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

This study describes the isolation and characterization of Fv fragments that recognize a T cell receptor V alpha (V alpha 1934.4). A VH gene repertoire from an immunized mouse was recombined with the anti-hen egg lysozyme (HEL) V kappa D1.3 gene as single chain (sc)Fvs, and an Fv with reasonable affinity for binding to V alpha 1934.4 isolated. The Fv (VH14/V kappa D1.3) does not bind to HEL, indicating that the heavy chain shuffling has converted an anti-HEL specificity to one that recognizes the unrelated V alpha 1934.4. The association constant for the Fv-V alpha 1934.4 interaction has been determined using surface plasmon resonance (SPR) and is 1.2 x 10(7) M-1. Recombinant antibodies of reasonable affinity can therefore be generated by combining a VH library with a 'fixed' V kappa. To improve the affinity further, light chain shuffling has been used to generate an Fv (VH14/V kappa 9) that has a 30-fold higher affinity for binding to V alpha 1934.4 than the parent (VH14/V kappa D1.3) Fv, and SPR measurements demonstrate that the affinity improvement is due to an increase in on-rate. Unexpectedly, V kappa 9 differs from V kappa D1.3 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.