Engineering soluble, high affinity receptor antagonists for bacterial exotoxins.

摘要

Superantigens, including many bacterial enterotoxins, are a family of proteins that act by inducing a hyperactive immune response. These proteins bind to cell surface molecules encoded by the major histocompatibility complex and to variable beta (Vbeta) regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, causing a condition known as toxic shock syndrome. Due to the lack of available therapeutics, we have developed high-affinity soluble Vbeta domains that neutralize superantigen activity by competing for the T cell binding site.; In Chapter two, the process of engineering a high-affinity receptor for the superantigen TSST-1 is described. Using random mutagenesis the human Vbeta2.1 region of a TCR was selected for enhanced display on the surface of yeast. These stabilized clones were subjected to two rounds of directed evolution to enhance binding to TSST-1. The location of the mutations isolated provides information on the molecular interactions leading to high-affinity.; In Chapter three, a Vbeta region with high-affinity for TSST-1 was further characterized. The contribution of individual residues was analyzed by alanine scanning mutagenesis of Vbeta2.1. Residues that were important for binding TSST-1 resided in two distinct hot regions. Additional mutational analysis performed by Eric Sundberg and Beenu Moza determined that mutations in these hot regions acted cooperatively, even though they were separated by 20A. A recently solved crystal structure provided further details about the TCR-superantigen complex.; Chapter four describes the engineering of murine Vbeta8.2 for picomolar affinity to staphylococcal enterotoxin B (SEB). As the known contact regions had already been heavily mutagenized, additional engineering of the Vbeta was performed through extension of the CDR1 loop. Soluble forms of the high-affinity Vbeta regions were tested for their ability to inhibit SEB-mediated T cell cytotoxicity in vitro. As the affinity of the Vbeta regions increased, the amount of protein needed to neutralize 50% of the toxin activity correspondingly decreased. These Vbeta regions were also tested in various rabbit models of toxic shock by Patrick Schlievert, and were remarkably effective at protecting rabbits from the lethal effects of the toxin.