TCR Discovery and Characterization
Cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR) is one of the important strategies employed in redirected T cell therapies, representing a new paradigm for cancer treatment. To facilitate the screening and characterization of new transgenic TCRs, we developed two TCRαβ-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR α and β chains into TCRαβ-null reporter T cell lines results in peptide-dependent TCR activation. The select expression of CD4 or CD8 in the TCRαβ-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.
We have developed a suite of T Cell Activation Bioassays that measure TCR signaling via NFAT or IL-2 driven expression of a luciferase reporter. To better enable researchers developing engineered T cell therapies, we also created a T Cell Activation Bioassay that lacks expression of endogenous TCRαβ chains. The T Cell Activation Bioassay (TCRαβ-KO) eliminates endogenous alpha and beta chain cross-pairing with transgenic TCRs and provides an improved assay window.
- Measure activity and potency of transgenic TCRs and CARs
- Bioassay cells lack endogenous TCRαβ expression
- Available in CD4+, CD8+, CD4+CD8+, CD4-CD8- formats
Activity of transgenic TCRs measured using the T Cell Activation Bioassay (TCRαβ-KO, CD4+). Panel A. TCRαβ-KO (CD4+) Cells were transfected with a Positive Control TCR Plasmid (or mock transfected) and then co-cultured with MHCII APC Cells (HLA-DR+ cells) and increasing concentrations of the Positive Control Peptide. Panel B. TCR-mediated T cell activation was measured using the T Cell Activation Bioassay (endogenous TCR, CD4+) and the T Cell Activation Bioassay (TCRαβ-KO, CD4+). The T Cell Activation Bioassay cells were co-cultured with HLA-DR-positive cells and increasing amounts of cognate peptide. The modified T cells are activated once they bind antigens on tumor cells, as indicated by luminescence. The data demonstrate that the T Cell Activation Bioassay can be used to screen transgenic TCRs used for T cell immunotherapy.
T Cell Activation and Cytokine Production
Cytokine production, especially IFN-γ and IL-2, is a characteristic feature of activated cytotoxic T cells. We have developed T cell cytokine immunoassays using the novel Lumit™ Immunoassay platform. These assays offer sensitive bioluminescent detection of the release of critical cytokines such as IL-2 and IFN-γ.
Lumit™ immunoassays measure IL-2 and IFN-γ production from activated CD8 T cells. Purified CD8+ T cells (effector cells) were combined with Raji B cells (target cells) and a serial dilution of Blincyto® (CD3xCD19 bispecific T cell engager). IFN-γ (Panel A) and IL-2 (Panel B) levels were measured with the corresponding Lumit™ Cytokine Immunoassays to detect cytokine release from the effector cells.
Target Cell Killing (TCK)
Targeted killing of tumor or infected cells by effector cells is the primary mechanism of action for many immunotherapy drugs. Thus, it is necessary to demonstrate robust and specific killing of target cells during drug development. Conventional cytotoxicity assays do not distinguish between target and effector cells. Further, target cell-specific assays are often laborious and require radioactive or fluorescent labeling.
The HiBiT Target Cell Killing bioassay platform is simple, homogenous, highly sensitive and provides a robust assay window. It enables highly sensitive and specific measurement of target cell killing induced by a variety of biologic drugs, including mAbs, bispecific Abs and CAR-T cells. These assays provide a choice of popular target cells expressing a HiBiT fusion protein. Upon target-cell killing, a bright luminescent signal is generated.
Extended assay incubation time reveals serial TCK activity. T cells transduced with CAR-19 or a GFP control lentivirus were combined with HiBiT Target cells (Ramos) at different effector:target (E:T) ratios. After incubation for 24 hours (Panel A) or 48 hours (Panel B), NanoBiT® Extracellular Detection Reagent was added, and luminescence was read on a GloMax® Discover plate reader. The EC50 shifts left over time, indicating serial TCK activity at lower E:T ratios.