Engineering T cells with improved TCR-CD3 interaction for optimal tumor killing

Abstract T cell activation requires extracellular stimulatory signals mainly mediated by T cell receptor complex (TCR/CD3). Obtaining detailed mechanistic knowledge of the TCR-mediated signaling pathway holds significant importance in understanding immune diseases, formulating new immunotherapies, and overcoming immunosuppression in current therapies. Here, we used a novel strategy to overcome ineffective anti-tumor responses by modifying the TCR interaction with CD3 subunits without affecting the binding affinity and/or specificity of the TCR-antigen interaction. Based on NMR and mutational studies, we introduced specific alanine mutations in the Cβ helix 3 and Cβ helix 4 – F strand of the TCR extracellular domain. We demonstrated varied functionality in terms of IL-2 production as well as altered bond lifetime measured by biomembrane force probe assay (BFP). Selected mutations retained or displayed enhanced CD3 binding ability in a CD3 tetramer assay. Based on these results we generated retroviral TCR libraries by random mutagenesis at specific extracellular TCR-CD3 interaction sites, expressed them in a T cell hybridoma system and selected TCRs with novel binding specificity using soluble CD3γɛ- and CD3δɛ-tetramers. We are currently determining the correlation between CD3 tetramer binding, bond lifetime, and functionality (kinase activity, IL-2 production, and tumor rejection) of selected TCR mutants. By integrating binding data, T cell signaling data, and functional outcomes, we expect to be able to manipulate the human immune system via the extracellular TCR-CD3 interaction site to be utilized in novel human T cell therapies.