The Research Of A Novel VEGF-target Antibody And The Molecular Mechanisms Underlying VEGF-induced Tumor Progression

VEGF plays a critical role in tumor initiation and progression. VEGF can interactwith VEGFR2on vascular endothelial cells membrane, and promote growth,migration, tubular formation of endothelial cells and enhance vascular permeability tomeet the needs of the oxygen and nutrients of tumor through activation ofdownstream signal. Blocking VEGF-VEGFR2signal pathway to inhibit abnormalangiogenesis is currently proven strategy for tumor therapy. Avastin is a recombinant,humanized monoclonal antibody targeted VEGF. Avastin has been approved by FDAfor the treatment of metastatic colorectal cancer and non-small-cell lung cancer incombination with chemotherapy. For both academic and applied purposes, it issignificant to construct a novel VEGF-target antibody of our own intellectualproperty.VEGF is an important proangiogenic factors, however, recent researches show thatthe function of VEGF is not limited to angiogenesis and vascular permeability intumor progression. VEGF interacts with cell membrane receptors of tumor cells, andpromote the growth, migration and invasion of cancer cells and formation of cancerstem cell through activation of downstream signal. VEGF also can contribute theinflammation by regulating the expression of miRNAs. However, whether VEGFinvolves in tumor progression thought regulating the expression of miRNAs remainsto be elucidated.In the present research work, on the basis of the antibody and antibody-likemolecular design platform, a novel anti-VEGF antibody MIL60was obtainedaccording to the3-D complex structure of VEGF and Avastin. The anti-angiogenicefficacy of MIL60was examined by biological experiments, furthermore, the molecular mechanisms on VEGF that promoting the process of tumor by regulatingthe expression of miR-21was studied.1. The design of a novel VEGF-target antibody and the investigation of biologicalfunctionThe interaction mode between MIL60and VEGF was studied theoretically basedon molecular modeling and dynamical docking methods. The result showed that thebinding mode of MIL60and VEGF was same as that of Avastin and VEGF. Thebiological experiment results show MIL60had the high specificity and affinity toVEGF. MIL60and Avastin had similar affinity to VEGF (the KDs of MIL60andAvastin were44.5pM and42.7pM, respectively), and recognized the same epitope.The results in vitro showed: MIL60could effectively blocked the binding ofVEGF to VEGFR-2of HUVEC, and inhibited VEGFR2phosphorylation anddownstream P38/IKK/NF-κB, ERK signal activation, and inhibited VEGF-inducedpro-angiogenic effects of HUVECs, such as proliferation, migration, and tubeformation. The results of carcinoma xenograft mouse model showed that the growthrate of tumor volume was significantly reduced, the tumor weight was significantlylow, and the microvessel density in the tumor decreased obviously by MIL60.The biological activity of MIL60was similar to that of Bevacizumab, whichshowed that MIL60could effectively inhibit tumor growth thought inhibitingangiogenesis.2. The research of the molecular mechanisms underlying VEGF-induced tumorprogressionMiRNAs microassay analysis showed that the expression of miRNAs hadsignificant change after treatment with VEGF targeted antibody, and the expression ofmiR-21was down-regulated significantly. The main mechanism of VEGF targetedantibody is binding VEGF, and blocking VEGF-VEGFR2signal pathway, therefore,we concluded that VEGF might involve in up-regulation the expression of miR-21.Further research showed VEGF induced by HIF-1a or exogenous can specificallyup-regulated the expression of miR-21, MIL60could reverse the effect, suggesting VEGF could up-regulate the expression of miR-21through an autocrine mechanism.VEGF need interact with tumor cell membrane specifically receptors to activatedownstream signal. Blocking the signaling by specifically antibody or siRNAinterference the expression of VEGFR1indicated that VEGF-VEGFR1signalingcould induced NF-κB phosphorylation and nuclear translocation, and up-regulate theexpression of miR-21.Over-expression of miR-21could promote the migration of HT-29cells, while notaffect their proliferation. Using Tarbase protein6.0database and clustering analysisby DAVID, further validated by means of biology indicated miR-21may promotedHT-29cells migration by targeting several tumor suppressor genes, such as PTEN,APC, TGF-βRⅢ.The innovation of this study is that: the MIL60can effectively inhibitangiogenesis and tumor growth through specifical blockage VEGF-VEGFR2signaling, and could become an anti-tumor antibody candidate. Our research alsoreveals the new mechanism on VEGF that promoting the process of tumor. VEGF canspecifically interacts with VEGFR1of tumor cells, activate VEGF-VEGFR1-NF-κBsignaling, and up-regulate the expression of miR-21; miR-21promoted HT-29cellsmigration by targeting several tumor suppressor genes. From another prospect thisstudy also indicate that VEGF targeted antibody not only can inhibit angiogenesis andtumor growth through specifical blockage VEGF-VEGFR2signaling, but alsodown-regulate the expression of miR-21, and inhibitor tumor metastsis throughblockage VEGF-VEGFR1signaling.