The Preparation Of Human Antibodies Against Aβ

Alzheimer's disease (AD) is a progressive brain disease that commonly diagnosed in the elderly. its symptoms include memory loss, drawback of cognition and motion ability. With the accelerated aging of global population, The risk of Alzheimer's disease is continuingly rising and this disease is causing more social problems. Right now, there isn't current cure for AD, so it has important significance to develop the special drug for AD. At this stage, it is not well understood about the cause of AD, but amyloid beta (Aβ) which is toxic to neurocyte deposits from soluble monomer are the fundamental cause of the disease. And there are research results showed that Aβis the common and key factor in the occurrence and progress of AD, and then Aβis thought to be the attractive target for AD therapy. Aβis formed throughβmetabolic pathway ofβ-amyloid precursor protein (APP) in the brain, it is made of 39-43 amino acids, has a secondary structure ofβ-sheet., and shows strong hydrophobicity, and tends to form the insoluble aggregations. In 1999, schenk etc got significant progress in AD therapy by immuninze mouse with Aβ1-42 in AD model. Since then, there were other reports showing that anti- Aβantibodies could be used in AD therapy. The AD vaccine, Betabloc(AN1792) developed by could effectively initiate the immune response and improve the patient's cognition ability in phase I clinic trial. But unfortunately, this research was terminated at phase II because of inflammation in the brain during treatment. Almost at the same time, other research showed anti- AβN terminal antibody could effectively relieve the AD symptoms and wouldn't ellicate trigger the Th1 cell immune response. The humanized antibody against Aβ(Bapineuzumab) developed by Elan and Wyth is now in the phase III clinical trial. In our work, we tried to confirm the human antibodies against N terminal (Aβ1-12 or Aβ1-15) of Aβby screening the phage-displayed human antibody library with huge size.Firstly, based on the structure of Aβ1-42 and characteristics of bacterial plagellum display system, Aβ1-42 was divided into four epitope peptides: Aβ1-12, Aβ12-23,Aβ21-32 and Aβ31-42, then these four recombinant plasmids respectively expression fusion gene of epitope peptide and bacterial flagellum genes were constructed, and then the expression of fusion genes was confirmed by SDS-PAGE and Western blot.Secondly, we conducted the screening of human antibody library with size of 1.35×1010. Different screening strategies were used in this work, including screening with bacterial displayed Aβ1-12, screening with coating Aβ1-42, screening with bacterial displayed and coating Aβ1-12 ,screening with coating Aβ1-15.Totally, about 2000 positive clones were characterized, and results showed there is low positive ratio while screening with bacterial displayed Aβ1-12, but if screening with coating peptides, the positive ratio could reach about 50%., and if combining bacterial displayed peptide with coating peptide , the positive ratio was bout 40%.Finally, one phage displayed antibody G10 was confirmed by screening with bacterial displayed Aβ1-12, two phage displayed antibodies 18 and 26 were confirmed by screening with coating Aβ1-42, two phage displayed antibodies H9 and B5 were confirmed by screening with bacterial displayed and coating Aβ1-12,and one phage displayed antibody 87 was confirmed by screening with coating Aβ1-15. It is interesting that sequencing results showed that 18 and H9 had the same sequence. These results showed that our screening strategies were reliable and feasible.Then, The selected phage displayed antibodies were reconstructed to whole antibody (IgG1) for further characterization of specificity. All five phage displayed scFv were reconstructed into whole antibody, try to express these antibodies in 293 transient expression system, but only three antibodies (G10, 26 and B5) were successfully expressed in this expression system. And further binding assay showed that G10 couldn't bind Aβ1-42, only 26 and B5 could specifically bind Aβ1-42.At last, the binding epitope and affinity of 26 and B5 were assayed. By testing the binding of these two antibodies to bacterial displayed different Aβepitope peptide, the results showed antibody 26 binds Aβ31-42, and antibody B5 binds Aβ1-12. And the affinity of B5 to Aβ1-12 was about KD=1.4×10-8mol/L.This work laid a base for further development of therapeutic antibody for AD.