Quantitative Multiplexed Immunohistochemistry Assays for Exploring CAR Modified T Cells and Checkpoint Inhibitors in Lymphoma Trials

Although there have been compelling advances in the cancer immunotherapy space recently in the form of chimeric antigen receptor (CAR) modified T-cells and checkpoint inhibitors, advanced tools to explore the therapeutic mechanisms of their combination are not adequately developed or widely available. To address this growing need, we developed a robust quantitative fluorescent immunohistochemistry platform using multiplex AQUA (Automated Quantitative Analysis) technology to evaluate checkpoint inhibitor expression, enumerate CAR T cells and determine the interaction between tumor cells and immune cells via novel co-localization algorithms. We explored utility of this method both in preclinical- and clinical model systems. In an immunodeficient mouse model of B-cell lymphoma, we evaluated homing of CAR T cells to malignant B-cells in primary lymphoid organs. We determined the phenotype and functional status of the CAR T cells via multiplex analyses of CD4, CD8, PD1 and FOXP3 expression. Additionally, to enable combination immunotherapies in Diffuse Large B-Cell Lymphoma (DLBCL) setting, we explored prevalence of adaptive immune resistance mechanisms in the form of PD1 and PD-L1 expression in immune- and tumor cell compartments via landmarks created by cytoplasmic and nuclear stains in both primary and secondary biopsies from DLBCL patients (n = 63). To support patient selection for CAR T trials, we quantified expression and prevalence of relevant tumor antigens that could not be scored reproducibly by traditional methods to yield objective cut points. We anticipate utilization of these quantitative multiplexed IHC methods for optimal selection of patients into upcoming novel combination immunotherapy trials