Methods of using prokaryotic phenylalanine ammonia-lyase

Abstract

The present disclosure provides methods of reducing blood phenylalanine concentration using a prokaryotic phenylalanine ammonia-lyase (PAL) in combination with methotrexate (MTX).

Description

METHODS OF USING PROKARYOTIC PHENYLALANINE AMMONIA-LYASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 712,153, filed October 25, 2024, the disclosure of which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “H808-547-228_SEQ_LISTING.xml”, was created on October 15, 2025, and is 7,305 bytes in size.FIELD OF THE DISCLOSURE

[0003] This disclosure relates to methods of reducing blood phenylalanine concentration with prokaryotic phenylalanine ammonia-lyase (PAL) in combination with methotrexate (MTX) or a pharmaceutically acceptable salt thereof.BACKGROUND OF THE DISCLOSURE

[0004] PAL is a non-mammalian enzyme widely distributed in plants (Koukol, et al., J. Biol. Chem. 236:2692-2698 (1961); Hanson, et al., The Enzymes 7:75-166 (1972); Poppe, et al., Curr. Org. Chem. 7:1297-1315 (2003)), some fungi (Rao, etal., Can. J. Biochem.4512:1863-1872 (1967); Abell, etal., Methods Enzymol. 142:242-253 (1987)) and bacteria (Bezanson, etal., Can. J. Microbiol. 16:147-151 (1970); Xiang, etal., J. Biol. Chem.277:32505-32509 (2002); Hill, etal., Chem. Commun. 1358-1359 (2003)) and can be recombinantly produced in Escherichia coli.

[0005] PAL from the cyanobacteria strains, Anabaena variabilis (Av), has been cloned and expressed in bacteria, and was shown to display PAL enzyme activity in vitro and in vivo (see e.g., U.S. Patent Nos. 7,531,341; 7,534,595; 7,537,923; and 7,560,263). Apegylated recombinant Anabaena variabilis PAL (rAvPAL-PEG) has also been produced, wherein the rAvPAL protein was derivatized by covalent attachment of polyethylene glycol (PEG) to increase its half-life and optimize its pharmacokinetic profile and / or reduce its immunogenicity (Id . Recently, rAvPAL-PEG was approved as an injectable product for treatment of Phenylketonuria (PKU) in adult subjects. There remains a need for newtherapies, including, for example, combinational therapies for modulating immune response during the treatment.SUMMARY OF DISCLOSURE

[0006] In one aspect, provided herein is a method for reducing blood phenylalanine concentration in a subject, the method comprising (1) administering to the subject a weekly dose of a formulation comprising an AvPAL variant, wherein the AvPAL variant comprises an amino acid sequence of SEQ ID NO:4; and (2) administering to the subject a weekly dose of methotrexate (MTX) or a pharmaceutically acceptable salt thereof. In some embodiments, the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered concurrently. In some embodiments, the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered in separate compositions. In some embodiments, the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered on the same day.

[0007] In some embodiments, the dosage of MTX is about 15 mg per week. In some embodiments, the dosage of MTX is about 10 mg per week. In some embodiments, MTX or a pharmaceutically acceptable salt thereof is administered orally. In some embodiments, the dosage of MTX is administered in a single dose. In some embodiments, the dosage of MTX is administered in multiple divided doses within a day. In some embodiments, the dosage of MTX is administered orally at 7.5 mg in the morning and at 7.5 mg in the evening of the same day.

[0008] In some embodiments, the weekly dose of the AvPAL variant is administered subcutaneously. In some embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 1 :6. In some embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 1:3. In some embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 2:3. In some embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 4:3. In some embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 8:3. In some embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 14:3. In some embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 28:3. In some embodiments, the weekly dose of the AvPAL variant is relative to the weekly dose of MTX at a ratio of about 56:3.

[0009] In some embodiments, the subject has not received a prior treatment with the AvPAL variant. In some embodiments, the subject has received a prior treatment with the AvPAL variant. In some embodiments, after the prior treatment with the AvPAL variant the subject has uncontrolled blood phenylalanine (Phe) concentration greater than about 600 micromol / L, and the prior treatment with the AvPAL variant has lasted for more than about 24 consecutive weeks. In some embodiments, the subject has been in the prior treatment with the AvPAL variant at the weekly dose as described herein, and the dosage of the AvPAL variant in the prior treatment has been higher than about 20 mg per day. In some embodiments, the subject in the prior treatment could not receive further dose escalation of the AvPAL variant. In some embodiments, the highest dosage of the AvPAL variant the subject received in the prior treatment has been between about 20 mg per day to about 40 mg per day.

[0010] In some embodiments, the dosage of the AvPAL variant is in the range of about 2 mg per week to about 10 mg per week. In some embodiments, the dosage of the AvPAL variant is in the range of about 10 mg per week to about 20 mg per week. In some embodiments, the dosage of the AvPAL variant is in the range of about 20 mg per week to about 40 mg per week. In some embodiments, the dosage of the AvPAL variant is in the range of about 40 mg per week to about 70 mg per week. In some embodiments, the dosage of the AvPAL variant is in the range of about 70 mg per week to about 140 mg per week. In some embodiments, the dosage of the AvPAL variant is in the range of about 140 mg per week to about 280 mg per week.

[0011] In some embodiments, the method provided herein further comprises administering to the subject folic acid, wherein the folic acid is administered orally about 1 mg per day. In some embodiments, the method further comprises administering to the subject folic acid, wherein the folic acid is administered orally about 2 mg per day. In some embodiments, the administration of folic acid starts from the day when MTX is administered.

[0012] In some embodiments, the AvPAL variant is administered once weekly. In some embodiments, the AvPAL variant is administered twice weekly. In some embodiments, the AvPAL variant is administered four times per week. In some embodiments, the AvPAL variant is administered seven times per week. In some embodiments, the AvPAL variant is administered daily.

[0013] In some embodiments, the method provided herein comprises administering to the subject the AvPAL variant at an induction dosage in the range of about 0.1 mg per week to about 10 mg per week, followed by administering to the subject the AvPAL variant at atitration dosage in the range of about 1 mg per week to about 200 mg per week, followed by administering to the subject the AvPAL variant at a maintenance dosage in the range of about 20 mg per week to about 280 mg per week. In some embodiments, the induction dosage is about 2.5 mg per week. In some embodiments, the titration dosage is in the range of about 5 mg per week to about 70 mg per week. In some embodiments, the maintenance dosage is in the range of about 140 mg per week to about 280 mg per week. In some embodiments, the induction dosage is administered for a duration of between about 2 week and about 6 weeks, the titration dosage is administered for a duration of between about 3 weeks and about 8 weeks, and the maintenance dosage is administered for a duration of between about 20 weeks and about 45 weeks. In some embodiments, the induction dosage is administered for a duration of about 4 weeks, the titration dosage is administered for a duration of about 5 weeks, and the maintenance dosage is administered for a duration of between about 24 weeks and 40 weeks.

[0014] In some embodiments, the weekly dose of MTX is administered for about 24 weeks. In some embodiments, the weekly dose of MTX is administered for about 28 weeks. In some embodiments, the administration of the weekly dose of MTX starts from four weeks before the administration of the AvPAL variant. In some embodiments, the weekly dose of the AvPAL variant is administered for about 24 weeks. In some embodiments, the administration of the weekly dose of MTX is discontinued at about 24 weeks and only the administration of the AvPAL variant continues for about another 24 weeks. In some embodiments, the administration of the weekly dose of MTX is discontinued at about 28 weeks and only the administration of the AvPAL variant continues for about another 24 weeks.

[0015] In some embodiments, the method provided herein further comprises assessing the blood phenylalanine concentration prior to administering MTX.

[0016] In some embodiments, the method further comprises assessing the blood phenylalanine concentration prior to administering the induction dosage of the AvPAL variant. In some embodiments, the method further comprises assessing the blood phenylalanine concentration after administration of one or more induction dosages, titration dosages, maintenance dosages, and / or extension dosages.

[0017] In some embodiments, the method further comprises adjusting the dosage based on the blood phenylalanine concentration. In some embodiments, the dosage is adjusted to attain a blood phenylalanine concentration of below about 600 pM. In some embodiments, the dosage is adjusted to attain a blood phenylalanine concentration of below about 360 pM.In some embodiments, the maintenance dosage is increased if blood phenylalanine concentration is greater than about 360 pM.

[0018] In some embodiments, the subject has phenylketonuria (PKU).

[0019] In some embodiments, the AvPAL variant is pegylated. In some embodiments, said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of at least 1.6 polyethylene glycol per lysine residue of AvPAL variant. In some embodiments, said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of at least 2.4 polyethylene glycol per lysine residue of AvPAL variant. In some embodiments, said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 3 polyethylene glycol per lysine residue of AvPAL variant. In some embodiments, said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 5 polyethylene glycol per lysine residue of AvPAL variant. In some embodiments, said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 6 polyethylene glycol per lysine residue of AvPAL variant. In some embodiments, said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 7 polyethylene glycol per lysine residue of AvPAL variant. In some embodiments, said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 8 polyethylene glycol per lysine residue of AvPAL variant. In some embodiments, said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 9 polyethylene glycol per lysine residue of AvPAL variant.

[0020] In some embodiments, the AvPAL variant is administered as a formulation comprising a pharmaceutically acceptable carrier comprising a stabilizer. In some embodiments, the stabilizer is L-phenylalanine or structural analog thereof. In some embodiments, the stabilizer is selected from the group consisting of L-phenylalanine, transcinnamic acid and benzoic acid. In some embodiments, the stabilizer is trans-cinnamic acid. In some embodiments, the formulation further comprises sodium chloride, and tromethamine and tromethamine hydrochloride

[0021] In some embodiments, an immune response of the subject to the AvPAL variant is suppressed. In some embodiments, tolerability of the subject to the AvPAL variant is improved. In some embodiments, efficacy of the AvPAL variant in the subject is improved.

[0022] In some embodiments, the AvPAL variant is pegvaliase.

[0023] In some embodiments, the subject is between 18 and 65 years of age. In some embodiments, the subject is a human subject.

[0024] Other features and advantages of the disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0025] FIG. 1 illustrates study schema for examining the concomitant use of methotrexate (MTX) with Palynziq® (pegvaliase or Palynziq). Palynziq dosing must be as prescribed by the participant’s treating physician. Participants in Cohort A may titrate their Palynziq dose following the Screening Period (during the entire study period) per the Palynziq USPI and as prescribed by their treating physician. Participants also take folic acid 1 mg orally every day beginning at the start of MTX Tolerability Period and continuing until the Week 25 Visit, a) Participants in Cohort A have never been treated with Palynziq (Palynziq naive). Participants in Cohort B have uncontrolled blood Phe > 600 pmol / L after U24 consecutive weeks of treatment with Palynziq, are on a current dose of A 20 mg / day and are unable to further dose escalate (Palynziq experienced), b) The MTX Tolerability Period is the first 4 weeks of MTX administration (Cohorts A and B). MTX, methotrexate; USPI, United States Prescribing Information.

[0026] FIG. 2 outlines the study assessments and procedures (i.e., Schedule of Activities (SoA)) of Cohort A (Palynziq Naive Participants). AE, adverse event; complement components C3 / C4; CBC, complete blood count; CIC, circulating immune complex;CTCAE, Common Terminology Criteria for Adverse Events; eCRF, electronic Case Report Form; ETV, Early Termination Visit; HRV, hypersensitivity reaction visit; Ig, immunoglobulin; LFT, liver function tests; MTX, methotrexate; NS AID, nonsteroidal antiinflammatory drug; PAH, phenylalanine hydroxylase; PE, physical examination; PEG, polyethylene glycol; Phe, phenylalanine; PK, pharmacokinetics; SAE, serious AE; SCV, Study Completion Visit; USPI, United States Prescribing Information, a) Scheduled visits take place at the hospital setting, clinic, or virtually if approved by the investigator or designee. For virtual visits, the investigator / designee performs assessments while online with the participant, apart from specimen collections and vital signs, which are performed by ahome healthcare personnel. Non clinic visits are to be performed via telephone. All assessments are performed pre dose unless otherwise specified, b) Potential participants enter the Screening Period, which lasts a total of 4 weeks. Following confirmation that both of the 2 blood Phe results are > 600 pmol / L and confirmation that all other eligibility criteria have been met, participants may proceed onto the MTX Tolerability Period. During the MTX Tolerability Period, participants takes oral MTX (15 mg / week) as a single dose each week. The participant is monitored weekly (remote visits via telephone call / video call) to assess compliance and side effects. If the participant has tolerated MTX treatment per protocol-defined clinical and laboratory criteria at the end of the MTX Tolerability Period and meets all other eligibility criteria, the participant may proceed to Day 1. c) If the investigator determines that the CTCAE Grade3 hypersensitivity reaction is related to administration with Palynziq, the participant should return to the clinic within 24 hours of event onset for the HRV. If CTCAE >3 hypersensitivity adverse drug reaction occurs, the participant should return to clinic within 24 hours for tryptase, high-sensitivity C-reactive protein, urine N methyl histamine, d) For participants who terminate from the study early, the ETV occurs within 4 weeks of the last dose of Palynziq or MTX or after withdrawal from study participation, whichever occurs last. Participants who discontinue from MTX or Palynziq early should continue to complete the remaining study assessments until as long as such continued participation will not detrimentally affect the health, safety, and welfare of the participant per investigator determination, e) Participants should take oral MTX at a dosage of 15 mg / week at the start of the MTX Tolerability Period through the Combination Treatment Period. Participants who are unable to tolerate MTX during the 4-week MTX Tolerability Period should stop MTX and be discontinued from the study. The time and day in which participants take their weekly dose of MTX should be consistent throughout the study, and MTX dosing should be separated from Palynziq dosing by at least 60 minutes. For nonclinic visits, participants should be asked about MTX administration in weekly telephone calls. MTX is administered at the clinic on scheduled visits. When participants are dosed at the site, they receive study treatments directly from the investigator or designee, under medical supervision. The date and time of each dose administered in the clinic is recorded in the source documents and recorded in the eCRF. On days when MTX is to be administered on the same day as Palynziq, there is no restriction with regard to the order of administration.I) Participants start Palynziq and follow the induction / titration / maintenance dosing regimen within the study at the start of the Combination Treatment Period according to the PalynziqUSPI and as directed by the investigator. Palynziq must be prescribed by the participant’s treating physician and as per labeled indication (uncontrolled blood Phe concentrations > 600 pmol / L on existing management) prior to study entry. Re-training on Palynziq administration should take place prior to the first dose administered and at the discretion of the investigator thereafter, g) Palynziq is administered in the clinic on Day 1. The time and day in which participants take their dose of Palynziq should be consistent throughout the study. Participants are required to premedicate with an Hl antagonist, an H2 antagonist, and / or an antipyretic approximately 2 to 3 hours prior to each dose of Palynziq until participants are able to achieve blood Phe600 pmol / L while on a stable dose of Palynziq (the Maintenance Phase). When an NSAID is administered as a premedication, it should be given with food. Premedication may be considered during the Maintenance Phase at the investigator’s discretion. When participants are dosed at the site, they receive study treatments directly from the investigator or designee, under medical supervision. The date and time of each dose administered in the clinic are recorded in the source documents and recorded in the eCRF. On days when MTX is to be administered on the same day as Palynziq, there is no restriction with regard to the order of administration but the time of day that each medication is taken should be consistent from day to day or week to week. For at least the first 6 months of Palynziq treatment when the participant is self-injecting (i.e., when administration is not under healthcare professional supervision), an observer must be present during and for at least 60 minutes after each administration. An observer is someone who is present during and after Palynziq administration, is able to recognize the signs and symptoms of an anaphylactic reaction, and can call for emergency medical support and administer adrenaline if warranted. After 6 months of Palynziq treatment, the need for an observer may be reconsidered, h) Participants take folic acid 1 mg orally every day beginning at Week -4 (the start of MTX) and continuing through Week 24 (to Day 168 or the next clinic visit), i) PKU history includes highest blood Phe level and age at the time of highest blood Phe. j) Participants with documented negative results within the last 4 weeks do not need to be retested, k) A full PE should be performed at screening, Day 1 and at the SCVZETV. A brief PE should be performed at other visits. A full PE includes, at a minimum, assessment of general appearance (head, eyes, ears, nose, and throat) and an assessment of cardiovascular, dermatologic, lymphatic, respiratory, gastrointestinal, musculoskeletal, and neurologic systems. A brief PE may be performed at the discretion of the investigator based on the participant’s clinical condition and may include, but is not limited to, general appearance andan assessment of cardiovascular, dermatologic, lymphatic, respiratory, gastrointestinal, musculoskeletal, and neurologic systems. 1) Clinical safety laboratory test includes CBC / differential, chemistry, LFTs, and urinalysis. It is recommended that urine samples be obtained as a first or second morning void. For in-clinic visits, testing should be performed prior to administration of any study drugs. In the event of elevated urinary protein result, a repeat urinalysis should be performed. This repeat urine sample must be performed in the morning at the first or second morning void to allow for accurate test results and may be performed by a home healthcare nurse. A blood sample is taken for local laboratory analysis at the Week -1 visit to assess for MTX issues and is used as part of the decision on if the participant should continue to the Combination Treatment Period, m) It is recommended that urine samples be obtained as a first or second morning void. Participants with a confirmed urine / albumin creatinine ratio of U 100 mg / g should be referred to a nephrologist for consultation if results are within normal range at baseline. Participants who had elevated results at baseline followed by a confirmed subsequent increase of 100 to 200 mg / g from baseline should also be referred to a nephrologist for consultation, n) Urine pregnancy test is done locally (if applicable). If urine pregnancy test is positive or equivocal, serum pregnancy test (central laboratory) must be done, o) If deemed necessary by the investigator, an X-ray should be performed for participants who have not had a chest X-ray within 2 years prior to screening. The chest X-ray is read locally by a radiologist. Data from a chest X-ray performed within 2 years prior to enrollment may be used provided the required data fields are available, p) Blood is collected once every 4 weeks before Palynziq administration (predose) within 2 hours prior to dosing, q) Blood is collected for plasma blood Phe analysis after fasting a minimum of 2.5 hours. Participants should be assessed for blood Phe concentration during screening with 2 measurements taken at least 5 days apart. The first screening sample should be collected after informed consent at the Week -8 visit (Day -56). The second screening sample should be taken and shipped 1 week later (by or on Day -49). Study eligibility criteria require each of the 2 screening blood Phe results to be >600 pmol / L. Baseline blood Phe is based on the average of the 2 blood Phe values at screening and the blood Phe result from Day 1. r) Blood is collected for tyrosine assessment after fasting for a minimum of 2.5 hours, s) Between scheduled clinic visits, the clinic staff contact the participant to monitor if the participant is experiencing problems with drug administration, to ask about any AEs or concomitant medications, and to answer questions. For non clinic visits, participants should be asked about Palynziq and MTX administration, and compliancewith study treatments should be assessed, t) AEs and concomitant medications should be noted whenever a participant is assessed by study personnel. Following signed informed consent, all SAEs are to be collected through the end of the study, u) All AEs, pregnancy events, and serious device events are collected following signed informed consent and first dose of study treatment through the end of the study.

