Preparation Of Neutralizing Monoclonal Antibody Against TIGIT And Establishment Of Screening System

In recent years,tumor immunotherapy has shown great potential.For example,AntiPD1（Programed death 1）which has attracted much attention,has achieved good clinical efficacy in the treatment of a variety of tumors;Anti-CTLA4（Cytotoxic T lymphocyteassociated protein 4）has been the first and most common agent used in combination therapy with anti-PD1,compared with monotherapy.Previous studies have shown that dual blockade of CTLA-4 and PD-1 can significantly prolong the survival of patients with melanomas.However,in clinical applications,some patients are not sensitive to PD-1 blockade therapy,and due to the extensive expression of CTLA-4 on T cells,antiCTLA-4 may cause strong toxic side effects.T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif（ITIM）domain（TIGIT）has gradually attracted people’s attention as an important immune checkpoint.Compared with CTLA-4,TIGIT is a receptor of IgG superfamily mainly expressed in exhausted T cells and NK cells,so anti-TIGIT has higher safety in tumor immunotherapy.TIGIT has become an important target of tumor immunotherapy.TIGIT as a higher affinity molecule could competitively bind to poliomyelitis receptor（PVR）with CD226,resulting in the inhibition of lymphocyte activation,directly leads to the down-regulation of TCR expression,and activates the expression of immunosuppressive molecule FGL2.Therefore,the preparation of neutralizing monoclonal antibodies against TIGIT and the screening of neutralizing antibodies against TIGIT are of great significance for the development of tumor immunotherapy drugs.This topic aims to prepare neutralizing monoclonal antibodies against TIGIT and construct a detecting system for screening TIGIT neutralizing antibody.The intact protein was usually used as immunogen in traditional preparation of neutralizing monoclonal antibody,and the antibody was obtained through a lot of screening and identification.However,the epitope targeted by the antibody was uncertain,and complex epitope identification work was required,which was difficult and inefficient.In order to solve the above problems,taking TIGIT as an example,this project designed a more optimized scheme for the preparation and screening of neutralizing antibodies.First of all,bioinformatics software was used to analyze the 3D structure of TIGIT protein,and the epitopes located in the binding site of TIGIT and its receptor PVR were screened.At the same time,hepatitis B virus core protein（HBc）and adenovirus type 5（Ad5）were used as immune vectors to present epitopes,so as to obtain better immune effect.After the preparation of immunogen,BALB/c mice were immunized by a variety of immunization methods,and then monoclonal antibodies were prepared by hybridoma technology.Monoclonal antibodies against TIGIT were identified by screening antigens.The antibodies were identified by specific detection,titer detection and other experiments.At the same time,based on the CAR-T cell activation,TIGIT neutralizing antibody screening system was established,and finally the monoclonal antibody with neutralizing activity was screened.The main contents are as follows:1.Screening of TIGIT B-cell epitopeTwo potential neutralizing B-cell epitopes were identified by bioinformatics websites,comparing several parameters and referring to the crystal diffraction results of TIGIT and PVR.The DNA sequences encoding the two epitopes were synthesized for subsequent antigen preparation:Epitopel（EP1）:VNWEQQDQLLAICNADL（V57-L73）Epitope2（EP2）:HTYPDGTYTGRIFLEVL（H111-L127）2.Expression and purification of immunogenHepatitis B virus core granule protein（HBc）and human adenovirus serotype 5（Ad5）were used as immune vectors to present epitope peptide,HBc-TIGIT EP1-2（EP1-2 represents EP1 or EP2）fusion proteins and TIGIT EP1-2 modified recombinant adenovirus were used as immunogen to prepare monoclonal antibody against TIGIT:（1）Expression and purification of HBc-TIGIT EP1-2 fusion proteinsTwo epitope peptides were inserted into the immunodominant region（MIR）of HBc by molecular cloning technique to construct prokaryotic expression vectors of pET-28a/HBc-TIGIT EP1-2 and the obtained prokaryotic expression vector was electroporated into E.coli Rosetta（DE-3）respectively.HBc-TIGIT EP1-2 fusion proteins expression were induced by IPTG and prepared by Ni-NTA affinity chromatography.