Abstract 3169: Development of an engineered T cell receptor-based system for the rapid detection of cancer biomarkers

Abstract

Abstract Biomarkers play a central role in cancer management by facilitating detection and characterization and are key to the development of personalized therapeutic strategies. We have developed a novel Surface Programmable Activation Receptor (SPAR) diagnostic platform which uses T cell-based technology to achieve the rapid and sensitive identification of cancer biomarkers. SPAR cells are T cells modified to express both an engineered T-cell receptor (TCR) and the luminescent reporter aequorin. Jurkat T cells are first transfected with the aequorin expression vector pEF1-Aeq introduced via random insertion, and stable transfectants selected using G418. The TCR complex is functionally modified via the gene fusion of enhanced monoavidin (eMA) with the CD3ϵ subunit of the human T cell receptor complex to form eMA-CD3ϵ and introduced as a homozygous insert into Jurkat/pEF1-Aeq T cells via electroporation, using CRISPR/Cas9 targeting to replace endogenous CD3ϵ. The modified TCR complex efficiently binds biotinylated target detection molecules (TDMs), which target specific biomarkers via an engineered protein binding domain and a biotinylated protein tag, and act as a ligand to program the SPAR TCR. Simultaneous binding of the TDM to the target and to the modified TCR causes receptor aggregation. Aggregation signals are amplified via the native T-cell signal cascade and result in rapid calcium release from the endoplasmic reticulum. Increased calcium concentrations activate aequorin, triggering the emission of detectible light. Initially developed for programmable pathogen detection, the system has demonstrated reliable and specific detection of the lymphoma marker CD19. Cultured human Burkitt’s lymphoma (Raji B) cells abundantly expressing the CD19 surface receptor were combined with SPAR cells programmed with a biotinylated anti-CD19 antibody as TDM. Binding of the anti-CD19 SPAR cells to cell surface CD19 resulted in TCR aggregation, signal transduction, and the generation of reporter signal. Repeat studies conducted in the presence of blood demonstrated equivalent sensitivity. We achieve similar results using the human melanoma markers GD2 and CD133, and mouse melanoma markers TRP1 and CD44 in cell lines known to express these markers. In these studies, a variant of the SPAR system was used to screen commercial monoclonal antibodies to identify those with biologically relevant activity against the cell surface biomarkers, thus demonstrating the utility of this system in optimizing useful antibody/ target combinations without requiring target protein purification. With optimized TDMs, no significant light signal could be detected with K562 cells used as control. The SPAR diagnostic platform is a rapid and effective method for the detection of known cancer biomarkers and may provide a direct screening tool for the optimization of interacting proteins used to guide cell therapies. Citation Format: Joseph D. Kittle, Joel Lwande, M Russell Williams, Shengwen Liang, Kyle McQuaid, Melissa Frenchmeyer, Yuanyuan Tang, Allison Neese, Jiangzhou Hua, Charles McBrairty. Development of an engineered T cell receptor-based system for the rapid detection of cancer biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3169.