The Single-chain Variable Fragment Antibodies Against Aβ42in Alzheimer’s Disease Arid Their Molecular Mechanism

Alzheimer’s disease (AD) is neurodegenerative diseases among older peoplewhich leads to memory loss and cognitive dysfunction and is difficult to heal after theonset. AD is characterized pathologically by the deposition β-amyloid protein (Aβ)which further formation of senile plaques (SP), and intracellular neurofibrillarytangles caused by hyperphosphorylation of Tau protein and neurons necrosis.Abundant evidences suggest that oligomeric forms of β-amyloid protein are the mainneurotoxins causing AD.Recently, significant efforts have been made towards the generation andassessment of conformation-dependent single chain fragment variable (scFv)antibodies capable of preventing and clearing amyloid aggregates as well aspreventing their synaptotoxic effects.To construct the human single-chain fragment variable (scFv) antibody libraryand obtain the specific scFv against soluble Aβ42(Amyloid-beta) oligomers byscreening the human scFv library, peripheral blood lymphocytes (PBL) wereseparated from the donors, and the total RNA was extracted from the PBL. Thevariable heavy (VH) and variable light (VL) genes were amplified by RT-PCR andthen the scFv was obtained through SOE-PCR. The scFv fragments were cloned intothe vector pET41b and transformed into competent E.coli BL21(DE3) cells, and thenestablished the scFv antibody library.Recombinant scFvs specific for Aβ42oligomers-positive clones were selected byELISA. The soluble scFv antibodies were expressed successfully, and displayedspecific binding to Aβ42oligomers and protofibrils by Dot-blot and Western blotanalysis. The binding affinity of soluble scFv antibodies to Aβ42oligomers were allabout10-6mol/L level. The scFvs reduced their level by blocking their formation orinducing their disassembly. Consequently, scFvs ameliorated or prevented theircytotoxicity and protected cell cultures from their damage in a concentration-dependent manner. Comparison of their cytotoxicity-inhibiting andcytotoxicity-neutralizing activities indicated that scFvs displayed their protectiveeffect on target cells mainly due to their cytotoxicityinhibitory activity though theycould also neutralize the cytotoxicity. In addition, scFvs efficiently passed through thein vitro BBB (blood-brain barrier) model with a delivery efficiency of over70%after60min post-administration.In conclusion, we obtained four Aβ42oligomers or immatureprotofibrils-specific scFv andtibodies, from the scFv antibody library in the presentstudy. Considering their unique and superior properties for AD immunotherapy, thefour scFvs or their fragments may be of interest as components of futureantibody-based therapeutics for AD as well as other neurodegenerative diseases.