David M. Kranz
Structure, Function and Engineering of T Cell Receptors
Introduction. Research in our laboratory is directed toward understanding a fundamental issue in immunology: how mammals can eliminate millions of different antigens that are "foreign" (e.g. viruses, bacteria) without destroying antigens that are "self" (e.g. one's own tissues). The specific focus of the lab is on the antigen-specific receptor expressed by T lymphocytes (T cell receptor, TCR). The TCR is an ?? heterodimer that recognizes foreign peptides that are bound to products of the major histocompatibility complex (MHC). Foreign peptides can be derived from viral or bacterial antigens or in some cases from tumor associated antigens. For example, cytotoxic T cells recognize and kill tumor cells that express on their surface peptides bound to class I products of the MHC. Down regulation of the peptide/class I complex is one mechanism that tumor cells use to escape recognition and destruction. It has also become clear that the aberrant reactivities of some ?? TCRs can have severe autoimmune consequences. Rheumatoid arthritis and multiple sclerosis are two examples of diseases that involve the activity of T cells. Studies of the molecular processes involved in the expression of the ?? TCR and the biochemical interactions between the TCR and its ligands are essential to understanding and eventually controlling such detrimental responses.
Characterization of the Interactions Between T Cell Receptors and Their Ligands. A complete understanding of T cell recognition will require knowledge about the binding properties of T cell receptors. Unfortunately, the major limitation to such biochemical analyses has been the lack of pure receptor protein. Our lab has engineered a "single-chain receptor" gene (scTCR) that contains the V? and V? genes from a T cell clone linked by a gene that encodes a flexible peptide. The scTCR has been overexpressed in E. coli and a product of the appropriate molecular weight (~27 kD) has been identified. The purified protein reacts with conformationally specific monoclonal antibodies and it binds to the specific peptide/MHC ligand. Our goals are to use these recombinant proteins to: 1) evaluate the binding affinities and specificities of the TCR, 2) use site-directed mutagenesis to map the binding site of the TCR, and 3) correlate the energy of TCR-peptide/MHC interactions with biological function.
In another project, the TCR has been displayed on the surface of yeast cells. The yeast display system has been used to evolve more stable forms of the soluble TCR and to evolve TCR with higher affinities for their ligands.
Engineering High Affinity Anti-Receptor Antibodies. Two different strategies toward controlling T cell activity are currently being explored. One involves generating high affinity anti-T cell receptor antibodies that inhibit detrimental T cells by preventing their recognition of the antigen. We have engineered a single-chain gene (VHVL) that encodes an anti-receptor antibody. After expression in E. coli, the refolded protein completely inhibits the cytolytic activity of the T cell. Efforts to increase its affinity using the yeast display system and to engineer additional single-chain antibodies against other key T cell surface molecules (such as CTLA-4) with high affinity are currently underway.
A second approach involves redirecting activity of T cells to tumor cells using engineered bispecific antibodies (one antibody is specific for the T cell receptor and the other antibody is specific for a tumor associated antigen). Recently, we have discovered a novel form of bispecific agent, folate coupled to an anti-TCR antibody. This agent targets tumors of the choroid plexus and ovary, which express high affinity folate receptors. Various folate conjugates are currently being tested in vitro and in vivo for their efficacy in recruiting a T cell response against the tumor.