         8 . The pharmaceutical composition of  claim 1 , wherein said PAL variant further comprises a water-soluble polymer. 
     
     
         9 . The pharmaceutical composition of  claim 8 , wherein the water-soluble polymer is a polyethylene glycol. 
     
     
         10 . The pharmaceutical composition of  claim 9 , wherein said PAL variant is  Anabaena variabilis  PAL (AvPAL) and the ratio of said AvPAL variant and the polyethylene glycol is about 1:3 (1:3 AvPAL:PEG). 
     
     
         11 . The pharmaceutical composition of  claim 10 , wherein the cysteine residues at positions 503 and 565 of said AvPAL variant have been substituted by serine residues. 
     
     
         12 . The pharmaceutical composition of  claim 1 , wherein the stabilizer is L-phenylalanine or structural analog thereof 
     
     
         13 . The pharmaceutical composition of  claim 1 , wherein the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid and benzoic acid. 
     
     
         14 . The pharmaceutical composition of  claim 13 , wherein the stabilizer is L-phenylalanine. 
     
     
         15 . The pharmaceutical composition of  claim 13 , wherein the stabilizer is trans-cinnamic acid. 
     
     
         16 . The pharmaceutical composition of  claim 13 , wherein the stabilizer is benzoic acid.