Abstract The breakthrough discoveries of checkpoint inhibitors in the field of tumor immunology have driven the clinical success of immunotherapies for cancer, despite their beneficial efficacy in only a small portion of patients. This is due in part to immuno-evasive mechanisms and the inability of the immune system to recognize tumor antigens as foreign. As a therapeutic approach to effectively present these tumor antigens in order to elicit an anti-tumor immune response, we previously designed and characterized an allogenic, gp96-Ig secreting, cell-based vaccine (ImPACT); currently being assessed in a phase II study in non-muscle invasive bladder cancer and a phase Ib study in non-small cell lung cancer – the latter, in combination with the PD-1 antagonist Nivolumab. We recently characterized a ‘next-generation’ vaccine (ComPACT) that combines the tumor antigen chaperone Gp96-Ig along with the T cell costimulator Fc-OX40L, which are both secreted from the same cell (Fromm et. al. Cancer Immunology Research. 2016). In preclinical assays, ComPACT is effective at stimulating CD4+ and CD8+ antigen-specific T cell expansion, the programing of a durable memory T cell phenotype, and the elimination of melanoma and colon tumors. This anti-tumor efficacy is enhanced when ComPACT is combined with checkpoint inhibition (anti-PD1 or anti-PDL1). To support manufacturing and clinical efforts of both ImPACT and ComPACT, in anticipation of phase III expansion and/or new trial initiation, we have developed novel potency assays to quantify the biologically active form of Gp96-Ig and the in vitro activity of Fc-OX40L on T cell costimulation. It has been shown that gp96 can interact with toll-like receptors (TLR) and that this interaction results in the activation of the NF-κB pathway. Since THP1 cells express abundant TLR2/4, we engineered a THP1 cell line to express luciferase that is regulated by NF-κB response elements. Furthermore, we utilized the human T cell line; Jurkat, as host cells in which we also express NF-κB-luciferase, to quantify Fc-OX40L costimulation. Jurkat/NF-κB-luciferase cells primed with either CD3/CD28 or TNFα, and subsequently cultured with ComPACT-secreted Fc-OX40L, results in a dose dependent increase in NF-κB (luciferase) expression. Our current data in both assays shows a linear correlation with the input of Gp96-Ig and Fc-OX40L, and may serve as an effective potency assay to facilitate the manufacturing of our vaccine product as it transitions into more advanced cancer immunotherapy clinical studies. Citation Format: Xin Xu, Louise E. Gonzalez, George J. Fromm, Suresh de Silva, Louise Giffin, Jason Rose, Taylor H. Schreiber. Potency of Gp96-Ig/Fc-OX40L cell-based combination vaccine in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2017-605