Effect And Mechanism Of Anti-oligomeric A?42 ScFv Antibody

Alzheimer's disease(AD)is the most common neurodegenerative disease in the elderly people.It is expected that by the year 2050,there will be more than 131.5million dementia patients worldwide.The most important pathological features of AD It is a senile plaque and neurofibrillary tangles(NFTs),the former being an extracellular aggregate of A?peptides and the latter being an aggregate of intracellular hyperphosphorylated Tau proteins,a microtubule-associated protein.The etiology and pathogenesis of AD remains controversial.The most influential hypothesis in recent years is the amyloid cascade hypothesis.This hypothesis assumes that the initial event of AD is the production of A?42 by amyloid precursor protein during cleavage and degradation by secretory enzymes,followed by a series of pathological changes,which in turn leads to the occurrence of AD.The theory originally thought that neuronal dysfunction and death were caused by the neurotoxic effect of total A?content,and it was recently proposed that not only A?peptide monomers,but also oligomeric forms of A?peptides(2 to 12 peptides)cause neuronal dysfunction.The small aggregates are actually more detrimental to brain function than A?high-aggregation forms such as senile plaques.In the previous study,our group obtained two anti-oligomeric A?42 single-chain Fv antibodies,MO6 and HT7,and found that scFv MO6 and scFv HT7 can specifically recognize A?42 oligomers and fibrils,can inhibit their aggregation and can promote their disaggregation.In addition,scFv MO6 and scFv HT7 have a significant inhibitory effect on the neurotoxicity of oligomeric A?42.However,scFv MO6 and scFv HT7 exhibit different efficacies on the toxic A?42 oligomers,such as the binding of HT7 to A?42 aggregates.Since scFv MO6 and HT7 have very similar heavy chain domains(VH)but different light chain domains(VL),it is speculated that this may be the reason for their functional differences.In order to character the difference in the interaction between the VH or VL domain and A?42,we systematically analyzed the effects of the VH and VL domains in recognizing and binding A?42 oligomers and in inhibiting their aggregation and cytotoxicity,and proposed the functional mechanisms,as well as the contribution of the VH and VL domains to the full scFv HT7.We have obtained the following results:(1)The recombinant expression vectors pET28a-scFv-VL and pET28a-scFv-VH were constructed by means of molecular cloning technique and the highly purified VH and VL proteins were prepared by using the expression engineering strains of VH and VL domains.(2)It was similar for the VH and VL domains to the full scFv HT7 to have specificity for the oligomeric forms of A?42(oligomers and fibrils),but not the fiber form of A?42.(3)The apparent affinity of the VH or VL domain to A?42 oligomers is higher than that of VH&VL by measuring the equilibrium dissociation constant(K_D)of the antigen and antibodies,the later was higher than the full scFv HT7.(4)The VL domains tend to interact with low molecular weight A?42 aggregates,whereas the VH domains tend to interact strongly with A?42 oligomers and protofibrils and inhibit their further aggregation.(5)The VH and VL domains were similar to the full scFv HT7 in inhibiting the cytotoxicity of A?42 by a concentration-associated manner and the VH&VL system also had the strongest inhibitory effect.Thus,the results of(2)to(5)above are consistent,both suggesting that the VH domain,although having a high affinity for A?42 oligomers,had a tendency of self-aggregation,while the VL domain,although having a slightly lower affinity for A?42 oligomers,has a lower tendency to self-aggregate.Therefore,the mixed system of VL and VH domain has a stronger inhibitory effect on the cytotoxicity of A?42,which was higher than that of the full scFv HT7.This also shows that the linker in the full scFv HT7 has a certain constraint for the function of the VH and VL domains.However,the existence of linker increased the complex effect of VH and VL domain,so the effective concentration of the full scFv HT7 is lower than VH&VL system.The results of this thesis laid the foundation for the further modification of anti-A?42 single-chain antibody at the molecular level,and also provided theoretical guidance for the development of more effective single-chain antibodies against cytotoxic A?42 and those promoting the metabolism of A?42.The results of this thesis also laid the theoretical and practical basis for the diagnosis and treatment of Alzheimer's disease.