Construction,Expression Optimization And Activity Analysis Of Therapeutic Single Chain Antibodies Against Alzheimer’s Disease

Alzheimer’s disease(AD),is a neurodegenerative disease,the main cause of dementia.Its main clinical manifestations are memory decline thinking ability decline and abnormal behavior.As the pathogenic factors of AD is complex,there is currently no drug that can cure or reverse the course of this disease.Therefore,AD remains one of the global public health and social care challenges facing humanity today and in the future.There are three characteristic pathological changes in AD patients: plaques formed by Amyloid-β(Aβ)aggregation,neurofibrillary tangles(NFT)formed by aggregation of highly phosphorylated microtubule-binding protein,and loss of neurons.At present,researchers generally believe that Aβ is the main pathogenic agent of AD.Therefore,through AD passive immunotherapy,injecting Aβ specific antibody into the patient to promote the clearance of Aβ in the patient’s brain is the most potential AD immunotherapy strategy.At present,some progress has been made in the study of AD therapeutic antibodies.In June 2021,Biogen received FDA approval for Aducanumab(Aduhelm)for the treatment of Alzheimer’s disease-related mild cognitive impairment(MCI)and mild Alzheimer’s disease.This is the first new drug approved by the FDA for the treatment of AD for the past 18 years,and has greatly encouraged researchers in the field of AD treatment.However,there are still some problems in the application of such drugs,such as the large molecular weight and the low blood-brain barrier permeability of the antibody,which limit its therapeutic effect.And the Fc fragment of the whole antibody,in the brain easy to cause the activation of immune cells,causing inflammation.Single chain fragment variable(scFv)is a genetically engineered antibody which contains only the variable region of the heavy chain and light chain of the antibody.Due to its small molecular weight,it can better cross the blood-brain barrier and bind to the pathogenic Aβ.In addition,scFv does not contain Fc fragment and is not likely to cause inflammatory response,which has unique advantages in the treatment of AD.In this study,two kinds of AD therapeutic antibodies(Aducanumab and Gantenerumab)were modified to construct specific single-chain antibodies Aducanumab-scFv and Gantenerumab-scFv which could recognize Aβ oligomer and pro-fibril.We used E.coli expression system for two single antibody expression,and optimized the conditions for its expression,determine the optimum culture condition,under the condition of the best training a large number of expressions,and USES the method of nickel column affinity chromatography for purification After get two kinds of high purity single antibody,we use ELISA method determined by MTT method The in vitro activity of two single chain antibodies was assayed.In addition,the in vivo activity of two single chain antibodies was assayed by immunohistochemistry and antibody injection.The results showed that two kinds of single-chain antibodies could bind to Aβ oligomer effectively and inhibit the cytotoxicity induced by Aβoligomer.In addition,two single-chain antibodies were able to bind to Aβ plaque deposition in the brain of AD model mice,showing excellent therapeutic potential to AD.In conclusion,we have successfully constructed two kinds of single chain antibodies for the treatment of Alzheimer’s disease,providing a new method for the immunotherapy of AD.