The Mechanism Of Passive Immunotherapy Of A Novel Anti-Aβ3-10 Monoclonal Antibody 7B8 In Alzheimer’s Disease

Objective: The basic pathological features of Alzheimer’s disease(AD)are deposition of beta-amyloid(Aβ)and neurofibrillary tangles formed by hyperphosphorylation of tau protein in the brain,accompanied by synaptic dysfunction and cognitive decline.Although passive immunotherapy targeting Aβ has failed to delay cognitive impairment in clinical trials,these antibody data provide important evidence for subsequent development.Aβ oligomers are the most important toxic substances in the brain and should be the primary binding target of monoclonal antibodies.The N-terminal truncated Aβ peptide modified with pyroglutamate(Aβp E3)represents all the important components of Aβ in the brain of patients with AD,and shows more toxic effects due to its easier aggregation and non-degradation properties.Based on our previous work,transgenic mice actively immunized with Aβ3-10-KLH showed the effect of eliminating Aβ oligomer to improve cognition.We screened Aβ3-10 monoclonal antibody and further studied its mechanism.Method: 1)Using Aβ3-10-KLH as antigen,myeloma cells were obtained through immunization,cell fusion,and multiple rounds of screening.Monoclonal antibody 7B8 was produced and purified by shaking flask,and its binding Aβ type was verified by in vitro binding experiments.2)Monoclonal antibody 7B8 was passive immunotherapy for three transgenic(3×Tg)AD mice.The changes of Aβ in the brain of mice were detected by immunohistochemical staining,western blot,and ELISA,the mechanism of Aβscavenging by 7B8 was investigated by immunofluorescence staining method,and the changes of cognitive function were detected by behavioral experiment.The effects of7B8 passive immunotherapy on the phosphorylation of tau protein and synaptic protein and intracerebral microbleeds were detected.Result: 1)Monoclonal antibody 7B8 was prepared and identified as mouse Ig G1 with heavy chain constant region and mouse κ chain with light chain constant region;2)ELISA,Western blot,and Dot blot showed that the prepared monoclonal antibody 7B8 was relatively weak in binding Aβ monomer,and had a strong binding effect on Aβ42oligomers,fibrils,and Aβp E3 oligomers,fibrils;3)Monoclonal antibody 7B8 can bind to loose and dense core senile plaques and intracellular Aβ;4)Passive immunotherapy with monoclonal antibody 7B8 reduced Th S positive(P<0.01)and 6E10 positive senile plaques(P<0.05)in the cortex and hippocampus of 3×Tg mice;5)Passive immunotherapy with monoclonal antibody 7B8 reduced intracellular Aβ oligomers and Aβp E3 oligomers in the brain of 3×Tg mice;6)Passive immunotherapy with monoclonal antibody 7B8 reduced the load of soluble Aβ42(P<0.05)and insoluble Aβ40,Aβ42(P<0.05),and the load of soluble(P<0.05)and insoluble(P<0.01)Aβp E3 in brain of 3×Tg mice;7)GFAP immunohistochemical staining showed that the load of astrocytes in the cortex(P<0.05)and hippocampus(P<0.01)of 3×Tg mice was significantly decreased,and Iba1 immunohistochemical staining showed that the load of microglia in the hippocampus of 3×Tg mice were decreased(P<0.05);8)7B8 passive immunotherapy reduced the levels of TNF-α and IL-6 in the hippocampus and cortex of3×Tg mice(P<0.05),but did not increase the overall levels of TNF-α,IL-6,and IL-1β in the brain of 3×Tg mice;9)Iba1-positive microglia near plaques in 3×Tg mice were significantly increased by passive immunotherapy with monoclonal antibody 7B8(P<0.05),and cerebral amyloid angiopathy(CAA)in the hippocampus was reduced by passive immunotherapy with monoclonal antibody 7B8(P<0.05);10)Passive immunotherapy with monoclonal antibody 7B8 increased synaptophysin levels in cortex and hippocampus(P<0.01),PSD95 level in cortex increased(P<0.05),but did not increase intracranial microbleeds(P=0.58)in 3×Tg mice;11)7B8 passive immunotherapy improved the nesting score of 3×Tg mice(P<0.05),and improved the discrimination index of the novel object recognition test(P<0.05);12)Passive immunotherapy with monoclonal antibody 7B8 reduced the levels of AT180,p Tau396,and p Tau404 in the hippocampus of 3×Tg mice(P<0.05),but no significant changes were observed in the levels of HT7 and AT8.Conclusion: 1)The novel monoclonal antibody 7B8 can bind Aβ42,Aβp E3 oligomers,and fibrils,but has a relatively weak ability to bind Aβ42 monomers;Monoclonal antibody 7B8 binds to the diffuse and dense core senile plaques and binds to the intracellular Aβ component.2)Passive immunotherapy with monoclonal antibody7B8 reduced Aβ oligomers and senile plaques in the brain of 3×Tg mice,cleared the total amount of Aβp E3 in soluble and insoluble brain components,and reduced CAA pathology;Passive immunotherapy with monoclonal antibody 7B8 reduced brain inflammation,reduced tau phosphorylation,protected synaptic function,did not increase intracerebral microbleeds,and improved the cognitive function of the mice;The mechanism of Aβ scavenging by passive immunotherapy with monoclonal antibody 7B8 is consistent with FcγR mediated microglia recruitment and phagocytosis and the hypothesis of peripheral deposition,and does not increase the level of inflammatory factors in the brain.