（2）Packaging and purification of adenovirus modified by TIGIT EP1-2An E1 region shuttle vector pAd5-E1/H1H2-HBc-TIGIT EP1-2 expressing a secreted HBc-TIGIT EP1-2 fusion protein and an adenovirus backbone vector pAd5/Hexon-TIGIT EP1-2/E3-CMV-Luciferase-T2A-eGFP modified by TIGIT EP1-2 in the HVR5 region of Hexon were constructe.The constructed shuttle vector and backbone vector were linearized by ScaI and ClaI,the adenovirus vector pAd5backbone/E1-CMV-H1H2-HBc-TIGIT EP1-2/Hexon-TIGIT EP1-2/E3-LuciferaseT2A-eGFP was obtained by transforming into E.coli BJ5183 competent cells.The vector was transfected into HEK293 cells.The packaged and purified adenovirus was named Ad5-El-CMV-H1H2-HBc-TIGIT EP1-2/Hexon-TIGIT EP1-2/E3-luciferaseT2A-eGFP.3.Expression and purification of monoclonal antibody screening antigenIn this project,the fusion proteins of Human progranulin E domain（GrnE）carrying TIGIT EP1-2 were used as monoclonal antibody screening antigen.The prokaryotic expression vectors pET-28a/GrnE-TIGIT EP1-2 were constructed,and the fusion proteins of GrnE-TIGIT EP1-2 were prepared to screen monoclonal antibody against TIGIT were expressed and purified respectively.4.Preparation and detection of monoclonal antibody against TIGIT epitopesFirstly,the monoclonal antibody was prepared,BALB/c mice were cross immunized with the above two kinds of immunogens.After immunization,the mice with high titer were killed three days later after booster immunization to prepare hybridoma cells.The positive cell lines were screened by screening antigen to prepare monoclonal antibody.The four antibodies against TIGIT EP1 and four antibodies against TIGIT EP2 were identified by Western blot,ELISA and immunofluorescence cytochemistry.5.Establishment of neutralizing monoclonal antibody screening system for TIGIT and detection of neutralizing activityThe extracellular segment of PVR gene was inserted into the lentiviral vector carrying the Chimeric Antigen Receptor（CAR）and transfected into HEK293 cells to prepare lentivirus.Jurkat-NFAT-Luci-PVR-CAR-T effector cell line was obtained by Puromycin from Jurkat cells infected with lentivirus carrying PVR extracellular（EX）and NFAT regulated luciferase reporter gene.CHO cells were infected with lentivirus carrying the extracellular segment of TIGIT gene,and the target cell line of TIGIT CHO was obtained by Puromycin screening.When these effector cells were co-incubated with target cells,PVR-CAR on the surface of effector cells was activated,and luciferase expression downstream of NFAT transcription factor was activated.The neutralizing antibody of TIGIT blocked the binding of TIGIT to PVR,and Jurkat cells could not be activated normally,which inhibited the expression of luciferase.Therefore,the system can be used to screen the neutralizing antibody of TIGIT sensitively and efficiently.Three neutralizing antibodies,anti-TIGIT EP1 No.1-1,No.5-1 and No.7-3,were screened out from the eight antibodies obtained in this project.Among them,antiTIGIT EP1 No.1-1 and No.7-3 had higher neutralization effect.In conclusion,the subject successfully screened two antigenic epitopes of TIGIT with potential neutralizing activity,and successfully prepared recombinant adenovirus and fusion protein carrying TIGIT epitopes;have successfully screened and identified four antibodies targeting TIGIT EP1 and four antibodies targeting TIGIT EP2;three strains of TIGIT neutralizing antibodies were screened by using the successfully constructed TIGIT neutralizing antibody screening system in this project.TIGIT was used as the target molecule to design a set of efficient epitope antibody preparation and sensitive antibody neutralization activity detection scheme,which not only provides a powerful tool for TIGIT molecular mechanism research,but also has important significance for the development of related immunotherapy drugs.At the same time,this scheme can also be used in the antibody preparation process of other target molecules,and has great application prospects.