Engineering human single-chain T cell receptors (2010)

The alpha-beta T cell receptor (TCR) is responsible for mediating T cell recognition of self and non-self tissues, through recognition between a complex of a peptide and a product of the major histocompatibility complex (pepMHC) on target cells. In the immune response to cancerous tissue, the immune repertoire of T cells is often insufficient to target pepMHC complexes associated with cancer cells, as these tumor antigens have often induced tolerance or the tumor microenvironment promotes immunosuppression of T cells. To improve the response to tumors, gene therapy with tumor specific T cell receptors provides an attractive approach to effectively arm patient???s T cells for cancer cell destruction. An inherent difficulty, however, is generation of T cell receptors of sufficient affinity to redirect both CD4+ and CD8+ T cells. This thesis describes the development of engineering strategies for human single-chain T cell receptor variable fragments (scTv), with the goal of understanding the properties that allow scTv to be expressed and deployed in a therapeutic mode. Stable scTv receptors can be used to generate high-affinity TCRs specific for disease-associated pepMHC complexes and produced in soluble expression systems for subsequent biochemical and biophysical characterization. This work also develops scTv and scFv (antibody variable fragments) as fusion proteins, collectively called chimeric antigen receptors, for cell mediated therapies to redirect T cells to specific antigens&#13;\nIn chapter 2, two human Valpha2+ T cell receptors specific for human immunodeficiency virus and human T cell lymphotrophic virus derived pepMHC complexes were engineered for improved stability as scTv proteins, consisting of only the variable domains of the T cell receptor attached by a flexible linker. High-affinity, stabilized scTv proteins could be expressed as soluble proteins in E. coli and used for detection of low levels of HIV pepMHC antigens, suggesting that these receptors have potential diagnostic applications for the detection of HIV infected cells. Finally, the results suggest that other V???2+ TCRs with different specificities can be engineered for enhanced affinity by yeast display. &#13;\n\tChapter 3 describes the development of chimeric antigen receptors, that consist of scTv-fusion proteins, for T cell targeting of tumor antigens. scTv proteins engineered for improved stability by yeast display were fused to the intracellular signaling domains of CD28,CD3zeta, and LCK and introduced into murine T cells. The high affinity scTv, called m33, that is specific for the pepMHC SIY/Kb was used to redirect T cells with similar antigen sensitivity to the full-length m33 TCR. An inherent problem with full-length TCR gene therapy is the generation of receptors of unknown specificity through mispairing between introduced and endogenous TCR, leading to graft versus host disease or autoimmunity. I show that the scTv-fusions avoided mispairing with endogenous alpha-beta TCRs and allowed for endogenous TCR surface expression at high levels. A human HIV-specific scTv (chapter 2) was also expressed as a fusion to intracellular signaling domains and it also mediated antigen specific T cell activity. &#13;\n\tIn chapter 4, the murine m33 scTv fusion was compared to an antibody derived chimeric antigen receptor called 237. The 237 antibody single-chain fragment variable (scFv) is specific for a tumor antigen resulting from a glycopeptide defect that is created by a mutant chaperone protein. Fusion of the 237 scFv to intracellular domains, as with the m33 scTv, mediated T cell activity against tumor cells that expressed the glycopeptide defect. Results with T cells transduced with chimeric antigen receptor in the absence or presence of coreceptor CD8, showed that CD8 could contribute to target cell recognition, presumably through its interactions with MHC molecules that are proximal to the antigen epitope.\t&#13;\nChapter 5 describes the engineering of a murine T cell receptor, called 3D, for enhanced affinity to the model Wilm???s tumor antigen (WT-1/Db). Using a novel T cell display system, mutated TCR libraries were displayed on the surface of T cell hybridomas in the absence of the coreceptor CD8. Selection with fluorescently labeled dimers of WT-1/Db resulted in the isolation of two high affinity 3D TCR variants, one which mediated T cell activity in the absence of CD8. Analysis of the TCR residues used by human and murine TCRs specific for the identical WT-1 peptide suggested that there is homology between human and mouse TCR CDR3 sequences, indicating that these residues have strong selective pressures to bind to the identical peptide epitope. Thus, one may be able to engineer high-affinity TCRs in the human TCR based on knowledge from the mouse TCRs.