Abstract 3428: Mechanistic insights into IFx-Hu2.0 responses in the first human trial after prior anti-PD-1 therapy failure

Introduction: IFx-Hu2.0 is a plasmid encoding Streptococcal Emm55 protein for transfection into mammalian cells. The first-in-human clinical trial of IFx-Hu2.0 for unresectable melanoma showed that intra-tumoral IFx-Hu2.0 is safe. Melanoma patients responded to anti-PD1-based treatments after IFx-Hu2.0 treatment despite being previously refractory to treatment. Antibodies to Emm55 protein were seen in patient-derived plasma (Markowitz et al., Mol Cancer Ther., 2024). We now report on TCRseq and BCRseq from patients who received IFx-Hu2.0 on study. We also report on murine studies investigating how IFx-Hu2.0 produces a systemic immune response. Methods: Samples were available pre/post therapy from 6/6 patients who completed the dose-limiting toxicity period on trial. TCRseq from peripheral blood and BCRSeq from peripheral blood and tumors were measured on purified RNA using Adaptive Biotechnologies™ company. The B16 murine model was utilized to study IFx-Hu2.0 systemic effects. Thirteen mice had tumors implanted in both right and left flanks. The mice received weekly injections of IFx-Hu2.0 or empty plasmid vehicle control into the right flank, and tumor growth curves were constructed. Tumor growth curves were compared using the CGGC permutation test. To determine whether antibodies are developed against Emm55, we transfected an unrelated cell line (EL4) with the Emm55 plasmid (EL4-Emm55) and exposed it to murine serum obtained from mice bearing B16 tumor, treated with IFx-Hu2.0 treatment using an empty plasmid as a control. Following this, we utilized a fluorescently tagged secondary goat anti-mouse IgG to assess whether antibodies in the serum bound to the cells, with measurements taken via flow cytometry. Results: TCRSeq and BCRSeq demonstrated unique T and B-Cell clones in melanoma patients treated with IFx-Hu2.0. Mice injected with IFx-Hu2.0 demonstrated a statistically significant reduction in tumor growth compared to control mice (mean tumor volume at Day 17; 104 ± 22 mm3 vs 702 ± 100 mm3, p < 0.01). Within mice treated with IFx-Hu2.0, uninjected tumors grew more slowly than tumors in uninjected mice (p = 0.03), supporting an abscopal effect in mice. Anti-emm55 IgG antibodies were measured via flow cytometry on EL4-emm55 cells pulsed with murine sera from mice treated with IFx-Hu2.0 and IgG did not bind to EL4 cells transfected with Emm55 but not pulsed with serum. Conclusions: The expression of Emm55 in a B16 murine melanoma model induces a direct anti-tumor and abscopal effect, with antibodies to Emm55 detected in murine sera. Further research is needed to assess the role of these antibodies in anti-tumor immunity. Unique clones from TCRSeq and BCRSeq suggest that IFx-Hu2.0 may be a personalized vaccine. Ongoing studies aim to understand how patients become re-sensitized to anti-PD-1 therapy, with subsequent clinical trials in development. Citation Format: Joseph Markowitz, Vishanna Balkaran, Andrew S. Brohl, Amod S. Sarnaik, Zeynep Eroglu, Nikhil I. Khushalani, Deanryan B. De Aquino, Pei-Ling Chen, Vernon K. Sondak, Ahmad A. Tarhini, Youngchul Kim, Shari Pilon-Thomas. Mechanistic insights into IFx-Hu2.0 responses in the first human trial after prior anti-PD-1 therapy failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3428.