Stabilization of ricombinant Fv fragment by base-loop interconnection and VH-LH permutation

We have developed a novel method to stabilize a recombinant antibody Fv fragment. The VH and VL domains of this Fv fragment, called pFv (permutated Fv), are covalently interconnected to each other at the two ``base-loops'' that normally connect VH beta strand 3 to 3b and VL beta strand 3 to 3b. To produce the base-loop stabilized Fv fragment, we connected the N-terminal half of the VL domain (VL 1-40) of murine antibody anti-Tac to the C-terminal half of VH (VH 42-115). We also fused the C terminus of VH by a (Gly4Ser)3 linker to the N-terminal half of VH (VH 1-40, thereby generating a permutated VH domain). Finally we connected the base loop of VH (N-terminal half) to the C-terminal half of VL (VH 42-115). The anti-Tac pFv fragment was fused to a truncated form of Pseudomonas exotoxin to generate a pFv-immunotoxin. Fvs with the correct structure were produced by refolding of recombinant inclusion body protein using a renaturation protocol that was originally developed for Fab and scFv fragments. Due to the arti®cially connected and permutated primary sequence, the folding pathway for the pFv structure may possibly be different from the conventional folding of antibody domains. Analysis of antigen binding of anti-Tac pFv, and of the speci®c cytotoxicity of pFv-immunotoxin towards antigen expressing cancer cells demonstrated that the anti-Tac pFv retained most of its af®nity and full speci®city when compared to anti-Tac scFv. Also anti-Tac pFv was relatively stable, retaining 25% of its binding activity after a 24 hour incubation in human serum at 37C. This indicates that connection of base loops can be a useful alternative to linker or disul®de stabilization of Fv fragments.