A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells

Alex Shepherd; Bigitha Bennychen; Zafer Ahmed; Risini D Weeratna; Scott McComb

doi:10.21769/BioProtoc.5021

A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells

Bio Protoc. 2024 Jun 20;14(12):e5021. doi: 10.21769/BioProtoc.5021.

Authors

Alex Shepherd^{1

2}, Bigitha Bennychen^{1

2}, Zafer Ahmed¹, Risini D Weeratna¹, Scott McComb^{1

3

2}

Affiliations

¹ Human Health Therapeutics Research Centre, National Research Council, Ottawa, ON, Canada.
² Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada.
³ Centre for Infection, Immunity and Inflammation, University of Ottawa, Ottawa, ON, Canada.

Abstract

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.

Keywords: CAR-T; Cell-to-cell interaction; Cellular avidity; Cellular kinetics; Flow cytometry; High throughput; Jurkat; Screening; T cell.