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

Abstract

Provided herein are 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. Further provided are compositions of prokaryotic PAL and biologically active fragments, mutants, variants or analogs thereof, as well as methods for the production, purification, formulation, and use of such compositions for industrial and therapeutic purposes, e.g., treating hyperphenylalaninemia, including phenylketonuria, and other disorders, including cancer.

1 .- 75 . (canceled) 
     
     
         76 . A pharmaceutical composition comprising
 (a) a pegylated  Anabaena variabilis  phenylalanine-ammonia lyase (AvPAL) variant having an amino acid sequence of SEQ ID NO:11, wherein the concentration of the pegylated AvPAL is from about 1 to 50 mg/mL, from about 5 to 20 mg/mL, or from about 5 to 15 mg/mL; and   (b) a pharmaceutically acceptable carrier comprising (i) NaCl, (ii) trans-cinnamic acid, (iii) Tris-HCl, (iv) L-phenylalanine (Phe), and (v) glycine (Gly).   
     
     
         77 . The pharmaceutical composition of  claim 76 , wherein the pegylated AvPAL variant comprises polyethylene glycol wherein the ratio of AvPAL variant and the polyethylene glycol is about 1:3. 
     
     
         78 . The pharmaceutical composition of  claim 76 , wherein the pegylated AvPAL variant comprises N-hydroxysuccinimide (NHS)-methoxypolyethylene glycol (PEG). 
     
     
         79 . The pharmaceutical composition of  claim 76 , wherein the concentration of the pegylated AvPAL is 5 mg/mL. 
     
     
         80 . The pharmaceutical composition of  claim 76 , wherein the concentration of the pegylated AvPAL is 20 mg/mL. 
     
     
         81 . The pharmaceutical composition of  claim 76 , wherein the pharmaceutically acceptable carrier comprises: (a) NaCl, trans-cinnamic acid, and Tris-HCl; or (b) NaCl, Tris-HCl, L-phenylalanine (Phe), and glycine (Gly). 
     
     
         82 . The pharmaceutical composition of  claim 76 , wherein (i) the concentration of Tris-HCl is from about 5 to 15 mM; (ii) the concentration of NaCl is from about 120 to 150 mM; (iii) the concentration of Phe is from about 0.5 to 1.5 mM; and/or (iv) the concentration of Gly is from about 1.0 to 100 mM, from about 1.0 to 20 mM, or from about 20 to 100 mM. 
     
     
         83 . The pharmaceutical composition of  claim 76 , wherein the pH of the pharmaceutical composition is from about pH 6.0 to 8.0, from about pH 6.5 to 7.5, or from about pH 7.0 to 7.6. 
     
     
         84 . The pharmaceutical composition of  claim 76 , wherein the pH of the pharmaceutical is from pH 6.6 to 7.4. 
     
     
         85 . A method for treating a subject having phenylketonuria (PKU), comprising administering to said subject the pharmaceutical composition of  claim 76 , wherein the administration of the AvPAL variant is effective to lower the phenylalanine concentration in the plasma of said subject as compared to said concentration in the absence of said administration. 
     
     
         86 . The method of  claim 85 , wherein the subject has above normal concentration of plasma phenylalanine prior to administration of the pharmaceutical composition, preferably wherein the subject has a plasma phenylalanine concentration greater than 180 μM, greater than 200 μM, greater than 360 μM, or greater than 600 μM prior to administration of the pharmaceutical composition. 
     
     
         87 . The method of  claim 86 , wherein the subject has a plasma phenylalanine concentration greater than 600 μM prior to administration of the pharmaceutical composition. 
     
     
         88 . The method of  claim 85 , wherein the subject is an adult.