Research On Effects Of Two Non-competitive Epitope Antibodies On The Endocytosis Of Epidermal Growth Factor Receptor And Related Mechanisms

As a member of ErbB family,epidermal growth factor receptor(EGFR)has become an important target of cancer therapy.In view of the developed monoclonal antibody drugs targeted to receptors of ErbB family,such as Cetuximab,Pertuzumab,Trastuzumab,etc.can bind with the extracellular domain of the epidermal growth factor receptor(EGFR)specifically,thereby blocking its combination with other ligands,preventing the activation of downstream signaling pathways,which is contributed to the inhibition of tumor growth.In animal and clinical trials,it has been reported that when two non-competitive epitopes antibodies are used together,the tumor inhibition rate will be significantly increased compared with the single use of antibodies.On the study of antibody combination use mechanisms,Yarden found that,compared with the single use of antibodies,after a combination use of two noncompetitive epitopes antibodies,more endocytosis of EGFR occurs on cell surface.This inhibits related intracellular recycling of EGFR and endosomes,promotes the degradation of intracellular EGFR,also enhances degradation of endosomes in the cell.This results in effective down-regulation on the number of EGFR on cell surface,thereby inhibiting growth signals in cells.It can be seen that a high strength endocytosis of EGFR in this process is the key to the mechanism,yet the reason is still unclear.Studies on clathrin-dependent internalization model in EGFR is mature,but it is unable to explain the internalization behavior of EGFR under the action of using two non-competitive epitope antibodies.According to the characteristics of the non-competitive antibodies,researchers has put forward a Lattice Model hypothesis that as a result of the non-overlapping of epitopes,two antibodies may bind with two EGFR on the membrane by two variable regions.The hypothesis speculated that the high strength EGFR internalization could be attributed to antibody-receptor complexes in high molecular weight that are formed on cell surfaces,thus to intense accumulation of EGFR on the cell membrane and internalization.However,there is still a lack of more experimental evidence to support the mechanism,especially the Lattice model observation on the cell membrane.In this paper,immunofluorescence method and 3D-SIM ultrahigh resolution microscope were mainly used in the experiment to provide evidence support for the existence of Lattice on the cell membrane,it further explored the confirmation of the Lattice hypothesis and determined the endocytosis way of EGFR when combined with two non-competitive antibodies.A variety of competitive and non-competitive EGFR antibodies were used and the cell actin was stained to observe the morphological changes of cytoskeleton to confirm the endocytosis way of EGFR induced by two noncompetitive antibodies is micropinocytosis.The Lattice on cell surface is observed successfully which provides evidence to support the existence of the Lattice.In addition,this study found that phosphatidylinositol-4,5-bisphosphate(PIP2)is another crux for high strength macropinocytosis induced by non-competitive antibodies.This study provides reliable cell biological data for the selection and design of antibodies,the promotion of clinical combination uses of antibodies,and the active selection of EGFR endocytosis way for EGFR internalization in large quantities.It also provides optimization direction and mechanism references for the antibody design of ADC drugs.