PROJECT SUMMARY T cells are responsible for the eradication infectious or cancerous cells in the body; however, cancer cells are capable of evading T cell recognition and activation. Bispecific molecules are an emerging treatment option for cancer immunotherapy that enhances the T cell’s ability to recognize cancerous cells and perform an immune response. Their mechanism of action is to increase T cell sensitivity to cancer cells and efficacy by binding to both T cells and the cancerous pMHC. Limitations in current designs include the inability to predict T cell activation after binding and in efficacy for treating cancer. The objective of this proposal is to expand our understanding of the factors that affect efficacy of bispecific molecules to enhance T cell performance. We hypothesize that force dependent binding and docking orientation impact the overall efficacy of bispecific molecules. The two specific aims are: Aim 1. Identify the determining factor(s) of the anti-tumor efficacy of bispecific molecules Aim 1A. Determine the 2D affinity and force dependent bond lifetime between several ImmTAC vs. pHLA and vs CD3, thereby providing a unique dataset only our lab is capable to obtain. Aim 1B. Examine the correlation, or the lack thereof, between the biophysical characteristics of the interactions measured in 1A with the biochemical characteristics and biological functions of the ImmTAC molecules, thereby shedding light on the determinant of their therapeutic efficacy Aim 2. Assess how the pMHC docking orientation affects anti-tumor efficacy of the TCR Aim 2A. Determine 2D affinity and force dependent bond lifetime for canonical and reversed orientation TCRs. Aim 2B. Determine if the absence of CD8 is responsible for low efficacy when TCR is in reversed orientation. Completing these aims will provide a new perspective on the considerations to be taken when designing bispecific molecules for cancer immunotherapy.