Optimizing CAR T Cells at the Level of Receptor Signaling
Determining responsiveness and safety using a multiplex quantitative protein-protein interaction platform
Currently, CAR T cells engineered to target cancer cells are tested for potential effectiveness using cytotoxicity assays. These tests measure the ability of a batch of CAR T cells to kill target cells in vitro. Another assay detects production of cytokines after T cell receptor stimulation with antigen. These tests measure downstream effects of the signal transduction pathways that are activated by T cell receptor stimulation. Even CAR T cells that seem like safe, effective therapeutic candidates based on these assays can be ineffective in practice or cause toxic side effects.
To study receptor responses at the level of receptor stimulation, Dr. Smith developed a quantitative multiplex immunoprecipitation (QMI) assay. Within hours, this technique can quantify dynamic interactions of proteins including hundreds of protein-protein associations that occur during T cell receptor stimulation. In QMI, proteins in complexes are co-immunoprecipitated from CAR T cells and quantified using mass spectrometry or labeled antibody probes detected by flow cytometry.
Using QMI, Dr. Smith’s group can provide a detailed analysis of the intracellular signalosome protein complex that forms at the T cell receptor upon stimulation of a CAR T cell. QMI can detect even small changes or disruptions to complexes that contain tens or hundreds of proteins.
In proof-of-concept experiments, Dr. Smith and colleagues detected donor-dependent variations in signalosomes in CD19-directed CAR T cells with a 4-1BB costimulatory domain. These variations correlated with standard CAR T cell effectiveness assays such as cytokine production.
These results show that QMI can detect meaningful differences in production batches of CAR T cells. Variations in protein-protein interaction patterns could be a valuable measure of potential CAR T cell effectiveness and safety. QMI analysis could also be used to determine how to further engineer T cell receptors for desired antigen-directed responsiveness with minimal adverse effects.
Dr. Smith is interested in industry partnerships that use QMI to map and quantitate CAR T cell signal transduction responses for more rational CAR T cell design. He is interested in collaborating to develop QMI as a standard assay for optimizing CAR T cells and selecting the most promising candidates for therapy.
Stage of Development
- Preclinical in vitro
- Collaborative research opportunity
- Drug development opportunity
- Sponsored research agreement
- Consultation agreement
- Ritmeester-Loy SA, Lautz JD, Zhang-Wong Y…Smith SEP. State-dependent protein-protein interactions mediating 4-1BB CAR signaling. bioRxiv 2022.06.07.495180; doi: https://doi.org/10.1101/2022.06.07.495180
- Neier SC, Ferrer A, Wilton KM, Smith SEP, et al. The early proximal αβ TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network. Sci Immunol. 2019;4(32):eaal2201.
- Brown EA, Neier SC, Neuhauser C…Smith SEP. Quantification of protein interaction network dynamics using multiplexed co-immunoprecipitation. J Vis Exp. 2019;(150):10.3791/60029.