Abstract 2750: Bioluminescent bioassays for the discovery and development of molecular and cellular T-cell redirecting cancer therapy
Authors: Gilden Julia , Grailer Jamison , Slater Michael , Stecha Pete , Hartnett Jim , Lazar Dan , Krishnan Gopal , Fan Frank , Cong Mei , Cheng Zhijie Jey 
Published: June 2022

Abstract
Redirected T cell therapies represent a new paradigm for cancer treatment. Two main approaches for T-cell-based therapies involve molecular T cell redirection by CD3 bispecific molecules such as bispecific T-cell engagers (BiTE) and cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). BiTEs redirect the cytotoxic activity of endogenous polyclonal T cells by simultaneously engaging CD3 on T cells and tumor antigens on target cells. Hitherto, BiTE potency studies have relied on primary cells, which measure target cell killing through redirected T cell cytotoxicity (RTCC) or cytokine release. However, these primary cell-based assays suffer from high donor-to-donor variability, as well as lengthy and hard to implement protocols. We have recently developed a new NanoBiT-based assay platform-based RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of CD3 bispecific antibodies or BiTEs and similar biologics that induce T Cell-dependent target cell killing and cytokine production. In this assay, preactivated, Thaw-and-Use cytotoxic T cells and target cells stably expressing HaloTag-HiBiT are co-incubated with a BiTE, which results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins then bind to LgBiT in the detection reagent and form functional NanoLuc Luciferase to generate luminescence. The assay is homogenous, highly sensitive, and has a robust assay window. Furthermore, BiTE-induced cytokine production (e.g IL-2 and IFN gamma) from the activated cytotoxic T cells can be quantitatively measured in the homogenous NanoBiT Immunoassays. Use of CAR-T has demonstrated promising results in treating leukemia, while the development of TCR-engineered T cells that can recognize intracellular tumor antigens, is still in early stages. To facilitate the screening and characterization of new transgenic TCRs, we used CRISPR/Cas9 to develop two TCRab-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR a and b chains into TCRab-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRab-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies.
Citation Format: Julia Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, Gopal Krishnan, Frank Fan, Mei Cong, Zhijie Jey Cheng. Bioluminescent bioassays for the discovery and development of molecular and cellular T-cell redirecting cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2750.