[0027] FIG. 3 outlines the study assessments and procedures (i.e., Schedule of Activities (SoA)) of Cohort B (Palynziq Experienced Participants). AE, adverse event; complement components C3 / C4; CBC, complete blood count; CIC, circulating immune complex;CTCAE, Common Terminology Criteria for Adverse Events; eCRF, electronic Case Report Form; ETV, Early Termination Visit; HRV, hypersensitivity reaction visit; Ig, immunoglobulin; LFT, liver function tests; MTX, methotrexate; NS AID, nonsteroidal antiinflammatory drug; PAH, phenylalanine hydroxylase; PE, physical examination; PEG, polyethylene glycol; Phe, phenylalanine; PK, pharmacokinetics; SAE, serious AE; SCV, Study Completion Visit; USPI, United States Prescribing Information, a) Scheduled visits take place at the hospital setting, clinic, or virtually if approved by the investigator or designee. For virtual visits, the investigator / designee performs assessments while online with the participant, apart from specimen collections and vital signs, which are performed by a home healthcare personnel. Non clinic visits are to be performed via telephone. All assessments are performed pre dose unless otherwise specified, b) Potential participants enter the Screening Period, which lasts 4 weeks and includes 2 assessments of blood Phe concentration 5 days apart. Following confirmation that both of the 2 blood Phe results are > 600 pmol / L and confirmation that all other eligibility criteria have been met, participants may proceed onto the MTX Tolerability Period / Combination Treatment Period. During the MTX Tolerability / Combination Treatment Period, participants take oral MTX (15 mg / week) as a single dose each week. The participant is monitored weekly (remote visits via telephone call / video call) to assess compliance and side effects. If the participant has tolerated MTX treatment per protocol-defined clinical and laboratory criteria at the end of the MTX Tolerability Period and meets all other eligibility criteria, the participant may continue in the Combination Treatment Period, c) If the investigator determines that the CTCAE Grade3 hypersensitivity reaction is related to administration with Palynziq, the participant should return to the clinic within 24 hours of event onset for the HRV. If CTCAE >3 hypersensitivity adverse drug reaction occurs, the participant should return to clinic within 24 hours for tryptase, high-sensitivity C-reactive protein, urine N methyl histamine, d) Forparticipants who terminate from the study early, the ETV occurs within 4 weeks of the last dose of Palynziq or MTX or after withdrawal from study participation, whichever occurs last. Participants who discontinue from MTX or Palynziq early should continue to complete the remaining study assessments until as long as such continued participation will not detrimentally affect the health, safety, and welfare of the participant per investigator determination, e) Participants should take oral MTX at a dosage of 15 mg / week at the start of the MTX Tolerability Period / Combination Treatment Period. Participants who are unable to tolerate MTX during the 4-week MTX Tolerability Period should be discontinued from MTX. The time and day in which participants take their weekly dose of MTX should be consistent, and MTX dosing should be separated from Palynziq dosing by at least 60 minutes. For nonclinic visits, participants should be asked about MTX administration in weekly telephone calls. MTX is administered at the clinic on scheduled visits. When participants are dosed at the site, they receive study treatments directly from the investigator or designee, under medical supervision. The date and time of each dose administered in the clinic are recorded in the source documents and recorded in the eCRF. On days when MTX is to be administered on the same day as Palynziq, there is no restriction with regard to the order of administration, f) Participants continue Palynziq treatment and follow the titration / maintenance dosing regimen within the study at the start of the Combination Treatment Period according to the Palynziq USPI and as directed by the investigator.Palynziq must be prescribed by the participant’s treating physician and as per labeled indication (uncontrolled blood Phe concentrations > 600 pmol / L on existing management) prior to study entry. Re-training on Palynziq administration should take place at the discretion of the investigator, g) Palynziq should continue to be administered as per prescribing physician throughout screening and on Day 1 and throughout the study. The time and day in which participants take their dose of Palynziq should be consistent throughout the study. Participants are required to premedicate with an Hl antagonist, an H2 antagonist, and / or an antipyretic approximately 2 to 3 hours prior to each dose of Palynziq until participants are able to achieve blood Phe600 pmol / L while on a stable dose of Palynziq (the Maintenance Phase). When an NSAID is administered as a premedication, it should be given with food. Premedication may be considered during the Maintenance Phase at the investigator’s discretion. When participants are dosed at the site, they receive study treatments directly from the investigator or designee, under medical supervision. The date and time of each dose administered in the clinic are recorded in the source documents andrecorded in the eCRF. On days when MTX is to be administered on the same day as Palynziq, there is no restriction with regard to the order of administration but the time of day that each medication is taken should be consistent from day to day or week to week. For at least the first 6 months of Palynziq treatment when the participant is self-injecting (i.e., when administration is not under healthcare professional supervision), an observer must be present during and for at least 60 minutes after each administration. An observer is someone who is present during and after Palynziq administration, is able to recognize the signs and symptoms of an anaphylactic reaction and can call for emergency medical support and administer adrenaline if warranted. After 6 months of Palynziq treatment, the need for an observer may be reconsidered, h) Participants take folic acid 1 mg orally every day from the start of MTX dosing, beginning at Week 1 (Day 1 to ; the start of MTX) and continuing through the Week 24 visit (Day 168 or next clinic visit), i) PKU history includes highest blood Phe level and age at the time of highest blood Phe. j) Participants with documented negative results within the last 4 weeks do not need to be retested, k) A full PE should be performed at screening, Day 1 and at the SCVZETV. A brief PE should be performed at other visits. A full PE will include, at a minimum, assessment of general appearance (head, eyes, ears, nose, and throat) and an assessment of cardiovascular, dermatologic, lymphatic, respiratory, gastrointestinal, musculoskeletal, and neurologic systems. A brief PE may be performed at the discretion of the investigator based on the participant’s clinical condition and may include, but is not limited to, general appearance and an assessment of cardiovascular, dermatologic, lymphatic, respiratory, gastrointestinal, musculoskeletal, and neurologic systems. 1) Clinical safety laboratory test includes CBC / differential, chemistry, LFTs, and urinalysis. It is recommended that urine samples be obtained as a first or second morning void. For in-clinic visits, testing should be performed prior to administration of any study drugs. In the event of elevated urinary protein result, a repeat urinalysis should be performed. This repeat urine sample must be performed in the morning at the first or second morning void to allow for accurate test results and may be performed by a home healthcare nurse. A blood sample is taken for local laboratory analysis at the Week 4 (approximately Day 22) visit to assess for MTX issues and is used as part of the decision on if the participant should continue in the Combination Treatment Period, m) It is recommended that urine samples be obtained as a first or second morning void. Participants with a confirmed urine / albumin creatinine ratio of 100 mg / g should be referred to a nephrologist for consultation if results are within normal range at baseline. Participants who had elevated results at baseline followed by a confirmedsubsequent increase of 100 to 200 mg / g from baseline should also be referred to a nephrologist for consultation, n) Urine pregnancy test is done locally (if applicable). If urine pregnancy test is positive or equivocal, serum pregnancy test (central laboratory) must be done, o) If deemed necessary by the investigator, an X-ray should be performed for participants who have not had a chest X-ray within 2 years prior to screening. The chest X-ray is read locally by a radiologist. Data from a chest X-ray performed within 2 years prior to enrollment may be used provided the required data fields are available, p) Blood is collected once every 4 weeks before Palynziq administration (predose) within 2 hours prior to dosing, q) Blood is collected for plasma blood Phe analysis after fasting a minimum of 2.5 hours. Participants should be assessed for blood Phe concentration during screening with 2 measurements taken at least 5 days apart. The first screening sample should be collected after informed consent at the Week -4 (Day-28) visit. The second screening sample should be taken and shipped by latest week -3 (Day -21). Study eligibility criteria require each of the 2 screening blood Phe results to be >600 pmol / L. Baseline blood Phe is based on the average of the 2 blood Phe values at screening and the blood Phe result from Day 1. r) Blood is collected for tyrosine assessment after fasting for a minimum of 2.5 hours, s) Between scheduled clinic visits, the clinic staff contact the participant to monitor if the participant is experiencing problems with drug administration, to ask about any AEs or concomitant medications, and to answer questions. For non clinic visits, participants should be asked about Palynziq and MTX administration, and compliance with study treatments should be assessed, t) AEs and concomitant medications should be noted whenever a participant is assessed by study personnel. Following signed informed consent, all SAEs are to be collected through the end of the study, u) All AEs, pregnancy events, and serious device events are collected following signed informed consent and first dose of study treatment through the end of the study.

[0028] FIG. 4A shows the sequence of wild type AvPAL (SEQ ID NO: 1). FIG. 4B shows sequences of three AvPAL variants (SEQ ID NOs:2-4).DETAILED DESCRIPTION OF THE DISCLOSURE

[0029] Antibody- and complement-mediated immune responses to an AvPAL variant or rAvPAL (e.g., pegvaliase) in a subject being treated with such protein can lead to increased protein clearance and to hypersensitivity reactions, which interfere with drug efficacy and result in poor tolerability. The present disclosure provides a novel solution for managing these undesirable immune responses induced by the present rAvPAL, by administering to apatient methotrexate (MTX) or a pharmaceutically acceptable salt thereof. The methods provided herein are based at least in part on the surprising finding of the effectiveness of methotrexate (MTX) on managing the subject immune responses even at a low oral dosage as well as in a patient population particularly difficult to manage.Definitions

[0030] Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Definition of standard chemistry terms can be found in reference works, including Carey and Sundberg, Advanced Organic Chemistry, 3rd Edition, Vols. A and B (Plenum Press, New York 1992). The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectroscopy, preparative and analytical methods of chromatography, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., 4th Edition, 2004); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).

[0031] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0032] “Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptidenucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence i.e., A, U, G, C) in which “U” replaces “T.”

[0033] “ cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0034] Conventional notation is used herein to describe polynucleotide sequences: the lefthand end of a single-stranded polynucleotide sequence is the 5 ’-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 ’-direction. The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand”; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5’ to the 5 ’-end of the RNA transcript are referred to as “upstream sequences”; sequences on the DNA strand having the same sequence as the RNA and which are 3’ to the 3’ end of the coding RNA transcript are referred to as “downstream sequences.”

[0035] “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide-binding partner of the second polynucleotide. Thus, the polynucleotide whose sequence 5’-TATAC-3’ is complementary to a polynucleotide whose sequence is 5’-GTATA-3’.

[0036] A nucleotide sequence is “substantially complementary” to a reference nucleotide sequence if the sequence complementary to the subject nucleotide sequence is substantially identical to the reference nucleotide sequence.

[0037] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription andtranslation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

[0038] “Recombinant polynucleotide” refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a “recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, e.g., a “recombinant polypeptide.” A recombinant polynucleotide can serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc. as well.

[0039] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.

[0040] “Amplification” refers to any means by which a polynucleotide sequence is copied and thus expanded into a larger number of polynucleotide molecules, e.g., by reverse transcription, polymerase chain reaction, and ligase chain reaction.

[0041] “Primer” refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, / .< ., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but can be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template towhich it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moi eties and used as detectable moieties.

[0042] “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term “protein” typically refers to large polypeptides. The term “peptide” typically refers to short polypeptides.

[0043] Conventional notation is used herein to portray polypeptide sequences: the lefthand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

[0044] “ Conservative substitution” refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Serine (S), Threonine (T);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).Amino acids can also be grouped as follows:(1) hydrophobic: Met, Ala, Vai, Leu, He;(2) neutral hydrophilic: Cys, Ser, Thr;(3) acidic: Asp, Glu;(4) basic: Asn, Gin, His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro; and(6) aromatic: Trp, Tyr, Phe.

[0045] The terms “identical” or percent “identity,” in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm described in U.S. Patent No. 7,553,653, which is herein incorporated by reference in its entirety, or by visual inspection.

[0046] The phrase “substantially homologous” or “substantially identical” in the context of two nucleic acids or polypeptides, generally refers to two or more sequences or subsequences that have at least 40%, 60%, 80%, 90%, 95%, 98% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The substantial identity can exist over a region of the sequences that is at least about 50 residues in length, such as over a region of at least about 100 residues, or over a region of at least about 150 residues. In certain embodiments, the sequences are substantially identical over the entire length of either or both comparison biopolymers.

[0047] “Substantially pure” or “isolated” means an object species is the predominant species present ( / .< ., on a molar basis, more abundant than any other individual macromolecular species in the composition), and a substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition is the purified species of interest. The object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition. In some embodiments, the prokaryotic PAL variant compositions are substantially pure or isolated. In some embodiments, the prokaryotic PAL variant compositions are substantially pure or isolated with respect to the macromolecular starting materials used in their synthesis. In some embodiments, the pharmaceutical compositions comprise a substantially purified or isolated prokaryotic PAL variant admixed with one or more pharmaceutically acceptable excipient.

[0048] “Naturally occurring” as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in anorganism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0049] “Wild-type” (wt) is a term referring to the natural genetic form of an organism. A wild-type is distinguished from a mutant form (an organism with a genetic mutation).

[0050] The terms “polypeptide” and “protein” refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, “polypeptide” as used herein refers to a protein, which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. Such polypeptides may be referred to as “mutants” herein. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations arising with hosts that produce the proteins or errors due to PCR amplification.

[0051] As used herein, “variant,” “analog,” or “derivative” is a compound, e.g., a peptide, having more than about 70% sequence but less than 100% sequence similarity with a given compound, e.g., a peptide. Such variants, analogs or derivatives can be comprised of non-naturally occurring amino acid residues, including by way of example and not limitation, homoarginine, ornithine, penicillamine, and norvaline, as well as naturally occurring amino acid residues. Such variants, analogs or derivatives can also be composed of one or a plurality of D-amino acid residues, and can contain non-peptide interlinkages between two or more amino acid residues.

[0052] As used herein, the “ratio” of a PAL polypeptide (e.g., AvPAL or variant thereof) and a water-soluble polymer (e.g., polyethylene glycol or PEG) refers to the reaction condition molar ratio between the PAL polypeptide and the water-soluble polymer. For example, a ratio of about 1:3 for AvPAL and polyethylene glycol (1:3 AvPAL:PEG) means that the chemically modified PAL was produced in a reaction condition with about 1 mol lysine residue on the AvPAL per 3 mol of polyethylene glycol. Because an AvPAL monomer has 18 lysine residues, a ratio of about 1:3 AvPAL:PEG corresponds to 1 mol AvPAL per 54 mol PEG in the pegylation reaction.

[0053] “ Treatment” or “treating” as used herein refers to prophylactic treatment or therapeutic treatment or diagnostic treatment. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of disease or pathology, z.e., a PKU, or exhibits only early signs for the purpose of decreasing the risk of developing pathology. The prokaryotic PAL compositions, including formulations, provided herein can be given as a prophylactic treatment to reduce the likelihood of developing a pathology, z.e., a PKU, or to minimize the severity of the pathology, if developed. A “therapeutic” treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology, / .< ., a PKU, for the purpose of diminishing or eliminating those signs or symptoms. The signs or symptoms can be biochemical, cellular, histological, functional, subjective or objective. The prokaryotic PAL compositions can be given as a therapeutic treatment or for diagnosis. “Diagnostic” means identifying the presence or nature of a pathologic condition, z.e., a PKU. Diagnostic methods differ in their specificity and selectivity. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0054] As used herein, the terms “prevent,” “preventing,” and “prevention” refer to the total or partial inhibition of the development, recurrence, onset or spread of a disease and / or symptom related thereto (e.g., a disease or symptom related thereto that is associated with elevated phenylalanine levels, such as PKU in a patient), resulting from the administration of a therapy or combination of therapies provided herein, e.g., AvPAL, AvPAL variant, or any derivative thereof.

[0055] “Pharmaceutical composition” refers to a composition suitable for pharmaceutical use in subject animal, including humans and mammals. A pharmaceutical composition comprises a pharmacologically effective amount of a prokaryotic PAL polypeptide and also comprises a pharmaceutically acceptable carrier. A pharmaceutical composition encompasses a composition comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions encompass any composition made by admixing a prokaryotic PAL polypeptide provided herein and a pharmaceutically acceptable carrier.

[0056] “Pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical excipients, vehicles, diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers, such as, for example and not for limitation, a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington’s Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Pharmaceutical carriers to be used can depend upon the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection; or topical, transdermal, or transmucosal administration).

[0057] By “pharmaceutically acceptable” or “pharmacologically acceptable” is meant a material which is not biologically or otherwise undesirable, i.e., the material can be administered to an individual without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0058] As used herein, the term “subject” encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or gender. As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human), most preferably a human. In some embodiments, the subject is a mammal, preferably a human, having been administered a PAL enzyme, such as AvPAL, or variants thereof (e.g., SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4 (FIG. 4B)) and / or any derivatives thereof (e.g., pegylated PAL) and / or any pharmaceutical compositions and / or any pharmaceutical compositions produced by any of the methods disclosed herein. In some embodiments of the methods and kits provided herein, the patient has a disease or symptom related thereto that is associated with elevated phenylalanine levels, such as HPA or PKU (e.g., classic PKU, severe PKU, moderate PKU or any subpopulation thereof). In some embodiments, the patient is a patient receiving EST (e.g., rAvPAL or rAvPAL-PEG) forelevated phenylalanine levels (e.g., a patient with PKU). In another embodiment of the methods provided herein, the patient is administered a low or modified protein diet, or a low or modified phenylalanine diet in combination with a pharmaceutical composition disclosed herein, such that plasma phenylalanine are decreased, e.g., by at least about 25%. See, e.g., U.S. Patent Nos. 7,531,341 and 7,534,595 for further information on the management of patient populations with elevated phenylalanine levels (e.g., HPA and PKU) with a PAL or PAL-PEG (e.g., AvPAL or rAvPAL-PEG, or any variant thereof), which, in certain embodiments, can be used in conjunction with the methods and kits provided herein. In some embodiments, the subject is a patient who has not received a prior treatment with the AvPAL variant. In another embodiments, the subject is a patient who has received a prior treatment with the AvPAL variant. In some embodiments, the subject after the prior treatment with the AvPAL variant has uncontrolled blood phenylalanine (Phe) concentration greater than about 600 micromol / L. In certain embodiments, the subject has uncontrolled blood Phe concentration greater than about 600 micromol / L after more than 24 consecutive weeks of the prior treatment with the AvPAL variant. In certain embodiments, the subject has been on the prior treatment with the AvPAL variant at the weekly dose provided herein. In certain embodiments, the subject has been on the prior treatment with the AvPAL variant at a dosage higher than about 20 mg per day. In certain embodiments, the subject in the prior treatment could not receive further dose escalation of the AvPAL variant. In certain embodiments, the highest dosage of the AvPAL variant the subject received in the prior treatment has been between about 20 mg per day to about 40 mg per day. In some embodiments, the subject is between 18 and 65 years of age. In some embodiments, the subject is a human subject.

[0059] As used herein, the term “therapy” refers to any protocol, method and / or agent that can be used in the prevention, management, treatment and / or amelioration of disease (or symptom related thereto) associated with elevated phenylalanine levels (e.g., PKU). In certain embodiments, the terms “therapies” and “therapy” refer to a combination therapy. In certain embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and / or other therapies useful in the prevention, management, treatment and / or amelioration of a disease associated with elevated phenylalanine levels (e.g., PKU) known to one of skill in the art such as medical personnel.

[0060] The term “tissue” as used herein refers to tissues that are obtained from a mammal, e.g., human. For example, a tissue may be from a biopsy sample, surgically removed tissue,or postmortem collection. Furthermore, the tissue may be homogenized and extracted to isolate the enzyme or antibodies from the tissue.

