Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators of T Cell Activation

Zhaoping Liu,Nina Senutovitch,John O’Rourke,Andrea Gomez-Donart,Caroline Weldon

doi:10.5772/intechopen.97304

Abstract

T cell activation plays a central role in inflammation, autoimmune diseases and cancer. Cancer immunotherapies, such as immune checkpoint inhibitor, bi-specific antibody, chimeric antigen receptor T (CAR T) cell, and adoptive tumor-infiltrating lymphocyte (TIL) therapies require the characterization and monitoring of T cell activation. Here we describe a novel, multiplex immune assay platform based on high-throughput flow cytometry technology and advanced computational algorithms for data analysis. The assay simultaneously measures T cell dynamics including phenotype, time-dependent expression of activation markers, secreted effector cytokines, and proliferation. The assay screened a kinase chemogenomic library and identified 25 kinase inhibitors with distinct inhibition profiles on early (CD69) and late (CD25) activation markers and the cytokines IFNγ and TNFα. We identified 5 kinase inhibitors with dissimilar effects on CD69 and CD25 expression, and a cluster of total 4 MEK1//2 inhibitors with similar activation profiles. The screening revealed 3 kinase inhibitors for PKC, IKK2, and MEK1/2 respectively, all with a phenotypic signature similar to ruxolitinib, a Jak1/2 inhibitor used to treat myelofibrosis disease. These results suggest this multiplexed assay platform, combined with a chemogenomic library screening, may be used as primary screen for phenotypic or target-based drug discovery, target identification, and potential drug repositioning.

Highlights

Unraveling the complex biochemistry of the immune system in search of innovative therapies is a new frontier of drug discovery with hundreds of autoimmune disease therapies and thousands of immuno-oncology therapies in the global development pipeline [1, 2].Hallmarks of autoimmune disease pathogenesis are abnormal CD4 and CD8 T cell activation [3]
Another type of cancer immunotherapy consists of bispecific antibodies designed to redirect immune cells to tumor sites where they induce immune synapse formation, immune cell activation, cytokine secretion and proliferation leading to tumor lysis [7]
This wide dynamic range ensures the detection of high levels of secreted IFNγ and TNFα after T cell activation and eliminates a sample dilution step

Summary

Introduction

Unraveling the complex biochemistry of the immune system in search of innovative therapies is a new frontier of drug discovery with hundreds of autoimmune disease therapies and thousands of immuno-oncology therapies in the global development pipeline [1, 2].Hallmarks of autoimmune disease pathogenesis are abnormal CD4 and CD8 T cell activation [3]. T helper cells —characterized by the expression of the surface molecule CD4—release cytokines that shape the immune response and pathology in autoimmune and inflammatory diseases [5]. Rather than directly attacking the tumor cell itself, the checkpoint inhibition strategy is removing inhibitory pathways by targeting the molecules involved in T cell regulation that block effective anti-tumor responses [6]. Another type of cancer immunotherapy consists of bispecific antibodies designed to redirect immune cells to tumor sites where they induce immune synapse formation, immune cell activation, cytokine secretion and proliferation leading to tumor lysis [7]

Methods

Results

Discussion

Conclusion