Recombinant human adenovirus type 5 (H101) combined with anti-PD-1 monoclonal antibody in the treatment of patients with advanced melanoma with previous immunotherapy failure: A single-site, single-arm, prospective study.

e21505 Background: Patients with melanoma who progress on immunotherapy have limited treatment options, especially for acral and mucosal melanoma.Here, we presented the efficacy and safety of H101 intra-tumor injection combined with anti-PD-1monoclonal antibody in the treatment of advanced melanoma with previous immunotherapy failure. Methods: In this single-site, single-arm, prospective study.10 checkpoint inhibitors (ICI) resistant patients with advanced melanoma received treatment of H101 combined with anti-PD-1 monoclonal antibody. The primary endpoint was set to ORR. The secondary endpoints include DOR,DCR,OS,QOL and AEs. For biomarker exploratory and mechanism-of-action purposes, we conducted single-cell spatial transcriptome(ST) profiling (Xenium in-situ 5K)side-by-side on all patient samples collected longitudinally during the treatment process. Results: Overall, 10 patients were enrolled (6 acral melanoma; 2 cutaneous melanoma;1 mucosal melanoma; 1 unknown primary melanoma). All patients received at least 2 cycles of the combination regimen. At the data cut-off on January 2025,overall median follow-up time was 18 months. Objective tumor responses were CR (n = 0), PR (n = 2), SD (n = 4) and PD (n = 4). ORR was 20.0% (2/10) and the median DOR was not reached and DCR was 60.0% (6/10). The one-year PFS was 30%, and the one-year OS was 60%. TRAE occurred in 10 patients (100.00%). The most frequently observed TRAEs were fever (100.00%), anemia (40.00%) and arrhythmia (20.00%), with all being classified as grade 1 or 2. No serious TRAEs were reported.In depth tumor microenvironment (TME) analysis using highly-resolved ST identified intrinsic response-associated TME characteristics of populated monocytic and potentially mono-derived macrophage subsets that co-presented with less infiltrated endothelial cells. In line with that, spatial neighborhood analysis identified colocalization patterns of tumor-adjacent cellular niche (CN6 and CN9) that were more prevalent in OV responders. As the treatment progressed, OV sensitive patients (n = 2) showed massive tumor regression with formation of an immunogenic spatial contexture: an antitumor T-cell subsets CN7 that was in direct contact with tumors. Whereas in OV-resistant patients, tumor persistence was observed without the presence of such tumor-reactive T-cell dominated spatial domain. Conclusions: The data showed that the combination of recombinant human adenovirus 5 injection plus Toripalimab demonstrated acceptable toxicity and promising antitumor efficacy in patients with advanced and refractory melanoma. Mechanistically, therapeutic response was directly regulated via specific mono/macro populations at tumor-immune boundary that may directly involve T cell recruitment and tumor killing. Clinical trial information: ChiCTR2200055931 .