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 . A formulation comprising:
 (a) a pegylated  Anabaena variabilis  phenylalanine ammonia-lyase (AvPAL) variant wherein the cysteine residues at positions 503 and 565 of said AvPAL variant have been substituted by serine residues (SEQ ID NO:11), said AvPAL variant comprising polyethylene glycol wherein the ratio of AvPAL variant and the polyethylene glycol is about 1:3, and   (b) a pharmaceutically acceptable carrier comprising: (i) Tris-HCl, (ii) NaCl, (iii) L-phenylalanine (Phe), and (iv) glycine (Gly).   
     
     
         2 . The formulation of  claim 1 , wherein the concentration of the pegylated AvPAL variant is from about 5 to 15 mg/mL. 
     
     
         3 . The formulation of  claim 2  wherein the concentration of Tris-HCl is from about 5 to 15 mM. 
     
     
         4 . The formulation of  claim 2 , wherein the concentration of NaCl is from about 120 to 150 mM. 
     
     
         5 . The formulation of  claim 2 , wherein the concentration of Phe is from about 0.5 to 1.5 mM. 
     
     
         6 . The formulation of  claim 2 , wherein the concentration of Gly is from about 1.0 to 100 mM, from about 1.0 to 20 mM, or from 20 to 100 mM. 
     
     
         7 . The formulation of  claim 2 , wherein the pH of the formulation is from about 6.5 to 7.5. 
     
     
         8 . The formulation of  claim 2 , wherein the pH of the formulation is from about 7.0 to 7.6. 
     
     
         9 . The formulation of  claim 2 , further comprising m-cresol as a preservative. 
     
     
         10 . The formulation of  claim 9 , wherein the concentration of m-cresol is from about 0.3% to 0.5% (w/v). 
     
     
         11 .- 18 . (canceled) 
     
     
         19 . The formulation of  claim 2 , wherein the shelf-life (T 90 ) of the pegylated AvPAL variant is increased between 1.5-fold and 10-fold at a temperature of between 4° C. and 42° C., as compared to a corresponding pegylated AvPAL variant formulation in the absence of the Phe. 
     
     
         20 .- 75 . (canceled) 
     
     
         76 . A method for purifying an  Anabaena variabilis  phenylalanine ammonia-lyase (AvPAL) variant with minimal aggregation comprising:
 (a) lysing bacterial cells containing the AvPAL variant by homogenization to generate a cell lysate;   (b) heating the cell lysate to 65° C. for 30 to 120 minutes;   (c) centrifuging the heated cell lysate, wherein a supernatant comprising the AvPAL variant is retained;   (d) filtering the supernatant to remove precipitates; and   (e) separating the AvPAL variant from contaminating proteins by sequential chromatography over an anion exchange (AIEX) column followed by a hydrophobic interaction (HIC) column, wherein the eluate from the HIC column comprises the AvPAL variant,   
       wherein the cysteine residues at positions 503 and 565 of said AvPAL variant have been substituted by serine residues (SEQ ID NO:11). 
     
     
         77 . The method of  claim 76  further comprising
 (f) ultrafiltering or ultrafiltering/diafiltering the eluate from the HIC column comprising the AvPAL variant; 
 (g) pegylating the AvPAL variant by mixing polyethylene glycol with the AvPAL variant; 
 (h) removing free polyethylene glycol from the pegylated AvPAL variant by ultrafiltration/diafiltration; and 
 (i) formulating the pegylated AvPAL variant 
 
       said AvPAL variant comprising polyethylene glycol. 
     
     
         78 . The method of  claim 77 , wherein the ratio of AvPAL variant and the polyethylene glycol is about 1:3. 
     
     
         79 . The method of  claim 77 , wherein the AIEX column is a Toyopearl Giga Cap Q 650M column and the HIC column is a Toyopearl Butyl 650M column. 
     
     
         80 . The method of  claim 77 , further comprising freezing and thawing the eluate from the HIC column comprising the AvPAL variant obtained in step (e), wherein one or more polyols or sugars selected from the group consisting of glycerol, sucrose, glucose, trehalose, mannitol and sorbitol are added to the HIC column eluate prior to freezing. 
     
     
         81 . The method of  claim 80 , wherein the polyol is glycerol. 
     
     
         82 . The method of  claim 81 , wherein the concentration of glycerol is 10% (v/v). 
     
     
         83 . The method of  claim 80 , wherein the sugar is sucrose. 
     
