Monoclonal Antibodies Targeting The Dimer Interface Of Epidermal Growth Factor Receptor

Epidermal growth factor receptor (EGFR) is one of the key members of the EGFRfamily, It is a transmembrane glycoprotein, including the extracellular domain thatcontaining the ligand binding region, the transmembrane region and the intracellulardomain that containing the tyrosine kinase active region. The statistical data represents thatEGFR overexpressed in30%of epithelial cancers, including non-small cell lung cancer,head and neck cancer, colorectal cancer, glioma, breast cancer, renal cancer, prostate cancer,ovarian cancer and gastric cancer,and sadly the kinds of cancer patients always associatedwith poor prognose, short survival and risestent to chemical therapy. So, EGFR is theattractive therapical target, also the first international cancermolecular target. There are thetwo very popular strategies for EGFR tumor: the EGFR monoclonal antibody targeting theextracellular domain and tyrosine kinase inhibitors against the intracellulardomain.However, statistics demonstrates that both drugs have very low respose rate (about10-30%),which is closely related to deletion or hypemutation of extracellular domain ofEGFR and intracellular domain.The “dimerization” played a crucial role in the activation ofthe EGFR family in most case, furthermore, The "interface region exposure " whichparticipates in dimerization mainly is a necessary for forming dimers,and it is highlyconservative, so targeting the dimer interface may promote the clinic response rates in the"anti-EGFR" therapeutic. Monoclonal antibody is the antibody targeting the single epitope,it is capable of specifically binding and killing of selective target cells. The experimentsintended to preparate a class of murine monoclonal antibodies targeting the EGFR dimerinterface, use the B cell epitope peptide-EGFR267-297that our laboratory isolated andidentificated EGFR B cell epitope peptide in preliminary work as the antigen, determine thebest construct throught analysising the immunogenics. Evaluating the antitumor activity in vitro and analyzing the biological characteristics to lay a good foundation. for humanizedmonoclonal antibody and in vivo experiments.Method:1Immunogenicity comparison of the immuse four different constructing forms ofEGFR267-297peptideObtaining the coupling peptide(BSA*EGFR267-297and OVA*EGFR267-297)inglutaraldehyde conjugated method, comparising the immunogenicity of the two fusionpeptide MVF-EGFR267-297, P64K-EGFR267-297and the coupling peptide P64K*EGFR267-297(the three forms constructed in previous work) and BSA*EGFR267-297by immunizingBalb/c mices and use OVA*EGFR267-297as detecting antigen to detect the serum titer inthe methond of indirect ELISA, choose the highest immunogenicity as the immunogen.2Production of monoclonal antibodies (mAb) targeting the dimmers interfaceImmunized Balb/c mices with the best immunogen, then assay immunogenicity inindirect ELISA, the titer1:8000is required. Established the mAb hybridoma cell lines inconventional methods,The Pure mAb was obtained by injecting Balb/c mices’ intraperitonto collect ascites,then purifing by PEG6000and Protein A.Detecting mAb titer inELISA,assaying kaff and identificating the monoclonal antibody subclass and light chain.3Cell growth inhibition assay of the mAb in vitroBioconversion of MTT was used to estimate whether the anti-peptide Abs can inhibitthe proliferation of the EGFR overexpression cell line human breast cancer (MDA-MB-231), and EGFR normal expression cell line mouse fibroblast (3T3).4Biology characterization of the mAbAnalysising the monoclonal antibody-induced MDA-MB-231cell apoptosis by flowcytometry;Observing the changes in cell morphologylaser after mAb induced MDA-MB-231cell apoptosis in aggregation; Observing the combination of mAb with MDA-MB-231cells and LLC cells surfaces and then internalization of mAb in cells.Results:1Immunogenicity comparison of the immuse four different constructing forms ofEGFR267-297peptideChoosed the coupling peptide BSA*EGFR267-297as the immuse peptide to preparatethe mAb because its’ immunogenicity is highest,andchoosed the OVA*E as the detectionof antigens in order to exclude background. 2Production of monoclonal antibodies (mAb) targeting the dimmers interface6stable hybridoma lines were established. The purity of the mAbs up to95%, mAbheavy chains was about50kDa, light chain25kDa, the highest monoclonal antibody titerhigher than1:2560000, the four high titers mAbs were high-affinity (K>108). Testsshowed the three mAbs’ subclass were IgG1and one was IgG2a, all light chains are Kappa.3Cell growth inhibition assay of the mAb in vitroMTT assay results showed that, The proliferation inhibition rates of EGFR267-297mAb(200μg/ml) and Cetuximab(200μg/ml) positive control group to MM-231cells are46.37%and51.3%separately,but no obvious inhibition was observed by3T3, indicatingthat the mAb can inhibite EGFR overexpressing cancer cells significantlyd and less sideeffects on normal cells.4Characterization of the mAbFlow cytometry analysis showed that low,middle,high concentrations group ofEGFR267-297mAb induced MDA-MB-231cells in early apoptosis rates were:41.3%,43.7%,57.4%, late apoptosis rates were:20.8%,28.6%,13.4%, and total apoptosis rates were:62.1%,72.3%,70.8%, the rate of apoptosis was higher than the cetuximab groups.Theconfocal observation declare that the mAb induced mAb MDA-MB-231cells toapoptotis,chromatin shrinkage, peripheralied,and there are scattered around the granularpoints; The confocal observations declare EGFR267-297mAb were capable to specificallybind to MM-231and LLC cells surface then internalized.Conclusion:6stable hybridoma lines were established successfully,the EGFR267-297mAb can inhibitthe proliferation of EGFR overexpression cell and induce EGFR overexpression cell toapoptosis significantly. The binding and internalization of the mAb to EGFRoverexpressing cell were observated in vitro,which layed a good foundation.forexperiments in vivo.