Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation

Ulrich Brinkmann, Angelina Di Carlo, George Vasmatzis, Natalya Kurochkina, Richard Beers, Byungkook Lee, Ira Pastan

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

We have developed a novel method to stabilize a recombinant antibody Fv fragment. The V(H) and V(L) domains of this Fv fragment, called pFv (permutated 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) beta strand 3 to 3b. To produce the base-loop stabilized Fv fragment, we 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 (Gly4Ser)3 linker to the N-terminal half of V(H) (V(H) 1-40, thereby generating a permutated V(H) domain). Finally we connected the base loop of V(H) (N-terminal half) to the C-terminal 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-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 artificially 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 specific cytotoxicity of pFv-immunotoxin towards antigen expressing cancer cells demonstrated that the anti-Tac pFv retained most of its affinity and full specificity 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 37°C. This indicates that connection of base loops can be a useful alternative to linker or disulfide stabilization of Fv fragments.

Original languageEnglish (US)
Pages (from-to)107-117
Number of pages11
JournalJournal of Molecular Biology
Volume268
Issue number1
DOIs
StatePublished - Apr 25 1997

Keywords

  • Anti-Tac
  • Antibody engineering
  • Cancer therapy
  • dsFv
  • scFv

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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