[0061] As used herein, the term “concurrently” in the context of administering two or more drugs or two or more treatment regimens means at the same time. For example, if the administrations of two drugs or two treatment regimens for a single patient are being conducted concurrently, then they are being conducted at the same time. It will be understood that administrations of two drugs or two treatment regimens happening at the same time, does not necessarily mean that actual delivery of two drugs happens at the same time, as each drug or regimen may call for a different dosing schedule and / or different delivery modes. For example, the deliveries of two drugs administered concurrently for a single patient can be performed within the same treatment cycle and / or performed within a certain period of time of each other. In some embodiments, the administrations of two drugs is separated from each other by at least 60 minutes. In some embodiments, the administrations of two drugs or two treatment regimens for a single patient are being conducted on the same day.Combinational Therapy for Treating Subjects with PKU

[0062] Palynziq® (pegvaliase or Palynziq) has been developed as a subcutaneous (SC) enzyme substitution treatment to reduce blood phenylalanine (Phe) levels in adult patients with PKU. Given that phenylalanine ammonia lyase (PAL) is a foreign, bacterium-derived protein, anti-drug antibody (ADA) responses to Palynziq are expected. Such antibody responses can affect blood Phe reduction in patients, which is likely primarily mediated by clearance of the drug from the plasma by the immune system. In addition, an association between antibody responses and adverse events (e.g., hypersensitivity) was observed in clinical studies. The presently disclosed combination therapy method provides a novel approach to modulating immune responses in these patients. The methods provided herein are based at least in part on the surprising finding of the effectiveness of methotrexate (MTX) on suppressing immune responses at a low oral weekly dose, in particular, comparing to the weekly dose of pegvaliase that is subcutaneously administered. Moreover, the methods provided herein not only benefit the patients who have not received a prior treatment with pegvaliase, also surprisingly benefit the patients who have received a prior treatment with pegvaliase but exhibited prolonged treatment time needed to achieve blood Phe reduction and difficulty in dose escalation that is likely associated with a more robust immune response.

[0063] The presently disclosed combination therapy method for reducing blood phenylalanine concentration in a subject comprises: (1) administering to the subject a weekly dose of a formulation comprising an AvPAL variant, wherein the AvPAL variant comprises an amino acid sequence of SEQ ID NO:4; and (2) administering to the subject a weekly dose of methotrexate (MTX) or a pharmaceutically acceptable salt thereof. In certain embodiments, the AvPAL variant and MTX are administered concurrently. In one embodiment, the AvPAL variant is administered before MTX. In another embodiment, the AvPAL variant is administered after MTX. In some embodiments, the administration of MTX is separated from the administration of Palynziq by at least 60 minutes. In certain embodiments, the AvPAL variant and MTX are administered on the same day. In certain embodiments, the AvPAL variant and MTX are administered in separate compositions. In some embodiments, the formulation comprising an AvPAL variant as disclosed herein and MTX or a pharmaceutically acceptable salt thereof as disclosed herein are administered concurrently. In one embodiment, the formulation comprising an AvPAL variant as disclosed herein is administered before MTX or a pharmaceutically acceptable salt thereof as disclosed herein. In another embodiment, the formulation comprising an AvPAL variant as disclosed herein is administered after MTX or a pharmaceutically acceptable salt thereof as disclosed herein. In some embodiments, the administration of MTX or a pharmaceutically acceptable salt thereof as disclosed herein is separated from the administration of the formulation comprising an AvPAL variant as disclosed herein by at least 60 minutes. In certain embodiments, the formulation comprising an AvPAL variant as disclosed herein and MTX or a pharmaceutically acceptable salt thereof as disclosed herein are administered on the same day. In some embodiments, the formulation comprising an AvPAL variant as disclosed herein and MTX or a pharmaceutically acceptable salt thereof as disclosed herein are administered in separate compositions.

[0064] In one aspect, a subject receiving the present combination treatment is a patient who has never been treated with pegvaliase (Palynziq naive). In another aspect, a subject receiving the present combination treatment is a patient who has been treated with pegvaliase (Palynziq experienced). In some embodiments, the subject after the prior treatment with the AvPAL variant has uncontrolled blood phenylalanine (Phe) concentration greater than about 600 micromol / L. In certain embodiments, the subject has uncontrolled blood Phe concentration greater than about 600 micromol / L after more than 24 consecutive weeks of the prior treatment with the AvPAL variant. In certain embodiments, the subject has been on theprior treatment with the AvPAL variant at the weekly dose provided herein. In certain embodiments, the subject has been on the prior treatment with the AvPAL variant at a dosage higher than about 20 mg per day. In certain embodiments, the subject in the prior treatment could not receive further dose escalation of the AvPAL variant. In certain embodiments, the highest dosage of the AvPAL variant the subject received in the prior treatment has been between about 20 mg per day to about 40 mg per day.

[0065] In certain embodiments, in the present combination therapy, for the subject who has not received a prior treatment with the AvPAL variant, the treatment with the AvPAL variant is initiated in the combination therapy on the induction / titration / maintenance (I / T / M) dosing regimen as described herein, in combination with the administration of MTX. In certain embodiments, the administration of the weekly dose of MTX starts at four weeks before the initiation of the treatment with the AvPAL variant. In certain embodiments, the administration of the weekly dose of MTX starts at four weeks before the combination treatment. In certain embodiments, in the present combination therapy, for the subject who has already received a prior treatment with the AvPAL variant, the treatment with the AvPAL variant continues in the combination therapy at the weekly dose as described herein, in combination with the administration of MTX. In certain embodiments, for the subject who has already received a prior treatment with the AvPAL variant, the AvPAL variant treatment continues and the combination therapy starts when the weekly dose of MTX is administered.

[0066] As described herein, a low oral dosage of MTX is administered concurrently with the administration of the AvPAL variant. In certain embodiments, MTX is administered orally from about 2.5 mg per week to about 20 mg per week. In certain embodiments, MTX is administered orally about 2.5 mg per week. In certain embodiments, MTX is administered orally about 5 mg per week. In certain embodiments, MTX is administered orally about 7.5 mg per week. In certain embodiments, MTX is administered orally about 10 mg per week. In certain embodiments, MTX is administered orally about 12.5 mg per week. In certain embodiments, MTX is administered orally about 15 mg per week. In certain embodiments, MTX is administered orally about 17.5 mg per week. In certain embodiments, MTX is administered orally about 20 mg per week. In certain embodiments, MTX is administered at a low oral dose of 15 mg per week. In certain embodiments, the oral dose of MTX is decreased from 15 mg per week to 10 mg per week during the combination therapy. In certain embodiments, the administration of MTX is temporarily discontinued during the combination therapy.

[0067] In certain embodiments, the weekly dose of MTX is administered orally once weekly. In certain embodiments, the weekly dose of MTX is administered orally twice weekly. In certain embodiments, the weekly dose of MTX is administered orally in a single dose. In certain embodiments, the weekly dose of MTX is administered orally in multiple divided doses. In certain embodiments, the weekly dose of MTX is administered orally in two divided doses. In certain embodiments, the divided doses of MTX are administered within a day. In certain embodiments, the divided doses of MTX are administered in the morning and evening of the same day, respectively.

[0068] In certain embodiments, the dosage of 15 mg per week is administered orally in a single dose. In certain embodiments, the dosage of 15 mg per week is administered orally in two divided doses within a day. In certain embodiments, the dosage of 15 mg per week is administered orally at 7.5 mg in the morning and at 7.5 mg in the evening of the same day.

[0069] In one aspect, MTX is administered at a low oral weekly dose. In certain embodiments, the low oral weekly dose of MTX comparing to the weekly dose of pegvaliase that is subcutaneously administered is presented as a ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX as described in the following section.

[0070] In certain embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 1 :6. In certain embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 1:3. In certain embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 2:3. In certain embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 4:3. In certain embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 8:3. In certain embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 14:3. In certain embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 28:3. In certain embodiments, the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 56:3. In certain embodiments, the weekly dose of MTX is administered orally. In certain embodiments, the weekly dose of the AvPAL variant is administered subcutaneously.

[0071] In certain embodiments, the weekly dose of MTX is administered for about 24 weeks to 28 weeks. In certain embodiments, the weekly dose of MTX is administered for about 24 weeks in the combination therapy methods as described herein. In certainembodiments, the weekly dose of MTX is administered for about 28 weeks. In certain embodiments, the administration of MTX can be temporarily discontinued in the combination therapy methods as described herein. In certain embodiments, the weekly dose of the AvPAL variant is administered for about 24 weeks in the combination therapy methods as described herein. In certain embodiments, the weekly dose of the AvPAL variant is administered for about 48 weeks. In certain embodiments, the administration of the weekly dose of MTX is discontinued at about 24 weeks and only the administration of the AvPAL variant continues for about another 24 weeks. In certain embodiments, the administration of the weekly dose of MTX is discontinued at about 28 weeks and only the administration of the AvPAL variant continues for about another 24 weeks.

[0072] In the present combination therapy methods, the AvPAL variant can be administered at the dosage described in the following section. In some embodiments, the dosage of the AvPAL variant is in the range of about 2 mg per week to about 10 mg per week. In some embodiments, the dosage is in the range of about 10 mg per week to about 20 mg per week. In some embodiments, the dosage is in the range of about 20 mg per week to about 40 mg per week. In some embodiments, the dosage is in the range of about 40 mg per week to about 70 mg per week. In some embodiments, the dosage is in the range of about 70 mg per week to about 140 mg per week. In some embodiments, the dosage is in the range of about 140 mg per week to about 280 mg per week.

[0073] In some embodiments, the AvPAL variant is administered once weekly. In some embodiments, the AvPAL variant is administered twice weekly. In some embodiments, the AvPAL variant is administered four times per week. In some embodiments, the AvPAL variant is administered seven times per week. In some embodiments, the AvPAL variant is administered fourteen times per week. In some embodiments, the AvPAL variant is administered daily.

[0074] In some embodiments, the method provided herein comprises administering to the subject the pegylated AvPAL variant at an induction dosage, followed by administering to the subject the pegylated AvPAL variant at a titration dosage, followed by administering to the subject the pegylated AvPAL variant at a maintenance dosage. In some embodiments, the induction dosage is administered for about 2 week to about 6 weeks, e.g., for 2, 3, 4, 5, or 6 weeks. In some embodiments, the titration dosage is administered for about 3 weeks to about 8 weeks, e.g., for 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the maintenance dosage isadministered for about 20 weeks to about 45 weeks, such as for 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 weeks.

[0075] In some embodiments, the method provided herein comprises administering to the subject the pegylated AvPAL variant at an induction dosage in the range of about 0.1 mg per week to about 10 mg per week, followed by administering to the subject the pegylated AvPAL variant at a titration dosage in the range of about 1 mg per week to about 200 mg per week, followed by administering to the subject the pegylated AvPAL variant at a maintenance dosage in the range of about 20 mg per week to about 280 mg per week. In some embodiments, the induction dosage is administered for about 2 week to about 6 weeks, e.g., for 2, 3, 4, 5, or 6 weeks. In some embodiments, the titration dosage is administered for about 3 weeks to about 8 weeks, e.g., for 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the maintenance dosage is administered for about 20 weeks to about 45 weeks, such as for 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 weeks.

[0076] In some embodiments, the method provided herein comprises administering to the subject the pegylated AvPAL variant at an induction dosage in the range of about 2.5 mg per week, followed by administering to the subject the pegylated AvPAL variant at a titration dosage in the range of about 5 mg per week to about 70 mg per week, followed by administering to the subject the pegylated AvPAL variant at a maintenance dosage in the range of about 140 mg per week to about 280 mg per week. In some embodiments, the induction dosage is administered for about 2 week to about 6 weeks, e.g., for 2, 3, 4, 5, or 6 weeks. In some embodiments, the titration dosage is administered for about 3 weeks to about 8 weeks, e.g., for 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the maintenance dosage is administered for about 20 weeks to about 45 weeks, such as for 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 weeks.

[0077] In some specific embodiments, the method provided herein comprises administering to the subject the pegylated AvPAL variant at an induction dosage in the range of about 2.5 mg per week for 4 weeks, followed by administering to the subject the pegylated AvPAL variant at a titration dosage in the range of about 5 mg per week to about 70 mg per week for 5 weeks, followed by administering to the subject the pegylated AvPAL variant at a maintenance dosage in the range of about 140 mg per week to about 280 mg per week for 24 to 40 weeks.

[0078] In some more specific embodiments, the method provided herein comprises administering to the subject the pegylated AvPAL variant according to the dosing regimen in Table 1.

[0079] The pegylated AvPAL variant is described in more detail in the sections below. In some specific embodiments, the pegylated AvPAL variant provided herein is composed of recombinant phenylalanine ammonia lyase (rAvPAL) conjugated to N-hydroxysuccinimide (NHS)-methoxypolyethylene glycol (PEG). The rAvPAL is a homotetrameric protein with a molecular weight of 62 kD per monomer. In some embodiments, to produce the pegylated AvPAL variant (rAvPAL-PEG), an average of nine (9) 20 kD PEG molecules are covalently bound (or conjugated) to each monomer of rAvPAL. In some embodiments, the total molecular weight of the pegylated rAvPAL is approximately 1000 kD. In some embodiments, the amino acid sequence of the rAvPAL monomer is SEQ ID NO:4, in which the serine residues at positions 503 and 565 are underlined:MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQ GIQASCDYINNAVESGEPIYGVTSGFGGMANVAISREQASELQTNLVWFLKTGAGNK LPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLV PLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMT GIAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMIS LLANSQLVRDELDGKHDYRDHELIQDRYSLRCLPQYLGPIVDGISQIAKQIEIEINSVT DNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASPEFSNG LPPSLLGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGY TSATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARASLSPATERLYSAV RHVVGQKPTSDRPYIWNDNEQGLDEHIARISADIAAGGVIVQAVQDILPSLH (SEQ ID NO:4).

[0080] The chemical structure of N-hydroxysuccinimide (NHS)-m ethoxypoly ethylene glycol (PEG) is as follows:# (Osi / CHSO4CH2CH2O f - (CH2)5CC> N$■ n \Phenylalanine Ammonia Lyase (PAL) and Variants thereof

[0081] As used herein, "bacterial PAL" and "prokaryotic PAL" are used interchangeably to mean (1) wild-type PAL from a prokaryotic organism, including but not limited to PAL from Streptomyces maritimus, Nostoc punctiforme, Anabaena variabilis, Anacystis nidulans (Lofflehardt, Z. Naturforsch. 31(11-12):693-9 (1976), Photorabdus luminescens TT01 (Williams, et al., Microbiology 151 :2543-2550 (2005), and Streptomyces verticillatus (Bezanson, et al., Can. J. Microbiol. 16(3): 147-51 (1970); (2) fragments, mutants, variants or analogs of such wild-type PAL enzymes that retain similar (i.e., at least 50%) catalytic activity for phenylalanine, and that can, for example, exhibit increased catalytic activity, greater biochemical stability, increased half-life, and / or decreased immunogenicity, and (3) chemically modified versions of such wild-type PAL enzymes or fragments, mutants, variants or analogs thereof that are linked to other chemical moieties that provide other advantageous effects, such as, for example and not for limitation, enhanced half-life and / or decreased immunogenicity. For example, any references to methods of making or using prokaryotic PAL, and fragments, mutants, variants, analogs or chemically modified versions thereof, and compositions of such enzyme(s), for therapeutic purposes, are meant to refer to methods of making, using or formulating all such wild-type prokaryotic PAL or fragments, mutants, variants, analogs or chemical modifications thereof.

[0082] One embodiment is a prokaryotic PAL from Anabaena variabilis (SEQ ID NO: 1) (see FIG. 4A) or biologically active fragment, mutant, variant or analog thereof.

[0083] The elucidation of a reliable three-dimensional structure or structural model for a specific macromolecule permits rational design to become a productive method for optimization of specific structure and / or function of said macromolecule. Methods of using a three-dimensional structure or structural model for optimizing PAL enzymes are described in U.S. Patent No. 7,553,653, which is herein incorporated by reference in its entirety. A high-resolution three-dimensional protein crystal structure of a prokaryotic PAL can be used in methods involving protein engineering to improve the biochemical and biophysical properties of a prokaryotic PAL, and to increase the in vivo therapeutic effectiveness of a prokaryotic PAL. In certain embodiments, provided herein are prokaryotic PAL variants with greater phenylalanine-converting activity and / or reduced immunogenicity as compared to a wild-type prokaryotic PAL. Also provided herein are prokaryotic PAL variants with greater biochemical stability and / or biochemical half-life as compared to a wild-type prokaryotic PAL.

[0084] Previous experiments have described modified forms of PAL, such as PAL mutants (Schuster, et al, FEBS Lett. 349(2):252-254 (1994); Schuster, et al, Proc Natl Acad Sci USA 92(18):8433-8437 (1995); Langer, et al, Biochemistry 36: 10867-10871 (1997); El- Batal, et al, Acta Microbiol Pol. 49(1): 51 -61 (2000); Rother, et al, Eur. J. Biochem. 269:3065-3075 (2002)) and HAL mutants (Taylor, et al, J. Biol. Chem. 269(44):27473- 27477 (1994);Baedeker, et al, Eur. J. Biochem. 269(6): 1790-1797 (2002)).

[0085] The biologically active sites of wild-type PAL provided herein can be modified to optimize PAL kinetic characteristics. Km, the concentration of substrate that gives half-maximal activity, is intimately associated with the therapeutic efficacy of PAL in maintaining Phe levels within an acceptable range, i.e., 120 pM to 240 pM. Km is the affinity of the enzyme for the substrate. By controlling affinity, one can limit or control the efficacy of any enzyme against substrate at different concentrations. For example, if Km is 1000 pM (e.g., PAL from Rhodosporidium toruloides), the activity of the enzyme will be reduced to about 12.5% at blood Phe levels of 240 pM and to about 3 % at blood Phe levels of 60 pM. If Km is 240 pM, the activity of the enzyme will be reduced to about 50%> at blood Phe levels of 240 pM and to about 12 % at blood Phe levels of 60 pM. If Km is 120 pM, the activity of the enzyme will be reduced to about 70%> at blood Phe levels of 240 pM and to about 35 %> at blood Phe levels of 60 pM. Optimally, a therapeutic objective would be to have an enzyme with sufficient activity to reduce but also maintain Phe within the optimal range of about 120 pM to about 240 pM. An enzyme with a high Km (i.e., 1000 pM) will lose activity rapidly as Phe levels drop to within normal range and will also require the impractical administration of highly concentrated or large volumes of doses. On the other hand, an enzyme with a very low Km can rapidly deplete Phe levels, which may be fatal for hyperphenylaninemias, but can be useful in the management of a disease or disorder.

[0086] In some embodiments, the biologically active modified PAL has a kcat of at least about 0.1 s-1 or greater than about 0.5 s-1. In other embodiments, the biologically active modified PAL has a kcat of at least about 0.2 s-1 or greater than about 1.0 s-1. In other embodiments, the biologically active modified PAL has a Km of between about 10 pM to about 1000 pM. In other embodiments, the biologically active modified PAL has a Km of between about 100 pM to about 1000 pM. In other embodiments, the biologically active modified PAL exhibits enzymatic activity that is from about two-fold to about 1000-fold times greater than that of the wild-type. In other embodiments, the biologically active modified PAL exhibits enzymatic activity that is from about 10% to about 100% higher thanthat of the wild-type. Such biological active modified PAL proteins can be formed using methods well known in the art, such as by site-directed mutagenesis.

[0087] A number of strategies are currently used to reduce protein immunogenicity. In certain embodiments, modifications that are introduced to minimize the immune response do not destroy the structure, function, or stability of the macromolecule. Effective strategies used include increasing human sequence content (chimeras and / or other humanization approaches), improving solution properties, removing antibody epitopes, introducing chemical derivatization (such as pegylation), and / or identifying and removing MHC agretopes.

[0088] Modification of antigenic surface protein regions reduces immunogenicity (Chirino, et al, Drug Discov. Today 9(2): 82-90 (2004)). One method of improvement involves the construction of smaller sized proteins that retain catalytic activity (e.g., an absorbance assay is used for activity measurement). Protein engineering coupled to ELISA screening, can also be used to identify mutants with reduced immunoreactivity. Another method introduces point mutations for additional surface Lys sites for pegylation derivatization, a method shown to reduce immunogenicity with the test enzyme purine nucleoside phosphorylase (Hershfield, et al. (1991), ibid.). An alternative pathway uses mutation of residues located in protein epitope regions to remove immunogenic sites (Yeung, et al, J. Immunol. 172(11):6658-6665 (2004)). In an approach that is analogous to antibody humanization, homologous loop regions and / or residues from human antibodies are substituted into the corresponding loop regions of a homologous protein.

