Development Of Full Human Anti-C5 Single-chain Variable Fragment For Optic Neuromyelitis

Ojective:Neuromyelitis optica?NMO?spectrum disorder?NMOSD?is an autoimmune disease of the central nervous system?CNS?inflammatory demyelinating disease,mainly involving the spinal cord and optic nerve,and may also involve the brain parenchyma.Aquaporin 4?AQP4?autoantibodies?AQP4-immunoglobulin G,AQP4-IgG?against astrocytes AQP4 lead to inflammatory responses,astrocyte loss,oligodendrocyte and neuronal death.At present,NMOSD therapies are including general immunosuppressive agents and plasma exchange,but patients still have high mortality and serious sequelae.Monoclonal antibodies have attention in the treatment of NMO.For example,rituximab,eculizumab,and tocilizumab have entered clinical trials that have good clinical results in the treatment of NMO;however,most of the monoclonal antibodies currently used in clinical are of a murine origin that may cause human anti-mouse anti-antibody?HAMA?for long-term application.Phage display is an important method for obtaining fully human monoclonal antibodies.We aimed to select fully human scFvs against complement C5 using phage display to treat NMO from semi-synthetic full-human scFv library.Method:We screened scFv bingding C5 by phage display from full-human scFv library(6×10¹⁰),and after 5 rounds of screening?adsorption-elution-amplification?,randomly selected 239.The phage clones were verified the ability of binding C5 by enzyme-linked immunosorbent assay?ELISA?.Some clones with high A450 nm values were selected for sequencing,and analysis sequence by DNAMAN.The three genes with the highest homology were picked and inserted into the prokaryotic expression vector pET30a?with his tag?.The prokaryotic expression vector with scFv was induced using Isopropyl?-D-Thiogalactoside?IPTG?at 16°C overnight in E.coli BL21.The cells were collected for protein purification.After a series of affinity chromatography,DEAE chromatography and Butyl chromatography purifications,soluble scFv with a purity of more than 90%was obtained.The protein concentration was concentrated to 40 mg/ml with an ultrafiltration tube.The protein was observed by SDS-PAGE protein electrophoresis.The specificity of soluble scFv and C5 was identified by Western Blot and ELISA.Biacore T200 was used to further determine the affinity of scFv and C5.In vitro,Using AQP4-IgG and complement mediated complement-induced cytotoxicity?CDC?in AQP4-transfected cells,and verifying its cytotoxicity by LIVE/DEAD staining method,In vivo,IgG from NMOSD seropositive patients(IgG_NMOSD)and human complement?hC?injected into mice by intraparenchymal injection.Related tissue staining to determine changes in lesions and inflammatory cells,further the protective effect of the screened single-chain antibody on NMO was determined.Result:1.After 5 rounds of phage screening,8 scFv sequences?C5B3,C5B35,C5B98,C5B106,C5A6,C5A102,C5A121,C5A152?were obtained,and the three sequences with the high homology were purified by prokaryotic expression.A series of purifications were performed to obtain the purity more than 90%.2.The soluble scFv?C5B3,C5A6,C5B35?was functionally verified.In vitro,LIVE/DEAD staining showed that C5B3 significantly reduced the complement-dependent cytotoxicity of CHO cells stably expressing AQP4,and reduced the formation of membrane attack complexes and the number of dead cells.The protective effect significantly dependent on the C5B3 concentration.3.In vivo,C5B3 significantly reduces the volume of lesions and infiltration of inflammatory cells that caused by injection of IgG_NMOSD and hC,and reduce the formation of membrane attack complexes.Conclusion:ScFv specifically binding complement C5 was obtained by phage library.In vitro and vivo it was confirmed that one scFv?C5B3?can reduce the cytotoxicity caused by AQP4-IgG and hC,and also play a protective role in NMO animal model.