A phase 1 dose-escalation and -expansion study of AFN0328, a novel mRNA-based immunotherapy, in patients with HPV16/18-associated high-grade squamous intraepithelial lesions.

5544 Background: Persistent infection with high-risk human papillomavirus (HPV), particularly HPV16 and HPV18, drives the development of high-grade squamous intraepithelial lesions (HSIL), an obligate precancerous condition. The standard care of cervical HSIL requires either surgical excision (LEEP or cold-knife conization), or in some circumstance, watchful waiting. AFN0328 is a novel mRNA-based immunotherapy encoding HPV16/18-related antigens, designed to elicit antigen-specific immune responses and interrupt viral antigen-driven disease progression. Methods: Safety, tolerability and preliminary efficacy of AFN0328 were assessed in patients with HPV16- and/or HPV18-associated HSIL (CIN2/3) in an ongoing, multicenter, open-label Phase 1 trial. The study comprised two sequential parts: Part 1 employed a classic 3+3 dose-escalation design to assess safety and determine the maximum tolerated dose (MTD); Part 2 is a dose-expansion cohort at selected doses (150 μg and 300 μg) to further characterize safety, tolerability, and preliminary efficacy. AFN0328 was administered intramuscularly at 0, 4, and 12 weeks. The primary endpoints included safety and tolerability (dose-limiting toxicities within 28 days post-first dose, adverse events graded per CTCAE v5.0). For Part 2, the efficacy endpoints included viral clearance rate at Week 24 and Week 36, and histopathological regression at Week 36. Secondary endpoints included pharmacokinetic parameters, HPV16/18-specific IgG antibody titers, and pharmacodynamic markers (cellular immune responses and serum cytokines). Results: As of December 29, 2025, 23 patients had received at least one dose of AFN0328. No DLTs, serious adverse events, or grade ≥3 treatment-related adverse events (TRAE) were observed. PK analyses of first and last doses showed that circulating mRNA encoding HPV-related antigens peaked within 48 hours and declined gradually, with detectable levels persisting for up to at least 4 weeks across dose levels. All evaluable patients developed specific IgG responses and viral clearance was observed in evaluable patients during follow-up. Conclusions: AFN0328 demonstrated favorable safety and tolerability with robust immune responses in HPV16/18-associated HSIL, supporting further clinical development as an immunotherapy for HPV-driven precancerous disease. Clinical trial information: CTR20244013. Summary of safety and immunogenicity outcomes. Outcome Result Patients treated 23 Dose-limiting toxicities 0 Grade ≥3 treatment-related AEs 0 Most common TRAEs Injection-site reactions, fever Specific IgG response rate 100%* Observed viral clearance Yes* *Observed in evaluable patients during interim follow-up.