Background 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. 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
Methods We have recently developed a new RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of BiTEs and similar biologics. In this assay, preactivated cytotoxic T cells and target cells (both in cryopreserved thaw-and-use format) stably expressing a HaloTag-HiBiT fusion protein are co-incubated with a BiTE, which results in lysis of the target cells and subsequent release of the Halotag-HiBiT protein. These HiBiT proteins then bind to extracellular LgBiT provided in the detection reagent and form functional NanoLuc Luciferase to generate luminescence.
Results The assay is homogenous, highly sensitive, and has a robust assay window. 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 TCRaß-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR a and ß chains into TCRaß-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRaß-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets.
Conclusions Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies.
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