Chimeric antigen receptor (CAR) T cell therapy is an engineered cell therapy where T cells are isolated and genetically modified to contain a synthetic CAR with specificity to a tumor cell antigen. Upon antigen binding, the CAR T cell will initiate signaling cascades that result in lysis of the associated tumor cell. Cytokine release syndrome (CRS) is the primary toxicity associated with CAR T cell therapy and remains a prominent safety issue with currently available commercial products. CRS is driven by interaction of the CAR T cells with endogenous monocytes and macrophages, which can lead to immune cell overactivation and an increase in certain cytokines to supraphysiological levels. Identifying the potential of any given CAR construct to drive toxicities in vivo should be assessed in preclinical models prior to human trials. While there are in vivo mouse models available for this purpose, these are often complex xenograft models available in few centers. Thus, there is a need to develop an in vitro assay for measuring the CRS potential of CAR T cells. The assay described here is a preclinical tool for assessing the propensity of any given CAR construct to produce potentially CRS-driving cytokines following tumor cell and monocyte interactions. This article provides a detailed protocol for target cell preparation and isolation of monocytes from peripheral blood mononuclear cells (PBMCs) autologous to the CAR T cells, as well as protocols for seeding the three cell types in a co-culture assay and collecting/analyzing the cytokines produced via an ELISA or multiplex bead array. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of K562 target cells Basic Protocol 2: Isolation of monocytes from autologous PBMCs Basic Protocol 3: Seeding of CAR T cells, monocytes, and K562 cells in 96-well plates Basic Protocol 4: Analysis of co-culture supernatants by single-cytokine ELISA Alternate Protocol: Analysis of co-culture supernatants by multiplex cytokine bead array.
Keywords:CAR T cells; CRS; chimeric antigen receptor; cytokine release syndrome; in vitro assay.