title: Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity.
abstract: Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the β-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the `empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure.
authors.forename: Jianya Y
authors.initials: JY
authors.lastname: Huan
authors.forename: Roberto
authors.initials: R
authors.lastname: Meza-Romero
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: Mooney
authors.forename: Yuan K
authors.initials: YK
authors.lastname: Chou
authors.forename: David M
authors.initials: DM
authors.lastname: Edwards
authors.forename: Cathleen
authors.initials: C
authors.lastname: Rich
authors.forename: Jason M
authors.initials: JM
authors.lastname: Link
authors.forename: Arthur A
authors.initials: AA
authors.lastname: Vandenbark
authors.forename: Dennis N
authors.initials: DN
authors.lastname: Bourdette
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Bächinger
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Burrows