Central amyloid-ß-specific single chain variable fragment ameliorates Aß aggregation and neurotoxicity.

Anti-amyloid-ß immunotherapies are a promising therapeutic approach for the treatment and prevention of Alzheimer's disease (AD). Single chain antibody fragments (scFv) are an attractive alternative to whole antibodies due to their small size, single polypeptide format and inability to stimulate potentially undesirable Fc-mediated immune effector functions. We have generated the scFv derivative of anti-Aß monoclonal antibody, 1E8, known to target residues 17-22 of Aß. Here we show that the soluble 1E8 scFv binds to the central region of Aß with an affinity of ~55 nM and significantly reduces fibril formation of Aß(1-42). Furthermore, 1E8 scFv ameliorates Aß(1-42)-mediated toxicity in the PC12 cell line and murine primary neuronal cultures. This ability to both target the central region of Aß and prevent Aß(1-42) neurotoxicity in vitro makes it a promising therapeutic antibody building block for further functionalization, toward the treatment of AD.

scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent.