The Discovery And Characterization Of An Anti-HCC Epitope On EGFR Loading Catastrophic Internalization

As a well-known therapeutic onco-target,epidermal growth factor receptor(EGFR)has played an important role in the treatment of various tumors,such as lung cancer.Two main anti-EGFR strategies are currently exploited: anti-EGFR monoclonal antibodies,such as Cetuximab,which bind directly to the ligand-binding site on the ectodomain,disturb the activation of downstream signaling pathways;tyrosine kinase inhibitors,such as Erlotinib,that could internalize into cells and inhibit the phosphorylated activation by binding with the intracellular tyrosine kinase site of EGFR.However,those anti-EGFR agents have not been proved positive evaluation in hepatocellular carcinoma(HCC)despite the expression of EGFR is commonly high.It has been reported that the complex signaling pathways is the main reason of anti-EGFR drug resistance in HCC cells.At present,the unique international recognized drug could benefit HCC patients is Sorafenib.Due to inhibiting multiple intracellular pathways,Sorafenib has several shortcomings such as insufferable side effects and short prolonging survival.Therefore,it still lacks more specific targeted drugs in HCC clinical therapy.The internalization of EGFR is extremely complicated and most studies focus on the pathway and intensity of its internalization.It has been reported that EGFR can enter cells by various endocytic pathways and the combinations of monoclonal antibodies induce more internalization of EGFR.A synergistic antibody combination containing two antibodies which bind to different epitopes of EGFR,called as Sym004,induced rapid internalization and degradation of EGFR that leads to down-regulation of EGFR and subsequent inhibition of cancer cell growth.In 2017,Sym004 was advanced through a Phase II trial in patients with metastatic colorectal cancer(mCRC)that have acquired resistance to anti-EGFR antibody therapies or mutations in RAS and BRAF and the data demonstrated that Sym004 could provide survival benefits or stabilization.It can be seen that rapid internalization and recycling blockage of EGFR may be a novel strategy to develop anti-EGFR agents for HCC therapy.In this study,the anti-tumor activity of an immunomodulatory protein from Ganoderma lucidum(rLZ-8)in HCC was investigated firstly.In vitro experiments,rLZ-8 could inhibit the growth of HCC cell line Hep3 B cells;in vivo experiments,rLZ-8 could significantly inhibit the tumor growth in nude mice orthotopic HCC models,and the inhibitory rate of tumor was improved compared with Sorafenib group.Besides,rLZ-8 could prolong the survival time of mice.Therefore,rLZ-8 possessed significant anti-tumor activity in HCC.Then the anti-tumor mechanism of rLZ-8 in HCC was studied.By super-resolution imaging and multiple intracellular imaging experiments,it was found that rLZ-8 could induce rapid and catastrophic internalization in Hep3 B cells by macropinocytosis.The results of intracellular localization tracking in Hep3 B cells demonstrated that rLZ-8 did not fuse with lysosomes after entering cells and the vesicles stayed in late endosome stage,which led to the blockade of plasma membrane recycling,Hep3 B cells were ruffling,contraction,rounding,cell burst and death finally.The high intensity internalization of rLZ-8 and the blockage of cell membrane recycling were the key points of the mechanism,and this catastrophic internalization was likely to be mediated by some kind of cell membrane receptor.The co-localized observation of cell membrane receptors with rLZ-8 showed that EGFR was the main receptor of rLZ-8 in HCC cells.The correlation analysis between the pharmacodynamics and EGFR expression in patient-derived tumor xenograft(PDX)models of HCC showed that the tumor inhibitory rate of rLZ-8 was correlative with the EGFR expression.By PLA dimerized detection and the phosphorylated detection of intracellular signaling pathway,it was found that rLZ-8 did not induce the dimerization and phosphorylation of EGFR.Previous results showed that rLZ-8 could cause cell death by high intensity internalization and blockage of cell membrane recycling,while other anti-EGFR antibodies did not induce this intense internalization.The reason for this difference seemed to be derived from the diverse binding sites on EGFR,thus the binding site of rLZ-8 and EGFR would be investigated.A new anti-EGFR antibody which possesses competitive epitope with rLZ-8 was obtained by antibody screening,the new antibody could induce catastrophic internalization in Hep3 B cells and the internalized quantity was far more than other anti-EGFR antibody such as mAb806 and Cetuximab.By chemical cross-linking coupled with mass spectrometry(CXMS)and molecular docking,the binding site of rLZ-8 and EGFR was analyzed and calculated,which the key sites on rLZ-8 may be(S18,D20,K41)and the one on EGFR may be(S196,S222,K269,S282).According to above results,the mutant proteins of rLZ-8 or EGFR were designed and expressed.Biacore assay demonstrated that the affinity of K41-based mutants of rLZ-8 and EGFR was reduced significantly.The internalization of these mutants in Hep3 B cells decreased comparing with that of rLZ-8,these mutants were almost not bind with EGFR on cell membrane,which indicated that K41 in rLZ-8 crystal structure is a key residue in binding with EGFR.Biacore analysis showed that the mutants of EGFR extracellular domain have lower ability to bind to rLZ-8 compared with EGFR.After NIH-3T3 cells expressing wild-type EGFR or mutants were constructed,it was found that the binding ability of rLZ-8 to EGFR mutants was weaker than that of wild-type EGFR.Thus S222/K269 in EGFR are the binding sites related to the internalization activity of rLZ-8.In summary,this study reveals that rLZ-8 has significant anti-tumor activity in HCC,and rLZ-8 could bind with the S222/K269 on EGFR and trigger an anti-tumor mechanism with catastrophic internalization and blockage of cell membrane recycling in HCC cells.Thus,this study finds and characterizes a novel epitope on EGFR for HCC therapy.The epitope could cause high intensity internalization of EGFR via macropinocytosis and recycling blockage of cell membrane.The novel oncotarget of EGFR in this study may facilitate the design and development of more effective anti-EGFR agents for HCC clinical therapy.The related characteristic indexes of the structural domain provide an optimized direction for monoclonal antibodies and antibody-drug conjugates designing targeting EGFR.These findings highlight rapid internalization of EGFR as a promising strategy to maximize EGFR inhibition that may induce more potent HCC tumor suppression than current clinically anti-EGFR agents.