[0089] Improving solution properties of proteins can increase specific enzyme activity and / or reduce immunogenicity. One solution property typical of bacterially expressed recombinant proteins is the formation of protein aggregates due, for example, to inter-chain disulfide bind formation, hydrophobic interactions and / or divalent cations (Chi, et al, Pharm. Res. 20(9): 1325-1336 (2003)). Aggregation of recombinantly expressed proteins can enhance the immune response (Hermeling, et al, Pharm. Res. 21(6): 897-903 (2004);Schellekens, Nephrol. Dial. Transplant. 20(suppl 6):vi3-9 (2005)). One method of improvement involves substituting surface cysteine residues with other amino acid residues (e.g., serine) to minimize the possibility of formation of inter-chain disulfide bonds. For example, substitution of two surface cysteine residues with serine residues reduced the aggregation of chorismate lyase with minor effects on enzyme activity (Holden, et al., Biochim. Biophys. Acta 1594(1): 160-167 (2002)).

[0090] Also provided herein are prokaryotic PAL variants that have the similar or greater phenylalanine-converting activity and / or reduced immunogenicity as compared to a wild-type PAL. Further provided herein are prokaryotic PAL variants that comprise one or more amino acids residues (e.g., cysteine) that have been substituted by another amino acid residues (e.g., serine) to reduce protein aggregation that can be associated with decreased enzyme activity, increased immunogenicity, and / or other disadvantageous effects, such as reduced bioavailability, in vivo. In some embodiments, provided herein are pharmaceutical compositions wherein one or more amino acid residues of the prokaryotic PAL variant have been substituted by another amino acid. In some embodiments, the substitution increases phenylalanine-converting activity and / or reduces immunogenicity as compared to the wildtype PAL.

[0091] In certain embodiments of the present methods or uses, the prokaryotic PAL variant is an Anabaena variabilis PAL (AvPAL) variant. In some embodiments, one or more amino acid residues of the AvPAL variant have been substituted by another amino acid residue. In some embodiments, one or more cysteine residues of the AvPAL variant have been substituted by a serine residue. In some embodiments, the one or more cysteine residues of the AvPAL variant that have been substituted by one or more serine residues are selected from the group consisting of cysteine residues at positions 503 and 565. In specific embodiments, the cysteine residue at position 503 of the AvPAL variant has been substituted by a serine residue (e.g., SEQ ID NO:2). In certain embodiments, the cysteine residue at position 565 of the AvPAL variant has been substituted by a serine residue (e.g., SEQ ID NO:3). In a certain embodiment, the cysteine residues at positions 503 and 565 of the AvPAL variant have been substituted by serine residues (e.g., SEQ ID NO:4).

[0092] Prokaryotic PAL variants can also include fusion proteins in which the PAL enzyme has been fused to another heterologous polypeptide, such as a native or modified constant region of an immunoglobulin or a fragment thereof that retains the salvage epitope, known in the art to increase half- life.Pegylated PAL

[0093] Macromolecule chemical modification can be performed in a non-specific fashion (leading to mixtures of derivatized species) or in a site-specific fashion (based on wild-type macromolecule reactivity-directed derivatization and / or site-selective modification using a combination of site-directed mutagenesis and chemical modification) or, alternatively, using expressed protein ligation methods (Hofmann, et al., Curr. Opin. Biotechnol. 13(4):297-303(2002)). In certain embodiments, chemical modification is used to reduce immunogenicity. Pegylation is a demonstrated method to reduce immunogenicity of proteins (Bhadra, et al., Pharmazie 57(l):5-29 (2002)), but glycosylation and other chemical derivatization procedures, using modification with phosphorylation, amidation, carboxylation, acetylation, methylation, creation of acid-addition salts, amides, esters, and N-acyl derivatives are also possible (Davis, Science 303:480-482 (2004)).

[0094] A series of different pegylation reactions on PAL, using a range of PEG chemical reagent to PAL protein ratios, will provide PEG-PAL derivatives for each modification method. The optimal degree of pegylation can be determined based upon the residual activity obtained for each derivatized PAL species using the absorbance assay in combination with PAGE and native gel analysis, or by using SE-HPLC with multiangle light scattering (MALS), to determine the extent of PEG derivatization. After initial ranges of optimal modification are determined, comparative kinetic analysis (including Vmax and Km determinations, binding constants of substrates, proteolytic stability, pH dependence of activity, temperature-dependence of activity) and immunoreactivity of optimal PEG-PAL species can be determined by ELISA, immunoprecipitation, and Western blot. Protein engineering can also be used to generate the most favorable PAL mutant for pegylation using the optimal derivatization conditions; by minimizing the size of the PAL protein and only modifying the most antigenic regions of the PAL surface, cost of PEG modification will be reduced while at the same time retaining the maximum amount of enzymatic activity and minimum amount of immunogenicity. Similarly, site-specific pegylation can be used to provide enzyme derivatives.

[0095] Other chemical modifications such as phosphorylation or other chemical modification of Lys, Arg, and Cys residues can be used to mask immunogenic regions and / or proteolytic sensitive regions. Such chemical modifications include the polymer addition method of Bednarsaki and the Altus Corporation cross-linking method for improving PAL stability, reducing immunogenicity, and improving protease resistance are representative examples. Bednarsaki demonstrated that polymer addition improves protein temperature stability (Wang, etal, J. Am. Chem. Soc. 114(l):378-380 (1992)), and Altus Corporation has found that glutaraldehyde cross-linking improves enzyme stability.

[0096] To discover if the in vivo therapeutic half-life of a protein such as PAL would benefit from pegylation, a variety of different PEG:PAL conjugates are synthesized, characterized in vitro and tested in vivo for L-Phe reduction. In order to both optimize thepotential effects of pegylation and to identify one or more sites of PEG attachment, a design strategy is employed wherein polymer length, conformation, and the degree of PEG attachment is varied. In some embodiments, methods for preparing the pegylated PAL generally comprise: (a) reacting PAL with polyethylene glycol under conditions whereby PAL becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s). Because the specific sites of PAL modification might significantly alter the intrinsic activity of the conjugate, different types and amounts of PEG were explored. The chemistry used for pegylation of PAL was the acylation of the primary amines of PAL using the NHS-ester of methoxy -PEG (O-[(N-Succinimidyloxycarbonyl)-methyl]-O’ -methylpolyethylene glycol). Acylation with methoxy-PEG-NHS or methoxy -PEG- SPA results in an amide linkage that eliminates the charge from the original primary amine.

[0097] The present methods provide for a substantially homogenous mixture of polymerprotein conjugate. “Substantially homogenous” as used herein means that only polymerprotein conjugate molecules are observed. The polymer: protein conjugate has biological activity and the present “substantially homogenous” pegylated PAL preparations provided herein are those which are homogenous enough to display the advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot to lot pharmacokinetics.

[0098] The polymer molecules contemplated for use in the pegylation approaches described herein can be selected from among water-soluble polymers or a mixture thereof. The water-soluble polymer can be selected from the group consisting of, for example, polyethylene glycol, monomethoxy-polyethylene glycol, dextran, poly-(N-vinyl pyrrolidone), propylene glycol homopolymers, a polypropylene oxide / ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), HPMA, Fleximer.TM., and polyvinyl alcohol, mono-(Cl-C10)alkoxy-PEG, aryloxy -PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonate PEG, cellulose, or other carbohydrate-based polymers. The polymer selected should be water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer can be branched or unbranched. In some embodiments, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable.

[0099] In some embodiments, a water-soluble polymer for use herein is polyethylene glycol, abbreviated PEG. As used herein, polyethylene glycol is meant to encompass any ofthe forms of PEG that have been used to derivatize other proteins, such as mono-(Cl-ClO) alkoxy- or aryloxy-polyethylene glycol.

[0100] The proportion of polyethylene glycol molecules to protein molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) will be determined by the molecular weight of the polyethylene glycol selected and on the number of available reactive groups (typically e amino groups) present. In general, the higher the molecular weight of the polymer used, the fewer number of polymer molecules which can be attached to the protein. Similarly, branching of the polymer can be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer: protein ratio. Several different linear PEG polymer lengths are contemplated, including but not limited to, 5 kDa and 20 kDa, conjugates of two-armed branched PEG polymers, including but not limited to 10 kDa and 40 kDa. In some embodiments, for the PEGylation reactions contemplated herein, the average molecular weight is about 2 kDa to about 100 kDa (the term “about” indicating + / -1 kDa). In other embodiments, the average molecular weight is about 5 kDa to about 40 kDa.

[0101] Examples 7 through 9 of co-owned U.S. Patent No. 7,531,341, which is herein incorporated by reference in its entirety, describe the effects of pegylated and nonpegylated forms of lysine mutant R91K PAL from Rhodosporidium toruloides (RtPAL), NpPAL and AvPAL on Phe levels in the ENU2 or BTBRenu2mouse. This animal model is a homozygous mutant at the PAH locus resulting in an animal with severe hyperphenylalaninemia. The high plasma Phe levels make this animal the appropriate model for evaluating the ability of PAL to reduce plasma Phe. Administration of pegylated forms of NpPAL and AvPAL resulted in greater reduction in Phe in the ENU2 mice as compared to unpegylated NpPAL and AvPAL, respectively. Such effects were maintained for NpPAL upon weekly injections over a ten-week period. These results show that pegylation of PAL from the cyanobacteria, Nostoc punctiforme and Anabaena variabdis, is essential in reducing Phe levels in PKU affected mice.

[0102] The effect of serine substitution of the cysteine residues (e.g., at positions 503 and 565) in the AvPAL polypeptide on Phe levels in ENU2 mice has also been shown. The administration of the pegylated AvPAL double cysteine mutant (at positions 503 and 565) AvPAL_C565SC503S results in a reduction in plasma Phe that is comparable to that achieved with pegylated wild-type AvPAL. It has been shown that AvPAL_C565SC503Shas in vivo PAL enzyme activity that is comparable to the pegylated wild-type AvPAL, and has reduced immunogenicity compared to the pegylated wild-type AvPAL.

[0103] Pegylated PAL variants with reduced immunogenicity are provided herein. One embodiment is a pegylated form of AvPAL variant with reduced immunogenicity. Specific embodiments contemplate AvPAL variants in which pegylation is achieved by reacting the AvPAL variant with a water-soluble polymer, e.g., polyethylene glycol (PEG). In some embodiments, pegylation is achieved by reacting the AvPAL variant once with PEG at a ratio of at least 1:1, at least 1:1.5, at least 1:2, at least 1:3, at least 1:4, at least 1:5, at least 1:6, at least 1 :7, at least 1 :8, at least 1 :9, or at least 1:10 (PAL:PEG). In one embodiment, the PAL variant is an AvPAL variant, and the pegylation is achieved using a PAL:PEG ratio of about 1 : 1 to about 1 :20. In another embodiment, the PAL variant is an AvPAL variant, and the pegylation is achieved using a PAL:PEG ratio of about 1 :3 to about 1 : 12. In yet another embodiment, the PAL variant is an AvPAL variant, and the pegylation is achieved using a PAL:PEG ratio of about 1 :5 to about 1:10. In yet another embodiment, the PAL variant is an AvPAL variant, and the pegylation is achieved using a PAL:PEG ratio of about 1 :9.

[0104] In certain embodiments, one or more lysine residues are introduced at and / or near the active site of a prokaryotic PAL variant to enhance catalytic activity, reduce immunogenicity and / or improve biochemical stability, in part by blocking potential pegylation of other amino acid residues (e.g., tyrosine) at and / or near the active site of the enzyme or by blocking potential pegylation of a lysine residue important for enzyme activity. Without being bound to a particular theory, it is hypothesized that a tyrosine residue at and / or near the active site of a prokaryotic PAL (z.e., position 78 or 314 in AvPAL) can be a site for pegylation, which reduces enzyme activity. In some embodiments, one or more amino acid residues at and / or near the active site of the prokaryotic PAL, which are not required for enzyme activity, are substituted by a lysine residue. In a certain embodiment, the prokaryotic PAL is AvPAL. In one embodiment, the AvPAL tyrosine residue at position 78 or 314 is not accessible for pegylation. Again without being bound to a particular theory, it is hypothesized that a lysine residue of a prokaryotic PAL (z.e., position 419 in AvPAL), which is normally blocked from pegylation due to pegylation of a neighboring lysine residue PAL (z.e., position 413 in AvPAL), can be a site for pegylation, which reduces substrate binding and / or catalytic activity. In some embodiments, one or more amino acid residues of the prokaryotic PAL are substituted by a lysine residue, such that a lysine residue important for the enzyme’s substrate binding and / or catalytic activity is not accessible for pegylation. In aspecific embodiment, the prokaryotic PAL is AvPAL. In one embodiment, the AvPAL lysine residue at position 419 is not accessible for pegylation.

[0105] In some embodiments, the composition comprises highly purified prokaryotic PAL variant derived from bacteria, or a biologically active fragment, mutant or analog thereof alone or in combination with a pharmaceutically suitable carrier. In some embodiments, preparations contain prokaryotic PAL variant with a purity greater than about 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In other embodiments, the relative specific activity of the prokaryotic PAL variant is at least about 50%, or greater than about 110%, of the specific activity of wild-type prokaryotic PAL.

[0106] Such prokaryotic PAL variants can be isolated and purified in accordance with the methods known in the art and is thereby present in amounts which enable using the prokaryotic PAL enzyme therapeutically. In some embodiments, a cDNA encoding for a complete or wild-type prokaryotic PAL is used. However, in other embodiments, a cDNA encoding for a biologically active fragment, mutant, variant or analog thereof can be used. Furthermore, provided herein are compositions of optimized prokaryotic PAL obtained by structure-based molecular engineering approaches and / or chemically-modified (e.g., pegylated) forms of PAL. Specific embodiments contemplate optimal compositions of prokaryotic PAL with improved specific activity, enhanced stability, reduced immunogenicity and / or proteolytic sensitivity appropriate for therapeutic use. In some embodiments, the PAL is a pegylated form of Anabaena variabilis PAL with improved specific activity, enhanced stability, reduced immunogenicity and / or proteolytic sensitivity.

[0107] In some embodiments, the pegylated prokaryotic PAL variant is an AvPAL variant and the cysteine residues at position 503 of AvPAL has been substituted with a serine residue (SEQ ID NO:2). In some embodiments, the pegylated prokaryotic PAL variant is an AvPAL variant and the cysteine residues at position 565 of AvPAL has been substituted with a serine residue (SEQ ID NO:3). In some embodiments, the pegylated prokaryotic PAL variant is an AvPAL variant and the cysteine residues at positions 503 and 565 of AvPAL have been substituted with serine residues (SEQ ID NO:4).Prokaryotic PAL Compositions., Pharmaceutical Compositions and Formulations

[0108] The present disclosure contemplates pharmaceutical compositions comprising therapeutically effective amounts of prokaryotic PAL compositions of the disclosure together with one or more pharmaceutically acceptable excipients, vehicles diluents, stabilizers,preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such pharmaceutical compositions include diluents of various buffer content (e.g., Tris-HCl, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Polysorbate 20, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); see, e.g., Remington's Pharmaceutical Sciences, 18thEdition (1990, Mack Publishing Co., Easton, Pa.) pages 1435:1712, which are herein incorporated by reference. An effective amount of active ingredient is a therapeutically, prophylactically, or diagnostically effective amount, which can be readily determined by a person skilled in the art by taking into consideration such factors as body weight, age, and therapeutic goal.

[0109] The prokaryotic PAL pharmaceutical compositions of the present disclosure may include a buffering agent to maintain the pH of the solution within a desired range. Preferred buffering agents include Tris-HCl, sodium acetate, sodium phosphate, and sodium citrate. Mixtures of these buffering agents may also be used. The amount of buffering agent useful in the composition depends largely on the particular buffer used and the pH of the solution. For example, acetate is a more efficient buffer at pH 5 than pH 6 so less acetate may be used in a solution at pH 5 than at pH 6. A more preferred buffering agent is Tris-HCl. A preferred pH range for the pharmaceutical compositions of the present disclosure is about pH 6.0-8.5. A more preferred pH range for the pharmaceutical compositions of the present disclosure is about pH 7.0-8.0. A most preferred pH range for the pharmaceutical compositions of the present disclosure is about pH 7.0-7.6.

[0110] The pharmaceutical compositions of the present disclosure may further include an isotonicity-adjusting agent to render the solution isotonic and more compatible for injection. A preferred agent is sodium chloride within a concentration range of 50-200 mM. A more preferred agent is sodium chloride within a concentration range of 100-150 mM. A most preferred agent is sodium chloride within a concentration range of 130-150 mM.

[0111] Pharmaceutically acceptable carriers or excipients may include stabilizers, which are molecules that stabilize the prokaryotic PAL compositions of the disclosure. The term “stabilize” as used herein, is meant to include, for example and not for limitation, increasing the shelf-life of a prokaryotic PAL enzyme, protecting the prokaryotic PAL enzyme from proteolytic digestion, maintaining the prokaryotic PAL enzyme in an active conformation, and preserving the prokaryotic PAL enzyme activity upon storage at elevated temperatures.

[0112] Stabilizers of the present disclosure include L-phenylalanine (Phe) and structural analogs thereof, such as trans-cinnamic acid (t-CA), benzoic acid, tyrosine (Tyr), and the like. Loss of activity of a plant PAL from Phaseolus vulgaris (PvPAL) has been shown upon removal of its substrate L-phenylalanine after affinity purification (Da Cunha, Eur. J.Biochem. 178:243-248 (1988)), and a yeast PAL from Rhodosporidium toruloides (RtPAL) has been shown to be protected from protease inactivation by tyrosine (Wang, et al., Mol. Genet. Metab. 86:134-140 (2005); Pilbak, eta!., FEBS J. 273:1004-1019 (2006)). As shown herein below, Phe and certain of its structural analogs have the ability to stabilize PEG:PAL conjugates of a prokaryotic PAL from Anabaena variabilis (AvPAL). Without being bound to a particular theory, it is hypothesized that the prokaryotic PAL enzyme is more stable as an enzyme-substrate complex, wherein the bound substrate Phe is converted to the product t-CA or to a transition state analog of t-CA. The t-CA remains bound to the otherwise highly reactive active site center (MIO group), thereby stabilizing the PAL enzyme. Accordingly, the PAL enzyme substrate, Phe, product, t-CA, or structural analogs thereof are stabilizers of the disclosure.

[0113] The disclosure contemplates a pharmaceutical composition comprising a prokaryotic PAL variant and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a stabilizer. The stabilizer is Phe or structural analog thereof. The stabilizer is selected from the group consisting of L-phenylalanine, transcinnamic acid and benzoic acid. A preferred range for the stabilizers of the disclosure is from about 0.1 to 20 moles of stabilizer per mole active site of prokaryotic PAL. A more preferred range for the stabilizers of the disclosure is from about 0.5 to 10 moles of stabilizer per mole active site of prokaryotic PAL. A most preferred range for the stabilizers of the disclosure is from about 1 to 10 moles of stabilizer per mole active site of prokaryotic PAL.

[0114] In some embodiments, the pharmaceutical composition comprises a prokaryotic PAL variant and a pharmaceutically acceptable carrier, wherein the prokaryotic PAL variant has a greater phenylalanine-converting activity and / or a reduced immunogenicity as compared to a wild-type PAL and is effective in reducing the Phe concentration in the blood, serum or plasma of the subject to a range from below the level of detection to between about 20 pM to 60 pM, preferably to less than about 20 pM, and even more preferably to less than about 10 pM, and wherein the pharmaceutically acceptable carrier comprises a stabilizer. In some embodiments, the stabilizer is Phe or structural analog thereof. In some embodiments,the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid and benzoic acid.

