STABILIZATION OF A RECOMBINANT FV FRAGMENT BY BASE-LOOP INTERCONNECTION AND V-H-V-L PERMUTATION

We have developed a novel method to stabilize a recombinant antibody F v fragment. The V-H and V-L domains of this Fv fragment, called pFv (p ermutated Fv), are covalently interconnected to each other at the two ''base-loops'' that normally connect V-H beta strand 3 to 3b and V-L b eta strand 3 to 3b. To produce the base-loop stabilized Fv fragment, w e connected the N-terminal half of the V-L domain (V-L 1-40) of murine antibody anti-Tac to the C-terminal half of V-H (V-H 42-115). We also fused the C terminus of V-H by a (Gly,Ser), Linker to the N-terminal half of V-H (V-H 1-40, thereby generating a permutated V-H domain). Fi nally we connected the base loop of V-H (N-terminal half) to the C-ter minal half of V-L (V-H 42-115). The anti-Tac pFv fragment was fused to a truncated form of Pseudomonas exotoxin to generate a pFv-immunotoxi n. Fvs with the correct structure were produced by refolding of recomb inant inclusion body protein using a renaturation protocol that was or iginally developed for Fab and scFv fragments. Due to the artificially connected and permutated primary sequence, the folding pathway for th e pFv structure may possibly be different from the conventional foldin g of antibody domains. Analysis of antigen binding of anti-Tac pFv, an d of the specific cytotoxicity of pFv-immunotoxin towards antigen expr essing cancer cells demonstrated that the anti-Tac pFv retained most o f its affinity and full specificity when compared to anti-Tac scFv. Al so anti-Tac pFv was relatively stable, retaining 25% of its binding ac tivity after a 24 hour incubation in human serum at 37 degrees C. This indicates that connection of base loops can be a useful alternative t o linker or disulfide stabilization of Fv fragments.