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Background We previously reported that administration of two intratumoral injections of CAN-2409 combined with prodrug (valacyclovir) prolonged median overall survival (mOS) in patients with unresectable stage III/IV non-small cell lung cancer (NSCLC), who had progressed on immune checkpoint inhibitor (ICI) treatment, doubling mOS compared to historical controls treated with docetaxel. We applied advanced analytics to characterize baseline features, including histologic subtype (non-squamous [NSQ] and squamous cell [SQ] type), immunological profile, and treatment-induced immunological changes following CAN-2409 administration to establish the relationship between biological response and clinical outcomes and inform patient selection for future development of CAN-2409 in NSCLC.Methods In a phase 2a open-label clinical trial ( NCT04495153), 41 patients with stage III/IV NSCLC with progressive disease despite ICI received per protocol treatment of 2 courses of intratumoral CAN-2409+valacyclovir with continued ICI treatment. We applied Multi-Omics Factor Analysis (MOFA) to integrate over 3,000 data points derived from flow cytometry, CyTOF, and proteomics analyses of longitudinal samples and identify associations between MOFA-derived factors, patient characteristics, and clinical outcome.Results mOS was 24.5 months, 1 markedly exceeding historical benchmarks.2 3 Biomarker analyses revealed robust systemic immune activation after the second CAN-2409 dose, with increased numbers of CD8+ central memory T cells and elevated levels of soluble granzymes A and B correlating with long-term survival (p = 0.0356, 0.0476, and 0.0261, respectively). MOFA identified a latent variable, Factor 2 (F2), which was negatively correlated with OS (r= –0.61, p < 0.0001) and strongly associated with SQ histology (p = 0.0344). F2 captured a broadly dysfunctional immune state present at both baseline and post-treatment, characterized by elevated frequencies of Tim-3+ central memory CD4+ T cells and CD11c+ naïve CD8+ T cells. In contrast, patients with NSQ histology exhibited low F2 and displayed greater expansion of effector and memory T cell populations following CAN-2409 treatment compared to SQ patients. This enhanced immunological response was associated with improved clinical outcomes: mOS in NSQ patients (n=33) was 25.4 vs. 13.3 months in SQ patients (n=8)(p = 0.1382); this difference was not statistically significant, possibly due to relatively small groups.Conclusions Experimental treatment with CAN-2409+prodrug was associated with prolonged survival in NSCLC patients with progressive disease despite ICI treatment. Multimodal analysis revealed that patients with NSQ histology displayed a more pronounced immunological response to CAN-2409 treatment compared to patients with SQ histology. Improved immune response was associated with improved survival, supporting development of CAN-2409 in non-SQ NSCLC.Acknowledgements This work could not have been performed without the participating patients and their families, as well as site research and clinical staff. We also relied upon support from the National Cancer Institute-supported Cancer Immune Monitoring and Analysis Centers-Cancer Immunologic Data Center (CIMAC-CIDC) and Sonrai Analytics. This project was funded by the Partnership for Accelerating Cancer Therapies (PACT). Scientific and financial support for the PACT project was made possible through funding support provided to the Foundation for the National Institutes of Health, Inc. (FNIH) by: AbbVie Inc., Amgen Inc., Boehringer-Ingelheim Pharma GmbH & Co. KG., Bristol-Myers Squibb, Celgene Corporation, Genentech Inc, Gilead, GlaxoSmithKline plc, Janssen Pharmaceutical Companies of Johnson & Johnson, Novartis Institutes for Biomedical Research, Pfizer Inc., and Sanofi.Trial Registration NCT04495153 ClinicalTrials.govReferences Aggarwal C, et al. CAN-2409 with continued immune checkpoint inhibitor (ICI) in patients with stage III/IV NSCLC with inadequate response to ICI. World Lung Conference on Lung Cancer 2025.Ahn MJ, et al. Datopotamab deruxtecan versus docetaxel for previously treated advanced or metastatic non-small cell lung cancer: the randomized, open-label phase III TROPION-Lung01 study. J Clin Oncol. 2025;43(3):260–272.Paz-Ares LG, et al. sacituzumab govitecan versus docetaxel for previously treated advanced or metastatic non-small cell lung cancer: the randomized, open-label phase III EVOKE-01 study. Journal of Clinical Oncology. 2024;42(24):2860–2872.Ethics Approval Patients participating in the study provided an IRB-approved informed consent at their participating institutions.