[0115] In some specific embodiments, the pharmaceutical composition comprises a prokaryotic PAL variant and a pharmaceutically acceptable carrier, wherein the prokaryotic PAL variant is an Anabaena variabilis PAL (AvPAL) variant, wherein the cysteine residues at positions 503 and 565 of the AvPAL variant have been substituted by serine residues, the AvPAL variant further comprises a water-soluble polymer of polyethylene glycol, wherein the ratio of AvPAL variant and the polyethylene glycol is about 1:3; and the AvPAL variant is effective in reducing the phenylalanine concentration in the blood, serum or plasma of the subject to a range from below the level of detection to between about 20 pM to 60 pM, preferably to less than about 20 pM, and even more preferably to less than about 10 pM, and wherein the pharmaceutically acceptable carrier comprises a stabilizer. In some embodiments, the stabilizer is Phe or structural analog thereof. In some embodiments, the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid and benzoic acid.

[0116] In some more specific embodiments, the pharmaceutical composition provided herein comprises an AvPAL variant comprising an amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and trans-cinnamic acid. In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the AvPAL variant comprises the amino acid sequence of SEQ ID NO:4. In some embodiments of the various pharmaceutical compositions described above, the pharmaceutical composition further comprises sodium chloride, and tromethamine and tromethamine hydrochloride.

[0117] As used herein, when contemplating prokaryotic PAL variant compositions, the term "therapeutically effective amount" refers to an amount that is effective to produce the intended beneficial effect on health of a patient. In some embodiments, a therapeutically effective amount of a prokaryotic PAL variant gives a decrease in blood, plasma or serum, preferably plasma, L-phenylalanine levels that provides benefit to the patient. The amount will vary from one individual to another and will depend upon a number of factors, including the overall physical condition of the patient, diet and disease state. The amount of prokaryotic PAL used for therapy gives an acceptable decrease in blood, plasma or serum, preferably plasma, L-phenylalanine levels, and maintains this value during PAL treatment ata beneficial level (typically in a range from less than about 5% to between about 35% and 100%, preferably in a range from less than about 5% to about 35%, and even more preferably in a range from less than about 5% to about 15% of the normal range of blood, plasma or serum, preferably plasma, L-phenyl alanine). In some embodiments, a therapeutically effective amount of a prokaryotic PAL variant reduces tumor growth, tumor size or tumor burden by greater than about 10%, 30%, 50%, 70%, 90%, 95%, 98% or 99% in a treated patient as compared to an untreated patient. In some embodiments, a therapeutically effective amount of a prokaryotic PAL variant maintains the tumor in static condition in a treated patient as compared to an untreated patient. In some embodiments, a therapeutically effective amount of a prokaryotic PAL variant increases survival time or disease-free time at least about 10%, 20%, 50%, 100%, 2-fold, 5-fold or 10-fold longer in a treated patient as compared to an untreated patient. A therapeutically effective amount of the prokaryotic PAL variant compositions of the disclosure may be readily ascertained by one skilled in the art using publicly available materials and procedures.

[0118] The disclosure provides for administering prokaryotic PAL variants less frequently than native PAL. The dosing frequency will vary depending upon the condition being treated, but in general will be about one time per week. It is understood that the dosing frequencies actually used may vary somewhat from the frequencies disclosed herein due to variations in responses by different individuals to the prokaryotic PAL variants; the term "about" is intended to reflect such variations. It is contemplated that the prokaryotic PAL variants are administered about two times per week, about one time per week, about one time every two weeks, about one time per month, or longer than about one time per month.

[0119] The present disclosure may thus be used to reduce blood, plasma or serum L-phenylalanine levels. Numerous conditions, where depletion of blood, plasma or serum L-phenylalanine levels would be beneficial, may be treated with the prokaryotic PAL variant pharmaceutical compositions of the disclosure.

[0120] The prokaryotic PAL pharmaceutical compositions prepared in accordance with the present disclosure are preferably administered by parenteral injection, either intravenously, intraperitoneally, subcutaneously, intramuscularly, intraarterially or intrathecally. However, it would be clear to one skilled in the art that other routes of delivery could also be effectively utilized using the pharmaceutical compositions of the present disclosure.

[0121] The methods described herein use prokaryotic PAL pharmaceutical compositions comprising the molecules described above, together with one or more pharmaceutically acceptable excipients, vehicles, diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers, and optionally other therapeutic and / or prophylactic ingredients. Such excipients include liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, cyclodextrins, modified cyclodextrins ( / .< ., sufobutyl ether cyclodextrins), etc. Suitable excipients for non-liquid formulations are also known to those of skill in the art.

[0122] Pharmaceutically acceptable salts can be used in the compositions of the present disclosure and include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington's Pharmaceutical Sciences, 18thEdition (Easton, Pennsylvania: Mack Publishing Company, 1990).

[0123] Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, may be present in such vehicles. A biological buffer can be virtually any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, / .< ., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank's buffered saline, and the like.

[0124] Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the prokaryotic PAL in combination with a pharmaceutically acceptable carrier and, in addition, may include other pharmaceutical agents, adjuvants, diluents, buffers, etc.

[0125] In general, the prokaryotic PAL pharmaceutical compositions of this disclosure will be administered as pharmaceutical formulations including those suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. Thepreferred manner of administration is intravenous using a convenient daily dosage regimen, which can be adjusted according to the degree of affliction.

[0126] For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., a prokaryotic PAL variant composition as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, tonicifying agents, and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, referenced above.

[0127] For oral administration, the composition will generally take the form of a tablet, capsule, or softgel capsule, or may be an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use will generally include one or more commonly used carriers such as lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. When liquid suspensions are used, the active agent may be combined with emulsifying and suspending agents. If desired, flavoring, coloring and / or sweetening agents may be added as well. Other optional components for incorporation into an oral formulation herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.

[0128] Parenteral formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid or lyophilized forms suitable for reconstitution, solubilization or suspension in liquid prior to injection, or as emulsions. Preferably, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation may also be a sterile injectable solution or a suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. Inaddition, parenteral administration may involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.

[0129] The prokaryotic PAL compositions of the disclosure described herein can be administered to a patient at therapeutically effective doses. The toxicity and therapeutic efficacy of such prokaryotic PAL compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as, for example, by determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDso ZEDso. Prokaryotic PAL compositions exhibiting large therapeutic indices are normally preferred.Methotrexate (MTX)

[0130] Methotrexate (MTX) is an anti-metabolite most commonly used in chemotherapy and immunosuppressant in auto-immune diseases. Methotrexate inhibits the enzyme, dihydrofolic acid reductase. This enzyme reduces dihydrofolates to tetrahydrofolates so that dihydrofolates can be utilized as carriers of one-carbon groups in the synthesis of purine nucleotides and thymidylate. Therefore, methotrexate, by inhibiting dihydrofolic acid reductase, can interfere with DNA synthesis, repair, and cellular replication. Actively proliferating tissues such as malignant cells, bone marrow, fetal cells, buccal and intestinal mucosa, and cells of the urinary bladder are in general more sensitive to this effect of methotrexate. This mechanism is utilized in the treatment of cancer because of its cytotoxic effect (Methotrexate, United States Prescribing Information (USPI), August 2020; Singh, et al, Eur J Pharmacol. 853: 264-274 (2019)). Methotrexate is also an FDA-approved folic acid antagonist indicated for the treatment of rheumatoid arthritis. In autoimmune diseases, different mechanisms are involved in choosing methotrexate as a drug of choice. For example, MTX inhibits enzyme AICAR transformylase, leading to adenosine accumulation, in which the anti-inflammatory action of adenosine leads to repression of T-cell activation, thereby down-regulation of B-cells and increasing activated CD-95 T cells sensitivity. MTX also causes repression of methyltransferase activity and inhibition of the binding of beta- 1 interleukin to its cell surface receptor.

[0131] Methotrexate (MTX) has the chemical name of 7V-[4-[[(2,4 diamino-6-pteridinyl) methyl]methylamino]benzoyl]-L-glutamic acid. The molecular formula is C20H22N8O5 and the molecular weight is 454.45 g / mol. MTX in this disclosure has the structural formula:In one aspect, provided herein is a method for reducing blood phenylalanine concentration in a subject, comprising administering to the subject a weekly dose of methotrexate (MTX) or a pharmaceutically acceptable salt thereof, in combination with a weekly dose of a formulation comprising an AvPAL variant as provided herein. In certain embodiments, MTX is administered as an oral tablet. In certain embodiments, the oral tablet is a 2.5 mg tablet. Each methotrexate tablet, USP contains 2.5 mg methotrexate equivalent to 2.74 mg methotrexate sodium and the following inactive ingredients: lactose (anhydrous), magnesium stearate, pregelatinized starch, sodium hydroxide pellets, sodium starch glycolate and purified water.Identifying and Monitoring Patient Populations

[0132] As discussed herein throughout, it will be necessary in various embodiments of the present disclosure to determine whether a given patient is responsive to PAL therapy, and to determine the phenylalanine concentrations of the patient both initially to identify the class of PKU patient being treated and during an ongoing therapeutic regimen to monitor the efficacy of the regimen. Exemplary such methods are described herein below.

[0133] BH4 Loading Test

[0134] The BH4 loading test allows discrimination between patients that have HPA due to a deficit in BH4 or through a deficiency in PAH.

[0135] The simplest BH4 loading test is one in which exogenous BH4 is administered and the effects of the administration on lowering of plasma Phe concentrations is determined.Intravenous loading of 2 mg / kg BH4 was initially proposed by Danks, et al., Lancet 1 : 1236 (1976), as BH4 of greater purity has become available it has become possible to perform the test using an oral administration of BH4 in amounts of about 2.5 mg / kg body weight.Ultimately, a standardized approach was proposed by Niederwieser et al. in which a 7.5 mg / kg single oral dose of BH4 is administered (Niederwieser, et al., Eur. J. Pediatr. 138:441 (1982)), although some laboratories do still use upwards of 20 mg BH4 / kg body weight.

[0136] In order for the simple BH4 loading test to produce reliable results, the blood Phe levels of the patient need to be higher than 400 pM. Therefore, it is often customary for the patient to be removed from the PKU diet for 2 days prior to performing the loading test. A BH4 test kit is available and distributed by Dr. Schircks Laboratories (Jona, Switzerland). This kit recommends a dosage of 20 mg BH4 / kg body weight about 30 minutes after intake of a normal meal.

[0137] Determination of Phe Concentrations

[0138] There are numerous methods for determining the presence of Phe in blood (Shaw et al., Analytical Methods in Phenylketonuria-Clinical Biochemistry, In Bickett et al. Eds., Phenylketonuria and Some Other Inborn Errors of Amino Acid Metabolism, Stuttgart, Georg Thiem Verlag, 47-56 (1971)). Typically, phenylalanine and tyrosine concentrations are determined from the serum of a patient using a fluorometric assay. This assay relies on the formation of fluorescent substance when phenylalanine is heated with ninhydrin in the presence of leucylalanine (McCaman, et al., J. Lab. Clin. Med. 59:885-890 (1962)).

[0139] The most popular method for determining Phe concentrations is the Guthrie test in which discs are punctured from filter paper that has been saturated with a blood sample from the patient. The uniform discs are incubated in a tray of agar that has been seededwith Bacillus subtilis and contains a specific inhibitor of Bacillus subtilis growth. As the phenylalanine transfers from the uniform discs onto the agar, the Phe reverse the inhibition of bacterial growth thereby yielding an area of bacterial growth that can be correlated to phenylalanine concentration by comparison to similar assays performed using discs containing known amounts of Phe.

[0140] Other methods of quantifying Phe concentration include HPLC, mass spectrometry, thin layer chromatography and the like. Such methods can be used to determine the plasma Phe concentration of a patient before the therapy and to monitor the Phe concentration during the therapeutic regimen to determine the efficacy thereof.

[0141] It is contemplated that the plasma Phe levels of the patients will be monitored at convenient intervals (e.g., daily, every other day or weekly) throughout the time course of the therapeutic regimen. By monitoring the plasma Phe levels with such regularity, the clinician will be able to assess the efficacy of the treatment and adjust the PAL and / or dietary protein requirements accordingly.Dietary Restriction

[0142] Certain methods of the invention involve the combined use of PAL and dietary protein restriction to effect a therapeutic outcome in patients with various forms of HPA. To achieve the appropriate therapeutic outcome in the combination therapies contemplated herein, one would generally administer to the subject the PAL composition and the dietary restriction in a combined amount effective to produce the desired therapeutic outcome (i.e., a lowering of plasma Phe concentration and / or the ability to tolerate greater amounts of Phe / protein intake without producing a concomitant increase in plasma Phe concentrations). This process may involve administering the PAL composition and the dietary protein therapeutic composition at the same time. This may be achieved by administering a single composition or pharmacological protein formulation that includes all of the dietary protein requirements and also includes the PAL within said protein formulation. Alternatively, the dietary protein (supplement or normal protein meal) is taken at about the same time as a pharmacological formulation (tablet, injection or drink) of PAL. PAL also may be formulated into a protein bar or other foodstuff such as brownies, pancakes, cake, suitable for ingestion.

[0143] In other alternatives, PAL treatment may precede or follow the dietary protein therapy by intervals ranging from minutes to hours. In embodiments where the protein and the PAL compositions are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that PAL will still be able to exert an advantageously effect on the patient. In such instances, it is contemplated that one would administer the PAL within about 2-6 hours (before or after) of the dietary protein intake, with a delay time of only about 1 hour being most preferred. In certain embodiments, it is contemplated that PAL therapy will be a continuous therapy where a daily dose of PAL is administered to the patient indefinitely. In other situations, e.g., in pregnant women having only the milder forms of PKU and HPA, it may be that PAL therapy is only continued for as long as the woman is pregnant and / or breast feeding.

[0144] Further, in addition to therapies based solely on the delivery of PAL and dietary protein regulation, the methods of the present invention also contemplate combinationtherapy with a third composition that specifically targets one or more of the symptoms of HPA. For example, it is known that the deficit in tyrosine caused by HPA results in a deficiency in neurotransmitters dopamine and serotonin. Thus, in the context of the present invention, it is contemplated that PAL and dietary protein based methods could be further combined with administration of L-dopa, carbidopa and 5-hydroxytryptophan neurotransmitters to correct the defects that result from decreased amounts of tyrosine in the diet.

[0145] As the administration of PAL would not generate tyrosine (unlike administration of PAH), such treatment will still result in tyrosine being an essential amino acid for such patients. Therefore, dietary supplementation with tyrosine may be desirable for patients receiving PAL in combination with the BH4 therapy.Dietary Protein

[0146] In addition to administering prokaryotic PAL compositions to the subjects, it is contemplated that the dietary protein of the patients also may be restricted or modified. Those of skill in the art are aware of various commercially available protein formulas for use in the treatment of PKU. Such formulas include MAXIMAID, PHENEX 1, PHENEX 2 (Ross Laboratories, Liverpool, UK), LOFENALAC, PHENYL-FREE (Mead- Johnson), and the like.

[0147] Those of skill in the art may use the referenced protein formulas, which are generally free of Phe concentrations. The protein formulas often are supplemented with amino acids that are deficient in PKU patients. Such amino acids include, for example, L-tyrosine, and L-glutamine.

[0148] Further, as it is known that L-carnitine and taurine, which are normally found in human milk and other foodstuffs of animal origin, also should be supplied in addition to the protein restriction. In certain embodiments, the L-camitine may be supplied as 20 mg / 100 g of protein supplement, and the taurine may be supplied as 40 mg / 100 g protein supplement in order to help supply amounts of these factors normally found in human milk and foods of animal origin.

[0149] In addition, those of skill in the art are referred to the 2000 National Academy of Sciences-National Research Council Dietary Reference Intakes for a further listing of other components, such as essential vitamins and minerals that should be supplied to the patient to ensure that other supplements are being provided despite the dietary protein restriction.

[0150] Referring to the discussion above regarding total protein amounts and desirable plasma Phe concentrations, one of skill in the art will be able to determine the amount of dietary protein restriction that is required and thus adjust the diet of the patient accordingly. Upon administering prokaryotic PAL to that subject, determining whether the methods of the disclosure are effective will entail determining the plasma Phe concentrations of the patient on a regular basis to ensure that the plasma Phe concentrations remain in a range from below the level of detection to between about 20 pM to 60 pM, preferably to less than about 20 pM, and even more preferably to less than about 10 pM. Tests for determining such concentrations are described below. Preferably, concentrations of less than the level of detection to between about 20 pM to 60 pM are achieved, more preferably to less than about 20 pM, and even more preferably to less than about 10 pM.

[0151] In certain embodiments, the disclosure provides a method for treating a subject comprising administering to a subject in need of such treatment a therapeutically effective amount of a pharmaceutical composition comprising a prokaryotic phenylalanine ammonialyase (PAL) variant and a pharmaceutically acceptable carrier, wherein the PAL variant has a greater phenylalanine-converting activity and / or a reduced immunogenicity as compared to a wild-type PAL, and is effective in reducing the phenylalanine concentration in the blood, serum or plasma of the subject to a range from below the level of detection to between about 20 pM to 60 pM, preferably to less than about 20 pM, and even more preferably to less than about 10 pM, and further comprising administering to the subject a protein-restricted ( / .< ., phenylalanine-free) diet.

[0152] To achieve the appropriate therapeutic outcome in the combination therapies contemplated herein, preferably one would generally administer to the subject the prokaryotic PAL composition and the dietary restriction in a combined amount effective to produce the desired therapeutic outcome ( / .< ., a lowering of plasma Phe concentration to a range from below the level of detection to optimally about 20 pM to 60 pM, preferably to less than about 20 pM, and even more preferably to less than about 10 pM, using standard detection methods well known in the art). This process may involve administering the prokaryotic PAL composition and the dietary protein therapeutic composition at the same time. This may be achieved by administering a single composition or pharmacological protein formulation that includes all of the dietary protein requirements and also includes the prokaryotic PAL within said protein formulation. Alternatively, the dietary protein (supplement or normal protein meal) is taken at about the same time as a pharmacological formulation (tablet, injection ordrink) of prokaryotic PAL. Prokaryotic PAL also may be formulated into a protein bar or other foodstuff such as brownies, pancakes, cake, suitable for ingestion.

[0153] As the administration of prokaryotic PAL would not generate tyrosine (unlike administration of PAH), such treatment will still result in tyrosine being an essential amino acid for such patients. Therefore, dietary supplementation with tyrosine may be desirable for patients receiving prokaryotic PAL alone in combination with the dietary protein therapy.