     
         84 . The method of  claim 83 , wherein the concentration of sucrose is 10% (v/v). 
     
     
         85 . The method of  claim 80 , wherein freezing and thawing the eluate from the HIC column comprising the AvPAL variant are performed in discrete temperature steps. 
     
     
         86 . The method of  claim 77 , wherein a diafiltration buffer in step (f) comprises potassium phosphate (KPi) and one or more agents consisting of trans-cinnamic acid (t-CA) and glycerol. 
     
     
         87 . The method of  claim 86 , wherein the diafiltration buffer comprises 50 mM KPi, 10 mM t-CA, 5% glycerol, pH 8.5. 
     
     
         88 . The method of  claim 77 , wherein a non-ionic detergent is added to the ultrafiltered or ultrafiltered/diafiltered eluate from the HIC column comprising the AvPAL variant obtained in step (f). 
     
     
         89 . The method of  claim 88 , wherein the non-ionic detergent is polysorbate 80 (PS80). 
     
     
         90 . The method of  claim 89 , wherein the concentration of PS80 is 0.02%. 
     
     
         91 . A method for treating a disease caused all or in part by a deficiency in phenylalanine hydroxylase (PAH), comprising administering to a human having a deficiency in PAH an effective amount of the formulation  claim 1 . 
     
     
         92 . The method of  claim 91 , wherein the disease is characterized by elevated phenylalanine levels. 
     
     
         93 . The method of  claim 92 , wherein said human has about 10% or less of a no mal PAH activity. 
     
     
         94 . The method of  claim 91 , wherein said human has a mutant PAH. 
     
     
         95 . The method of  claim 94 , wherein said mutant PAH comprises a mutation in the catalytic domain of PAH. 
     
     
         96 . The method of  claim 95 , wherein said mutation comprises one or more mutations selected from the group consisting of F39L, L48S, 165T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241c, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S, and Y414C. 
     
     
         97 . A method for treating a subject having phenylketonuria (PKU), comprising administering to said subject the formulation of  claim 1 , wherein the administration of the AvPAL variant is effective to lower the phenylalanine concentration in the plasma of said human as compared to said concentration in the absence of said administration. 
     
     
         98 . The method of  claim 97 , wherein said human has a mutant PAH. 
     
     
         99 . The method of  claim 98 , wherein said mutant PAH comprises a mutation in the catalytic domain of PAH. 
     
     
         100 . The method of  claim 99 , wherein said mutation comprises one or more mutations selected from the group consisting of F39L, L485, 165T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241c, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S, and Y414C. 
     
     
         101 . The method of  claim 97 , wherein the human is an infant between 0 and 3 years of age, and wherein the plasma phenylalanine concentration of said infant is between about 360 μM to about 4800 μM before administration of the variant. 
     
     
         102 . A method for the treating a pregnant female having hyperphenylalaninemia (HPA), comprising administering to said female the formulation of  claim 1  in combination with a protein-restricted diet, wherein the combined administration of the formulation and protein-restricted diet is effective to lower the plasma phenylalanine concentration of said female as compared to said concentration in the absence of said combined administration. 
     
     
         103 . A method of treating elevated plasma phenylalanine concentrations, comprising administering to a human having elevated plasma phenylalanine the formulation of  claim 1  in an amount effective to produce a decrease in the plasma phenylalanine concentration of said human. 
     
     
         104 . A method for decreasing plasma phenylalanine concentration of a human, comprising administering to said human an effective amount of the formulation of  claim 1 . 
     
     
         105 . The method of  claim 104 , wherein the plasma phenylalanine concentration of the human is greater than about 200 μM prior to administration of the formulation. 
     
     
         106 . The method of  claim 105 , wherein the effective amount decreases the plasma phenylalanine concentration of the human by 10% or more. 
     
     
         107 . The method of  claim 104 , wherein the human has a deficiency in PAH activity. 
     
     
         108 . The method of  claim 107 , wherein the human has a mutant PAH. 
     
     
         109 . The method of  claim 108 , wherein the mutant PAH comprises a mutation in the catalytic domain of PAH. 
     
     
         110 . The method of  claim 109 , wherein said mutation comprises one or more mutations selected from the group consisting of F39L, L48S, 165T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241c, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S, and Y414C. 
     
     
         111 . The method of  claim 104 , further comprising administering to the human a protein-restricted diet. 
     
     
         112 . The method of  claim 104 , wherein the human has PKU. 
     
