VH shuffling can be used to convert an Fv fragment of anti-hen egg lysozyme specificity to one that recognizes a T cell receptor Vα

Abstract

This study describes the isolation and characterization of Fv fragments that recognize a T cell receptor Vα (Vα 1934.4). A VH gene repertoire from an immunized mouse was recombined with the anti-hen egg lysozyme (HEL) VκD1.3 gene as single chain (sc)Fvs, and an Fv with reasonable affinity for binding to Vα1934.4 isolated. The Fv (VH14/VκD1.3) does not bind to HEL, indicating that the heavy chain shuffling has converted an anti-HEL specificity to one that recognizes the unrelated Vα1934.4. The association constant for the Fv-Vα1934.4 interaction has been determined using surface plasmon resonance (SPR) and is 1.2 × 10 7 M −1. Recombinant antibodies of reasonable affinity can therefore be generated by combining a VH library with a ‘fixed’ Vκ. To improve the affinity further, light chain shuffling has been used to generate an Fv (VH14/Vκ9) that has a 30-fold higher affinity for binding to Vα1934.4 than the parent (VH14/VκD1.3) Fv, and SPR measurements demonstrate that the affinity improvement is due to an increase in on-rate. Unexpectedly, Vκ9 differs from VκD1.3 by only two amino acids at positions 30 and 91 and, consistent with the change in binding affinity, both of these residues are located in CDRs.