A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin

T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.     

Limitations of yeast surface display in engineering proteins of high thermostability

Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein alpha3D were studied using yeast surface display. A library of alpha3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type alpha3D. Two other related sequences (alpha3A and alpha3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.