Abstract B067: An antigen-specific t cell stimulation and expansion platform for immunological investigation of the Ad5.F35-GUCY2C-PADRE vaccine in gastrointestinal cancers

Abstract Background: Collectively, cancers of the esophagus, stomach, pancreas, and colorectum (“GI cancers”) are the leading cause of cancer-related death. Despite standard of care, 30-90% of GI cancer patients experience tumor recurrence. In colorectal cancer (CRC), guanylyl cyclase C (GUCY2C), a transmembrane heat-stable enterotoxin receptor, is overexpressed in more than 95% of colorectal tumors and in subsets of gastroesophageal and pancreatic tumors. We are conducting a Phase 2A, dose-finding, single-center, open-label, randomized trial (NCT04111172) to evaluate the safety and immunogenic activity of three dose levels of the Ad5.F35-GUCY2C-PADRE vaccine in adult patients with select solid tumors (colorectal, pancreatic, or gastroesophageal) who are at high risk of relapse following definitive surgery and adjuvant therapy. To further investigate the immunogenicity of the GUCY2C-targeting vaccine across diverse patient populations and facilitate downstream laboratory-based studies—such as autologous T cell expansion, TCR sequencing and cloning, and in vivo patient-derived xenograft (PDX) models—we explored the feasibility of a protocol for the stimulation and expansion of antigen-specific T cells derived from peripheral blood mononuclear cells (PBMCs). Methods: PBMCs from healthy donors were used as a pilot to optimize this protocol and assess its efficacy. PBMCs were cultured in vitro for approximately 10 days with adjuvants, cytokine cocktails, and antigen-specific peptides. The enriched T-cell populations were then evaluated for antigen-specific targeting using flow cytometry and ELISPOT analysis. Results and Conclusion: The stimulation protocol successfully expanded both CD4+ and CD8+ effector T cells. The final T cell population was predominantly CD8+ T cells, which secreted IFNγ following CEF (CMV, EBV, Influenza), but not MOG (self-antigen myelin oligodendrocyte glycoprotein) peptide stimulation, indicating antigen-specific reactivity of the expanded T cells. Further studies, including cytotoxicity assays and antigen-presenting cell (APC)-based longitudinal expansion, are necessary to support the high-throughput application of this protocol for future preclinical and clinical investigations. Citation Format: Zhengyang Sun, Jagmohan Singh, Miao Cao, Anne Glowacki, Tingting Zhan, Babar Bashir, Scott A Waldman, Adam E Snook. An antigen-specific t cell stimulation and expansion platform for immunological investigation of the Ad5.F35-GUCY2C-PADRE vaccine in gastrointestinal cancers [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B067.