The Role And Mechanisms Of A Bispecific Antibody Fusion Protein Simultaneously Targeting The Complement System And VEGF In Choroidal Neovascularization

Purpose: Neovascular age related macular degeneration(neovascular AMD)is characterized by choroidal neovascularization(CNV).Intravitreal injection of antivascular endothelial growth factor(VEGF)agents is the mainstay of therapy for neovascular AMD.However,as a mono-target therapy,anti-VEGF therapy could benefit parts of patients in the short term.Both genetic and pathological studies have demonstrated that aberrant activation of the complement system plays an important role in the pathogenesis of AMD.Hence,we designed a novel bispecific antibody fusion protein called IBI302 that can inhibit VEGF and the complement system simultaneously.We explored the role and mechanisms of IBI302 in CNV,which may provide novel strategies for the treatment of neovascular AMD.Methods: 1.Human umbilical vein endothelial cells(HUVEC)were cultured in vitro and divided into blank control group,VEGF-induced angiogenesis group,IBI302 treatment group(experimental group),aflibercept treatment group(anti-VEGF group)and CID treatment group(complement inhibition group).The antiangiogenic potential of IBI302 in HUVEC was assessed by proliferation,migration,and tube formation assays.2.CNV was induced by laser photocoagulation in C57BL/6J mice.Mouse eyes were randomly divided into four groups treated with IBI302,aflibercept,CID or PBS through intravitreal administration.Fundus fluorescein angiography,immunofluorescence of choroid flat mounts and hematoxylin and eosin(H&E)staining of histopathological sections were performed to evaluate therapeutic efficacy of IBI302 on CNV formation.3.In laser-induced CNV model of mice,IBI302,aflibercept,CID or PBS was administered through intravitreal injection respectively.Western blot,ELISA,and immunofluorescence were performed to clarify the activation of complement system,and evaluate the expression of pro-angiogenic factors and pro-inflammatory cytokines in normal tissues and tissues after laser injury treated with different agents.4.The C57BL/6J mice receiving intravitreal injection of agents were evaluated by electroretinogram and H&E staining of histological sections to investigate the influence on retinal function and structure.Results: IBI302 significantly inhibited VEGF-induced proliferation,migration and tube formation of HUVEC,which was superior to that of CID.In the aspect of proliferation,IBI302 was superior to aflibercept.Compared with the aflibercept,CID,or PBS intervention group,IBI302 inhibited the formation and leakage of CNV more effectively.IBI302 diminished the deposition of C3 d,reduced the level of C5 a and suppressed the formation of membrane attack complex(MAC)to effectively inhibit complement system activation.IBI302 down-regulated the expression of proangiogenic factors and pro-inflammatory cytokines,such as VEGF,IL-1?,IL-18,COX-2,and TFG-?2.Furthermore,intravitreal injection of IBI302 did not induce retinal toxicity in mice.Conclusion: By dual inhibition of VEGF and the complement system,IBI302 synergistically exerts anti-angiogenic and anti-inflammatory effects,and might be considered as a promising therapeutic candidate for the treatment of neovascular AMD,which commands the future in its confidence.