     
         113 . The method of  claim 112 , wherein the PKU is classic severe PKU. 
     
     
         114 . The method of  claim 104 , wherein the human is a pregnant female. 
     
     
         115 . The method of  claim 114 , further comprising administering to the female a protein-restricted diet. 
     
     
         116 . The method of  claim 104 , wherein the human is between 0 and 3 years of age. 
     
     
         117 . A method for treating a disease caused all or in part by a deficiency in phenylalanine hydroxylase (PAH), comprising administering to a human having a deficiency in PAH an effective amount of a formulation comprising the pegylated AvPAL variant purified according to the method of  claim 77 . 
     
     
         118 . The method of  claim 117 , wherein the disease is characterized by elevated phenylalanine levels. 
     
     
         119 . The method of  claim 118 , wherein said human has about 10% or less of a normal PAH activity. 
     
     
         120 . The method of  claim 117 , wherein said human has a mutant PAH. 
     
     
         121 . The method of  claim 120 , wherein said mutant PAH comprises a mutation in the catalytic domain of PAH. 
     
     
         122 . The method of  claim 121 , wherein said mutation comprises one or more mutations selected from the group consisting of F39L, L48S, 165T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241c, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S, and Y414C. 
     
     
         123 . A method for treating a subject having phenylketonuria (PKU), comprising administering to said subject a formulation comprising the pegylated AvPAL variant purified according to the method of  claim 77 , wherein the administration of the AvPAL variant is effective to lower the phenylalanine concentration in the plasma of said human as compared to said concentration in the absence of said administration. 
     
     
         124 . The method of  claim 123 , wherein said human has a mutant PAH. 
     
     
         125 . The method of  claim 124 , wherein said mutant PAH comprises a mutation in the catalytic domain of PAH. 
     
     
         126 . The method of  claim 125 , wherein said mutation comprises one or more mutations selected from the group consisting of F39L, L48S, 165T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241c, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S, and Y414C. 
     
     
         127 . The method of  claim 123 , wherein the human is an infant between 0 and 3 years of age, and wherein the plasma phenylalanine concentration of said infant is between about 360 μM to about 4800 μM before administration of the variant. 
     
     
         128 . A method for the treating a pregnant female having hyperphenylalaninemia (HPA), comprising administering to said female a formulation comprising the pegylated AvPAL variant purified according to the method of  claim 77  in combination with a protein-restricted diet, wherein the combined administration of the formulation and protein-restricted diet is effective to lower the plasma phenylalanine concentration of said female as compared to said concentration in the absence of said combined administration. 
     
     
         129 . A method of treating elevated plasma phenylalanine concentrations, comprising administering to a human having elevated plasma phenylalanine a formulation comprising the pegylated AvPAL variant purified according to the method of  claim 77  in an amount effective to produce a decrease in the plasma phenylalanine concentration of said human. 
     
     
         130 . A method for decreasing plasma phenylalanine concentration of a human, comprising administering to said human an effective amount of a formulation comprising the pegylated AvPAL variant purified according to the method of  claim 77 . 
     
     
         131 . The method of  claim 130 , wherein the plasma phenylalanine concentration of the human is greater than about 200 μM prior to administration of the formulation. 
     
     
         132 . The method of  claim 131 , wherein the effective amount decreases the plasma phenylalanine concentration of the human by 10% or more. 
     
     
         133 . The method of  claim 130 , wherein the human has a deficiency in PAH activity. 
     
     
         134 . The method of  claim 133 , wherein the human has a mutant PAH. 
     
     
         135 . The method of  claim 134 , wherein the mutant PAH comprises a mutation in the catalytic domain of PAH. 
     
     
         136 . The method of  claim 135 , wherein said mutation comprises one or more mutations selected from the group consisting of F39L, L48S, 165T, R68S, A104D, S110C, D129G, E178G, V190A, P211T, R241c, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S, and Y414C. 
     
     
         137 . The method of  claim 130 , further comprising administering to the human a protein-restricted diet. 
     
     
         138 . The method of  claim 130 , wherein the human has PKU. 
     
     
         139 . The method of  claim 138 , wherein the PKU is classic severe PKU. 
     
     
         140 . The method of  claim 130 , wherein the human is a pregnant female. 
     
     
         141 . The method of  claim 140 , further comprising administering to the female a protein-restricted diet. 
     
     
         142 . The method of  claim 130 , wherein the human is between 0 and 3 years of age.