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 fragment s 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 e gg 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 rec ognizes the unrelated V alpha 1934.4. The association constant for the Fv-V alpha 1934.4 interaction has been determined using surface plasm on resonance (SPR) and is 1.2 x 10(7) M(-1). Recombinant antibodies of reasonable affinity can therefore be generated by combining a VH libr ary with a 'fixed' Vn. To improve the affinity further, light chain sh uffling 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 (V H14/V kappa D1.3) Fv, and SPR measurements demonstrate that the affini ty 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 a nd 91 and, consistent with the change in binding affinity, both of the se residues are located in CDRs.