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|Title:||Characterization of a single-chain anti-T cell receptor antibody and a single-chain T cell receptor|
|Author(s):||Schodin, Beth Arlene|
|Doctoral Committee Chair(s):||Kranz, David M.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
Health Sciences, Immunology
|Abstract:||The T cell receptor (TCR) is a cell surface protein that specifically binds to a foreign antigen on the surface of another cell. The TCR is made up of two disulfide-linked chains (called $\alpha$ and $\beta$) each containing a variable and a constant domain. The tertiary structure and binding properties (e.g. affinity and kinetics) of the TCR have not been solved. Additionally, the T cell has been implicated in many autoimmune diseases and some reports show that antibodies to the TCR may possibly control these diseases.
It has been shown that antibodies to the TCR can either interfere with binding to the ligand (antagonist) or directly activate a T cell (agonist). To begin to control these opposing properties of anti-TCR antibodies, a single-chain form of the anti-TCR antibody 1B2 was engineered by linking the variable regions genes of the heavy and light chains. Monomeric and noncovalently-linked dimeric forms of the scFv 1B2 were isolated from E. coli inclusion bodies. The binding affinities of the scFv 1B2 monomer and dimer were 20- and 2-fold lower than the 1B2 Fab fragments, respectively. Both forms of the scFv 1B2 were able to inhibit the activity of the T cell, but the dimer was significantly more effective than the monomer. Furthermore, when the scFv 1 B2 was presented in a multivalent form on the surface of another cell it acted as an agonist in that it mediated killing of the 'targeted' cell. These results showed that the scFv could potentially be used for inhibiting detrimental in vivo T cell activity and mediating lysis of a detrimental cell such as a tumor.
To study the structure and binding properties of the TCR, a single-chain form of the TCR from a T cell clone was examined. The linked V$\beta$ and V$\alpha$ regions were expressed in E. coli and isolated from inclusion bodies. The concentration of active scV$\beta$V$\alpha$ in the solubilized, folded protein was low but detectable. Various expression vectors, solubilization, and folding conditions were evaluated to improve this yield. In the optimum system, the concentration of active, properly folded scV$\beta$V$\alpha$ was increased 100-fold. These concentrations do not allow high resolution structural analysis but are sufficient for binding studies.
|Rights Information:||Copyright 1995 Schodin, Beth Arlene|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9543717|