Increase of spacer sequence yields higher dimer (Fab-spacer-toxin)₂ formation

The divalent antibody-toxins are expected to have increased binding avidities to target cells because of the two cell-binding domains. However, previous studies showed that the refolding yield of divalent antibody-toxin is very low, and it is assumed that homodimer formation of antibody-toxin is strongly interfered by the repulsion between the two large toxin domains that come close to each other during dimer formation. In this study, B3 antibody was used as a model antibody, and its Fab domain was used to construct three different kinds of Fab divalent molecules, [B3(Fab)-toxin]₂. The monomer Fab-toxin molecules were made by fusing the Fab domain of monoclonal antibody B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE), and a connecting sequence that contained spacer amino acid sequence (G4S)n (n=1, 2, 3) was inserted between Fab and PE38. The prepared divalent molecules were [Fab-S1, 2, 3-PE38]₂ (=[Fab-SKPCIST-KAS(G₄S)nGGPE-PE38]₂ (n=1, 2, 3)), and they are derivatives of previously studied [Fab-H2cys-PE38]₂ (=[Fab-SKPCIST-KASGGPE-PE38]₂). In [Fab-S1, 2, 3-PE38]₂, two Fab-S1, 2, 3-PE38 monomers were covalently linked by the disulfide bond bridge made from cysteine in the -SKPCIST-sequence. The insertion of spacer amino acids after the disulfide bridge resulted in a 12-18 fold higher yield of dimer formation than previously constructed [Fab-H1cys-PZ38]₂ [7], 3-4-fold higher than [Fab-ext-PZ38]₂ [25]. These two molecules have less amino acid spacer sequence between the disulfide bridge and PE38 domain. The design of [Fab-PE38]₂ in this study gave molecules with a higher refolding yield. The results of cytotoxicity assay showed a higher cytotoxic effect of these divalent molecules than that of the monovalent scFv-PE38 molecule.