Phase 2 Evaluation of RLY-2608 in PIK3CA-Related Overgrowth Spectrum and PIK3CA Mutation-Driven Malformations in Adult and Pediatric Populations

The primary objective of this Phase 2 study is to determine the recommended Phase 2 dose (**RP2D**) for Groups 1, 2, and 3, and to evaluate the safety and tolerability of **RLY-2608** in patients with **PIK3CA Related Overgrowth Spectrum** and malformations driven by **PIK3CA mutation**. Additionally, the study aims to assess the efficacy of RLY-2608 compared to placebo, as measured by the volumetric response rate. These objectives are clinically relevant as they aim to establish an effective and safe dosing regimen for RLY-2608, which could potentially improve treatment outcomes for patients with these genetic conditions.

Secondary objectives include:

• Assessing the pharmacokinetics (PK) of RLY-2608.
• Characterizing the preliminary efficacy of RLY-2608.
• Evaluating the **PIK3CA** mutational status in lesional fluid and/or tissue.
• Assessing changes in clinical reported outcome assessments in participants treated with RLY-2608 compared to placebo.
• Evaluating changes in the sum of target lesion volume over time in participants treated with RLY-2608 compared to placebo.
• Assessing the duration of response in participants treated with RLY-2608 compared to placebo.
• Determining the safety and tolerability of RP2D RLY-2608.

The clinical trial involves a total of **248 participants** diagnosed with **PIK3CA Related Overgrowth Spectrum** and malformations driven by PIK3CA mutation. The study population includes both male and female subjects, encompassing a wide age range from children to adults, as indicated by the age range categories 2, 3, and 4. Participants were selected based on specific inclusion criteria, such as having a clinical diagnosis of PROS or a malformation within the ISSVA classifications, and possessing one or more documented activating PIK3CA mutations. The trial includes a vulnerable population, and participants are required to have a Lansky performance status of at least 50 for those under 16 years old, or a Karnofsky performance status of at least 50 for those 16 years and older. Lifestyle considerations such as diet, physical activity, or habits are not specified in the available data. The selection process ensures that participants agree to provide archived lesional fluid and/or tissue or are willing to undergo a pretreatment lesional biopsy to assess PIK3CA status, if considered safe and medically feasible.

The clinical trial is designed to evaluate the **safety** and **efficacy** of the investigational drug RLY-2608, a mutant-selective PI3Kα inhibitor, in individuals with PIK3CA Related Overgrowth Spectrum and Malformations Driven by PIK3CA Mutation. This study is structured as a randomized, double-blind, controlled trial, with an estimated duration extending until July 2031. The trial is divided into three parts, each with specific objectives. Parts 1 and 2 aim to determine the recommended Phase 2 dose (RP2D) for different groups and assess the safety and tolerability of RLY-2608. Part 3 focuses on evaluating the efficacy of RLY-2608 compared to placebo, as measured by the volumetric response rate at Week 24.

Participants will undergo a series of study visits, beginning with an inclusion (screening) visit to confirm eligibility based on criteria such as performance status and the presence of a PIK3CA mutation. Following successful screening, participants will be randomized to receive either RLY-2608 or placebo. Regular follow-up visits will be scheduled to monitor safety, collect pharmacokinetic data, and assess treatment response. These visits will include evaluations of adverse events, changes in vital signs, and laboratory tests. The end-of-study visit will occur after the final treatment cycle, where comprehensive assessments will be conducted to determine the overall impact of the treatment.

The expected length of participant involvement in the trial is approximately 12 months, with the possibility of early termination if significant adverse events occur or if the participant withdraws consent. The primary endpoints include determining the RP2D and assessing the overall safety profile of RLY-2608. Secondary endpoints involve measuring the percentage of participants with a volumetric response at specified intervals, changes in lesion volume, and duration of response. The trial will also evaluate plasma concentrations and pharmacokinetic parameters of RLY-2608. Participants' involvement may be discontinued if they experience unacceptable toxicity, non-compliance with study procedures, or if the study sponsor decides to terminate the trial for any reason.

The clinical trial involves the administration of the experimental medication **RLY-2608**, a mutant-selective PI3Kα inhibitor. **RLY-2608** is provided in a **capsule** form and is intended for oral administration. The active substance, also named **RLY-2608**, is of chemical origin. The medication is not formulated for pediatric use and is not classified as an orphan drug. The maximum treatment period for **RLY-2608** is 12 months. The specific dosage and frequency of administration are determined based on the study's objectives to establish the recommended Phase 2 dose (RP2D) and assess safety and tolerability. The trial aims to evaluate the efficacy of **RLY-2608** in participants with PIK3CA Related Overgrowth Spectrum and malformations driven by PIK3CA mutation.

In addition to the experimental treatment, a **placebo** is used as a comparator in Part 3 of the study to assess the efficacy of **RLY-2608**. The placebo is administered in a manner consistent with the experimental medication to ensure blinding and maintain the integrity of the study. Compliance with the dosing schedule is monitored throughout the trial to ensure adherence to the protocol and to accurately assess the outcomes related to the investigational product.

The efficacy of the investigational product **RLY-2608** will be assessed in a clinical trial involving participants with PIK3CA Related Overgrowth Spectrum and malformations driven by PIK3CA mutation. The primary endpoint for Part 3 of the study is the percentage of participants with a volumetric response at Week 24. Secondary endpoints include the percentage of participants with a volumetric response at Weeks 12 and 24, percent change from baseline in lesion volume by blinded independent central review (BICR), and duration of response, defined as the time from first documented response to the date of first documented disease progression or death due to any cause.

Additional secondary endpoints for Part 3 include the percentage of participants with improvement compared to baseline based on Patient Global Impression of Severity (PGI-S), Patient Global Impression of Change (PGI-C), and Investigator Global Impression of Change (IGIC) of **RLY-2608** compared to placebo. Changes from baseline will also be measured using age-appropriate PROMIS Profile, EQ-5D, EQ-5D-Y, or EQ-5D-Y Proxy. Pharmacokinetic parameters such as plasma concentrations, area under the concentration-time curve (AUC), Cmax, tmax, terminal half-life (t1/2), and total body clearance following oral dose (CL/F) will be evaluated for **RLY-2608**.