[0154] In other alternatives, prokaryotic PAL treatment may precede or follow the dietary protein therapy by intervals ranging from minutes to hours. In embodiments where the protein and the prokaryotic PAL compositions are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that PAL will still be able to exert an advantageously effect on the patient. In such instances, it is contemplated that one would administer the PAL within about 2-6 hours (before or after) of the dietary protein intake, with a delay time of only about 1 hour being most preferred. In certain embodiments, it is contemplated that PAL therapy will be a continuous therapy where a daily dose of PAL is administered to the patient indefinitely.Folic Acid

[0155] Folate deficiency may result in an increased risk of methotrexate adverse reactions. Folic acid or folinic acid can be supplemented to the combination therapy methods as provided herein to reduce such risk. In certain embodiments, folic acid or folinic acid is administered during the combination therapy. In certain embodiments, folic acid or folinic acid is administered before the combination therapy. In certain embodiments, folic acid or folinic acid is administered four weeks before the combination therapy. In certain embodiments, folic acid or folinic acid is administered 1 mg orally daily. In certain embodiments, folic acid or folinic acid is administered 2 mg orally daily. In certain embodiments, folic acid or folinic acid is administered 7.5 mg orally daily. In certain embodiments, folic acid or folinic acid is administered in a single dose. In certain embodiments, folic acid or folinic acid is administered in multiple divided doses. In certain embodiments, folic acid or folinic acid is administered in multiple divided doses in the morning and evening of the same day. In certain embodiments, folic acid or folinic acid is administered 1 mg orally daily for about 24 weeks to 28 weeks. In certain embodiments, folic acid or folinic acid is administered 1 mg orally daily for about 24 weeks. In certain embodiments, folic acid or folinic acid is administered 1 mg orally daily for about 28 weeks.Premedication

[0156] To mitigate the risk of hypersensitivity reactions, premedication is applied before each administration of the AvPAL variant, including the premedication of an Hl antagonist, an H2 antagonist, and / or an antipyretic. Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) can be used as premedication for the administration of the AvPAL variant. In certain embodiments, premedication with an Hl antagonist, an H2 antagonist, and / or an antipyretic is administered about 2 to 3 hours prior to each dose of the AvPAL variant until the subject is able to achieve blood Phe600 pmol / L. In certain embodiments, premedication with an Hl antagonist, an H2 antagonist, and / or an antipyretic is applied to a subject who is on a stable dose of the AvPAL variant (the maintenance phase). In certain embodiments, premedication is considered during the maintenance phase. In certain embodiments, NSAID administered as a premedication should be given with food.Therapeutic Uses

[0157] Provided herein are methods of reducing blood phenylalanine concentration in a subject comprising administering to the subject a weekly dose of a formulation comprising an AvPAL variant as described here in and methotrexate (MTX) or a pharmaceutically acceptable salt thereof. In various embodiments, the disease or disorder treated by a combination therapy method described herein is associated with an elevated blood phenylalanine (Phe) concentration. In various embodiments, the disease or disorder associated with the elevated blood Phe concentration treated by a combination method described herein is a phenylketonuria (PKU).

[0158] In the combination treatment comprising administering an AvPAL variant (e.g., pegvaliase) or rAvPAL as described herein, antibody- and complement-mediated immune responses to this foreign protein can lead to an increased protein clearance and to hypersensitivity reactions in a subject being treated with such protein, which interfere with drug efficacy and result in poor tolerability. In various embodiments, the combination therapy method described herein can modulate immune response in the subject, for example, suppressing immune responses to the AvPAL variant e.g., pegvaliase), thereby improving the tolerability and efficacy of the AvPAL variant (e.g., pegvaliase) in the subject with PKU.

[0159] In certain embodiments, the combination treatment methods described herein apply to a subject who has not received a treatment with the AvPAL variant. It is contemplated that certain methods provided herein will allow the concurrent MTXadministration to align with the immune response to the AvPAL variant. In certain embodiments, the combination treatment methods described herein apply to a subject who has already received a prior treatment with the AvPAL variant. This subject has completed a minimum of 24 consecutive weeks of treatment with the AvPAL variant with persistent uncontrolled blood Phe >600 pmol / L, who are on a current dose of A 20 mg / day and could not further dose escalate. It is contemplated that by applying certain methods provided herein, this subject may benefit from modulation of immune response given the less responsiveness and the difficulty in dose escalation that is likely associated with a more robust immune response.

[0160] In various embodiments, diseases or disorders treated by a combination therapy method described herein are provided below.

[0161] Various Forms of Hyperphenylalaninemia (HP A)

[0162] Provided herein are combination therapy methods of treating a variety of HP A patient populations comprising the combinational use of pharmaceutical compositions provided herein (e.g., pegvaliase together with MTX), either the combination alone or in combination with other therapeutic regimens, for managing HPA and / or PKU. In particular, it is contemplated that the pharmaceutical compositions provided herein can be used to treat the patient population with phenylalanine concentrations that are low enough that dietary intervention is not normally used (i.e., patients with mild HPA), patients with moderate PKU, patients with classic or severe PKU, and any subpopulations thereof.

[0163] Certain embodiments are directed to treating classic severe PKU by administering to the subject a protein-restricted diet in combination with a composition comprising prokaryotic PAL variant or a biologically active variant, mutant, or fragment thereof together with a composition comprising MTX or a pharmaceutically acceptable salt thereof, wherein the combined administration of the protein-restricted diet and prokaryotic PAL variant together with MTX is effective to lower the phenylalanine concentration in the plasma of said subject as compared to said concentration in the absence of said combined administration. In specific embodiments, therapy is contemplated for a patient who manifests Phe levels greater than 420 pM. In another specific embodiments, therapy is contemplated for a patient who manifests Phe levels greater than 500 pM. In yet another specific embodiments, therapy is contemplated for a patient who manifests Phe levels greater than 550 pM. In yet specific embodiments, therapy is contemplated for a patient who manifests Phe levels greater than600 pM. In specific embodiments, therapy is contemplated for a patient who manifests Phe levels greater than 650 pM.

[0164] Other embodiments entail administering the pharmaceutical composition comprising prokaryotic PAL variant provided herein and the pharmaceutical composition comprising MTX or a pharmaceutically acceptable salt thereof provided herein to any individual that has HP A, characterized by a plasma Phe concentration greater than 180 pM prior to the administration of prokaryotic PAL variant, in an amount effective to produce a decrease in such a plasma Phe concentration of the patient.

[0165] Characteristics of Severe Classical PKU and Methods of Treatment Thereof

[0166] Severe PKU manifests in a plasma Phe concentration greater than 1200 pM and can be found to be as high as 4800 pM. Patients that have this disorder must be treated with a Phe-free diet in order to bring their plasma Phe concentrations down to a level that is clinically acceptable (typically, less than 600 pM or less than 300 pM). These patients are only able to tolerate a maximum of between 250-350 mg dietary Phe per day (Spaapen et al., Mol. Genet Metab. 78:93-99 (2003)). As such, these patients are started on a Phe-restricted formula diet between 7-10 days after birth and are burdened with this dietary restriction for the remainder their lifespan. Any alleviation of the strict dietary restrictions that these individuals are encumbered with would be beneficial.

[0167] The tests used for the diagnosis of individuals with classical Phe are described in further detail here. These tests have revealed that patients with classical severe PKU require a low phenylalanine diet (Lucke et al, Pediatr. Neurol. 28:228-230 (2003)). Thus, it is contemplated that certain methods provided herein will entail determining that the patient is suffering from classical PKU by monitoring the plasma Phe concentration of the individual. The patient can then be treated by administering the pharmaceutical composition comprising the prokaryotic PAL variant provided herein alone or a combined regimen of a low protein diet and PAL variant such that there is produced at least a 25% decrease in the plasma Phe concentrations of the patient. In some embodiments, the method will produce a 30% decrease in the plasma Phe concentration. In other embodiments, the method will produce a 40%, 50%, 60%, 70%, 80%, 90% or greater decrease in the plasma Phe concentration of the individual (for example, where a patient with severe classical PKU has a Phe concentration of 4800 pM a 90% decrease in the Phe concentration will produce a plasma Phe concentration of 480 pM, a concentration that is sufficiently low to require little dietary restriction). Ofcourse, it should be understood that the treatment methods provided herein, whether for treating severe classical PKU or any other HPA described herein, should attempt to lower the plasma Phe concentrations of the patient to levels as close to a range of about 120 pM to about 360 pM ± 15 pM as possible, or to an optimal range of about 120 pM to about 240 pM.

[0168] In some embodiments, the plasma Phe concentrations of the classical PKU patient being treated is reduced from any amount of unrestricted plasma Phe concentration that is greater than 1000 pM to any plasma Phe level that is less than 600 pM. Of course, even if the combined treatment with prokaryotic PAL variant and the protein-restricted diet produces a lesser decrease in plasma Phe concentration, e.g., to a level of between 800 pM to about 1200 pM, this will be viewed as a clinically useful outcome of the therapy because patients that have a plasma Phe concentration in this range can manage the disease by simply restricting the amount of protein in the diet as opposed to eating a Phe-restricted formula, thereby resulting in a marked improvement in the quality of life of the individual, as well as leading to greater patient compliance with the dietary restriction.

[0169] Any increase in the amount of dietary Phe levels that can be tolerated by the patient as a result of the treatment will be considered to be a therapeutically effective outcome. For example, it is contemplated that as a result of administering the prokaryotic PAL variant therapy, the patient will be able to increase his / her intake of dietary Phe from 250-350 mg / day to 350-400 mg / day (i.e., the Phe tolerance phenotype of the patient is altered from that of a classic PKU patient to a moderate PKU patient). It would be desirable that the therapeutic intervention taught herein would allow the patient to increase his / her intake of dietary Phe from 250-350 mg / day to 400-600 mg / day (i.e., the Phe tolerance phenotype of the patient is altered from that of a classic PKU patient to a mild PKU patient), or in some cases, to allow the patient to have an intake of greater than 600 mg Phe / day (i.e., normal dietary intake).

[0170] Characteristics of BH4-non-responsive PKU Patients and Methods of Treatment Thereof

[0171] A second group of patients that can be treated with the pharmaceutical compositions and methods provided herein are those individuals that have been determined to have an elevated plasma Phe concentrations i.e., any concentration that is greater than 200 pM, but have been diagnosed to be non-responsive to BH4 therapy (as determined by the BH4 loading test described below). Such patients can include those individuals that have mildPKU (i.e., plasma Phe concentrations of up to 600 pM), individuals that have moderate PKU (i.e., plasma Phe concentrations of between 600 pM to about 1200 pM), as well as patients that have classic severe PKU (i.e., plasma Phe concentrations that are greater than 1200 pM).

[0172] In some embodiments, patients that are non-responsive to BH4 therapy are given PAL variant in combination with a reduced amount of protein in their diet in order to decrease the plasma Phe concentrations of the patient. The administration of prokaryotic PAL variant can produce a greater decrease in the plasma Phe concentrations of the patient as compared to the decrease that is produced with the same dietary protocol administered in the absence of prokaryotic PAL variant therapy. The dietary restrictions can be a diet that restricts the Phe intake by providing a synthetic medical protein formula that has a diminished amount of Phe or alternatively, the dietary restriction can be one which simply requires that the patient limit his / her overall protein intake but nevertheless allows the patient to eat normal foodstuffs in limited quantities.

[0173] The therapeutic outcomes discussed for classical PKU patients are incorporated into the present section by reference. For example, the therapeutic outcomes for patients with moderate PKU (i.e., patients that has an unrestricted plasma Phe concentration of 600 pM to 1200 pM) can include at least a 25% decrease in the plasma Phe concentrations of the patient. In some embodiments, the method will produce a 30% decrease in the plasma Phe concentration. In other embodiments, the method will produce a 40%, 50%, 60%, 70%, 80%, 90% or greater decrease in the plasma Phe concentration of the individual (for example, where a patient with moderate classical PKU has a Phe concentration of 1000 pM, a 90% decrease in the Phe concentration will produce a plasma Phe concentration of 100 pM, a concentration that is sufficiently low to require little or no dietary restriction).

[0174] In some embodiments, the plasma Phe concentrations of the moderate PKU patient being treated is reduced from any amount of unrestricted plasma Phe concentration that is between 600 pM to 1200 pM to any plasma Phe level that is less than 300 pM. In one embodiment, treatment with prokaryotic PAL variant (either alone or in combination with a dietary restriction) produces a decrease in plasma Phe concentration, e.g., to a level of between 200 pM to about 400 pM, which will be viewed as a clinically useful outcome of the therapy because patients that have a plasma Phe concentration in this range can manage the disease by simply restricting the amount of protein in the diet as opposed to eating a Phe-restricted formula. Indeed, in many studies, it is taught that such patients can even eat a normal diet.

[0175] Any increase in the amount of dietary Phe levels that can be tolerated by the patient as a result of the treatment will be considered to be a therapeutically effective outcome. For example, it is contemplated that as a result of administering the prokaryotic PAL variant therapy (either alone or in combination with other therapeutic intervention), the patient will be able to increase his / her intake of dietary Phe from 350-400 mg / day to 400-600 mg / day (i.e., the Phe tolerance phenotype of the patient is altered from that of a moderate PKU patient to a mild PKU patient). Of course, it would be desirable that the therapeutic intervention taught herein would allow the patient to increase his / her intake of dietary Phe from 350-400 mg / day to have an intake of greater than 600 mg Phe / day (i.e., normal dietary intake).

[0176] A patient manifesting only mild PKU, i.e., has a dietary allowance of 400-600 mg Phe intake / day, can be treated using the compositions and methods provided herein and can benefit from the prokaryotic PAL variant-based therapies because it is desirable to produce a normalized plasma Phe concentration that is as close to 360 pM ± 15 pM as possible. For such patients, an advantageous therapeutic outcome will include at least a 25% decrease in the plasma Phe concentrations of the patient. In one embodiment, the method will produce a 30% decrease in the plasma Phe concentration. In another embodiment, the method will produce a 40%>, 50%>, 60%>, or greater decrease in the plasma Phe concentration of the individual (for example, where a patient with mild PKU has a Phe concentration of 600 pM, a 60% decrease in the Phe concentration will produce a plasma Phe concentration of 360 pM, i.e., an acceptable, normal concentration of plasma Phe).

[0177] In some embodiments, the plasma Phe concentrations of the mild PKU patient being treated is reduced from any amount of non-restricted plasma Phe concentration that is between 400 pM to 600 pM to any plasma Phe level that is less than 100 pM. Of course, even if the treatment with prokaryotic PAL variant (either alone or in combination with a dietary restriction) produces a lesser decrease in plasma Phe concentration, e.g., to a level of between 200 pM to about 400 pM, this will be viewed as a clinically useful outcome of the therapy.

[0178] Any increase the amount of dietary Phe levels that can be tolerated by the patient as a result of the treatment will be considered to be a therapeutically effective outcome. For example, it is contemplated that as a result of administering prokaryotic PAL variant therapy (either alone or in combination with other therapeutic intervention), the patient will be able to increase his / her intake of dietary Phe from 400-600 mg / day (i.e., the Phe tolerance phenotype of the patient is altered from that of a mild PKU patient to a mild HPA patient) to allow thepatient to have an intake of greater than 600 mg Phe / day (i.e., normal dietary intake).Furthermore, even if the patient is one who only manifests the symptoms of non PKU HP A, i.e., has an elevated plasma Phe concentration of up to 600 pM, but is otherwise allowed to eat a normal protein diet will benefit from prokaryotic PAL variant therapy because it has been shown that elevated Phe concentrations have significant effects on the IQ of such individuals.Kits

[0179] Also provided herein are kits comprising a AvPAL variant provided herein and MTX or a pharmaceutically acceptable salt thereof provided herein, packaged into suitable packaging material. A kit optionally includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.

[0180] The term “packaging material” refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0181] Kits provided herein can include labels or inserts. Labels or inserts include “printed matter,” e.g., paper or cardboard, separate or affixed to a component, a kit or packing material (e.g., a box), or attached to, for example, an ampoule, tube, or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM / RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic / optical storage media, FLASH media, or memory type cards. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location, and date.

[0182] Kits provided herein can additionally include other components. Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. Kits can be designed for cold storage. Kits can also be maintained under appropriate storage conditions until ready to use.

[0183] For the sake of conciseness, certain abbreviations are used herein. One example is the single letter abbreviation to represent amino acid residues. The amino acids and their corresponding three letter and single letter abbreviations are as follows:alanine Ala (A)arginine Arg (R)asparagine Asn (N)aspartic acid Asp (D)cysteine Cys (C)glutamic acid Glu (E)glutamine Gin (Q)glycine Gly (G)histidine His (H)isoleucine He (I)leucine Leu (L)lysine Lys (K)methionine Met (M)phenylalanine Phe (F)proline Pro (P)serine Ser (S)threonine Thr (T)tryptophan Trp (W)tyrosine Tyr (Y)valine Vai (V)

[0184] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein.

[0185] Particular embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Upon reading the foregoing description, variations of the disclosed embodiments may become apparent to individuals working in the art, and it is expected that those skilled artisans may employ such variations as appropriate. Accordingly, it is intended that the invention be practiced otherwise than asspecifically described herein, and that the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0186] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure and to the same extent as if each individual publication, patent application, patent, or other reference was specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.EMBODIMENTS

[0187] Embodiment 1. A method for reducing blood phenylalanine concentration in a subject, the method comprising: (1) administering to the subject a weekly dose of a formulation comprising an AvPAL variant, wherein the AvPAL variant comprises an amino acid sequence of SEQ ID NO:4; and (2) administering to the subject a weekly dose of methotrexate (MTX) or a pharmaceutically acceptable salt thereof.

[0188] Embodiment 2. The method of embodiment 1, wherein the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered concurrently.

[0189] Embodiment 3. The method of embodiment 1 or 2, wherein the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered in separate compositions.

[0190] Embodiment 4. The method of any one of embodiments 1-3, wherein the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered on the same day.

[0191] Embodiment 5. The method of any one of embodiments 1-4, wherein the dosage of MTX is about 15 mg per week.

[0192] Embodiment 6. The method of any one of embodiments 1-4, wherein the dosage of MTX is about 10 mg per week.

[0193] Embodiment 7. The method of any one of embodiments 1-6, wherein MTX or a pharmaceutically acceptable salt thereof is administered orally.

[0194] Embodiment 8. The method of any one of embodiments 1-7, wherein the dosage of MTX is administered in a single dose.

[0195] Embodiment 9. The method of any one of embodiments 1-7, wherein the dosage of MTX is administered in multiple divided doses within a day.

[0196] Embodiment 10. The method of embodiment 9, wherein the dosage of MTX is administered orally at 7.5 mg in the morning and at 7.5 mg in the evening of the same day.

[0197] Embodiment 11. The method of any one of embodiments 1-10, wherein the weekly dose of the AvPAL variant is administered subcutaneously.

[0198] Embodiment 12. The method of any one of embodiments 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 1 :6.

[0199] Embodiment 13. The method of any one of embodiments 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 1 :3.

[0200] Embodiment 14. The method of any one of embodiments 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 2:3.

[0201] Embodiment 15. The method of any one of embodiments 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 4:3.

[0202] Embodiment 16. The method of any one of embodiments 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 8:3.

[0203] Embodiment 17. The method of any one of embodiments 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 14:3.

[0204] Embodiment 18. The method of any one of embodiments 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 28:3.

[0205] Embodiment 19. The method of any one of embodiments 1-11, wherein the weekly dose of the AvPAL variant is relative to the weekly dose of MTX at a ratio of about 56:3.

[0206] Embodiment 20. The method of any one of embodiments 1-19, wherein the subject has not received a prior treatment with the AvPAL variant.

[0207] Embodiment 21. The method of any one of embodiments 1-19, wherein the subject has received a prior treatment with the AvPAL variant.

[0208] Embodiment 22. The method of embodiment 21, wherein after the prior treatment with the AvPAL variant the subject has uncontrolled blood phenylalanine (Phe) concentration greater than about 600 micromol / L, and wherein the prior treatment with the AvPAL variant has lasted for more than about 24 consecutive weeks.

[0209] Embodiment 23. The method of embodiment 21 or 22, wherein the subject has been in the prior treatment with the AvPAL variant at the weekly dose of embodiment 1, further wherein the dosage of the AvPAL variant in the prior treatment has been higher than about 20 mg per day.

[0210] Embodiment 24. The method of embodiment 23, wherein the subject in the prior treatment could not receive further dose escalation of the AvPAL variant.

[0211] Embodiment 25. The method of any one of embodiments 21-24, wherein the highest dosage of the AvPAL variant the subject received in the prior treatment has been between about 20 mg per day to about 40 mg per day.

