Class Switching Of An Anti-fluB Broad-spectrum IgM MAb Via Activation-induced Cytidine Deaminase (AID) Activity And The Preliminary Functional Study Of These IgG MAbs

Influenza virus is one of the most important public health issues with high impact on human health,and Influenza B(FluB)is one of the main pathogens causing epidemics.While the rapid evolution of influenza virus making the development of broadly applicable antiviral drugs challenging.Broadly neutralizing monoclonal antibodies against Flu B,especially IgG antibodies,may show great potential use for therapeutic purposes of the dieases caused by influenza B virus.We have previously obtained an anti-Flu B broadly neutralizing monoclonal antibody,mouse immunoglobulin(Ig)M,7G1.Compared with the traditional IgG therapeutic antibodies,manufacturing challenges surround the use of IgM mAbs,which include more complicated process of production,weaker antibody-mediated cell killing effect and shorter half-life.In this study,we described an "cloning-free"approach to generate IgG class switching hybirdma cells in vitro.This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase(AID)to induce class switch recombination(CSR)coupled with high-throughput FACS of hybridoma cells to isolate IgG class switched cells.Next,characterization of the IgG mAbs was done in vivo and in vitro,and finally we obtained the functional IgG monoclonal antibodies.This thesis consists of three parts.The first part was to estalish a 7G1 hybridoma stable cell line with controllable expression of AID.Firstly,a 7G1 hybridoma cell line stably expressing tTA was established via pLenti-CMV-rtTA lentiviral transduction.Secondly,we designed a tetracycline-inducible AID expression cassette(Teton-AID-HA-2A-EGFP)into the lentiviral Tet-on vector pLenti-TRE3G.After the transduction,we successfully obtained a 7G1-Tet-on-AID hybridoma cell line with inducible expression of AID.This stable cell line incubated with Dox displayed dose-dependent induction of eGFP,corresponding to inducible expresion of AID.The second part of this study aimed to isolate IgG class switched 7G1 hybirdoma cells.We enriched the membrane IgG(mIgG)positive cells by FACS.After several rounds of FACS,mAbs with different IgG isotypes were observed in the culture supernatant via IgG isotyping ELISA,which include IgG1,IgG2a,IgG2b and IgG3.Then single mlgG positive cell was sorted into 96-well plates.Finally,we obtained 380 IgG positive hybridoma cell lines(IgG3:65%,IgG1:24%,IgG2b:10%,IgG2a:1%).The third part focused on characterization and comparison of the 7G1-IgG mAbs and the original IgM mAb in vivo and in vitro.we first identified and compared the in vitro binding activity of 7G1-IgM and 7G1-IgG.3-4 cell lines for each isotype were randomly selected to be tested,including in vitro hemagglutination inhibition(HI)assays and micropore neutralization(MN)assays,we found that the response-spectrum of some IgG mAbs was wider compared with IgM mAb;in addition,the representative mouse ascites purified 7G1-IgG mAbs,also showed a broader spectrum of viral reactivity than 7G1-IgM mAb.Moreover,in vitro ELISA binding assay,the binding activity of 7G1-IgG mAbs was significantly better than that of 7G1-IgM mAb.In addition,we found that IgG2a and IgG2b of 7G1 mAb had Fc mediated ADCC activity,while IgM mAb did not possess this ability to eliminate the virus.Meanwhile,the protective efficacy of these IgG mAbs was also evaluated preliminarily in vivo by virus challenge in the BALB/c mice model.All the IgG and IgM mAbs could provide mice complete protection against B/FIorida/4/2006(Yamagata lineage)and B/Brisbane/60/2008(Victoria lineage)with high-dose administration.Therefore,in order to distinguish the therapeutic activity of each 7G1 mab in vivo,we reduced the dosage of antibody.However,in the case of low dose administration,the protective efficacy of IgG2a-7G1 was the best,then IgG1.In addition,the in vivo therapeutic activity of low dose IgG2b and IgG3 was better than that of IgM mab.At the same time,we also evaluated the protective effects of 7G1-IgG and IgM mAbs in different ways of administration,we found that IgM mab can only play a protective role by intraperitoneal administration,but can not be protected by tail vein administration,while IgG mabs either intraperitoneal administration or intravenous administration,can play a good protective effect in vivo.Therefore,in the choice of route of administration,IgG mabs also showed an advantage.In conclusion,in this thesis,we generated class switching hybirdma cells from IgM to IgG with same binding properties and enhancer protective efficacy without the need of carry out antibody engineering and recombinant protein expression.The frequency of immunoglobulin class switching in hybridoma cells was increased via Tet-on based controllable expression of AID.Our study indicates that this approach may help generate IgG class-switched antibodies from IgM hybridoma cells not only in anti-Flu B but also in other pathogen mAb development.