Engineering and characterization of high-affinity T cell receptors

Engineering high-affinity TCRs would provide novel reagents for the study of TCR:pepMHC interactions and could eventually lead to the use of soluble TCRs as antigen-specific targeting agents. The focus of this thesis work has been to engineer TCRs with high affinity for pepMHC and to characterize the biochemical and functional properties of these TCRs.;In Chapter 2, a stabilized scTCR served as a scaffold for the directed evolution of mutant scTCRs with up to 100-fold higher affinity (KD∼9 nM). An analysis of fine specificity showed that the high-affinity TCRs were peptide specific.;In Chapter 3, the function of T cells expressing a high-affinity TCR was examined. T cell hybridomas that were transfected with a high-affinity TCR showed increased sensitivity to antigen as compared to T cell hybridomas that expressed the wild-type receptor. In addition, studies with peptide variants demonstrated that pepMHC stability plays a dominant role in T cell activation.;In Chapter 4, the cross-reactivity and specificity of several high-affinity TCRs were examined. A high level of cross-reactivity among cognate pepMHC was observed. T cells that expressed a TCR selected for high affinity on a foreign peptide also were stimulated by a self-peptide, indicating that TCRs with high affinity for foreign pepMHC have a high probability of reacting to self-pepMHC. Binding data and structural modeling of mutant TCRs suggested that high-affinity binding was a product of stabilization of the CDR loops through interactions with other CDRs.;In Chapter 5, the possible therapeutic value of engineered TCRs was explored. It was demonstrated that improving TCR affinity could abrogate the need for the co-receptor molecule, CD8. Also, raising the affinity to a self-peptide resulted in T cell activity against a syngeneic tumor line, thus breaking tolerance.;In Chapter 6, the role of TCR affinity in T cell activation was studied using CD8-negative T cells, as well as T cells expressing the co-receptor CD8. The peptide sensitivity of T cells that expressed the highest-affinity TCRs reached a plateau, suggesting the existence of an affinity maximum for T cell activation. Furthermore, CD8 requirements were directly related to the affinity of the TCR.