Peptide-based B-cell Epitope Vaccine Targeting Dimer Interface Of Epidermal Growth Factor Receptor (EGFR)

Targeting is the key to develop the agent having high therapeutic index and low adverseeffect. Despite expressing in normal tissue, epidermal growth factor receptor (EGFR) isover-expressing in most epidermoid manligent and associated with poor prognose, shortsorvivor and risestent to chemical therapy. Activation of the EGFR signaling pathway incancer cells has been linked with increased cell proliferation, angiogenesis, and metastasis,and decreased apoptosis, which can be avoided by inhibition the activation of the receptor.So the EGFR is the attractive therapical target. To date, the agents targeting the receptor aremonoclonal antibodies (mAbs) and TKI. Despite having significant therapy effect in sometypical cases and low adverse effect observed, both drugs have very low respose rate (about10-25%), which is closely related to hypermutation of the extracellular and intracellulardomain of the EGFR. It is necessary to develop a new strategy targeting to EGFR. Largestudies showed that “dimerization” played a crucial role in the activation of the receptor.Furthermore, the dimer interface is highly conservative, so targeting the dimer interfacemay promote the clinic response rate and broaden the anti-tumor spectrum of theanti-EGFR agents. The B-cell epitope peptide vaccin, having attractive applicationperspective, took effect by immunization of low dosage and limited times and has the sameeffect of mAb that need to repeatedly and chronically administer high dose antibody. Theaim of this study is to contruct a novel B-cell epitope peptide vaccine targeting the dimerinterface of EGFR.Methods:1. Prediction of the B-cell epitope from EGFR dimer interface The dimer interface was selected accoding to the crystal structure of EGFR displayedby the Cn3D-4.1software, then the B-cell epitopes in this dimerization domain werepredicted according to the characteristic of hydrophilicity, accessibility, antegenicpropensity and turns of the amino acids residual.2. Design of the B cell epitope antigen peptideThe selected B cell epitopes were respectively fused to the C terminal of promiscuousT cell epitope derived from the measles virus fusion protein via a four-residue turnsequence (GPSL).3. Clone, expression and purification of B cell epitope antigen peptideThe DNA sequence coding the antigen peptide was amplificated by SOE-PCR, thencloned into pET32a to be expressed, fused to Trx and His6tag, under the control of the T7promoter. The fusion proteins were purified by Ni2+chelating chromatography. The antigenpeptides were recovered from enterokinase-cutting production of fusion protein by Ni2+chelating chromatography.4. Immunological analysis of B cell epitope antigen peptideBalb/c mice were used to analyzed the immunogenicity of the antigen peptide. Theanti-peptide Abs were captured from the serum obtained from rabbit immuned with the3rdpeptide by immune affinity chromatography. The specific antibody IgG was monitored byELISA.5. Cell growth inhibition assay of Anti-peptide AbsBioconversion of MTT was used to estimate whether the anti-peptide Abs can inhibitthe proliferation of the EGFR overexpression cell line (A431,A549,LLC), and EGFR lowexpression cell line(3T3,SKBR-3).6. Assay of anti-peptide Abs binding to native EGFRThe specific binding capability of the anti-peptide Abs to A431cell from rabbitinduced by peptide were evaluated by Flow cytometry and Immunofluorescence. Therelationship between binding capability and growth inhibition were established too. Theinfluence of serum-starvation and EGF-stimulation in binding capability of anti-peptideAbs to A431cell were used to evaluated whether the Abs can target the EGFR dimer Interface.7. Transplantable tumor challenge modelsThe C57BL6was used as LLC transplantable tumor model to evaluated the growthinhibition and survival prolongation effect of antigen peptide immunization.Results:1. B cell epitope peptides from the dimer interface of EGFRThree B cell epitope peptides were selected in the rich region of disulfide-bond locatedin dimerization domain of EGFR. Of these peptides, EGFR237-267selected from theβ-hairpin extension, which directly contact with another mono EGFR, was53%sequencehomology with the B cell epitope of the anti-HER2mAb(pertuzumab) and100%sequencehomology among human and mouse. The EGFR287-303was100%sequence homologywith the anti-EGFRvIII mAb806and80%sequence homology among human and mouse.The EGFR237-303contain EGFR237-267aa and EGFR287-303with a20aa residueinterval.2. Design and preparation of antigen peptideThe coding sequences of antigen peptides were successfully synthesised by SOE-PCR.The antigen peptide genes can’t be expressed independently when cloned in pET21a, butover-expressed when fused to C-terminal of Trx in pET32a. The antigen peptide wasrecovered from the fusion protein by enterokinase and purified to a purity of95%.3. The immunogenicity of antigen peptidesAfter vaccinated with antigen peptides specific IgG antibodies can be detected inserum with the highest titers above1︰64000. The titers induced by3rd peptide in rabbit ishigher than1︰500000. The purity anti-peptide were captured from serum by immuneaffinity chromatography.4. Effects of purified antipeptide Abs on the proliferation of tumor cell.Bioconversion of MTT showed that the inhibition effect of the3rd rabbit anti-peptideAbs to A431cell is dose-dependent with23.5%inhibition rate(400μg/ml). At the does of200μg/ml, the Abs have litter effect on the proliferation of SKBR-3and LLC cells (8.5%and8%inhibition rate, respectively), no obvious inhibition was observed by3T3and A549 cells.5. Biological properties of the anti-peptide antibodiesThe result of Flow cytometry showed that the binding capability of the3rdrabbitanti-peptide Abs to A431is72.3%and98.5%with50and500μg/ml does specific. Whilethe binding capability of the rabbit anti-peptide Abs to serum-starved A431cell is11.7%,31.8%and50.9%with the stimulation of0μg/ml，5μg/ml and50μg/ml EGF. The3rdrabbitAnti-peptide Abs showed marked membrane staining of normally cultivated A431cancercells and FCS starved cells activated by EGF compared to seldom binding to starved A431cells.6. The experimental therapeutic effect of the antigen peptide vaccineThe growth rate and volume of tumor of the3rdpeptide immunized mouse are smallerthan negative control in the preliminary experiment. Furthmore, the peptide cansignificantly prolong the survival time of the tumor challenge mice.Conclusion:1. Three B cell epitope peptides from the dimer interface of the EGFR weresuccessfully selected. Moreover, the EGFR237-267is a novel B cell epitope and wasapplied for patents of inventions.2. We successfully established a preparation method for antigen peptide, which fuse apromiscuous Th cell epitope to B cell epitope from EGFR dimer interface, using DNArecombiant technology.3. Antigen peptide constructs can break the immune tolerance of host to autoantigenand elicit high titers specific antibody.4. The anti-peptide Abs raised by rabbit can specifically bind to EGFR on the surfaceof cell and inhibit the proliferation of EGFR overexpression cancer cell. The bindingcapability of anti-peptide Abs is associated with its proliferation inhibition.5. The binding of anti-peptide Abs to EGFR on the surface cell is synergistic with theEGF stimulation, So, the anti-peptide Abs is targeting the EGFR dimer interface, which iscoincidencing with design.