Study 165-401 is a Phase 4, open-label study designed to examine the concomitant use of methotrexate (MTX) to suppress immune responses to Palynziq and improve tolerability and efficacy in adults with PKU.

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 adults with phenylketonuria (PKU). Up to 12 participants will be enrolled into the study across 2 cohorts, with a target enrollment of 6 participants into each cohort:

• Cohort A: Individuals with uncontrolled blood phenylalanine (Phe) > 600 μmol/L on existing management who have never been treated with Palynziq (Palynziq naïve)
• Cohort B: Individuals who have uncontrolled blood Phe > 600 μmol/L after ≥ 24 consecutive weeks of treatment with Palynziq, are on a current dose of≥ 20mg/day, and are unable to further dose escalate (Palynziq experienced)

Palynziq will be initiated (Cohort A) or will continue (Cohort B) as indicated in the Palynziq United States Prescribing Information (USPI) and as prescribed by the participant's treating physician. The decision to initiate or continue Palynziq must be made independently of participation in this study. Palynziq and MTX will not be supplied by BioMarin as part of participation in this study.

All participants will be administered MTX at an oral dose of 15 mg/week concurrent with Palynziq:

• Cohort A participants will have a 4-week Screening Period, followed by a 4-week MTX Tolerability Period during which only MTX will be administered. If MTX is tolerated during the MTX Tolerability Period, the participant will then enter the 24-week Combination Treatment Period during which MTX + Palynziq will be administered concurrently.
• Cohort B participants will have a 4-week Screening Period. Participants will then have 24 weeks of concurrent MTX + Palynziq treatment (Combination Treatment Period): participants will be 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 will be discontinued for both cohorts and Palynziq only will continue for another 24 weeks (to Week 49; Follow-up Period).

Participants will be assessed for safety and MTX tolerability. If a participant in either Cohort A or B is unable to tolerate MTX, the participant will be discontinued from MTX treatment. Participants who discontinue from MTX 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

Inclusion Criteria:

• Adults between 18 and 65 years old
• Have a confirmed diagnosis of phenylketonuria (PKU)
• Are in generally good health based on medical evaluation
• Are willing and medically eligible to receive Palynziq and methotrexate (MTX) Cohort A: Have never taken Palynziq before and are willing to start it during the study Cohort B: Have blood > 600 μmol/L after taking Palynziq for at least 24 weeks, are on a daily dose of at least 20mg and unable to increase the dose further
• Agree to use required contraception if they or their partner could become pregnant
• Are willing to carry two epinephrine devices at all times during Palynziq treatment

Exclusion Criteria:

• Pregnant, breastfeeding, planning to become pregnant, planning to father a child, or not using effective birth control if applicable
• Have a known severe allergy or hypersensitivity reaction to methotrexate (MTX), Palynziq, or other PEG-containing medications
• Have a serious active infection or a history of severe or recurrent infections
• Have significant medical conditions that may affect safety or participation (such as serious heart, lung, liver, kidney, immune, neurological, psychiatric, or cancer-related conditions)
• Have a history of substance or alcohol abuse within the past 12 months
• Have had an organ transplant or are taking chronic immunosuppressive medications
• Are currently taking medications that are not allowed in the study, including other PKU treatments besides Palynziq
• Are using, or plan to use, injectable PEG-containing medications other than Palynziq during the study
• Have major surgery planned during the study participation period
• Are currently participating in another clinical study involving Palynziq
• In the opinion of the study doctor, are not a suitable candidate for the study or may have difficulty complying with study requirements