[0212] Embodiment 26. The method of any one of embodiments 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 2 mg per week to about 10 mg per week.

[0213] Embodiment 27. The method of any one of embodiments 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 10 mg per week to about 20 mg per week.

[0214] Embodiment 28. The method of any one of embodiments 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 20 mg per week to about 40 mg per week.

[0215] Embodiment 29. The method of any one of embodiments 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 40 mg per week to about 70 mg per week.

[0216] Embodiment 30. The method of any one of embodiments 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 70 mg per week to about 140 mg per week.

[0217] Embodiment 31. The method of any one of embodiments 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 140 mg per week to about 280 mg per week.

[0218] Embodiment 32. The method of any one of embodiments 1-31, wherein the method further comprises administering to the subject folic acid, wherein the folic acid is administered orally about 1 mg per day.

[0219] Embodiment 33. The method of any one of embodiments 1-31, wherein the method further comprises administering to the subject folic acid, wherein the folic acid is administered orally about 2 mg per day.

[0220] Embodiment 34. The method of embodiment 32 or 33, wherein the administration of folic acid starts from the day when MTX is administered.

[0221] Embodiment 35. The method of any one of embodiments 1-34, wherein the AvPAL variant is administered once weekly.

[0222] Embodiment 36. The method of any one of embodiments 1-34, wherein the AvPAL variant is administered twice weekly.

[0223] Embodiment 37. The method of any one of embodiments 1-34, wherein the AvPAL variant is administered four times per week.

[0224] Embodiment 38. The method of any one of embodiments 1-34, wherein the AvPAL variant is administered seven times per week.

[0225] Embodiment 39. The method of any one of embodiments 1-34, wherein the AvPAL variant is administered daily.

[0226] Embodiment 40. The method of any one of embodiments 1-39, wherein the method comprises: (a) administering to the subject the AvPAL variant at an induction dosage in the range of about 0.1 mg per week to about 10 mg per week, followed by (b) administering to the subject the AvPAL variant at a titration dosage in the range of about 1 mg per week to about 200 mg per week, followed by (c) administering to the subject the AvPAL variant at a maintenance dosage in the range of about 20 mg per week to about 280 mg per week.

[0227] Embodiment 41. The method of embodiment 40, wherein the induction dosage is about 2.5 mg per week.

[0228] Embodiment 42. The method of embodiment 40 or 41, wherein the titration dosage is in the range of about 5 mg per week to about 70 mg per week.

[0229] Embodiment 43. The method of any one of embodiments 40 to 42, wherein the maintenance dosage is in the range of about 140 mg per week to about 280 mg per week.

[0230] Embodiment 44. The method of any one of embodiments 40 to 43, wherein the induction dosage is administered for a duration of between about 2 week and about 6 weeks, the titration dosage is administered for a duration of between about 3 weeks and about 8 weeks, and the maintenance dosage is administered for a duration of between about 20 weeks and about 45 weeks.

[0231] Embodiment 45. The method of embodiment 44, wherein the induction dosage is administered for a duration of about 4 weeks, the titration dosage is administered for a duration of about 5 weeks, and the maintenance dosage is administered for a duration of between about 24 weeks and 40 weeks.

[0232] Embodiment 46. The method of embodiment 1, wherein the weekly dose of MTX is administered for about 24 weeks.

[0233] Embodiment 47. The method of embodiment 1, wherein the weekly dose of MTX is administered for about 28 weeks.

[0234] Embodiment 48. The method of embodiment 47, wherein the administration of the weekly dose of MTX starts from four weeks before the administration of the AvPAL variant.

[0235] Embodiment 49. The method of any one of embodiments 1-48, wherein the weekly dose of the AvPAL variant is administered for about 24 weeks.

[0236] Embodiment 50. The method of embodiment 46, wherein the administration of the weekly dose of MTX is discontinued at about 24 weeks and only the administration of the AvPAL variant continues for about another 24 weeks.

[0237] Embodiment 51. The method of embodiment 47 or 48, wherein the administration of the weekly dose of MTX is discontinued at about 28 weeks and only the administration of the AvPAL variant continues for about another 24 weeks.

[0238] Embodiment 52. The method of any one of embodiments 1 to 51, wherein the method further comprises assessing the blood phenylalanine concentration prior to administering MTX.

[0239] Embodiment 53. The method of any one of embodiments 40 to 52, wherein the method further comprises assessing the blood phenylalanine concentration prior to administering the induction dosage of the AvPAL variant.

[0240] Embodiment 54. The method of embodiment any one of embodiments 40 to 53, wherein the method further comprises assessing the blood phenylalanine concentration after administration of one or more induction dosages, titration dosages, maintenance dosages, and / or extension dosages.

[0241] Embodiment 55. The method of embodiment 54, wherein the method further comprises adjusting the dosage based on the blood phenylalanine concentration.

[0242] Embodiment 56. The method of embodiment 55, wherein the dosage is adjusted to attain a blood phenylalanine concentration of below about 600 pM.

[0243] Embodiment 57. The method of embodiment 55, wherein the dosage is adjusted to attain a blood phenylalanine concentration of below about 360 pM.

[0244] Embodiment 58. The method of embodiment 56, wherein the maintenance dosage is increased if blood phenylalanine concentration is greater than about 360 pM.

[0245] Embodiment 59. The method of any one of embodiments 1 to 58, wherein the subject has phenylketonuria (PKU).

[0246] Embodiment 60. The method of any one of embodiments 1 to 59, wherein the AvPAL variant is pegylated.

[0247] Embodiment 61. The method of embodiment 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of at least 1.6 polyethylene glycol per lysine residue of AvPAL variant.

[0248] Embodiment 62. The method of embodiment 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of at least 2.4 polyethylene glycol per lysine residue of AvPAL variant.

[0249] Embodiment 63. The method of embodiment 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 3 polyethylene glycol per lysine residue of AvPAL variant.

[0250] Embodiment 64. The method of embodiment 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 5 polyethylene glycol per lysine residue of AvPAL variant.

[0251] Embodiment 65. The method of embodiment 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 6 polyethylene glycol per lysine residue of AvPAL variant.

[0252] Embodiment 66. The method of embodiment 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 7 polyethylene glycol per lysine residue of AvPAL variant.

[0253] Embodiment 67. The method of embodiment 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 8 polyethylene glycol per lysine residue of AvPAL variant.

[0254] Embodiment 68. The method of embodiment 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 9 polyethylene glycol per lysine residue of AvPAL variant.

[0255] Embodiment 69. The method of any one of embodiments 1 to 68, wherein the AvPAL variant is administered as a formulation comprising a pharmaceutically acceptable carrier comprising a stabilizer.

[0256] Embodiment 70. The method of embodiment 69, wherein the stabilizer is L-phenylalanine or structural analog thereof.

[0257] Embodiment 71. The method of embodiment 70, wherein the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid and benzoic acid.

[0258] Embodiment 72. The method of embodiment 71, wherein the stabilizer is transcinnamic acid.

[0259] Embodiment 73. The method of embodiment 72, wherein the formulation further comprises sodium chloride, and tromethamine and tromethamine hydrochloride.

[0260] Embodiment 74. The method of any one of embodiments 1-73, wherein an immune response of the subject to the AvPAL variant is suppressed.

[0261] Embodiment 75. The method of any one of embodiments 1-74, wherein tolerability of the subject to the AvPAL variant is improved.

[0262] Embodiment 76. The method of any one of embodiments 1-75, wherein efficacy of the AvPAL variant in the subject is improved.

[0263] Embodiment 77. The method of any one of embodiments 1-76, wherein the AvPAL variant is pegvaliase.

[0264] Embodiment 78. The method of any one of embodiments 1-77, wherein the subject is between 18 and 65 years of age.

[0265] Embodiment 79. The method of any one of embodiments 1-78, wherein the subject is a human subject.

[0266] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the descriptions in the Experimental section are intended to illustrate but not limit the scope of invention described in the claims.EXAMPLES EXAMPLE 1 : Clinical Evaluation for Immune Modulation during Treatment with AvPAL Variant in Adults with Phenylketonuria (PKU)

[0267] Studies are performed to investigate the immune modulation during a treatment with a PEGylated form of an AvPAL polypeptide variant (e.g., Palynziq® (pegvaliase or Palynziq), which has serine substitution of the cysteine residues at positions 503 and 565 (SEQ ID NO:4)) on adult PKU patients aged 18 to 65 years.

[0268] Methods of preparing pegylated AvPAL double cysteine mutant AvPAL_C565SC503S are described in co-owned US7534595B2, which is herein incorporated by reference in its entirety. The pegylated AvPAL double cysteine mutant AvPAL_C565SC503S was prepared as described in Example 10 of US7534595B2.

[0269] The present Study 165-401 is a Phase 4, open-label study designed to examine the concomitant use of methotrexate (MTX) with Palynziq®. The overall objective of the study is to evaluate the ability of MTX, when co-administered with Palynziq®, to suppress immune responses to Palynziq® and thus improve the tolerability and efficacy of Palynziq® in adultswith phenylketonuria (PKU). An overview of the 165-401 study design, study periods, and study duration is presented in FIG. 1.Study Rationale

[0270] Palynziq® (pegvaliase or Palynziq) is a subcutaneous (SC) enzyme substitution treatment to reduce blood phenylalanine (Phe) levels in adult patients with PKU. It is a daily treatment that are administered in an Induction / Titration / Manousakis (I / T / M) regimen described in Table 1. During pegvaliase clinical development, this dosing regimen was found to help mitigate the onset and severity of hypersensitivity reactions while substantially reducing blood Phe concentrations. Previous clinical studies have also suggested that the primary clearance mechanism of pegvaliase from plasma is via formation of circulating immune complexes (CICs) leading to complement activation and removal of the drug by phagocytosis, indicating pegvaliase plasma exposure was driven by immune response. The antibody-mediated and complement-mediated immune responses to pegvaliase lead to increased pegvaliase clearance (interfering with efficacy) and to hypersensitivity reactions (leading to poor tolerability). It is hypothesized that co-administration of methotrexate (MTX) with Palynziq improves the clinical effects associated with the immune response and enables improved tolerability and efficacy. In this clinical trial, immune modulation (MTX) is to be administered concurrently with Palynziq to adults with PKU.Table 1: Palynziq Dosing RegimenTreatment Palynziq Dosage Duration*Induction 2.5 mg once weekly 4 weeksTitration 2.5 mg twice weekly 1 week10 mg once weekly 1 week10 mg twice weekly 1 week10 mg four times per week 1 week10 mg once daily 1 weekMaintenance 20 mg once daily 24 weeksMaximumf 40 mg once daily 16 weeksj* Additional time may be required prior to each dosage escalation based on patient tolerability.f Individualize treatment to the lowest effective and tolerated dosage. Consider increasing to a maximum of 40 mg once daily in patients who have not achieved a response with 20 mg once daily continuous treatment for at least 24 weeks.{ Discontinue Palynziq treatment in patients who have not achieved a response after 16 weeks of continuous treatment with the maximum dosage of 40 mg once daily.

[0271] Based on the Palynziq clinical experience to date, patients who have never been treated with Palynziq (treatment naive) and patients who have completed a minimum of 24 consecutive weeks of Palynziq treatment with persistent uncontrolled blood Phe >600 pmol / L, who are on a current dose of J>20 mg / day and are unable to further dose escalate (treatment experienced), may benefit from this immune modulation approach to decrease the time to reach efficacy and increase tolerability, improving clinical outcomes during Palynziq (pegvaliase) treatment and optimizing the therapeutic experience.

[0272] Patients naive to Palynziq treatment were chosen as an appropriate study population for inclusion in this study as this allows MTX administration to align with the overall timing of the peak Palynziq immune response as demonstrated in pegvaliase clinical trials. It is hypothesized that MTX co-administration during this period may blunt safety and PK impacts associated with the early immune response (increase in HAEs and increased Palynziq clearance). In addition, Palynziq experienced patients who have completed a minimum of 24 consecutive weeks of treatment with persistent uncontrolled blood Phe >600 pmol / L, who are on a current dose of J>20 mg / day and are unable to further dose escalate were chosen as an appropriate second study cohort. Treatment experienced patients may benefit from modulation of immune response for the same reasons as the treatment naive group given the prolonged treatment time needed to achieve blood Phe reduction and the difficulty in dose escalation that is likely associated with a more robust immune response.

[0273] Study 165-401 was designed to limit the period of MTX exposure due to the multiple potential toxicities associated with MTX. The Follow-up Period determines the durability of impact of early immune tolerance induction therapy after treatment with MTX is withdrawn.Study Objectives

[0274] Primary Objectives. The primary objective is to evaluate the ability of MTX to modulate the immune response to Palynziq in adults with PKU. In addition, the primary objective is to evaluate blood Phe response in adults with PKU co-administered Palynziq with MTX for 6 months.

[0275] Secondary Objectives. The secondary objective is to evaluate the safety of coadministration of Palynziq with MTX in adults with PKU. Further, the secondary objective is to characterize the pharmacokinetics (PK) of Palynziq during co-administration with MTX in adults with PKU.

[0276] Exploratory Objectives. The exploratory objectives are to evaluate formation of circulating immune complex (CIC) and activation of complement pathway.Study Endpoints

[0277] Primary Endpoints. The primary endpoint includes detections of anti-PEG IgG, IgM, anti-PAL IgG, IgM levels at Weeks 5, 9, 25, and 49 of the combination treatment. The primary endpoint also includes the change from baseline in blood Phe levels at Weeks 25 and 49. Baseline blood Phe is based on the average of the 2 blood Phe values at screening and the blood Phe on Day 1.

[0278] Secondary Endpoints. The secondary endpoints include incidence of treatment-emergent adverse events (TEAEs) and detection of trough Palynziq plasma PK. A TEAE is defined as any AE that newly appeared, increased in frequency, or worsened in severity following initiation of the study drug (MTX or Palynziq) administration.

[0279] Exploratory Endpoints. The exploratory endpoints include change from baseline in IgG-C3d CIC, C3, C4 levels at Weeks 9, 25, and 49. Baseline is defined as the last nonmissing measurement prior to the first dose of MTX.Study Design

[0280] Population. The Phase 4, open-label study collects data from up to 12 participants with phenylketonuria (PKU) who are A l 8 and A 65 years of age. They are enrolled into the study across 2 cohorts, Cohort A and Cohort B. Cohort A includes individuals who have never been treated with Palynziq (Palynziq naive). For Palynziq naive participants (Cohort A), Palynziq is initiated during the study per the induction / titration / maintenance (I / T / M) dosing regimen indicated in the Palynziq USPI and as prescribed by their treating physician. Palynziq administration is initiated in the clinic on Day 1 and continues per the USPI and per the participant’s treating physician throughout the study. Cohort B includes individuals who have uncontrolled blood Phe >600 pmol / L after A 24 consecutive weeks of treatment with Palynziq, are on a current dose of A 20 mg / day, and are unable to further dose escalate (Palynziq experienced). For Palynziq experienced participants (Cohort B), Palynziq dosing should be stable and remain as prescribed by their treating physician prior to study entry and throughout the Screening Period. Participants in Cohort B then continue with Palynziq for the duration of the study per the USPI and per their treating physician.

[0281] Study Assessments and Procedures. The decision to initiate or continue Palynziq must be made independent of participation in this study. To ensure a comprehensive data set for analysis, participants who withdraw early from the study may be replaced. All participants are administered MTX at an oral dose of 15 mg / week concurrent with Palynziq. Cohort A participants have a 4-week Screening Period, followed by a 4-week MTX Tolerability Period during which only MTX is administered. If MTX is tolerated during the MTX Tolerability Period, the participant then enter the 24-week Combination Treatment Period during which MTX + Palynziq are administered concurrently. Cohort B participants have a 4-week Screening Period. Participants then have 24 weeks of concurrent MTX + Palynziq treatment (Combination Treatment Period), in which participants are assessed for MTX tolerability during the first 4 weeks of MTX + Palynziq (MTX Tolerability Period) and then receive another 20 weeks of MTX + Palynziq. After completion of Week 24 (Combination Treatment Period), MTX is discontinued for both cohorts and Palynziq only continues for another 24 weeks (to Week 49; Follow-up Period).

[0282] MTX is to be sourced and dispensed by local pharmacies; a prescription must be in place during the Screening Period, following participant consent to participate in the study, so as to allow MTX to be administered in the MTX Tolerability Period and throughout the Combination Treatment Period. Participants are assessed for safety and MTX tolerability throughout the study. If a participant in either Cohort A or B is unable to tolerate MTX, the participant is discontinued from MTX treatment.

[0283] Participants also take folic acid 1 mg orally every day beginning on the first day of MTX treatment and continuing until the Week 25 Visit (i.e., the end of the Combination Treatment Period) as folate deficiency may increase MTX adverse reactions. Folic acid is to be supplied by local pharmacies.

[0284] Participants are required to premedicate with an Hl antagonist, an H2 antagonist, and / or an antipyretic approximately 2 to 3 hours prior to each dose of Palynziq until participants are able to achieve blood Phe <600 pmol / L while on a stable dose of Palynziq (the maintenance phase). Premedication may be considered during the maintenance phase at the investigator’s discretion. When a nonsteroidal anti-inflammatory drug (NSAID) is administered as a premedication, it should be given with food.

[0285] Participants should maintain dietary Phe intake consistent with their baseline diet at enrollment for the duration of the study (including during the Screening Period) unlessclinically indicated, with all participants following their individual dietary management plan as directed by the investigator (or delegated dietician) to support individual nutritional needs during study participation.

[0286] Study procedures and their timing are summarized in the Schedule of Activities (SoA) as shown in FIG. 2 (Cohort A) and FIG. 3 (Cohort B).Study Treatment

[0287] The study treatments administered during this study are Palynziq + MTX (also referred to collectively as study drug) as displayed in FIG. 1. The time and day on which participants take their weekly dose of MTX and dose of Palynziq should be kept consistent throughout the study, and MTX dosing should be separated from Palynziq dosing by at least 60 minutes. On days when MTX is to be administered on the same day as Palynziq, there is no restriction with regard to the order of administration.

[0288] Palynziq must be administered at an individualized dose and dosing frequency as prescribed by the participant’s treating clinician. All participants receiving Palynziq must be premedicated with an Hl antagonist, and an H2 antagonist, and / or an antipyretic approximately 2 to 3 hours prior to each dose until they reach the maintenance phase, defined as when participants achieve blood Phe600 pmol / L while they are on a stable dose of Palynziq. Premedication may be considered during the maintenance phase at the investigator’s discretion, e.g., approximately 2 to 3 hours prior to study drug for 1 week upon reintroduction of study drug, upon resolution of an AE, following any dose interruption of4 days. Participants may also be premedicated at any other time in the study per investigator discretion.

