Generation And Characterization Of A Human Neutralizing Bispecific Antibody Against Rabies Virus

Rabies is a fatal nervous system diseasewhich takes place in Asia and Africa mostly.Once central nervous infection occurs and symptoms develop,the case fatality rate approaches 100%.We usually combinerabies immunoglobulin(RIG)and rabies immunization for treatment after exposure of rabies virus.Rabies immunoglobulin used in clinic mainly comes from human rabies immunoglobulin or equine rabies immunoglobulin(HRIG、ERIG)therefore the output of antibodies is limited and there are some problems in security of antibodies.With the development and improvement of engineering antibody technology,it is a trend for human genetically engineered antibody to replace blood-borne antibodies.However virus antigen mutation immune escape mechanism and defect of micromolecule antibody make therapeutic effency of rabies virus neutralizing antibody failed to 100%.It’s confirmed that human bispecific antibodies have extensive application prospect in disease treatment.Human bispecific antibodies are highly targeted and can specifically combine with different epitopes,producing superposition or synergistic effect to enhance ability of interdicting combination between virus and receptors.Dock-and-Lock(DNL)utilizes specific interaction characteristics between RII’s dimerization and docking domain(DDD)of protein kinase A(PKA)and anchoring domain(AD)of the protein PKA anchoring to develop bispecific antibodies for different epitopes.Anti-rabies virus bispecific antibodies can be produced by DNL and used for rabies treatment.In early experiments,the groupfiltrated some strains of anti-rabies virus Fab with high combining vitality from the human phageantibody library(6.7×108).Cloned strains Fab 094 and Fab 091 show high neutralization activity.This article produced anti-rabies virus antibodies Fab 094-DDD and IgG 091-AD using eukaryotic carrier expression system through genetic engineering and antibody engineering technology,identifying immunologic activity of purified antibodies,and produced anti-rabies virusbispecific antibodies by DNL,identifying neutralization activity of purified antibodies.These works lay a foundation for further developing rabiestheraputic antibody drug with high specificity and neutralization activity.Methods:1.Generation and characterization of anti-rabies virusFab 094-DDDLinker-C-DDD sequence was optimized and synthesized according to DDD gene sequence in Dock-and-Lock(DNL);the Fd section and the light chain variable region(Vκ)of anti-rabies virus Fab 094 were amplified,the Fd section of Fab 094 and linkerC-DDD gene were recombined using overlap PCR,Fd-DDD and Vκ were cloned into eukaryotic expression vectors and both vectors were transfected into 293Free style cells,Fab 094-DDD antibody was expressed and purified.The immunological features of Fab 094-DDD was detected by SDS-PAGE test,Western blotting assay,enzyme-linked immunosorbent assay(ELISA),Co-IP,affinity test and indirect immunofluorescence test.2.Generation and characterization of anti-rabies virus IgG 091-ADLinker-C-AD-C sequence was optimized and synthesized according to AD sequence(binding domain of Protein kinase A)in Dock-and-Lock(DNL);Genes of VH and light chains(λ chain)were amplified by the model of anti-rabies virusIgG 091 carrier.Genes of Λ chain and Linker-C-AD-C were recombined and connected through gene splicing by overlap extension PCR.Eukaryotic expression vectors of human anti-rabies virus IgG 091-AD were constructed by InFusion PCR and transfected into 293Free style cells,IgG 091-AD antibody was expressed and purified.The immunological features of IgG 091-AD was detected byW estern blotting assay,enzyme-linked immunosorbent assay(ELISA)and indirect immunofluorescence test.3.Generation and neutralizing ability analysis of anti-rabies virus bispecific antibodiesMix anti-rabies virus Fab 094-DDD and IgG 091-AD produced in earlier stage at the proportion of 1:1.1,next add reduced glutathione(final concentration 1mM)to the mixture.After 12-24h at room temperature mix oxidized glutathione(final concentration 2mM),then wait 12-24h at room temperature to promote intermolecular disulfide bond formation.Produce relevant protein through chromatographic purification of reagents above mentioned by Protein A affinity chromatography column and molecular sieve,and check the protein by SDS-PAGE.The immunological features of bispecific antibodies was detected by Western blotting assay and enzyme-linked Immunosorbent assay(ELISA).The neutralizing ability of Fab 094、IgG 091 and bispecific antibodies in vitro was detected by fluorescent antibody virus neutralization test(FAVN).Results:1.Generation and characterization of anti-rabies virusFab 094-DDD094 Fd genes(786 bp)and linker-C-DDD genes(204bp)were amplified by PCR.094 Fd-DDD genes(957bp)were amplified by overlap PCR.094 Vκ genes(405bp)were amplified and connected with linearizing pFUSEss-CHIg-hAl and pFUSE2ssCLIg-hk plasmids.Anti-rabies virus Fab 094-DDD were expressed by 293 Free style Expression System.After purified,antibodies Fab 094-DDD could specifically combine with rabies virus.2.Generation and characterization of anti-rabies virusIgG 091-AD091 λ genes(684bp)and linker-C-AD-C genes(141bp)were amplified by PCR.091 λ-AD genes(795bp)and VH genes(411bp)were amplified by overlap PCR.Antirabies virus IgG 091-AD was constructed successfully after pFUSE-CHIg-hGl-VH and pFUSE-CLIg-hL-λ-AD achieved by InFusion PCRtransforming escherichia coliDH5α.Anti-rabies virusIgG 091-AD was expressed by 293 Free style Expression System.According to detection,the system expressed IgG 091-AD antibodies with high obtained rate(about 4mg/100mL)and purity.IgG 091-AD antibodies we got had high affinity with rabies virus and it could specifically combine with rabies virus.3.Generation and activity analysis of bispecific antibodiesAnti-rabies virus bispecific antibodies were produced by DNL and we got high purity of antibodies after purification.According to detection,the bispecific antibodies could specifically combine with rabies virus and neutralization titer was 253.5 IU/mg.Neutralization titers of Fab 094-DDD and IgG 091-AD to rabies virus were respectively 213.2IU/mg and 30.77 IU/mg.So,the bispecific antibodies have obvious improvements in neutralizing activity to rabies virus.Conclusions:1.We successfully generated Fab 094-DDD and IgG 091-AD antibodies against rabies virus and the antibodies showed high binding activity.2.We successfully generated bispecific antibodies for different epitopes using Fab 094-DDD and IgG 091-AD.The antibodies had higher neutralizing activity and important application value for rabies treatment.