The Nature Of The Anti-a¦Â Monoclonal Antibody, Biological Effects And Humanized Transformation Research

Alzheimer's disease(AD) is a common progressive degenerative disease of the centralnervous system. AD is the most prevalent forms of dementia. It is histopathologicallycharacterized by beta-amyloid-containing plaques, tau-containing neurofibrillary tangles,reduced synaptic density and neuronal loss in selected brain areas. So far, there is no specifictherapy for AD. The accumulation of beta-amyloid (Aβ) peptides in the senile plaques is oneof the hallmarks of the progression of AD, and has been considered to be the main cause forneuronal injury and impairment of memory. Both active and passive immunological therapieshave been proved successful in animal trials, which can reduce amyloid load and reversememory deficits in AD mouse model. Thus, antibodies against Aβmay be a potentialtherapeutic strategy for AD. Anti-Aβmonoclonal antibody studying may provide a hopefulapproach for AD therapy.Based on the new therapy strategy of AD with mAb agaist Aβ, our study is dedicated topreparing chimeric antibody agaist Aβ_1-42 by gene engineering and discussing issues about itshumanization. Three parts is included in the work: preparation of mAb agaist Aβ_1-42, itstherapeutic effect study in AD model animals and primary study of its humanization.1. Anti-AβmAb preparation, and characterizationAβ_1-42 is a kind of peptide with low molecular weight(MW), about 4 kDa. It is veryhard to prepare corresponding mAb for its weak immunogenecity. At first, we preparemAb with regular way, but only got IgM, with low titres and hard in purification. Wechanged the immunological strategy and finally got IgG, whose subclass is IgG1, withtitres of 1:1×10⁶ and K_aff of 1×10⁹ M(-1). Using Protein A affinity chromatography, themAb with high purity (＞90%) was obtained.2. Study of therapeutic effect of anti-Aβ_1-42 mAb in AD mouse modelIt has been reported by foreign researchers that anti-Abeta mAb can reduce Aβload and improve the memory impairment in AD mouse model. But there is no relatedarticles available in our country. To prove whether our anti-Aβ_1-42 mAb is effective intreating AD, we performed animal trials. BALB/c mice were divided into four groups:positive control(AD mouse model), negative control, mAb therapy group, group of falseoperation. AD mouse model was construct by injecting Aβ_1-40 (10μg/mouse)into hippocampus of mouse brain. Anti-Aβ_1-42 mAb was administered (500μg/mouse)byintraperitoneal(IP) 3 days before intracerebral injection of Aβ_1-40. and will beadministered every 3 days until the forth injection 8 days Morris water maze behavioraltrial was performed 12 days after model establishment and the results statisticallyanalyzed indicated: for positive control, the mean latent time for searching hiddenplatform is longer than the negative groups(P＜0.05); for mAb therapy group, thememory impairment was improved(P＜0.05). According to behavioral study, anti-Aβ_1-42mAb is effective in improving the memory deficits of AD mouse model in vivo.3. Humanization of anti-Aβ_1-42 monoclonal antibodyMouse monoclonal antibody can not be used in clinical research unless behumanized. The main purpose of our study is to construct human-mouse chimericantibody gene and establish reasonable expression system. First, collect 10⁷ hybridomacells, and extract cell total RNA using Trizol method. Then perform RT-PCR foracquirement of the first strand of cDNA. Finally, amplify the target antibody gene byusing specific primers. We acquired human-mouse chimeric antibody gene byperforming overlap PCR, then cloned the gene into pcDNA3.1 vector, with the purposeto express it in mammalian cells. In the humanization research work we mainlyconstruct the human-mouse chimeric antibody gene, and have set an very importantbasis for further study in this work.In this study, we have successfully made two kind ofanti-Aβ_1-42 mAb, with titres of1:1×10⁶ and K_aff of 1×10⁹ M^-1. The mAb can recognize and bind Aβ_1-42 specifically.Animal trials have proved that our anti-Aβ_1-42 mAb can improve the memory deficit ofthe AD mouse model. At the same time, we have successfully cloned and studied thevariable region of the antibody's gene. We also constructed the human-mouse chimericantibody gene, and will be good for further study in humanization. Our antibody will bea powerful tool for AD therapeutic study. But further experiment should be performed totest how our antibody work in vivo and whether it can be used in clinical study.