[0289] MTX should be administered as a single oral dose of 15 mg / week as outlined in FIG. 1. Participants also take folic acid 1 mg orally every day beginning on the first day of MTX treatment and continuing until the Week 25 Visit (i.e., the end of the Combination Treatment Period) as folate deficiency may increase MTX adverse reactions. During the first 4 weeks of MTX treatment (MTX Tolerability Period), if minor clinical symptoms emerge (mild stomatitis, mild GI discomfort), the investigator may increase the folic acid dose (e.g., 2 mg daily) or recommend a divided dose of MTX (e.g., 3 tablets of 2.5 mg in the morning and evening on the day of dosing). If symptoms improve, the participant is not considered a screen failure on the basis of that symptom. After the first 4 weeks of dosing with MTX,MTX dose guidance based on new laboratory findings or new symptoms is as outlined in Table 2.Table 2: MTX Dose Modifications: Abnormal Laboratory Values Laboratory Parameter Value MTX Dose Change 3.0 x 109 / L to ~ <3.5 x 109 / L Decrease to 10 mg WBC<3.0 x 109 / L Temporarily discontinue Platelets <50 x 109 / L Temporarily discontinue Hematocrit <27% Temporarily discontinue Between 1.5 to ~ 2 x ULN Decrease to 10 mg AST or ALT>2 x ULN Temporarily discontinue eGFR <30 ml / min / 1.73 m2Temporarily discontinue New clinically importantYes Temporarily discontinue symptoms / signs?aALT, alanine aminotransferase; AST, aspartate aminotransferase; eGFR, estimated glomerular filtration rate; ULN, upper limit of normal; WBC, white blood cell count.aNew clinically important symptoms or important medical events include rash or oral ulceration; persistent nausea, vomiting and diarrhea; new or increasing dyspnea or dry cough, or unexplained cough with fever; severe sore throat, abnormal bruising; severe headaches, fatigue, and problems concentrating; any other important medical events that might increase methotrexate toxicity or predispose to new or worsening infection (e.g., undergoing surgery, hospitalization, being treated with antibiotics, having a clinical infection, developing new clinically significant pericardial / pleural effusion or ascites).

[0290] At this stage, if minor clinical symptoms emerge (mild stomatitis, mild GI discomfort), the investigator may increase the folic acid dose to 2 mg daily or recommend a divided dose of MTX (e.g., 3 tablets of 2.5 mg in the morning and evening on the day of dosing) and monitor for symptom resolution. Provided below is guidance for increasing MTX back towards 15 mg / week after dose reduction, based on improvement or resolution of abnormal liver enzymes (>2 x ULN): (1) when liver enzymes return to values1.5 x ULN, increase MTX dose by 2.5 mg and reassess in 2 weeks; or (2) if liver enzymes remain ^ 1.5 x ULN, increase MTX dose by 2.5 mg and reassess in 2 weeks. Improvement of other laboratory abnormalities potentially attributed to MTX may also warrant titration back up to 15 mg weekly, based on investigator judgement and in discussion with the medical monitor.Continued Access to Study Drug after Study Completion

[0291] Palynziq prescription is independent of study participation. Study participants may continue Palynziq treatment as prescribed by their treating physician following completion ofthe study (or early termination). MTX is not provided to participants following completion of the Combination Treatment Period. Modifications to Palynziq dosing are per the USPI and the participant’s treating physician. Modifications to MTX are not permitted during the MTX Tolerability Period. If participants are unable to tolerate the weekly 15 mg MTX dosing during the MTX Tolerability Period, they should be discontinued from MTX.Study Assessments and Procedures

[0292] Assessments and procedures during the Screening Period are outlined in FIG. 2 (Cohort A) and FIG. 3 (Cohort B). In brief, potential participants enter the Screening Period, which lasts 4 weeks and includes 2 assessments of blood Phe concentration 5 days apart. The second screening sample should be taken and shipped by Day -49 for Cohort A and Day -15 for Cohort B. Assessment of dietary intake including protein and Phe should be performed for the 3 days preceding each of the blood Phe assessments. Following confirmation that each of the 2 blood Phe results are > 600 pmol / L and confirmation that all other eligibility criteria have been met, participants may proceed onto the MTX Tolerability Period, which is 4 weeks in duration. During the MTX Tolerability Period, participants take oral MTX (15 mg / week) as a single dose each week, either alone (Cohort A) or in conjunction with ongoing Palynziq (Cohort B). The participant is monitored weekly (remote visits via telephone call / video call) to assess compliance and side effects. Additionally, clinical safety laboratory testing is performed at Week -1 for Cohort A and at Week 4 for Cohort B. If the participant has tolerated MTX treatment at the end of the MTX Tolerability Period and continues to meet all other eligibility criteria, the participant may proceed to start MTX + Palynziq on Day 1 (Cohort A) or continue MTX + Palynziq on Day 29 (Cohort B).

[0293] Further assessments and procedures are outlined in FIG. 2 (Cohort A) and FIG. 3 (Cohort B), including efficacy and immunogenicity assessments, safety assessments, adverse events and serious adverse events, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity assessments.

[0294] Efficacy and Immunogenicity Assessments. Planned timepoints for all efficacy and immunogenicity assessments are provided in the SoA. Immunogenicity assessments, which are primary endpoints of this study, are discussed below in immunogenicity assessments. The primary efficacy endpoint is change in blood Phe concentration from baseline (defined as the average of the 2 blood Phe measurements at screening and the measurement on Day 1) to Week 24 and Week 48. Blood samples for blood Phe analysis are to be drawn according to the SoA. Monitoring of blood Phe levels is the cornerstone ofdiagnosis and treatment of PKU patients. Guidelines for the treatment of PKU are based on blood Phe levels (Vockley et al., Genet Med. 16:1 13 (2014); van Wegberg et al., Orphanet J Rare Dis. 12:162 (2017)), and clinicians and patients use blood Phe concentration to assess and monitor PKU disease progression and control. Samples for blood Phe concentration are analyzed by a central laboratory.

[0295] Safety Assessments. Planned time points for all safety assessments are provided in the SoA. Study participants undergo physical examinations, which include assessment of height and weight, throughout the study. A full physical examination includes, at a minimum, general appearance (head, eyes, ears, nose, and throat), cardiovascular, dermatologic, lymphatic, respiratory, gastrointestinal (GI), musculoskeletal, and neurologic systems. A brief physical examination includes general appearance, cardiovascular, dermatologic, respiratory, gastrointestinal (GI), musculoskeletal, and neurologic assessments. Brief physical examinations may be performed at the discretion of the investigator based on the participant’s clinical condition. Investigators should pay special attention to clinical signs related to previous serious illnesses, as well as clinical signs of MTX toxicity. Physical examinations (full or brief) should be performed at the timepoints indicated in the SoA.Height and weight are also measured and recorded at the timepoints indicated in the SoA.

[0296] Change from baseline vital signs is assessed during the study to monitor participant safety. Vital signs include seated systolic blood pressure (SBP) and diastolic BP, heart rate, respiration rate, and temperature, recorded before blood collection for laboratory tests if required. Any clinically significant change in vital signs is recorded as an AE. Blood pressure and pulse measurements are assessed (seated) with a completely automated device. Manual techniques are used only if an automated device is not available. Blood pressure and pulse measurements should be preceded by at least 5 minutes of rest for the participant in a quiet setting without distractions (e.g., television, cell phones). Vital signs (to be taken before blood collection for laboratory tests) consist of 1 pulse and 3 BP measurements (3 consecutive BP readings are recorded at intervals of at least 1 minute). The average of the 3 BP readings are recorded on the eCRF.

[0297] If deemed necessary by the investigator, a chest X-ray should be performed for participants who have not had a chest X-ray within 2 years prior to screening. The chest X-ray is read locally by a radiologist. Data from a chest X-ray performed within 2 years prior to enrollment may be used provided the required data fields are available. Additional chest X-rays should be obtained only if clinically indicated during the study.

[0298] Change from baseline laboratory and urinalysis test values is assessed during the study to monitor participant safety. The timing and frequency are outlined in the SoA. The investigator must review each laboratory and urinalysis report and document this review. The laboratory and urinalysis reports must be filed with the source documents. If known, the underlying diagnosis associated with abnormal clinical laboratory test results that are considered clinically significant by the investigator should be recorded on the AE eCRF. Clinically significant abnormal laboratory or urinalysis findings are those which are not associated with the underlying disease, unless judged by the investigator to be more severe than expected for the participant's condition. All laboratory and urinalysis tests with values considered clinically significantly abnormal during participation in the study or within 30 days after the last dose of study treatment should be repeated until the values return to normal or baseline or are no longer considered clinically significant by the investigator or medical monitor. In the event of elevated urinary protein on a test result, a repeat urinalysis should be performed. This repeat urine sample must be performed in the morning at the first or second morning void to allow for accurate test results and may be performed by a home healthcare nurse. Participants with a confirmed urine / albumin creatinine ratio of100 mg / g should be referred to a nephrologist for consultation if results are within normal range at baseline. Participants who had elevated results at baseline followed by a confirmed subsequent increase of 100 mg / g from baseline should also be referred to a nephrologist for consultation. If clinically significant values do not return to normal / baseline within a period of time judged reasonable by the investigator, the etiology should be identified and the sponsor notified. All protocol-required laboratory and urinalysis assessments must be conducted in accordance with the laboratory manual and the SoA.

[0299] In addition to the standard clinical safety laboratory test panels, the safety laboratory assessments on HIV antibody, Hepatitis B surface antigen, and Hepatitis C antibody are performed as part of this study at screening as indicated in the SoA.

[0300] Methotrexate can cause fetal death, embryotoxicity, abortion, or teratogenic effects when administered to a pregnant woman. Women of childbearing potential should not be started on MTX until pregnancy has been excluded and should be counselled on the serious risk to the fetus should they become pregnant while undergoing treatment. In addition to contraception requirements, females of child-bearing potential are required to have urine pregnancy tests performed at the timepoints specified in the SoA.

[0301] Pharmacokinetics. Whole blood samples are collected once every 4 weeks predose for measurement of trough plasma concentrations of Palynziq as specified in the SoA. Blood for PK samples (pre-dose) is collected within 2 hours prior to Palynziq administration. Plasma trough concentrations (Ctrough) of pegvaliase are reported based on once every 4 weeks pre-dose blood sample collection. Instructions for the collection and handling of biological samples are provided by the sponsor. The actual date and time (24-hour clock time) of each sample are recorded. Samples are used to evaluate the PK of Palynziq.Samples collected for analyses of plasma concentration may also be used to evaluate safety or efficacy aspects related to concerns arising during or after the study.

[0302] Pharmacodynamics. Planned timepoints for pharmacodynamic assessments are provided in the SoA. Blood Phe concentrations are measured every 2 weeks during the Combination Treatment Period and Follow-up Period. The primary efficacy endpoint is change in blood Phe concentration from baseline (defined as the average of the 2 blood Phe measurements at screening and the measurement on Day 1) to Week 25 and Week 49. Blood Phe and tyrosine are measured using validated assays.

[0303] Immunogenicity Assessments. A primary efficacy endpoint is change in immune analyte levels from baseline. Sampling for immunogenicity testing is performed routinely throughout the study to provide a comprehensive assessment of the immune response against Palynziq. Antibodies against Palynziq are evaluated in all participants according to the SoA. Samples are tested by the sponsor or sponsor's designee. Other analyses may be performed to verify the stability of antibodies and / or further characterize immunogenicity of Palynziq.

[0304] Numerous modifications and variations in the disclosure as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently only such limitations as appear in the appended claims should be placed on the disclosure.

Claims

CLAIMSWHAT IS CLAIMED:

1. A method for reducing blood phenylalanine concentration in a subject, the method comprising:(1) administering to the subject a weekly dose of a formulation comprising an AvPAL variant, wherein the AvPAL variant comprises an amino acid sequence of SEQ ID NO:4; and(2) administering to the subject a weekly dose of methotrexate (MTX) or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered concurrently.

3. The method of claim 1 or 2, wherein the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered in separate compositions.

4. The method of any one of claims 1-3, wherein the AvPAL variant and MTX or a pharmaceutically acceptable salt thereof are administered on the same day.

5. The method of any one of claims 1-4, wherein the dosage of MTX is about 15 mg per week.

6. The method of any one of claims 1-4, wherein the dosage of MTX is about 10 mg per week.

7. The method of any one of claims 1-6, wherein MTX or a pharmaceutically acceptable salt thereof is administered orally.

8. The method of any one of claims 1-7, wherein the dosage of MTX is administered in a single dose.

9. The method of any one of claims 1-7, wherein the dosage of MTX is administered in multiple divided doses within a day.

10. The method of claim 9, wherein the dosage of MTX is administered orally at 7.5 mg in the morning and at 7.5 mg in the evening of the same day.

11. The method of any one of claims 1-10, wherein the weekly dose of the AvPAL variant is administered subcutaneously.

12. The method of any one of claims 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 1 :6.

13. The method of any one of claims 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 1 :3.

14. The method of any one of claims 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 2:3.

15. The method of any one of claims 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 4:3.

16. The method of any one of claims 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 8:3.

17. The method of any one of claims 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 14:3.

18. The method of any one of claims 1-11, wherein the ratio of the weekly dose of the AvPAL variant to the weekly dose of MTX is about 28:3.

19. The method of any one of claims 1-11, wherein the weekly dose of the AvPAL variant is relative to the weekly dose of MTX at a ratio of about 56:3.

20. The method of any one of claims 1-19, wherein the subject has not received a prior treatment with the AvPAL variant.

21. The method of any one of claims 1-19, wherein the subject has received a prior treatment with the AvPAL variant.

22. The method of claim 21, wherein after the prior treatment with the AvPAL variant the subject has uncontrolled blood phenylalanine (Phe) concentration greater than about 600 micromol / L, and wherein the prior treatment with the AvPAL variant has lasted for more than about 24 consecutive weeks.

23. The method of claim 21 or 22, wherein the subject has been in the prior treatment with the AvPAL variant at the weekly dose of claim 1, further wherein the dosage of the AvPAL variant in the prior treatment has been higher than about 20 mg per day.

24. The method of claim 23, wherein the subject in the prior treatment could not receive further dose escalation of the AvPAL variant.

25. The method of any one of claims 21-24, wherein the highest dosage of the AvPAL variant the subject received in the prior treatment has been between about 20 mg per day to about 40 mg per day.

26. The method of any one of claims 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 2 mg per week to about 10 mg per week.

27. The method of any one of claims 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 10 mg per week to about 20 mg per week.

28. The method of any one of claims 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 20 mg per week to about 40 mg per week.

29. The method of any one of claims 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 40 mg per week to about 70 mg per week.

30. The method of any one of claims 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 70 mg per week to about 140 mg per week.

31. The method of any one of claims 1-11 or 20-25, wherein the dosage of the AvPAL variant is in the range of about 140 mg per week to about 280 mg per week.

32. The method of any one of claims 1-31, wherein the method further comprises administering to the subject folic acid, wherein the folic acid is administered orally about 1 mg per day.

33. The method of any one of claims 1-31, wherein the method further comprises administering to the subject folic acid, wherein the folic acid is administered orally about 2 mg per day.

34. The method of claim 32 or 33, wherein the administration of folic acid starts from the day when MTX is administered.

35. The method of any one of claims 1-34, wherein the AvPAL variant is administered once weekly.

36. The method of any one of claims 1-34, wherein the AvPAL variant is administered twice weekly.

37. The method of any one of claims 1-34, wherein the AvPAL variant is administered four times per week.

38. The method of any one of claims 1-34, wherein the AvPAL variant is administered seven times per week.

39. The method of any one of claims 1-34, wherein the AvPAL variant is administered daily.

40. The method of any one of claims 1-39, wherein the method comprises:a. administering to the subject the AvPAL variant at an induction dosage in the range of about 0.1 mg per week to about 10 mg per week, followed byb. administering to the subject the AvPAL variant at a titration dosage in the range of about 1 mg per week to about 200 mg per week, followed byc. administering to the subject the AvPAL variant at a maintenance dosage in the range of about 20 mg per week to about 280 mg per week.

41. The method of claim 40, wherein the induction dosage is about 2.5 mg per week.

42. The method of claim 40 or 41, wherein the titration dosage is in the range of about 5 mg per week to about 70 mg per week.

43. The method of any one of claims 40 to 42, wherein the maintenance dosage is in the range of about 140 mg per week to about 280 mg per week.

44. The method of any one of claims 40 to 43, wherein the induction dosage is administered for a duration of between about 2 week and about 6 weeks, the titration dosage is administered for a duration of between about 3 weeks and about 8 weeks, and the maintenance dosage is administered for a duration of between about 20 weeks and about 45 weeks.

45. The method of claim 44, wherein the induction dosage is administered for a duration of about 4 weeks, the titration dosage is administered for a duration of about 5 weeks, and the maintenance dosage is administered for a duration of between about 24 weeks and 40 weeks.

46. The method of claim 1, wherein the weekly dose of MTX is administered for about 24 weeks.

47. The method of claim 1, wherein the weekly dose of MTX is administered for about 28 weeks.

48. The method of claim 47, wherein the administration of the weekly dose of MTX starts from four weeks before the administration of the AvPAL variant.

49. The method of any one of claims 1-48, wherein the weekly dose of the AvPAL variant is administered for about 24 weeks.

50. The method of claim 46, wherein the administration of the weekly dose of MTX is discontinued at about 24 weeks and only the administration of the AvPAL variant continues for about another 24 weeks.

51. The method of claim 47 or 48, wherein the administration of the weekly dose of MTX is discontinued at about 28 weeks and only the administration of the AvPAL variant continues for about another 24 weeks.

52. The method of any one of claims 1 to 51, wherein the method further comprises assessing the blood phenylalanine concentration prior to administering MTX.

53. The method of any one of claims 40 to 52, wherein the method further comprises assessing the blood phenylalanine concentration prior to administering the induction dosage of the AvPAL variant.

54. The method of claim any one of claims 40 to 53, wherein the method further comprises assessing the blood phenylalanine concentration after administration of one or more induction dosages, titration dosages, maintenance dosages, and / or extension dosages.

55. The method of claim 54, wherein the method further comprises adjusting the dosage based on the blood phenylalanine concentration.

56. The method of claim 55, wherein the dosage is adjusted to attain a blood phenylalanine concentration of below about 600 pM.

57. The method of claim 55, wherein the dosage is adjusted to attain a blood phenylalanine concentration of below about 360 pM.

58. The method of claim 56, wherein the maintenance dosage is increased if blood phenylalanine concentration is greater than about 360 pM.

59. The method of any one of claims 1 to 58, wherein the subject has phenylketonuria (PKU).

60. The method of any one of claims 1 to 59, wherein the AvPAL variant is pegylated.

61. The method of claim 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of at least 1.6 polyethylene glycol per lysine residue of AvPAL variant.

62. The method of claim 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of at least 2.4 polyethylene glycol per lysine residue of AvPAL variant.

63. The method of claim 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 3 polyethylene glycol per lysine residue of AvPAL variant.

64. The method of claim 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 5 polyethylene glycol per lysine residue of AvPAL variant.

65. The method of claim 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 6 polyethylene glycol per lysine residue of AvPAL variant.

66. The method of claim 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 7 polyethylene glycol per lysine residue of AvPAL variant.

67. The method of claim 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 8 polyethylene glycol per lysine residue of AvPAL variant.

68. The method of claim 60, wherein said pegylation is achieved by reacting the AvPAL variant with NHS-activated polyethylene glycol at a ratio of 9 polyethylene glycol per lysine residue of AvPAL variant.

69. The method of any one of claims 1 to 68, wherein the AvPAL variant is administered as a formulation comprising a pharmaceutically acceptable carrier comprising a stabilizer.

70. The method of claim 69, wherein the stabilizer is L-phenyl alanine or structural analog thereof.

71. The method of claim 70, wherein the stabilizer is selected from the group consisting of L-phenylalanine, trans-cinnamic acid and benzoic acid.

72. The method of claim 71, wherein the stabilizer is trans-cinnamic acid.

73. The method of claim 72, wherein the formulation further comprises sodium chloride, and tromethamine and tromethamine hydrochloride.

74. The method of any one of claims 1-73, wherein an immune response of the subject to the AvPAL variant is suppressed.

75. The method of any one of claims 1-74, wherein tolerability of the subject to the AvPAL variant is improved.

76. The method of any one of claims 1-75, wherein efficacy of the AvPAL variant in the subject is improved.

77. The method of any one of claims 1-76, wherein the AvPAL variant is pegvaliase.

78. The method of any one of claims 1-77, wherein the subject is between 18 and 65 years of age.

79. The method of any one of claims 1-78, wherein the subject is a human subject.

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