The Study Of Anti-tumor Efficacy Of Anti-c-MET VHHs

Receptor tyrosine kinases(RTKs)are high affinity receptors on cell surface,they belong to protein tyrosine kinases family and their ligands are growth factors,cytokines,and hormones.RTKs can not only promote normal celluar processes but also play a critical role in the development and progression of many types of cancer.Mutations in RTKs affect the expression level of different proteins and celluar processes because of activation of a series of signaling cascades.C-MET,encoded by MET gene,is a member of RTKs family.C-MET is a single transmembrane protein and its ligand is hepatocyte growth factor(HGF).C-MET is normally expressed by cells of epithelial,while expression of HGF is restricted to cells of mesenchymal.When HGF binds c-MET it induces c-MET dimerization and its activation.C-MET and HGF play crucial roles in many human malignancies.C-MET activation in cancer is related to poor prognosis.Abnormal c-MET activation triggers tumor growth,angiogenesis and metastasis and deregulation of HGF/c-MET signaling is observed in many types of malignancies including cancers of kidney,liver,stomach and breast,which makes c-MET become a crucial target in treatment of cancers.Blockage of binding between HGF and c-MET is a prime strategy for HGF/c-MET targeted cancer therapy.Antibody drugs are more attractive in targeted therapy than chemical drugs because of their low side effect.VHH is the recombinant variable region of camelid heavy-chain antibody,it shows excellent stability and it's easier to tailor than conventional antibody.As a smaller antigen-binding unit,VHH has become an appealing drug candidate in cancer therapy.In this study,a novel strategy to construct an anti-MET VHH pool against the whole ecto-domain of MET was used.Prime results of the thesis are listed below:1.Generation of c-MET specific VHHsPrevious studys shown,c-MET has a semaphorin domain through which it binds to HGF.To develop antibodies block the binding between them,we prepared semaphorin domain and extracelluar domain of c-MET for animal inoculation and antibody screening.After the inoculation,a VHH library with 1×10~8 capacity was constructed successfully.Taking advantage of phage display and bio-panning,33 unique specific antigen binders were obtained by ELISA.Then the specific VHHs were subjected to HepG2 cell.The data shows that 18 of 33 VHHs have inhibitory effect on HepG2,and10 of 18 VHHs have stronger function than the rest.2.Prokaryotic expression,characterization and functional analysis of c-MET specific VHHsTen VHHs were subcloned into pET28a(+)vector.After transformation into E.coli BL21(DE3),10 VHHs were expressed and purified.K_Ds of each VHH to c-MET and epitope binning assay were determined by bio-layer interferometry.Western blot data shows that individual VHH and TOP10MIX(10 VHHs were mixed at equal molar)promote c-MET dephosphorylation in HepG2 cell,TOP10MIX shows the strongest function.Subsequently,the function of TOP10MIX in different types of cancer cell were tested,the data shows that TOP10MIX has outstanding function.To verify the fuction of subgroups of TOP10MIX,narrow-down experiment of TOP10MIX was performed.5G7,4G3,3G4 and 2G1 were wined out according to their HepG2 cell inhibitory effect.In western blot analysis,2G1 shows good ability to promote c-MET degradation and dephosphorylation in HepG2 cell.But compared to TOP10MIX,2G1acts unpromising inhibitory function of cancer cell proliferation,viability and colony formation.3.Function and machenism dissection of TOP10MIX in xenograft tumor modelsAbout 200mg purified VHHs were obtained.C-MET degradation and dephosphorylation were observed in different types of cancer cell.Compared to therapeutical drug one-armed 5D5,TOP10MIX acts more powerful on Xenograft tumor models in promoting c-MET degradation,p-MET dephosphorylation and suppression of tumor growth.Endocytosis of memberane c-MET is mainly mediated by clathrin.TOP10MIX can interact with c-MET and clathrin,and the interaction of TOP10MIX and clatherin is c-MET dependent.